Patent Application
20220334101
The present invention provides for methods for identifying modulators of extracellular matrix (ECM) movement towards a site requiring deposition of ECM. Such modulators can be applied for use in a method for the modulation of ECM movement towards a site requiring deposition of ECM, e.g. a wound, thereby allowing treatment of a condition involving ECM deposition. Since the modulator may either be an inhibitor or promoter, either excessive or insufficient ECM deposition could be dealt with by the means and methods of the present invention.
In mammals, scars are formed when a specialized population of fibroblasts immigrates into wounds to locally deposit plugs of connective tissue matrix at sites of injury1. The origin of scar-producing fibroblasts, myofibroblasts, in wounds is unclear and so, by extension, is the mechanism by which they act2. Myofibroblasts are suggested to emanate from various sources, such as papillary (upper) and reticular (lower) dermal layers3, pericytes4, adipocytes5-6, and from bone-marrow derived circulating monocytes7.
The provenance of scar, myofibroblasts, and the mechanism by which they gain this unique capacity are thus still obscure despite scars being an extensively studied major clinical challenge. Indeed, when normal scarring fails, the result is either non-healing chronic wounds or aggravating scarring and fibrosis8-10. Impaired wounds and excessive scarring are a tremendous burden for patients and for the global healthcare system and they cost tens of billions of dollars per year, just in the US11. Understanding this fundamental patching process is therefore critical to restore and preserve the normal functions of injured adult organs.
It was previously demonstrated that all scars in the back-skin come from a distinct fibroblast lineage expressing the Engrailed-1 gene in embryogenesis12-13. This cell lineage is present not only in the skin, but also in the strata underneath the skin, called subcutaneous fascia. The subcutaneous fascia is a gelatinous viscoelastic membranous sheet of matrix that creates a frictionless gliding interface between the skin and the body's rigid structure below. For example, in the murine back-skin, the subcutaneous fascia is a single connective sheet that is separated from the skin by the Panniculus carnosus (PC) muscle, whereas in humans there is no intervening muscle and the subcutaneous fascia is relatively thick, consisting of several membranous sheets that are continuous with the upper skin layers. In humans the facia layers incorporate fibroblasts, lymphatics, adipose tissue, neurovascular sheets and sensory neurons14-15.
A major component of scars is extracellular matrix (ECM). Excessive as well as insufficient deposition of ECM is undesired, since it may result, e.g. in fibroproliferative diseases or chronic wounds, respectively. Many attempted are made in the prior art to deal with medical conditions concerning excessive or insufficient ECM deposition in the scar-process, but the process is still not clearly understood which hampers the development of beneficial treatment options. Hence, there is still a need to provide further options in the treatment of excessive or insufficient scar formation.
It is therefore desired to satisfy the need to provide further options in the treatment of excessive or insufficient scar formation.
The present invention addresses this need and provides options in the treatment of conditions involving ECM deposition, e.g. excessive or insufficient scar formation. Such conditions may be either excessive deposition of ECM at a site requiring ECM deposition or insufficient deposition of ECM at a site requiring ECM deposition.
Accordingly, in a first aspect, the present invention relates to a method for identifying modulators of extracellular matrix (ECM) movement towards a site requiring deposition of ECM, comprising (a) contacting extracellular matrix of organ tissue obtainable by biopsy from a mammalian subject with a label; (b) contacting said labelled extracellular matrix of organ tissue with a compound of interest; (c) determining whether said compound of interest modulates ECM movement towards said site requiring deposition of ECM in comparison to labelled extracellular matrix of organ tissue obtainable by biopsy from a mammalian subject which is not contacted with said compound of interest, wherein modulation of ECM movement towards said site requiring deposition of ECM is indicative for said compound of interest to be a modulator of said ECM movement.
The present invention may also comprise the method as described elsewhere herein, wherein modulation is inhibition.
Further, the present invention may also comprise the method as described elsewhere herein, wherein modulation is promotion.
Further being envisaged herein is the method as described elsewhere herein, wherein said organ tissue comprises fascia matrix, serosa and/or adventitia.
The present invention may also comprise the method as described elsewhere herein, wherein fascia matrix, serosa and/or adventitia comprises macrophages, neutrophils, mesothelial cells and/or fibroblasts.
The present invention may also encompass the method as described elsewhere herein, wherein ECM comprises proteins, polysaccharides and/or proteoglycans.
Also comprised by the present invention may be the method as described elsewhere herein, wherein the label is a dye or tag. Preferably, the dye is a fluorescent dye.
Additionally, the present invention may encompass the method as described elsewhere herein, wherein primary amine groups of extracellular matrix components are labelled.
Also envisaged herein is the method as described elsewhere herein, wherein the label is covalently coupled to extracellular matrix components.
Further, the present invention may also comprise the method as described elsewhere herein, wherein contacting extracellular matrix of organ tissue obtainable by biopsy from said mammalian subject with a label is achieved by contacting said extracellular matrix with a paper-like material comprising the label.
The present invention may also envisage the method as defined elsewhere herein, wherein fluid of said mammalian's body cavity is present during step (a), (b) and/or (c).
The present invention may also encompass the method as described elsewhere herein, further comprising step (a′) contacting said organ tissue obtainable by biopsy from said mammalian subject with a label visualizing cells comprised in the ECM.
It may also be comprised herein the method as described elsewhere herein, wherein the organ tissue is from skin, kidney, lung, heart, liver, bone, peritoneum, intestine, diaphragm or pleura.
According to a second aspect, the present invention relates to a method for identifying a biomarker associated with extracellular matrix (ECM) movement towards a site requiring deposition of ECM, comprising (a) contacting extracellular matrix of organ tissue obtainable by biopsy from a mammalian subject with a label; (b) isolating proteins from said labelled ECM which move towards said site requiring deposition of ECM; (c) determining at least a partial amino acid sequence of said proteins, thereby identifying said proteins as a biomarker associated with ECM movement.
Additionally, according to a third aspect, the present invention refers to a compound for use in a method for the modulation of extracellular matrix (ECM) movement towards a site requiring deposition of ECM, preferably in the treatment of a condition involving ECM deposition.
The present invention may also comprise the compound for the use as described elsewhere herein, wherein ECM movement is mediated by fascia matrix.
The present invention may also encompass the compound for the use as described elsewhere herein, wherein fascia matrix, serosa and/or adventitia comprises macrophages, neutrophils, mesothelial cells, and/or fibroblasts.
Also comprised by the present invention is the compound for the use as described elsewhere herein, wherein fascia matrix, serosa and/or adventitia comprises fibroblasts.
Also envisaged herein is the compound for the use as described elsewhere herein, wherein ECM comprises proteins, polysaccharides and/or proteoglycans.
Further, the present invention may also comprise the compound for the use as described elsewhere herein, wherein the site requiring deposition of ECM is a wound.
Additionally, the present invention may also encompass the compound for the use as described elsewhere herein, wherein modulation is inhibition. Preferably, inhibition of ECM movement towards a site requiring deposition of ECM prevents excessive deposition of ECM at said site. Even more preferably, excessive deposition of ECM is associated with fibroproliferative disease.
Further, the present invention may also envisage the compound for the use as described elsewhere herein, wherein the condition involving ECM deposition is excessive deposition of ECM. Preferably, excessive deposition of ECM is associated with fibroproliferative disease.
Additionally, the present invention may also encompass the compound for the use as described elsewhere herein, wherein modulation is promotion. Preferably, promotion of ECM movement towards a site requiring deposition of ECM prevents insufficient deposition of ECM at said site. Even more preferably, insufficient deposition of ECM is associated with chronic wounds.
Also envisaged herein is the compound for the use as described elsewhere herein, wherein the condition involving ECM deposition is insufficient deposition of ECM. Preferably, insufficient deposition of ECM is associated with chronic wounds.
Additionally, the present invention may also encompass the compound for the use as described elsewhere herein, wherein said compound is obtainable by the method for identifying modulators of extracellular matrix (ECM) movement towards a site requiring deposition of ECM as described elsewhere herein.
a) NHS-FITC labelling reveals surface ECM structures of liver, peritoneum and cecum. Representative images of three biological replicates SHG: second harmonic generation. Scale bars: 15 μM. Representative immunofluorescence image of a histological section showing NHS-FITC penetration depth. Scale bars: 10 μM. b) Patch mediated fate tracing of liver surface ECM in local electroporation injury model. c) Liver surface matrix flows upon injury response. Stereomicroscopic images of mouse livers 24 hours after electroporation against undamaged control. Representative images of three biological replicates. Scale bar overview: 2000 μM; High magnification: 100 μM. d) Fluid matrix is restructured during wound healing on liver surfaces. Representative H&E histology and multiphoton images of livers 24 hours and 14 days after electroporation. Scale bar overview: 50 μM; High magnification: 15 μM. e) Patch mediated fate tracing of peritoneal surface and local injury by brushing reveals motion of surrounding fibrous elements (NHS-FITC+) into wound areas (NHS-AF568+) after 30 minutes. Representative stereomicroscope images of three biological replicates. Scale bar overview: 1000 μM; High magnification: 100 μM. f) Peritoneal surface matrix flows laparotomy closure response. Stereomicroscopic images of mouse peritoneas 1 minute and 24 hours after laparotomy closure. Representative images of three biological replicates. Scale bar overview: 2000 μM. g) Fluid matrix currents flow into wounds for three days. FITC intensity of liver and peritoneal wound lysates after the indicated time points, n=three biological replicates. One-way ANOVA, multiple comparison Tukey's test, 95% Cl. h) Fluid matrix closes peritoneal laparotomy closures with net like structures. Representative H&E histology and multiphoton images of three biological replicates. Scale bar overview: 50 μM; High magnification: 15 μM.
a) Overview of patch mediated in vivo crosslink assay (see methods). b) Increasing FITC intensity of Streptavidin Pulldown samples reveals growing crosslinking over time in liver wounds. n=three biological replicates. One-way ANOVA, multiple comparison Tukey's test, 95% Cl. c) Four-week old organ fusions between Peritoneum (AF568+) and Cecum (FITC+). Representative images of n>three biological replicates. d) Matrix fusions between peritoneum and cecum in four-week old adhesions. Immunolabeling shows contribution of peritoneal Collagen 1 in cecum repair. Representative images of n>three biological replicates. e) Peritoneal matrix fuses livers and flows onto surfaces. Representative images of n>three biological replicates. Scale bar: 50 μM. f) Crosslinking between organ matrix fractions starts after two weeks. Cecum: NHS-FITC. Peritoneum: NHS-EZ-LINK-Biotin. n=three biological replicates.
a) Workflow of proteomic based identification of fluid matrix systems. b) Fluid matrix originates from multiple organ depths and layers. c) Fluid matrix fractions consist mostly of Collagens and ECM glycoproteins. d) Abundance of single proteins of fluid matrix vary between organs. e) Fluid matrix of the liver inherits pro regenerative, peritoneal fluid more pro fibrotic elements. Classification was based on uniport entries. f) Liver fluid matrix proteins are linked to metabolic regulation whereas peritoneal and cercal fluid elements are linked to fibrotic reactions.
a) Visualization of cell populations upon liver electroporation. b) Liver cell populations show distinct ECM surface receptor expression patterns upon liver electroporation. c) Fast migrating cell populations upregulate a limited number of surface receptor genes. d) Crossing scheme of Lyz2Cre; Ai14 transgenic mouse line, Lyz2+ cells express dTomato. e) Snapshots of extended video 7 showing Lyz2+ cells transport matrix elements across liver surfaces. Arrows highlight single cells. Representative image of three biological replicates. Scale bar: 50 μM. f) Swarms of Lyz2+ cells accumulate FITC+fluid matrix. Representative image of three biological replicates. Scale bar: 50 μM. g) Lyz2+ cells transport FITC+fluid elements in a no phagocytic form. Representative image of three biological replicates. Scale bar: 5 μM. h) Accumulations of FITC+fluid matrix elements are rich with Ly6g+ positive cells. Representative immunolabeling image of three biological replicates. Scale bar: 5 μM. i) Fluid matrix flows are mediated Ly6g+ positive cells and can be directed with local application of Lipoxin. Representative stereomicroscope of three biological replicates. Scale bar: 500 μM. j) Neutrophils upregulate CD11b, CD18 and NOS reactome upon injury. k) Targeted inhibition of neutrophil ECM receptors, swarming mediators and NOS stress enzymes blocks fluid matrix flows after liver electroporation. n=4 One-way ANOVA, Dunnett's multiple comparisons, 95% Cl. ***, p=0.0001.
a) Representative immunofluorescence images of histological sections of murine and human abdominal postsurgical adhesions. Murine peritonea were labeled with NHS-AF568 cecums with NHS-FITC, mice were sacrificed 4 weeks after surgery. Scale bar: 20 μM.
a) Percentage of individual cell population compared to the total number during liver injury. b) Time dependent abundance of cell populations during liver injury. c) Visualization of cellular abundances post liver electroporation. d) Sub clustering of activated neutrophil populations. e) Activated neutrophils show a consistent higher expression of CD11b and CD18 post injury.
a) Crossing scheme of a transgenic mouse line; Lyz+ cells express dTomato. b) Snapshots of extended video 7 showing Lyz2+ cells transport matrix elements across peritoneal surfaces. Arrows highlight single cells. Representative image of three biological replicates. Scale bar: 50 μM. c) Swarms of Lyz2+ cells accumulate FITC+fluid matrix on peritoneal surfaces. Representative image of three biological replicates. Scale bars: Overview: 500 μM; High magnifications: 50 μM. d) Majority of Lz2 positive cells carry FITC+Elements 24 hours after organ injury. One-way ANOVA, Dunnett's multiple comparisons, 95% Cl. ***, p=0.0001. e) Accumulations of FITC+fluid matrix elements on peritoneums are rich with Ly6g+positive cells. Representative immunolabeling image of three biological replicates. Scale bar: 5 μM. f) Targeted inhibition of neutrophil ECM receptors, swarming mediators and NOS stress enzymes blocks fluid matrix flows after peritoneal injury. n=4 One-way ANOVA, Dunnett's multiple comparisons, 95% Cl. ***, p=0.0001.
a) Overview of treatment regime in the liver electroporation setup (see methods). b) Inhibition of fluid matrix influx leads to impaired wound healing in liver electroporation sites. Representative immunofluorescence images of three >=biological replicates. Scale bar: 50 μM. One-way ANOVA, Dunnett's multiple comparisons, 95% Cl. ***, p=0.0001. c) Overview of treatment regime in the peritoneal-cercal setup (see methods). d) Treatment regime inhibits matrix flows in peritoneal and cercal injury sites, n>=4 biological replicates. Representative immunofluorescence images of histological sections FITC-NHS marked livers seven days after electroporation. Scale bar: 50 μM. One-way ANOVA, Dunnett's multiple comparisons, 95% Cl. e) Inhibition of matrix flows blocks adhesion formation in vivo, n>=3 biological replicates. Representative immunofluorescence images of histological sections FITC-NHS marked peritoneas five days post injury. Scale bar: 100 μM. One-way ANOVA, Dunnett's multiple comparisons, 95% Cl. ***, p=0.0001.
a) Scheme of experimental Setup. Livers of mice were electroporated. On day 2 an intra peritoneal injection of NHS-Rhodamine or control PBS injection was performed. 30 minutes later the organs were harvested. b) Representative stereomicroscopic images of livers.
a) After intra pleural injection of NHS esters, bleomycin is installed. Organs and blood are taken 14 days later. b) Histological sections of NHS-FITC labelled mouse lungs 14 days after bleomycin installation. c) Extract of proteins identified in mouse lungs treated with bleomycin after 14 days. d) Extract of protein found in the blood of mice 14 days after bleomycin.
a) Pictures of the setup. Liver and peritoneal tissues were locally labelled after injury. Organs were harvested after 24 h, wound sites lysed and FITC amounts measured. b) Quantifications of inhibitor experiments. n=4.
(A) Workflow of pleural matrix fate tracing setup. Mice were intra-pleurally injected with N-Hydroxysuccinimide-fluorescein isothiocyanate (NHS-FITC) labelling mix and two weeks later lungs were harvested. (B) Surface matrix stays stable over 2 weeks. Light sheet images of murine lungs (n=6). Scale bars: 500 μM. (C) Pleural matrix fate tracing reveals pools of extra cellular matrix. Multiphoton images of murine lung surfaces (n=6). Scale bars: 100 μM (overview) and 15 μM (high magnification). (D) Workflow of the bleomycin induced injury model. Mice were intra-pleurally injected with NHS-FITC labelling mix. The next day bleomycin was instilled and two weeks later lungs were harvested. (E) and (F) Pleural surface matrix invades deep into the interstitium upon bleomycin injury. Light sheet microscopy and histology images of murine lungs two-week post-bleomycin injury (n=6). Statistical comparison was by unpaired t-test. Scale bars: whole organ 500 μM, histology 500 μM (overview) and 15 μM (high magnification). (G) Interstitial fibrotic plaques are filled with invaded matrix. H&E and fluorescence images of murine lungs two-weeks post-bleomycin injury (n=6). Statistical comparison was by unpaired t-test. Scale bars: 100 μM. (H) Pleural matrix pools are depleted bleomycin induced injury. Multiphoton images of murine lung surfaces two-weeks post-bleomycin injury (n=3). Scale bars: 100 μM (overview) and 15 μM (high magnification).
(A) Workflow of murine ex vivo fluid scar tracing assay. (B) Immune cells enhance loss of protein from pleural surfaces 48 hours after incubation with immune cells (n=3). Control=mouse lungs without immune cells; healthy=mouse lungs supplemented with immune cells from healthy human volunteers; IPF=mouse lungs supplemented with immune cells from humans with lung disease. Scale bars: 100 μM. Data represented are mean±SD. One-way ANOVA was used for the multiple comparison (control 48 h vs. healthy and IPF immune cells, *P<0.05; *Healthy vs. IPF, *P<0.05, **P<0.01, ***P<0.001). (C) Immune cells accelerate interstitial fluid scar invasion of murine lung biopsies 48 hours after incubation. Plots for the NHS ester labelled ECM movement in the mouse lung biopsies. Data represented are mean±SD. One-way ANOVA was used for the multiple comparison (control 48 h vs. healthy and IPF immune cells, *P<0.05; *Healthy vs. IPF, *P<0.05, **P<0.01, ***P<0.001).
(A) Fluid matrix invasion in human lung tissues (n=2). Scale bars: 200 μM and 100 μM (i). (B) Fluid matrix accumulates in interstitial structures (n=2). Scale bars: 100 μM and 20 μM (i). (C) Workflow of proteomic identification of human fluid matrix systems. (D) Human pleural fluid matrix fractions have abundant fibrous elements (n=5). (E) GO enrichment reveals invading matrix resembles atrophic scar tissue with abnormal elastic tissue morphology. (F) Invading matrix harbors fibrous building blocks and crosslinking enzymes.
(A) Fluid scar tissue inherits SRC dependent tyrosine kinase signaling. GO enrichment of human proteomic signaling. (B) Workflow of Nintedanib treatment regime in the bleomycin-induced lung fibrosis model. (C) Nintedanib rescues pleural matrix pools after bleomycin-induced injury (n=5). (D) Immunofluorescence and H&E images of murine lungs two weeks after bleomycin injury (n=5). Statistical comparison was performed by unpaired t-test. Scale bars: 500 μM and 100 μM (Immunostainings).
(A) Bleomycin-induced pneumonia increased local thickness in murine lung surfaces after 14 days (n=3). (B) Fibrotic lungs have more complex surface structures (n=3).
(A) Schema of the mass spectrometry experiment. (B) Fluid matrix consists mostly of collagenous elements (n=5). (C) Pleural fluid matrix fractions contain fibrillar and basement elements (n=5). (D) Fluid matrix composition resembles atrophic scar tissue. (n=5)
A) Calculation of fluidity factor. B) Fluid scar elements show distinct fluidity profiles (n=5).
In order to overcome some of the shortcomings of the means described so far in the prior art that there is still a need to provide further options in the treatment of excessive or insufficient scar formation, the inventors of the present invention surprisingly discovered that chronic and excessive skin wounds may be attributed to the mobility of the fascia matrix. Thus, the inventors provide herein promising new methods for identifying modulators of extracellular matrix (ECM) movement towards a site requiring deposition of ECM.
The present inventors have now surprisingly found that scars originate from prefabricated matrix in the fascia, e.g. subcutaneous fascia that home into sites requiring deposition of extracellular matrix (ECM), such as wounds. The identification of fascia as the source for, e.g. dermal scars allowed the present inventors to identify the mechanism of scar formation, by using matrix-tracing techniques, live-imaging, genetic lineage-tracing and anatomic fate-mapping models. Strikingly, the present inventors found that scars originate from, inter alia, fascia fibroblasts bundled with its prefabricated matrix. Upon injury, this assembly homes into open wounds as a movable sealant that not only drags plugs of matrix, but also vasculature, immune cells and nerves, upwards into the outer skin. Accordingly, the present inventors observed that fascia fibroblasts rise to the site requiring patching after wounding, thereby dragging their surrounding extracellular jelly-like matrix, including embedded blood vessels, macrophages, and peripheral nerves, to form a scar. Genetic ablation of fascia fibroblasts prevented matrix from homing into wounds and resulted in poor scars, whereas placing an impermeable film beneath the skin, to prevent fascia fibroblasts migrating upwards, led to chronic open wounds. Thus, fascia contains a specialised prefabricated kit of, inter alia, sentry fibroblasts, embedded within a movable sealant, that preassemble together all the cell types and matrix components needed to heal wounds. The findings of the present inventors suggest that chronic and excessive skin wounds may be attributed to the mobility of the fascia matrix.
While prior art focuses on end-point phenotypes regarding fibrosis or keloid, the present invention allows focusing on the starting point. Indeed, the present inventors succeeded for the first time in in vivo labelling of ECM and could thus observe in real-time its movement towards a site of injury. This allows interfering with ECM deposition at a much earlier point in time than known before and, thus, opens new avenues for treatment options as described herein. Accordingly, the present invention relates to a method for identifying modulators of extracellular matrix (ECM) movement towards a site requiring deposition of ECM, comprising (a) contacting extracellular matrix of organ tissue obtainable by biopsy from a mammalian subject with a label; (b) contacting said labelled extracellular matrix of organ tissue with a compound of interest; (c) determining whether said compound of interest modulates ECM movement towards said site requiring deposition of ECM in comparison to labelled extracellular matrix of organ tissue obtainable by biopsy from a mammalian subject which is not contacted with said compound of interest, wherein modulation of ECM movement towards said site requiring deposition of ECM is indicative for said compound of interest to be a modulator of said ECM movement.
The method for identifying modulators of ECM movement towards a site requiring deposition of ECM includes labelling of the ECM. Hence, by labelling ECM, the ECM is visualized for being observed. Observation of ECM movement allows the identification of modulators of ECM movement, since a modulator may either decrease or accelerate ECM movement. As explained, visualization of the movement of labelled ECM allows the identification of a modulator being an inhibitor of ECM movement on the basis of decreasing ECM movement, while a modulator being a promoter of ECM movement can be identified on the basis of accelerating ECM movement. Without being bound by theory, it is assumed that decreasing ECM movement will result in a decreased deposition of ECM at a site requiring ECM deposition, such as a wound, while accelerating ECM movement will result in an accelerated deposition of ECM at a site requiring ECM deposition, such as a wound.
Decreasing ECM movement when used herein is equivalent to inhibition of ECM movement. Inhibition of ECM movement towards a site requiring ECM deposition preferably prevents excessive deposition of ECM at said site.
Accelerating ECM movement when used herein is equivalent to promotion of ECM movement. Promotion of ECM movement towards a site requiring ECM deposition preferably prevents insufficient deposition of ECM at said site.
“Identifying modulators of ECM movement” or “identification of modulators of ECM movement” includes screening such modulators and, once identified or screened, isolating, i.e. providing such modulators.
Step (a)
An “extracellular matrix (ECM)” according to the present invention refers to a collection of extracellular molecules secreted by cells. The ECM of the organ tissue of the present invention may be composed of collagen fibrils, microfibrils, and elastic fibers, embedded in hyaluronan and proteoglycans. Preferably, said ECM comprises proteins, polysaccharides and/or proteoglycans. Those components may refer to ECM components according to the present invention. Such ECM components may be covalently coupled to said label which is used to contact the ECM, in particular the ECM components of organ tissue are obtainable by biopsy from a mammalian subject. Preferably, ECM may also comprise cells of fascia matrix, serosa and/or adventitia as described herein, such as macrophages, neutrophils, mesothelial cells and/or fibroblasts, with fibroblasts being preferred. ECM proteins, such as labelled ECM proteins described herein, are used herein preferably as surrogate marker for ECM movement.
The organ tissue which is used for contacting the ECM of said tissue with a label may refer to a tissue sample/piece comprising cells from an organ as defined elsewhere herein. The organ tissue may also refer to a biopsy punch, which is created with a biopsy puncher from said tissue sample/piece. In this context, a “biopsy punch” refers to a small, roundish organ tissue sample created with a tool important in medical diagnostics—also called biopsy puncher—which is able to punch out/stamp out small pieces of said organ tissue with cleanly defined diameter. Preferably, a disposable, round biopsy puncher with 2 mm in diameter may be used. It generates uniform round shape biopsies (punch biopsies) that reduce variability. However, the organ tissue which is used for contacting the ECM of said tissue with a label can also be a whole organ as defined elsewhere herein such as an organ withdrawal. Said organ tissue, when it refers to a tissue sample/piece as defined above may be obtainable/obtained by biopsy from a mammalian subject. A biopsy according to the present invention is a medical test involving extraction of organ tissue(s) from a mammalian subject for examination to identify modulators of said ECM movement according to the method of the present invention. The technique being applied when the organ tissue may be obtainable by biopsy is known to a person skilled in the art. According to the method of the present invention, said organ tissue obtainable by biopsy from a mammalian subject may be a healthy or a diseased organ tissue.
Preferably, said organ tissue obtainable/obtained by biopsy from said mammalian subject according to the method of the present invention is from skin, kidney, lung, heart, liver, bone, peritoneum, intestine, diaphragm or pleura. More preferably, said organ tissue obtainable/obtained by biopsy from said mammalian subject according to the method of the present invention is skin. Said mammalian subject may be any mammal known to a person skilled in the art. Preferably, said mammalian subject is a human. Thus, in a preferred embodiment of the present invention an organ tissue may be obtainable by biopsy from a human, preferably an adult.
“Obtainable by biopsy” or “obtained by biopsy” is not limited to classical biopsy. It may even be a whole organ withdrawal. However, classical biopsy and biopsy as described herein is encompassed. When referring to “biopsy” in the context of the present invention, it is meant that during or at biopsy or after biopsy an organ tissue is injured, e.g., due to brushing or any other stimulus such that a site requiring ECM deposition is generated, unless the organ tissue may already have one or more of such sites requiring ECM deposition. The latter may be fulfilled in case of a diseased organ tissue, e.g. where ECM deposition may be excessive or insufficient.
Preferably, said organ tissue according to the method of the present invention comprises fascia matrix, serosa and/or adventitia. Even more preferably, said organ tissue according to the method of the present invention comprises fascia matrix. Fascia matrix, serosa and/or adventitia being used in the method of the present invention preferably comprise macrophages, neutrophils, mesothelial cells and/or fibroblasts.
Fascia matrix may be characterized by containing cells expressing α-SMA, CD90, ER-TR7, PDGFRα, Sca1, βIIITubulin, CD31, MOMA-2, F4/80, CD24, CD34, CD26, Dlk1, Fn1, Col14a1, Emilin2, Gsn and/or Nov. In this context, the term “expressing” refers to cells “expressing” a surface or cytoplasmic marker such as α-SMA, CD90, ER-TR7, PDGFRα, Sca1, βIIITubulin, CD31, MOMA-2, F4/80, CD24, CD34, CD26, Dlk1, Fn1, Col14a1, Emilin2, Gsn and/or Nov or said term refers to cells “having expressed” when referring to a lineage marker such as En1.
The Engrailed-1-lineage-positive fibroblasts or Engailed1-history-past fibroblasts (EPFs) are the main contributor of scar tissue development in murine back skin and cranial dermis, whereas Wnt1 lineage positive fibroblasts are the main contributor of scar tissue development in murine oral cavity. This embryonic lineage within the dorsal dermis possesses many of the functional attributes and characteristics such as the similar spindle-shaped morphology commonly associated with the term “fibroblast”. However, this lineage is not only present in the skin but also in the underlying superficial fascia. These fibroblast lineages (e.g., EPFs) responsible for scar deposition are derived from circulating fibroblast-like cells. EPFs may refer to En1-lineage-positive fibroblasts, meaning the ancestor/progenitors expressed En1 in the history during embryogenesis, but EPFs most likely do not express Engrailed-1 (En1) at stage of E18.5-P10, the developmental stages where the skin tissues may be collected from mice.
Engrailed-1 (and Wnt1) is expressed only transiently during embryonic development. En1 is a transcription factor, it turns on very early during development and regulates the expression of several downstream target genes. The En1 gene marks a lineage of cells. Once it is turned on, the cells and its progeny are EPFs, no matter whether En1 is expressed or not in the cells. Therefore, En1 is not a surface marker to mark the cells, but a lineage marker, thus defining an embryonic lineage.
In the wild type mouse system or in human, there is no direct way to mark EPFs. Therefore, surrogate markers such as CD26 or other fibroblast markers as mentioned below may be used for marking EPFs. CD26 labels a large percentage of EPFs (94%) and offers the highest-fold enrichment of EPFs over ENFs that have never expressed Engrailed in the history. ENFs do not participate in scar tissue formation. By transplanting adult ENFs & EPFs, separately, in different anatomical locations, it has been determined that the difference in the capacity of EPFs & ENFs to form a scar is cell-intrinsic, and permanent, and that these are in vivo behaviours of two distinct fibroblastic cell types (Rinkevich et al., 2015, Science 348 (6232)).
For human samples, the bellow pan markers for fibroblasts may further be used, such as N-Cadherin, alpha-smooth muscle actin (α-SMA), fibroblast specific protein 1 (FSP1), and/or platelet derived growth factor receptors alpha (PDGFRα) and beta (PDGFRβ), all important indicators and markers of scar formation. The bellow pan markers for fibroblasts as mentioned above may also be used in mice as well.
When in step a) of the method of the present invention the term “contact” or “contacting” is used, it means that said ECM of organ tissue as defined elsewhere herein is brought into contact with said label, which covalently couples to said ECM components. In a preferred embodiment, the term “contact” or “contacting” refers to “selectively contact” or “contacting”. In this context, “selectively contacting” means that not the whole ECM of the organ tissue is contacted with said label as defined elsewhere herein, but one or more portion of said ECM of said organ tissue. In other words, when the term “selectively contacting” is used herein, a confined very specific spot of the ECM of said organ tissue is contacted with said label as defined elsewhere herein, thus performing a locally ECM labelling on the organ tissue of the present invention. Preferably, proteins comprised by ECM are labelled. However, it is also envisioned that other components of ECM may be labelled, such as carbohydrates.
A “label” is a molecule or material that can produce a detectable (such as visually, electronically or otherwise) signal that indicates the presence and/or concentration of the label in a sample from an organ tissue. Thereby, e.g., the presence, location and/or concentration of a labelled molecule in a sample can be detected by detecting the signal produced by the (detectable) label. A label can be detected directly or indirectly. It will be appreciated that the label may be attached to or incorporated into a molecule, for example, a protein, polypeptide, or other entity, at any position. It will be appreciated that, in certain embodiments, a label may react with a suitable substrate (e.g., a luciferin) to generate a detectable signal. In particular, the detectable label can be a fluorophore, an enzyme (peroxidase, luciferase), a radioisotope, a fluorescent protein. Other detectable labels include chemiluminescent labels, electrochemiluminescent labels, bioluminescent labels, polymers, polymer particles, metal particles, haptens, and dyes.
A “fluorophore” (or fluorochrome) is a fluorescent chemical compound that can re-emit light upon light excitation. Examples of fluorophores include 5-(and 6)-carboxyfluorescein, 5- or 6-carboxyfluorescein, 6-(fluorescein)-5-(and 6)-carboxamido hexanoic acid, fluorescein isothiocyanate, rhodamine, tetramethylrhodamine, and dyes such as Cy2, Cy3, and Cy5, optionally substituted coumarin including AMCA, PerCP, phycobiliproteins including R-phycoerythrin (RPE) and allophycoerythrin (APC), Texas Red, Princeton Red, inorganic fluorescent labels such as particles based on semiconductor material like coated CdSe nanocrystallites.
Examples for fluorescent proteins include Exemplary fluorescent proteins include, e.g., Sirius, Azurite, EBFP, EBFP2, TagBFP, mTurquoise, ECFP, Cerulean, CyPet, TagCFP, mTFPI, mUkGI, mAGI, AcGFPI, TagGFP2, EGFP, GFP, mWasabi, EmGFP, YFP, TagYPF, Ypet, EYFP, Topaz, SYFP2, Venus, Citrine, mKO, mK02, mOrange, mOrange2, TagRFP, TagRFP-T, mStrawberry, mRuby, mCherry, mRaspberry, mKate2, mPlum, mNeptune, mKalama2, T-Sapphire, mAmetrine, mKeima, UnaG, dsRed, eqFP611, Dronpa, KFP, EosFP, Dendra, and IrisFP.
Examples of enzymes used as enzymatic labels include horseradish peroxidase (HRP), alkaline phosphatase (ALP or AP), β-galactosidase (GAL), glucose-6-phosphate dehydrogenase, β-N-acetylglucosamimidase, β-glucuronidase, invertase, Xanthine Oxidase, firefly luciferase and glucose oxidase (GO).
Examples of radioactive labels include radioactive isotopes of hydrogen, iodide, cobalt, selenium, tritium, carbon, sulfur and phosphorous. 2H, 3H, 13C, 14C, 15N, 18F, 31P, 32P, 35S, 67Ga, 76Br, 99mTc (Tc-99m), mIn, 123I, 125I, 131I, 153Gd, 169Yb, and 186Re.
According to the present invention, said label is preferably a dye or a tag. When said label is a dye, a fluorescent dye is preferred. A fluorescent dye refers to a reagent coupled to a fluorophore. In particular, said reagent refers to N-Hydroxysuccinimide ester or Succinimidyl esters (NHS) or sulfodichlorophenol (SDP)-esters. When used in the present invention NHS ester means N-hydroxysuccinimide ester or Succinimidyl esters. NHS or SDP-esters react with extracellular amines, like N-termini of proteins and lysines labelling ECM-components. NHS/SDP esters conjugated with fluorophores such as Alexa 488, Alexa 568, Alexa 647, Fluorescein, Fluorescein isothiocyanate (FITC), Pacific Blue, are used to visualize ECM.
As apparent from the above a NHS ester is sufficient to label extracellular amines. An essential step in untangling the phenomenon of ECM movement is the possibility to crosslink of moved material in the wound areas. Primary amines of proteins and peptides of distinct protein classes are covalently linked. Since the NHS esters also mark primary amines, the inventors asked themselves whether the restructuring in wound areas has led to an increase in free amine groups and whether they can visualize these via intraperitoneal application of NSH-Esters which was the case. The discovery the inventors have made here has many potential implications. The data shows that there is an accumulation of primary amines in abdominal wound areas which can be labelled via NHS-linked reaction (
In one preferred embodiment the NHS ester of the present invention may be used to label primary amines. In another embodiment the NHS ester of the present invention may be used to label amines and primary amines in a wound as defined herein. The NHS ester labeling might be used in a diagnostic approach. A diagnostic approach might to monitoring wound healing or wound progression. In this scenario it might be advantageous to combine NHS ester with a further reporter molecule as described above. In one preferred embodiment the NHS ester stain might be combined with any kind of reporter or fluorescent dye.
Preferably, such fluorescent dye include, but is not limited to, Alexa Fluor 488 NHS-ester, NHS-Fluorescein (5/6-carboxyfluorescein succinimidyl ester), Alexa Fluor 568 NHS-ester, Pacific Blue Succinimidly Ester, Alexa Fluor 647 NHS-ester (N-hydroxysuccinimide ester or Succinimidly Ester), Alexa Fluor 488 5-SDP-ester or NHS-Rhodamine (5/6-carboxy-tetramethyl-rhodamine succinimidyl ester). Each of the abovemetioned fluorescent dyes are able to label the ECM components of the ECM matrix from each organ tissue described elsewhere herein.
A method for diagnosing the healing progress of wounds wherein the method comprises administering NHS ester systemically to a patient and thereby labeling amines in the wounds. The method for diagnosing the healing of wounds wherein the NHS ester is combined with a reporter molecule.
In addition to imaging wounds, effector molecules could also be coupled to NHS esters, and thereby targeting wound areas with a single global injection. Such effector molecules might be therapeutic compounds. In case NHS ester is coupled or linked to a compound, any kind of compound might be suitable. However, preferred are therapeutic compounds for the treatment of chronic wounds. The compound coupled to NHS ester might a modulator of the extracellular matrix (ECM) movement as described herein.
Said NHS ester might be administered systemically or locally as
A therapeutic compound comprising NHS ester administered to a patient in the need thereof wherein the patient is suffering from chronic wounds. A therapeutic compound comprising NHS ester for use in treating chronic wounds wherein the compound is administered systemically meaning it is injected into the blood stream. A NHS ester for use in diagnosing wounds wherein the NHS ester is administered systemically and thereby labels wounds. The NHS ester for use in diagnosing wounds wherein the NHS ester is further combined with a reporter molecule. Due to the NHS ester being capable of labeling primary amines of a wound when injected into the blood stream, it can be used to monitor the extend or healing process of wounds.
The herein described NHS ester labeling of wounds which can be established by injecting the NHS ester stain into the blood flow and thereby marking primary extracellular amines, which might be used during or after surgery to monitor the extend or healing progression of a surgery wound (
A therapeutic compound comprising NHS ester and a modulator of extracellular matrix (ECM) movement administered to a patient in the need thereof wherein the patient is suffering from chronic wounds. A therapeutic compound comprising NHS ester and a modulator of extracellular matrix (ECM) movement for use in treating chronic wounds wherein the compound is administered systemically as injection into the blood stream.
Also comprised herein is that the label used in the method of the present invention is a tag. A “tag” can be an affinity tag (also called purification tag), such as a Biotin tag, histidine tag, Flag-tag, streptavidin tag, strep II tag, an intein, a maltose-binding protein, an IgA or IgG Fc portion, protein A or protein G. Preferably, said tag which is used in the method of the present invention and also conjugates with NHS/SDP esters is a Biotin tag. Such tags as defined elsewhere herein can thus also be used to analyze ECM components via protein biochemistry, like western blotting or mass spectrometry.
Depending on the ester, which may be used as a reagent of the fluorescent dye, specific reaction buffers may be used. When a NHS-ester is used as a reagent of the fluorescent dye 100 mM pH 9.0 BiCarbonate buffer is preferably used. It has been surprisingly shown that ester-reaction buffer mixtures can be applied on each organ as defined elsewhere herein without detectable toxic side effects.
When contacting ECM of organ tissue, which is obtainable by biopsy from said mammalian subject, with a label as defined elsewhere herein, a paper-like material comprising the label is used. Thus, the label, in particular the labelling solution which may be comprised by the label and the reaction buffer, can be applied locally (on one or more portions/spots of the ECM) onto the ECM of organ tissue as defined elsewhere herein preferably using such small paper pieces. Such paper-like material should be a non-reactive material meaning that said material itself does not interact/react with said ECM components of said organ tissue and/or said paper-like material should comprise an alcalic pH. Said paper-like material is thus able to soak up the label, in particular the labelling solution, leading to a local ECM labelling on the organ tissue. Examples of paper-like material include, but are not limited to, Whatman filter paper. In a preferred embodiment, 2 mm Whatman filter paper is used and an amount of 0.3 μl of the labelling solution is applied/added onto said filter paper before said paper is put onto the ECM of said organ tissue.
In particular, said label as defined elsewhere herein targets primary amine groups of ECM components as defined elsewhere herein. In other words, primary amine groups of ECM components are preferably labelled when applying the method of the present invention. Amines are compounds and functional groups that contain a basic nitrogen atom with an ion pair. They can be classified according to the nature and number of substituents on nitrogen. In nature there are primary, secondary and tertiary amines. Primary amines (also called primary amine groups) arise when one of three hydrogen atoms in ammonia is replaced by an alkyl or aromatic group. Important primary alkyl amines include, methylamine, most amino acids, while primary aromatic amines include aniline. According to the method of the present invention, primary amine groups of certain amino acids of said ECM components as defined elsewhere herein are labelled by said label as described above. In a preferred embodiment, primary amine groups of lysine of said ECM components as defined elsewhere herein are labelled.
An amine staining by Succinimidyl (NHS)-ester labelling has its effect in labelling all amine-containing ECM components and is not selective like antibodies which label one specific targets. The staining was developed for dead tissue and needs an alkaline pH, thus was assumed to damage living tissue. The inventors now surprisingly found out that living ex vivo tissue can be stained without damage. Thus, currently there are no reports on NHS/SDP-ester usage on living tissue, so no methods exists to visualize all amine-containing ECM molecules on organs.
After said ECM of said organ tissue as defined elsewhere herein has been contacted with said label, preferably contacted with a paper-like material comprising the label according to the present invention, said organ tissue may further be stamped with a biopsy punch into biopsy punches as described elsewhere herein. The contacting step of the ECM of said organ tissue with said label (labelling step) as described above followed by the punching step into small biopsy punchies can also be done in the other order.
It is also envisioned that components of the ECM, in particular proteins already comprise, e.g. non-canonical amino acids which enable a reaction with a label as described herein. For example, transgenic animals are available which express proteins comprising unusual or non-canonical amino acids which enable a reaction with a label as described herein.
The method of the present invention may also be extended by further comprising step (a′) namely contacting said organ tissue obtainable by biopsy from said mammalian subject with a label visualizing cells comprised in the ECM. In this context, said label refers to a lipophilic membrane fluorescent dye that spread through lateral diffusion capturing the entire cells. The additional labelling step may be performed before or after contacting the ECM of organ tissue with the first label as described elsewhere herein. Such membrane staining may be helpful to better identify/trace the ECM movement towards a site requiring deposition of ECM.
Step (b)
When in step b) of the method of the present invention the term “contact” or “contacting” is used, it refers that said compound of interest that is tested whether it modulates ECM movement towards a site requiring deposition of ECM is added directly onto the organ tissue which is placed in medium or said compound is added into the medium where the organ tissue is placed into. By simply adding the compounds of interest of the present invention either way (into the cultivation medium or explicitly onto the organ tissue), first ECM dynamics can be observed after about one hour or overnight. However, when ever it is deemed necessary contacting may further comprise adding compound or labeled compound into the blood stream. Such an administration may be a systemic administration, namely an injection.
The term “compound of interest” refers to a compound which is tested in the method of the present invention in order to identify whether said compound is a modulator of said ECM movement. Such modulator can be an inhibitor, thus inhibiting said ECM movement towards a site requiring deposition of ECM, once the inhibitor is contacted with said labelled ECM of organ tissue. However, such modulator may also refer to a promoter/an inducer, thus promoting/inducing said ECM movement towards a site requiring deposition of ECM, once the promoter is contacted with said labelled ECM of organ tissue. Preferably, the compound of interest may be an inhibitor. Even more preferably, said compound of interest refers to a protease inhibitor. A protease in general comprises metalloprotease, elastase or cathepsin and the like. Metalloproteases (metalloproteinase) can be divided into metalloendopeptidases, such as matrix-metallopeptidases (MMP1, 2, 3, 8, and 9), and metalloexopeptidases. In the present invention it has been shown that the metalloprotease (MMP) inhibitor GM6001 reacts specifically to Collagenases (MMP 1, 8), Gelatinases (MMP 2, 9) and Stromelysins (MMP 3).
When the compound of interest is identified as an inhibitor of ECM movement on the basis of decreasing ECM movement, which results in a decreased deposition of ECM at a site requiring ECM deposition, such compound of interest may have an anti-fibrotic phenotype. Such compound of interest refers, but is not limited to GM6001, a metalloprotease (MMP) inhibitor; 1400W and L-Name, iNOS inhibitors; LY255283 and CP-105696, a leukotriene B4 receptor antagonists; and Cath-G inhibitor, a Cathepsin-G inhibitor (
When the compound of interest is identified as a promoter of ECM movement on the basis of accelerating ECM movement, which results in an accelerated deposition of ECM at a site requiring ECM deposition, such compound of interest may have a pro-fibrotic phenotype. Such compound of interest refers, but is not limited to Elastial, an Elastase inhibitor, having a pro-fibrotic phenotype (
The term “modulate” or “modulating” as used herein and described elsewhere herein in more detail means “inhibit” or “inhibiting”, if the compound may be an inhibitor of said ECM movement towards a site requiring deposition of ECM or “promote/induce”, if the compound may be a promoter/an inducer of said ECM movement towards a site requiring deposition of ECM
Step (c)
When the term “to determine” or “determining” in step c) of the method of the present invention is used herein, it may be done or achieved by visual inspection or protein biochemistry methods. The term “visual inspection” refers to the visualization whether said compound of interest indeed modulates ECM movement as defined elsewhere herein by using a microscope, preferably by using a fluorescence stereomicroscope. This determination/examination by visual inspection or even by any protein biochemistry methods known to a person skilled in the art is performed in comparison to an ECM of organ tissue, also obtainable by biopsy as defined elsewhere herein from a mammalian subject (or from the same mammalian subject as already used for taking the organ tissue for step a) of the method of the present invention), which has been labeled according to the present invention, however which has not been contacted with said compound of interest as described elsewhere herein.
During step (a), (b) and/or (c) of the method of the present invention as defined above fluid of said mammalian's body cavity may be present. For step (a) said fluid may be present in said labelling solution as described above when the ECM of organ tissue is contacted with said label comprised in said solution. When said fluid is present in step (b) of the method of the present invention, it may be added to the medium, where the organ tissue already having a labelled ECM is placed into for culturing, which may also comprise the compound of interest.
A body cavity is a space created in an organism which houses organs. It is lined with a layer of cells and is filled with the fluid being preferably used in the method of the present invention, to protect the organs from damage as the organism moves around. Said fluid of said mammalian's body cavity may enhance the labelling or culturing effect in the method of the present invention.
According to a second aspect, the present invention relates to a method for identifying a biomarker associated with extracellular matrix (ECM) movement towards a site requiring deposition of ECM, comprising (a) contacting extracellular matrix of organ tissue obtainable by biopsy from a mammalian subject with a label; (b) isolating proteins from said labelled ECM which move towards said site requiring deposition of ECM; (c) determining at least a partial amino acid sequence of said proteins, thereby identifying said proteins as a biomarker associated with ECM movement.
Examples of biomarkers for the ECM of different organs can be found in table 2 to 4.
Step (a) is carried out as described herein in the context of the methods for identifying modulators of ECM movement towards a site requiring deposition of ECM.
Step (b) is carried out by applying means and methods generally known to isolate proteins from, e.g. surfaces of membranes, paper-like material, etc. Indeed, since in step (a) preferably proteins comprised by ECM are labelled, it is possible to visualize such proteins moving towards a site requiring ECM deposition. Such ECM proteins are used as surrogate for movement of ECM towards a site requiring ECM deposition. Hence, any of such ECM proteins which move within ECM towards a site requiring deposition of ECM may be a suitable biomarker associated with ECM movement towards a site requiring deposition of ECM. Put differently, such an ECM protein identified as described herein may be indicative of ECM movement. In case of a pathological medical condition, such as fibrosis or chronic wounds, the presence, the amount, or absence of such a biomarker may be indicative of the degree or extent of the pathological medical condition.
Step (c) is carried out by applying means and methods generally known to determine at least a partial amino acid sequence of one or more proteins isolated in step (b), such as MALDI-TOF, HPLC, etc.
Thus far, the understanding in the prior art is that mammals form scars to quickly patch up wounds and ensure survival by an incompletely understood mechanism. Indeed, current wound healing models propose that fibroblasts migrate into sites of wounds where they locally initiate matrix deposition that is then remodeled into a mature scar. Based on their finding, the present inventors propose a revised model (see
The prevailing scientific view is that the body's connective tissues serves merely as a passive support framework for cells and organs and that this connective fibrous acellular network known as the ECM is stationary. The inventors disprove this idea by uncovering a fluid matrix system that radiates across internal organs. They show that injury induces gushes of fluid matrix across visceral and parietal organs. This immature fluid matrix is then cross linked, on site, to establish rigid frames thereby regenerating breaches in the structural continuums of organs and preserving organ integrity and function.
These findings challenge several widely held notions. The first dogma the inventors can dispense with is the idea that ECM is static. Secondly, they have now seen that new anatomies do not only emerge from de novo deposition of that rigid matrix, but rather from a mixture of new and shuttled fluid matrix. Finally, the data demonstrates that fibroblasts are no longer the major contributors for tissue reconstruction, but it is rather the job of the relative underappreciated immune-competent cells, the neutrophils. To recapitulate, the inventors findings indicate that rigid anatomies emerge from reservoirs of fluid matrix that are maneuvered into tissue construction sites by organ wide invasion of neutrophils. Fluid matrix then serves in injured organs as building blocks for new rigid anatomies and tissue repair.
Thus, in one preferred embodiment the presence of neutrophils might be determined or targeted when monitoring wound healing progression. In another embodiment it might be advantageous to stimulate neutrophils to move into a wound to increase the ECM movement toward a site requiring ECM deposition. Such stimulation might be established by the use of a compound or cytokines.
Fluid matrix is a pool of raw ingredients for fibrotic scars and regeneration and the inventors speculate that specific protein composition of the fluid matrix determines the diverging rigid anatomies that develop during adult tissue repair. Indeed, the composition of fluid elements varies from organ to organ. While in the liver many enzymes and pro-regenerative proteins are part of the fluid fraction, peritoneal elements consist mostly of fibrous and profibrotic elements, which are building blocks for scars.
The inventors also uncover a new exciting link between inflammation and tissue repair by showing the central role for neutrophils in piloting this fluid matrix material into wounds, and they do so in various ways. Immune cells transport cloudy matrix elements across organ surfaces within minutes. Whereas the transcriptomics analysis indicates that neutrophils are transcriptionally primed for this endeavor by activating multiple pathways. One pathway involves upregulation of the collagen binding integrins CD11b and CD18, which play an essential role in matrix movement, as blocking antibodies reduced the matrix currents. Another pathway involves LTB4 and nitric oxide synthase, and locally placing LTB4 forms new deposits of matrix, whereas inhibiting nitric oxide inhibits matrix flows. Neutrophils regulate therefore all facets of adult organ repair.
Thus, in another scenario it might be advantageous to block the ECM movement. By using the herein established reasoning an inhibition of the ECM movement might be established by using an neutrophil neutralizing antibody. A preferred neutrophil neutralizing antibody might be directed against Ly6g, CD11b or CD18.
The inventors have seen that mobilization of fluid matrix is a general principle of wound repair in adult tissues and organs, and they speculate it is in fact even more general. I.e. that flows are likely involved in development (organogenesis). They further speculate that emergence of new rigid frames during embryonic development is enacted by a similar fluid matrix process, and that ‘fluid matrix reservoirs & currents’ are newly emerged biological principles of the body plan. Their findings that matrix currents are absent in healthy adult organs suggests there are strong forces that hold-off matrix reservoirs from flowing, whereas counter-balancing forces activate matrix flows throughout adult life.
To the best of the present inventors' knowledge, the extent and magnitude of matrix movements documented in the present application; see the Examples, have never been observed during injury or regenerative settings.
The findings of the present inventors reveal an unprecedented dynamic and scale of motion for composite tissue matrix during injury that is mediated, inter alia, by specialized fibroblasts of the fascia. Thus, fascia serves as an externum repono for scar-forming matrix, and these findings indicate that matrix steering into wounds is the principle response of the fascia to large injuries.
The findings of the present inventors that fascia contributes to large scars and that its blockage leads to chronic open wounds, indicates that the ranges of chronic and excessive wound healing phenotypes of skin, such as diabetic and ulcerative wounds, as well as hypertrophic and particularly keloid scars might all be attributed to the fascia. Indeed, the superficial fascia varies widely in different species, sex, age, and anatomic skin locations24. In some mammals, the superficial fascia is loose, whereas in man, dog and horse, the superficial fascia is thicker with larger connective tissue bands. The superficial fascia of human skin further varies in thickness on different regions of the body25. For example, lower chest, back, thigh and arm have much thicker and multi-layered membranous sheets, and it is these anatomic sites that are prone to form large and keloid scars. Whereas other sites such as the foot have a much thinner or inexistent fascia.
Additionally, according to a third aspect, the present invention refers to a compound for use in a method for the modulation of extracellular matrix (ECM) movement towards a site requiring deposition of ECM, preferably in the treatment of a condition involving ECM deposition.
“A condition involving ECM deposition” is a medical condition which requires ECM deposition. As found by the present inventors, ECM can deliver components which, when deposited at a site requiring ECM deposition, aid in scar formation, preferably effect scar formation. Sometimes it is desired to modulate ECM movement and thereby ECM deposition and thus scar formation.
Accordingly, the present invention provides means and methods both for identifying modulators and ECM movement towards a site requiring ECM deposition and medical applications for the modulation, e.g. inhibition or promotion, of ECM movement towards a site requiring ECM deposition, preferably in the treatment of a condition involving ECM deposition.
Indeed, if scar is generated excessively, such a condition is undesired. Accordingly, the present invention provides for medical applications for the inhibition of ECM movement towards a site requiring ECM deposition, preferably in the treatment of a condition involving ECM deposition. Such a condition is, e.g., excessive deposition of ECM which may be associated with fibroproliferative disease.
Similarly, if scar is generated insufficiently, such a condition is undesired, too. Accordingly, the present invention provides for medical applications for the promotion of ECM movement towards a site requiring ECM deposition, preferably in the treatment of a condition involving ECM deposition. Such a condition is, e.g., insufficient deposition of ECM which may be associated with chronic wounds.
“A site requiring deposition of ECM”, or “a site requiring ECM deposition” as also used herein, is a site within organ tissue which signals a mammal's body the requirement for ECM deposition. The signal is triggered by, e.g. an injury caused, e.g. by a wound. Usually, ECM deposition is required for patching a wound. Thus, a site requiring ECM deposition is preferably a wound. A “wound” is a break in the continuity of any mammalian bodily tissue due to, e.g. violence, where violence is understood to encompass any action of external agency, including, for example, surgery. Said term includes open and closed wounds.
ECM movement as described herein and which can be visualized as described herein is, in accordance with the findings of the present inventors, mediated by fascia matrix. Fascia matrix, serosa and/or adventitia may comprise macrophages, neutrophils, mesothelial cells and/or fibroblasts. In particular, fascia matrix, serosa and/or adventitia may comprise fibroblasts.
A compound for use in a method for the modulation of ECM movement towards a site requiring deposition of ECM (equivalent to ECM deposition) can be any compound, such as a small molecule or the like. Such a compound includes cells or material from cells.
The effect of the compound on modulation of ECM movement may, for example, be tested in accordance with the methods of the present invention as described herein. Briefly, extracellular matrix of organ tissue obtainable by biopsy from a mammalian subject is contacted with a label; said labelled extracellular matrix of organ tissue is contacted with a compound of interest, i.e. a potential compound for use in a method for the modulation of ECM movement towards a site requiring ECM deposition; it is determined whether said compound of interest modulates ECM movement towards said site requiring deposition of ECM in comparison to labelled extracellular matrix of organ tissue obtainable by biopsy from a mammalian subject which is not contacted with said compound of interest, wherein modulation of ECM movement towards said site requiring deposition of ECM is indicative for said compound of interest to be a modulator of said ECM movement. As a modulator the compound of interest may inhibit ECM movement or may promote ECM movement.
Preferably, in accordance with the methods for identifying modulators, e.g. inhibitors or promoters, of ECM movement towards a site requiring ECM deposition as described herein, a compound for use in a method for the modulation of ECM movement towards a site requiring ECM deposition, preferably in the treatment of a condition involving ECM deposition may be identified. A thus-identified compound may then be used in a method for the modulation of ECM movement towards a site requiring ECM deposition, preferably in the treatment of a condition involving ECM deposition. Accordingly, the present invention provides for a compound which is obtainable/obtained by the methods or identifying modulators, e.g. inhibitors or promoters, of ECM movement towards a site requiring ECM deposition as described herein for the modulation of ECM movement towards a site requiring ECM deposition, preferably in the treatment of a condition involving ECM deposition.
However, although preferred, it is not necessary that a compound for use in the method for the modulation of EM movement of the present invention is tested in accordance with the methods for identifying such modulators as provided by the present inventors. Indeed, any compound can be used as long as it modulates ECM movement towards a site requiring ECM deposition. If needed, ECM movement towards a site requiring ECM deposition may be tested as described herein, e.g. as described hereinabove.
Preferably, a compound is for use in a method for the modulation of ECM movement towards a site requiring ECM deposition in the treatment of a condition involving ECM deposition. Since the present inventors found for the first time that ECM movement delivers components for scar formation to a site requiring ECM deposition, the present invention provides for an early as possible treatment of a condition involving ECM deposition. Accordingly, the treatment of a condition involving ECM deposition allows thus preferably the prevention of either excessive deposition of ECM at a site requiring ECM deposition or insufficient deposition of ECM at a site requiring ECM deposition.
Indeed, inhibition of ECM movement towards a site requiring deposition of ECM prevents excessive deposition of ECM at said site. Accordingly, a modulator of ECM movement towards a site requiring ECM deposition may preferably be an inhibitor. That said, the present invention relates to a compound for use in a method for the inhibition of extracellular matrix (ECM) movement towards a site requiring deposition of ECM, preferably in the treatment of a condition involving ECM deposition. It is preferred that inhibition of ECM movement towards a site requiring deposition of ECM prevents excessive ECM deposition at said site. An example of excessive deposition of ECM is associated with fibroproliferative diseases.
A “fibrotic” disease or a “fibroproliferative” disease refers to a disease characterized by scar formation and/or the over production of extracellular matrix by connective tissue. Fibrotic disease may occur as a result of tissue damage. It can occur in virtually every organ of the mammalian body. Examples of fibrotic or fibroproliferative diseases include, but are not limited to, idiopathic pulmonary fibrosis, fibrotic interstitial lung disease, interstitial pneumonia, fibrotic variant of non-specific interstitial pneumonia, cystic fibrosis, lung fibrosis, silicosis, asbestosis, asthma, chronic obstructive pulmonary lung disease (COPD), pulmonary arterial hypertension, liver fibrosis, liver cirrhosis, renal fibrosis, glomerulosclerosis, x kidney fibrosis, diabetic nephropathy, heart disease, fibrotic valvular heart disease, systemic fibrosis, rheumatoid arthritis, excessive scarring resulting from surgery, e.g., surgery to fix hernia, chemotherapeutic drug-induced fibrosis, radiation induced fibrosis, macular degeneration, retinal and vitreal retinopathy, atherosclerosis, and restenosis. Fibrotic disease or disorder, fibroproliferative disease or disorder and, as sometimes used herein, fibrosis, are used interchangeably herein.
For livers and in the case of peritoneas the laparotomy section as local injury (
Using the inventors signaling pathway analysis, they identified multiple molecules that inhibited or amplified matrix flows. Interestingly, some effector molecules like Blebbistatin and ciliobrevin effected matrix currents of only one organ.
For the peritoneum the lysyl oxidase BAPN and the proteases elastase inhibitor II and Elastatinal increased the ECM movement of the surrounding tissue. Thus, in a preferred embodiment BAPN and Elastatinal might be used to increase the ECM movement of the tissue surrounding the peritoneum.
A lysyl oxidase inhibitor for use in increasing the ECM movement of the tissue surrounding the peritoneum. The lysyl oxidase inhibitor for use in increasing the ECM movement wherein the lysyl oxidase inhibitor is BAPN. A lysyl oxidase inhibitor for use in increasing the wound healing capacity of the peritoneum tissue. The lysyl oxidase inhibitor for use in increasing the wound healing capacity of the peritoneum tissue.
The motor protein Ciliobrevin D and (S)-nitro-Blebbistatin, the heat shock factor Quercetin, KNK437 and the proteases cathepsin B inhibitor, GM6001 and BESTATIN decreased the ECM movement around the peritoneum.
In a preferred embodiment Ciliobrevin D, (S)-nitro-Blebbistatin, Quercetin, KNK437, cathepsin B inhibitor, GM6001 and BESTATIN might be used to decrease the ECM movement around the peritoneum.
An inhibitor of motor protein, a heat shock factor, or a protease for use in decreasing fibrosis in the peritoneum tissue.
For the liver the lysyl oxidase BAPN and the proteases elastase inhibitor II and Elastatinal increased the ECM movement. Thus, in a preferred embodiment BAPN and Elastatinal might be used to increase the ECM movement of the tissue surrounding the liver.
A lysyl oxidase inhibitor for use in increasing the ECM movement of the tissue surrounding the liver. The lysyl oxidase inhibitor for use in increasing the ECM movement of the tissue surrounding the liver wherein the lysyl oxidase inhibitor is BAPN. A lysyl oxidase inhibitor for use in increasing the wound healing capacity of the liver tissue. The lysyl oxidase inhibitor for use in increasing the wound healing capacity of the liver tissue.
The heat shock protein Qercetin and KNK437, the proteases cathepsin B inhibitor, MMP12 and Cathepsin G inhibitor and GM6001 decreased the matrix movement. In yet another embodiment Qercetin, KNK437, cathepsin B inhibitor, MMP12, Cathepsin G inhibitor and GM6001 might be used to decrease the ECM movement around the liver.
An inhibitor of motor protein, a protease for use in decreasing fibrosis in the liver tissue, wherein the motor protein inhibitor is Quercetin or KNK437, wherein the protease inhibitor is cathepsin B inhibitor, MMP12, Cathepsin G inhibitor or GM6001. Quercetin for use in decreasing fibrosis in the liver tissue. KNK437 for use in decreasing fibrosis in the liver tissue. Cathepsin B inhibitor for use in decreasing fibrosis in the liver tissue. MMP12 for use in decreasing fibrosis in the liver tissue. Cathepsin G inhibitor for use in decreasing fibrosis in the liver tissue. GM6001 for use in decreasing fibrosis in the liver tissue.
Since the composition of the fluid matrix differs from organ to organ, organ-specific modulators of the matrix currents could be applied after identification of appropriate biomarkers.
In summary, the Inventors show a new method to attach molecules to wounds, new potential markers for pulmonary fibrosis and signaling pathways to modulate matrix movements.
Bleomycin-induced pulmonary fibrosis has different degrees of severity. Robust biomarkers should therefore show different abundancies depending on the severity of pulmonary fibrosis.
Until now it was assumed that lungs scar from newly synthesized connective tissue in response to injury. The inventors data presented herein paint a new picture by revealing a system of fluid scar tissue that, after activation, migrates from the pleura into the interstitium. This new fluid scar brings fibrous building blocks as well as the corresponding enzymes to mature the tissue into fixed scar tissue on site. Thus, lungs scar primarily by restructuring pre-existing connective tissue.
It still remains possible that fibroblasts deposit matrix to contribute to scarring, as a secondary response to irrigation. Indeed, the experiments show that in the absence of matrix irrigation, fibroblasts remain dormant and remain inactive. Thus, it still remains possible that matrix irrigation stiffens the connective tissues surrounding the fibroblasts, which in turn activates them to further secrete or remodel the new matrix surrounding them.
The proteomics data of the fluid matrix from mouse and humans indicates that irrigation of human lungs leads to a much greater reduction of surface elasticity. These findings also uncover the link between inflammation and fibrosis. Monocytes and lymphocytes, not only trigger the invasion of fluid scar tissue they can accelerate the inward movement and accretion of new connective tissue. The fact that immune cells of patients with chronic lung diseases are ‘primed’ for matrix maneuvering and further enhance this effect even more has exciting therapeutic implications. This indicates that the rate of fluid movements and fibrosis depends on the ‘priming’ state of individual immune cells and that a deeper understanding of this ‘priming’ mechanism could open new therapeutic and diagnostic opportunities to combat fibrosis onset.
Taken together, the inventors findings presented herein on lungs, and the previous findings on skin (REF) that show loose connective tissues serve as a source for dermal scars, imply for matrix movements as a germinal scarring mechanism and response to injury across the body.
First mass spectrometric analyses of lung tissue found varying amounts of proteins in the lungs of bleomycin versus control animals. This indicates that the primarily labelled proteins undergo changes due to the stimulus. Proteins such as fibrinogen are known to form net-like structures. It could be that fibrinogen is covalently bound to the primary labelled proteins. In fact, the inventors were also able to identify proteins of varying abundance of the initially labelled lung matrix in the blood of the animals (
From the abovementioned
Different markers were found in blood samples. As shown in
In summary, the Inventors show here that fluid elements enter the blood stream during mobilization of the lung matrix during fibrotic events. These elements can be detected and could serve as biomarkers for fibrotic events.
In contrast, promotion of ECM movement towards a site requiring deposition of ECM prevents insufficient deposition of ECM at said site. Accordingly, a modulator of ECM movement towards a site requiring ECM deposition may preferably be a promoter. That said, the present invention relates to a compound for use in a method for the promotion of extracellular matrix (ECM) movement towards a site requiring deposition of ECM, preferably in the treatment of a condition involving ECM deposition. A compound promoting the ECM movement is for example the lysyl oxidase inhibitor BAPN or a chemokine attracting neutrophils to a site requiring ECM deposition, preferably the chemokine Lipoxin A4. As the inventors have found that neurtrophils are one of the first cells moving into a site requiring ECM deposition and that neutrophils are capable of recruiting ECM material for the deposition at that site, it is apparent that a chemokine attracting neutrophils may be able to initiate the ECM deposition and thus a natural healing process. This may be advantageous for chronic wounds which are not closed following the natural pathway or to fasten the closure of wounds. It may also be advantageous for other inflammatory diseases where healing of damaged tissue is wanted.
It is preferred that promotion of ECM movement towards a site requiring deposition of ECM prevents insufficient ECM deposition at said site. An example of insufficient deposition of ECM is associated with chronic wounds. A “chronic wound” is a wound (preferably as defined herein) that does not heal in an orderly set of stages and in a predictable amount of time the way most wounds do; wounds that do not heal within about two to three months are usually considered chronic. For example, chronic wounds often remain in the inflammatory stage for too long and remain as opening in the skin and sometimes the deeper tissue. Chronic wounds may never heal or may take years to do so.
While prior art focuses on end-point phenotypes regarding, e.g., fibrosis or keloid, the present invention—due to the findings of the present inventors—allows focusing on the starting point. Indeed, the present inventors succeeded for the first time in in vivo labelling of ECM and could thus observe in real-time its movement towards a site requiring ECM deposition, such as a wound, e.g. caused by an injury. This allows interfering with ECM deposition at a much earlier point in time than known before which opens up new treatment options which were not available before. Put differently, the treatment methods of the present invention which apply compounds which modulate ECM movement towards a site requiring ECM deposition allow preferably a prevention of either excessive or insufficient deposition of ECM at a site requiring ECM deposition, since the present inventors elucidated the mechanism which a mammal's body uses to patch wounds—by ECM movement.
Accordingly, the methods of the present invention relating to treatment aspects described herein are preferably for the prevention of either excessive deposition of ECM at a site requiring ECM deposition or insufficient deposition of ECM at a site requiring ECM deposition. Prior to the present invention, such an early (preventative) treatment was not possible, since the mechanism elucidated by the present inventors was neither known nor understood. However, thanks to the present invention, the mechanism of ECM movement is understood and, therefore, new treatment options are available, in particular a preventative treatment of either excessive deposition of ECM at a site requiring ECM deposition or insufficient deposition of ECM at a site requiring ECM deposition. Indeed, a modulator which is an inhibitor of ECM movement towards a site requiring ECM deposition may ideally prevent excessive deposition of ECM due to inhibiting ECM movement, while a modulator which is a promoter of ECM movement towards a site requiring ECM deposition may ideally prevent insufficient deposition of ECM due to promoting ECM movement.
By following the teachings of the present invention, the present inventors could already identify compounds for use in a method for the modulation of ECM movement towards a site requiring ECM deposition, preferably in the treatment of a condition involving ECM deposition as described herein. In particular, matrix metalloprotease inhibitors, such as GM6001; serine protease inhibitors, such as cathepsin G inhibitors; iNOS inhibitors, such as W1400; or leukotriene B4 receptor antagonists, such as LY255283 were identified and applied in vivo. As is shown in
Accordingly, matrix metalloprotease inhibitors, serine protease inhibitors, iNOS inhibitors or leukotriene B4 receptor antagonists, heat shock inhibitors, inhibitors of motor proteins and neutrophil neutralizing antibodies are preferred compounds for use in a method for the modulation of ECM movement towards a site requiring ECM deposition, preferably in the treatment of a condition involving ECM deposition as described herein. Matrix metalloprotease inhibitors, serine protease inhibitors, iNOS inhibitors or leukotriene B4 receptor antagonists, heat shock inhibitors, inhibitors of motor proteins and neutrophil neutralizing antibodies are in the sense of the present invention inhibitors of ECM movement. As such they have an anti-fibroproliferative effect.
In particular, elastase inhibitors, such as elastial was identified and applied in vivo. As is shown in
Accordingly, elastase inhibitors are preferred compounds for use in a method for the modulation of ECM movement towards a site requiring ECM deposition, preferably in the treatment of a condition involving ECM deposition as described herein. Elastase inhibitors are in the sense of the present invention promoters of ECM movement. As such they have a pro-fibroproliferative effect.
It is noted that as used herein, the singular forms “a”, “an”, and “the”, include plural references unless the context clearly indicates otherwise. Thus, for example, reference to “a reagent” includes one or more of such different reagents and reference to “the method” includes reference to equivalent steps and methods known to those of ordinary skill in the art that could be modified or substituted for the methods described herein.
Unless otherwise indicated, the term “at least” preceding a series of elements is to be understood to refer to every element in the series. The term “at least one” refers to one or more such as two, three, four, five, six, seven, eight, nine, ten or more. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.
The term “and/or” wherever used herein includes the meaning of “and”, “or” and “all or any other combination of the elements connected by said term”.
The term “less than” or in turn “more than” does not include the concrete number.
For example, less than 20 means less than the number indicated. Similarly, more than or greater than means more than or greater than the indicated number, e.g. more than 80% means more than or greater than the indicated number of 80%.
Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integer or step. When used herein the term “comprising” can be substituted with the term “containing” or “including” or sometimes when used herein with the term “having”. When used herein “consisting of” excludes any element, step, or ingredient not specified.
The term “including” means “including but not limited to”. “Including” and “including but not limited to” are used interchangeably.
The term “about” means plus or minus 10%, preferably plus or minus 5%, more preferably plus or minus 2%, most preferably plus or minus 1%.
Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.
It should be understood that this invention is not limited to the particular methodology, protocols, material, reagents, and substances, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims.
All publications cited throughout the text of this specification (including all patents, patent application, scientific publications, instructions, etc.), whether supra or infra, are hereby incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention. To the extent the material incorporated by reference contradicts or is inconsistent with this specification, the specification will supersede any such material.
The content of all documents and patent documents cited herein is incorporated by reference in their entirety.
A better understanding of the present invention and of its advantages will be gained from the following examples, offered for illustrative purposes only. The examples are not intended to limit the scope of the present invention in any way.
The present invention may also be characterized by the following items:
Material and Methods being Used in the Present Invention
Mice and Genotyping.
All mouse strains (C57BL/6J, En1Cre, R26VT2/GK3, R26mtmg, R26iDTR, Rag2−/−, and Fox Chase SCID) were either obtained from Jackson laboratories, Charles River, or generated at the Stanford University Research Animal Facility as described previouslyl2. Animals were housed at the Helmholtz Center Animal Facility rooms were maintained at constant temperature and humidity with a 12-h light cycle. Animals were supplied with food and water ad libitum. All animal experiments were reviewed and approved by the Government of Upper Bavaria and registered under the projects 55.2-1-54-2532-61-2016 and 55.2-2532-02-19-23 and conducted under strict governmental and international guidelines. This study is compliant with all relevant ethical regulations regarding animal research. Cre-positive (Cre+) animals from double-transgenic reporter mice were identified by detection of relevant fluorescence in the dorsal dermis. Genotyping was performed to distinguish mouse lines containing a 200-base pair (bp) Cre fragment (Cre+/−) from the wild-type (Cre−/−). Genomic DNA from the ear-clips was extracted using QuickExtract DNA extraction solution (Epicenter) following the manufacturer's guidelines. DNA extract (1 μl) was added to each 24 μl PCR. The reaction mixture was set up using Taq PCR core kit (Qiagen) containing 1× coral buffer, 10 mM dNTPs, 0.625units Taq polymerase, 0.5 μM forward primer “Cre_genotype_4F”-5′ ATT GCT GTC ACT TGG TCG TGG C-3′″ (SEQ ID NO: 2, Sigma) and 0.5 μM reverse primer “Cre_genotype_4R”-5′ GGA AAA TGC TTC TGT CCG TTT GC-3′ (SEQ ID NO: 3, Sigma). PCRs were performed with initial denaturation for 10 min at 94° C., amplification for 30 cycles (denaturation for 30 s at 94° C., hybridization for 30 s at 56° C., and elongation for 30 s at 72° C.) and final elongation for 8 min at 72° C., and then cooled to 4° C. In every experiment, negative controls (non-template and extraction) and positive controls were included. The reactions were carried out in an Eppendorf master cycler. Reactions were analyzed by gel electrophoresis.
Viral Particle Production.
Adeno-associated virus serotype 6 (AAV6) expressing GFP or Cre recombinase were produced by transfecting the AAVpro® 293T Cell Line (Takara Bio, 632273) with pAAV-U6-sgRNA-CMV-GFP (Addgene, 8545142) or pAAV-CRE Recombinase vector (Takara Bio, 6654), pRC6 and pHelper plasmids procured from AAVpro Helper Free System (Takara Bio, 6651). Transfection was performed with PEI transfection reagent and vires were harvested 72 h later. AAV6 viruses were extracted and purified with an AAVpro® purification kit (Takara Bio, 6666) and titer was calculated using real-time PCR.
Human Skin Samples.
Fresh human skin and scar biopsies, from various anatomic locations, were collected from donors between 18-65 years of age, through the Section of Plastic and Aesthetic Surgery, Red Cross Hospital Munich (reference number 2018-157), and by the Department of Dermatology and Allergology, Klinikum rechts der Isar Technical University Munich (reference number 85/18S). Informed consent was obtained from all subjects prior to skin biopsies. Upon collection, these samples were directly processed for tissue culture or fixed with PFA and then processed for cryosection or paraffin section followed by histological or immunofluorescent analyses.
Fascia In Vitro Culture.
Two in vitro systems were used. To visualize the changes in matrix architecture in real time, 2 mm-diameter biopsies were excised from P0 C57BL/6J neonates and processed for live imaging (SCAD assay, Patent Application No. PLA17A13). To determine the effectiveness of the DT treatment, muscle+fascia was manually separated from the rest of the skin in the chimeric grafts experiments and incubated with DT at different concentrations for 1 h at ambient temperature. Next, samples were washed with PBS and incubated in DMEM/F12 (Thermo Fisher) supplemented with 10% Serum (Thermo Fisher), 1% penicillin/streptavidin (Thermo Fisher), 1% GlutaMAX (Thermo Fisher) and 1% non-essential amino acids solution (Thermo Fisher) in a 37° C., 5% CO2 incubator. Medium was routinely exchanged every other day. Samples were fixed at day 6 of culture with 2% paraformaldehyde and processed for histology.
Histology.
Tissue samples were fixed overnight with 2% paraformaldehyde in PBS at 4° C. Samples were rinsed three times with PBS, embedded in optimal cutting temperature (OCT, Sakura Finetek) and flash-frozen on dry ice. 6-micron sections were made in a Cryostar™ NX70 cryostat (Thermo fisher). Masson's trichrome staining was performed with a Sigma-Aldrich Trichrome stain kit, according to the manufacturer's guidelines. For immuno-labeling, sections were air-dried for 5 min and fixed with −20° C.-chilled acetone for 20 min. Sections were rinsed three times with PBS and blocked for 1h at room temperature with 10% serum in PBS. Then, the sections were incubated with primary antibody in blocking solution for 3h at ambient temperature. Sections were then rinsed three times with PBS and incubated with secondary antibody in blocking solution for 60 min at ambient temperature. Finally, sections were rinsed three times in PBS and mounted with fluorescent mounting media with 4,6-diamidino-2-phenylindole (DAPI). Primary antibodies used: goat-anti-αSMA (1:50, Abcam), rabbit-anti-TUBB3 (1:100, Abcam), rat-anti-THY1(CD90) (1:100, Abcam), rat-anti-CD24 (1:50, BD biosciences), rabbit-anti-DPP4(CD26) (1:150, Abcam), rabbit-anti-PECAM1(CD31) (1:10, Abcam), rat-anti-CD34 (1:100, Abcam), rabbit-anti-COLLAGEN I (1:150, Rockland), rabbit-anti-COLLAGEN III (1:150, Abcam), rabbit-anti-COLLAGEN VI (1:150, Abcam), rabbit-anti-DLK1 (1:200, Abcam), rat-anti-ERTR7 (1:200, Abcam), rat-anti-F4/80 (1:400, Abcam), rabbit-anti-LYVE1 (1:100, Abcam), rat-anti-MOMA2 (1:100, Abcam), goat-anti-PDGFRA (1:50, R & D systems), rat-anti-LY6A(Sca1) (1:150, Biolegend), rat-anti-CD44 (1:100, Abcam), rabbit-anti-NOV/CCN3 (1:20, Elabscience), sheep-anti-FAP (1:100, R&D systems). PacificBlue-, AlexaFluor488-, AlexaFluor568, or AlexaFluor647-conjugated antibodies (1:500, Life technologies) against suitable species were used as secondary antibodies.
Microscopy.
Histological sections were imaged using a using a ZEISS AxioImager.Z2m (Carl Zeiss). For whole-mount 3D imaging of wounds, fixed samples were embedded in 35-mm glass bottom dishes (Ibidi) with low-melting point Agarose (Biozym) and left to solidify for 30 min. Imaging was performed using a Leica SP8 multi photon microscope (Leica, Germany). For live imaging of fascia cultures, samples were embedded as just above. Attention was paid to mount the samples with the fascia facing up towards the objective. Imaging medium (DMEM/F-12; SiR-DNA 1:1,000) was then added. Time-lapse imaging was performed over twenty hours under the multi photon microscope. A modified incubation system, with heating and gas control (ibidi 10915 & 11922), was used to guarantee physiologic and stable conditions during imaging. Temperature control was set to 35° C. with 5% CO2-supplemented air. Second harmonic generation signal and green auto-fluorescence as a reference were recorded every hour. 3D and 4D data was processed with Imaris 9.1.0 (Bitplane) and ImageJ (1.52i). Contrast and brightness were adjusted for better visibility.
Image Analysis.
Histological images were analyzed using ImageJ. For quantification of labeled cells in the fate mapping experiments, the Inventors manually defined the wound, surrounding dermis, and adjacent fascia areas. The Inventors defined the wound as the area flanked by the near hair follicles on both sides, extending from the base of the epidermis down to the level of the hair follicles bulges. Surrounding dermis area was defined as the 200 microns immediately adjacent to the wound bed on both sides. Fascia area was defined as the tissue immediately below the wound. The number of labeled cells in each area was determined by quantifying the particles that were double-positive for DAPI and for the desired label (eg. Dil, GFP, etc) channels. The coverage of the labeled matrix in the wound area was determined by quantifying the area that was double-positive for the labeled matrix and the COLLAGEN I+III+VI staining signal. Cell density of En1Cre; R26iDTR cultures treated with DT was quantified by dividing the total cells (DAPI) by the matrix area (COLLAGEN I+III+VI), Collagens density was calculated as the collagens area coverage of the entire section area. Matrix movements in live imaged cultures were determined by tracking the length of the two furthest points along the sample in both the second harmonic generation (SHG) and auto-fluorescence channels. Length measurements were normalized to the original length at time 0. Wound size was normalized for each time point using the original area at day 0. Scar length was quantified from randomly selected sections taken from the middle of the scar using as a reference the two flanking hair follicles. Relative fluorescence intensity (RFI) was calculated by measuring the mean gray value and normalizes to the dermis images. Fractal analysis was performed using the ImageJ plug-in ‘FracLac’29 (FracLac2015Sep090313a9390) using the same settings and preprocessing as previously described.
Dil Labeling of Fascia in Animals.
Two 5 mm-diameter full-thickness excisional wounds were created on the back of 8-10 weeks old C57BL6/J mice with a biopsy punch. 10-20 μl of the lipophilic “Vybrant™ Dil” dye (Life technologies, V22885) were injected into the exposed fascia directly above the dorsal muscles. Wounded tissue was harvested on day 9 and day 14 post-wounding and processed for histology and imaging by fluorescence microscopy.
Chimeric Skin Transplantations.
Full-thickness 6 mm-diameter biopsies were collected from the back-skin of either R26mtmg, R26VT2/GK3, En1Cre; R26mtmg, En1Cre; R26iDTR, or C57BL6/J adult mice. Using the Panniculus carnosus muscle layer as an anatomical reference, the fascia together with the muscle layer were carefully separated from the dermis and epidermis using Dumont #5 forceps (Fine Science Tools) and a 26G needle under the fluorescent stereomicroscope (M205 FA, Leica). EPFs from fascia+muscle samples of En1Cre; R26iDTR mice were ablated by incubation with 20 pg/ml of diphteria toxin (Sigma-Aldrich, D0564) or only DMEM/F12 as vehicle for 1 h at ambient temperature followed by 3 washing steps with PBS. At this point, the matrix samples were labeled by incubation with 100 μM Alexa Fluor™ NHS Ester (Life technologies, A20006) or Pacific Blue Succinimidyl Ester (Thermo Fisher, P10163) in PBS for 1 h at ambient temperature followed by 3 washing steps with PBS. Chimeras were made by placing the epidermis+dermis portion of a mouse strain on top of the muscle+fascia of another strain and left to rest for 20 min at 4° C. inside a 35 mm culture dish with 2 ml of DMEM/F12. Special attention was paid on preserving the original order of the different layers (Top to bottom: Epidermis->Dermis->Muscle->Fascia). Then, a 2 mm “deep” full-thickness was excised from the chimeric graft using a biopsy punch in the middle of the biopsy. To create “superficial” wounds, the 2 mm excision was done only in the epidermis+dermis half, prior to reconstitution with the bottom part. “Wounded” chimeric grafts were then transplanted into freshly-made 4 mm-diameter full-thickness excisional wounds in the back of either RAG2−/− or Fox Chase SCID immunodeficient 8-10 weeks-old mice. Precautions were taken to clean out the host blood from the fresh wound before the transplant and to leave the graft to dry for at least 20 min before ending the anesthesia, to increase the transplantation success. To prevent mice from removing the graft, a transparent dressing (Tegaderm, 3M) was placed on top of the grafts.
In Situ Matrix Tracing and EdU Pulses.
C57BL6/J mice received subcutaneous 20 microliter injections of 10 mg/mi FITC NHS ester in physiological saline with 0.1M sodium bicarbonate pH9 (46409, Life technologies) four and two days before wounding. At 2, 6, or 13 days post-wounding, mice received 200 μl i.p. injections of 1 mg/ml EdU in PBS. Samples were collected 24 hours after the EdU pulse and processed for cryosection and imaging by fluorescence microscopy.
Flow Cytometry.
Fascia and dermis were physically separated from the back-skin of C57BL6/J or En1Cre; R26mTmG mice under the fluorescence stereomicroscope as before. Harvested tissue was minced with surgical scissors and digested with an enzymatic cocktail containing 1 mg/ml Collagenase IV, 0.5 mg/ml Hyaluronidase, and 25 U/ml DNase I (Sigma-Aldrich) at 37° C. for 30 min. The resulted single cell suspension was filtered and incubated with conjugated/unconjugated primary antibodies (dilution 1:200) at 4° C. for 30 min, followed by an incubation with a suitable secondary antibody when needed at 4° C. for 30 min. Cells were washed and stained with Sytox blue dye (dilution 1:1000. Life technologies, S34857) for dead cell exclusion. Cells were subjected to flow cytometric analysis using a FACSAria III (BD Bioscience). Primary antibodies used: anti-DLK1 (Abcam), anti-CD9 (Santa Cruz), anti-CD271(LNGFR) (Miltenyi), anti-F4/80 (Abcam), AlexaFluor790-anti-NG2 (Santa Cruz), FITC-anti-DPP4(CD26) (eBioscience), PerCP-eFluor710-anti-ITGB1(CD29) (eBioscience), anti-CD34 (Abcam), PerCP-Cy5.5-anti-CD24 (eBioscience), APC-Fire750-anti-CD34(Biolegend), APC-anti-ITGA7 (R&D systems), PerCP-Cy5.5-anti-LY6A(Sca1) (eBioscience), PE-Vio770-anti-PDGFRA (Miltenyi), PerCP-Vio700-anti-CD146 (Miltenyi), APC-anti-PECAM1(CD31) (eBioscience), eFluor660-anti-LYVE1 (Thermo fisher), APC-LY76(TER119), APC-anti-EPCAM(CD326), and APC-anti-PTPRC(CD45). Secondary antibodies used: AlexaFluor488 Goat anti-Rabbit (Life technologies), AlexaFluor568 Goat anti-Rat (Life technologies).
Scanning Electron Microscopy.
Skin biopsies of adult C57BL6/J mice were collected, and the fascia was manually separated as before. Samples were then fixed overnight with paraformaldehyde and glutaraldehyde, 3% each, in 0.1% sodium cacodylate buffer pH 7.4 (Electron Microscopy Sciences, Germany). Samples were dehydrated in gradual ethanol and dried by the critical-point method, using CO2 as the transitional fluid (Polaron Critical Point Dryer CPC E3000; Quorum Technologies) and observed by scanning electron microscopy (JSM 6300F; JEOL, Germany).
ePTFE Membrane Implants.
Two 6 mm-diameter full-thickness excisional wounds were created with a biopsy punch on the back of 8-week old En1Cre; R26VT2/GK3 or C57BL6/J mice. Sterile 8 mm-diameter ePTFE impermeable membranes (Dualmesh®, GORE®) were implanted between the surrounding skin and the dorsal skeletal muscle underneath, to cover the open wound on the right side. For this, the surrounding skin was loosen using Dumont #5 forceps and spatula (10090-13, Fine Science Tools). The dual-surface membrane was implanted with the attaching face facing out, so to promote dermal cell attachment, while the smooth surface was in direct contact with the fascia. The left sham control wound underwent the same procedure without implanting any membrane. Each wound was photographed at indicated time points, and wound areas were measured using ImageJ. Wound sizes at any given time point after wounding were expressed as percentage of initial (day 0) wound area. At 7 or 63 days post-wounding, samples were collected and processed for histology.
Released Fascia Injury in Adult Mice.
Two 5 mm-diameter full-thickness excisional wounds were created with a biopsy punch on the back of 8-week old male C57BL6/J mice. The skin around the wound on the left side was separated from the underneath skeletal muscle using a sterilized gold-plated 3×5 mm genepaddles (Harvard Apparatus, #45-0122) to release the fascial layer. The right wound served as a control. Each wound was digitally photographed at indicated time points, and wound areas were measured using Photoshop (Adobe Systems, San Jose, Calif.). Wound sizes at any given time point after wounding were expressed as percentage of initial (day 0) wound area. The harvested tissue at the indicated time points was processed for cryosection and Masson's trichrome staining for histology.
Fascial Cells Ablation with AAV6-Cre Viral Particles and DT Treatment in Pups.
Two 3 mm-diameter full-thickness excisional wounds were created with a biopsy punch on the back of postnatal day 11 (P11) R26iDTR mice. 20 μl of Cre-expressing adeno-associated virus type 6 (AAV6-Cre) or control eGFP-expressing AAV6 (AAV6-EGFP) at viral titre of 5×1011/ml were injected subcutaneously at the area between the two wounds. Diphtheria toxin (DT) solution at 1 ng/μl in PBS was intraperitoneally injected to each mouse once per day for 7 days at the dosage of 5 ng/g. Tissue was harvested on 7 days after wounding.
Statistics.
All plots depict mean value and error bars represent SEM. Statistical analyses were performed using GraphPad Prism software (version 6.0, GraphPad). Statistical test and p values are specified in the figure legends and in the corresponding plots. For ease, p values below 0.0001 were stated equal as 0.0001.
To map the origins of all cells that contribute to wounds the Inventors developed a fate mapping technique by transplanting chimeric, skin and fascia, grafts into living animals (
At 14 days post wounding (dpw), 80.04±3.443% of the labeled cells in the wound bed were GFP+, indicating a fascia origin (
Next the Inventors took an independent in vivo labeling approach by injecting Dil-dye directly into the fascia (see ‘Methods’ and
Collectively, the two-independent fate-mapping approaches demonstrate that fascia is the major reservoir for the fibroblasts, endothelial, macrophages, and peripheral nerves that populate wounds at dermal surfaces.
The inventors previously showed that all scar-forming fibroblasts express Engrailed-1 (En1) early on in embryogenesis and the Inventors refer to these cells as Ent-lineage positive fibroblasts or EPFs. By crossing the En1-Cre recombinase driver (En1Cre) to a double-color fluorescent reporter (R26mtmg) the Inventors could lineage-trace all GFP+ EPFs across dermal and fascial compartments12-13. The Inventors then analyzed the cellular makeup of the fascia compared to dermis using En1Cre; R26mTmG double transgenic mice. Fibroblasts were the predominant fascia cell type (71.1% of the total living cells), while dermis had a significant lower fraction of total fibroblasts (56.4%,
The inventors then used two-photon microscopy to generate high-resolution 3D images of the whole back-fascia layer. EPFs were wedged in a specialized multilayered conformation within the fascia. EPFs were aligned in monolayers of consecutive perpendicular sheets across the dorsal-ventral axis (
The deeper an injury, the more severe the resulting scar. The Inventors therefore investigated if this correlation can be attributed to fascia by analyzing the extent of fibroblast contributions from the fascia and dermis in deep vs. superficial wounds.
To track the fibroblast contribution even more precisely, the Inventors combined the genetic lineage-tracing approach with anatomic fate-mapping by performing chimeric skin transplants using these mice (En1Cre; R26mtmg). The Inventors used fascia or dermis with traceable EPFs and the untraceable complementary tissue, and then made either a superficial wound through just the dermis and not the fascia below, or a deep excision through both tissues (
Fourteen days later, wound sizes in deep injuries were 1.7-times larger than superficial injuries (
To determine the final fate of fascial fibroblasts in wounds, the Inventors performed long-term tracing of chimeric grafts with traceable fascial EPFs. After 10 weeks, fascial EPFs were completely absent from the wound bed (
Having established that fascial EPFs are the primary cells that direct and enact wounding, the Inventors sought to place fascial EPFs in the framework of previously reported fibroblast lineage markers by co-immunostaining. Markers previously used to define other sources and lineages of wound fibroblasts CD24, CD34, DPP4 (CD26), DLK1, and LY6A (Sca1) were all more prominent in fascial-EPFs compared to dermal EPFs, and all five markers were surprisingly downregulated upon entering the wounds (
The inventors then looked at the fascia gel itself. Second harmonic generation (SHG) signal and scanning electro-micrographs both revealed profuse collagen fibrils in a coiled arrangement in the fascia, indicative of a relaxed and immature matrix reservoir (
The immaturity of the fascia matrix itself motivated us to check if it could work as a repository for provisional matrix tissue in skin wounds. To answer this question, the Inventors first developed an incubation chamber that enabled live imaging of the fascia matrix over days (see ‘Methods’). Remarkably, recording of SHG signal over 30 hours illuminated steering of the whole matrix across the fascia at a rate of 11.4 μm/hour (
To test if fascia matrix shoots upwards into wounds in vivo, the Inventors developed a technique to trace and fate map the fascia matrix using the chimeric grafts. In this new assay, the Inventors excised the fascia and fluorescently tagged its matrix using an Alexa Fluor 647 NHS Ester. The Inventors combined the labeled fascia with unlabeled wounded dermis and transplanted the chimeric graft into the host back-skin (
The inventors then asked whether dermal matrix can be steered as well. Using the chimera experiments, the Inventors labeled both dermis and fascia with different fluorescent NHS esters. Only the fascia matrix was able to plug open wounds (
To definitively prove that fascia matrix is steered into and clog open wounds, the Inventors labeled the fascia matrix in situ with FITC NHS ester prior to injury (
To test if matrix steering from fascia is caused by EPFs, the Inventors generated deep excisional wounds, and physically separated fascia from upper skin by implanting an impermeable dual surface ePTFE membrane between the fascia and the PC muscle layer (
The inventors then asked whether mechanical separation between dermis and fascia alone, without barrier implants, would affect matrix steering and scar formation. To address this question, the Inventors performed full excisional wounds in wild type mice and physically released the fascia below the PC muscle surrounding the fresh wound (
To definitively link fascial EPFs to matrix steering into wounds, the Inventors genetically ablated fascial EPFs using two separate strategies. First, the Inventors used a transgenic line that expresses the diphtheria toxin receptor (DTR) in a Cre-dependent manner (R26iDTR). This line allowed us to deplete cells expressing Cre recombinase upon diphtheria toxin (DT) exposure. The Inventors thus generated Cre-expressing adeno-associated viral particles (AAV6-Cre) and injected them into the fascia of R26iDRT pups underneath freshly made full excisional wounds (
In a second independent approach, the Inventors used En1Cre; R26iDTR double transgenic mice in which DTR expression is restricted to EPFs, making them susceptible to DT-mediated ablation. To corroborate the ablation of EPFs in the fascia, the Inventors cultured fascia biopsies from En1Cre; R26iDTR for 6 days after an acute exposure to DT for 1 h ex vivo. A single exposure of 2 μg/ml DT prevented the normal increase in collagen fiber density observed in control samples and in wounds (fiber contraction and deposition,
Next, the Inventors created chimeric grafts using dermis from wild-type mice and fascia from En1Cre; R26iDTR mice. The Inventors ablated fascial EPFs using DT as before, then fluorescently labeled the matrix, and transplanted the skin grafts into the back-skin (
To check if fibroblastic proliferations preceded and was needed for matrix steering (
Human keloids are abnormal scars with clinical features of early and unresolved wounds (e.g. itchiness, inflammation, and pain) that progressively grow beyond the injury site27. These unresolved features in human scars motivated us to investigate the presence of fascia and fascia fibroblasts in human skin and keloid tissue. The Inventors found bands of connective tissue in the subcutaneous space of human skin across multiple anatomic skin locations (
Scars form by mobilizing fascia to sites of injury. The mechanism of this patch-repair is still obscure despite wounds being an extensively studied major clinical challenge. Here, the Inventors reveal a unique cellular mechanism of fibroblast sprouting and webbing that enact fascia mobilization and scarring. The Inventors screen live fascia explants with a library of 1280 small molecules and unearth a phenotypic class of chemicals with negligible effects on matrix biogenesis, yet completely inhibit scar formation by halting fascia mobilization, termed matrix motion inhibitors. The Inventors show that matrix motion inhibitors alter fibroblast sprouting and webbing. Inhibiting sprouting and webbing by either Thiostrepton, Fluvastatin sodium salt and Itraconazole reduced fascia jelly movements and led to a reduced scaring of wounds in animals. The findings place sprouting and webbing as a germinal mechanism of fibrotic scar formation, and a novel therapeutic space where matrix motion inhibitors provide a novel class of therapeutic treatments for the range of human fibrotic conditions.
Methods
Mouse Lines and Animal Experiments
En1Cre; R26mTmG, En1Cre; R26mcherry, C57BL/6J were purchased from Charles River or Jackson laboratories or generated in Research Animal Facility at the Stanford University as previously described (Rinkevich Y et al., 2015). Animals were housed in Animal Facility at Helmholtz Zentrum München at constant temperature and humidity with a 12-hour light cycle. Food and water were provided ad libitum. All animal experiments were reviewed and approved by the Government of Upper Bavaria and registered under the project 55.2-1-54-2532-61-16 and conducted under strict government and international guidelines. This study is compliant with all relevant ethical regulations regarding animal research.
For the chemical treatment study in live mice, splinted wounds were made in wildtype mouse back skin. Splinting rings were prepared from a 0.5-mm silicone sheet (CWS-S-0.5, Grace Bio-Labs) by cutting rings with an outer diameter of 12 mm and an inner diameter of 6 mm. After washing with detergent and rinsing with water, the splints were sterilized with 70% ethanol for 30 min and air dried in a cell culture hood and kept in a sterile bottle. Mice were anaesthetized with 100 μl MMF (medetomidine, midazolam and fentanyl). Dorsal hair was removed by a hair clipper, followed by hair removal cream for 3-5 min. Two full-thickness excisional wounds were created with a 5-mm diameter biopsy punch (Stiefel). One side of a splint was applied with silicone elastomer super glue (Kwik-Sil Adhesive, World Precision Instruments) and placed around the wound. The splint was secured with 5 sutures of 6.0 nylon, 75 μl per wound saline diluted chemical solutions with a final concentration of 250 μM were injected intra-dermally immediately after suture. Mice were recovered from anesthesia with an MMF antagonist and were supplied with metamizole (500 mg metamizole/250 ml drinking water) as postoperative analgesia. Scar samples were collected on D21.
For the fascia labelling study, 2 mg/ml NHS-fluorescein dye was subcutaneously injected into dorsal skin of wildtype mice at 4 days and 2 days prior to the surgery. After labelling, excisional wounds were made in mouse back-skin and treated with 75 μl saline diluted chemical solutions with a final concentration of 250 μM three times a week from the surgery day onwards. Scar samples were collected on D7.
Ex Vivo Skin Explant Assay
Post born Day 0 (P0) neonates of C57BL/6J wild type mouse were first sacrificed by decapitation. Then dorsal back skin was isolated to make 2 mm full thickness biopsies (0 2 mm, Stiefel) that included the epidermis, dermis and deep subcutaneous fascia layers. The whole skin tissue explant system is termed as scar-in-a-dish (SCAD) assay (Patent Application no. PLA17A13). The SCAD tissue was maintained in DMEM/F12 cell culture medium (Life Technologies) supplemented with 10% FBS (Life Technologies), 1% non-essential amino acid (Thermo Fisher), 1% Glutamax (Thermo Fisher), 1% penicillin and streptomycin (Thermo Fisher) in a 37° C. incubator supplied with 95% O2 and 5% CO2. Medium was changed every other day until day 5 when SCADs were collected and fixed for histology analysis. To be noticed, the excised skin was submerged dermis-side up in culture media, which confined the scar-prone fibroblasts to the explant and discouraged their adherence to the tissue culture plate.
Prestwick Chemical Library® (PCL) and Automatic Screening of Chemicals
The Prestwick library contains 1280 approved (by FDA, European Medicines Agency (EMA) or other agencies) small molecules covering a range of major anatomical therapeutic classes including central nervous system (19%), cardiovascular system (11%), metabolism (24%) and infectious diseases (16%). The purity of the compounds was >90% as reported by the provider of the compounds. The PCL provides an additional advantage as all chemicals are of stable physicochemical properties, show a high range of chemical diversity, and are with known bioavailability and safety data in humans. All these information helps to reduce the probability of screening low-quality hits and save the costs of preliminary screening process.
In order to accommodate to medium scale screening approach, the Inventors adapted the SCAD explant system into 96-well plate (Falcon) formats, with each well contained one biopsy. The novel 96-well SCAD pipeline was then combined with the 1280 approved small molecules from the Prestwick chemical library. Plate and liquid handling were performed using a high-throughput screening platform system composed of a Sciclone G3 Liquid Handler from PerkinElmer (Waltham, Mass., USA). On Day 0 tissues were treated either with the respective compound (1 mM stock solution) dissolved in 100% dimethyl sulfoxide (DMSO, Carl Roth) or DMSO alone. 0.5 μl of compounds/DMSO were transferred with a 96-array head to 200 μl DMEM/F12 medium per well to keep the final DMSO concentrations at 2.5 μM. Tissues were then incubated (37° C.; 5% CO2) for 72 h prior to a second round of compound treatment, which was performed by exchanging cell culture medium per well and transferring 2.5 μM of compounds/DMSO into the fresh medium. After an incubation time of another 48 h (37° C.; 5% CO2) the tissues were harvested and fixed for histological processing and imaging.
Ex Vivo Fascia Three-Dimensional (3D) Culture-Fascia Invasion Assay
To create a 3D environment that mimics the physiological environments in vivo, the Inventors established a fascia Matrigel (Corning) system. In order to observe the dynamic changes of fascia fibroblasts, the inventors isolated dorsal skin from P4 to P6 neonates of En1Cre; R26mTmG mouse lines. Cre positive neonates from this double transgenic mouse line were detected by green fluorescent signal in dorsal skin with a Leica M205 FA stereo microscope. Matrigel was prepared by diluting the stock aliquots with DMEM/F12 medium to a concentration of 6 mg/ml. Then 150 μl prepared gel was added in the center of a 35 mm cell culture dish (Ibidi). 4 mm biopsies were made from the dorsal skin and fascia tissues were then isolated from the dorsal skin tissues. Isolated fascia tissues were then embedded into the gel and were allowed to be solidified for one hour at 37° C. Then the tissue-gel system was maintained in DMEM/F12 medium (Life Technologies) supplemented with 10% FBS (Life Technologies), 1% non-essential amino acid (Thermo Fisher), 1% Glutamax (Thermo Fisher), 1% penicillin and streptomycin (Thermo Fisher) in a 37° C. incubator supplied with 95% O2 and 5% CO2. Medium was changed every other day until day 4 when tissues were collected and fixed. For fixation with Matrigel system, tissues were fixed in 2% paraformaldehyde (VWR) with 0.1% glutaraldehyde (Sigma) for 1 hour and then washed three times with phosphate buffered saline (PBS, Life Technologies) and stored in PBS at 4° C.
Invasion Index and Contraction Index Measurement
Fascia tissues were recorded everyday by a brightfield microscope to check the invasion and contraction state of the tissues. The invasion index was calculated with the following formula: Invasion index=(SD4−SD0)/SD0
SD4 and SD0 represent tissue size (including the migrated area) on Day 4 and Day 0, respectively. The contraction index was calculated with the following formula: Contraction index=(TD0−TD4)/TD0
TD4 and TD0 represent original tissue size (excluding the migrated area) on Day 4 and Day 0 respectively.
Histology
Except otherwise stated, all the samples were fixed overnight in 2% paraformaldehyde (VWR) in PBS at 4° C. and washed three times with PBS. Samples were then embedded in optimal cutting temperature compound (OCT, Sakura Finetek) and snap frozen on dry ice. 6 μm frozen sections were made by a cryostat (Cryostar NX70, Thermo fisher) and frozen section slides were stored at −20° C. Masson's trichrome staining was applied using a trichrome stain kit (Sigma-Aldrich) according to the manufacturer's instructions. Images were recorded by a ZEISS AxioImager. Z2m (Carl Zeiss) with brightfield channel. In Masson's trichrome staining, muscle fibers and keratin are stained as red color, collagen is stained as blue, cytoplasm is stained as light red and cell nuclei is stained as black.
3D Staining and Whole Mount Imaging
In order to characterize the properties of fascia samples cultured in Matrigel, the Inventors fixed the whole gel (with fascia tissue embedded inside) and conducted 3D staining. Samples were immersed overnight in PBSGT (lx PBS implemented with 0.2% gelatin (Sigma), 0.5% Triton X-100 (Sigma) and 0.01% thimerosal (Sigma)) at room temperature and incubated with primary antibodies diluted in PBSGT for three days at room temperature. The tissues were then washed three times with PBSGT for at least 30 min each time and incubated with secondary antibodies diluted in PBSGT for one day. Finally, tissues were rinsed three times with PBSGT and stored in PBS at 4° C. until imaging. 3D whole mount imaging was conducted with a Leica SP8 multi-photon microscope.
Primary and secondary antibodies applied in 3D staining: αSMA (ab21027, Abcam), Ki 67 (ab16667, Abcam), Cleaved Caspase-3 (9661S, Cell signalling), Gli1 (ab49314, Abcam), Donkey anti rabbit AF647 (A-31573, Life Technologies), Donkey anti goat AF647 (A-21447, Life Technologies).
Live Imaging
Fascia tissue cultured in Matrigel was fixed in 2% low-melting agarose (Biozym) and left at room temperature to be solidified. DMEM/F12 medium without phenol red was then added to keep the tissues alive during imaging. Four-dimension (4D) time-lapse images were performed by a Zeiss AxioObserver Z1 microscope for tissues obtained from En1Cre; R26mCherry mouse line or a Leica SP8 multi-photon microscope for tissues obtained from En1Cre; R26mTmG mouse line. Samples were placed in a qualified incubator with heating and gas control (Ibidi). The incubator temperature was adjusted to 35° C. and was supported with 5% CO2 during imaging. Brightfield and mCherry signals were recorded for tissues from En1Cre; R26 mCherry; green fluorescent protein (GFP) and tdTomato signals were recorded for tissues from En1Cre; R26mTmG.
Cell Tracking
4D time-lapsed imaging was subjected to maximum intensity projection in Imaris 9.3.1 (Bitplane) software. The projected data sets were proceeded to cell migration and cell-tracking analysis using Trackmate function of ImageJ. Variables, such as blob diameter, threshold, and segmentation detector were adjusted to suit the nature of the data and the samples. For the fourth dimension of the tracks, color ramp was applied to the individual tracks as a function of time (blue, first time point of the track; orange, last time point of the track).
Data Analysis
3D images and time series videos were processed with Imaris 9.3.1 (Bitplane). Brightness and contrast were modified to exclude false positive signal and to obtain better visibility. Fractal analysis was conducted using the ImageJ plug in ‘FracLac’29 (FracLac 2015Sep090313a9390) (Karperien A, 1999-2003). Fractal dimension (DF) values and Lacunarity (Lac) values were calculated using the box counting approach (slipping and tighten grids were set at default sample sizes, threshold of minimum pixel density was set as 0.40).
Statistics and Reproducibility
Statistical analysis was performed using GraphPad Prism software (Version 7.0, GraphPad). Statistical significance was determined using analysis of variance (ANOVA) with Tukey's or Dunnett's multiple comparison test, as indicated in the corresponding figure legends. Until otherwise stated, all results were repeated with at least three independent experiments or three biological samples with consistent results. Cell tracking were derived with three single movies. 3D staining was performed on two samples and images were recorded at three different sites of the samples.
Anti-Fibrotic and Pro-Fibrotic Agents Identified Through Compound Screening
To identify the mechanism of how fascia is mobilized/centralized in wounds to form scars, the Inventors took advantage of whole skin-fascia explants, in which uniform centralized scars develop ex vivo (Correa-Gallegos et al., 2019). Briefly, the Inventors excised 2-mm full thickness skin biopsies that included epidermis, dermis, and deep subcutaneous fascia layers from mouse backs (see Methods). Skin-fascia explants were submerged fascia-side up in culture media, to confine scar-prone fibroblasts to the explant and discourage their adherence to the tissue culture plate. Under these conditions, explants develop uniform scars over a course of 5 days that contract and fold the skin (
In order to discover novel inhibitors of fascia mobilization and scar formation, the Inventors combined the skin-fascia explant system with 1280 FDA-approved small molecules (Prestwick library) via a high-throughput screening platform. The Prestwick library has selected a diverse array of chemicals with strong physicochemical properties, with known bioavailability and clinical safety data. The library is an ideal place to start screening as it covers all major therapeutic chemical classes such as central nervous system, cardiovascular system, metabolism, and infectious diseases.
Our initial phenotypic screening of the 1280 small molecules identified 122 chemicals (9.53% ‘hits’) that consistently changed the extent of mobilization/centralization of the fascia connective tissue and of scar size and severity (part A as table 1 and part B as
Part B of table 1 is depicted as
The inventors categorised the scar-active hits into 18 distinct phenotypic groups, based on overall scar dimension and morphology. For example, certain explant groups gave exuberant scars that extended beyond the skin explant boarders, with contraction and bending similar to hypertrophic scars (
To further classify and grade the ranges of scar phenotypes and severities, the Inventors performed fractal analysis on all sample groups, by determining the fractal dimensions (DF) and lacunarity (porosity) values of the ECM lattice organisation (Jiang et al., 2018). Fractal porosity and lacunarity are measures of the general organization of the extracellular matrix, with scars having higher fractal dimensions (FD) and lower lacunarity (L) values than normal healthy skin (
The combined histo-morphometric and fractal analysis, together, allowed us to extend the phenotypic groups of chemicals into 26 distinct grades/severities, each with unique scarring phenotypes. Out of the 26, ten compounds gave more severe scars, whereas sixteen reduced scarring with measurable anti-fibrotic effects (
Fascia Fibroblasts Form Reticulations Ex-Vivo
To visualize the early mechanisms that mobilize/centralize fascia connective tissue, the Inventors crossed fibroblastic lineage reporter mice (En1Cre) with transgenic reporter mice (R26mTmG). Double transgenic offspring (En1Cre; R26mTmG) express GFP under the En1 promoter, thereby genetically tagging fibrogenic lineage cells (EPFs) in the fascia with GFP. The Inventors excised fascia explants from back-skin of this double-transgenic mice and performed high-resolution live imaging to follow individual EPFs throughout scarring. EPFs within the fascia jelly became increasingly connected to one another within the jelly (
To better visualize the dynamics of sprouting and reticulation of fascia fibroblasts, the Inventors crossed the fibroblast lineage-specific promoter (En1) mice to a nuclear mCherry reporter line, allowing to computationally track and map all fibroblast dynamics. Live imaging of fascia explants from En1Cre; R26mcherry double transgenic mice from day 2 to day 5 revealed cell-cell connections formed a network of cell clusters. Automatic tracking data also revealed a formed network of fibroblasts that constantly sprout new filaments, which anastomose to create a web of interconnected fibroblasts (
Three Top Anti-Scar Agents were Identified by Secondary Screen on Fascia
The inventors then went on to determine the anti-scarring actions of the small molecules. The Inventors isolated fascia from back-skin of En1Cre; R26mTmG double-transgenic mice, separately incubated whole fascia explants with 26 small molecules, and measured the invasion index (Si)=(SD4−SD0)/SD0 (see Methods) as a proxy measure of sprouting and reticulation. The change of invasion index in the presence of the 26 small molecules precisely mimicked both the anti- and pro-scarring effects from the original screen (
Among the five top anti-scarring chemicals, Fenbendazole (210) and Pyrvinium pamoate (1040) showed toxic effects to fascia tissues, so the Inventors focused on the other three for mechanism study. The three anti-fibrotic agents Itraconazole (1139), Thiostrepton (522), and Fluvastatin sodium salt (859) all significantly perturbed the magnitude of inter-cell connectivity and completely inhibited sprouting and reticulations within the fascia, whereas control fascia showed extensive sprouting and reticulations followed by a massive increase in invasion speed and change in overall fascia dimensions (
Anti-Scar Agents Inhibit Fibroblast Reticulation Through Sonic-Hedgehog
To better understand the molecular basis of reticulations that leads to scarring, the Inventors took a closer look at the molecular targets of the ‘hit’ chemicals. All three anti-scarring chemicals, including the pro-scarring chemical, all effectively targeted hedgehog pathway signaling, in multiple ways and Gli-1 nuclear protein expression was significantly decreased in the presence all three anti-scarring treatments (
The hedgehog pathway was also reported to induce proliferation and to depress apoptosis. Ki67 and caspase 3 staining showed that cell proliferation activity of Thiostrepton, Itraconazole and Fluvastatin treated samples decreased (
Anti-Scar Agents Inhibit Fascia Mobility In Vivo
Having discovered sprouting and reticulations are altered by the compounds, the Inventors went on to check how these effect fascia mobilization in physiologic wounds in live animals. First, the Inventors labelled the subcutaneous fascia layer with a FITC-NHS ester dye in animals, then made full thickness excisional wounds on the backs of fascia-labelled mice, and followed wound healing under the presence of a weekly injection regimen using the above 3 separate compounds. Skin wounds were collected at Day 7 after wounding to determine the extent of fascia mobilization and also at 21 days post-injury to determine final wound size and scar severity. In DMSO control groups, the fascia matrix jelly was mobilized into the wound from all sides of the wound, and wounds at 7 days were completely clogged with large patches of labelled matrix jelly. All three anti-scarring drugs, on the other hand, significantly reduced fascia mobilization into wounds. Fluvastatin completely inhibited matrix jelly movements. Itraconazole and Thiostrepton treatments also inhibited fascia jelly movements, with only marginal connective tissue fragments relocating into wounds, and without the massive shift seen in controls (
Anti-Scar Agents Inhibit Scar Formation In Vivo
Next, the Inventors tested the three chemicals in an in vivo splinted wound model to document their overall effects on scarring. Trichrome staining showed that all the three anti-scarring chemicals gave smaller scars on day 21 after wounding (
Damaged organs repair injuries by forming new connective tissue, reestablishing structural and mechanical continuums that ensure survival, but it has been unclear how connective tissues repopulate and rebuild the injured site. Specifically, it was believed that local fibroblasts secrete new extracellular matrix.
Here the Inventors focus on three different internal organs to reveal the basis of damage repair. By separately tagging extracellular matrix of liver, cecum and peritoneum before injury in live mice the Inventors demonstrate that the matrix itself plays the primary role in the damage response. Thus, the Inventors identify reservoirs of fluid-like matrix in connective tissues that gush across organs to repair liver, cecal and peritoneal wounds.
Using proteomics analysis, the Inventors uncover distinct compositions of fluid matrix that lead to regeneration or scarring and fibrous adhesions. Using single cell analysis and mechanistic studies, the Inventors uncover neutrophils orchestrate matrix flows and are functionally primed for matrix transportation in multiple ways. Blocking neutrophil adherence, their chemotactic or nitric oxide signaling inhibited matrix flows, and curbed postsurgical adhesions and liver regeneration. The finding of a body-wide reservoir of fluid matrix reconfigures the traditional view of wound repair, and provides a wide potential novel therapeutic space to treat impaired wounds and excessive scarring across a range of human diseases/conditions.
Methods
All mouse lines were obtained (C57BL/6J, B6.129P2-Lyz2tm1(cre)lfo/J (Lyz2Cre), B6; 129S6-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J (Ai14)) from Jackson Laboratories or Charles River and bred and maintained in the Helmholtz Animal Facility in accordance with EU directive 2010/63. Animals were housed in individual ventilated cages (IVC) and animal housing rooms were maintained at constant temperature and humidity with a 12-h light cycle. Animals were supplied with water and chow ad libitum. All animal experiments were reviewed and approved by the Government of Upper Bavaria and registered under the project number ROB-55.2-2532.Vet_02-19-133 or ROB-2532.Vet_02-19-148 and conducted under strict governmental and international guidelines. This study is compliant with all relevant ethical regulations regarding animal research.
Thirty minutes before surgery mice received a preemptive subcutaneous injection with Metamizole (200 mg/kg bw). Anesthesia was supplied by an intraperitoneal injection of a Medetomidin (500 μg/kg), Midazolam (5 mg/kg) and Fentanyl (50 pg/kg) cocktail, hereafter referred to as MMF. Monitoring anesthetic depth was assessed by toe reflex. Eyes were covered with Bepanthen-cream to avoid dehydration, and the abdomen was shaved and disinfected with betadine and sterile phosphate buffered saline (PBS). Animals were kept on their backs on a heating plate at 39° C. A midline laparotomy (1-1.5 cm) was performed through the skin and peritoneum. Four hooks, positioned around the incision and fixed to a retractor and magnetic base plate, allowed for clear access to the abdominal cavity and liver.
Local damage to the liver surface was induced via electroporation tweezers by applying 30V 50 ms pulses at 1s interval for 8 cycles. Before closure of the incision, Buprenorphine (0.1 mg/kg) was pipetted in the abdomen to allow for initial post-surgical analgesia. For long-term analgesia, Metamizole (Novalgin, 200 mg/kg) was provided through daily injections. The peritoneum and skin were closed with two separate 4-0 silk sutures (Ethicon). Upon closure of the incision, mice were woken up by antagonizing Medetomidin and Midazolam through a subcutaneous cocktail injection of Atipamezol (1 mg/kg) and Flumazenil (0.25 mg/kg). Mice were allowed to recover on a heating pad, after which they were single housed. Mice were sacrificed after indicated time points and liver tissue was obtained. In the peritoneal model, surgical procedure was as described above, but the peritoneal areas were marked.
To induce adhesions between liver and peritoneum, abrasion was applied to the electroporated side of the liver and to the opposite side of the peritoneum. In the peritoneal-cecal adhesion model, surfaces of cecum and peritoneum were injured with a brush, two surgical knots were placed and talcum powder was applied onto wound sides of both organs. Immune cell knockout was performed as follows: inhibitors were injected intraperitoneally 2 hours before surgery at a concentration of 10 μM in sterile PBS. Neutralizing antibodies (Bio X Cell) were applied at a concentration of 200 pg/20g body weight. Clodronate Liposomes (Liposoma), CP-105,696 and LY255283 (Sigma Aldrich), TD139 (Probechem), W1400 (Enzo) and L-NAME (Biocat) were applied at a concentration of 10 μM. Lipoxin (Merck Millipore) was applied locally by soaking the reagent in a sterile filter paper with 100 nM solution and applying the filter paper over the liver surface for 5 minutes.
All human samples were obtained from surgery at the Department of Surgery, Klinikum rechts der Isar, Technical University of Munich, following approval of the local ethics committee of the Technical University of Munich, Germany (Nr. 173/18 S). Adhesions were intraoperatively diagnosed and dissected from the respective organs and prepared for further analysis.
Succinimidyl esters (NHS-esters; Thermo Fisher) were diluted in DMSO to a concentration of 25 mg/ml and stored at −80° C. To obtain ectopic labeling of matrix, the Inventors generated a labelling solution by mixing NHS-ester 1:1 with 100 mM pH 9.0 sodium bicarbonate buffer. Sterile Whatman filter paper (Sigma Aldrich) biopsy punches where soaked in NHS-labelling solution, and locally placed on the liver surface. After one minute, the labelling punch was removed. For global abdominal labelling, 20 μl of NHS-labelling solution were mixed with 100 μl sterile PBS and injected i.p. For kinetic measurements organ surfaces were marked with either a 1.0 cm (near) or 2 cm (far) 2 mm filter patch with NHS-FITC.
Upon organ excision, organs were fixed overnight at 4° C. in 2% formaldehyde. The next day, fixed tissues were washed three times in Dulbecco's phosphate buffered saline (DPBS, GIBCO, #14190-094), and depending on the purpose, either embedded, frozen in optimal cutting temperature compound (Sakura, #4583) and stored at −20° C., or stored at 4° C. in PBS containing 0.2% gelatin (Sigma Aldrich, #G1393), 0.5% Triton X-100 (Sigma Aldrich, #X100) and 0.01% Thimerosal (Sigma Aldrich, #T8784) (PBS-GT). Fixed tissues were embedded in optimal cutting temperature (OCT) compound and cut with a Microm HM 525 (Thermo Scientific) by the standard protocol. In short, In short, sections were fixed in ice-cold acetone for 5 min at −20° C., and then washed with PBS. Sections were then blocked for non-specific binding with 10% serum in PBS for 60 minutes at room temperature, and then incubated with primary antibody in blocking solution O/N at 4° C. The next day, following washing, sections were incubated in PBS with fluorescent secondary antibody, for 120 min at RT. Finally, sections were washed and incubated with Hoechst 33342 nucleic acid stain (Invitrogen, #H1399), washed in ddH2O, mounted with Fluoromount-G® (Southern Biotech, #0100-01), and stored at 4° C. in the dark. Primary antibodies: rabbit-anti-collagen I (1:150, Rockland), rabbit-anti-Cytokeratin (1:100, Sigma Aldrich), rabbit-anti-Ki67 (1:100, Abcam), rabbit-anti-Fibronectin (1:100, Abcam), rabbit-anti-HSP70 (1:100, Elabscience), rabbit-anti-HSPG2 (1:100, Elabscience), rabbit-anti-Keratin9 (1:100, Elabscience), rabbit-anti-Ki67 (1:100, Abcam), rabbit-anti-cleaved Caspase 3 (1:100, Abcam), rabbit-anti-Laminin (1:100, Abcam), rabbit-anti-HSP70 (1:100, Elabscience), hamster-anti-PDPNα (1:100, Abcam), rat-anti-LY6G(Sca1) (1:100, Abcam), rabbit-anti-MMP23(1:100, Elabscience), rabbit-anti-Vitronectin (1:100, Elabscience) and rabbit-anti-WT1 (1:100, Abcam). Alexa Fluor 488-, Alexa Fluor 568- or Alexa Fluor 647-conjugated antibodies (1:500, Life technologies) against suitable species were used as secondary antibodies. H&E staining was performed according to manufacturer's protocol (Sigma).
Histological sections were imaged under a M205 FCA Stereomicroscope (Leica). For whole-mount 3D imaging of tissues, fixed samples were embedded in 35-mm glass bottom dishes (Ibidi) with low-melting point agarose (Biozym) and left to solidify for 30 min. Imaging was performed with a Leica SP8 multi photon microscope (Leica, Germany). For time-lapse imaging liver and peritoneal tissues, samples were embedded as just above. Imaging medium (DMEM/F-12) was then added. Time-lapse imaging was performed under the M205 FCA Stereomicroscope. A modified incubation system, with heating and gas control (ibidi, catalogue nos. 10915 and 11922), was used to guarantee physiologic and stable conditions during imaging. Temperature control was set to 35° C. with 5% CO2-supplemented air. 2D, 3D and 4D data was processed with Imaris 9.1.0 (Bitplane) and ImageJ (1.52i). Contrast and brightness were adjusted for better visibility.
Tissues were snap frozen and grinded using a tissue lyser (Qiagen). Pulverised tissues were resuspended in lysis buffer (20 mM Tris-HCl pH 7.5, 1% Triton X-100, 2% SDS, 100 mM NaCl, 1 mM sodium orthovanandate, 9.5 mM sodium fluoride, 10 mM sodium pyruvate, 10 mM beta-glycerophosphate), and supplemented with protease inhibitors (complete protease inhibitor cocktail, Pierce) and kept 10 min on ice. Samples were sonicated and spun down for 5 minutes at 10,000g. Supernatants were stored at −80° C. Protein concentrations were determined via BCA-Assay according to the manufacturer's protocol (Pierce).
Protein pulldown was as follows. Lysates were diluted with a pulldown buffer (20 mM Tris-HCl pH 7.5, 1% Triton X-100, 100 mM NaCl, supplemented with protease and phosphatase inhibitors) and incubated overnight with dynabeads (Thermo Fisher) according to the manufacturer's instructions at 4° C. on a rotator. The next day, the samples were each diluted twice with Wash Buffer 1 (20 mM Tris-HCl pH 7.5, 1% Triton X-100, 2% SDS, 100 mM NaCl and supplemented with protease and phosphatase inhibitors) and then with Wash Buffer 2 (20 mM Tris-HCl pH 7.5, 0.5% Triton X-100, 100 mM NaCl) and supplemented with protease and phosphatase inhibitors and finally washed twice with Wash Buffer 3 (20 mM Tris-HCl pH 7.5 and 100 mM NaCl). Beads were then resuspended in Elution Buffer (20 mM Tris-HCl pH 7.5, 100 mM NaCl and 50 mM DTT) and incubated for 30 minutes at 37° C. Finally, the samples were boiled for 5 minutes at 98° C. and the supernatants were stored at −80° C. Fluorescence intensities of lysates were measured in a Fluostar optima fluorometer (BMGlabtech).
Tissues were marked locally with an EZ-LINK-NHS 100:1 FITC-NHS mixture. After 24 hours the organs were removed. Tissue pieces from the original marking were separated from moved matrix fractions and snap frozen. Tissue lysis was performed as described above. Samples were digested by a modified FASP procedure23. After reduction and alkylation using DTT and IAA, the proteins were centrifuged on Microcon® centrifugal filters (Sartorius Vivacon 500 30 kDa), washed thrice with 8 M urea in 0.1 M Tris/HCl pH 8.5 and twice with 50 mM ammoniumbicarbonate. The proteins on the filter were digested for 2 hours at room temperature using 0.5 μg Lys-C (Wako Chemicals, Neuss, Germany) and for 16 hours at 37° C. with 1 pg trypsin (Promega, Mannheim, Germany). Peptides were collected by centrifugation (10 min at 14,000 g), acidified with 0.5% TFA and stored at −20° C. until measurements. The digested peptides were loaded automatically onto an HPLC system (Thermo Fisher Scientific) equipped with a nano trap column (100 μm ID×2 cm, Acclaim PepMAP 100 C18, 5 μm, 100 Å/size, LC Packings, Thermo Fisher Scientific, Bremen, Germany) in 95% buffer A (2% ACN, 0.1% formic acid (FA) in HPLC-grade water) and 5% buffer B (98% ACN, 0.1% FA in HPLC-grade water) at 30 μl/min. After 5 min, the peptides were eluted and separated on the analytical column (nanoEase MZ HSS T3 Column, 100 Å, 1.8 μm, 75 μm×250 mm, Waters) at 250 nl/min flow rate in a 105 minute non-linear acetonitrile gradient from 3 to 40% in 0.1% formic acid. The eluting peptides were analyzed online in a Q Exactive HF mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) coupled to the HPLC system with a nano spray ion source and operated in the data-dependent mode. MS spectra were recorded at a resolution of 60,000 and after each MS1 cycle, the 10 most abundant peptide ions were selected for fragmentation. Raw spectra were imported into Progenesis QIsoftware (version 4.1, Nonlinear Dynamics, Waters). After feature alignment and normalization, spectra were exported as Mascot Generic files and searched against the SwissProt mouse database (16,872 sequences) with Mascot (Matrix Science, version 2.6.2) with the following search parameters: 10 ppm peptide mass tolerance and 0.02 Da fragment mass tolerance, two missed cleavages allowed, carbamidomethylation was set as fixed modification, camthiopropanoyl, methionine and proline oxidation were allowed as variable modifications. A Mascot-integrated decoy database search calculated an average false discovery of <1% when searches were performed with a mascot percolator score cut-off of 13 and an appropriate significance threshold p.
Peptide assignments were re-imported into the Progenesis QI software and the abundances of all unique peptides allocated to each protein were summed up. The resulting normalized abundances of the individual proteins were used for calculation of protein ratios and p-values (ANOVA) between sample groups using a nested design. Gene ontology analysis was performed using the EnrichR webtool24,25. Extracellular elements were identified through a database search against http://matrisomeproject.mit.edu/.
Three livers per experimental group were pooled for each sequencing run. For each liver, the electroporated area was punched out with a circular 4 mm biopsy punch, and subsequently minced with fine scissors into small pieces (approximately 1 mm2). The equivalent, but non-injured area was used in control livers. The resulting fragments were further processed by enzymatic digestion in 5 mL enzyme mix consisting of dispase (50 caseinolytic units/ml), collagenase (2 mg/ml), and DNase (30 pg/ml), for 30 min at 37° C. under constant agitation (180 rpm). Enzyme activity was inhibited by adding 5 ml of phosphate-buffered saline (PBS) supplemented with 10% fetal bovine serum (FBS). Dissociated cells in suspension were passed through a 70 μm strainer and centrifuged at 500×g for 5 min at 4° C. Red blood cell lysis (Thermo Fisher 00-4333-57) was performed for 2 min and stopped with 10% FBS in PBS. After another centrifugation step, the cells were counted in a Neubauer chamber and critically assessed for single-cell separation and viability. A total of 250,000 cells were aliquoted in 2.5 ml of PBS supplemented with 0.04% of bovine serum albumin and loaded for DropSeq at a final concentration of 100 cells/μL. DropSeq experiments were performed as described previously26. In brief, using a microfluidic PDMS device (Nanoshift), single cells were co-encapsulated in droplets with barcoded beads (Chemgenes Corporation, Wilmington, Mass.) at a final concentration of 120 beads/uL. Droplets were collected for 15 min/sample. After droplet breakage, beads were harvested, washed, and prepared for on-bead mRNA reverse transcription (Maxima RT, Thermo Fisher). Following an exonuclease I (New England Biolabs) treatment for the removal of unused primers, beads were counted, aliquoted (2000 beads/reaction, equals ˜100 cells/reaction), and pre-amplified by 13 PCR cycles (primers, chemistry, and cycle conditions identical to those described previously26). PCR products were pooled and purified twice on 0.6× clean-up beads (CleanNA). Prior to tagmentation, cDNA samples were loaded on a DNA High Sensitivity Chip on the 2100 Bioanalyzer (Agilent) to ensure transcript integrity, purity, and quantity. For each sample, 1 ng of pre-amplified cDNA from an estimated 1000 cells was tagmented by Nextera XT (Illumina) with a custom P5 primer (Integrated DNA Technologies). Single cell libraries were sequenced in a 100 bp paired-end run on the Illumina HiSeq4000 using 0.2 nM denatured sample and 5% PhiX spike-in. For priming of read 1, 0.5 μM Read1CustSeqB was used (primer sequence:
Results
Damaged tissues rebuild with a complex mixture of tissue and matrix, the provenance of which has remained obscure. The Inventors recently demonstrated in skin that loose connective tissue (matrix) serves a source for dermal scars. They therefore set out to test the possibility that fluid-like matrix systems might also be mobilized in response to injury in the internal organs.
For this, the Inventors locally tagged and fate-mapped the matrix lining the visceral (serosa) and parietal (adventitia) organ surfaces of live mice using a N-hydroxysuccinimide ester fluorescein (NHS-FITC,
The inventors then set out to test the mobility of these volumes of immature matrix by locally applying dye ester as before and ‘fate mapping’ these local pools of matrix (
To study if a similar fluid-like matrix system exists in other organs, the Inventors used a second clinically relevant model of organ injury using clinical incision (laparotomy) and local abrasion of the peritoneum. Similar to the findings in liver, peritoneal injury induced gushes of labeled matrix across the entire peritoneal surface. Foci of labeled matrix extensively intermixed, and gushed into wounds in a matter of minutes (
Fluid Matrix is Transformed into Rigid Frames in Wounds
To investigate whether fluid matrix matures into rigid frames the Inventors tested if transported fluid elements undergo fibrilar cross-linking in wounds. The Inventors marked live mouse liver surfaces at two distinct locations, one with NHS-EZ-LINK-Biotin and another with NHS-FITC-ester (
To functionally prove crosslinking occurs between elements derived from two separate organs (cecum and peritoneum), the Inventors marked the peritoneal matrix with a distinct EZ-LINK-NHS-Biotin ester type and the cecum with another distinct FITC-NHS ester type and induced local adhesions between these two organs in a remote location by local abrasion (
Next, the Inventors sought to define the protein constituents of the fluid matrix in the injury models by mass-spectrometry. Briefly, the Inventors tagged pools of matrix using modified Biotin-conjugated EZ-link sulfo-NHS esters on liver, peritoneum, and cecum, and subjected them to injury models. Twenty-four hours post-injury, the Inventors collected matrix from wound sites and purified mobile matrix proteins via Streptavidin followed by proteomics of all tagged peptides (
These analyses indicate that while fluid matrix provides building blocks for multiple steps of the repair process, its composition is organ-specific and an indicator of the ensuing repair response, to either regenerate or scar.
Next, the Inventors sought to compare if the findings translate to human wounds. The Inventors took samples from patients who have developed postoperative adhesions and determined their protein composition by immunofluorescence. Importantly the Inventors found they are composed of the same adventitial protein elements found in the mouse peritoneal fluid matrix fractions (
Neutrophils Pilot Fluid Matrix into Wounds
Next, the Inventors checked for a possible link between ECM movement and inflammatory onset, as both act during the early phases of the wound response. To comprehensively explore all possible cellular agents in matrix movement, the Inventors employed highly parallel single-cell transcriptomics of electroporated liver.
Single cell RNA sequencing of over 25,054 cells (see methods) across healthy liver at 1 and 7 days post electroporation revealed 17 distinct cell populations within wounds, predominantly of myeloid lineage such as monocytes, macrophages and neutrophils (
To investigate if neutrophils are responsible for matrix mobilization, the Inventors employed a chemical cell depletion strategy in combination with matrix fate mapping and organ injury. Depleting or halting neutrophils with Ly6g neutralizing antibodies completely blocked fluid matrix flows in both liver and peritoneum, whereas chemically depleting macrophages with Clodronate had no effect on matrix mobilization (
In the absence of matrix mobilization wounds failed to heal. In the liver, blocking matrix mobilization into wounds led to a complete block of regeneration. Liver wounds were enlarged, failed to close and lacked structural organization (
The inventors conclude that organs posses reservoirs of fluid matrix within connective tissues, and that injury triggers organ-wide mobilization of fluid matrix into new tissue construction sites where they fuel tissue repair and regeneration. Further, that neutrophils have a newly found and essential role in executing, piloting and depositing matrix into wounds.
Mus
musculus GN = Rps4x PE = 1 SV = 2
musculus GN = Gdi1 PE = 1 SV = 3
musculus GN = Cct2 PE = 1 SV = 4
musculus GN = Cth PE = 1 SV = 1
musculus GN = Foxe3 PE = 1 SV = 1
musculus GN = Hsp90aa1 PE = 1 SV = 4
musculus GN = Krt8 PE = 1 SV = 4
musculus GN = Egfr PE = 1 SV = 1
musculus GN = Hspa9 PE = 1 SV = 3
musculus GN = Sod1 PE = 1 SV = 2
musculus GN = Itch PE = 1 SV = 2
musculus GN = Krt17 PE = 1 SV = 3
musculus GN = Tst PE = 1 SV = 3
musculus GN = Gstm1 PE = 1 SV = 2
musculus GN = Ppia PE = 1 SV = 2
musculus GN = Hspa1l PE = 1 SV = 4
musculus GN = Blvrb PE = 1 SV = 3
musculus GN = Ephx2 PE = 1 SV = 2
musculus GN = Pygl PE = 1 SV = 4
musculus GN = Tpi1 PE = 1 SV = 4
musculus GN = Hsp90ab1 PE = 1 SV = 3
musculus GN = Inmt PE = 1 SV = 1
musculus GN = Scp2 PE = 1 SV = 3
musculus GN = Mtco2 PE = 1 SV = 1
musculus GN = Prdm16 PE = 1 SV = 1
musculus GN = Tufm PE = 1 SV = 1
musculus GN = Selenbp1 PE = 1 SV = 2
musculus GN = Aco2 PE = 1 SV = 1
musculus GN = Eef1a1 PE = 1 SV = 3
musculus GN = Pgk1 PE = 1 SV = 4
musculus GN = Gnmt PE = 1 SV = 3
musculus GN = Acsl1 PE = 1 SV = 2
musculus GN = Ldha PE = 1 SV = 3
musculus GN = Aco1 PE = 1 SV = 3
musculus GN = Cbs PE = 1 SV = 3
musculus GN = Krt10 PE = 1 SV = 3
musculus GN = Comt PE = 1 SV = 2
musculus GN = Akr1c6 PE = 1 SV = 1
musculus GN = Cyp2d26 PE = 1 SV = 1
musculus GN = Akr1a1 PE = 1 SV = 3
musculus GN = Hbb-b1 PE = 1 SV = 2
musculus GN = Hgd PE = 1 SV = 2
musculus GN = Fdps PE = 1 SV = 1
musculus GN = Krt18 PE = 1 SV = 5
musculus GN = Cct8 PE = 1 SV = 3
musculus GN = Krt72 PE = 3 SV = 1
musculus GN = Krt42 PE = 1 SV = 1
musculus GN = Ass1 PE = 1 SV = 1
musculus GN = Me1 PE = 1 SV = 2
musculus GN = Gdi2 PE = 1 SV = 1
musculus GN = Krt75 PE = 1 SV = 1
musculus GN = Aldob PE = 1 SV = 3
musculus PE = 1 SV = 1
musculus GN = Hist1h2bc PE = 1 SV = 3
musculus GN = Cs PE = 1 SV = 1
musculus GN = Tcp1 PE = 1 SV = 3
musculus GN = Pipox PE = 1 SV = 1
musculus GN = Ak2 PE = 1 SV = 5
musculus GN = Ndufab1 PE = 1 SV = 1
musculus GN = Pgam1 PE = 1 SV = 3
musculus GN = Krt5 PE = 1 SV = 1
musculus GN = Fbp1 PE = 1 SV = 3
musculus GN = Prdx5 PE = 1 SV = 2
musculus GN = Krt14 PE = 1 SV = 2
musculus GN = Tpm3 PE = 1 SV = 3
musculus GN = Gstp1 PE = 1 SV = 2
musculus GN = Ehhadh PE = 1 SV = 4
musculus GN = Mif PE = 1 SV = 2
musculus GN = Selenbp2 PE = 1 SV = 2
musculus GN = Copg2 PE = 1 SV = 1
musculus GN = Amdhd1 PE = 1 SV = 1
musculus GN = Adh5 PE = 1 SV = 3
musculus GN = Hacl1 PE = 1 SV = 2
musculus GN = Gpt PE = 1 SV = 3
musculus GN = Ttpa PE = 1 SV = 1
musculus GN = Rplp0 PE = 1 SV = 3
musculus GN = Krt1 PE = 1 SV = 4
musculus GN = Esd PE = 1 SV = 1
musculus GN = Eif4a1 PE = 1 SV = 1
musculus GN = Ugdh PE = 1 SV = 1
musculus GN = Mycn PE = 2 SV = 2
musculus GN = Slc27a5 PE = 1 SV = 2
musculus GN = Col6a6 PE = 1 SV = 2
musculus GN = Sec13 PE = 1 SV = 3
musculus GN = Krt77 PE = 1 SV = 1
musculus GN = Col4a2 PE = 1 SV = 4
musculus GN = Cct3 PE = 1 SV = 1
musculus GN = Aldoa PE = 1 SV = 2
musculus GN = Col6a2 PE = 1 SV = 3
musculus GN = Gstz1 PE = 1 SV = 1
musculus GN = Krt76 PE = 1 SV = 1
musculus GN = Hoxa4 PE = 2 SV = 4
musculus GN = Rrbp1 PE = 1 SV = 2
musculus GN = Krt79 PE = 1 SV = 2
musculus GN = Fabp1 PE = 1 SV = 2
musculus GN = Iigp1 PE = 1 SV = 2
musculus GN = Cct4 PE = 1 SV = 3
avidinii PE = 1 SV = 1
musculus GN = Rpf1 PE = 2 SV = 2
musculus GN = Sec31a PE = 1 SV = 2
musculus GN = Col5a2 PE = 1 SV = 1
musculus GN = Krt19 PE = 1 SV = 1
musculus GN = Cbr1 PE = 1 SV = 3
musculus GN = Psmb3 PE = 1 SV = 1
musculus GN = Ecm1 PE = 1 SV = 2
musculus GN = Col7a1 PE = 1 SV = 3
musculus GN = Lamc1 PE = 1 SV = 2
musculus GN = Pah PE = 1 SV = 4
musculus GN = Lta4h PE = 1 SV = 4
musculus GN = Col6a4 PE = 1 SV = 2
musculus GN = Krt16 PE = 1 SV = 3
musculus GN = Rpl15 PE = 2 SV = 4
musculus GN = Myl12b PE = 1 SV = 2
musculus GN = Rab35 PE = 1 SV = 1
musculus GN = Senp5 PE = 2 SV = 1
musculus GN = Trim33 PE = 1 SV = 2
musculus GN = F13b PE = 1 SV = 2
musculus GN = Urgcp PE = 2 SV = 1
musculus GN = Agmat PE = 1 SV = 1
musculus GN = Acaca PE = 1 SV = 1
musculus GN = Col6a5 PE = 1 SV = 4
musculus GN = Cdkn2aip PE = 1 SV = 1
musculus GN = Glyat PE = 1 SV = 1
musculus GN = Rplp1 PE = 1 SV = 1
musculus GN = Dpf1 PE = 1 SV = 2
musculus GN = Insrr PE = 1 SV = 2
musculus GN = P4hb PE = 1 SV = 2
musculus GN = Ppa1 PE = 1 SV = 1
musculus GN = Rplp2 PE = 1 SV = 3
musculus GN = Col4a1 PE = 1 SV = 4
musculus GN = Serpina3m PE = 1 SV = 2
musculus GN = Saa2 PE = 1 SV = 1
musculus GN = Mtch2 PE = 1 SV = 1
musculus GN = Ugt1a1 PE = 1 SV = 2
musculus GN = Aldh3a2 PE = 1 SV = 2
musculus GN = Banf1 PE = 1 SV = 1
musculus GN = Col3a1 PE = 1 SV = 4
musculus GN = Acta2 PE = 1 SV = 1
musculus GN = Ywhab PE = 1 SV = 3
musculus GN = Col15a1 PE = 1 SV = 2
musculus GN = Chat PE = 2 SV = 2
musculus GN = F13a1 PE = 1 SV = 3
musculus GN = Tfap2d PE = 1 SV = 1
musculus GN = Nedd4 PE = 1 SV = 3
musculus GN = Rai1 PE = 1 SV = 3
musculus GN = Pabpc1 PE = 1 SV = 2
musculus GN = Col11a1 PE = 1 SV = 2
musculus GN = Ddt PE = 1 SV = 3
musculus GN = Acacb PE = 1 SV = 1
musculus GN = Krt7 PE = 1 SV = 1
musculus GN = Col2a1 PE = 1 SV = 2
musculus GN = Col6a1 PE = 1 SV = 1
musculus GN = Eef1g PE = 1 SV = 3
musculus GN = Col4a4 PE = 2 SV = 1
musculus GN = Gys2 PE = 1 SV = 2
musculus GN = Rnf43 PE = 2 SV = 1
musculus GN = Pcbp1 PE = 1 SV = 1
musculus GN = Fh PE = 1 SV = 3
musculus GN = Col18a1 PE = 1 SV = 4
musculus GN = Bcl9 PE = 1 SV = 3
musculus GN = Col1a2 PE = 1 SV = 2
musculus GN = Calml3 PE = 2 SV = 1
musculus GN = Krt35 PE = 1 SV = 1
musculus GN = Myo16 PE = 1 SV = 2
musculus GN = Zc3h12a PE = 1 SV = 2
musculus GN = Hdgf PE = 1 SV = 2
musculus GN = Krt71 PE = 1 SV = 1
musculus GN = Efl1 PE = 1 SV = 1
musculus GN = Lasp1 PE = 1 SV = 1
musculus GN = Pdlim1 PE = 1 SV = 4
musculus GN = Zfhx3 PE = 1 SV = 1
musculus GN = Col8a1 PE = 1 SV = 3
musculus GN = Pc PE = 1 SV = 1
musculus GN = Prg2 PE = 1 SV = 1
musculus GN = Serpina3k PE = 1 SV = 2
musculus GN = Krt4 PE = 1 SV = 2
musculus GN = Col14a1 PE = 1 SV = 2
musculus GN = Col1a1 PE = 1 SV = 4
musculus GN = Ltk PE = 1 SV = 3
musculus GN = Col16a1 PE = 1 SV = 2
musculus GN = Col5a1 PE = 1 SV = 2
musculus GN = Krt6a PE = 1 SV = 3
musculus GN = Col12a1 PE = 2 SV = 3
musculus GN = Ces2a PE = 1 SV = 1
musculus GN = Arhgap5 PE = 1 SV = 2
musculus GN = Hspa5 PE = 1 SV = 3
musculus GN = Urah PE = 1 SV = 1
musculus GN = Rpl27a PE = 1 SV = 5
musculus GN = Elk4 PE = 2 SV = 2
musculus GN = Pdia3 PE = 1 SV = 2
musculus GN = Atp5b PE = 1 SV = 2
musculus GN = Tinagl1 PE = 1 SV = 1
musculus GN = Pygm PE = 1 SV = 3
musculus GN = Mdh2 PE = 1 SV = 3
musculus GN = Aldoa PE = 1 SV = 2
musculus GN = Tnnt3 PE = 1 SV = 3
musculus GN = Aco2 PE = 1 SV = 1
musculus GN = Hspa8 PE = 1 SV = 1
musculus GN = Ldha PE = 1 SV = 3
musculus GN = Efl1 PE = 1 SV = 1
musculus GN = Klhdc9 PE = 1 SV = 1
musculus GN = Rev3l PE = 1 SV = 3
musculus GN = Csf2rb PE = 1 SV = 2
musculus GN = Mfap4 PE = 1 SV = 1
musculus GN = Filip11 PE = 1 SV = 2
musculus GN = Fhdc1 PE = 1 SV = 3
musculus GN = Serpina3m PE = 1 SV = 2
musculus GN = Efhc1 PE = 1 SV = 1
musculus GN = Cxcr1 PE = 1 SV = 1
musculus GN = Cilp2 PE = 1 SV = 1
musculus GN = Kmt2a PE = 1 SV = 3
musculus GN = Capza1 PE = 1 SV = 4
musculus GN = Chrnd PE = 2 SV = 1
musculus GN = Pc PE = 1 SV = 1
musculus GN = Npepps PE = 1 SV = 2
musculus GN = Arhgap5 PE = 1 SV = 2
musculus GN = Mvb12a PE = 1 SV = 1
musculus GN = Insrr PE = 1 SV = 2
musculus PE = 1 SV = 1
musculus GN = Comp PE = 1 SV = 2
musculus GN = ltih4 PE = 1 SV = 2
musculus GN = Krtap3-1 PE = 3 SV = 1
musculus GN = Tmem184c PE = 2 SV = 1
musculus GN = Apobec2 PE = 1 SV = 1
musculus GN = Tnni2 PE = 2 SV = 2
musculus GN = Lef1 PE = 1 SV = 1
musculus GN = Aldh2 PE = 1 SV = 1
musculus GN = Dock8 PE = 1 SV = 4
musculus GN = Kiaa1109 PE = 1 SV = 4
musculus GN = Krtap15-1 PE = 2 SV = 1
musculus GN = Hsp90ab1 PE = 1 SV = 3
musculus GN = Krt6a PE = 1 SV = 3
musculus GN = Ddx25 PE = 1 SV = 2
musculus GN = Ppia PE = 1 SV = 2
musculus GN = Met PE = 1 SV = 1
musculus GN = Aldob PE = 1 SV = 3
musculus GN = Dgcr2 PE = 2 SV = 1
musculus GN = Krt75 PE = 1 SV = 1
musculus GN = Trim33 PE = 1 SV = 2
musculus GN = Acaa1b PE = 1 SV = 1
musculus GN = Brd3 PE = 1 SV = 2
musculus GN = Riok3 PE = 1 SV = 3
musculus GN = Alms1 PE = 1 SV = 2
musculus GN = Krt76 PE = 1 SV = 1
musculus GN = Krt17 PE = 1 SV = 3
musculus GN = Mrps2 PE = 1 SV = 1
musculus GN = Mybpc2 PE = 1 SV = 1
musculus GN = Mtco2 PE = 1 SV = 1
musculus GN = Tmprss6 PE = 1 SV = 4
musculus GN = Bmp2k PE = 1 SV = 1
musculus GN = Gsk3a PE = 1 SV = 2
musculus GN = Krt2 PE = 1 SV = 1
musculus GN = Krtap19-5 PE = 2 SV = 1
musculus GN = Zmym3 PE = 1 SV = 1
musculus GN = Upf1 PE = 1 SV = 2
musculus GN = Epg5 PE = 1 SV = 2
musculus GN = Golga4 PE = 1 SV = 2
musculus GN = Krt6a PE = 1 SV = 3
musculus GN = Trerf1 PE = 1 SV = 1
musculus GN = Cep295nl PE = 1 SV = 1
musculus GN = lnsrr PE = 1 SV = 2
musculus GN = Pigr PE = 1 SV = 1
musculus GN = Runx1 PE = 1 SV = 1
musculus GN = Pc PE = 1 SV = 1
musculus GN = Usp18 PE = 1 SV = 2
musculus GN = Mfap5 PE = 1 SV = 1
musculus PE = 1 SV = 1
musculus GN = Hspd1 PE = 1 SV = 1
musculus GN = Zg16 PE = 1 SV = 1
musculus GN = Tinagl1 PE = 1 SV = 1
musculus PE = 1 SV = 1
musculus GN = Pglyrp1 PE = 1 SV = 1
musculus GN = Psmb7 PE = 1 SV = 1
musculus GN = Krt79 PE = 1 SV = 2
musculus GN = ltih4 PE = 1 SV = 2
musculus GN = Nup214 PE = 1 SV = 2
musculus GN = Krt2 PE = 1 SV = 1
musculus GN = Ppia PE = 1 SV = 2
musculus GN = Ykt6 PE = 1 SV = 1
musculus GN = Krt17 PE = 1 SV = 3
musculus GN = Krt75 PE = 1 SV = 1
musculus GN = Krt71 PE = 1 SV = 1
musculus GN = Hspa2 PE = 1 SV = 2
musculus PE = 1 SV = 1
musculus GN = Tmprss13 PE = 2 SV = 2
musculus GN = Rgs8 PE = 1 SV = 1
musculus GN = Psmb4 PE = 1 SV = 1
musculus GN = Krt80 PE = 1 SV = 1
musculus GN = Hsp90ab1 PE = 1 SV = 3
musculus GN = Srcin1 PE = 1 SV = 2
musculus GN = Psmb2 PE = 1 SV = 1
musculus GN = Eif6 PE = 1 SV = 2
musculus GN = Fabp5 PE = 1 SV = 3
musculus GN = Psmb5 PE = 1 SV = 3
musculus GN = Hspa5 PE = 1 SV = 3
musculus GN = Aldoa PE = 1 SV = 2
musculus GN = Krt73 PE = 1 SV = 1
musculus GN = Psma1 PE = 1 SV = 1
musculus GN = Ccdc6 PE = 1 SV = 1
musculus GN = Psma3 PE = 1 SV = 3
musculus GN = Psma6 PE = 1 SV = 1
musculus GN = Psma7 PE = 1 SV = 1
An essential step in the phenomenon of ECM movement is crosslinking of moved material in in wound areas. Primary amines of proteins and peptides of distinct protein classes are covalently linked. Since the NHS esters also mark primary amines, the Inventors asked ourselves whether the restructuring in wound areas has led to an increase in free amine groups and whether the Inventors can visualize these via intraperitoneal application of NSH-Esters.
All mouse lines were obtained (C57BL/6J, B6.129P2-Lyz2tm1(cre)lfo/J (Lyz2Cre), B6; 129S6-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J (Ai14)) from Jackson Laboratories or Charles River and bred and maintained at Helmholtz Animal Facility in accordance to the EU directive 2010/63. Animals were housed in individual ventilated cages (IVC) and animal housing rooms were maintained at constant temperature and humidity with a 12-h light cycle. Animals were supplied with water and chow ad libitum. All animal experiments were reviewed and approved by the Government of Upper Bavaria and registered under the project number ROB-55.2-2532.Vet_02-19-133 or ROB-2532.Vet_02-19-148 and conducted under strict governmental and international guidelines. This study is compliant with all relevant ethical regulations regarding animal research.
Mice received 30 minutes before surgery a preemptive subcutaneous injection with Metamizole (200 mg/kg bw). Anesthesia was supplied by an intraperitoneal injection of a Medetomidin (500 pg/kg), Midazolam (5 mg/kg) and Fentanyl (50 pg/kg) cocktail, hereafter referred to as MMF. Monitoring anesthetic depth was assessed by toe reflex. Eyes were covered with Bepanthen-cream to avoid dehydration, and the abdomen was shaved and disinfected with betadine and sterile phosphate buffered saline (PBS). Animals were kept on their backs on a heating plate at 39° C. A midline laparotomy (1-1.5 cm) was performed through the skin and peritoneum. Four hooks, positioned around the incision and fixed to a retractor and magnetic base plate, allowed for clear access to the abdominal cavity and liver.
Labelling of liver surfaces was performed. Local damage to the liver surface was induced via electroporation tweezers by applying electric voltage: 30V, pulse: 50 ms, interval: 1 second, cycles: 8. Before closure of the incision, Buprenorphine (0.1 mg/kg) was pipetted in the abdomen to allow for initial post-surgical analgesia. For long-term analgesia, Metamizole (Novalgin, 200 mg/kg) was provided through daily injections. The peritoneum and skin were closed with two separate 4-0 silk sutures (Ethicon). Upon closure of the incision, mice were woken up by antagonizing Medetomidin and Midazolam through a subcutaneous cocktail injection of Atipamezol (1 mg/kg) and Flumazenil (0.25 mg/kg). Mice were allowed to recover on a heating pad, after which they were single housed. Mice where sacrificed after indicated time point and liver tissue was obtained. In the peritoneal model, surgical procedure was as described above, but the peritoneal areas were marked.
Inhibitors were injected 2 hours before surgery with a concentration of 10 μM of the corresponding small molecules dissolved in sterile PBS i.p.
Succinimidyl esters (NHS-esters; Thermo Fisher) were diluted in DMSO to 25 mg/ml and stored at −80° C. For local matrix staining labelling solution was generated by mixing NHS-ester 1:1 with 100 mM pH 9.0 sodium bicarbonate buffer. For global abdominal labelling, 20 μl of NHS-labelling solution were mixed with 100 μl sterile PBS and injected i.p.
Upon organ excision, organs were fixed overnight at 4° C. in 2% formaldehyde. The next day, fixed tissues were washed three times in Dulbecco's phosphate buffered saline (DPBS, GIBCO, #14190-094), and depending on the purpose, either embedded, frozen in optimal cutting temperature compound (Sakura, #4583) and stored at −20° C., or stored at 4° C. in PBS containing 0.2% gelatin (Sigma Aldrich, #G1393), 0.5% Triton X-100 (Sigma Aldrich, #X100) and 0.01% Thimerosal (Sigma Aldrich, #T8784) (PBS-GT). Fixed tissues were embedded in optimal cutting temperature (OCT) and cut with a Microm HM 525 (Thermo Scientific). In short, sections were fixed in ice-cold acetone for 5 min at −20° C., and then washed with PBS. Sections were then blocked for non-specific binding with 10% serum in PBS for 60 minutes at room temperature, and then incubated with primary antibody in blocking solution O/N at 4° C. The next day, following washing, sections were incubated in PBS with fluorescent secondary antibody, for 120 min at RT. Finally, sections were washed and incubated with Hoechst 33342 nucleic acid stain (Invitrogen, #H1399), washed in ddH2O, mounted with Fluoromount-G® (Southern Biotech, #0100-01), and stored at 4° C. in the dark. Primary antibodies: rabbit-anti-collagen I (1:150, Rockland), rabbit-anti-Cytokeratin (1:100, Sigma Aldrich), rabbit-anti-Ki67 (1:100, Abcam), rabbit-anti-Fibronectin (1:100, Abcam), rabbit-anti-HSP70 (1:100, Elabscience), rabbit-anti-HSPG2 (1:100, Elabscience), rabbit-anti-Keratin9 (1:100, Elabscience), rabbit-anti-Ki67 (1:100, Abcam), rabbit-anti-cleaved Caspase 3 (1:100, Abcam), rabbit-anti-Laminin (1:100, Abcam), rabbit-anti-HSP70 (1:100, Elabscience), hamster-anti-PDPNα (1:100, Abcam), rat-anti-LY6G(Sca1) (1:100, Abcam), rabbit-anti-MMP23(1:100, Elabscience), rabbit-anti-Vitronectin (1:100, Elabscience) and rabbit-anti-WT1 (1:100, Abcam). Alexa Fluor 488-, Alexa Fluor 568- or Alexa Fluor 647-conjugated antibodies (1:500, Life technologies) against suitable species were used as secondary antibodies. H&E stainings where performed according to MMM.
Histological sections were imaged using a using a M205 FCA Stereomicroscope (Leica). 2D, 3D and 4D data was processed with Imaris 9.1.0 (Bitplane) and ImageJ (1.52i). Contrast and brightness were adjusted for better visibility.
Tissues were snap frozen and grinded using a tissue lyser (Quiagen). Pulverised tissues were resuspended in lysis buffer (20 mM Tris-HCl pH 7.5, 1% Triton X-100, 2% SDS, 100 mM NaCl, 1 mM sodium orthovanandate, 9.5 mM sodium fluoride, 10 mM sodium pyruvate, 10 mM beta-glycerophosphate, and supplemented with protease inhibitors (complete protease inhibitor cocktail, Pierce) and kept 10 min on ice. Samples were sonicated and spinned down for 5 minutes with 10.000g. Supernatants were stored at −80° C. Protein concentration was determined via BCA-Assay according to manufactures protocol (Pierce).
Protein pulldown was as follows. Lysates were diluted with a pulldown buffer (20 mM Tris-HCl pH 7.5, 1% Triton X-100, 100 mM NaCl and supplemented with protease and phosphatase inhibitors) and incubated overnight with dynabeads (Thermo Fisher) according to manufacturer's instructions at 4° C. on a rotator. The next day, the samples were each diluted twice with Wash Buffer 1 (20 mM Tris-HCl pH 7.5, 1% Triton X-100, 2% SDS, 100 mM NaCl and supplemented with protease and phosphatase inhibitors) and then with Wash Buffer 2 (20 mM Tris-HCl pH 7).5, 0.5% Triton X-100, 100 mM NaCl and supplemented with protease and phosphatase inhibitors) and finally washed twice with Wash Buffer 3 (20 mM Tris-HCl pH 7.5 and 100 mM NaCl). Beads were then resuspended in Elution Buffer (20 mM Tris-HCl pH 7.5, 100 mM NaCl and 50 mM DTT) and incubated for 30 minutes in Elution Buffer at 37° C. Finally, the samples were boiled for 5 minutes at 98° C. and the supernatants were stored at −80° C. Fluorescence intensities of lysates were measured were measured in a Fluostar optima fluorometer (BMGlabtech).
Tissues were marked locally with an EZ-LINK-NHS 100:1 FITC-NHS mixture. After 24 hours the organs were removed, tissue pieces of the original marking were separated from moved matrix fraction and snap frozen. Tissue lysis was performed as described above. Samples were digested using a modified FASP procedure25. After reduction and alkylation using DTT and IAA, the proteins were centrifuged on Microcon® centrifugal filters (Sartorius Vivacon 500 30 kDa), washed thrice with 8 M urea in 0.1 M Tris/HCl pH 8.5 and twice with 50 mM ammoniumbicarbonate. The proteins on filter were digested for 2 hours at room temperature using 0.5 pg Lys-C (Wako Chemicals, Neuss, Germany) and for 16 hours at 37° C. using 1 pg trypsin (Promega, Mannheim, Germany). Peptides were collected by centrifugation (10 min at 14000 g), acidified with 0.5% TFA and stored at −20° C. until measurements. The digested peptides were loaded automatically to a HPLC system (Thermo Fisher Scientific) equipped with a nano trap column (100 μm ID×2 cm, Acclaim PepMAP 100 C18, 5 μm, 100 Å/size, LC Packings, Thermo Fisher Scientific, Bremen, Germany) in 95% buffer A (2% ACN, 0.1% formic acid (FA) in HPLC-grade water) and 5% buffer B (98% ACN, 0.1% FA in HPLC-grade water) at 30 μl/min. After 5 min, the peptides were eluted and separated on the analytical column (nanoEase MZ HSS T3 Column, 100 Å, 1.8 μm, 75 μm×250 mm, Waters) at 250 nl/min flow rate in a 105 minutes non-linear acetonitrile gradient from 3 to 40% in 0.1% formic acid. The eluting peptides were analyzed online in a Q Exactive HF mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) coupled to the HPLC system with a nano spray ion source and operated in the data-dependent mode. MS spectra were recorded at a resolution of 60,000 and after each MS1 cycle, the 10 most abundant peptide ions were selected for fragmentation. Raw spectra were imported into Progenesis QIsoftware (version 4.1, Nonlinear Dynamics, Waters). After feature alignment and normalization, spectra were exported as Mascot Generic files and searched against the SwissProt mouse database (16,872 sequences) with Mascot (Matrix Science, version 2.6.2) with the following search parameters: 10 ppm peptide mass tolerance and 0.02 Da fragment mass tolerance, two missed cleavages allowed, carbamidomethylation was set as fixed modification, camthiopropanoyl, methionine and proline oxidation were allowed as variable modifications. A Mascot-integrated decoy database search calculated an average false discovery of <1% when searches were performed with a mascot percolator score cut-off of 13 and an appropriate significance threshold p.
Peptide assignments were re-imported into the Progenesis QI software and the abundances of all unique peptides allocated to each protein were summed up. The resulting normalized abundances of the individual proteins were used for calculation of protein ratios and p-values (ANOVA) between sample groups using a nested design. Gene ontology analysis was performed using EnrichR webtool26,27.
Electroporation of murine livers with a tweezer electrode leads to local damage of the dorsal and ventral side. These round sides of electroporation could be visualized with an NHS-Rhodamine ester by intra peritoneal injection (
The discovery the Inventors have made here has many potential implications. The data show that there is an accumulation of primary amines in abdominal wound areas. These can be labelled via NHS-linked reaction. This would allow abdominal wounds to be marked by a simple intra peritoneal injection. By using an NHS ester coupled to deeper wavelength reporters, this would open a new dimension of wound visualization in the clinics. In addition to image wounds, effector molecules, like drugs, could also be coupled to NHS esters to target wound areas with a global injection.
Fibrotic processes take place over long periods of time and are usually identified too late. To date, there are no meaningful biomarkers for early stage fibrotic processes.
ECM movement takes place at rapid kinetics. Therefore, the Inventors asked ourselves the question whether parts of the mobilized elements are transferred into the circulating blood stream and whether the Inventors can detect these fluid elements in the blood. These fluid elements can provide information about the stage of a fibrotic process. Since the Inventors have observed that the fluid fractions are organ-specific, the protocol could even provide organ-specific biomarkers.
All mouse lines were obtained (C57BL/6J, B6.129P2-Lyz2tm1(cre)lfo/J (Lyz2Cre), B6; 129S6-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J (Ai14)) from Jackson Laboratories or Charles River and bred and maintained at Helmholtz Animal Facility in accordance to the EU directive 2010/63. Animals were housed in individual ventilated cages (IVC) and animal housing rooms were maintained at constant temperature and humidity with a 12-h light cycle. Animals were supplied with water and chow ad libitum. All animal experiments were reviewed and approved by the Government of Upper Bavaria and registered under the project number ROB-55.2-2532.Vet_02-19-133 or ROB-2532.Vet_02-19-148 and conducted under strict governmental and international guidelines. This study is compliant with all relevant ethical regulations regarding animal research.
Mice received 30 minutes before surgery a preemptive subcutaneous injection with Metamizole (200 mg/kg bw). Anesthesia was supplied by an intraperitoneal injection of a Medetomidin (500 pg/kg), Midazolam (5 mg/kg) and Fentanyl (50 pg/kg) cocktail, hereafter referred to as MMF. Monitoring anesthetic depth was assessed by toe reflex. Eyes were covered with Bepanthen-cream to avoid dehydration, and the abdomen was shaved and disinfected with betadine and sterile phosphate buffered saline (PBS). Animals were kept on their backs on a heating plate at 39° C. A midline laparotomy (1-1.5 cm) was performed through the skin and peritoneum. Four hooks, positioned around the incision and fixed to a retractor and magnetic base plate, allowed for clear access to the abdominal cavity and liver.
Labelling of liver surfaces was performed. Local damage to the liver surface was induced via electroporation tweezers by applying electric voltage: 30V, pulse: 50 ms, interval: 1 second, cycles: 8. Before closure of the incision, Buprenorphine (0.1 mg/kg) was pipetted in the abdomen to allow for initial post-surgical analgesia. For long-term analgesia, Metamizole (Novalgin, 200 mg/kg) was provided through daily injections. The peritoneum and skin were closed with two separate 4-0 silk sutures (Ethicon). Upon closure of the incision, mice were woken up by antagonizing Medetomidin and Midazolam through a subcutaneous cocktail injection of Atipamezol (1 mg/kg) and Flumazenil (0.25 mg/kg). Mice were allowed to recover on a heating pad, after which they were single housed. Mice where sacrificed after indicated time point and liver tissue was obtained. In the peritoneal model, surgical procedure was as described above, but the peritoneal areas were marked.
Inhibitors were injected 2 hours before surgery with a concentration of 10 μM of the corresponding small molecules dissolved in sterile PBS i.p.
Succinimidyl esters (NHS-esters; Thermo Fisher) were diluted in DMSO to 25 mg/ml and stored at −80° C. For local matrix staining labelling solution was generated by mixing NHS-ester 1:1 with 100 mM pH 9.0 sodium bicarbonate buffer. For global abdominal labelling, 20 μl of NHS-labelling solution were mixed with 100 μl sterile PBS and injected i.p.
Upon organ excision, organs were fixed overnight at 4° C. in 2% formaldehyde. The next day, fixed tissues were washed three times in Dulbecco's phosphate buffered saline (DPBS, GIBCO, #14190-094), and depending on the purpose, either embedded, frozen in optimal cutting temperature compound (Sakura, #4583) and stored at −20° C., or stored at 4° C. in PBS containing 0.2% gelatin (Sigma Aldrich, #G1393), 0.5% Triton X-100 (Sigma Aldrich, #X100) and 0.01% Thimerosal (Sigma Aldrich, #T8784) (PBS-GT). Fixed tissues were embedded in optimal cutting temperature (OCT) and cut with a Microm HM 525 (Thermo Scientific). In short, sections were fixed in ice-cold acetone for 5 min at −20° C., and then washed with PBS. Sections were then blocked for non-specific binding with 10% serum in PBS for 60 minutes at room temperature, and then incubated with primary antibody in blocking solution O/N at 4° C. The next day, following washing, sections were incubated in PBS with fluorescent secondary antibody, for 120 min at RT. Finally, sections were washed and incubated with Hoechst 33342 nucleic acid stain (Invitrogen, #H1399), washed in ddH2O, mounted with Fluoromount-G® (Southern Biotech, #0100-01), and stored at 4° C. in the dark. Primary antibodies: rabbit-anti-collagen I (1:150, Rockland), rabbit-anti-Cytokeratin (1:100, Sigma Aldrich), rabbit-anti-Ki67 (1:100, Abcam), rabbit-anti-Fibronectin (1:100, Abcam), rabbit-anti-HSP70 (1:100, Elabscience), rabbit-anti-HSPG2 (1:100, Elabscience), rabbit-anti-Keratin9 (1:100, Elabscience), rabbit-anti-Ki67 (1:100, Abcam), rabbit-anti-cleaved Caspase 3 (1:100, Abcam), rabbit-anti-Laminin (1:100, Abcam), rabbit-anti-HSP70 (1:100, Elabscience), hamster-anti-PDPNα (1:100, Abcam), rat-anti-LY6G(Sca1) (1:100, Abcam), rabbit-anti-MMP23(1:100, Elabscience), rabbit-anti-Vitronectin (1:100, Elabscience) and rabbit-anti-WT1 (1:100, Abcam). Alexa Fluor 488-, Alexa Fluor 568- or Alexa Fluor 647-conjugated antibodies (1:500, Life technologies) against suitable species were used as secondary antibodies. H&E stainings where performed according to MMM.
Histological sections were imaged using a using a M205 FCA Stereomicroscope (Leica). 2D, 3D and 4D data was processed with Imaris 9.1.0 (Bitplane) and ImageJ (1.52i). Contrast and brightness were adjusted for better visibility.
Tissues were snap frozen and grinded using a tissue lyser (Quiagen). Pulverised tissues were resuspended in lysis buffer (20 mM Tris-HCl pH 7.5, 1% Triton X-100, 2% SDS, 100 mM NaCl, 1 mM sodium orthovanandate, 9.5 mM sodium fluoride, 10 mM sodium pyruvate, 10 mM beta-glycerophosphate, and supplemented with protease inhibitors (complete protease inhibitor cocktail, Pierce) and kept 10 min on ice. Samples were sonicated and spinned down for 5 minutes with 10.000g. Supernatants were stored at −80° C. Protein concentration was determined via BCA-Assay according to manufactures protocol (Pierce).
Protein pulldown was as follows. Lysates were diluted with a pulldown buffer (20 mM Tris-HCl pH 7.5, 1% Triton X-100, 100 mM NaCl and supplemented with protease and phosphatase inhibitors) and incubated overnight with dynabeads (Thermo Fisher) according to manufacturer's instructions at 4° C. on a rotator. The next day, the samples were each diluted twice with Wash Buffer 1 (20 mM Tris-HCl pH 7.5, 1% Triton X-100, 2% SDS, 100 mM NaCl and supplemented with protease and phosphatase inhibitors) and then with Wash Buffer 2 (20 mM Tris-HCl pH 7).5, 0.5% Triton X-100, 100 mM NaCl and supplemented with protease and phosphatase inhibitors) and finally washed twice with Wash Buffer 3 (20 mM Tris-HCl pH 7.5 and 100 mM NaCl). Beads were then resuspended in Elution Buffer (20 mM Tris-HCl pH 7.5, 100 mM NaCl and 50 mM DTT) and incubated for 30 minutes in Elution Buffer at 37° C. Finally, the samples were boiled for 5 minutes at 98° C. and the supernatants were stored at −80° C. Fluorescence intensities of lysates were measured were measured in a Fluostar optima fluorometer (BMGlabtech).
Tissues were marked locally with an EZ-LINK-NHS 100:1 FITC-NHS mixture. After 24 hours the organs were removed, tissue pieces of the original marking were separated from moved matrix fraction and snap frozen. Tissue lysis was performed as described above. Samples were digested using a modified FASP procedure25. After reduction and alkylation using DTT and IAA, the proteins were centrifuged on Microcon® centrifugal filters (Sartorius Vivacon 500 30 kDa), washed thrice with 8 M urea in 0.1 M Tris/HCl pH 8.5 and twice with 50 mM ammoniumbicarbonate. The proteins on filter were digested for 2 hours at room temperature using 0.5 pg Lys-C (Wako Chemicals, Neuss, Germany) and for 16 hours at 37° C. using 1 pg trypsin (Promega, Mannheim, Germany). Peptides were collected by centrifugation (10 min at 14000 g), acidified with 0.5% TFA and stored at −20° C. until measurements. The digested peptides were loaded automatically to a HPLC system (Thermo Fisher Scientific) equipped with a nano trap column (100 μm ID×2 cm, Acclaim PepMAP 100 C18, 5 μm, 100 Å/size, LC Packings, Thermo Fisher Scientific, Bremen, Germany) in 95% buffer A (2% ACN, 0.1% formic acid (FA) in HPLC-grade water) and 5% buffer B (98% ACN, 0.1% FA in HPLC-grade water) at 30 μl/min. After 5 min, the peptides were eluted and separated on the analytical column (nanoEase MZ HSS T3 Column, 100 Å, 1.8 μm, 75 μm×250 mm, Waters) at 250 nl/min flow rate in a 105 minutes non-linear acetonitrile gradient from 3 to 40% in 0.1% formic acid. The eluting peptides were analyzed online in a Q Exactive HF mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) coupled to the HPLC system with a nano spray ion source and operated in the data-dependent mode. MS spectra were recorded at a resolution of 60,000 and after each MS1 cycle, the 10 most abundant peptide ions were selected for fragmentation. Raw spectra were imported into Progenesis QIsoftware (version 4.1, Nonlinear Dynamics, Waters). After feature alignment and normalization, spectra were exported as Mascot Generic files and searched against the SwissProt mouse database (16,872 sequences) with Mascot (Matrix Science, version 2.6.2) with the following search parameters: 10 ppm peptide mass tolerance and 0.02 Da fragment mass tolerance, two missed cleavages allowed, carbamidomethylation was set as fixed modification, camthiopropanoyl, methionine and proline oxidation were allowed as variable modifications. A Mascot-integrated decoy database search calculated an average false discovery of <1% when searches were performed with a mascot percolator score cut-off of 13 and an appropriate significance threshold p.
Peptide assignments were re-imported into the Progenesis QI software and the abundances of all unique peptides allocated to each protein were summed up. The resulting normalized abundances of the individual proteins were used for calculation of protein ratios and p-values (ANOVA) between sample groups using a nested design. Gene ontology analysis was performed using EnrichR webtool26,27.
Pulmonary fibrosis is a disease that is usually fatal for humans, with no treatment or biomarker options. Therefore, the Inventors tested the biomarker hypothesis in a murine pulmonary fibrosis model. After application of bleomycin, fibrotic plaques develop within the lung in the course of 2 weeks. First, the Inventors checked whether there is mobilization of fluid matrix elements in the model. The Inventors therefore followed 2 setups (
Bleomycin-induced pulmonary fibrosis has different degrees of severity depending on the animal. Robust biomarkers should therefore show different abundancies depending on the severity of pulmonary fibrosis. First mass spectrometric analyses of lung tissue found varying amounts of proteins in the lungs of bleomycin versus control animals. This indicates that the primarily labelled proteins undergo changes due to the stimulus. Proteins such as fibrinogen are known to form net-like structures. It could be that fibrinogen is covalently bound to the primary labelled proteins. In fact, the Inventors were also able to identify proteins of varying abundance of the initially labelled lung matrix in the blood of the animals. In summary, the Inventors show here that fluid elements enter the blood stream during mobilization of the lung matrix during fibrotic events. These elements can be detected and could serve as biomarkers for fibrotic events.
Since the ECM movement is a global phenomenon, the Inventors wanted to find out which signaling pathways and mediators play a role in Matrix currents. Here the Matrix studied currents in livers and peritoneas. Here the Inventors investigated matrix currents in livers and peritoneas.
All mouse lines were obtained (C57BL/6J, B6.129P2-Lyz2tm1(cre)lfo/J (Lyz2Cre), B6; 129S6-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J (Ai14)) from Jackson Laboratories or Charles River and bred and maintained at Helmholtz Animal Facility in accordance to the EU directive 2010/63. Animals were housed in individual ventilated cages (IVC) and animal housing rooms were maintained at constant temperature and humidity with a 12-h light cycle. Animals were supplied with water and chow ad libitum. All animal experiments were reviewed and approved by the Government of Upper Bavaria and registered under the project number ROB-55.2-2532.Vet_02-19-133 or ROB-2532.Vet_02-19-148 and conducted under strict governmental and international guidelines. This study is compliant with all relevant ethical regulations regarding animal research.
Mice received 30 minutes before surgery a preemptive subcutaneous injection with Metamizole (200 mg/kg bw). Anesthesia was supplied by an intraperitoneal injection of a Medetomidin (500 μg/kg), Midazolam (5 mg/kg) and Fentanyl (50 μg/kg) cocktail, hereafter referred to as MMF. Monitoring anesthetic depth was assessed by toe reflex. Eyes were covered with Bepanthen-cream to avoid dehydration, and the abdomen was shaved and disinfected with betadine and sterile phosphate buffered saline (PBS). Animals were kept on their backs on a heating plate at 39° C. A midline laparotomy (1-1.5 cm) was performed through the skin and peritoneum. Four hooks, positioned around the incision and fixed to a retractor and magnetic base plate, allowed for clear access to the abdominal cavity and liver.
Labelling of liver surfaces was performed. Local damage to the liver surface was induced via electroporation tweezers by applying electric voltage: 30V, pulse: 50 ms, interval: 1 second, cycles: 8. Before closure of the incision, Buprenorphine (0.1 mg/kg) was pipetted in the abdomen to allow for initial post-surgical analgesia. For long-term analgesia, Metamizole (Novalgin, 200 mg/kg) was provided through daily injections. The peritoneum and skin were closed with two separate 4-0 silk sutures (Ethicon). Upon closure of the incision, mice were woken up by antagonizing Medetomidin and Midazolam through a subcutaneous cocktail injection of Atipamezol (1 mg/kg) and Flumazenil (0.25 mg/kg). Mice were allowed to recover on a heating pad, after which they were single housed. Mice where sacrificed after indicated time point and liver tissue was obtained. In the peritoneal model, surgical procedure was as described above, but the peritoneal areas were marked.
Inhibitors were injected 2 hours before surgery with a concentration of 10 μM of the corresponding small molecules dissolved in sterile PBS i.p.
Succinimidyl esters (NHS-esters; Thermo Fisher) were diluted in DMSO to 25 mg/ml and stored at −80° C. For local matrix staining labelling solution was generated by mixing NHS-ester 1:1 with 100 mM pH 9.0 sodium bicarbonate buffer. For global abdominal labelling, 20 μl of NHS-labelling solution were mixed with 100 μl sterile PBS and injected i.p.
Upon organ excision, organs were fixed overnight at 4° C. in 2% formaldehyde. The next day, fixed tissues were washed three times in Dulbecco's phosphate buffered saline (DPBS, GIBCO, #14190-094), and depending on the purpose, either embedded, frozen in optimal cutting temperature compound (Sakura, #4583) and stored at −20° C., or stored at 4° C. in PBS containing 0.2% gelatin (Sigma Aldrich, #G1393), 0.5% Triton X-100 (Sigma Aldrich, #X100) and 0.01% Thimerosal (Sigma Aldrich, #T8784) (PBS-GT). Fixed tissues were embedded in optimal cutting temperature (OCT) and cut with a Microm HM 525 (Thermo Scientific). In short, sections were fixed in ice-cold acetone for 5 min at −20° C., and then washed with PBS. Sections were then blocked for non-specific binding with 10% serum in PBS for 60 minutes at room temperature, and then incubated with primary antibody in blocking solution O/N at 4° C. The next day, following washing, sections were incubated in PBS with fluorescent secondary antibody, for 120 min at RT. Finally, sections were washed and incubated with Hoechst 33342 nucleic acid stain (Invitrogen, #H1399), washed in ddH2O, mounted with Fluoromount-G® (Southern Biotech, #0100-01), and stored at 4° C. in the dark. Primary antibodies: rabbit-anti-collagen I (1:150, Rockland), rabbit-anti-Cytokeratin (1:100, Sigma Aldrich), rabbit-anti-Ki67 (1:100, Abcam), rabbit-anti-Fibronectin (1:100, Abcam), rabbit-anti-HSP70 (1:100, Elabscience), rabbit-anti-HSPG2 (1:100, Elabscience), rabbit-anti-Keratin9 (1:100, Elabscience), rabbit-anti-Ki67 (1:100, Abcam), rabbit-anti-cleaved Caspase 3 (1:100, Abcam), rabbit-anti-Laminin (1:100, Abcam), rabbit-anti-HSP70 (1:100, Elabscience), hamster-anti-PDPNα (1:100, Abcam), rat-anti-LY6G(Sca1) (1:100, Abcam), rabbit-anti-MMP23(1:100, Elabscience), rabbit-anti-Vitronectin (1:100, Elabscience) and rabbit-anti-WT1 (1:100, Abcam). Alexa Fluor 488-, Alexa Fluor 568- or Alexa Fluor 647-conjugated antibodies (1:500, Life technologies) against suitable species were used as secondary antibodies. H&E stainings where performed according to MMM.
Histological sections were imaged using a using a M205 FCA Stereomicroscope (Leica). 2D, 3D and 4D data was processed with Imaris 9.1.0 (Bitplane) and ImageJ (1.52i). Contrast and brightness were adjusted for better visibility.
Tissues were snap frozen and grinded using a tissue lyser (Quiagen). Pulverised tissues were resuspended in lysis buffer (20 mM Tris-HCl pH 7.5, 1% Triton X-100, 2% SDS, 100 mM NaCl, 1 mM sodium orthovanandate, 9.5 mM sodium fluoride, 10 mM sodium pyruvate, 10 mM beta-glycerophosphate, and supplemented with protease inhibitors (complete protease inhibitor cocktail, Pierce) and kept 10 min on ice. Samples were sonicated and spinned down for 5 minutes with 10.000g. Supernatants were stored at −80° C. Protein concentration was determined via BCA-Assay according to manufactures protocol (Pierce).
Protein pulldown was as follows. Lysates were diluted with a pulldown buffer (20 mM Tris-HCl pH 7.5, 1% Triton X-100, 100 mM NaCl and supplemented with protease and phosphatase inhibitors) and incubated overnight with dynabeads (Thermo Fisher) according to manufacturer's instructions at 4° C. on a rotator. The next day, the samples were each diluted twice with Wash Buffer 1 (20 mM Tris-HCl pH 7.5, 1% Triton X-100, 2% SDS, 100 mM NaCl and supplemented with protease and phosphatase inhibitors) and then with Wash Buffer 2 (20 mM Tris-HCl pH 7).5, 0.5% Triton X-100, 100 mM NaCl and supplemented with protease and phosphatase inhibitors) and finally washed twice with Wash Buffer 3 (20 mM Tris-HCl pH 7.5 and 100 mM NaCl). Beads were then resuspended in Elution Buffer (20 mM Tris-HCl pH 7.5, 100 mM NaCl and 50 mM DTT) and incubated for 30 minutes in Elution Buffer at 37° C. Finally, the samples were boiled for 5 minutes at 98° C. and the supernatants were stored at −80° C. Fluorescence intensities of lysates were measured were measured in a Fluostar optima fluorometer (BMGlabtech).
Tissues were marked locally with an EZ-LINK-NHS 100:1 FITC-NHS mixture. After 24 hours the organs were removed, tissue pieces of the original marking were separated from moved matrix fraction and snap frozen. Tissue lysis was performed as described above. Samples were digested using a modified FASP procedure. After reduction and alkylation using DTT and IAA, the proteins were centrifuged on Microcon® centrifugal filters (Sartorius Vivacon 500 30 kDa), washed thrice with 8 M urea in 0.1 M Tris/HCl pH 8.5 and twice with 50 mM ammoniumbicarbonate. The proteins on filter were digested for 2 hours at room temperature using 0.5 μg Lys-C (Wako Chemicals, Neuss, Germany) and for 16 hours at 37° C. using 1 μg trypsin (Promega, Mannheim, Germany). Peptides were collected by centrifugation (10 min at 14000 g), acidified with 0.5% TFA and stored at −20° C. until measurements. The digested peptides were loaded automatically to a HPLC system (Thermo Fisher Scientific) equipped with a nano trap column (100 μm ID×2 cm, Acclaim PepMAP 100 C18, 5 μm, 100 Å/size, LC Packings, Thermo Fisher Scientific, Bremen, Germany) in 95% buffer A (2% ACN, 0.1% formic acid (FA) in HPLC-grade water) and 5% buffer B (98% ACN, 0.1% FA in HPLC-grade water) at 30 μl/min. After 5 min, the peptides were eluted and separated on the analytical column (nanoEase MZ HSS T3 Column, 100 Å, 1.8 μm, 75 μm×250 mm, Waters) at 250 nl/min flow rate in a 105 minutes non-linear acetonitrile gradient from 3 to 40% in 0.1% formic acid. The eluting peptides were analyzed online in a Q Exactive HF mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) coupled to the HPLC system with a nano spray ion source and operated in the data-dependent mode. MS spectra were recorded at a resolution of 60,000 and after each MS1 cycle, the 10 most abundant peptide ions were selected for fragmentation. Raw spectra were imported into Progenesis QIsoftware (version 4.1, Nonlinear Dynamics, Waters). After feature alignment and normalization, spectra were exported as Mascot Generic files and searched against the SwissProt mouse database (16,872 sequences) with Mascot (Matrix Science, version 2.6.2) with the following search parameters: 10 ppm peptide mass tolerance and 0.02 Da fragment mass tolerance, two missed cleavages allowed, carbamidomethylation was set as fixed modification, camthiopropanoyl, methionine and proline oxidation were allowed as variable modifications. A Mascot-integrated decoy database search calculated an average false discovery of <1% when searches were performed with a mascot percolator score cut-off of 13 and an appropriate significance threshold p.
Peptide assignments were re-imported into the Progenesis QI software and the abundances of all unique peptides allocated to each protein were summed up. The resulting normalized abundances of the individual proteins were used for calculation of protein ratios and p-values (ANOVA) between sample groups using a nested design. Gene ontology analysis was performed using EnrichR webtool26,27.
To analyze the ECM movement the Inventors chose the electroporation model for livers and in the case of peritoneas the laparotomy section as local injury (
Using the inventors signaling pathway analysis, they identified multiple molecules that inhibited or amplified matrix flows. Interestingly, some effector molecules like Blebbistatin and ciliobrevin effected matrix currents of only one organ. Since the composition of the fluid matrix differs from organ to organ, organ-specific modulators of the matrix currents could be applied after identification of appropriate biomarkers.
In summary, they show a new method to attach molecules to wounds, new potential markers for pulmonary fibrosis and signaling pathways to modulate matrix movements.
Lung disease leads to organ failure from inflammation, connective tissue matrix accretion and fibrotic scarring. Although the mechanism of fibrosis is poorly understood, it is thought to occur through de novo synthesis and deposition of extra cellular matrix by fibroblasts. Here the Inventors discover that lungs are actually sheathed by a ready-made adventitial reservoir of fluid-like matrix and that injury triggers a massive flow of this reservoir into mouse lungs, culminating in fibrotic scars. Furthermore, these adventitial reservoirs of fluid matrix also exist and flow in ex vivo human diseased lung samples. Using mass spectrometric analysis of mouse and human lungs, the Inventors uncover that fluid matrix irrigation liberates basement membranes, elastic and collagenous fibers and crosslinking enzymes, decreasing lung surface elasticity while stiffening the lungs.
Addressing the mechanism in mice, the Inventors demonstrate that immune cells trigger fluid matrix irrigation and this effect is exacerbated by immune cells from patients with lung disease. By uncovering how inflammation liberates matrix reservoirs the findings reveal a new phenomenon that fundamentally changes the view of fibrotic processes. This study thus creates new therapeutic and diagnostic avenues to treat a variety of incurable, and hard to diagnose, lung diseases.
Methods
Patient derived PFA and ST Tissue
All tissues (PFA, ST) used in this study were obtained with properly informed consent of patients. All experimental procedures were performed in accordance with the Research Ethics Boards (REB 1000055059) at The Hospital for Sick Children (Toronto, Canada). Primary tumor cultures used in this study are from patients that have not undergone radiotherapy or chemotherapy prior to surgical resection.
All samples used in this study were obtained with properly informed consent of patients. All experimental procedures were performed in accordance with the Research Ethics Boards at The Hospital for Sick Children (Toronto, Canada). Patient derived PFA-ependymoma cell lines (MDT-PFA1, MDT-PFA2, MDT-PFA3, MDT-PFA4, MDT-PFA5, MDT-PFA7 MDT-PFA8, MDT-PFA9, MDT-PFA13, MDT-PFA15) and supratentorial ependymoma cell lines (MDT-ST1, MDT-ST4) were established in this study. GBM and DIPG, K27M cell cultures were obtained from Dr. Peter Dirks (The Hospital for sick Children, Canada) and Dr. Nada Jabado (McGil University, Canada) respectively. All cell cultures were confirmed to match original tumors by STR fingerprinting, where tumor tissues were available. The following PFA and ST cell cultures were derived from male patients: MDT-PFA1, MDT-PFA2, MDT-PFA3, MDT-PFA5, MDT-PFA7, MDT-PFA8, MDT-PFA9, MDT-PFA13, MDT-PFA15, MDT-ST4. The following PFA and ST cell cultures were derived from female patients: MDT-PFA4, MDT-ST1. Human fetal neural stem cells, fNSC (HF7450, HF6562) and immortalized normal human astrocytes (iNHA) were obtained from Dr. Peter Dirks (The Hospital for sick Children, Canada) and Dr. Nada Jabado (McGil University, Canada) respectively.
All mouse breeding and procedures were performed as approved by The Centre for Phenogenomics (Toronto). Pairs of C57BL/6J mice were obtained from The Jackson laboratory for mouse breeding. Embryos of mated C57BL/6J female mice were dissected to collect hindbrain tissue from E10, E12, E14, E16 and E18 gestational time points. Hindbrain of C57BL/6J pups was dissected to collect tissue from P0, P5, P7 and P14 postnatal time points.
The inventors generated a labelling solution by mixing 5 μl NHS-ester (25 mg/ml) with 5 μl of 100 mM pH 9.0 sodium bicarbonate buffer, combining with 40 μl PBS to a total volume of 50 μl. The labelling solution was applied intra pleural under isoflurane anaesthesia by using a 30G cannula.
The oropharyngeal administration of bleomycin for the induction of pulmonary fibrosis was carried out in an antagonistic anesthesia in C57BL/6J mice of both sexes (6-8 weeks age). After the toe-pinch reflex was absent, the mouse was placed on the incisors of the upper jaw and thus kept in an upright position. The tongue was carefully fixed and held to the side with tweezers while the nose of the animal is covered with tweezers. By keeping the nose closed, the mouse was forced to breathe through the mouth. With the help of a pipette, bleomycin was dissolved in a dosage of 2 units/kg KGW in 80 μl PBS carefully into the throat. As soon as the animal has inhaled the solution, it was tansferred to a Hot plate (duration approx. 30 to 60 seconds). After antagonization animals were housed for 14 days. Nintedanib was added 1 hour before bleomycin installation and every other day intra peritoneal 10 μM.
C57BL/6J male mice (6-8 weeks age) were used to study the movement of the lung matrix. After the organ withdrawal 4 mm biopsy punches of murine lungs were generated. To obtain ectopic labeling of matrix, the inventors generated a labelling solution by mixing NHS-ester 1:1 with 100 mM pH 9.0 sodium bicarbonate buffer. Sterile Whatman filter paper (Sigma Aldrich) biopsy punches where soaked in NHS-labelling solution, and locally placed on the lung biopsy surface. After one minute, the labelling punch was removed. Mouse lung biopsies were cocultured in the RPMI medium (10% FBS with 1% Pen/Strep and 0.1% AmB) consist of different sub types of immune cells (0.1×106 cells/biopsy) isolated from the healthy and idiopathic pulmonary fibrosis (IPF) donors. Mouse lung biopsies with immune cells were then cultured in the ex vivo condition and provided with 5% CO2 at 37° C.
After 48 hours, mouse lung biopsies were fixed with the 4% formalin and incubated for overnight at 40 C followed by PBS wash. Human lung tissues where obtained, labelled, and cultivated for 24 hours as described above.
Upon organ excision, organs were fixed overnight at 4° C. in 2% formaldehyde. The next day, fixed tissues were washed three times in Dulbecco's phosphate buffered saline (DPBS, GIBCO, #14190-094), and depending on the purpose, either embedded, frozen in optimal cutting temperature compound (Sakura, #4583) and stored at −20° C., or stored at 4° C. in PBS containing 0.2% gelatin (Sigma Aldrich, #G1393), 0.5% Triton X-100 (Sigma Aldrich, #X100) and 0.01% Thimerosal (Sigma Aldrich, #T8784) (PBS-GT). Fixed tissues were embedded in optimal cutting temperature (OCT) and cut with a Microm HM 525 (Thermo Scientific). In short, sections were fixed in ice-cold acetone for 5 min at −20° C., and then washed with PBS. Sections were then blocked for non-specific binding with 10% serum in PBS for 60 minutes at room temperature, and then incubated with primary antibody in blocking solution O/N at 4° C. The next day, following washing, sections were incubated in PBS with fluorescent secondary antibody, for 120 min at RT. Finally, sections were washed and incubated with Hoechst 33342 nucleic acid stain (Invitrogen, #H1399), washed in ddH2O, mounted with Fluoromount-G® (Southern Biotech, #0100-01), and stored at 4° C. in the dark.
For multi-photon imaging, samples were embedded in a 4% NuSieve GTG agarose solution (Lonza, #50080). Imaging was performed using a 25× water-dipping objective (HC IRAPO L 25×/1.00W) coupled to a tunable pulsed laser (Spectra Physics, Insight DS+). Multi-photon excited images were recorded with external, non-descanned hybrid photo detectors (HyDs). Following band pass (BP) filters were used for detection: HC 405/150 BP for Second Harmonic Generation (SHG) and a ET 525/50 BP for green channel Tiles were merged using Leica Application suite X (v3.3.0, Leica) with smooth overlap blending and data were visualized with Imaris software (v9.1.3, Bitplane).
Whole-mount samples were stained and cleared with a modified 3DISCO protocol (Ertürk et al., 2012). Samples were dehydrated in an ascending tetrahydrofuran (Sigma Aldrich, #186562) series (50%, 70%, 3×100%; 60 minutes each), and subsequently cleared in dichloromethane (Sigma Aldrich, #270997) for 30 min and eventually immersed in benzyl ether (Sigma Aldrich, #108014). Cleared samples were imaged whilst submerged in benzyl-ether with a light-sheet fluorescence microscope (LaVision BioTec). Whilst submerged in benzyl-ether, specimens were illuminated on two sides by a planar light-sheet using a white-light laser (SuperK Extreme EXW-9; NKT Photonics). Optical sections were recorded by moving the specimen chamber vertically at 5-mm steps through the laser light-sheet. Three-dimensional reconstructions were obtained using Imaris imaging software (v9.1.3, Bitplane).
Histological sections were imaged under a M205 FCA Stereomicroscope (Leica) and ZEISS AxioImager Z2m (Carl Zeiss). Murine biopsy punches were imaged under a M205 FCA Stereomicroscope (Leica). Data was processed with Imaris 9.1.3 (Bitplane) and ImageJ (1.52i). Contrast and brightness were adjusted for better visibility. Thundering was performed with fluoromount and standard parameter settings for histology cuts.
Human whole peripheral blood from healthy control and interstitial lung disease patients was collected in EDTA—tubes and processed within two hours of. PBMCs were isolated using density gradient centrifugation (Stemcell, Catalog #07851). The PMBC layer and additionally the white cells directly above the red blood cells (RBC) were collected for isolation of the different immune cell subsets.
The PBMCs layer was split in half and underwent autoMACS® (Miltenyi Biotec, Catalog #130-092-545) bead isolations for the different cellular subtypes.
a) One half was used for monocyte isolation following the protocol provided by the Pan monocyte isolation kit (Miltenyi Biotec, Catalog #130-096-537).
b) The other half was used to isolate lymphocytes. (i) First isolation was performed with CD19 microbeads (Miltenyi Biotec, Catalog #130-050-301) and the positive fraction corresponding to B cells was collected. (ii) Then, using the negative fraction, T cells were isolated according to the protocol provided with the human Pan T isolation kit (Miltenyi Biotec, Catalog #130-096-535).
In parallel with the isolations above, the white cell pellet of the RBCs was used to isolate the different granulocyte subtypes. Initially, the cells were resuspended in PBS and centrifuged at 300g for 15 minutes. In the next step, red blood cell lysis was performed on the pellet using the TQ-Prep Workstation (Beckam Coulter, Catalog #6605429). The lysed pellet was washed with PBS and centrifuged at 300g for 10 minutes, to procure all granulocytic cells.
To be able to separate the different granulocyte subtypes, the inventors proceeded with CD16 microbeads (Miltenyi Biotec, Catalog #130-045-701) following the protocol for magnetic isolation using autoMACS®. The resulting negative fraction corresponded to granulocytes and the positive fraction a mixed population of neutrophils and basophils. The quality of the different cell types was determined by flow cytometry.
Tissues were snap frozen and grinded using a tissue lyser (Quiagen). Pulverised tissues were resuspended in lysis buffer (20 mM Tris-HCl pH 7.5, 1% Triton X-100, 2% SDS, 100 mM NaCl, 1 mM sodium orthovanandate, 9.5 mM sodium fluoride, 10 mM sodium pyruvate, 10 mM beta-glycerophosphate), and supplemented with protease inhibitors (complete protease inhibitor cocktail, Pierce) and kept 10 min on ice. Samples were sonicated and spun down for 5 minutes at 10,000g. Supernatants were stored at −80° C. Protein concentration was determined via BCA-Assay according to manufactures protocol (Pierce).
Protein pulldown was as follows. Lysates were diluted with a pulldown buffer (20 mM Tris-HCl pH 7.5, 1% Triton X-100, 100 mM NaCl and supplemented with protease and phosphatase inhibitors) and incubated overnight with Dynabeads at 4° C. on a rotator according to the manufacturer's instructions. The next day, the samples were each diluted twice with Wash Buffer 1 (20 mM Tris-HCl pH 7.5, 1% Triton X-100, 2% SDS, 100 mM NaCl, supplemented with protease and phosphatase inhibitors) and then with Wash Buffer 2 (20 mM Tris-HCl pH 7.5, 0.5% Triton X-100, 100 mM NaCl, supplemented with protease and phosphatase inhibitors) and finally washed twice with Wash Buffer 3 (20 mM Tris-HCl pH 7.5 and 100 mM NaCl). Beads were then resuspended in Elution Buffer (20 mM Tris-HCl pH 7.5, 100 mM NaCl and 50 mM DTT) and incubated for 30 minutes at 37° C. Finally, the samples were boiled for 5 minutes at 98° C. and the supernatants were stored at −80° C.
Tissue lysis was performed as described above. Samples were digested using a modified FASP procedure as described by Wiśniewski et al., 2009. After reduction and alkylation using DTT and IAA, the proteins were centrifuged on Microcon® centrifugal filters (Sartorius Vivacon 500 30 kDa), washed thrice with 8 M urea in 0.1 M Tris/HCl pH 8.5 and twice with 50 mM ammonium bicarbonate. The proteins on filters were digested for 2 hours at room temperature using 0.5 μg Lys-C (Wako Chemicals) and for 16 hours at 37° C. with 1 μg trypsin (Promega). Peptides were collected by centrifugation (10 min at 14000 g), acidified with 0.5% TFA and stored at −20° C. until measurements. The digested peptides were loaded automatically on a HPLC system (Thermo Fisher Scientific) equipped with a nano trap column (100 μm ID×2 cm, Acclaim PepMAP 100 C18, 5 μm, 100 Å/size, LC Packings, Thermo Fisher Scientific) in 95% buffer A (2% ACN, 0.1% formic acid (FA) in HPLC-grade water) and 5% buffer B (98% ACN, 0.1% FA in HPLC-grade water) flowing at 30 μl/min. After 5 min, the peptides were eluted and separated on the analytical column (nanoEase MZ HSS T3 Column, 100 Å, 1.8 μm, 75 μm×250 mm, Waters) for 105 minutes at 250 nl/min flow rate in a 3 to 40% non-linear acetonitrile gradient in 0.1% formic acid. The eluting peptides were analyzed online in a Q Exactive HF mass spectrometer (Thermo Fisher Scientific) coupled to the HPLC system with a nano spray ion source, operated in the data-dependent mode. MS spectra were recorded at a resolution of 60,000 and after each MS1 cycle, the 10 most abundant peptide ions were selected for fragmentation. Raw spectra were imported with Progenesis QI software (version 4.1, Nonlinear Dynamics, Waters). After feature alignment and normalization, spectra were exported as Mascot Generic files and searched against the SwissProt mouse database (16,872 sequences) with Mascot (Matrix Science, version 2.6.2) with the following search parameters: 10 ppm peptide mass tolerance and 0.02 Da fragment mass tolerance, two missed cleavages allowed, carbamidomethylation was set as fixed modification, camthiopropanoyl, methionine and proline oxidation were allowed as variable modifications. A Mascot-integrated decoy database search calculated an average false discovery of <5% when searches were performed with a mascot percolator score cut-off of 13 and a significance threshold p-value.
Peptide assignments were re-imported into the Progenesis QI software and the abundances of all unique peptides allocated to each protein were summed. The resulting normalized abundances of the individual proteins were used for calculation of protein ratios and p-values (ANOVA) between sample groups using a nested design. Extracellular elements were identified through a database search against a matrisomal database (Shao et al., 2020). Gene ontology analysis was performed using EnrichR webtool (Chen et al., 2013; Kuleshov et al., 2016).
Results
A Pleuro-Vascular Axis Irrigates Injured Lungs with Pre-Existing Fluid Matrix
Damaged and inflamed lungs rebuild with a complex mixture of tissue and matrix, the provenance of which has remained obscure. The Inventors recently demonstrated in skin that loose connective tissue (matrix) serves a source for dermal scars (Correa-Gallegos et al., 2019). The Inventors therefore set out to test the possibility that preexisting matrix translocates in, another injured tissue namely, lung. For this, the Inventors locally tagged and fate-mapped the matrix lining the lungs (pleura) of live mice with N-hydroxysuccinimide ester fluorescein isothiocyanate (
Foci of labeled pleura matrix clearly coincided with the second harmonic signal, indicating the extracellular collagenous fibers were correctly labeled. The second harmonic signal also revealed a rigid intertwined framework of large mature collagen fibers in lung pleura. The fibers formed a web across the lung surface with large gaps with no signal. Gaps and distances between adjacent mature fibers were filled with a matrix of minute fibrils and multi-fibril aggregates in an immature arrangement (
To test the mobility of the immature matrix in response to disease in mice, the Inventors instilled Bleomycin in trachea (
Next, the Inventors sought to define the protein constituents of the fluid matrix from pleural reservoirs by mass-spectrometry. Briefly, the Inventors injected a Biotin-conjugated EZ-link sulfo-N-Hydroxysuccinimide ester into the pleural space, tagging pools of matrix reservoirs on pleural lung surfaces as the Inventors did before. The Inventors followed up by subjecting mice to Bleomycin-induced injury (
Lung fibrosis implicates a wide variety of immune cells although any causality and or mechanisms remain to be established. Motivated by this, the Inventors set out to investigate the influence of distinct immune cell populations on matrix invasion in lungs. The Inventors purified populations of lymphocytes (B and T cells), monocytes, and granulocytes (neutrophils, eosinophils, basophils) from healthy human volunteers. Lung explant fluid matrix reservoirs were labeled on pleural surfaces with dye ester as before, and they were individually cultivated with subsets of immune cells obtained from healthy human volunteers (
As immune cells from healthy volunteers triggered matrix invasion into the lungs, the Inventors next closely analyzed the impact of lung disease patient immune cells on matrix movement. For this the Inventors isolated immune cells directly from idiopathic pulmonary fibrosis patients and added them to the lung explant cultures. All types of patient immune cells augmented vigorous movements of matrix from pleural surfaces. This led to decreased surface fluorescence intensity, from 49 to 20, that was accompanied by a high inward invasion index of fluid matrix from 60 mm to 110 mm. Fluorescence histologic images of lung explants revealed that monocytes and lymphocytes from idiopathic pulmonary fibrosis patients induced the most significant invasion of pleural matrix into the alveolar and interstitial space, which remarkably resembled the initial findings in Bleomycin-treated animals.
These findings strongly suggest that both monocytes and lymphocytes play key roles in liberating fluid matrix from pleural surfaces and irrigating the lungs. Moreover, the Inventors can deduce that immune cells from diseased patients are ‘primed’ for this task, much more then in healthy individuals.
To study if human diseased lungs undergo matrix movements in the same way as in the mouse model, the Inventors adapted the labeling technique to human lung samples from diseased patients (
Labelled human pleural reservoirs, underwent dramatic inward movement of fluid matrix within 24 hours. Importantly the Inventors were able to detect matrix currents into the interior of injured human lung tissue (
Two-photon images of labeled diseased lung biopsies showed protein-rich fluid and fibers completely irrigated the interstitial spaces surrounding the bronchioles and blood vessels of injured lungs, down to the major bronchus. Here, the Inventors observed multiple accretion layers of connective tissue fibers laid down and intermingled within the tunica adventitia, media and intima, generating a thickened bronchial wall and rim replete with new fibers, as the Inventors initially found in Bleomycin-treated mice (
To summarize, the Inventors observed the same protein-rich matrix movements in human diseased lungs that the Inventors observed above in a mouse model of lung disease. Furthermore these movements were also induced in healthy lung samples from mouse after cultivating with immune cells from diseased patients. Thus, immune cells trigger irrigation of protein-rich matrix from reservoirs on pleural surfaces. These data therefore establish a functional link between inflammation and downstream fibrosis.
To study the composition of this protein-rich fluid matrix in human lungs, the Inventors tagged diseased biopsy pleura, and incubated them for 24 hours. The Inventors then separated pleural and interstitial tissues for protein extraction, followed by mass spectrometry proteomics (
By analyzing the relative fractions of proteins remaining on lung surfaces versus those removed, the Inventors found that individual proteins had vastly distinct translocation profiles (
Having discovered that immune cells trigger matrix translocations, the Inventors went on to study if an anti-fibrotic anti-inflammatory drug inhibits matrix motions in animals. The pan-tyrosine kinase inhibitor Nintedanib is currently one of only two anti-fibrotic drugs on the market that have been approved for pulmonary fibrosis, as it has anti-fibrosis and anti-inflammatory activities that impede disease progression. To check a possible effect of Nintedanib on matrix reservoirs, the Inventors performed a global kinase enrichment assay across the entire fluid matrix proteome. Indeed, the Inventors were encouraged to find that the fluid matrix proteome was highly enriched in tyrosine kinase and therefore significantly affected by its activity (
To directly answer if Nintedanib's anti-fibrotic actions are mediated through its effects on fluid matrix movements, the Inventors labeled fluid matrix reservoirs on lung surfaces as before, induced lung inflammation and fibrosis with Bleomycin, and followed these mice with daily injections of Nintedanib (
In sum, the Inventors reveal here the connection between inflammation and downstream fibrogenesis. The Inventors demonstrate that inflammation mobilizes protein-rich fluid matrix from pleural reservoirs to irrigate lungs with scar tissue, and that Nintedanib acts by inhibiting fluid matrix irrigation, thereby improving disease progression. Matrix irrigation is likely a general principle of organ injury and disease with potential clinical ramifications to many human fibrotic conditions.
| Number | Date | Country | Kind |
|---|---|---|---|
| 19191998.4 | Aug 2019 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2020/073008 | 8/17/2020 | WO |
