Patent Application
20070238663
Throughout this application, various references or publications are cited. Disclosures of these references or publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.
HMGB1 (High mobility group 1 protein) is both a nuclear factor and a secreted protein. In the cell nucleus it acts as an architectural chromatin-binding factor that binds DNA and promotes protein assembly on specific DNA targets (Bustin, Mol. Cell. Biol., 19, 5237-5246, 1999). Outside the cell, it binds with high affinity to RAGE (receptor for advanced glycation endproducts) (Horiet al., J. Biol. Chem. 270, 25752-25761, 1995), and is a potent mediator of inflammation (Wang et al., Science 285, 248-251, 1999; Abraham et al., J. Immunol., 165, 2950-2954, 2000; Andersson et al., J. Exp. Med., 192, 565-570, 2000). HMGB1 is actively secreted by activated monocytes and macrophages (Wang et al., Science 285, 248-251, 1999), and is passively released by necrotic or damaged cells (Degryse et al., J. Cell Biol. 152: 1197-2006, 2001; Muller et al. EMBO J., 16, 4337-4340, 2001; Falciola et al., J. Cell Biol. 137, 19-26, 1997).
Wang et al. (Science, 285, 248-251, 1999) identified HMGB1 as a late mediator of endotoxin lethality in mice. They showed that monocytes/macrophages stimulated by LPS, TNF or IL-1 secreted HMGB1 as a delayed response. In mice, administration of anti-HMGB1 antibodies attenuated LPS-induced endotoxemia; conversely, injection of HMGB1 caused toxic shock. Moreover, septic patients showed increased serum levels of HMGB1, which correlated with the severity of the infection (U.S. Pat. No. 6,303,321; WO 00/47104).
Subsequently, HMGB1 was also shown to cause acute lung inflammation when administered intratracheally (Abraham et al., J. Immunol., 165, 2950-2954, 2000). Antibodies against HMGB1 decreased lung edema and neutrophile migration, whereas they did not reduce the levels of the other proinflammatory cytokines TNF-alpha, IL-lbeta or macrophage-inflammatory-protein-2 (MIP2). Pituicytes, which provide an important link between the immune and the neuroendocrine s-ystems, release HMGB1 in response to specific stimuli like TNF-alpha and IL-lbeta, suggesting that HMGB1 also participates in the regulation of neuroendocrine and immune responses to inflammatory processes (Wang et al., Surgery, 126, 389-392, 1999). However, most cells (including lymphocytes, adrenal cells or kidney cells) are not able to secrete HMGB1.
The phenomena investigated by the groups of Wang and Andersson depend from the active (if unconventional) secretion of HMGB1 by specific cells, that respond to specific stimulation by proinflammatory cytokines. HMGB1 secretion requires at least 16 hours after cell stimulation, and thus is a late event in inflammation. The role of secreted HMGB1 is thus to reinforce and prolong inflammation that was initiated by some other event.
In contrast, the authors have now demonstrated that HMGB1 released by necrotic cells can be the initial trigger for inflammatory responses, and that released HMGB1 itself can activate inflammatory cells. HMGB1 release and diffusion can take place in a matter of seconds or minutes, and is thus a very early event in inflammation. The ability of certain cell types to secrete HMGB1 without dying must therefore be a type of molecular mimicry: these cells have evolved the ability to secrete the same molecule whose extracellular presence signals tissue damage.
The authors of the instant invention report that Hmgb1−/− necrotic cells have a greatly reduced ability to promote inflammation, proving that the release of HMGB1 can signal the demise of a cell to its neighbors, and tissue damage to rest of the organism. Moreover, apoptotic cells do not release HMGB1 even after undergoing secondary necrosis and partial autolysis, and thus fail to promote inflammation even if not promptly cleared by phagocytic cells. In apoptotic cells HMGB1 is firmly bound to chromatin because of generalized chromatin modification, and is released in the extracellular medium promoting inflammation) if chromatin deacetylation is prevented. Thus, cells undergoing apoptosis are programmed to withhold the signal broadcast by cells damaged or killed by trauma. Therefore, though HMGB1 is an abundant component of chromatin, it also has a role as a soluble, extracellular protein.
The following succession of events could be envisaged:
Necrosis->primary HMGB1 release->inflammatory cell activation->secretion of cytokines->further activation and recruitment of inflammatory cells->secretion of HMGB1 by inflammatory cells->loop continues with secretion of cytokines and further activation and recruitment of inflammatory cells.
This loop will create a positive feedback, with strong inflammatory responses. The loop must be broken at some point to interrupt inflammatory responses.
Besides triggering inflamrnation, HMGB1 also activates other systems of tissue protection or repair. It is shown in the present invention that HMGB1 can promote the migration and the proliferation of cells whose function is to repair tissue damage. Foremost among these cells are stem cells. As an example of stem cell recruitment and proliferation, the authors have demonstrated the effect in vitro and in vivo on vessel-associated stem cells, the mesoangioblasts.
As an example of immediate practical application for HMGB1 as a signal for tissue damage, the authors have investigated the regeneration and functional recovery of myocardial tissue after infarction of the heart.
Advantageously HMGB1 promotes regeneration and functional recovery of infarcted heart. Myocardial infarction leads to loss of tissue and impairment of cardiac performance. One of the major therapeutic goals of modern cardiology is to design strategies aimed at minimizing myocardial necrosis and optimizing cardiac repair following myocardial infarction. Although promising results have been obtained with transplantation and mobilization of bone marrow cells to the area of the infarction (Orlic et al., Circ Res. 27, 1092-1102, 2002), signals that regulate stem cells homing to areas of tissue injury were not well characterized so far.
WO02/074337 discloses that HMGB1 may be active in connective tissue regeneration. Connective tissue cells are not embriologically related to target cells of the instant invention.
WO00/47104 claims a pharmaceutical composition for treating conditions (diseases) characterized by activation of the inflammatory cytokines cascade, as sepsis and obesity, comprising an effective amount of an antagonist or inhibitor of HMGB1.
It is therefore an object of the instant invention to provide a method of promoting stem cell migration and/or proliferation in cell culture or in vivo comprising the step of exposing such cells to an effective amount of the HMGB1 protein or its ABbt fragment. Preferably the stem cell is an adult stem cell or an an embryonic stem cell. More preferably, the stem cell is a resident cardiac or circulating stem cell.
It is another object of the invention to provide a method of promoting the proliferation of cardiomyocites in cell culture or in vivo comprising the step of exposing such cells to an effective amount of the HMGB1 protein or its ABbt fragment.
The authors have demonstrated that HMGB1 is released by necrotic cells, but not by apoptotic cells. In such a way, HMGB1 can signal tissue damage, since tissue damage produces unprogrammed cell death (necrosis).
The first effects of HMGB1 released by necrotic cells (in a matter of seconds or minutes) will be the activation of nearby cells, the loss of barrier function of endothelia, the recruitment and activation of myeloid cells, and the triggering of inflammation. As a more delayed response, HMGB1 (either directly released by necrotic cells, or actively secreted by activated inflammatory cells) will promote migration of certain cell types, including stem cells, to the site of tissue damage, and their proliferation.
Specifically, both adult and embryonic vessel-associated stem cells (mesoangioblasts) proliferate when stimulated with HMGB1. HMGB1 was also able to induce mesoangioblast migration as well as transmigration through an endothelial barrier. In vivo, heparin beads containing HMGB1 injected into the tibialis muscle were able to attract meso-angioblasts carrying nuclear lacZ injected into the femoral artery.
In vivo data show that HMGB1 promotes regeneration of infarcted hearts. As a matter of fact HMGB1 induces newly formed myocytes into the infarcted heart. The newly formed tissue is visible by staining with hematoxylin and eosin and expresses cardiac molecular markers such as alpha-sarcomeric actin, connexin 4, MEF2 and others. The newly formed tissue is highly proliferative as seen by the expression of Ki67. HMGB1 thus induces cardiomyocyte proliferation and differentiation in the infarcted heart. Moreover, HMGB1 treated hearts improved myocardial function as tested by echocardiographic and hemodynamic parameters.
The effect of HMGB1 on ischemic heart recovery could be due to recruitment/proliferation of resident cardiac stem cells present in the heart and/or to the recruitment/proliferation of circulating stem cells, and/or to the induction of cardiomyocyte proliferation.
HMGB1-induced cardiac tissue regeneration and its effect on myocardial function recovery is comparable to those observed after stem cell transplantation of hematopoietic stem cells (HSC) injected directly into the heart (Orlic et al., Nature, 410, 701-705, 2001). The invention represents a potential novel approach to be used in the treatment of myocardial infarction.
HMGB1 maybe used alone (or in conjunction with cellular therapy) for all types of tissue regeneration. This represents an advantage compared to stem cell-based regeneration procedures, which require GMP extraction and expansion, are subject to transplant rejection/immune response, require bioethical approval and are not easily packaged into an industrial product.
HMGB1 may have the unique capacity of inducing proliferation of cardiomyocytes and thus therapeutic application in several cardiac pathologies.
Such cardiac pathologies include Heart failure which is a clinical syndrome resulting from a cardiac disease which compromises ventricular systolic or diastolic function or both. Heart failure results when the heart is unable to generate a cardiac output sufficient to meet the demands of the body without unduly increasing diastolic pressure. Heart failure may be manifested by symptoms of poor tissue perfusion alone (e.g., fatigue, poor exercise tolerance, confusion) or by both symptoms of poor tissue perfusion and congestion of vascular beds (e.g., dyspnea, chest rales, pleural effusion, pulmonary edema, distended neck veins, congested liver, peripheral edema). Applications to Congestive Heart Failure (CHF) are also contemplated. CHF is a clinical syndrome caused by heart disease, characterized by breathlessness and abnormal sodium and water retention, and resulting in edema. This term is used when there is congestion of pulmonary or systemic vascular beds.
HMGB1 may be administered as a recombinant protein injected in the contracting wall bordering the infarct or in heparin beads in the tibialis muscle. However, due to the short mean life of the protein the administration scheme may need high or repeated doses to attain therapeutic effects. Therefore, a gene therapy approach could be advantageously used. A nucleic acid coding for HMGB1 may be carried into cells in appropriate vectors (plasmids, viruses).
Dosage is also a factor to be controlled since HMGB1 is an inflammatory molecule. In the current study on ischemia, 200 ng of protein was injected in the anterior and posterior aspects of the viable mouse myocardium bordering the infarct. This dose is around 2000 times lower than the dose required for causing systemic inflammation in mouse.
Effective amount of HMGB1 depends by the dosage unit form, by the route of administration and by other factors known in the art.
The invention will be now described with reference to following Figures:
The data represent the mean±SE, The statistical significance of the result is P<0.0001 in an ANOVA model. Treatment with HMGB1 plus anti-HMGB1 antibody gave results that did not differ statistically from the unstimulated control. Treatment of D18 cells with the anti-HMG1 antibody alone, or an unspecific antibody, also were indistinguishable from the unstimulated control.
Nomenclature
High mobility group proteins have been renamed recently. Previous/alternative names for HMGB1 are High mobility group 1, HMG1, HMG-1, amphoterin, and p30.
In this study the authors refer to HMGB1 accession number NP—037095 (NCBI) having the following sequence (SEQ ID No. 1):
The following HMGB1 fragments were also studied:
ABbt: from aa 1 to 187 of SEQ ID No. 1
AB: from aa 1 to 176 of SEQ ID No. 1
Box A: from aa 1 to 89 of SEQ ID No. 1
Box B (SEQ ID No. 2):
It should be noted that the sequences of ABbt, AB, Box A and Box B are identical in all mammals.
Cloning, expression and purification of HMGB1 protein and fragments
The plasmids HMGB1 and fragments thereof have been described (Müller et al., Biochemistry, 40, 10254-10261, 2001). Protein concentrations were determined spectroscopically using the method of Gill and von Hippel (Analyt. Biochem., 182, 319-326, 1989). The following extinction coefficients have been used for the native protein: Box A: A280=9.98 103 M−1 cm−1, Box B: A280=1.15 104 M−1 cm−1, AB, ABbt and full-length HMGB1: A280=2.14 104 M−1 cm−1.
Constructs and Cells.
Plasmid pEGFP-HMGB1 was generated by inserting the coding sequence of the CDNA for rat HMGB1 into pEGFP-N1 (Clontech) using the EcoRI and SacII restriction sites. pEGFP-H1c, pEGFP-NF1, pEGFP-HMGN2, and pEF-flag-mICAD were generously provided by A. Gunjan, N. Bhattacharyya, R. Hock, M. Bustin and S. Nagata (Phair and Misteli, Nature, 404, 604-609, 2000; Misteli et al., Nature 408, 877-881, 2000; Enari et al., Nature 391, 43-50, 1998). HeLa cells and fibroblasts (line VA1, Hmgb1+/+, and line C1, Hmgb1−/−) were grown as described (Calogero et al., Nature Genet. 22, 276-280, 1999). HeLa cells were electroporated with pEGFP-HMGB1 and were observed 18 h later. The average amount of HMGB1-GFP in the cell population was between 1 and 3% of HMGB1 (by immunoblotting with anti-HMGB1 antibodies). At the single cell level, the amount of HMGB1-GFP varied at most tenfold between different cells; care was taken to always use for analysis cells with a moderate fluorescence level.
Apoptosis was induced by treating the cells for 16 h with 2 ng/ml human TNF-alpha and 35 μM cycloheximide. Necrosis was induced by treatment for 16 h either with 5 μM ionomycin and 20 μM CCCP, or 6 mM deoxyglucose and 10 mM sodium azide, or by 3 cycles of freezing and thawing.
Three different clones of HeLa cells stably transfected with pEF-flag-mICAD were stained with TUNEL (Apoptosis detection system, Promega), and their chromosomal DNA was extracted and electrophoresed on a 1.5% agarose gel.
Indirect immunofluorescence was performed as described (Degryse et al., J. Cell Biol., 152, 1197-2006, 2001) using an anti-HMGB1 polyclonal antibody (Pharmingen) at 1:1600 dilution, and FITC- or TRIC-conjugated anti-rabbit antibodies (Boehringer) at 1:300 dilution.
In Vivo Microscopy and FLIP.
Cells were plated and observed in LabTek II chambers (Nalgene) with an Axiovert 135M microscope (Zeiss). FLIP experiments were carried out on a Leica TCS-SP confocal microscope using the 488 nm excitation line of an Ar laser and detection at 500-575 nm as described (Phair and Misteli, Nature, 404, 604-609, 2000). Cells were bleached (in a spot of 1 μm in radius, 20 mW nominal output, 200-500 ms) and imaged (at 0.2 mW nominal output) at intervals of 6 s.
Binding of Recombinant HMGB1 to Chromatin.
Hmgb−/− fibroblasts were treated with 2 ng/ml hTNF-alpha and 35 μM cycloheximide. After 16 h, apoptotic cells were recovered by gentle flushing of the dish. Ten million apoptotic Hmgb−/− fibroblasts and a control population of non-apoptotic ones were resuspended in 50 μl PBS containing 0.32 M sucrose, 0.5% NP-40 and 1 μM bacterially produced HMGB1, either fluorescently labelled with Cy5 (Pharmacia) or unlabelled. Average labelling was 2.3 Cy5 molecules per HMGB1 molecule. After 30 minutes at room temperature, sample cells were mixed and mounted on slides using Vectashield (Vector Laboratories) containing 1.5 μg/ml DAPI, and observed on an Axiophot microscope (Carl Zeiss) with TRITC filter. The two pools of cells incubated with unlabelled HMGB1 were layered onto discontinuous gradients formed by 5 ml of 1.16 M sucrose in PBS and a 6 ml cushion of 2 M sucrose in PBS, and centrifuged at 30,000 g for 90 minutes in a SW27 Beckman rotor. Apoptotic and non-apoptotic chromatin free from membrane debris was recovered from the bottom of the tubes, and applied to a 12% SDS-PA gel. The amount of recombinant HMGB1 bound to apoptotic and non-apoptotic chromatin was determined by immunoblotting using an anti-HMGB1 antibody (Pharmingen) at 1:3000 dilution. Aliquots of apoptotic and non-apoptotic chromatin were also probed with anti-acetyl-histone H4 (R10, a gift from B. Turner), anti-acetyl-Histone H3 (Lys 9, Biolabs) and anti-acetyl-lysine antibody (Biolabs).
Inflammation assays. To measure TNF-alphaproduction in vitro, bone marrow was recovered from the hind legs of female C56B16 mice, diluted to 5·106 cells/ml in Optimem and dispensed in 96-well microtiter plates (120 μl per well). Necrotic cells (lysed by 3 cycles of freeze-thawing) or apoptotic cells were added to the indicated final concentration into the wells and incubated at 37° C. for 18 hours. TNF-alpha in the supernatant was assayed by ELISA (Quantikine M, R&D Systems). TSA was added at 200 ng/ml together with TNF-alpha, when indicated, and was washed away before mixing the apoptotic cells with bone marrow cells.
To measure inflammation in vivo, one day old mice (weighing 1.1±0.1 grams) were injected intraperitoneally with 20 μl of PBS containing 320 μg of acetaminophen (Sigma), and 320 μg of antibodies (Pharmingen BD) where indicated. After 9 hours the mice were analyzed for serum ALT activity with the GP-Transaminase kit (Sigma) and for MPO activity in liver extracts as described (Kato et al., Am. J. Pathol. 157: 297-302, 2000). Statistical analysis was performed with the non-parametric Mann-Whitney test on MPO/ALT ratios. Similar results were obtained using the t-test on the MPO levels of mice paired so as to minimize the difference in ALT levels.
HMGB1 is loosely bound to chromatin of both interphase and mitotic cells, and it is rapidly leaked out into the medium when membrane integrity is lost in permeabilized or necrotic cells (Degryse et al., J. Cell Biol. 152: 1197-2006, 2001; Müller et al., EMBO J., 16, 4337-4340, 2001; Falciola et al., J. Cell Biol. 137, 19-26, 1997). These results suggested that in living cells HMGB1 associates and dissociates rapidly from chromatin. To prove this, HMGB1 was tagged with GFP at its C terminus, forming a chimeric protein that was equivalent to the unperturbed HMGB1 in enhancing the expression of a HOXD9-responsive reporter gene in transfection assays (Zappavigna et al., EMBO J., 15, 4981-4991, 1996). HeLa cells expressing the fusion protein were easily detectable by the uniform green fluorescence of their nuclei. Cells undergoing mitosis showed a diffuse cytoplasmic fluorescence, but also a distinct association of HMGB1-GFP to condensed chromosomes, that lasted throughout M phase (
When HMGB1-GFP transfected HeLa cells were permeabilized with NP-40, most lost their fluorescence after a few seconds, confirming the loose association of HMGB1 to chromatin. However, a few cells retained a bright fluorescence. From the characteristically fragmented appearance of their nuclei, these cells appeared apoptotic. HeLa cells were then forced to undergo apoptosis by treatment with TNF-alpha and cycloheximide, permeabilized, and immunostained for their endogenous, unmodified HMGB1. Whereas control non-apoptotic cells leaked all HMGB1 into the medium (
To quantify the dynamic properties of HMGB1-GFP within single cells, a technique called fluorescence loss in photobleaching (FLIP) was used (Phair and Misteli, Nature, 404, 604-609, 2000). Repeated bleaching of the same area leads to fluorescence loss from the rest of the nucleus, with kinetics dependent on the overall mobility of the fluorescent protein. If a fraction of the protein pool is at any given time bound to chromatin, the loss of its fluorescence will be slowed. Bleaching of total nuclear HMGB1-GFP was rapidly obtained (
The authors also assessed the diffusion rate of HMGB1-GFP associated to condensed chromosomes of living HeLa cells during mitosis. Repeated bleaching of cytoplasmic HMGB1-GFP led to rapid and parallel loss of fluorescence from condensed chromosomes and from the cytoplasm (
To the other extreme, HMGB1-GFP appeared almost immobile in apoptotic cells (
Hmgb1−/− cells offered us the opportunity to test whether the binding of HMGB1 to apoptotic chromatin was due to alterations of HMGB1, or of nuclei undergoing apoptosis. Embryonic fibroblasts obtained from Hmgb1−/− and Hmgb1+/+ mice Calogero et al., Nature Genet., 22, 276-280, 1999) were equally susceptible to apoptosis (not shown), indicating that freezing of HMGB1 onto chromatin is a consequence of apoptosis, but not a requisite. Hmgb1−/− fibroblasts were treated with TNF-alpha and cycloheximide, and apoptotic cells were recovered from the flask by gentle flushing. This cell population, and a control population of non-apoptotic Hmgb1−/− fibroblasts, were permeabilized with detergent and exposed to bacterially produced, Cy5-labelled HMGB1. HMGB1 bound to apoptotic nuclei, but not to non-apoptotic ones (
The authors next investigated the nature of the chromatin modification allowing the stable binding of HMGB1. Since HMGB1 binds tightly to in vitro reconstructed mononucleosomes (Falciola et al., J. Cell Biol., 137, 19-26, 1997), the authors tested whether the fragmentation of chromatin to oligo- and mononucleosomes that occurs in the later stages of apoptosis would provide stable binding sites for HMGB1. HeLa cells were stably transfected with a construct expressing ICAD, the inhibitor of the CAD nuclease that fragments DNA during apoptosis. HeLa cells overexpressing ICAD underwent apoptosis, but their DNA showed little if any fragmentation (Enari, et al., Nature, 391, 43-50, 1998) (
Alteration of the acetylation status of chromatin was tested next. TSA, a general deacetylase inhibitor, was added to the medium of HeLa cells just prior to the induction of apoptosis: in this case, HMGB1 freezing onto chromatin was suppressed (
The authors thus showed that HMGB1 binding to chromatin depends on the viability of the cell and clearly distinguishes necrotic from apoptotic cells. The differential release of HMGB1 might be exploited as a cue to nearby cells to activate the appropriate responses to unprogrammed and programmed cell death. Unprogrammed death is usually the result of trauma, poisoning or infection, all events that require prompt reaction and damage containment and/or repair. Inflammation is the primary tissue damage response in mammals, and HMGB1 has already been reported to be a mediator of inflammation (Wang et al., Science, 285, 248-251, 1999; Abraham et al. J. Immunol., 165, 2950-2954, 2000; Andersson et al., J. Exp. Med. 192, 565-570, 2000). To test directly whether the release of HMGB1 by necrotic cells can be the immediate trigger for an inflammatory response, the authors challenged wild type bone marrow cells with Hmgb1−/− or wild type (+/+) dead fibroblasts. As expected, wild type necrotic cells triggered the production of the proinflammatory cytokine TNF-alpha, whereas wild type apoptotic cells were much less effective (
The authors could not test whether Hmgb1−/− mice have a reduced inflammatory response following tissue necrosis, because these mice survive only a few hours after birth (Calogero et al., Nature Genet., 22, 276-280, 1999). To provide evidence in an animal model for the significance of HMGB1 release, massive hepatocyte necrosis was induced in wild type mice and the inflammatory response was measured. Both in humans and rodents, an overdose of the analgesic acetaminophen (AAP, also known as paracetamol) produces large areas of liver necrosis, concomitant with local inflammation, Kupffer cell activation and the recruitment and sequestration of neutrophils and macrophages into the injured tissue (Lawson et al., Toxicol Sci., 54, 509-516, 2000; Thomas, Pharmacol. Ther., 60, 91-120, 1993). Levels of liver damage and neutrophil sequestration are strictly proportional until most hepatocytes become necrotic between 12 and 24 hours after AAP poisoning (Lawson et al., Toxicol Sci., 54, 509-516, 2000). The authors administered 300 mg/kg AAP with a single intraperitoneal injection to young mice, and 9 hours later estimated liver injury by measuring alanine transaminase (ALT) activity in serum, and inflammatory cell sequestration by measuring myeloperoxidase (MPO) activity in total liver extracts. One group of mice (n=8) received no AAP, one group (n=10) received AAP alone, another (n=6) AAP and affinity purified anti-HMGB1 antibodies (300 mg/kg), and the last one (n=8) AAP and irrelevant rabbit antibodies (300 mg/kg). All 3 groups injected with AAP had elevated ALT levels in comparison to sham-treated controls, but the differences between the 3 treated groups were not statistically significant. Thus, antibodies do not protect against liver damage, at least at the onset of the inflammatory response. The authors then used the MPO/ALT ratio to compare inflammatory cell recruitment, normalized to the level of liver damage (
In conclusion, the authors have shown that the passive release of an abundant chromatin component can serve as a diffusible signal of unprogrammed death, that can be used as a cue to nearby cells. Core histones, though more abundant, would probably not be good signals of necrosis, as they remain anchored to the insoluble chromatin of necrotic cells. Apoptotic cells are not the result of a present and immediate danger and do not trigger inflammation in physiological conditions. They retain nuclear components until cleared by macrophages or nearby cells that act as semi-professional phagocytes, that they attract and activate by displaying “eat me” signals (Ren and Savill, Cell Death Different., 5, 563-568, 1998). However, apoptotic cells that escape prompt clearance undergo secondary necrosis, lead to an increased level of nuclear autoantibodies (Scott et al., Nature, 211, 201-211, 2001), and have been proposed to play an important pathogenetic role in autoimmune diseases, such as lupus (Herrmann et al., Arthritis Reum., 41, 1241-1250, 1998). Thus, the retention of HMGB1 by apoptotic cells undergoing secondary necrosis represents an additional safeguard against confusing necrotic and apoptotic cells.
The authors also demonstrated that HMGB1 has chemotactic activity on D18 mouse mesoangioblasts derived from fetal aorta cells (Minasi et al., Development. 129, 2773-83, 2002). Mesoangioblastic stem cells can be derived from mouse fetal aorta cells but also from umbilical chord cells, peripheral blood vessels and bone marrow ckit±cells in post-natal mice. Once derived from these original cell populations with a proprietary method (described in Minasi et al., Development. 129, 2773-83, 2002), mesangioblastic cells, of which D18 are an example, are ‘naturally’ immortalized (they grow indefinitely). D18 cells serve as precursor of the following mesodermal tissue types: bone, cartilage, skeletal smooth and cardiac muscle, endothelial cells, monocytes, macrophages and osteoclasts. They are also capable of generating hepatocytes and neurons. Clone D18 was deposited according to the Budapest Treaty at CBA, Centro Biotecnologie Avanzate, Genova, Italy, N. PD02005). Chemotaxis assays were performed using modified Boyden chambers. The value of 1 corresponds to the number of cells migrating in the absence of any stimulator (random cell migration).
The data represent the mean±SE. The statistical significance of the result is P<0.0001 in an ANOVA model. Treatment with HMGB1 plus anti-HMGB1 gave results that did not differ statistically from the unstimulated control. Treatment of D18 cells with the anti-HMGB1 antibody alone, or an unspecific antibody, also were indistinguishable from the unstimulated control.
Stem cells are expected to proliferate at the site where tissue repair must take place. The authors then tested whether HMGB1 could also stimutate stem cell proliferation. Stem cells in RPMI with no serum did not divide, while cells in RPMI with no serum but in the presence of HMGB1 divided actively for at least 24 hours, and then more slowly due to nutrient exhaustion. At day 3, nonetheless, all plates with D18 cells stimulated with HMGB1 (at all concentrations) contained some dividing cells, as evaluated by microscopic inspection.
Cells in RPMI alone did not divide, while cells in RPMI plus HMGB1 divided actively for at least 24 hours, and then more slowly due to nutrient exhaustion. At day 3, nonetheless, all plates with D18 cells stimulated with HMGB1 (all concentrations) contained some dividing cells, as evaluated by microscopic inspection (
The experiment has been repeated with mesangioblasts derived from adult mouse capillaries (stem cells from adult), showing the same proliferation inducing effect.
All of experiments shown hereinabove were also repeated with another stem cell clone, named D16, with similar results, as illustrated in the following Example 3.
Extracellular HMGB1, a Signal of Tissue Damage, Induces Mesoangioblast Migration and Proliferation
Cells
Bovine Aorta Endothelial Cells (BAEC) were isolated from a section of the thoracic aorta of a freshly slaughtered calf as described (Palumbo al., Arterioscler. Thromb. Vasc. Biol., 22, 405-11, 2002). Mesoangioblasts were isolated from the dorsal aorta of mouse embryos and from juvenile arteries as previously described (De Angelis et al., J. Cell Biol., 147, 869-78, 1999). After cloning, cells were expanded on a feeder layer of mitomycin C-treated STO fibroblasts. Clones showing the mesoangioblast gene expression pattern (presence of CD34, Kit, Flk1 and MEF2D) were used for the in vitro and in vivo experiments. Embryonic mesoangioblasts (clone D16) were transduced with a lentiviral vector encoding for nuclear LacZ, while adult mesoangioblasts (clone G1) were labeled with DiI and then injected into the femoral artery of mice.
HMGB1 and Antibodies
Expression and purification of the full-length HMGB1 protein and fragments thereof was performed as described previously (Müller et al., Biochemistry, 40, 10254-10261, 2001). Endotoxins were removed by passage through Detoxy-Gel columns (Pierce).
Rabbit polyclonal anti-HMGB1 antibodies raised against the peptide 166-181 were from Pharmingen BD, polyclonal antibodies against box A were from MBL. The anti-RAGE goat antibody was from Chemikon. Western blots were performed as described (Degryse et al., J. Cell Biol., 152, 1197-2006, 2001).
Proliferation Assay
Cells were seeded in 6-well plates (1×105 cells/well) and grown in RPMI supplemented with 20% FCS. After 24 hours the medium was replaced with serum-free RPMI for 16 hours. Subsequently the cells were grown with medium alone, or medium with the addition of 20% FCS or HMGB1 at the concentration of 1, 3, 10, and 30 ng/ml. Cells were counted after 1, 2 and 3 days, and Trypan blue dye exclusion was used as indicator of cell viability. All experiments were performed three times in duplicate.
Chemotaxis Assay
Cell migration was assayed using Boyden chambers (Degryse et al., J. Cell Biol., 152, 1197-2006, 2001). Briefly, PVP-free polycarbonate filters with 8 μm pores (Costar) were coated with 5 μg/ml porcine skin gelatin (Sigma). Serum-free RPMI (negative control), RPMI containing 10, 50 or 100 ng/ml HMGB1, and RPMI with 20% serum (positive control) were placed in the lower chambers. D16 cells were grown in RPMI plus 10% FCS, starved overnight, washed twice with PBS to eliminate any floating cells, and harvested with trypsin. Fifty thousand cells rsuspended in ration Assay
BAEC cells were grown in DMEM plus 10% FCS on polycarbonate transwell inserts (3 μm pores; Costar) for 5 days until they formed a monolayer. The inserts were then placed between chambers in Boyden apparatuses, and the tightness of monolayers was checked by measuring the diffusion of BSA between chambers. Mesoangioblasts (105 cells in 100 μl RPMI) were placed in the upper compartments and RPMI containing HMGB1 or VEGF (mature 121 amino acid variant of human VEGF expressed in E. coli, from R&D Systems) was placed in the lower compartments (500 μl). After 8 hours at 37° C. the filters were removed, and the result was evaluated as described for the chemotaxis assay. These experiments were carried out three times in duplicate.
Flow Cytometry
Mesoangioblasts starved overnight were grown in RPMI medium alone, RPMI plus 20% FCS or RPMI plus 100 ng/ml HMGB1 for 6, 12, 24 or 48 hours. Cells were washed, fixed in 70% ethanol, stained with 50 μg/ml propidium iodide (PI) in PBS plus 50 μg/ml RNase A and incubated for 30 min at room temperature. The DNA content was measured by flow cytometry (FACScan; Becton-Dickinson) using the Standard CellQuest software.
Estimation of the Number of Cell Divisons
Ten million embryonic or adult mesoangioblasts were seeded on 100 mm dishes in RPMI supplemented with 20% FCS. After 24 hours the cells were starved in RPMI alone overnight. Cells were then washed in PBS, and CEDASE (Molecular Probes) was added to the final concentration of 2.5 μM for 8 minutes at room temperature. The staining was quenched by the addition of 10% FCS and cells were washed in RPMI. Fluorescently labeled cells were then grown in RPMI alone, RPMI plus 100 ng/ml HMGB1 or RPMI plus 20% FCS and harvested after 48 hours. Cells were then analyzed on FACScan.
Cytoskeleton Visualization
BAEC cells were grown on glass coverslips until filly confluent. After the treatments described in the text, the cells were washed with PBS and fixed with 4% paraformaldehyde at room temperature for 10 minutes. Cells were then stained with FITC-conjugated phalloidin (Sigma) to visualize the actin cytoskeleton as described (Degryse et al., J. Cell Biol., 152, 1197-2006,2001).
Preparation of HMGB1-Loaded Beads.
Heparin beads (34 μm diameter) were recovered from a HiTrap Heparin HP column (Pharmacia) and extensively washed in PBS. Beads (20 μl packed volume) were then incubated for 1 hour at 4° C. with 60 μg HMGB1, harvested by centrifugation, washed twice with PBS and resuspended in PBS. SDS-PAGE was performed to check the amount of HMGB1 on the beads. All of the added HMGB1 was found to have adsorbed to the beads.
Intra-artery Delivery of Mesoangioblasts in Mice.
Heparin beads (a slurry containing 3 μg beads in 20 μl PBS), either laoded with HMGB1 or not, were injected with an insulin syringe into tibialis anterior muscles of 6-week old female CD-1 mice (3 per group). The total amount of HMGB1 injected (where indicated) was approximately 9 μg. After 1 hour mesoangioblasts (4×105 cells/animal) were injected through the femoral artery as previously described (Torrente et al., J. Cell Biol., 152, 335-48, 2001); animals were sacrified 24 hours later. For histochemistry analysis, samples of tibialis anterior muscles were frozen in liquid nitrogen-cooled isopentane and cryostat-sectioned. Serial muscle sections of 10 μm thickness were stained with X-gal for the experiment with LacZ labeled cells (Totsugawa et al., Cell Transplant., 11, 481-8, 2002), or visualized directly for the experiment with DiI. DiI was from Molecular Probes.
HMGB1 Stimulates the Proliferation of Vessel-associated Embryonic Stem Cells
Stem cells isolated from mouse dorsal aorta of E9.5 embryos (mesoangioblasts) were cultured in vitro and tested for the presence of the CD34, Kit, Flk1 and MEF2D cellular markers (Minasi et al., Development, 129, 2773-2783, 2002). The authors used one of these clones (called D16) to assess whether HMGB1 can act as a mitogen.
D16 cells were seeded in RPMI medium with 20% FCS and then starved for 16 hours in the absence of serum to synchronize the cell population. Increasing concentrations of HMGB1 were then added to the medium without serum, and cells were counted after 1, 2, and 3 days.
The authors investigated in more detail the proliferative response of D16 cells to HMGB1: cells were exposed for 6, 12, and 24 hours to RPMI medium alone (negative control), or medium containing 30 ng/ml HMGB1 or 20% FCS, and analyzed for DNA content by FACS after propidium iodide staining. After six hours of stimulation with HMGB1, the majority of mesoangioblasts had entered the cell cycle; after 24 hours, most cells appeared to have a diploid DNA content and thus to be in G1 or G0 (
These data indicate that HMGB1 induces a limited number of cell divisions. This might be due to a specific program of mesoangioblasts, or to the depletion of HMGB1 in the medium. The authors therefore added 30 ng/ml HMGB1 at time 0, and additional HMGB1 at 12, 36 and 60 hours. Mesoangioblasts continually exposed to HMGB1 continued to divide (
HMGB1 Induces Mesoangioblast Migration
The authors have previously shown that HMGB1 is a chemoattractant for rat smooth muscle cells (RSMC) (Degryse et al., J. Cell Biol., 152, 1197-2006, 2001). They then investigated whether HMGB1 is a chemoattractant for D16 cells too. In a chemotaxis assay using modified Boyden chambers, HMGB1 stimulated migration of D16 cells in a concentration-dependent manner (
The authors also tested the individual domains of HMGB1 for the ability to induce D16 cell migration (
Huttunen et al. (Cancer Res., 62, 4805-4811, 2002) have identified residues 150-183 as the HMGB1 segment that interacts with RAGE. Remarkably, the anti-HMGB1 antibodies that block D16 migration (
HMGB1 induces Mesoangioblast Migration Across Endothelial Monolayers
Mesoangioblasts are vessel-associated stem cells that can migrate to damaged tissues through the general circulation (Sampaolesi et al., Science, 301, 487-492, 2003), and have the ability to transit through the endothelial barrier. The authors then tested whether HMGB1 could also promote the transmigration of stem cells across an endothelial monolayer grown on the septum between the chambers of a Boyden apparatus. When the authors added 100 ng/ml HMGB1 to the medium in the lower chamber, the number of D16 cells crossing the monolayer increased 8-fold when compared to medium without any addition (
After stimulation with HMGB1 for 5 to 30 minutes, endothelial cells were fixed, labeled with fluorescein-conjugated phalloidin and examined by immunofluorescence. Both HMGB1 and VEGF caused stress fiber formation and disaggregation of endothelial cells, suggesting that HMGB1 can dramatically increase the permeability of the endothelial lining of vessels (
HMGB1 Directs Mesoangioblast Homing in vivo
The authors finally assessed HMGB1's ability to control mesoangioblast migration in vivo. Heparin beads were loaded with HMGB1 at the concentration of 3 μg/ml and then injected with a fine needle into the tibialis anterior muscle of mice. D16 cells transduced by a lentiviral vector causing the expression of nuclear LacZ were injected after 30 min through the proximal femoral artery (see materials and methods). The mice were sacrificed after 24 hours and the tibialis anterior muscle was removed, sectioned, and analyzed by immunohistochemistry. Muscles injected with HMGB1-loaded beads showed a considerable swelling compared to both sham-injected muscles (not shown) and muscles injected with unloaded heparin beads (
Muscle sections were stained with X-gal and blue cells were scored using computer-assisted imaging techniques (
These observations clearly suggest that HMGB1 is able to recruit mesoangioblasts in vivo.
The Biological Action of HMGB1 on Adult Mesoangioblasts
These findings identify a role for extracellular HMGB1, a proinflammatiory cytokine, on the migration and proliferation of embryonic mesoangioblasts. However, inflammation does not occur in the embryo even in the presence of tissue damage. The authors then asked whether HMGB1 had an effect on adult mesoangioblasts as well.
The authors repeated the experiments described previously on adult mesoangioblasts isolated from bone marrow (G1 clone, see materials and methods).
Similar effects were also observed in additional experiments with different adult mesoangioblast lines derived from the aorta of 8-week old mice (data not shown).
Immunoistochemical Analysis
Hearts were arrested in diastole at 1 week from the infarction, perfused retrogradely with 10% (vol/vol) formalin, embebbed in paraffin and sectioned at 4 μm. Sections were stained with hematoxilin and eosin (H&E) for histologic examination or processed for immunoistochemical analysis to identify newly formed cardiac cells. The following antibodies were used to assess cardiac differentiation: rabbit polyclonal connexin 43 antibody (Sigma St. Louis, Mo., USA), mouse monoclonal anti α-sarcomeric actin (clone 5C5; Santa Cruz Biotechnology, Santa Cruz, Calif., USA), rabbit polyclonal anti-MEF-2 (C-21; Santa Ciuz). Ki67 was detected by using mouse monoclonal antibody Ki67 (clone MIB5 Novocastra Laboratories, UK). FITC conjugated goat anti-rabbit and TRITC conjugated goat anti mouse (Sigma) were used as secondary antibodies.
Evaluation of Myocardial Function
Echocardiography was performed in conscious mice with a Sequoia 256c instrument equipped with a 13-MHz linear transducer. Two dimensional images and M-mode tracings were recorded from the parasternal short axis view at the level of papillary muscle. From M-mode tracings, anatomical parameters in diastole and systole were obtained (Orlic et al., Nature, 98, 10344-10349, 2001). For hemodynamic studies, mice were anesthetized with chloral hydrate (400 mg/kg body weight), the right carotid artery was cannulated with a microtip pressure transducer (Millar 1.4F) and the Left Ventricular pressures (LV, LV+and −dP/dt), were measured.
HMGB1 Promotes the Formation of New Myocytes in the Infarcted Heart
Myocardial infaction was induced in C57BL/6 mice by ligating the left coronary artery (Li et al., J. Clin. Invest., 100, 1991-1999, 1997) under anesthesia. After 4 hrs animals were re-operated and 200 ng of purified HMGB1 was administered in the peri-infarcted area in the left ventricle. Control animals consisted of infarcted mice injected with 200 ng of an unrelated protein (GST).
Injection of HMGB1 in the peri-infarcted left ventricle results in the formation of a compact tissue characterized by aligned cells occupying most of the damaged area, as showed by hematoxylin and eosin staining (
To characterize the newly formed tissue, the authors probed the expression of α-sarcomeric actin, a specific marker of cardiac differentiation. Immunoistochemical analysis showed that the tissue present in HMGB1-injected hearts (n=8) consists of α-sarcomeric actin positive cells (
Myocardial Function after Infaction is Improved by HMGB1
One week following infarction, the authors investigated echocardiographic and hemodynamic parameters in the surviving mice. In comparison with sham-operated mice, the infarcted animals whether treated or not with HMGB6b 1 showed indices of cardiac failure. In comparison to infacted but untreated mice, however, mice treated with HMGB1 showed a significant recovery of cardiac performance. Ejectirmation, improved left ventricular performance in the infarcted heart.
| Number | Date | Country | Kind |
|---|---|---|---|
| PCT/IT03/00265 | Apr 2003 | IT | national |
This application claims the benefit of priority of U.S. Ser. No. 10/519,427, filed May 31, 2005. The entire content and disclosure of the preceding application are incorporated by reference into this application.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 10519427 | May 2005 | US |
| Child | 11748632 | May 2007 | US |
