Patent Application
20060148695
The invention relates to a composition for promoting angiogenesis, for controlling DNA synthesis of a cell, and for controlling chemotactic motility of a cell. The invention also relates to a method for treating ischemia diseases.
Thrombomodulin is an anticoagulant, endothelial-cell-membrane glycoprotein. A recombinant thrombomodulin domain containing six epidermal growth factor-like structures exhibits mitogenic activity.
Although several molecules are known in the art as useful therapeutic agents for the treatment of diseases and disorders having as a symptom thereof abnormal clotting in critical blood vessels, there remains a need in the art for compositions and methods which are useful for the treatment of such diseases as well as for the treatment of diseases and disorders having as symptoms abnormally high or abnormally low muscle cell contractility or undesirable angiogenic activity. Such diseases and conditions include the following: cardiovascular diseases and conditions such as hypotension, hypertension and atherosclerosis; thrombotic conditions such as stroke, heart attack and post angioplasty stenting; angiogenic disorders; respiratory diseases and conditions such as pulmonary fibrosis and asthma; diseases and disorders related to tumor cell invasion, angiogenesis and metastasis; wound healing and clotting disorders and reproductive disorders such as premature uterine contraction and impotence.
In present day, a number of biomolecules, which induce or promote angiogenesis in tissues, have been identified. The most prominent of these are: growth factors such as vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), epidermal growth factor (EGF), platelet-derived growth factors (PDGFs) and transforming growth factors (TGFs) Healy A M., et al, Absence of the blood-clotting regulator thrombomodulin causes embryonic lethality in mice before development of a functional cardiovascular system. Proc Natl Acad Sci USA. 1995;92:850-854; and nitric oxide (NO) see, eg, J P Cooke et al, Nitric oxide and angiogenesis. Circulation 105 (2002) 2133-5. The current approaches aimed at promoting angiogenesis in the related field can be summarized into three main categories: (i) delivery of angiogenic growth factors using synthetic and natural polymeric scaffolds; (ii) delivery of plasmids containing DNA that encodes for angiogenic proteins; and (iii) combined delivery of angiogenic molecules and endothelial cell transplantation.
In accordance to the related arts, the invention proposes that loading the delivery systems with human recombinant thrombomodulin molecules instead of growth factors. It is proposed that the loaded human recombinant thrombomodulin can be released under physiological conditions, thus promoting localized, rapid angiogenesis.
Term Definition
The following definitions are offered for purposes of illustration, not limitation, in order to assist with understanding the discussion that follows.
In accordance with the present invention there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within persons skilled in the art. Such techniques are explained fully in the literature.
“Angiogenesis”—is generally thought to be heavily regulated by growth factors and other ligands. Angiogenesis, and the concurrent tissue development and regeneration, depends on the tightly controlled processes of endothelial cell proliferation, migration, differentiation and survival. Both stimulator and inhibitor ligands appear to interact, directly or indirectly, with cellular receptors during these processes. Angiogenesis begins with the erosion of the basement membrane by enzymes released by endothelial cells and leukocytes. The endothelial cells, which line the lumen of blood vessels, then protrude through the basement membrane. Angiogenic stimulators induce endothelial cells to migrate through the eroded basement membrane. The migrating cells then form a “sprout” off the parent blood vessel, where the endothelial cells undergo mitosis and proliferate. The endothelial sprouts merge with each other to form capillary loops, creating the new blood vessel.
The term “extracellular matrix” is a complex structure that contains collagen, proteoglycan, glycosaminoglycan, glycoproteins (fibronectin, chondronectin, laminin) and in some tissues, elastin (Hay, E. D., J. Cell Biol., 91:205-223 (1981)).
The term “Matrix metalloproteinases (MMP's)” is a protein family constitutes the major group of zinc-binding endopeptidases that degrade extracellular matrix proteins, for example connective tissue, collagen and gelatin, during remodeling of connective tissue during normal physiological and some pathological processes. The unrestrained activity of MMP's may result in extensive tissue damage, and these enzymes have been implicated in a variety of disease processes, including tumor cell invasion, tumor angiogenesis and rheumatoid arthritis (Okada, Y., et al., J. Biol. Chem., 261:14245-14255 (1986)). The MMP's are secreted from cells as inactive zymogens and their activity in the extracellular environment is regulated by various activators and inhibitors (Matrisian, L. M., Trends Genet., 6:121-125 (1990)).
A “nucleic acid” or “nucleotide sequence” refers to the phosphate ester polymeric form of ribonucleosides (adenosine, guanosine, uridine or cytidine; “RNA molecules”) or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine; “DNA molecules”) in either single stranded form, or a double-stranded helix. Double stranded DNA-DNA, DNA-RNA and RNA-RNA helices are possible. The term nucleic acid, and in particular DNA or RNA molecule, refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary or quaternary forms. Thus, this term includes double-stranded DNA found, inter alia, in linear or circular DNA molecules (e.g., restriction fragments), plasmids, and chromosomes. In discussing the structure of particular double-stranded DNA molecules, sequences may be described herein according to the normal convention of giving only the sequence in the 5′ to 3′ direction along the nontranscribed strand of DNA (i.e., the strand having a sequence homologous to the mRNA). A “recombinant DNA” is a DNA molecule that has undergone a molecular biological manipulation.
“Operably linked”, when referring to DNA segments, indicates that the segments are arranged so that they function in concert e.g. the transcription process takes place via the RNA-polymerase binding to the promoter segment and proceeding with the transcription through the coding segment until the polymerase stops when it encounters a transcription terminator segment.
As used herein the term “nucleic acid fragment” is intended to indicate any nucleic acid molecule of cDNA, genomic DNA, synthetic DNA or RNA origin. The term “fragment” is intended to indicate a nucleic acid segment which may be single- or double-stranded, and which may be based on a complete or partial naturally occurring nucleotide sequence encoding a polypeptide of interest. The fragment may optionally contain other nucleic acid segments.
The nucleic acid fragment of the invention encoding the polypeptide of the invention may suitably be of genomic or cDNA origin, for instance obtained by preparing a genomic or cDNA library and screening for DNA sequences coding for all or part of the polypeptide by hybridization using synthetic oligonucleotide probes in accordance with standard techniques.
The polypeptides may be detected using methods known in the art that are specific for the polypeptides. These detection methods may include use of specific antibodies, formation of an enzyme product, or disappearance of an enzyme substrate. For example, an enzyme assay may be used to determine the activity of the polypeptide.
The polypeptides of the present invention may be purified by a variety of procedures known in the art including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic procedures (e.g., preparative isoelectric focusing (IEF), differential solubility (e.g., ammonium sulfate precipitation), or extraction.
An “antisense RNA” refers to a specific sequence of RNA which can be used to inactivate gene expression. An antisense gene codes for an (antisense) RNA that has a complementary sequence to an RNA that is its target. Antisense genes block expression of their targets when introduced into eukaryotic cells. Base pairing offers a powerful means for one RNA to control the activity of another. There are many cases in both prokaryotes and eukaryotes where a (usually rather short) single-stranded RNA base pairs with a complementary region of an mRNA, and as a result prevents expression of the mRNA. One of the early illustrations of this effect was provided by an artificial situation, in which antisense genes were introduced into eukaryotic cells. This technique offered an early approach for turning off genes at will; for example, the function of a regulatory gene can be investigated by introducing an antisense version. Using the same principle, now we have more sophisticated possibilities as the result of the discovery that small RNAs can inhibit expression of complementary sequences.
RNAi is normally triggered by long stretches of double stranded RNA (dsRNA). This is then processed in the cell by an RNase III-like enzyme called Dicer to form short dsRNAs 21-25 nt in length, known as short interfering RNAs (siRNA). When working with mammalian systems, long dsRNA cannot be added to cells without inducing an interferon response, so siRNAs are added instead. This can either be in the form of chemically synthesised siRNA or transcribed from a siRNA expression vector to form a short hairpin RNA (shRNA).
Once processed by Dicer, these siRNAs can enter a multi-enzyme complex known as the RNA-induced silencing complex (RISC). RISC then unwinds the siRNA, incorporating one of the two strands and it is this strand that is then used by RISC to identify a complementary mRNA target. The final result is the degradation of the mRNA target.
Oligonucleotide-mediated antisense and siRNA-mediated RNA interference (RNAi) offer a precise and specific means of knocking down expression of a target gene in any biological system. Gene expression relies on a genomic DNA sequence being transcribed into a messenger RNA (mRNA) that is in turn translated into protein. An antisense or siRNA recognises a specific mRNA target and blocks or degrades it, preventing its translation. Any protein encoded by the target gene thus depletes as a function of its half-life and a rapid and specific knockdown is achieved.
The present invention provides a composition for promoting angiogenesis, comprising a composition including a polypeptide comprising amino acid sequence or a conservative variant thereof having EGF-like domain of thrombomodulin.
The present invention also provides a composition for controlling DNA synthesis of a cell, comprising an agent antagonizing specifically to a fragment of polypeptide or nucleotide derived from thrombomodulin.
The present invention also provides a composition for controlling chemotactic motility of a cell, comprising an agent antagonizing specifically to a fragment of polypeptide or nucleotide derived from thrombomodulin.
Another significant object of the invention is to offer a method for treating ischemia associated with angiogenesis comprising administering to a patient in need of the treatment with a therapeutically effective amount of a polypeptide comprising amino acid sequence or a conservative variant thereof having EGF-like domain of thrombomodulin.
Other objects and features of the present invention will become apparent from the following detailed description considered in conjunction with the accompanying drawings. It is to be understood, however, that the drawings are designed solely for purposes of illustration and not as a definition of the limits of the invention, for which reference should be made to the appended claims. It should be further understood that the drawings are not necessarily drawn to scale and that, unless otherwise indicated, they are merely intended to conceptually illustrate the structures and procedures described herein.
In the drawings:
Thrombomodulin is an anticoagulant, endothelial-cell-membrane glycoprotein. A recombinant thrombomodulin domain containing six epidermal growth factor-like structures exhibits mitogenic activity. The invention relates to the recombinant domain's novel angiogenic effects using in vitro and in vivo models.
In the present invention, human recombinant thrombomodulin containing six epidermal growth factor-like structures (TMD2) and TMD2 plus a serine and threonine-rich domain (TMD23) were prepared using the Pichia pastoris expression system. Combined with purified TMD2 or TMD23, thrombin effectively activated protein C. TMD23 had higher activity than TMD2 in stimulating DNA synthesis in cultured human umbilical vein endothelial cells (HUVECs). Additionally, TMD23 stimulated chemotactic motility and capillary-like tube formation in HUVECs, an effect mediated through phosphorylation of extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinase and the phosphatidylinositol-3 kinase/Akt/endothelial nitric oxide synthase pathway. TMD23 also stimulated endothelial-cell expression of matrix metalloproteinases and plasminogen activators, which mediated extracellular proteolysis leading to endothelial-cell invasion and migration during angiogenesis. Furthermore, TMD23-containing implants in rat cornea induced ingrowth of new blood vessels from the limbus. Using the murine angiogenesis assay, TMD23 not only induced neovascularization coinjected with Matrigel and heparin but also enhanced angiogenesis in Matrigel containing melanoma A2058 cells in nude mice.
The recombinant thrombomodulin domain TMD23 enhanced the angiogenic response in vitro and in vivo, suggesting that thrombomodulin fragments may play a role in formation of new vessels. These findings may provide a new therapeutic option for treating ischemic diseases.
The present invention provides a composition for promoting angiogenesis, comprising a composition including a polypeptide comprising amino acid sequence or a conservative variant thereof having EGF-like domain of thrombomodulin. In the preferred embodiment, the composition of the invention further comprises an operably linked polypeptide comprising amino acid sequence or a conservative variant of serine-threonine rich domain of thrombomodulin. In the preferred embodiment, the promotion of angiogenesis is related to stimulate new capillary formation from endothelial cells. In a more preferred embodiment, the new capillary forms vessel.
The composition of the invention could be used in a cell-based therapy for treating ischemia related diseases. In the preferred embodiment, the endothelial cells express matrix metalloproteinases or plasminogen activators. In the more preferred embodiment, the matrix metalloproteinases or plasminogen activators mediate extracellular proteolysis leading to endothelial-cell invasion and migration during angiogenesis.
In the clinical use, the composition of the invention could help wound healing or reconstructive surgery.
In the present invention, the ischemia related disease is such as myocardial ischemia, peripheral ischemia, cerebral ischemia or deep vein thrombosis.
In the invention, the stimulation of new capillary formation is mediated through phosphorylation of extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinase and phosphatidylinositol-3 kinase/Akt/endothelial nitric oxide synthase pathway.
The composition of the invention further comprises additional carriers or excipients such a gel, cream, paste, lotion, spray, suspension, solution, dispersion salve, hydrogel or ointment formulation.
The present invention also provides a composition for controlling DNA synthesis of a cell, comprising an agent antagonizing specifically to a fragment of polypeptide or nucleotide derived from thrombomodulin. In the preferred embodiment, the polypeptide further comprises an operably linked polypeptide comprising amino acid sequence or a conservative variant of serine-threonine rich domain of thrombomodulin.
In the preferred embodiment, the DNA synthesis is directed to the result of abnormal cell growth. In the more preferred embodiment, the abnormal cell is a result of tumor cell proliferation. In the most preferred embodiment, the tumor is directed to angioma.
The present invention further provides a composition for controlling chemotactic motility of a cell, comprising an agent antagonizing specifically to to a fragment of polypeptide or nucleotide derived from thrombomodulin. In the preferred embodiment, the fragment is sufficient to produce an effect of chemotactic motility on a cell. In the more preferred embodiment, the polypeptide further comprises an operably linked polypeptide comprising amino acid sequence or a conservative variant of serine-threonine rich domain of thrombomodulin.
In the further more preferred embodiment, the composition controlling cell chemotactic motility is in the form of antibody, RNAi, siRNA or antisense RNA. In the more preferred embodiment, the chemotactic motility is directed to the result of tumor metastasis. In particular, the chemotactic motility of the cell is mediated through phosphorylation of extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinase and phosphatidylinositol-3 kinase/Akt/endothelial nitric oxide synthase pathway.
The present invention also provides a method for treating ischemia associated with angiogenesis comprising administering to a patient in need of the treatment with a therapeutically effective amount of a polypeptide comprising amino acid sequence or a conservative variant thereof having EGF-like domain of thrombomodulin. In the preferred embodiment, the polypeptide of the composition further comprises an operably linked polypeptide comprising amino acid sequence of serine-threonine rich domain of thrombomodulin.
The administration of the polypeptide is by route of intravenous (iv), subcutaneous (sc), intraperitoneal (ip), or intramuscular (im). In the preferred embodiment, the administration of the polypeptide is by means of gene transfer.
For further understanding of the diseases related to the present invention, by which offering inhibitors or antagonist of angiogenesis via the mechanisms against TMD2 or TMD23 as a therapy is disclosed thereafter. Such diseases and processes, may for example include, but are not limited to, preventing tumor growth, inhibiting tumor growth, reduction in tumor volume, elimination of tumors, inhibiting metastasis, preventing metastasis, inducing dormancy of tumors and metastasis, preventing arteriosclerosis, inhibiting atherosclerosis, reducing existing plaque in blood vessels, reducing intimal plaque neovascularization, hemangioma, solid tumors, leukemia, myocardial angiogenesis, neovascular glaucoma, diabetic retinopathy, retrolental fibroplasia, arthritis, diabetic neovascularizaton, macular degeneration, wound healing, peptic ulcer, fractures, keloids, hematopoiesis, ovulation, menstruation, or placentation.
The present invention will be described in detail below.
Material and Processes:
Expression and Purification of Recombinant TMD Proteins
The pPICZaA vector (Invitrogen Corporation) was used for expression and secretion of recombinant human TMD2 and TMD23 in the Pichia pastoris protein expression system. Briefly, DNA fragments coding for TMD2 (residues Ala224-Cys462) and TMD23 (residues Ala224-Ser497) were obtained by a polymerase chain reaction of human umbilical vein endothelial cell cDNA using primers as described previously. Han HS., et al. Effect of thrombomodulin on plasminogen activation. Fibrinolysis and Proteolysis. 2000;14:221-228. Fermentation medium containing expressed TMD2 or TMD23 was applied to a nickel-chelating Sepharose column (Amersham Pharmacia Biotech AB), and TMD-containing fractions were eluted with imidazole.
Thrombomodulin Activity Assay
Cofactor activity of TMD2 and TMD23 for thrombin-dependent protein C activation was measured as described previously. Huang H C., et al Thrombomodulin-mediated cell adhesion: Involvement of its lectin-like domain. J Biol Chem. 2003;278:46750-46759.
Cell Proliferation Assay
To evaluate the effect of TMD proteins on DNA synthesis in human umbilical vein endothelial cells (HUVECs), we performed a 5-bromo-2′-deoxyuridine (BrdU) incorporation assay with a commercial quantification kit (Roche Diagnostic GmbH) and manufacturer's protocol. Mitogenic effects induced by TMD proteins were also determined in the presence of polyclonal rabbit anti-TMD23 antibody (prepared by GlycoNex, Inc., Taiwan) that was purified by protein G-Sepharose column (Amersham Pharmacia Biotech AB).
Chemotaxis Assay
The chemotactic motility of HUVECs was assayed using Transwell (Costar) with 6.5-mm diameter polycarbonate filters (8-μm pore size). The lower filter surface was coated with 10 μg gelatin (Sigma-Aldrich). Various concentrations of TMD23 in the absence or presence of 10 mg/mL polyclonal rabbit anti-TMD23 antibody or 10 ng/mL vascular endothelial growth factor (VEGF) (R & D Systems, Inc.) in M199 were placed in the lower wells. One hundred mL cell suspension containing 1×105 cells was loaded into each of upper well. The chamber was incubated at 37° C. for 4 h. Cells were fixed with methanol and stained with 4′,6′-diamidino-2-phenylindole (DAPI). Chemotaxis was quantified with optical microscopy (Leica) at 100× magnification using MetaMorph imaging software (Universal Imaging Corporation) by counting the cells that migrated to the filter's lower side. Five random fields in each well were counted. Each sample was assayed in triplicate, and assays were repeated three times. To determine the role of extracellular signal-regulated kinase 1/2 (ERK1/2), p38 mitogen-activated protein kinase (MAPK), and phosphatidylinositol 3-kinase (PI3 kinase)-protein kinase B/Akt (Akt) in TMD23-mediated cell migration, assays were performed in the presence of U0126, a MAPK/ERK kinase (MEK) inhibitor, SB 203580, a p38 MAPK inhibitor, and LY 294002, a PI3 kinase inhibitor. Cell viability as determined by trypan blue staining was not altered by treatment. U0126 was purchased from Promega, with SB 203580 and LY 294002 from Calbiochem.
Assay of ERK1/2, p38 MAPK, Akt, and Endothelial Nitric Oxide Synthase (eNOS) Phosphorylation
HUVECs were cultured to confluence in a 6-cm-diameter dish and incubated in M199 containing 1% FBS for 18 h. Cells were washed and incubated with serum-free M199 for 6 h, and treated with the indicated concentration of TMD23. Cell lysates were separated by SDS-PAGE, and the levels of phospho-ERK1/2 (Tyr 204) and total ERK1/2, phospho-p38 (Thr-180/Tyr-182) and p38, phosphor-Akt (Ser-473) and Akt, and phospho-eNOS (Ser-1173) and eNOS were detected by Western blotting with specific antibodies (Cell Signaling Technology).
Gelatin and Casein Zymographies and Plasminogen Activator Inhibitor (PAI) Activity Assay
HUVECs were seeded at 80% confluence on 100-mm dishes in M199 medium. After 24 h, cells were rinsed with serum-free M199 twice and incubated in serum-free M199 with various concentrations of TMD23, VEGF (10 ng/mL), or PMA (70 ng/mL) for 12 h. The conditioned medium containing 5 mg secreted proteins was analyzed by gelatin- and casein-based zymographies. The digested area appeared clear on a blue background, indicating the location of matrix metalloproteinase (MMP) activity. To further verify MMP caseinolytic activity, the casein-containing gel was incubated with incubation buffer containing 10 mM EDTA, an inhibitor of MMPs, to observe disappearance of the clear band. Plasminogen activator inhibitor (PAI) activity was measured by titrating samples with increasing amounts of tissue-type plasminogen activator (t-PA) (Boehringer Ingelheim) into a fixed volume of endothelial-cell-conditioned media.
In Vitro Matrigel Angiogenesis Assay
Growth-factor-reduced Matrigel (BD Biosciences) was thawed at 4° C. A total of 200 mL Matrigel was added to wells of a 48-well plate and polymerized 30 min at 37° C. Two hundred mL cell suspension containing various concentrations of VEGF, TMD23, or TMD23 in the presence of polyclonal rabbit anti-TMD23 antibody (10 mg/mL) or various signaling pathway inhibitors were plated onto Matrigel-coated wells at a density of 2.4×104 cells/well in M199 containing 10% FBS for 24 h at 37° C. in a 5% CO2 humidified atmosphere. Each concentration was tested in triplicate in the same plate, with wells photographed using a Leica camera (40× magnification). Tube length was quantified by measuring tubes in five randomly chosen fields from each well using MetaMorph software. Experiments were repeated at least three times.
Rat Corneal Angiogenesis Assay
The rat corneal assay was performed as described elsewhere. See eg., Kenyon B M., et al A model of angiogenesis in the mouse cornea. Invest Ophthalmol Vis Sci. 1996;37:1625-1632. Uniformly sized pellets of Hydron (polyhydroxyethylmethacrylate [poly HEMA]) (Sigma-Aldrich) containing either TMD23 or basic fibroblast growth factor (bFGF) and sucralfate (Sigma-Aldrich) were implanted into the rat corneal stroma adjacent to the temporal limbus. Briefly, sterile-saline suspensions containing 5-40 mg TMD23 or bFGF plus 10 mg sucralfate were made and speed vacuumed for 5 min; 12% Hydron in ethanol was added as described. The suspension was deposited onto an autoclaved, sterilized, 15-mm×15-mm nylon mesh. In some preparations, polyclonal anti-TMD23 IgG (10 mg/mL) was included during pellet preparation to specifically inhibit TMD23 activity. The eyes of Sprague Dawley rats (250-300 g) were topically anesthetized and a single pellet was inserted into a surgically created pocket in the corneal stroma. Corneas were examined daily with a dissecting microscope for up to 24 days for capillary growth. Maximal vessel length, clock h, and vascular areas of corneas of all rats were measured with a slit-lamp stereomicroscope.
Murine Angiogenesis Assay
To assess angiogenic effects in vivo, growth-factor-reduced liquid Matrigel (0.5 mL) containing heparin (60 U/mL) and various concentrations of TMD23, bFGF or VEGF was subcutaneously injected into FVB mice near the abdominal midline. Matrigel with PBS plus heparin served as the negative control, with bFGF and VEGF plus heparin as the positive control. Four days after injection, mice were sacrificed and Matrigel plugs were surgically removed. For macroscopic analysis of angiogenesis, hemoglobin content in Matrigel was measured using Drabkin reagent 525 (Sigma-Aldrich). For histological analysis, rat anti-mouse CD31 (PECAM-1, platelet endothelial cell adhesion molecule-1) antibody (Pharmingen) was used. The same specimens were used for hematoxylin and eosin (H&E) staining.
Effect of TMD23 on Tumor Neoangiogenesis
To assess TMD23 effects on tumor neoangiogenesis, 1×106 human melanoma A2058 cells (ATCC CRL-11147) were mixed with Matrigel (0.5 mL) containing heparin (60 U/mL) and various concentrations of TMD23 and subcutaneously injected into athymic nude mice. Matrigel plugs were harvested after 18 days and frozen in liquid nitrogen. Blood vessels were detected by immunohistochemistry using anti-CD31 antibody as described above. Microvessel density (MVD) was calculated by counting six highly vascularized fields in each section (200× magnification; MetaMorph software).
Statistical Analysis
Data are expressed as mean±standard deviation (SD). Statistical significance was analyzed by unpaired Student's t test. Differences between more than two groups was compared by one-way analysis of variance (ANOVA) followed by a Bonferroni's post hoc test, with P<0.05 considered statistically significant.
The pPICZaA vector (Invitrogen Corporation) was used for expression and secretion of recombinant human TMD2 and TMD23 in the Pichia pastoris protein expression system. Briefly, DNA fragments coding for TMD2 (residues Ala224-Cys462) and TMD23 (residues Ala224-Ser497) were obtained by a polymerase chain reaction of human umbilical vein endothelial cell cDNA using primers as described previously. Han HS., et al Effect of thrombomodulin on plasminogen activation. fibrinolysis and proteolysis. 2000;14:221-228. Fermentation medium containing expressed TMD2 or TMD23 was applied to a nickel-chelating Sepharose column (Amersham Pharmacia Biotech AB), and TMD-containing fractions were eluted with imidazole.
Recombinant TMD2 (Ala224-Cys462) containing six EGF-like structures (D2) and TMD23 (Ala224-Ser497) containing D2 and the serine and threonine (Ser/Thr)-rich domain D3 (molecular masses of 47 and 50 kDa, respectively) were prepared with the Pichia pastoris expression system. TMD2 or TMD23 purified from fermentation medium was homogeneous as judged by SDS-PAGE and Western blotting with monoclonal anti-TM antibody (
Cofactor activity of TMD2 and TMD23 for thrombin-dependent protein C activation was measured as described previously. Huang H C., et al Thrombomodulin-mediated cell adhesion: Involvement of its lectin-like domain. J Biol Chem. 2003;278:46750-46759.
Electrophoresis
Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was performed with Laemmli's procedure using a 12.5% separating gel under reduced conditions. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970;227:680-685.
Quantification of Recombinant TMD Proteins
TMD protein concentration was determined using the BCA kit (Pierce) with BSA as standard.
Sequence Analysis
Amino-acid sequence determinations were performed automatically by Edman degradation using a Model 477A sequencer (Applied Biosystems).
Cell Cultures
Endothelial cells were isolated from human umbilical cord veins with previously described methods and cells from the second passage were used in all experiments. Shi G Y., et al., Plasmin and the regulation of tissue-type plasminogen activator biosynthesis in human endothelial cells. J. Biol Chem.
Cell Proliferation Assay
To evaluate the effect of TMD proteins on DNA synthesis in human umbilical vein endothelial cells (HUVECs), a 5-bromo-2′-deoxyuridine (BrdU) incorporation assay were performed with a commercial quantification kit (Roche Diagnostic GmbH) and manufacturer's protocol. Mitogenic effects induced by TMD proteins were also determined in the presence of polyclonal rabbit anti-TMD23 antibody (prepared by GlycoNex, Inc., Taiwan) that was purified by protein G-Sepharose column (Amersham Pharmacia Biotech AB).
It showed that TMD2 and TMD23 stimulated DNA synthesis in HUVECs and TMD23 had higher mitogenic activity than TMD2 (
Chemotaxis Assay
The chemotactic motility of HUVECs was assayed using Transwell (Costar) with 6.5-mm diameter polycarbonate filters (8-μm pore size). The lower filter surface was coated with 10 μg gelatin (Sigma-Aldrich). Various concentrations of TMD23 in the absence or presence of 10 mg/mL polyclonal rabbit anti-TMD23 antibody or 10 ng/mL vascular endothelial growth factor (VEGF) (R & D Systems, Inc.) in M199 were placed in the lower wells. One hundred mL cell suspension containing 1×105 cells was loaded into each of upper well. The chamber was incubated at 37° C. for 4 h. Cells were fixed with methanol and stained with 4′,6′-diamidino-2-phenylindole (DAPI). Chemotaxis was quantified with optical microscopy (Leica) at 100× magnification using MetaMorph imaging software (Universal Imaging Corporation) by counting the cells that migrated to the filter's lower side. Five random fields in each well were counted. Each sample was assayed in triplicate, and assays were repeated three times. To determine the role of extracellular signal-regulated kinase 1/2 (ERK1/2), p38 mitogen-activated protein kinase (MAPK), and phosphatidylinositol 3-kinase (PI3 kinaseyprotein kinase B/Akt (Akt) in TMD23-mediated cell migration, assays were performed in the presence of U0126, a MAPK/ERK kinase (MEK) inhibitor, SB 203580, a p38 MAPK inhibitor, and LY 294002, a PI3 kinase inhibitor. Cell viability as determined by trypan blue staining was not altered by treatment. U0126 was purchased from Promega, with SB 203580 and LY 294002 from Calbiochem.
TMD23 Induces HUVEC Migration. The effect of TMD23 on migration of HUVECs was evaluated using the Transwell assay. As shown in
HUVECs were cultured to confluence in a 6-cm-diameter dish and incubated in M199 containing 1% FBS for 18 h. Cells were washed and incubated with serum-free M199 for 6 h, and treated with the indicated concentration of TMD23. Cell lysates were separated by SDS-PAGE, and the levels of phospho-ERK1/2 (Tyr 204) and total ERK1/2, phospho-p38 (Thr-180/Tyr-182) and p38, phosphor-Akt (Ser-473) and Akt, and phospho-eNOS (Ser-1173) and eNOS were detected by Western blotting with specific antibodies (Cell Signaling Technology).
TMD23 Induces ERK1/2, p38 MAPK, Akt, and eNOS Phosphorylation. The ERK pathway, which is activated by many growth factors, may mediate endothelial-cell proliferation and migration. It has also been reported that p38 MAPK activation by VEGF mediates actin reorganization and cell migration in HUVECs, and thus may be an important regulator of angiogenesis. Furthermore, it has been demonstrated that the PI3 kinase/Akt/eNOS signaling axis plays critical roles in angiogenesis. Kawasaki K., et al Activation of the phosphatidylinositol 3-kinase/protein kinase Akt pathway mediates nitric oxide-induced endothelial cell migration and angiogenesis. Mol Cell Biol. 2003;23:5726-5737. To validate whether ERK1/2, p38, Akt, and eNOS activity are involved in TM-induced HUVEC proliferation and migration, the effect of TMD23 on ERK1/2, p38, Akt, and eNOS phosphorylation were studied by Western blotting. As shown in
HUVECs were seeded at 80% confluence on 100-mm dishes in M199 medium. After 24 h, cells were rinsed with serum-free M199 twice and incubated in serum-free M199 with various concentrations of TMD23, VEGF (10 ng/mL), or PMA (70 ng/mL) for 12 h. The conditioned medium containing 5 mg secreted proteins was analyzed by gelatin- and casein-based zymographies. Lee O H, et al., Identification of angiogenic properties of insulin-like growth factor II in in vitro angiogenesis models. Brit J Cancer. 2000;82:385-391. The digested area appeared clear on a blue background, indicating the location of matrix metalloproteinase (MMP) activity. To further verify MMP caseinolytic activity, the casein-containing gel was incubated with incubation buffer containing 10 mM EDTA, an inhibitor of MMPS, to observe disappearance of the clear band. Plasminogen activator inhibitor (PAI) activity was measured by titrating samples with increasing amounts of tissue-type plasminogen activator (t-PA) (Boehringer Ingelheim) into a fixed volume of endothelial-cell-conditioned media. Shi GY., et al., Plasmin and the regulation of tissue-type plasminogen activator biosynthesis in human endothelial cells. J Biol Chem. 1992;267:19363-19368.
MMPs secreted by various cells including endothelial cells are responsible for degradation of extracellular matrix components, thus facilitating cell migration. Because endothelial-cell migration was stimulated by TMD23. Gelatin and casein zymographies were performed to examine the activities of MMP-1, MMP-2, MMP-3, and MMP-9. TMD23 had no significant effect on expression of MMP-2 and MMP-9, but stimulated expression of MMP-1/3 as shown by gelatin zymography (
Because MMP and plasminogen activator (PA)-plasmin systems play important roles in angiogenesis, we measured the PAI activity of conditioned TMD23-treated cell medium. PAI activity in conditioned media of HUVECs treated with 100 or 1000 ng/mL TMD23, 70 ng/mL PMA, or 10 ng/mL VEGF for 12 h decreased significantly (
Growth-factor-reduced Matrigel (BD Biosciences) was thawed at 4° C. A total of 200 mL Matrigel was added to wells of a 48-well plate and polymerized 30 min at 37° C. Two hundred mL cell suspension containing various concentrations of VEGF, TMD23, or TMD23 in the presence of polyclonal rabbit anti-TMD23 antibody (10 mg/mL) or various signaling pathway inhibitors were plated onto Matrigel-coated wells at a density of 2.4×104 cells/well in M199 containing 10% FBS for 24 h at 37° C. in a 5% CO2 humidified atmosphere. Each concentration was tested in triplicate in the same plate, with wells photographed using a Leica camera (40× magnification). Tube length was quantified by measuring tubes in five randomly chosen fields from each well using MetaMorph software. Experiments were repeated at least three times.
TMD23 Promotes Vascular Tube Formation on Matrigel
Control culture developed small amounts of tube formation and incomplete networks within 24 h (
The rat corneal assay was performed as described elsewhere. Uniformly sized pellets of Hydron (polyhydroxyethylmethacrylate [poly HEMA]) (Sigma-Aldrich) containing either TMD23 or basic fibroblast growth factor (bFGF) and sucralfate (Sigma-Aldrich) were implanted into the rat corneal stroma adjacent to the temporal limbus. Briefly, sterile-saline suspensions containing 5-40 mg TMD23 or bFGF plus 10 mg sucralfate were made and speed vacuumed for 5 min; 12% Hydron in ethanol was added as described. The suspension was deposited onto an autoclaved, sterilized, 15-mm×15-mm nylon mesh. In some preparations, polyclonal anti-TMD23 IgG (10 mg/mL) was included during pellet preparation to specifically inhibit TMD23 activity. The eyes of Sprague Dawley rats (250-300 g) were topically anesthetized and a single pellet was inserted into a surgically created pocket in the corneal stroma. Corneas were examined daily with a dissecting microscope for up to 24 days for capillary growth. Maximal vessel length, clock hour, and vascular areas of corneas of all rats were measured with a slit-lamp stereomicroscope.
TMD23-induced Angiogenesis in Rat Corneas
Samples were implanted in a vascular rat cornea to allow blood vessel ingrowth from the limbus. Control pellets without growth factors did not induce an angiogenic response (
To assess angiogenic effects in vivo, growth-factor-reduced liquid Matrigel (0.5 mL) containing heparin (60 U/mL) and various concentrations of TMD23, bFGF or VEGF was subcutaneously injected into FVB mice near the abdominal midline. Matrigel with PBS plus heparin served as the negative control, with bFGF and VEGF plus heparin as the positive control. Four days after injection, mice were sacrificed and Matrigel plugs were surgically removed. For macroscopic analysis of angiogenesis, hemoglobin content in Matrigel was measured using Drabkin reagent 525 (Sigma-Aldrich). For histological analysis, rat anti-mouse CD31 (PECAM-1, platelet endothelial cell adhesion molecule-1) antibody (Pharmingen) was used. The same specimens were used for hematoxylin and eosin (H&E) staining.
TMD23 Induces Angiogenesis in the Matrigel Plug Assay in Vivo
To further evaluate TMD23 angiogenesis effects in vivo, liquid Matrigel containing various amounts of TMD23 (10,100, or 1,000 ng), bFGF (250 ng), or VEGF (50 ng) plus heparin (30 U), or heparin alone was injected into FVB mice.
Histological examinations revealed cellular invasion. Capillary-like structures were found in Matrigel plugs with TMD23 and heparin (
To assess TMD23 effects on tumor neoangiogenesis, 1×106 human melanoma A2058 cells (ATCC CRL-11147) were mixed with Matrigel (0.5 mL) containing heparin (60 U/mL) and various concentrations of TMD23 and subcutaneously injected into athymic nude mice. Matrigel plugs were harvested after 18 days and frozen in liquid nitrogen. Blood vessels were detected by immunohistochemistry using anti-CD31 antibody as described above. Microvessel density (MVD) was calculated by counting six highly vascularized fields in each section (200× magnification; MetaMorph software).
TMD23 Stimulating Neoangiogenesis in Tumors
Angiogenesis is an essential step for many physiological and pathological processes, TMD23 effects on tumor neoangiogenesis were assessed. A2058 cells were mixed in Matrigel supplemented with TMD23 and heparin and injected subcutaneously into athymic nude mice. The tumor size developed by Matrigel plugs containing A2058 cells and TMD23 (or bFGF) was bigger than that for A2058 cells alone (
Neovascularization encompasses both angiogenesis and vasculogenesis. Vasculogenesis is defined as creation of blood vessels by differentiation of endothelial precursor cells (EPCs), whereas angiogenesis represents formation of new capillaries from pre-existing mature endothelial cells, a process including migration and proliferation of endothelial cells, extracellular matrix degradation, and capillary tube formation. Many molecules have been identified that induce vessel formation both in vitro and in vivo including acidic FGF, bFGF, transforming growth factor (TGF)-a, TGF-b, hepatocyte growth factor (HGF), tumor necrosis factor-a, angiogenin, interleukin-8, and angiopoietins. Conversely, thrombospondin, platelet factor-4, angiostatin, arresten, restin, endostatin, and tumstatin have been identified as important endogenous anti-angiogenic molecules. Protease cleavage of collagen types IV, XV, and XVIII generates arresten (and tumstatin), restin, and endostatin, respectively, while plasminogen proteolysis yields angiostatin. Several other molecules and their proteolytic cleavage products have the potential to exert opposing effects on angiogenesis. For example, certain parental proteins including prolactin, growth hormone, and placental lactogen can promote angiogenesis. However, after proteolytic processing the same proteins generate peptide fragments that have anti-angiogenic properties. Corbacho A M., et al., Roles of prolactin and related members of the prolactin/growth hormone/placental lactogen family in angiogenesis. J Endocrin. 2002;173:219-238. The present invention provides a novel angiogenic-promoting function of TM domains 2 and 3. Recombinant TM fragment TMD23 can stimulate endothelial migration, proliferation, and tube formation in vitro, a process that may be mediated via phosphorylation of ERK1/2, p38, Akt, and eNOS. It has been found that TMD23 stimulates endothelial-cell expression of MMPs and PAs that mediate extracellular proteolysis, leading to endothelial-cell invasion and migration during the early stages of angiogenesis. Using a murine Matrigel model and rat corneal neovascularization assays, it has been found that a TMD23-dependent induction of angiogenesis in vivo. It suggests that TMD23 could become a novel therapeutic agent for some ischemia-related diseases.
The D3 of TM is rich in Ser and Thr, which can support post-translational attachment of a chondroitin sulfate moiety. Bourin M C., et al., Isolation and characterization of the glycosaminoglycan component of rabbit thrombomodulin proteoglycan. J Biol Chem. 1990;265:15424-15431. The present invention demonstrates that TMD23 was more effective than TMD2 in stimulating proliferation of HUVECs (
During the course of the murine angiogenesis assays, it has been found that Matrigel supplemented with either TMD23, bFGF, or VEGF produced a variable angiogenic reaction, but that the magnitude of the reaction was considerably greater in gels that were supplemented with both angiogenic factors and heparin. These results are consistent with previous findings. Matrigel, a solution of basement membrane proteins isolated from the Engelbreth-Holm-Swarm tumor, can serve as a vehicle for slow release of angiogenic factors, with heparin promoting the binding, modulation, and sustained release of these factors. Passaniti A., et al., A simple quantitative method for assessing angiogenesis and antiangiogenic agents using reconstituted basement membrane, heparin, and fibroblast growth factor. Lab Invest. 1992;67:519-528. However, heparin is not necessary to obtain the angiogenic response in the rat corneal neovascularization assay because the combination of angiogenic factors with sucralfate and incorporation into Hydron achieves stabilization of angiogenic factors and sustains release over a prolonged time. Kenyon B M., et al., A model of angiogenesis in the mouse cornea. Invest Ophthalmol Vis Sci. 1996;37:1625-1632.
It has been demonstrated that cell surface proteoglycan syndecan-4 participates in mediating the effects of FGF-2 on cell function. It suggests that the biological activities of TM domains could be mediated by binding of the peptide to its specific site on the cell surface. However, TM domains interact with other membrane molecules such as syndecan or synectin which can mediate or trigger the effects of TM on cell function, as in the case of FGF-2. Therefore, it is possible that TM domains act indirectly.
Expression of TM proteins has been documented in a myriad of tumors and cell lines. An inverse relationship of TM expression and rate of tumor-cell proliferation has been reported in hepatocellular carcinoma, ovarian carcinoma, and esophageal squamous carcinoma. It suggests that various soluble TM fragments released from the cell surface by in situ proteolytic enzymes including PAs-plasmin, elastase, and MMPs may function as paracrine substances to modulate angiogenesis in tumors. Soluble forms of TM derived from the molecule's extracellular domain have been found in the plasma and urine of healthy subjects, suggesting that it is cleaved under physiological conditions. The increase of plasma TM fragments in patients with various diseases suggests that TM release from endothelial cells is accelerated by proteolytic activity generated on the surface of the endothelium. The proteases responsible for TM shedding are still unknown, although neutrophil-derived enzymes including elastase, proteinase-3, and cathepsin G have been implicated.
In the present invention, it demonstrated the TMD23 can promote angiogenesis in vitro and in vivo. In the in vitro Matrigel assay, TMD23 could promote the morphological differentiation of HUVECs to form tube-like structures with well-connected network. Therefore, TMD23 could induce vasculogenesis.
This application claims priority of U.S. Provisional Application Ser. No. 60/641,213 filed on Jan. 3, 2005.
| Number | Date | Country | |
|---|---|---|---|
| 60641213 | Jan 2005 | US |
