Regulation of in vitro capillary tube formation by anti-integrin antibodies.

Human endothelial cells are induced to form an anastomosing network of capillary tubes on a gel of collagen I in the presence of PMA. We show here that the addition of mAbs, AK7, or RMAC11 directed to the alpha chain of the major collagen receptor on endothelial cells, the integrin alpha 2 beta 1, enhance the number, length, and width of capillary tubes formed by endothelial cells derived from umbilical vein or neonatal foreskins. The anti-alpha 2 beta 1 antibodies maintained the endothelial cells in a rounded morphology and inhibited both their attachment to and proliferation on collagen but not on fibronectin, laminin, or gelatin matrices. Furthermore, RMAC11 promoted tube formation in collagen gels of increased density which in the absence of RMAC11 did not allow tube formation. Neither RMAC11 or AK7 enhanced capillary formation in the absence of PMA. Lumen structure and size were also altered by antibody RMAC11. In the absence of antibody the majority of lumina were formed intracellularly from single cells, but in the presence of RMAC11, multiple cells were involved and the lumen size was correspondingly increased. Endothelial cells were also induced to undergo capillary formation in fibrin gels after PMA stimulation. The addition of anti-alpha v beta 3 antibodies promoted tube formation in fibrin gels and inhibited EC adhesion to and proliferation on a fibrinogen matrix. The enhancement of capillary formation by the anti-integrin antibodies was matrix specific; that is, anti-alpha v beta 3 antibodies only enhanced tube formation on fibrin gels and not on collagen gels while anti-alpha v beta 1 antibodies only enhanced tubes on collagen and not on fibrin gels. Thus we postulate that changes in the adhesive nature of endothelial cells for their extracellular matrix can profoundly effect their function. Anti-integrin antibodies which inhibit cell-matrix interactions convert endothelial cells from a proliferative phenotype towards differentiation which results in enhanced capillary tube formation.

Exposure of the cryptic Arg-Gly-Asp sequence in thrombospondin-1 by protein disulfide isomerase.

Thrombospondin-1 is a matrix protein that inhibits proliferation, motility and sprouting of endothelial cells in vitro and angiogenesis in vivo. One mechanism by which thrombospondin-1 may influence endothelial cell biology is through interaction with the endothelial cell alphav beta3 integrin receptor. This interaction is mediated via a cryptic Arg-Gly-Asp sequence in the C-terminal Ca2+-binding region of thrombospondin-1. Exposure of the Arg-Gly-Asp sequence is controlled by disulfide interchange events in the Ca2+-binding loops and C-globular domain. Limited reduction of thrombospondin-1 by dithiothreitol exposes the Arg-Gly-Asp sequence which can bind to the alphav beta3 integrin receptor and support endothelial cell spreading (X. Sun, K. Skorstengaard, D.F. Mosher, J. Cell Biol. 118 (1992) 693-701). Our aim was to identify possible physiological reductants that can mediate Arg-Gly-Asp exposure. We now report that protein disulfide isomerase, which is known to catalyze disulfide interchange in thrombospondin-1 and change its enzyme inhibitory properties and its binding to monoclonal antibodies, was secreted by bovine aortic endothelial cells and deposited on the cell surface. There was an average of approximately 2.2 fg of protein disulfide isomerase on the surface of a bovine aortic endothelial cell. Treatment of thrombospondin-1 with purified protein disulfide isomerase enhanced adhesion of endothelial cells to thrombospondin-1 in an Arg-Gly-Asp-dependent manner through the alphav beta3 integrin receptor and supported cell spreading. Both Ca2+-depleted and Ca2+-replete thrombospondin-1 were substrates for protein disulfide isomerase. These results suggest that endothelial cell derived protein disulfide isomerase may regulate Arg-Gly-Asp-dependent binding of thrombospondin-1.

Identification of monoclonal antibodies that recognize different disulfide bonded forms of thrombospondin 1.

Thrombospondin 1 (TSP1) is a multidomain glycoprotein from platelets and cells which functions in cell-cell and cell-matrix interactions. The structure of TSP1 is regulated by sulfhydryl-disulfide interchange in the carboxy-terminal Ca2(+)-binding loops and globular domain which markedly influence its interaction with cell surface integrins and its inhibition of neutrophil enzymes. We have identified murine monoclonal antibodies that recognized different disulfide-bonded forms of TSP1, made by preparing TSP1 in buffers containing either 0.1 mM or 2 mM Ca2+. Antibody HB8432 recognizes TSP1 prepared in buffers containing either 0.1 or 2 mM Ca2+, while antibodies D4.6 and A65M recognized only TSP1 prepared in buffers containing 0.1 mM Ca2+. The antibodies recognize these different TSP1 preparations either adsorbed to plastic or extracellular matrix. Immunohistochemistry of human rheumatoid synovial tissue using HB8432 resulted in staining of numerous blood vessel walls and matrix cells, while D4.6 and A65M stained a subset of the HB8432 positive blood vessels and only occasionally stained matrix cells. These results suggested that different disulfide-bonded forms of TSP1 were being expressed in different areas of inflamed tissue.

Generation of angiostatin by reduction and proteolysis of plasmin. Catalysis by a plasmin reductase secreted by cultured cells.

Extracellular manipulation of protein disulfide bonds has been implied in diverse biological processes, including penetration of viruses and endotoxin into cells and activation of certain cytokine receptors. We now demonstrate reduction of one or more disulfide bonds in the serine proteinase, plasmin, by a reductase secreted by Chinese hamster ovary or HT1080 cells. Reduction of plasmin disulfide bond(s) triggered proteolysis of the enzyme, generating fragments with the domain structure of the angiogenesis inhibitor, angiostatin. Two of the known reductases secreted by cultured cells are protein disulfide isomerase and thioredoxin, and incubation of plasmin with these purified reductases resulted in angiostatin fragments comparable with those generated from plasmin in cell culture. Thioredoxin-derived angiostatin inhibited proliferation of human dermal microvascular endothelial cells with half-maximal effect at approximately 0.2 microg/ml. Angiostatin made by cells and by purified reductases contained free sulfhydryl group(s), and S-carbamidomethylation of these thiol group(s) ablated biological activity. Neither protein disulfide isomerase nor thioredoxin were the reductases used by cultured cells, because immunodepletion of conditioned medium of these proteins did not affect angiostatin generating activity. The plasmin reductase secreted by HT1080 cells required a small cofactor for activity, and physiologically relevant concentrations of reduced glutathione fulfilled this role. These results have consequences for plasmin activity and angiogenesis, particularly in the context of tumor growth and metastasis. Moreover, this is the first demonstration of extracellular reduction of a protein disulfide bond, which has general implications for cell biology.

Lumen formation during angiogenesis in vitro involves phagocytic activity, formation and secretion of vacuoles, cell death, and capillary tube remodelling by different populations of endothelial cells.

BACKGROUND: We have utilised an in vitro model of angiogenesis to investigate the morphological changes which occur during the formation of a lumen in capillary tubes. METHODS AND RESULTS: On collagen 1 gel in the presence of phorbol myristate acetate (PMA) and anti-alpha 2 beta 1 antibody, cell aggregation and alignment takes place within two hours of plating. The initial apparently homogeneous population of endothelial cells (EC) actually display at least three distinct phenotypes. One population, characterised by a phagocytic phenotype, migrated through the gel creating channels and defines the extent of the capillary network. These are later enveloped by a second population of cells characterised by intracellular vacuoles. The ultimate fate of these vacuoles is fusion with the plasma membrane. By 12 hours the original phagocytic cell population undergoes cell death, which morphologically appears apoptotic in nature. A consequence of the secretion of vacuoles and programmed cell death is the extensive remodelling of the capillary tubes, resulting in expansion of the intercellular space into a lumen. The remodelling results in 45% of the EC membrane contacting the lumenal surface at the expense of EC-EC and EC-matrix contact. A third population of cells implant between the EC involved in lumen formation and thus expand the size of the capillary tube. CONCLUSION: Thus, in the formation of a mature multicellular lumen we have identified a number of key events. First, cell-cell contact is essential in order to define the intercellular space. Second, at least three morphologically distinct subpopulations of ECs are involved. Third, vacuole formation and programmed cell death are required for expansion of the intercellular space which ultimately becomes the lumen.

Angiostatin formation involves disulfide bond reduction and proteolysis in kringle 5 of plasmin.

Plasmin is processed in the conditioned medium of HT1080 fibrosarcoma cells producing fragments with the domain structures of the angiogenesis inhibitor, angiostatin, and microplasmin. Angiostatin consists of kringle domains 1-4 and part of kringle 5, while microplasmin consists of the remainder of kringle 5 and the serine proteinase domain. Our findings indicate that formation of angiostatin/microplasmin involves reduction of plasmin by a plasmin reductase followed by proteolysis of the reduced enzyme. We present evidence that the Cys461-Cys540 and Cys511-Cys535 disulfide bonds in kringle 5 of plasmin were reduced by plasmin reductase. Plasmin reductase activity was secreted by HT1080 and Chinese hamster ovary cells and the human mammary carcinoma cell lines MCF-7, MDA231, and BT20 but not by the monocyte/macrophage cell line THP-1. Neither primary foreskin fibroblasts, blood monocyte/macrophages, nor macrovascular or microvascular endothelial cells secreted detectable plasmin reductase. In contrast, cultured bovine and rat vascular smooth muscle cells secreted small but reproducible levels of plasmin reductase. Reduction of the kringle 5 disulfide bonds triggered cleavage at either Arg529-Lys530 or two other positions C-terminal of Cys461 in kringle 5 by a serine proteinase. Plasmin autoproteolysis could account for the cleavage, although another proteinase was mostly responsible in HT1080 conditioned medium. Three serine proteinases with apparent Mr of 70, 50, and 39 were purified from HT1080 conditioned medium, one or more of which could contribute to proteolysis of reduced plasmin.