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We report the results of an immunogold electron microscopical analysis on microfibrils from the arterial subendothelium showing that thrombospondin (TSP) is present on 40 nm-diameter structures joining 8-10 nm-diameter microfibrils containing fibrillin. They differ from type VI collagen which forms 3-5 nm-diameter microfibrils. TSP containing microfibrils (TSP-MF) extracted from human umbilical arteries did not contain fibrillin or type VI collagen. Blood platelet interactions with TSP-MF were not modified by anti-fibrillin or anti-type VI collagen antibodies. In situ, vWF was bound to cross-linked microfibrils, at the level of their 40 nm junction, and a double-labeling with the anti-thrombospondin and anti-vWF antibodies was observed. In vitro, vWF binding to TSP-MF was not inhibited by anti-fibrillin or anti-type VI collagen antibodies. These results suggest a structural and functional heterogeneity of microfibrils and emphasize the role of TSP-MF in the thrombogenicity of the subendothelium.

We examined the effects of proinflammatory cytokines on the expression of two extracellular matrix proteins, e.g., thrombospondin (TSP) and fibronectin (FN) b cultured human umbilical vein endothelial cells (HUVECs). Treatment of HUVECs with human recombinant interleukin-1 beta (IL-1 beta) or human tumor necrosis factor-alpha (TNF-alpha) caused a time-and dose-dependent decline in TSP production whereas FN production was not modified. At low concentrations, IL-1 beta and TNF-alpha in combination ha a greater effect than either agent alone. Interferon-gamma (IFN-gamma) was without effect. The decline in TSP synthesis resulted in a decreased secretion of this glycoprotein into the extracellular matrix. Endothelial cell monolayers cultured on porous filters were used to study the polarity of TSP secretion. Approximately two thirds of the synthesized protein was secreted to the apical side medium and one third to the basal side medium and both types of secretion were inhibited to a similar extent by cytokine treatment. Immunoprecipitation experiments revealed no apparent degradation of secreted TSP, either in the apical or in the basal compartment. Treatment of HUVECs with lL-1 beta, either alone or in combination with TNF-alpha, had no significant effect on the steady-state TSP mRNA levels, suggesting a posttranscriptional regulation. Our results indicate that IL-1 beta decreasing TSP deposition and suggest different regulatory mechanisms for the expression of various secreted proteins by endothelial cells.

We report herein the characterization of a mouse monoclonal antibody (Mab) raised against the recombinant NH2-terminal heparin-binding domain (rHBD) of human endothelial cell thrombospondin (TSP). The antibody, a IgG1 (kappa), hereafter referred to as V58A4, reacted with two rHBD, TSPN18 and TSPN28 (i.e. 18 kDa and 28 kDa, respectively) with an affinity constant of 1.33 x 10(-8) M. However, V58A4 failed to recognize native or deglycosylated forms of TSP purified from platelets or endothelial cells, as well as a 25-30 kDa HBD fragment produced by limited proteolysis of native TSP. In contrast, Mab V58A4 was shown to react with larger HBD fragments (50-60 kDa) that were present in platelet or endothelial cell extracts and could be retained on a heparin-Sepharose column at low salt concentrations. These fragments also reacted with MA-II, a mouse Mab (IgG1), which recognizes both rHBD and HBD as well as intact TSP. Thus, V58A4 Mab appears to selectively recognize naturally occurring HBD fragments of TSP and may thus prove to be useful for detecting TSP proteolysis in situ under various physiopathological conditions.

The role of thrombospondin on the adhesion of endothelial cells in primary culture was studied using a serum-free defined medium or thrombospondin-depleted fetal bovine serum. Under these conditions, only 6% of the cells adhered to gelatin-coated dishes, whereas cells adhering to gelatin in the presence of normal fetal bovine serum were considered as 100% adhesion. The percentage of cells attached to fibronectin or thrombospondin-coated dishes in thrombospondin-depleted serum was 66 and 32%, respectively. The addition of purified platelet thrombospondin to thrombospondin-depleted serum increased the adhesion of endothelial cells to gelatin and to thrombospondin, up to 32 and 59%, respectively, and restored the attachment to fibronectin to the same extent as that observed in the presence of normal serum. In contrast to the attachment, the spreading of the adhering cells was not further influenced by the addition of soluble thrombospondin. Subcultured cells did not require any protein for adhering to gelatin substrata. These observations indicate that thrombospondin plays a major role in the adhesion of endothelial cells in primary culture.