The activation of endothelial cells is thought to contribute to the host response to infection and to the pathogenesis of autoimmune disease. It was recently shown that antibody and complement can activate endothelial cells leading to cleavage and release of heparan sulfate from the cells. We show here that release of heparan sulfate from endothelial cells is mediated by antibody and the complement fragment C5a and that assembly of the membrane attack complex and lysis of endothelial cells is not necessarily involved. These data suggest that the generation of C5a in conditions such as autoimmunity and infection in which anti-endothelial cell antibodies may also be present, might amplify tissue injury by a novel mechanism involving endothelial cell activation and loss of heparan sulfate mediated by antibody and C5a.
Heparan sulfate is rapidly degraded by an endoglycosidase (heparanase) secreted by activated platelets. Since the cleavage and release of heparan sulfate would profoundly alter the local physiology of the endothelium, platelet heparanase activity should be tightly regulated. Consistent with this hypothesis, platelet heparanase was found to degrade endothelial cell heparan sulfate at pH 6.0 but not at pH 7.4, even though 25% of maximum activity was detected at pH 7.4. Loss of heparanase activity occurred rapidly (t1/2 is approximately equal to 20 min) and reversibly at physiologic pH but did not occur at acidic pH (<7.0). Inactivation of heparanase at pH 7.4 did not affect heparin binding and was reversed by 0.5 M NaCl or by heparan sulfate but not by chondroitin sulfate, suggesting inactive heparanase could be tethered on cell surfaces and the function regulated by heparan sulfate. Heparanase was gradually inactivated by trypsin and urokinase (t1/2 = 5 h) but resisted cleavage by leukocyte cathepsin G, leukocyte elastase, plasmin, and thrombin. These findings are consistent with a model in which platelet heparanase is active at the low pH of inflammation but inactive under physiologic conditions preventing inadvertent cleavage of heparan sulfate and loss of physiologic functions of endothelial cells.
Activation of endothelial cells by cytokines and endotoxin causes procoagulant and pro-inflammatory changes over a period of hours. We postulated that the same functional state might be achieved more rapidly by changes in the metabolism of heparan sulfate, which supports many of the normal functions of endothelial cells. We previously found that binding of anti-endothelial cell antibodies and activation of complement on endothelial cells causes the rapid shedding of endothelial cell heparan sulfate. Here we report the biochemical mechanism responsible for the release of the heparan sulfate. Stimulation of endothelial cells by anti-endothelial cell antibodies and complement resulted in the release of 35S-heparan sulfate proteoglycan and partially degraded 35S-heparan sulfate chains. Degradation of the 35S-heparan sulfate chains was not necessary for release since heparin and suramin prevented cleavage of the heparan sulfate but did not inhibit release from stimulated endothelial cells. The 35S-heparan sulfate proteoglycan released from endothelial cells originated from the cell surface and had a core protein similar in size (70.5 kD) to syndecan-1. Release was due to proteolytic cleavage of the protein core by serine and/or cysteine proteinases since the release of heparan sulfate was inhibited 87% by antipain and 53% by leupeptin. Release of heparan sulfate coincided with a decrease of approximately 7 kD in the mass of the protein core and with a loss of hydrophobicity of the proteoglycan, consistent with the loss of the hydrophobic transmembrane domain. The cleavage and release of cell-surface 35S-heparan sulfate proteoglycan might be a novel mechanism by which endothelial cells may rapidly acquire the functional properties of activated endothelial cells.
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