Shedding of heparan sulfate proteoglycan by stimulated endothelial cells: Evidence for proteolysis of cell-surface molecules

Abstract

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.

The membrane attack complex of complement contributes to plasmin‐induced synthesis of platelet‐activating factor by endothelial cells and neutrophils

“…It is well known that the interaction of MAC with EC can result in a number of pro‐inflammatory actions and it triggers changes similar to those induced by cytokines. MAC induces, within minutes, the formation of intracellular gaps, 27 the expression of P‐selectin 28 and the activation of proteinases that cleave and release heparan sulphate proteoglycan from EC surfaces 29 . In addition, MAC stimulates, over a period of hours, the up‐regulation of tissue factor, cyclooxygenase‐2 and chemokines 22,30–32 .…”

Inflammation- and ischemia-induced shedding of venular glycocalyx

“…Syndecan-1 has been shown to be shed after treatment by thrombin and epidermal growth factors, and its shedding can be mediated by G protein-coupled receptors (GPCRs), protein tyrosine kinase receptors, and a tissue inhibitor of the metalloproteinase-3-sensitive pathway (9,34). HS is shed after the treatment of cultured ECs with complement (13) and endotoxin (5). Prior studies suggested that stimulation of the GPCRs with N-formylmethionyl-leucyl-phenylalanine (fMLP) results in enhanced WBC adhesion in postcapillary venules in mesentery (rat) because of shedding of HS proteoglycans that in turn exposes greater amounts of ICAM-1 to facilitate adhesion of the CD11a,b/CD18 receptor on the leukocyte (20).…”

Surface modification of pig endothelial cells with a branched heparin conjugate improves their compatibility with human blood

“…Due to the heterogeneity of its composition and its location, the glycocalyx plays a critical role as a protective barrier in maintaining vessel wall permeability and modulating blood cell-vessel wall interactions 2–5 . The transplant process activates and injures graft endothelium, causing shedding of the glycocalyx and loss of regulators, creating a pro-inflammatory, pro-coagulant environment 6 . While the graft eventually recovers, this early insult can contribute to primary non-function and cause long-term detrimental effects.…”