Abstract
Human aortic endothelial cells (HAEC) and human umbilical vein endothelial cells (HUVEC) were labeled with 35SO(4)2- for 48 h. The membrane-associated proteoglycans were solubilized from these monolayers with detergent and purified by ion-exchange chromatography on Mono Q, incorporation in liposomes, and gel filtration. The liposome-intercalated proteoglycans were 125I-iodinated and treated with heparitinase before SDS-polyacrylamide gel electrophoresis. Radio-labeled proteins with apparent molecular masses of 130, 60, 46, 35, and 30 kDa (HAEC) and 180, 130, 62, 43, and 35 kDa (HUVEC) were detected by autoradiography. Further characterization by affinity chromatography on immobilized monoclonal antibodies and by Northern blot analysis provided evidence for the expression of syndecan, glypican, and fibroglycan in human endothelial cells. Most of the heparan sulfate which accumulated in the subendothelial matrix was implanted on a 400-kDa core protein. This protein was immunologically related to perlecan and bound to fibronectin. Binding studies on immobilized antithrombin III suggested that all membrane-associated heparan sulfate proteoglycan forms had the capacity to bind to antithrombin III but that high affinity binding was more typical for glypican. Most of the proteoglycans isolated from the extracellular matrix also bound only with low affinity to antithrombin III. These results imply that glypican may specifically contribute to the antithrombotic properties of the vascular wall.
Highlights
From the Center for Human Genetics and the WCenterfor Thrombosis and Vascular Research, University of Leuuen, B-3000 Leuuen, Belgium
Radiolabeled proteins with apparent molecular masses of 130,60, 46,35, and 30 kDa (HAEC) and 180,130, 62, 43,and 35 kDa (HUVEC) were detected by autoradiographyF. urthecr haracterizationbyaffinity chromatography on immobilized monoclonal antibodfactors (Rapraegeret al., 1991;Yayon et al, 1991),and anchors several enzymes, including lipoprotein lipase to thecell layer (Saxena et al, 1990).The mostextensively studied interaction of heparin and heparan sulfate, is that with antithrombin 111.High affinity binding to this proteinase inhibitor which results in a n anticoagulant effect depends on the presies and by Northern blot analysis provided evidence ence of a specific pentasaccharide sequence in these glycosafor theexpression of syndecan, glypican, and fibrogly-minoglycans (Thunberg et al, 1982)
This proteoglycan is related to perlecan, thperoteoglycan which accumulates in basement membranes, but is not related to thecell surface-associated heparan sulfate proteoglycans of these cells which are characterizedby core proteins of 125 kDa, 80 kDa, 64 kDa, 48 kDa and 35 kDa (Lories et al, 1989)
Summary
Isolation of the Membrane-associated and Matrix-associated Proteoglycans-The membrane-associated heparansulfate proteoglycans from confluent human aortic (HAEC) and human umbilical vein endothelial cells (HUVEC) were metabolically labeled with ""SO:- for 48 h, extracted with Triton X-. HUVEC and 100- and 80-kDa proteins in HAEC (Fig. 6, A the expression of cell surface proteoglycans in and B ) .The 80-kDacore protein was recognizedby mAb 7D8, human vascular endothelial cellswas studied at the an antibody which reacts with a CD44-related CSPG HSPG purified on mAb 2E9, which in -2.6 kb (Fig. 9) These results confirmed the expression of fibroblasts reacts with syndecan and one other HS proteogly- syndecan, glypican, and fibroglycan in vascular endothelial can (Lories et al, 1992), yielded a core protein of 130 kDa cells and suggested that the endothelial and fibroblastic proafter heparitinase digestion. A_lv- cans. isolated from HAEC ( A ) and HUVEC ( R )were labeled with '251.'Nontreated, heparitinase-, and/or chondroitinase ABC-treated samples were analyzed by electrophoresis in SDS-polyacrylamide gradient gels (1.5 X lo3cpm/ lane for HAEC; 5 X lo3cpm/lane for HUVEC) and detected by autoradiography
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