Alterations in heparan sulfate proteoglycan synthesis and sulfation and the impact on vascular endothelial function

Abstract

The glycocalyx attached to the apical surface of vascular endothelial cells is a rich network of proteoglycans, glycosaminoglycans, and glycoproteins with instrumental roles in vascular homeostasis. Given their molecular complexity and ability to interact with the intra- and extracellular environment, heparan sulfate proteoglycans uniquely contribute to the glycocalyx's role in regulating endothelial permeability, mechanosignaling, and ligand recognition by cognate cell surface receptors. Much attention has recently been devoted to the enzymatic shedding of heparan sulfate proteoglycans from the endothelial glycocalyx and its impact on vascular function. However, other molecular modifications to heparan sulfate proteoglycans are possible and may have equal or complementary clinical significance. In this narrative review, we focus on putative mechanisms driving non-proteolytic changes in heparan sulfate proteoglycan expression and alterations in the sulfation of heparan sulfate side chains within the endothelial glycocalyx. We then discuss how these specific changes to the endothelial glycocalyx impact endothelial cell function and highlight therapeutic strategies to target or potentially reverse these pathologic changes.

Keywords: ACE2, Angiotensin-converting enzyme 2; CLP, cecal ligation and puncture; COVID-19, Coronavirus disease 2019; EXT, Exostosin; EXTL, Exostosin-like glycosyltransferase; FFP, Fresh frozen plasma; FGF, Fibroblast growth factor; FGFR1, Fibroblast growth factor receptor 1; GAG, Glycosaminoglycan; GPC, Glypican; Gal, Galactose; GlcA, Glucuronic acid; GlcNAc, N-actetyl glucosamine; Glycocalyx; HLMVEC, Human lung microvascular endothelial cell; HS, Heparan sulfate; HS2ST, Heparan sulfate 2-O-sulfotransferase; HS3ST, Heparan sulfate 3-O-sulfotransferase; HS6ST, Heparan sulfate 6-O-sulfotransferase; HSPG, Heparan sulfate proteoglycan; HUVEC, Human umbilical vein endothelial cell; Heparan sulfate proteoglycan; LPS, lipopolysaccharide; NDST, N-deacetylase/N-sulfotransferase; SARS-CoV-2, Severe acute respiratory syndrome coronavirus 2; SDC, Syndecan; Sulf, Endosulfatase; Sulfation; Synthesis; TNFα, Tumor necrosis factor alpha; UA, Hexuronic acid; VEGF, Vascular endothelial growth factor; Vascular endothelium; XYLT, Xylosyltransferase; Xyl, Xylose; eGCX, Endothelial glycocalyx; eNOS, Endothelial nitric oxide synthase.

Fig. 1

Heparan sulfate (HS) disaccharide structural modification/sulfation process. Polymerized HS chains attached to core proteoglycans destined for the endothelial surface may undergo several sulfation modifications within the endothelial cell Golgi apparatus prior to apical surface expression. First, N-deacetylase/N-sulfotransferase replaces the acetyl group for a sulfate at the amino linkage to the second carbon of a glucosamine residue. Next, HS 2-O-sulfotransferase may attach a sulfate group to the hexuronic acid residue (either d-glucuronic acid or l-iduronic acid). Glucosamine residues within the various disaccharides may undergo further sulfation by HS 3-O-sulfotransferase and/or 6-O-sulfotransferase.

Fig. 2

Shear stress increases heparan sulfate (HS) expression within the glycocalyx on human primary lung microvascular endothelial cells (HLMVEC), concurrent with increased gene expression of HS-synthesizing enzymes. A) Stacked confocal micrographs of HLMVECs incubated for 48 h in either static conditions (top panel) or 15 dynes/cm² of laminar shear stress (bottom panel) demonstrating staining for HS 10E4 epitope (cells demarcated by β-catenin with DAPI overlay). Scale bar represents 20 µm. B) Relative staining intensity for HS 10E4 epitope on HLMVECs following 48 h of static culture or laminar flow (from 4 representative 20x images). Data are presented as mean ± SEM and analyzed by Mann-Whitney U test. C) Heat map displaying the relative fold change in gene expression for select HS-synthesizing enzymes measured in HLMVECs incubated in static or flow conditions (n = 3 per group) by an nCounter (NanoString, Seattle, WA) digital multiplexed gene expression array. Overall, shear stress promoted increased mRNA expression for xylotransferases, exostosin-like glycosyltransferases, and exostosin 2 while also increasing mRNA expression for most sulfotransferases. Flow conditioning decreased mRNA expression for sulfatases. D) Interestingly, flow conditioning promoted higher expression of GPC1 mRNA while uniformly reducing SDC1-4 mRNA in HLMVECs.

Fig. 3

Cartoon thematically depicting the impact of sulfation on heparan sulfate (HS)-mediated leukocyte trafficking, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infectivity, antithrombin activity, and growth factor signaling at the luminal surface of the vascular endothelium. A) Inflammatory cytokines promote upregulation of N-deacetylase/N-sulfotransferase (NDST) and HS 6-O-sulfotransferase (HS6ST) activity within the Golgi of endothelial cells. This results in increased endothelial glycocalyx (eGCX) expression of proteoglycan HS side chains with N- and 6-O-sulfation of N-acetyl glucosamine residues (represented by a green ring in inset; blue ring represents the hexuronic acid), respectively, that support leukocyte adhesion and transmigration. B) Furin preactivation of SARS-CoV-2 virion spike proteins (represented by the change in spike protein color to yellow) is enhanced by interaction with eGCX HS molecules that are enriched in N- and 3-O-sulfation by NDST and HS 3-O-sulfatotransferases (HS3ST). Following furin preactivation, HS molecules further support virion spike protein adherence to surface angiotensin-converting enzyme 2 (ACE2) receptors that is essential for cell infectivity. C) Antithrombin binding to 3-O-sulfated HS moieties within the eGCX may promote a conformational change that increases its efficiency to inactive thrombin, thereby contributing to coagulation regulation at the endothelial surface. Further, antithrombin binding to 3-O-sulfated HS side chains in the endothelial glycocalyx promotes homeostatic, anti-inflammatory endothelial cell signaling. D) The eGCX harbors fibroblast growth factor 2 (FGF2) near HS regions enriched in 2-O-sulfation by HS 2-O-sulfotransferase (HS2ST) . Moreover, eGCX HS side chains enriched in 6-O-sulfation by HS 6-O-sulfotransferase (HS6ST) synergize cognate receptor binding with FGF2 (and other FGF variants) and vascular endothelial growth factor (VEGF) at the endothelial surface with putative effects on angiogenesis and vascular permeability. For simplicity, the proteoglycan depicted in all panels is syndecan; however, it is currently unknown how sulfation varies within the HS side chains of specific proteoglycans within the eGCX. Figure generated using Biorender. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)