Alteration Of Heparan Sulfate Research Articles

Hemolysis is associated with altered heparan sulfate of the endothelial glycocalyx and with local complement activation in thrombotic microangiopathies

The complement system plays a key role in the pathophysiology of kidney thrombotic microangiopathies (TMA), as illustrated by atypical hemolytic uremic syndrome. But complement abnormalities are not the only drivers of TMA lesions. Among other potential pathophysiological actors, we hypothesized that alteration of heparan sulfate (HS) in the endothelial glycocalyx could be important. To evaluate this, we analyzed clinical and histological features of kidney biopsies from a monocentric, retrospective cohort of 72 patients with TMA, particularly for HS integrity and markers of local complement activation. The role of heme (a major product of hemolysis) as an HS-degrading agent invitro, and the impact of altering endothelial cell (ECs) HS on their ability to locally activate complement were studied. Compared with a positive control, glomerular HS staining was lower in 57 (79%) patients with TMA, moderately reduced in 20 (28%), and strongly reduced in 37 (51%) of these 57 cases. Strongly reduced HS density was significantly associated with both hemolysis at the time of biopsy and local complement activation (C3 and/or C5b-9 deposits). Using primary endothelial cells (HUVECs, Glomerular ECs), we observed decreased HS expression after short-term exposure to heme, and that artificial HS degradation by exposure to heparinase was associated with local complement activation. Further, prolonged exposure to heme modulated expression of several key genes of glycocalyx metabolism involved in coagulation regulation (C5-EPI, HS6ST1, HS3ST1). Thus, our study highlights the impact of hemolysis on the integrity of endothelial HS, both in patients and in endothelial cell models. Hence, acute alteration of HS may be a mechanism of heme-induced complement activation.

MO071: Hemolysis is Associated with Altered Heparan Sulfate of Endothelial Glycocalyx And with Local Complement Activation in Thrombotic Microangiopathies

Abstract BACKGROUND AND AIMS The complement system plays a key role in the pathophysiology of renal thrombotic microangiopathies (TMA), as illustrated by atypical hemolytic uremic syndrome (HUS) characterized by a high rate of mutations in complement proteins. However, complement abnormalities are not fully sufficient to drive to TMA lesions. Among other suspected pathophysiological actors, we hypothesize that the alteration of heparan sulfate (HS) of endothelial glycocalyx—a cell membrane glycoaminoglycans and glycoproteins matrix able of regulating complement by binding factor H—may play a significant role. METHOD We recorded clinical and histological features in native kidney biopsies from a monocentric retrospective cohort of 72 TMA-patients. Glomerular HS integrity and local complement activation markers (C3 and C5b9 staining) were analyzed by immunofluorescence (IF) and immunohistochemistry. Using an in vitro endothelial cells model (human umbilical vein endothelial cells, HUVECs), we studied the impact of heme exposure (30’, 37°C)—a major product of hemolysis—on endothelial HS integrity by IF and flow cytometry (FCM). Heme-induced HS disruption was studied by slot blot of HS core-protein Syndecan (SDC) 1 and 4 in cells supernatant. Artificial enzymatic degradation of HS was achieved after exposure of HUVECs to a combination of heparinases I, II and III (30', 37°C), and we studied its effect on the ability of the HS-free cell membrane to locally activate complement by analyzing C3b deposition by IF and FCM, and the release of complement anaphylatoxins C3a and soluble attack complex membrane s-C5b9 in the cell supernatants by ELISA. The HS remodeling process induced by heme exposure (16 h, 37°C) were indirectly analyzed via a transcriptional profiling of the expression of the key enzymes involved in HS synthesis by RT–qPCR. RESULTS Glomerular HS staining decreased in 57 TMA-patients (79%), moderately (2+) or strongly (0–1+) in 20 (28%) and 37 (51%) cases, respectively. Reduced HS density was significantly associated with hemolysis at the time of biopsy (P = 0.008) and local complement activation (C3d and/or C5b-9 deposits, P < 0.001). Hemolysis at the time of biopsy was associated with more severe glomerular TMA lesions (P = 0.025), the presence of vascular thrombi (P = 0.007) and local complement activation (C3d and/or C5b-9 deposits, P = 0.006). On endothelial cells (HUVECs), we confirmed that HS expression was decreased after heme exposure in IF and FCM (P < 0.01). Coherently, HS core-proteins SDC-1 and 4 were significantly increased in HUVECs supernatant (P < 0.05), suggesting a negative heme-driven impact on HS integrity. Artificial HS degradation increased C3b membrane deposition on HUVECs on average ∼ 3 fold by FCM and ∼ 10 by IF, and we observed an increased release of complement C3a and s-C5b9 in cells supernatant (P < 0.05). Finally, we showed that prolonged exposure to heme induced a decreased of genic expression of C5-Epimerase and an increase in 6-OST1 and 3-OST1 expression (P < 0.05), the very three crucial genes of HS remodeling specifically and sequentially involved in the generation of anti-thrombin pattern, the well-known key regulator of the HS/heparin-driven inhibition of coagulation. CONCLUSION This study highlights the impact of hemolysis on the integrity of endothelial glycocalyx HS, both in kidney biopsy of TMA-patients and in an in vitro endothelial cell model, and suggests that acute alteration of HS is a mechanism of heme-induced complement activation. The endothelial glycocalyx appears then to be an important key in the understanding of TMA and could lead to the development of therapeutics targeting its preservation and/or reconstitution, in order to better control the disease.

Use of flow cytometry for characterization and fractionation of cell populations based on their expression of heparan sulfate epitopes.

The ability to characterize alterations in heparan sulfate (HS) structure during development or as a result of loss or mutation of one or more components of the HS biosynthetic pathway is essential for broad understanding of the effects these changes may have on cell/tissue function. The use of anti-HS antibodies provides an opportunity to study HS chain composition in situ, with a multitude of different antibodies having been generated that recognize subtle differences in HS patterning, with the number and positioning of sulfate groups influencing antibody binding affinity. Flow cytometry is a valuable technique to enable the rapid characterization of the changes in HS-specific antibody binding in situ, allowing multiple cell types to be directly compared. Additionally fluorescent-activated cell sorting (FACS) allows fractionation of cells based on their HS-epitope expression.

Alteration of heparan sulfate 2‐O‐sulfation in endothelial cells enhances neutrophil infiltration in mice

Inactivation of the enzyme N‐acetylglucosamine N‐deacetylase–N‐sulfotransferase‐ 1 (Ndst1) in endothelial cells results in diminished overall sulfation of heparan sulfate and reduced neutrophil infiltration in various inflammation models. In this report, we examined the contribution of uronic acid 2‐O‐sulfation to neutrophil infiltration by inactivation of uronyl 2‐O‐sulfotransferase (Hs2st) in endothelial cells. Surprisingly, deletion of Hs2st had the opposite effect of deleting Ndst1, resulting in accentuated neutrophil trafficking in acute inflammatory models. Enhanced neutrophil infiltration was partially due to reduced rolling velocity in vitro and in vivo, correlating with stronger binding of L‐selectin to mutant endothelial cells. Hs2st‐deficient endothelial cells also displayed greatly enhanced binding of interleukin‐8. The gain‐of‐function phenotype in Hs2st‐deficient mice apparently results from enhanced glucosamine N‐sulfation and 6‐O‐sulfation that occurs with inhibition of 2‐O‐sulfation. Thus, specific sulfation patterns of endothelial heparan sulfate can have distinct effects on neutrophil recruitment during inflammation, a property that might be exploited for treating inflammatory disorders.

Epigenetics: methylation‐associated repression of heparan sulfate 3‐O‐sulfotransferase gene expression contributes to the invasive phenotype of H‐EMC‐SS chondrosarcoma cells

Heparan sulfate proteoglycans (HSPGs), strategically located at the cell-tissue-organ interface, regulate major biological processes, including cell proliferation, migration, and adhesion. These vital functions are compromised in tumors, due, in part, to alterations in heparan sulfate (HS) expression and structure. How these modifications occur is largely unknown. Here, we investigated whether epigenetic abnormalities involving aberrant DNA methylation affect HS biosynthetic enzymes in cancer cells. Analysis of the methylation status of glycosyltransferase and sulfotransferase genes in H-HEMC-SS chondrosarcoma cells showed a typical hypermethylation profile of 3-OST sulfotransferase genes. Exposure of chondrosarcoma cells to 5-aza-2'-deoxycytidine (5-Aza-dc), a DNA-methyltransferase inhibitor, up-regulated expression of 3-OST1, 3-OST2, and 3-OST3A mRNAs, indicating that aberrant methylation affects transcription of these genes. Furthermore, HS expression was restored on 5-Aza-dc treatment or reintroduction of 3-OST expression, as shown by indirect immunofluorescence microscopy and/or analysis of HS chains by anion-exchange and gel-filtration chromatography. Notably, 5-Aza-dc treatment of HEMC cells or expression of 3-OST3A cDNA reduced their proliferative and invading properties and augmented adhesion of chondrosarcoma cells. These results provide the first evidence for specific epigenetic regulation of 3-OST genes resulting in altered HSPG sulfation and point to a defect of HS-3-O-sulfation as a factor in cancer progression.

Alterations in Heparan Sulfate in the Vessel in Response to Vascular Injury in the Mouse

Heparan sulfate (HS) is ubiquitous throughout the human body. The backbone of HS is composed of many types of sugars. HS serves as a docking site for a vast array of protein ligands. Recent evidence suggests a unique diversity in HS structure that alters protein binding and protein function. This diversity in HS structure has been overlooked till now. The goal of this study was to determine whether femoral artery wire injury modified HS structure. Femoral artery wire injury was performed in 16-week-old male C57BL6 mice. Transcript levels of a panel of enzymes that regulate HS fine structure, including N-deacetylase-N-sulfotransferases (Ndst) 1 and 2, exostoses (Ext) 1 and 2, C5 epimerase, and 2-O and 6-O sulfotransferases, were quantified with real-time quantitative polymerase chain reaction at 7 and 14 days post injury. All enzymes showed significant alterations in messenger RNA expression in response to injury. Ndst1, the most prevalent isoform, exhibited a 20-fold increase in response to injury. Injury induced significant alterations in fine structure specially increases in N-sulfated disaccharides at 14 days post injury. Vascular injury invokes transcriptional regulation of the enzymes that regulate HS structure, as well as changes in the pattern of HS chains in the vessel wall 14 days post injury. These findings may be important as the foundation of altered growth factor and chemokine binding in the process of vascular remodeling.

Heparan sulfate stimulates extracellular matrix protein synthesis by mesangial cells in culture.

Heparan sulfate, a glycosaminoglycan which is a component of the glomerular basement membrane and the mesangial matrix, has been demonstrated to inhibit mesangial cell growth. The present studies investigated the effects of heparan sulfate on extracellular matrix protein synthesis by cultured rat mesangial cells. Mesangial cells were metabolically labeled with 35S-methionine and specific extracellular matrix proteins were immunoprecipitated from supernatant and cellular fractions. The following extracellular proteins were assayed: type I collagen, type IV collagen, type V collagen, laminin and fibronectin. Western blotting was performed to demonstrate the specificity of the immunoprecipitates. Heparan sulfate decreased cell number by 23.5%, p < 0.05, while chondroitin sulfate, another glycosaminoglycan, increased cell number by 11.5%, p < 0.05. Heparan sulfate significantly increased type V collagen and laminin in the cellular material and significantly increased laminin and fibronectin in the supernatant material. Chondroitin sulfate had no effect on matrix protein synthesis. Thus, heparan sulfate has effects on both mesangial cell growth and synthesis of matrix proteins and alterations in heparan sulfate may play a role in the development of glomerulosclerosis.

Altered synthesis of heparan sulfate proteoglycans at low sulfate concentration

The structural alterations in heparan sulfate produced by sulfate deprivation were studied in cell cultures of the Engelbreth-Holm-Swarm tumor. Tumor cells were labeled in vitro with [ 3H]glucosamine and/or [ 35S]sulfate in media containing either 300 μ m MgSO 4 or no added carrier sulfate, and the newly synthesized proteoglycans isolated by chromatography on DEAE-Sephacel. The proteoglycans isolated from low sulfate cultures showed a reduced affinity for the column eluting at lower salt concentrations compared with the proteoglycans isolated from cultures containing sulfate, suggesting that the former were undersulfated. Analysis of the isolated heparan sulfate side chains indicated that two pools of heparan sulfate were present which differed in their degree of sulfation. Both pools were synthesized by both high sulfate and low sulfate cultures, but the highly sulfated pool was the predominant form produced in sulfate containing cultures, while the undersulfated pool was the predominant form synthesized in low sulfate cultures. The more sulfated pool contained more N-sulfate than the less sulfated pool. Few if any free amino groups were detected in either pool, suggesting that the initial deacetylation step in the biosynthesis of heparan sulfate is tightly coupled to the N-sulfation step in the cells.

Age-related changes in sulfation of basement membrane glycosaminoglycans

Alterations in heparan sulfate, the principal glycosaminoglycan species in glomerular basement membrane, have been proposed as contributory to permeability changes in nephropathic disorders. In the present study, the extent of sulfation of glycosaminoglycans isolated from pronase digests of human glomerular basement membrane, obtained from subjects 15–89 years old, was examined. The sulfate to hexuronic acid ratio significantly diminished as a function of increasing age. These results suggest that decreased sulfation of glycosaminoglycans and consequent reduction in net negative charge alters permselective properties of the glomerular basement membrane with aging.