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Isolation and Characterization of Tissue-Type Plasminogen Activator–Binding Proteoglycans From Human Umbilical Vein Endothelial Cells
- Thomas Böhm,
- Margarethe Geiger and
- Bernd R. Binder
Arteriosclerosis, Thrombosis, and Vascular Biology Volume 16, Issue 51 May 1996

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Abstract We analyzed the tissue-type plasminogen activator (TPA)–binding proteoglycans (PGs) on human umbilical vein endothelial cells (HUVECs), which were metabolically labeled with [³⁵S]Na₂SO₄. Cell extracts were then prepared and subjected to affinity chromatography on diisopropyl fluorophosphate (DFP)–inactivated TPA–Sepharose 4B. Approximately 6% of the incorporated ³⁵S radioactivity bound to DFP-treated TPA–Sepharose 4B and was eluted with 2 mol/L NaCl. In addition to NaCl, heparin, arginine, and lysine but not glycine, ε-amino-n-caproic acid, or aspartic acid inhibited this binding and eluted the bound ³⁵S radioactivity. Urea-containing polyacrylamide gel electrophoresis of the eluted material consistently revealed two main signals of ³⁵S radioactivity (one with an M_r between 600 000 and 750 000 [PGA] and the other with an M_r between 120 000 and 180 000 [PGC]). Occasionally a less intense signal with an M_r between 340 000 and 440 000 (PGB) was seen. Heparitinase treatment markedly decreased the intensities of both ³⁵S signals (PGA and PGB), and chondroitinases AC and ABC abolished the ³⁵S signal of PGC, indicating that most of the HUVEC-incorporated radioactivity with an affinity for TPA could be attributed to heparan sulfate– and chondroitin sulfate–like structures. Reductive elimination, which was performed to separate the possible glycosaminoglycan moieties from the core proteins, confirmed the PG-like nature of this material and again revealed heparan sulfate and chondroitin sulfate as the major glycosaminoglycan components. We therefore conclude that HUVECs synthesize TPA-binding, heparan sulfate– and chondroitin sulfate–containing PGs. In vivo, similar PGs may play a role in TPA binding to endothelial cells and thereby possibly influence TPA activity and/or provide an intravascular storage pool of TPA.
Role of Macrophage Glycosaminoglycans in the Cellular Catabolism of Oxidized LDL by Macrophages
- Marielle Kaplan,
- Kevin Jon Williams,
- Hanna Mandel and
- Michael Aviram
Arteriosclerosis, Thrombosis, and Vascular Biology Volume 18, Issue 41 April 1998

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Abstract—Macrophage binding sites for oxidized LDL (Ox-LDL) include class A scavenger receptors (SR-As), the CD-36 molecule, and an additional but hitherto unidentified binding site. Because cell-surface glycosaminoglycans (GAGs) were previously shown to be involved in the cellular uptake of native LDL and lipoprotein(a), several strategies to assess the participation of heparan sulfate (HS) and chondroitin sulfate (CS) in macrophage catabolism of Ox-LDL were used. First, incubation of J-774 A.1 macrophage-like cells with either heparinase or chondroitinase, or with both enzymes together, reduced the binding, uptake, and degradation of ¹²⁵I–Ox-LDL by 20% to 45%, in comparison with control nontreated cells, while catabolism of ¹²⁵I-labeled acetylated LDL (Ac-LDL) and native LDL were unaffected. Second, the proteoglycan (PG) cellular content was increased by cell enrichment with exogenous GAGs or by using human monocyte-derived macrophages from two patients with Sanfilippo mucopolysaccharidosis, which are characterized by cellular HS accumulation. In these macrophages, cellular uptake of ¹²⁵I–Ox-LDL increased, while catabolism of ¹²⁵I–Ac-LDL and native LDL were unaffected. Experiments using conditioned media from control, heparinase-digested, or chondroitinase-digested macrophages indicated that neither secreted GAGs nor released digestion products played any role in Ox-LDL catabolism. To evaluate potential interactions between cell-surface GAGs and known receptors for Ox-LDL, we used excess unlabeled Ac-LDL to block SR-As or anti–CD-36 antibodies to block CD-36, and then examined the catabolism of ¹²⁵I–Ox-LDL by GAG-enriched or -depleted macrophages. Both excess unlabeled Ac-LDL and anti–CD-36 antibodies reduced ¹²⁵I–Ox-LDL catabolism, but only excess unlabeled Ac-LDL completely abolished the increase in ¹²⁵I–Ox-LDL catabolism on GAG enrichment of the cells, indicating a cooperation between exogenous GAGs and cell-surface SR-As in the catabolism of OX-LDL. Moreover, the addition of GAGases to macrophages that were preincubated with anti–CD-36 antibodies and excess Ac-LDL further reduced macrophage degradation of Ox-LDL in comparison with cells that were pretreated only with anti–CD-36 antibodies and Ac-LDL, indicating a more complex role for endogenous GAGs. Overall, these studies demonstrate a substantial contribution of macrophage-associated GAGs in the catabolism of Ox-LDL, which is mediated in part by a cooperation between GAGs and cell-surface SR-As.
Age-Related Changes in Populations of Aortic Glycosaminoglycans : Species With Low Affinity for Plasma Low-Density Lipoproteins, and Not Species With High Affinity, Are Preferentially Affected
- Ana M. F. Tovar,
- Davi C. F. Cesar,
- Graziela C. Leta and
- Paulo A. S. Mourão
Arteriosclerosis, Thrombosis, and Vascular Biology Volume 18, Issue 41 April 1998

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Abstract—Glycosaminoglycans were extracted from the intima and media layers of normal human thoracic aortas from donors of different ages. The arterial segments were devoid of macroscopically visible lesions obtained from patients who had no clinically evident cardiovascular disease. Total glycosaminoglycan content increases during the first 40 years of life. Changes in the content of hyaluronic acid and heparan sulfate are less noticeable. The content of chondroitin sulfate (mainly the 6-isomer) increases, whereas dermatan sulfate remains constant. Plasma LDL-affinity chromatography of dermatan sulfate+chondroitin 4/6-sulfate fractions allowed the separation of LDL high- and low-affinity glycosaminoglycan species. Remarkably, only glycosaminoglycan species with low affinity for plasma LDL increase with age in the disease-free areas of human thoracic aortas studied. These results suggest that age-related changes in glycosaminoglycan composition of the arterial wall do not contribute to increased deposition of plasma LDL. However, the alternative explanation that individuals with arterial glycosaminoglycans that avidly bind LDL would develop early and severe cardiovascular disease and would thus be excluded from our analysis cannot be ruled out.
The Heparin-Binding Proteins Apolipoprotein E and Lipoprotein Lipase Enhance Cellular Proteoglycan Production
- Joseph C. Obunike,
- Sivaram Pillarisetti,
- Latha Paka,
- Yuko Kako,
- Mathew J. Butteri,
- Yuan-Yaun Ho,
- William D. Wagner,
- Nobuhiro Yamada,
- Theodore Mazzone,
- Richard J. Deckelbaum and
- Ira J. Goldberg
Arteriosclerosis, Thrombosis, and Vascular Biology Volume 20, Issue 11 January 2000

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Abstract—Apolipoprotein E (apoE) and lipoprotein lipase (LPL), key proteins in the regulation of lipoprotein metabolism, bind with high affinity to heparin and cell-surface heparan sulfate proteoglycan (HSPG). In the present study, we tested whether the expression of apoE or LPL would modulate proteoglycan (PG) metabolism in cells. Two apoE-expressing cells, macrophages and fibroblasts, and LPL-expressing Chinese hamster ovary (CHO) cells were used to study the effect of apoE and LPL on PG production. Cellular PGs were metabolically labeled with ³⁵[S]sulfate for 20 hours, and medium, pericellular PGs, and intracellular PGs were assessed. In all transfected cells, PG levels in the 3 pools increased 1.6- to 3-fold when compared with control cells. Initial PG production was assessed from the time of addition of radiolabeled sulfate; at 1 hour, there was no difference in PG synthesis by apoE-expressing cells when compared with control cells. After 1 hour, apoE-expressing cells had significantly greater production of PGs. Total production assessed with [³H]glucosamine was also increased. This was due to an increase in the length of the glycosaminoglycan chains. To assess whether the increase in PGs was due to a decrease in PG degradation, a pulse-chase experiment was performed. Loss of sulfate-labeled pericellular PGs was similar in apoE and control cells, but more labeled PGs appeared in the medium of the apoE-expressing cells. Addition of exogenous apoE and anti-human apoE antibody to both non–apoE-expressing and apoE-expressing cells did not alter PG production. Moreover, LPL addition did not alter cell-surface PG metabolism. These results show that enhanced gene expression of apoE and LPL increases cellular PG production. We postulate that such changes in vascular PGs can affect the atherogenic potential of arteries.
Aging Influences Development and Progression of Early Aortic Atherosclerotic Lesions in Cholesterol-Fed Rabbits
Arteriosclerosis, Thrombosis, and Vascular Biology Volume 20, Issue 41 April 2000

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Abstract—The arterial wall in aged animals shows an increased susceptibility to develop atherosclerotic lesions, although the mechanisms by which aging acts are still unclear. We investigated early aortic lesions in aged rabbits (5 to 6 years old, AH group) and young rabbits (2 months old, YH group) after 2 months of 0.2% cholesterol feeding. Fatty streaks or spots mainly in the proximal segments occupied a relative surface area that was greater in AH than in YH rabbits, although plasma cholesterol and lipoprotein levels did not differ. YH lesions showed an irregular endothelial profile mainly from accumulations of large, rounded, RAM 11–positive macrophagic foam cells. There was a higher percentage of myocytic, CD-5–positive, proliferating, and terminal deoxynucleotidyl transferase–mediated dUTP nick end-labeling (TUNEL)-positive cells and larger accumulation of glycosaminoglycans in AH fatty streaks than in YH lesions. Ligation-mediated polymerase chain reaction confirmed differences in apoptosis. Early fibromuscular coats and subendothelial plasma-like insudate were also observed in AH lesions. Aged-matched normocholesterolemic rabbits showed a diffuse aortic intimal thickening composed of myocytic cells with a synthetic phenotype and extracellular matrix rich in glycosaminoglycans. In addition, in aged rabbits, we observed a spontaneous increase of monocytes adhering to the endothelial surface and a reduced expression of endothelial nitric oxide synthase in areas distant from the branches. These plasma cholesterol–independent spontaneous changes in the aortic wall of aged rabbits seem to act as a multiple atherogenic risk factor. Moreover, age-related differences in the distribution, composition, and proliferative and apoptotic rates represent crucial events during the progression of early fatty streaks to advanced plaques.
Repair in Arterial Tissue : MORPHOLOGICAL AND BIOCHEMICAL CHANGES IN RABBIT AORTA AFTER A SINGLE DILATATION INJURY
- P. Helin,
- I. Lorenzen,
- C. Garbarich and
- M. E. Matthienen
Circulation Research Volume 29, Issue 51 November 1971

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Male albino rabbits were subjected to a single mechanical dilatation of the descending thoracic aorta with a balloon catheter. The animals were killed 3, 6, 14, 30, and 60 days later. Between the sixth and the fourteenth days after injury, all the aortas which had been dilated developed severe, gross arteriosclerosis. Microscopic examination showed destruction and degeneration in the form of necrosis and calcification as well as regeneration and repair including new formation of cells, intercellular substance, and fibers. Biochemical and histochemical analyses revealed an early increase in hyaluronic acid and water followed by an increase in chondroitin-4, 6-sulfate and a later increase in heparan sulfate, dermatan sulfate, and collagen. These alterations were related to the focal lesions in the aortic wall. The permeability of the aorta to ¹²⁵I-albumin increased to a maximum 3 days after the dilatation, whereupon it decreased rapidly. The alterations were interpreted as nonspecific processes of repair in the vascular connective tissue. The pronounced dependence of the alterations on the time elapsed after injury must be considered in the study of vascular diseases where injury and repair may be involved.
Reduction of Intimal Hyperplasia by Naroparcil, a 4-Methylumbelliferyl β-d-Xyloside Analogue, After Arterial Injury in the Hypercholesterolemic Rabbit
- Philippe Gabriel Steg,
- Marianne Ziol,
- Ouafae Tahlil,
- Claude Robert,
- Philippe Masson,
- Didier Pruneau,
- Patrick Bruneval and
- Pierre Bélichard
Circulation Research Volume 77, Issue 51 November 1995

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Abstract 4-Methylumbelliferyl β-D-xylosides (β-d-xylosides) inhibit proteoglycan synthesis, and this is associated with reduced proliferation and extracellular matrix production by vascular smooth muscle cells. This study evaluated whether treatment with naroparcil, a β-d-xyloside analogue, reduced intimal hyperplasia after arterial injury in the hypercholesterolemic rabbit. Forty-two rabbits were assigned to three groups that received either a 1% cholesterol-enriched diet (group 1, n=15) or the same diet with added 100 mg · kg⁻¹ naroparcil (group 2, n=15) or 300 mg · kg⁻¹ naroparcil (group 3, n=12). All animals underwent iliac artery endothelial abrasion at day 14 and were killed at day 56. At the time of death, the angiographic minimal luminal diameter was significantly larger in both treated groups. Morphometric analysis showed a larger luminal area in treated rabbits (groups 2 and 3) compared with control rabbits (group 1) (0.75±0.54 and 0.85±0.61 mm² versus 0.32±0.25 mm², respectively; P<.05), with a decreased intimal thickness in groups 2 and 3 (average reduction of 37% and 39%, respectively, compared with group 1; P<.05) but without changes in medial area. Total vessel area was comparable among all groups. In the media, immunohistochemistry suggested reduced infiltration by macrophages and a larger fractional area of smooth muscle cells. There were no differences in plasma or arterial wall cholesterol content between groups. Plasma levels of glycosaminoglycans and dermatan sulfate content were increased only in group 3. In this model, oral treatment with naroparcil appears to preserve the arterial lumen and reduce intimal thickness after arterial injury.
Mineral Surface in Calcified Plaque Is Like That of Bone: Further Evidence for Regulated Mineralization
- Melinda J. Duer,
- Tomislav Friščić,
- Diane Proudfoot,
- David G. Reid,
- Michael Schoppet,
- Catherine M. Shanahan,
- Jeremy N. Skepper and
- Erica R. Wise
Arteriosclerosis, Thrombosis, and Vascular Biology Volume 28, Issue 111 November 2008

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Objectives— Cell biological studies demonstrate remarkable similarities between mineralization processes in bone and vasculature, but knowledge of the components acting to initiate mineralization in atherosclerosis is limited. The molecular level microenvironment at the organic-inorganic interface holds a record of the mechanisms controlling mineral nucleation. This study was undertaken to compare the poorly understood interface in mineralized plaque with that of bone, which is considerably better characterized.Methods and Results— Solid state nuclear magnetic resonance (SSNMR) spectroscopy provides powerful tools for studying the organic-inorganic interface in calcium phosphate biominerals. The rotational echo double resonance (REDOR) technique, applied to calcified human plaque, shows that this interface predominantly comprises sugars, most likely glycosaminoglycans (GAGs). In this respect, and in the pattern of secondary effects seen to protein (mainly collagen), calcified plaque strongly resembles bone.Conclusion— The similarity between biomineral formed under highly controlled (bone) and pathological (plaque) conditions suggests that the control mechanisms are more similar than previously thought, and may be adaptive. It is strong further evidence for regulation of plaque mineralization by osteo/chondrocytic vascular smooth muscle cells.NMR techniques show that bone and vascular calcifications are very similar with respect to constituents of the mineral/matrix interface, a predominant component being glycosaminoglycans. This implies that regulation of both is very similar, supporting cell biological studies showing pathological calcification of vascular smooth muscle cells to be a regulated process.
Induction of Anti-Anti-Idiotype Antibodies Against Sulfated Glycosaminoglycans Reduces Atherosclerosis in Apolipoprotein E–Deficient Mice
- Víctor Brito,
- Katia Mellal,
- Simon Giroux Portelance,
- Arlenis Pérez,
- Yosdel Soto,
- Denis deBlois,
- Huy Ong,
- Sylvie Marleau and
- Ana María Vázquez
Arteriosclerosis, Thrombosis, and Vascular Biology Volume 32, Issue 121 December 2012

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Objective—The pathogenesis of atherosclerosis is associated with the early retention of low-density lipoproteins that are trapped in the extracellular matrix of the arterial intima by interaction with glycosaminoglycan side chains of proteoglycans. Mutant mouse/human chimeric antibodies of the murine monoclonal antibody P3, which react with N-glycolyl–containing gangliosides and sulfated glycosaminoglycans, were tested for their potentially antiatherogenic properties through the induction of an idiotypic antibody network that may specifically interfere with the binding of low-density lipoproteins to proteoglycan side chains, low-density lipoprotein modification, and foam cell formation.Methods and Results—Apolipoprotein E–deficient mice fed a high-fat, high-cholesterol diet received 5 to 6 doses of chP3R99 or chP3S98 mutant antibodies, showing high and low reactivity, respectively, against their respective antigens. Both chimeric antibodies elicited an immunodominant anti-idiotypic response in the absence of adjuvant. A striking (40%–43%) reduction (P<0.01) in total lesion areas was observed in 18-week-old mice immunized with chP3R99, but not chP3S98, compared with PBS-treated mice. The antiatherosclerotic effect was associated with increased mice sera reactivity against heparin and sulfated glycosaminoglycans, including chondroitin and dermatan sulfate. In addition, purified IgG from chP3R99-immunized mice blocked the retention of apolipoprotein B–containing lipoproteins within the arterial wall of apolipoprotein E^−/− mice.Conclusion—The present study supports use of active immunization and the mounting of an idiotypic antibody network response against glycosaminoglycans as a novel approach to target atherosclerosis.
PKC (Protein Kinase C)-δ Modulates AT (Antithrombin) Signaling in Vascular Endothelial Cells
- Sumith R. Panicker,
- Indranil Biswas,
- Hemant Giri,
- Xiaofeng Cai and
- Alireza R. Rezaie
Arteriosclerosis, Thrombosis, and Vascular Biology Volume 40, Issue 71 July 2020

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Objective:Native and latent conformers of AT (antithrombin) induce anti-inflammatory and proapoptotic signaling activities, respectively, in vascular endothelial cells by unknown mechanisms. Synd-4 (syndecan-4) has been identified as a receptor that is involved in transmitting signaling activities of AT in endothelial cells.Approach and Results:In this study, we used flow cytometry, signaling assays, immunoblotting and confocal immunofluorescence microscopy to investigate the mechanism of the paradoxical signaling activities of high-affinity heparin (native) and low-affinity heparin (latent) conformers of AT in endothelial cells. We discovered that native AT binds to glycosaminoglycans on vascular endothelial cells via its heparin-binding D-helix to induce anti-inflammatory signaling responses by recruiting PKC (protein kinase C)-δ to the plasma membrane and promoting phosphorylation of the Synd-4 cytoplasmic domain at Ser179. By contrast, the binding of latent AT to endothelial cells to a site(s), which is not competed by the native AT, induces a proapoptotic effect by localizing PKC-δ to the perinuclear/nuclear compartment in endothelial cells. Overexpression of a dominant-negative form of PKC-δ resulted in inhibition of anti-inflammatory and proapoptotic signaling activities of both native and latent AT.Conclusions:These results indicate that the native and latent conformers of AT may exert their distinct intracellular signaling effects through differentially modulating the subcellular localization of PKC-δ in endothelial cells.
Major Reservoir for Heparin-Releasable TFPIα (Tissue Factor Pathway Inhibitor α) Is Extracellular Matrix
- Julie A. Peterson,
- Susan A. Maroney,
- Nicholas D. Martinez and
- Alan E. Mast
Arteriosclerosis, Thrombosis, and Vascular Biology Volume 41, Issue 61 June 2021

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Graphical AbstractObjective:Human endothelial cells produce 2 alternatively spliced TFPI (tissue factor pathway inhibitor) isoforms that maintain anticoagulant properties of the vasculature. TFPIβ is glycosylphosphatidylinositol anchored on the cell surface. TFPIα has a basic C terminus sharing homology with VEGF (vascular endothelial growth factor) and is a heparin-releasable protein, suggesting it binds glycosaminoglycans on the endothelium surface. However, this is unclear because TFPIα is not on the surface of cultured endothelial cells. This study identifies the source of heparin-releasable TFPIα.Approach and Results:ELISA assays localized heparin-releasable TFPIα to the extracellular matrix (ECM) of Ea.hy926 cells and human umbilical vein endothelial cells. Immunofluorescence microscopy for TFPIα showed punctate intracytoplasmic staining and ECM staining beneath individual cells. Flow cytometry identified TFPIβ but not TFPIα on the cell surface. TFPIα localization to ECM was confirmed with ELISA and immunohistochemistry studies of umbilical cord veins. The TFPIα C terminus interacted with Ea.hy926 ECM glycosaminoglycans, and a homologous VEGF peptide competed for this binding, suggesting these interactions modulate VEGF responses. Immobilized TFPIα C-terminal peptide bound to several ECM proteoglycans in Ea.hy926 conditioned media. Immunofluorescence studies of human kidney colocalized TFPIα with 4 of these proteoglycans surrounding the microvasculature: glypican-1, syndecan-4, thrombospondin, and laminin-5. The absence of TFPIα on the surface of endothelial cells and its co-localization with specific ECM proteins suggests TFPIα binds to unique proteoglycan structures.Conclusions:ECM contained the primary vascular pool of heparin-releasable TFPIα. By localizing to ECM, TFPIα is positioned to inhibit the procoagulant activity of tissue factor surrounding the vasculature.
pH-Dependent Protonation of Histidine Residues Is Critical for Electrostatic Binding of Low-Density Lipoproteins to Human Coronary Arteries
- Lars Glise,
- Mikael Rutberg,
- Liliana Håversen,
- Malin C. Levin,
- Max Levin,
- Anders Jeppsson,
- Jan Borén and
- Per Fogelstrand
Arteriosclerosis, Thrombosis, and Vascular Biology Volume 42, Issue 81 August 2022

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Background:The initiating step in atherogenesis is the electrostatic binding of LDL (low-density lipoprotein) to proteoglycan glycosaminoglycans in the arterial intima. However, although proteoglycans are widespread throughout the intima of most coronary artery segments, LDL is not evenly distributed, indicating that LDL retention is not merely dependent on the presence of proteoglycans. We aim to identify factors that promote the interaction between LDL and the vessel wall of human coronary arteries.Methods:We developed an ex vivo model to investigate binding of labeled human LDL to human coronary artery sections without the interference of cellular processes.Results:By staining consecutive sections of human coronary arteries, we found strong staining of sulfated glycosaminoglycans throughout the arterial intima, whereas endogenous LDL deposits were focally distributed. Ex vivo binding of LDL was uniform at all intimal areas with sulfated glycosaminoglycans. However, lowering the pH from 7.4 to 6.5 triggered a 35-fold increase in LDL binding. The pH-dependent binding was abolished by pretreating LDL with diethyl-pyrocarbonate, which blocks the protonation of histidine residues, or cyclohexanedione, which inhibits the positive charge of site B on LDL. Thus, both histidine protonation and site B are required for strong electrostatic LDL binding to the intima.Conclusions:This study identifies histidine protonation as an important component for electrostatic LDL binding to human coronary arteries. Our findings show that the local pH will have a profound impact on LDL’s affinity for sulfated glycosaminoglycans, which may influence the retention and accumulation pattern of LDL in the arterial vasculature.Graphical Abstract
Myocardial Extracellular Matrix: An Ever-Changing and Diverse Entity
Circulation Research Volume 114, Issue 528 February 2014

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The cardiac extracellular matrix (ECM) is a complex architectural network consisting of structural and nonstructural proteins, creating strength and plasticity. The nonstructural compartment of the ECM houses a variety of proteins, which are vital for ECM plasticity, and can be divided into 3 major groups: glycoproteins, proteoglycans, and glycosaminoglycans. The common denominator for these groups is glycosylation, which refers to the decoration of proteins or lipids with sugars. This review will discuss the fundamental role of the matrix in cardiac development, homeostasis, and remodeling, from a glycobiology point of view. Glycoproteins (eg, thrombospondins, secreted protein acidic and rich in cysteine, tenascins), proteoglycans (eg, versican, syndecans, biglycan), and glycosaminoglycans (eg, hyaluronan, heparan sulfate) are upregulated on cardiac injury and regulate key processes in the remodeling myocardium such as inflammation, fibrosis, and angiogenesis. Albeit some parallels can be made regarding the processes these proteins are involved in, their specific functions are extremely diverse. In fact, under varying conditions, individual proteins can even have opposing functions, making spatiotemporal contribution of these proteins in the rearrangement of multifaceted ECM very hard to grasp. Alterations of protein characteristics by the addition of sugars may explain the immense, yet tightly regulated, variability of the remodeling cardiac matrix. Understanding the role of glycosylation in altering the ultimate function of glycoproteins, proteoglycans, and glycosaminoglycans in the myocardium may lead to the development of new biochemical structures or compounds with great therapeutic potential for patients with heart disease.
Sulodexide for the Prevention of Recurrent Venous Thromboembolism: The Sulodexide in Secondary Prevention of Recurrent Deep Vein Thrombosis (SURVET) Study: A Multicenter, Randomized, Double-Blind, Placebo-Controlled Trial
and on behalf of the SURVET Study Investigators*

Circulation Volume 132, Issue 2017 November 2015

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Background—Patients with a first episode of unprovoked venous thromboembolism have a high risk of recurrence after discontinuation of anticoagulant therapy. Extending anticoagulation reduces the risk of recurrence but is associated with increased bleeding. Sulodexide, a glycosaminoglycan, exerts antithrombotic and profibrinolytic actions with a low bleeding risk when administered orally, but its benefit for preventing recurrent venous thromboembolism is not well known.Methods and Results—In this multicenter, double-blind study, 615 patients with first-ever unprovoked venous thromboembolism who had completed 3 to 12 months of oral anticoagulant treatment were randomly assigned to sulodexide 500 lipasemic units twice daily or placebo for 2 years, in addition to elastic stockings. The primary efficacy outcome was recurrence of venous thromboembolism. Major or clinically relevant bleeding was the primary safety outcome. Venous thromboembolism recurred in 15 of the 307 patients who received sulodexide and in 30 of the 308 patients who received placebo (hazard ratio, 0.49; 95% confidence interval [CI], 0.27–0.92; P=0.02). The analysis in which lost to follow-up was assigned to failure yielded a risk ratio among treated versus control subjects of 0.54 (95% confidence interval, 0.35–0.85; P=0.009). No major bleeding episodes occurred; 2 patients in each treatment group had a clinically relevant bleeding episode. Adverse events were similar in the 2 groups.Conclusion—Sulodexide given after discontinuation of anticoagulant treatment reduced the risk of recurrence in patients with unprovoked venous thromboembolism, with no apparent increase of bleeding risk.Clinical Trial Registration—URL: https://www.clinicaltrialsregister.eu/. Identifier: EudraCT number 2009-016923-77.
Targeting Chondroitin Sulfate Glycosaminoglycans to Treat Cardiac Fibrosis in Pathological Remodeling
- Rong-Rong Zhao,
- Matthew Ackers-Johnson,
- Justus Stenzig,
- Chen Chen,
- Tao Ding,
- Yue Zhou,
- Peipei Wang,
- Shi Ling Ng,
- Peter Y. Li,
- Gavin Teo,
- Pauline M. Rudd,
- James W. Fawcett and
- Roger S.Y. Foo
Circulation Volume 137, Issue 235 June 2018

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Background:Heart failure is a leading cause of mortality and morbidity, and the search for novel therapeutic approaches continues. In the monogenic disease mucopolysaccharidosis VI, loss-of-function mutations in arylsulfatase B lead to myocardial accumulation of chondroitin sulfate (CS) glycosaminoglycans, manifesting as myriad cardiac symptoms. Here, we studied changes in myocardial CS in nonmucopolysaccharidosis failing hearts and assessed its generic role in pathological cardiac remodeling.Methods:Healthy and diseased human and rat left ventricles were subjected to histological and immunostaining methods to analyze glycosaminoglycan distribution. Glycosaminoglycans were extracted and analyzed for quantitative and compositional changes with Alcian blue assay and liquid chromatography–mass spectrometry. Expression changes in 20 CS-related genes were studied in 3 primary human cardiac cell types and THP-1–derived macrophages under each of 9 in vitro stimulatory conditions. In 2 rat models of pathological remodeling induced by transverse aortic constriction or isoprenaline infusion, recombinant human arylsulfatase B (rhASB), clinically used as enzyme replacement therapy in mucopolysaccharidosis VI, was administered intravenously for 7 or 5 weeks, respectively. Cardiac function, myocardial fibrosis, and inflammation were assessed by echocardiography and histology. CS-interacting molecules were assessed with surface plasmon resonance, and a mechanism of action was verified in vitro.Results:Failing human hearts displayed significant perivascular and interstitial CS accumulation, particularly in regions of intense fibrosis. Relative composition of CS disaccharides remained unchanged. Transforming growth factor–β induced CS upregulation in cardiac fibroblasts. CS accumulation was also observed in both the pressure-overload and the isoprenaline models of pathological remodeling in rats. Early treatment with rhASB in the transverse aortic constriction model and delayed treatment in the isoprenaline model proved rhASB to be effective at preventing cardiac deterioration and augmenting functional recovery. Functional improvement was accompanied by reduced myocardial inflammation and overall fibrosis. Tumor necrosis factor–α was identified as a direct binding partner of CS glycosaminoglycan chains, and rhASB reduced tumor necrosis factor–α—induced inflammatory gene activation in vitro in endothelial cells and macrophages.Conclusions:CS glycosaminoglycans accumulate during cardiac pathological remodeling and mediate myocardial inflammation and fibrosis. rhASB targets CS effectively as a novel therapeutic approach for the treatment of heart failure.
Proteoglycans Synthesized by Arterial Smooth Muscle Cells in the Presence of Transforming Growth Factor-β1 Exhibit Increased Binding to LDLs
- Peter J. Little,
- Lisa Tannock,
- Katherine L. Olin,
- Alan Chait and
- Thomas N. Wight
Arteriosclerosis, Thrombosis, and Vascular Biology Volume 22, Issue 11 January 2002

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The “response-to-retention” hypothesis of atherogenesis states that atherogenic lipoproteins, such as low density lipoprotein (LDL), are retained in vessels by proteoglycans and undergo proatherosclerotic modifications. Transforming growth factor (TGF)-β1 has been identified in atherosclerotic vessels and has been shown to stimulate the synthesis of chondroitin sulfate– and dermatan sulfate–containing proteoglycans by arterial smooth muscle cells (ASMCs), but whether it promotes lipid retention has not been addressed. We investigated whether TGF-β1 modulates the biosynthesis of proteoglycans by ASMCs in a manner that promotes binding to LDL. Proteoglycans isolated from TGF-β1–treated ASMCs exhibited enhanced binding to native LDL compared with the binding of proteoglycans isolated from control cultures (K_d 18 μg/mL LDL versus 81 μg/mL LDL, respectively). The increase in proteoglycan-LDL binding caused by TGF-β1 could be attributed primarily to the glycosaminoglycan portion of the proteoglycans, since the glycosaminoglycan chains liberated from the core proteins of these proteoglycans synthesized in the presence of TGF-β1 exhibited increased LDL binding as well. Furthermore, glycosaminoglycan chains initiated on xyloside (an initiator of glycosaminoglycan synthesis) in the presence of TGF-β1 were longer and displayed enhanced binding to LDL compared with the LDL binding of xyloside-initiated glycosaminoglycan chains from control cultures. These results indicate that TGF-β1 promotes LDL-proteoglycan interaction primarily by its effects on the glycosaminoglycan synthetic machinery of the ASMCs. Therefore, this study supports a proatherogenic role for TGF-β1.
Myeloperoxidase and Hypochlorite, but Not Copper Ions, Oxidize Heparin-Bound LDL Particles and Release Them From Heparin
Arteriosclerosis, Thrombosis, and Vascular Biology Volume 21, Issue 121 December 2001

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Abstract— A key factor in atherosclerosis is the retention of low density lipoprotein (LDL) in the extracellular matrix of the arterial intima, where it binds to the negatively charged glycosaminoglycan chains of proteoglycans. Oxidation may lead to modification of the lysine residues of apolipoprotein B-100 of LDL, which normally mediate the binding of LDL to glycosaminoglycans. Here, we studied whether various modes of oxidation can release LDL from heparin, a glycosaminoglycan with a strong negative charge, in vitro. We found that copper ions were unable to oxidize heparin-bound LDL particles because of their redox inactivation by the glycosaminoglycans. In contrast, myeloperoxidase and hypochlorite, a product of myeloperoxidase, were able to oxidize heparin-bound LDL, and this oxidation led to the release of the oxidized particles from heparin. When the released LDL particles were compared with the residual heparin-bound LDL particles, the released particles were more electronegative and contained more modified lysine residues than did the particles that remained bound. Because human atherosclerotic lesions contain catalytically active myeloperoxidase and (lipo)proteins modified by hypochlorite, the results suggest that myeloperoxidase-secreting monocytes/macrophages in the arterial intima can oxidize and extract LDL from the extracellular matrix with ensuing uptake by the macrophages of the oxidized and released LDL, with eventual formation of foam cells.
Multiple Soluble Components of the Glycocalyx Are Increased in Patient Plasma After Ischemic Stroke
- Brian DellaValle,
- Henrik Hasseldam,
- Flemming F. Johansen,
- Helle K. Iversen,
- Jørgen Rungby and
- Casper Hempel
Stroke Volume 50, Issue 101 October 2019

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Background and Purpose—The GLX (glycocalyx) is a protein/polysaccharide meshwork at the cellular surface. Consisting largely of glycosaminoglycans and proteoglycans, the GLX can shed in response to stress. In this study, we assay 11 components of the GLX in plasma from patients with ischemic stroke from a longitudinal cohort.Methods—Plasma samples from healthy individuals (N=8), and patients with ischemic stroke day ≥3, day 7, and day 90 (N=9–14) were immunoassayed for diverse components of the GLX.Results—Median stroke severity was mild (National Institutes of Health Stroke Scale 2.0 (range, 0–6) at day ≤3). Three (keratan-chondroitin-heparan-sulfate) of 4 glycosaminoglycans and CD44 (proteoglycan) were increased at day 7 and returned to baseline at day 90. Proteoglycan syndecan (Syn)-3 increased and Syn-2 levels decreased, significantly.Conclusions—Individual GLX components are often assayed as stand-alone biomarkers for endothelial health. This study suggests a full assessment of GLX components is more indicative of the endothelial health of an individual and represents a complex GLX signature that may be valuable as a composite biomarker of disease.

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