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Differential Accumulation of Proteoglycans and Hyaluronan in Culprit Lesions: Insights Into Plaque Erosion
- Frank D. Kolodgie,
- Allen P. Burke,
- Andrew Farb,
- Deena K. Weber,
- Robert Kutys,
- Thomas N. Wight and
- Renu Virmani
Arteriosclerosis, Thrombosis, and Vascular Biology Volume 22, Issue 101 October 2002

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Objective— The importance of the extracellular matrix molecules versican, biglycan, decorin, and hyaluronan in plaque instability has not been recognized.Methods and Results— Coronary lesions with acute thrombi and stable plaques were examined for the accumulation and distribution of specific proteoglycans and hyaluronan at culprit sites. The cell surface receptor for hyaluronan, CD44, and smooth muscle (SM) cell maturation markers were also assessed. Proteoglycans and hyaluronan accumulated in distinct patterns depending on plaque type. The fibrous cap of stable lesions was enriched in versican and biglycan, with considerably less staining for decorin and hyaluronan, whereas picrosirius red revealed a heavy accumulation of collagen type I. In contrast, intense staining for hyaluronan and versican was found in erosions at the plaque/thrombus interface, with weak staining for biglycan and decorin; collagen content was predominantly type III. Rupture sites showed little immunoreactivity for proteoglycans or hyaluronan. CD44 was localized along the plaque/thrombus interface in erosions, whereas in ruptures and stable plaques, it was mostly confined to inflammatory cells. Positive immunostaining for immature SM cells (SM myosin heavy chain SM1 and SMemb) was present in stable and eroded plaques, whereas the presence of SM2 and smoothelin was weak or nonexistent.Conclusions— Specific accumulation of versican, hyaluronan, and CD44 at the sites of plaque erosion implicates an involvement of these molecules in events associated with acute coronary thrombosis.
Association Between Complement Factor H and Proteoglycans in Early Human Coronary Atherosclerotic Lesions: Implications for Local Regulation of Complement Activation
- Riina Oksjoki,
- Hanna Jarva,
- Petri T. Kovanen,
- Petri Laine,
- Seppo Meri and
- Markku O. Pentikäinen
Arteriosclerosis, Thrombosis, and Vascular Biology Volume 23, Issue 41 April 2003

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Objective— Complement activation has been suggested to play a role in atherogenesis. To study the regulation of complement activation in human coronary atherosclerotic lesions, we examined the spatial relationships between the major complement inhibitor, factor H, and the complement activation products C3d and C5b-9.Methods and Results— In early lesions (American Heart Association types II and III), factor H was immunohistochemically found in the superficial proteoglycan-rich layer in association with numerous macrophages and C3d, whereas C5b-9 was found deeper in the intima, where factor H was virtually absent. In vitro experiments involving surface plasmon resonance and affinity chromatography analyses demonstrated that isolated human arterial proteoglycans bind factor H, and functional complement assays showed that glycosaminoglycans inhibit the complement activation induced by modified low density lipoprotein or by a foreign surface.Conclusions— The present observations raise the possibility that proteoglycans, because of their ability to bind the major complement inhibitor factor H, may inhibit complement activation in the superficial layer of the arterial intima. In contrast, deeper in the intima, where factor H and proteoglycans are absent, complement may be activated and proceed to C5b-9. Thus, the superficial and the deep layers of the human coronary artery appear to differ in their ability to regulate complement activation.
Evidence for a Proinflammatory and Proteolytic Environment in Plaques From Endarterectomy Segments of Human Carotid Arteries
- Marilena Formato,
- Miriam Farina,
- Rita Spirito,
- Marco Maggioni,
- Anna Guarino,
- Gian Mario Cherchi,
- Paolo Biglioli,
- Celina Edelstein and
- Angelo M. Scanu
Arteriosclerosis, Thrombosis, and Vascular Biology Volume 24, Issue 11 January 2004

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Objectives Based on previous observations on apolipoprotein(a), apo(a), in human unstable carotid plaques, we explored whether in the inflammatory environment of human atheroma, proteolytic events affect other hepatic and topically generated proteins in relation to the issue of plaque stability.Methods and Results Forty unstable and 24 stable plaques from endarterectomy segments of affected human carotid arteries were extracted with buffered saline (PBS) and then 6 mol/L guanidine-hydrochloride (GdHCl) to identify loosely and tightly bound products, respectively. The extracts were studied before and after ultracentrifugation at d 1.21 g/mL. In the extracts, the concentrations of interleukin (IL)-6, −8, and −18 were significantly higher in the unstable plaques and correlated to those of MMP-2 and MMP-9. By Western blots, both apoB and apo(a) were highly fragmented and mostly present in the d 1.21 bottom that also contained fragments of apoE (10 and 22 kDa), decorin, biglycan, and versican. Fragmentation was higher in the unstable plaques. In baseline plasmas, concentrations of lipids, lipoproteins, and ILs did not differ between patients with unstable and stable plaques.Conclusions In unstable and to a lesser extent in stable plaques, there is a proinflammatory and proteolytic microenvironment with the generation of fragments with potential pathobiological significance that requires investigation.
Molecular Mechanism for Changes in Proteoglycan Binding on Compositional Changes of the Core and the Surface of Low-Density Lipoprotein–Containing Human Apolipoprotein B100
- Christofer Flood,
- Maria Gustafsson,
- Robert E. Pitas,
- Lorenzo Arnaboldi,
- Rosemary L. Walzem and
- Jan Borén
Arteriosclerosis, Thrombosis, and Vascular Biology Volume 24, Issue 31 March 2004

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Objective— The aim of this study was to investigate the molecular mechanism for changes in proteoglycan binding and LDL receptor affinity on two compositional changes in LDL that have been associated with atherosclerosis: cholesterol enrichment of the core and modification by secretory group IIA phospholipase A2 (sPLA₂) of the surface.Methods and Results— Transgenic mice expressing recombinant apolipoprotein (apo) B and sPLA₂ were generated. Recombinant LDL were isolated and tested for their proteoglycan and LDL receptor-binding activity. The results show site A (residues 3148-3158) in apoB100 becomes functional in sPLA₂-modified LDL and that site A acts cooperatively with site B (residues 3359-3369), the primary proteoglycan-binding site in native LDL, in the binding of sPLA₂-modified LDL to proteoglycans. Our results also show that cholesterol enrichment of LDL is associated with increased affinity for proteoglycans and for the LDL receptor. This mechanism is likely mediated by a conformational change of site B and is independent of site A in apoB100.Conclusion— Site A in apoB100 becomes functional in sPLA₂-modified LDL and acts cooperatively with site B resulting in increased proteoglycan-binding activity. The increased binding for proteoglycans of cholesterol-enriched LDL is solely dependent on site B.
Molecular Interactions Leading to Lipoprotein Retention and the Initiation of Atherosclerosis
- Maged F. Khalil,
- William D. Wagner and
- Ira J. Goldberg
Arteriosclerosis, Thrombosis, and Vascular Biology Volume 24, Issue 121 December 2004

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Atherosclerosis is distinguished by the accumulation of lipoprotein lipid within the arterial wall. An ionic interaction of positively charged regions of apolipoprotein (apo) B with matrix proteins, including proteoglycans, collagen, and fibronectin, is thought to initiate this process. Proteoglycans are complex glycoproteins containing highly negatively charged carbohydrate chains. These proteins are abundant in atherosclerosis lesions, and they associate with apoB-containing lipoproteins. Several specific regions of apoB may mediate this process. Other lipoprotein-associated proteins, including apoE and lipases, might also participate in this process. In addition, retention may occur via lipoprotein association with other matrix molecules or as a consequence of intra-arterial lipoprotein aggregation.Atherosclerosis is initiated by the accumulation of lipoprotein lipid within the artery wall. This process is thought to involve an ionic interaction between apoB and proteoglycans. Lipoprotein aggregation and association with other matrix proteins are likely additional routes to lipoprotein retention.
LDL Receptor–Related Protein and the Vascular Wall: Implications for Atherothrombosis
- Vicenta Llorente-Cortés and
- Lina Badimon
Arteriosclerosis, Thrombosis, and Vascular Biology Volume 25, Issue 31 March 2005

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LDL receptor–related protein 1 (LRP1) is highly expressed in the vascular wall and is mainly associated with macrophages and vascular smooth muscle cells (VSMCs). Overexpression of LRP1 in atherosclerotic lesions has been demonstrated in several animal models and human lesions. Clinical studies have suggested a relation between alterations in LRP1 expression and coronary heart disease. Indeed, it has been demonstrated that LRP1 gene expression is increased in blood mononuclear cells from patients with coronary obstruction and that the LRP1 mRNA-protein expression ratio is altered in coronary patients. Taken together, these results seem to suggest that LRP1 may be a pivotal receptor in the etiology of atherosclerosis. Our group has contributed to the elucidation of the physiopathologic role of LRP1 in the vascular wall by demonstrating that LRP1-mediated, matrix-retained LDL internalization could be crucial for VSMC–foam cell formation, that LRP1 is upregulated by lipid during human atherosclerotic lesion progression, and that LRP1-mediated aggregated LDL uptake causes the prothrombotic transformation of the vascular wall. Therefore, LRP1 seems to play a pathologic function during atherosclerotic lesion progression; however, LRP1 also seems to be essential for embryonic development and for the maintenance of vascular integrity. The protective effect of LRP1 in the vessel wall seems to be mainly due to its role in controlling certain signaling pathways. In this review, we will focus on the description of the main physiopathologic functions of LRP1 in the vascular wall.Our group has contributed to elucidate the physiopathological role of LRP1 in vascular wall by demonstrating that LRP1-mediated matrix-retained LDL internalization is crucial for VSMC-foam cell formation, that LRP1 is upregulated by lipid during human atherosclerotic lesion progression and that LRP1-mediated agLDL uptake causes prothrombotic transformation of the vascular wall.
Serum Amyloid A and Lipoprotein Retention in Murine Models of Atherosclerosis
- Kevin D. O’Brien,
- Thomas O. McDonald,
- Vidya Kunjathoor,
- KimLi Eng,
- Eleanor A. Knopp,
- Katherine Lewis,
- Roland Lopez,
- Elizabeth A. Kirk,
- Alan Chait,
- Thomas N. Wight,
- Frederick C. deBeer and
- Renee C. LeBoeuf
Arteriosclerosis, Thrombosis, and Vascular Biology Volume 25, Issue 41 April 2005

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Objective— Elevated serum amyloid A (SAA) levels are associated with increased cardiovascular risk in humans. Because SAA associates primarily with lipoproteins in plasma and has proteoglycan binding domains, we postulated that SAA might mediate lipoprotein retention on atherosclerotic extracellular matrix.Methods and Results— Immunohistochemistry was performed for SAA, apolipoprotein A-I (apoA-I), apolipoprotein B (apoB), and perlecan on proximal aortic lesions from chow-fed low-density lipoprotein receptor (LDLR)^−/− and apoE^−/− mice euthanized at 10, 50, and 70 weeks. SAA was detected on atherosclerotic lesion extracellular matrix at all time points in both strains. SAA area correlated highly with lesion areas (apoE^−/−, r=0.76; LDLR^−/−, r=0.86), apoA-I areas (apoE^−/−, r=0.88; LDLR^−/−, r=0.80), apoB areas (apoE^−/−, r=0.74; LDLR^−/−, r=0.89), and perlecan areas (apoE^−/−, r=0.83; LDLR^−/−, r=0.79) (all P<0.0001). In vitro, SAA enrichment increased high-density lipoprotein (HDL) binding to heparan sulfate proteoglycans, and immunoprecipitation experiments using plasma from apoE^−/− and LDLR^−/− mice demonstrated that SAA was present on both apoA-I–containing and apoB-containing lipoproteins.Conclusions— In chow-fed apoE^−/− and LDLR^−/− mice, SAA is deposited in murine atherosclerosis at all stages of lesion development, and SAA immunoreactive area correlates highly with lesion area, apoA-I area, apoB area, and perlecan area. These findings are consistent with a possible role for SAA-mediated lipoprotein retention in atherosclerosis.In chow-fed apoE^−/− and LDLR^−/− mice, serum amyloid A (SAA) is deposited at all stages of atherosclerotic lesion development and SAA immunoreactive area correlates highly with areas for lesions, apoA-I, apoB, and perlecan. These findings are consistent with the hypothesis that SAA may participate in atherosclerotic lesion lipoprotein retention.
Fatty Acids Cause Alterations of Human Arterial Smooth Muscle Cell Proteoglycans That Increase the Affinity for Low-Density Lipoprotein
- Mariam Rodríguez-Lee,
- Gunnel Östergren-Lundén,
- Boel Wallin,
- Jonatan Moses,
- Göran Bondjers and
- Germán Camejo
Arteriosclerosis, Thrombosis, and Vascular Biology Volume 26, Issue 11 January 2006

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Objective— The dyslipidemia of insulin resistance, with high levels of albumin-bound fatty acids, is a strong cardiovascular disease risk. Human arterial smooth muscle cell (hASMC) matrix proteoglycans (PGs) contribute to the retention of apoB lipoproteins in the intima, a possible key step in atherogenesis. We investigated the effects of high NEFA levels on the PGs secreted by hASMCs and whether these effects might alter the PG affinity for low-density lipoprotein.Methods and Results— hASMC exposed for 72 hours to high concentrations (800 μmol/L) of linoleate (LO) or palmitate upregulated the core protein mRNAs of the major PGs, as measured by quantitative PCR. Insulin (1 nmol/L) and the PPARγ agonist rosiglitazone (10 μmol/L) blocked these effects. In addition, high LO increased the mRNA levels of enzymes required for glycosaminoglycan (GAG) synthesis. Exposure to NEFA increased the chondroitin sulfate:heparan sulfate ratio and the negative charge of the PGs. Because of these changes, the GAGs secreted by LO-treated cells had a higher affinity for human low-density lipoprotein than GAGs from control cells. Insulin and rosiglitazone inhibited this increase in affinity.Conclusions— The response of hASMC to NEFA could induce extracellular matrix alterations favoring apoB lipoprotein deposition and atherogenesis.We examined the effects of increased NEFA and insulin on the proteoglycans secreted by hASMC and whether these effects might affect LDL binding. The results indicate that increased fatty acids could induce qualitative and quantitative alterations of the intima extracellular matrix proteoglycans favoring LDL retention and possibly atherogenesis.
Antioxidants Inhibit the Ability of Lysophosphatidylcholine to Regulate Proteoglycan Synthesis
- Mary Y. Chang,
- Chang-Yeop Han,
- Thomas N. Wight and
- Alan Chait
Arteriosclerosis, Thrombosis, and Vascular Biology Volume 26, Issue 31 March 2006

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Objective— We previously have shown that lysophosphatidylcholine (lysoPC) regulates proteoglycan synthesis by vascular smooth muscle cells (SMCs). Given the accumulating evidence for reactive oxygen species (ROS) as mediators of a variety of effects of lysoPC, the present study evaluates the potential role of ROS as intermediate molecules in the regulation of proteoglycan synthesis by lysoPC.Methods and Results— LysoPC (10 μmol/L) was found to stimulate rapid and sustained generation of ROS by SMC, as indicated using a fluorescent probe for measuring intracellular oxidants and fluorescence-activated cell sorting. This was not associated with cytotoxicity, as evaluated by fluorescence microscopy using MitoTracker Red or propidium iodide, cell number, cell protein, or lactate dehydrogenase release. Pretreatment with catalase or superoxide dismutase, specific scavengers of hydrogen peroxide and superoxide, respectively, blocked the ability of lysoPC to stimulate both accumulation of ROS and proteoglycan synthesis. Most importantly, these enzymatic antioxidants prevented lysoPC from stimulating the synthesis of proteoglycans with enhanced lipoprotein-binding properties, as quantified by a gel shift binding assay.Conclusions— These findings strongly suggest that ROS are key mediators in the ability of lysoPC to regulate proteoglycan synthesis and that these effects can be inhibited by antioxidants.We previously have shown that lysoPC regulates proteoglycan synthesis by vascular smooth muscle cells. Current findings strongly suggest that reactive oxygen species are key mediators in this ability of lysoPC and that these effects can be inhibited by antioxidants.
Secretory Phospholipase A₂ Group V: Lesion Distribution, Activation by Arterial Proteoglycans, and Induction in Aorta by a Western Diet
Arteriosclerosis, Thrombosis, and Vascular Biology Volume 26, Issue 71 July 2006

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Objective— To study the distribution of group V secretory phospholipase A₂ (sPLA₂) in human and mouse lesions and compare its expression by human vascular cells, its activity toward lipoproteins, and the interaction with arterial proteoglycans (proteoglycans) with those of sPLA₂-IIA. In addition, we also investigated the effect of a Western diet and lipopolysaccharide challenge on the aortic expression of these enzymes in mouse models.Methods and Results— Immunohistochemistry showed sPLA₂-V in human and mouse lesions to be associated with smooth muscle cells and also surrounding foam cells in lipid core areas. mRNA of the enzyme was expressed in human lesions and human vascular cells, supporting the immunohistochemistry data. sPLA₂-V but not sPLA₂-IIA was active on lipoproteins in human serum. The association with proteoglycans enhanced 2- to 3-fold sPLA₂-V activity toward low-density lipoproteins but not that of the group IIA enzyme. Experiments in mouse models showed that treatment with a Western diet induced expression of sPLA₂-V but not that of sPLA₂-IIA in aorta. On the other hand, lipopolysaccharide-induced acute inflammation augmented the expression of sPLA₂-IIA but not that of sPLA₂-V.Conclusions— These results indicate that these phospholipases could have different roles in atherosclerosis.SPLA₂-V was observed in human and mouse lesions associated with smooth muscle cells and surrounding foam cells in lipid cores. Proteoglycans increased the activity of sPLA₂-V toward low-density lipoproteins. Western diet induced sPLA₂-V expression in mouse aorta but not that of sPLA₂-IIA. These enzymes may contribute to atherosclerosis by different pathways.
Differentiated Macrophages Synthesize a Heparan Sulfate Proteoglycan and an Oversulfated Chondroitin Sulfate Proteoglycan That Bind Lipoprotein Lipase
- Iris J. Edwards,
- Hongzhi Xu,
- Joseph C. Obunike,
- Ira J. Goldberg and
- William D. Wagner
Arteriosclerosis, Thrombosis, and Vascular Biology Volume 15, Issue 31 March 1995

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Abstract Lipoprotein lipase (LpL), which facilitates lipoprotein uptake by macrophages, associates with the cell surface by binding to proteoglycans (PGs). Studies were designed to identify and characterize specific PGs that serve as receptors for LpL and to examine effects of cell differentiation on LpL binding. PG synthesis was examined by radiolabeling THP-1 monocytes and macrophages (a cell line originally derived from a patient with acute monocytic leukemia) with [³⁵S]sodium sulfate and [³H]serine or [³H]glucosamine. Radiolabeled PGs isolated from the cell surface were purified by chromatography and identified as chondroitin-4–sulfate (CS) PG and heparan sulfate (HS) PG. A sixfold increase in CSPG and an 11-fold increase in HSPG accompanied cell differentiation. Whereas HS glycosaminoglycan chains from both monocytes and macrophages were 7.5 kD in size, CS chains increased in size from 17 kD to 36 kD with cell differentiation, and contained hexuronyl N-acetylgalactosamine-4,6-di-O sulfate disaccharides. LpL binding was sevenfold higher to differentiated cells, and affinity chromatography demonstrated that two cell surface PGs bound to LpL: HSPG and the oversulfated CSPG produced only by differentiated cells. We conclude that differentiation-associated changes in cell surface PG of human macrophages have functional consequences that could increase the atherogenic potential of the cells.
Interferon Gamma Binds to Extracellular Matrix Chondroitin-Sulfate Proteoglycans, Thus Enhancing Its Cellular Response
- Eva Hurt Camejo,
- Birgitta Rosengren,
- Germán Camejo,
- Peter Sartipy,
- Gunnar Fager and
- Göran Bondjers
Arteriosclerosis, Thrombosis, and Vascular Biology Volume 15, Issue 91 September 1995

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Abstract The amino acid sequence of interferon gamma (IFN-γ) has basic amino acid clusters similar to the heparin-binding consensus sequences found in other proteins that bind to proteoglycans (PGs). We investigated whether recombinant human IFN-γ could bind to extracellular matrix (ECM) PGs secreted by human arterial smooth muscle cells (HASMCs) in vitro and whether the interaction affected the cellular response to IFN-γ. As an in vitro model of ECM we used the basement membrane from HASMCs in culture. The binding of ¹²⁵I-IFN-γ to ECM was reduced significantly by pretreatment of ECM with chondroitinase ABC, an enzyme that degrades chondroitin-sulfate glycosaminoglycans. IFN-γ binding to ECM was reduced by increasing concentrations of chondroitin-6-sulfate. ¹²⁵I-IFN-γ (0.05 to 2 ng/mL) binding data indicated an apparent K_d of 2×10⁻¹¹ mol/L and a maximum binding of 1.6×10⁶ IFN-γ molecules bound per square millimeter of ECM. Experiments with synthetic peptides suggested that residues 127 through 135 (AKTGKRKRS) are involved in the binding. The binding to chondroitin-sulfate PGs was confirmed by affinity chromatography of isolated [³⁵S]chondroitin-sulfate PGs from ECM and cell-culture medium on immobilized IFN-γ. The binding was abolished by treatment with chondroitinase ABC. ECM-bound IFN-γ was more effective in inducing the expression of class II major histocompatibility antigens such as HLA-DR in HASMCs and human arterial endothelial cells than soluble IFN-γ. These results suggest a role for chondroitin-sulfate PGs in immobilizing IFN-γ in the ECM compartment and enhancing the cellular response to IFN-γ.
Heparin-Binding Domain of Fibrin Mediates Its Binding to Endothelial Cells
Arteriosclerosis, Thrombosis, and Vascular Biology Volume 16, Issue 121 December 1996

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Spreading of human umbilical vein endothelial cells (ECs) on fibrin requires thrombin cleavage of fibrinopeptide B (FPB) and subsequent exposure of the new β15-42 N-terminus. To further understand the interactions between ECs and fibrin β15-42 sequences, binding of fibrin(ogen) to EC monolayers was measured with polyclonal anti-fibrinogen (FBG) in parallel with monoclonal anti-FBG (18C6, β1-21; J88B, γ63-78) and anti-fibrin (T2G1, β15-21) antibodies in an indirect enzyme-linked immunosorbent assay. To accomplish this, large, soluble fragments of fibrin were prepared by cyanogen bromide (CNBr) cleavage (fibrin-CNBr); CNBr-cleaved FBG (FBG-CNBr) served as the control ligand. N-terminal fibrin-CNBr bound to EC monolayers and cells in suspension in a dose-dependent and saturable manner. By contrast, FBG-CNBr bound only 50% as well to EC monolayers, with no significant binding of intact FBG, C-terminal FBG plasmic fragment D, or N-terminal plasmic fragment E, which lacks β1-53. ECs bound the peptide β15-42–bovine serum albumin (BSA) conjugate but neither a scrambled β15-42 peptide conjugate nor conjugates of β24-42, β18-27, or β18-31. Binding of fibrin-CNBr was inhibited 54% by the β15-42–BSA conjugate and 17% by the Bβ1-42-BSA conjugate but not by free β15-42 peptide or RGDS-cell binding peptide. Binding of fibrin-CNBr was inhibited >95% by heparin in a concentration-dependent manner; the same concentrations of heparin inhibited binding of β15-42–BSA by >75% but not the dose-dependent binding of fibronectin to ECs. These data suggest that in their native conformation, FBG Bβ15-42 sequences are unavailable for binding to ECs and that thrombin-induced exposure of β15-42 is required for binding by a heparin-dependent, RGD-independent mechanism at the new N-terminus of fibrin.
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.
Distinct Rat Aortic Smooth Muscle Cells Differ in Versican/PG-M Expression
- Joan M. Lemire,
- Susan Potter-Perigo,
- Keith L. Hall,
- Thomas N. Wight and
- Stephen M. Schwartz
Arteriosclerosis, Thrombosis, and Vascular Biology Volume 16, Issue 61 June 1996

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Abstract Smooth muscle cells (SMCs) with distinct phenotypes are present in blood vessels, and distinct culture types appear when SMCs are maintained in vitro. For example, cultured SMCs from rat adult media grow as bipolar cells, which differ in gene expression from the predominantly cobblestone-shaped SMCs from rat pup aortas and rat neointimas that we call π SMCs. Since proteoglycans are present at different concentrations in the normal intima and media and are elevated in atherosclerotic plaque, we sought to determine whether π and adult medial SMC types synthesize different or unique proteoglycans that are characteristic of each phenotype. [³⁵S]sulfate-labeled proteoglycans were purified by ion-exchange chromatography. An adult medial SMC line synthesized a large proteoglycan (0.2 K_av on Sepharose CL-2B) that was not detectable in a π SMC line. Digestion of this proteoglycan with chondroitin ABC lyase revealed three core glycoproteins of 330, 370, and 450 kD. By Western blot analysis, the two smallest of these reacted with two antibodies to the human fibroblast proteoglycan versican. RNAs hybridizing to versican probes were found only in adult medial–type SMCs, including an adult medial type clone from pup aorta, by Northern blot analysis. Both SMC types synthesize RNAs that hybridize to probes for other proteoglycans, such as perlecan, biglycan, and decorin. We conclude that rat π SMC cultures, unlike monkey, human, and rat adult medial SMC cultures, express little or no versican. This difference in expression may be responsible for the different morphologies and growth properties of the two cell types.
Arterial Smooth Muscle Cell Heparan Sulfate Proteoglycans Accelerate Thrombin Inhibition by Heparin Cofactor II
- Rebecca A. Shirk,
- Frank C. Church and
- William D. Wagner
Arteriosclerosis, Thrombosis, and Vascular Biology Volume 16, Issue 91 September 1996

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Heparin cofactor II (HCII) is a potent thrombin inhibitor in the presence of heparin and dermatan sulfate, glycosaminoglycans that accelerate the inhibition reaction. HCII is postulated to be an extravascular thrombin inhibitor that is stimulated physiologically by dermatan sulfate proteoglycans. To understand how thrombin activity may be downregulated within the artery wall, cultured monkey aorta smooth muscle cell (SMC) proteoglycans were tested for their ability to accelerate thrombin inhibition by HCII. Early confluent SMC monolayers increased thrombin-HCII inhibition rates 2-fold to 4-fold compared with reactions in cell-free control wells (7.3±0.5 versus 2.7±0.2×10⁴ mol·L⁻¹·min⁻¹, with and without SMCs, respectively; n=7 experiments). Extracellular matrix obtained by cell monolayer removal also accelerated the thrombin-HCII inhibition reaction 3-fold to 5-fold. Rate increases were abolished by Polybrene or protamine sulfate. Pretreatment of monolayers with heparitinase I (and of extracellular matrix with HNO₂) to degrade heparan sulfate blocked the thrombin-HCII inhibition rate increase. In contrast, pretreatment with chondroitinase ABC in the presence of proteinase inhibitors had no effect. “Pericellular” (cell surface– and extracellular matrix–derived) SMC heparan sulfate proteoglycans (HSPGs) were purified and fractionated by charge on DEAE-Sephacel. At a concentration of 1 μg/mL hexuronic acid, high-charge HSPG stimulated a 7-fold thrombin-HCII inhibition rate increase relative to reactions without proteoglycan, whereas low-charge HSPG induced a 2-fold rate increase. In comparison, an 18-fold rate increase was observed with 1 μg/mL dermatan sulfate proteoglycan purified from SMC culture media. These results indicate that SMC HSPG could contribute significantly to thrombin inhibition by HCII in the artery wall.
Cellular Consequences of the Association of ApoB Lipoproteins With Proteoglycans: Potential Contribution to Atherogenesis
- Eva Hurt-Camejo,
- Urban Olsson,
- Olov Wiklund,
- Göran Bondjers and
- Germán Camejo
Arteriosclerosis, Thrombosis, and Vascular Biology Volume 17, Issue 61 June 1997

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Effects of Contraceptive Estrogen and Progestin on the Atherogenic Potential of Plasma LDLs in Cynomolgus Monkeys
- James M. Manning,
- Iris J. Edwards,
- William D. Wagner,
- Janice D. Wagner,
- Michael R. Adams and
- John S. Parks
Arteriosclerosis, Thrombosis, and Vascular Biology Volume 17, Issue 71 July 1997

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Abstract This study was designed to determine the effect of oral contraceptive treatment (estrogen and progestin), alone or in combination, on LDL composition and atherogenic potential in cynomolgus monkeys fed an atherogenic diet. Groups (n=8 each) of monkeys were untreated (control) or treated with ethinyl estradiol (EE), levonorgestrel (LNG), or triphasic oral contraceptive (EE+LNG) for 1.5 years before plasma LDLs were isolated for characterization. Total plasma cholesterol concentrations were unaffected by the treatments. LDL particle size (measured as LDL molecular weight, g/μmol) was significantly smaller in the EE (4.61±0.09) and EE+LNG (4.43±0.09) treatment groups compared with the control (4.99±0.09) or LNG (5.29±0.17) groups and contained fewer molecules of free and esterified cholesterol. Both the EE and EE+LNG groups had significantly less cholesterol and apolipoprotein B distributed in the d=1.015 to 1.025 g/mL subfraction and correspondingly more in the d=1.025 to 1.035 g/mL subfraction of LDL compared with the control and LNG groups. The apolipoprotein E content (molecules/particle) of LDL was significantly less in the EE (0.35±0.1) and EE+LNG (0.28±0.1) groups compared with the control (0.86±0.2) and LNG (0.99±0.2) groups, and this trend was apparent in all three LDL subfractions. The atherogenic potential of LDL was tested using an in vitro binding assay to arterial proteoglycans. Twice as much LDL bound to arterial proteoglycans in the LNG group (11.3±1.8% of total LDL cholesterol in the incubation) compared with the control (6.4±1.9%), EE (5.5±1.5%), or EE+LNG (5.2±1.2%) groups. We conclude that EE and EE+LNG treatment alters the composition of LDL toward a less atherogenic particle that is smaller and more dense, contains less cholesterol and less apolipoprotein E, and is less reactive with arterial proteoglycans compared with LNG treatment. The inclusion of EE in the triphasic oral contraceptive treatment was sufficient to negate the potentially atherogenic effects of LNG on LDL composition. (Arterioscler Thromb Vasc Biol. 1997;17:1216-1223.)
Lipoprotein Lipase Can Function as a Monocyte Adhesion Protein
- Joseph C. Obunike,
- Swarnalatha Paka,
- Sivaram Pillarisetti and
- Ira J. Goldberg
Arteriosclerosis, Thrombosis, and Vascular Biology Volume 17, Issue 71 July 1997

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Abstract Lipoprotein lipase (LPL) is made by several cell types, including macrophages within the atherosclerotic lesion. LPL, a dimer of identical subunits, has high affinity for heparin and cell surface heparan sulfate proteoglycans (HSPGs). Several studies have shown that cell surface HSPGs can mediate cell binding to adhesion proteins. Here, we tested whether LPL, by virtue of its HSPG binding, could mediate monocyte adhesion to surfaces. Monocyte binding to LPL-coated (1-25 μg/mL) tissue culture plates was 1.4- to 7-fold higher than that of albumin-treated plastic. Up to 3-fold more monocytes bound to the subendothelial matrix that had been pretreated with LPL. LPL also doubled the number of monocytes that bound to endothelial cells (ECs). Heparinase and heparitinase treatment of monocytes or incubation of monocytes with heparin decreased monocyte binding to LPL. Heparinase/heparitinase treatment of the matrix also abolished the LPL-mediated increase in monocyte binding. These results suggest that LPL dimers mediate monocyte binding by forming a “bridge” between matrix and monocyte surface HSPGs. Inhibition of LPL activity with tetrahydrolipstatin, a lipase active-site inhibitor, did not affect the LPL-mediated monocyte binding. To assess whether specific oligosaccharide sequences in HSPGs mediated monocyte binding to LPL, competition experiments were performed by using known HSPG binding proteins. Neither antithrombin nor thrombin inhibited monocyte binding to LPL. Next, we tested whether integrins were involved in monocyte binding to LPL. Surprisingly, monocyte binding to LPL-coated plastic and matrix was inhibited by ≈35% via integrin-binding arginine-glycine–aspartic acid peptides. This result suggests that monocyte binding to LPL was mediated, in part, by monocyte cell surface integrins. In summary, our data show that LPL, which is present on ECs and in the subendothelial matrix, can augment monocyte adherence. This increase in monocyte-matrix interaction could promote macrophage accumulation within arteries.
Phospholipase A₂ Type II Binds to Extracellular Matrix Biglycan : Modulation of Its Activity on LDL by Colocalization in Glycosaminoglycan Matrixes
- Peter Sartipy,
- Göran Bondjers and
- Eva Hurt-Camejo
Arteriosclerosis, Thrombosis, and Vascular Biology Volume 18, Issue 121 December 1998

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Abstract—We recently reported the presence of secretory, nonpancreatic phospholipase A₂ type II (snpPLA₂; EC 3.1.1.4) in human atherosclerotic arteries (Hurt-Camejo et al, Arterioscler Thromb Vasc Biol. 1997;17:300–309). SnpPLA₂ may generate the proinflammatory products lysophospholipids and free fatty acids, thus contributing to atherogenesis when acting on low density lipoproteins (LDLs) retained in the arterial wall. Immunohistochemical studies showed that smooth muscle cells (SMCs) in human arterial tissue are the main sources of snpPLA₂. In cultures of human arterial SMCs, snpPLA₂ interacts with versican and smaller heparan/chondroitin sulfate proteoglycans (PGs) secreted as soluble components into the medium. In the present study, we investigated the binding of snpPLA₂ to extracellular matrix (ECM) PGs produced by SMCs. The results show that snpPLA₂ can bind to the ECM at physiological salt concentrations. ECM-bound snpPLA₂ was active, hydrolyzing phosphatidylcholine-containing micelles. Soluble chondroitin-6-sulfate at concentrations >1 μmol/L, but not heparin or heparan sulfate, was able to release ECM-bound snpPLA₂. The PG mainly involved in the binding of snpPLA₂ was identified as biglycan. Perlecan was also present in the ECM synthesized by SMCs, but it contributed less to the binding of snpPLA₂. Experiments with immobilized glycosaminoglycans indicated that snpPLA₂ hydrolyzed 7-fold more LDL phospholipids when the lipoprotein and the enzyme were colocalized in a matrix with chondroitin-6-sulfate compared with one with heparin. These data suggest that retention of snpPLA₂ in ECMs of different composition may modulate the enzymatic activity of snpPLA₂ toward LDL. The results presented in this work support the hypothesis of the potential contribution of snpPLA₂ to atherosclerosis.

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