Ian P. Hayward

Arterial heparan sulfate proteoglycans inhibit vascular smooth muscle cell proliferation and phenotype change in vitro and neointimal formation in vivo

PURPOSE The aim of this study was to determine whether heparan sulfate proteoglycans (HSPGs) from the normal arterial wall inhibit neointimal formation after injury in vivo and smooth muscle cell (SMC) phenotype change and proliferation in vitro. METHODS Arterial HSPGs were extracted from rabbit aortae and separated by anion-exchange chromatography. The effect of HSPGs, applied in a periadventitial gel, on neointimal formation was assessed 14 days after balloon catheter injury of rabbit carotid arteries. Their effect on SMC phenotype and proliferation was measured by point-counting morphometry of the cytoplasmic volume fraction of myofilaments (Vvmyo) and 3H-thymidine incorporation in SMCs in culture. RESULTS Arterial HSPGs (680 microg) reduced neointimal formation by 35% at 14 days after injury (P=.029), whereas 2000 microg of the low-molecular-weight heparin Enoxaparin was ineffective. HSPGs at 34 microg/mL maintained subconfluent primary cultured SMCs with the same high Vvmyo (52.1%+/-13.8%) after 5 days in culture as did cells freshly isolated from the arterial wall (52.1%+/-15.1%). In contrast, 100 microg/mL Enoxaparin was ineffective in preventing phenotypic change over this time period (Vvmyo 38.9%+/-14.6%, controls 35.9%+/-12.8%). HSPGs also inhibited 3H-thymidine incorporation into primary cultured SMCs with an ID50 value of 0.4 microg/mL compared with a value of 14 microg/mL for Enoxaparin (P< .01). CONCLUSION When used periadventitially in the rabbit arterial injury model, natural arterial HSPGs are effective inhibitors of neointimal formation. In vitro, the HSPGs maintain SMCs in a quiescent state by inhibiting phenotypic change and DNA synthesis. This study suggests that HSPGs may be a natural agent for the treatment of clinical restenosis.

Blockade of vascular smooth muscle cell proliferation and intimal thickening after balloon injury by the sulfated oligosaccharide PI-88: phosphomannopentaose sulfate directly binds FGF-2, blocks cellular signaling, and inhibits proliferation.

Abstract— Percutaneous transluminal coronary angioplasty is a frequently used interventional technique to reopen arteries that have narrowed because of atherosclerosis. Restenosis, or renarrowing of the artery shortly after angioplasty, is a major limitation to the success of the procedure and is due mainly to smooth muscle cell accumulation in the artery wall at the site of balloon injury. In the present study, we demonstrate that the antiangiogenic sulfated oligosaccharide, PI-88, inhibits primary vascular smooth muscle cell proliferation and reduces intimal thickening 14 days after balloon angioplasty of rat and rabbit arteries. PI-88 reduced heparan sulfate content in the injured artery wall and prevented change in smooth muscle phenotype. However, the mechanism of PI-88 inhibition was not merely confined to the antiheparanase activity of this compound. PI-88 blocked extracellular signal-regulated kinase-1/2 (ERK1/2) activity within minutes of smooth muscle cell injury. It facilitated FGF-2 release from uninjured smooth muscle cells in vitro, and super-released FGF-2 after injury while inhibiting ERK1/2 activation. PI-88 inhibited the decrease in levels of FGF-2 protein in the rat artery wall within 8 minutes of injury. PI-88 also blocked injury-inducible ERK phosphorylation, without altering the clotting time in these animals. Optical biosensor studies revealed that PI-88 potently inhibited (Ki 10.3 nmol/L) the interaction of FGF-2 with heparan sulfate. These findings show for the first time the capacity of this sulfated oligosaccharide to directly bind FGF-2, block cellular signaling and proliferation in vitro, and inhibit injury-induced smooth muscle cell hyperplasia in two animal models. As such, this study demonstrates a new role for PI-88 as an inhibitor of intimal thickening after balloon angioplasty. The full text of this article is available online at http://www.circresaha.org.

Matrix metalloproteinases can facilitate the heparanase-induced promotion of phenotypic change in vascular smooth muscle cells

Previous studies from this laboratory have shown that degradation of heparan sulphate proteoglycan by both living macrophages and macrophage lysosomal heparanase induces phenotypic change of vascular smooth muscle cells (SMC) from a high volume fraction of myofilaments (V(v)myo) to a low V(v)myo [Campbell et al. Exp Cell Res 1992; 200: 156-167]. The aim of this study was to determine whether matrix metalloproteinase (MMP) activity is also involved in the induction of SMC phenotypic change by macrophages. A specific inhibitor of MMPs (BB94) was able to block macrophage-induced SMC phenotypic change and subsequent DNA synthesis in freshly dispersed SMC seeded in primary culture at confluent density. The inhibitor did not block these SMC changes when SMC were seeded at low density without macrophages nor did it block heparanase activity directly. We also determined whether heparanase and MMP activities are upregulated together in vivo. Artery homogenates were analysed in a heparanase enzyme assay and for MMPs using zymograms. Increased heparanase activity was observed 3-14 days following balloon catheter injury of rabbit carotid arteries, and returned to control levels 6 weeks after injury. Active MMP2 was induced with heparanase after injury. MMP9 induction was also apparent 6 h after injury. Immunohistology on sections of these arteries showed the presence of MMP1, 2, 3 and 9 with these MMPs being strongly induced in the intima 7 days after balloon catheter injury. Both heparanase and MMP activities were also present in human end-stage complex lesions from coronary arteries, carotid endarterectomies and abdominal aortic aneurysms. Because MMPs and heparanase are expressed at the same time, it is possible that MMPs facilitate heparanase activity in promotion of phenotypic modulation of SMC in vivo during neointimal thickening following injury and in atherosclerotic lesions

Effects of collagen gel configuration on behavior of vascular smooth muscle cells in vitro: association with vascular morphogenesis

SummaryThe growth, behavior, and contractile protein expression of rabbit aortic smooth muscle cells (SMC) grown on, between layers, or within a collagen gel was investigated by confocal laser scanning fluorescence microscopy and Western analysis. SMC grown on collagen gel behaved similarly to those on conventional culture dishes. However, when a second layer of collagen was overlaid, cells underwent an elongated quiescent phase before onset of proliferation and a more than threefold lower logarithmic growth rate was observed. These cells self-organized into a network with ring-like structures. With increasing culture time, some of the rings developed into funnel-like, incomplete or complete tubular structures. If a tubular template preexisted within the gel, the SMC established a cylinder-shaped tube with several circularly arranged muscular layers (similar to an artery wall). This behavior mimicked endothelial cells during angiogenesis in vitro. A similar phenomenon occurred in cultures in which SMC were randomly mixed in a collagen gel, but here their behavior and morphology varied with their position within the gel. Western blot analysis showed that the SMC differentiation marker, smooth muscle myosin heavy chain-2 (SM-2), rapidly decreased, disappearing by day 10 in SMC grown on collagen, but was still detectable until day 25 in cells cultured between or within the same gel. These findings indicate that like endothelial cells, vascular SMC can display blood vessel formation behavior in vitro when an appropriate three-dimensional matrix environment is provided to keep them in a relatively higher-differentiated and low-proliferative state.

Inhibition of neointimal formation by natural heparan sulfate proteoglycans of the arterial wall.

Coronary angioplasty has been used clinically for more than a decade to treat primary atherosclerotic plaque. The initial promise of angioplasty as an alternative to coronary bypass surgery has only partially been filled because of a high rate of post-operative restenosis within the first six months.’,2 Post-mortem studies on patients dying several months after apparently successful angioplasty show clear evidence of plaque fracturing and medial dissection with neointimal proliferative tissue filling the crevices overlying the plaque and obstructing the coronary l~men.3 .~ It is believed that exposure of subintimal components of the vessel wall result in deposition of platelets and thrombus formation with release of enzymes and cytokines from platelets, circulating monocytes, endothelial cells, and perhaps vascular smooth muscle cells (SMC) within the vessel waL5 The resulting intimal hyperplasia leads to luminal restenosis consisting predominantly of SMC in a loose connective tissue matrix of sparse collagen fibrils and proteoglycans. While injury is accompanied by the liberation of cytokines, many of which are mitogenic for SMC, there may be more subtle control over SMC migration and proliferation than simply the presence or absence of cytokines. In the uninjured artery, SMC may be actively maintained in a quiescent state by inhibitory molecules. In such a “balance” model for control of cell proliferation, injury would not only present numerous cytokines to the SMC, but also remove the natural inhibitors. Maintaining these natural inhibitors could prove an effective therapeutic tool to prevent restenosis in the clinical setting. Heparan sulfate proteoglycans (HSPG) are a diverse family of molecules present both pericellularly and extracellularly around all c e k 6 The heparan sulfate sidechains from both endothelid’ and SMC8 proteoglycans have been shown to be similarly or more potently inhibitory of SMC proliferation in vitro as the closely related glycosaminoglycan heparin, and are thus good candidates to be naturat inhibitors of SMC proliferation in the normal artery wall. To examine this possibility, proteoglycans were extracted from the aortas of healthy rabbits by a two-step digestion process. The elution profile of the proteoglycans from DEAE-cellulose anion exchange chromatography showed two distinct

Expression of Heparan Sulphate N-deacetylase/N-sulphotransferase by Vascular Smooth Muscle Cells

Heparan sulphate is an important mediator in determining vascular smooth muscle cell (SMC) phenotype. The sulphation pattern of the heparan sulphate chains is critical to their function. We have examined the initial step in the biosynthesis of the sulphated domains mediated by the enzyme heparan sulphate N-deacetylase/N-sulphotransferase (NDST). Rabbit aortic SMC in primary culture exhibited NDST enzyme activity and expressed NDST-1 in their Golgi apparatus, with maximal expression in SMC 2 days after dispersal in primary culture confirmed by Western blot analysis. Endothelial cells, macrophages and fibroblasts expressed NDST-1 but had generally less intense staining than SMC, although SMC expression decreased with culture. The uninjured rat aorta also showed widespread expression of NDST-1. After balloon de-endothelialisation, NDST-1 could not be detected in SMC of the neointima in the early stages of neointimal formation, but was re-expressed at later time points (after 12 weeks). In human coronary arteries, SMC of the media and the diffuse intimal thickening expressed NDST-1, while SMC in the atherosclerotic plaque were negative for NDST-1. We conclude that SMC may regulate their heparan sulphate sulphation at the level of expression of the enzyme heparan sulphate NDST in a manner related to their phenotypic state.

alpha(1)-Adrenoceptors on rabbit aortic smooth muscle cells in culture and in experimental intimal thickening

1. This study has defined α1‐adrenoceptors and their reactivity in rabbit aorta, following removal of the endothelium and formation of a myointimal thickening, and also in smooth muscle cells (SMC) in cell culture which had undergone serial passaging and changes in phenotype.

Inhibition of phenotype modulation, growth, and migration of vascular smooth muscle cells by a guanosine-rich 30-mer phosphorothioate oligodeoxynucleotide.

The testing of a 30‐mer dG‐rich phosphorothioate oligodeoxynucleotide (LG4PS) for effects on the behaviour of vascular smooth muscle cells (VSMC)in vitroandin vivois described. LG4PS at 0.3μminhibited significantly the phenotype modulation of freshly isolated rabbit VSMC, and cell outgrowth from pig aortic explants was inhibited ∼80% by 5μmLG4PS. The growth of proliferating rabbit and pig VSMC was inhibited ∼70% by 0.3μmand 5μmLG4PS, respectively. Though less marked, the antiproliferative effects of LG4PS on human VSMC were comparable to those obtained with heparin. The cytotoxic effects of LG4PS on VSMCin vitrowere low. Despite these promising results, adventitial application of 2–200nmol LG4PS in pluronic gel failed to reduce vascular hyperplasia in balloon‐injured rabbit carotid arteries, and the highest dose caused extensive mortality.