Melanie E. Ivey

Endothelin-1 signalling in vascular smooth muscle: Pathways controlling cellular functions associated with atherosclerosis

Atherosclerosis is the primary ischaemic vascular condition underlying a majority of cardiovascular disease related deaths. Endothelin-1 is a vasoactive peptide agent upregulated in atherosclerosis and in conjunction with its G protein-coupled receptors exerts diverse actions on all cells of the vasculature in particular vascular smooth muscle cells (VSMC). The effects of endothelin-1 include cell proliferation, migration and contraction, and the induction of extracellular matrix components and growth factors. VSMC as the major component of the neointima in atherosclerotic plaques accordingly play a key role in atherogenesis. In this review we examine classic and novel signalling pathways activated by endothelin-1 in VSMC (including phospholipase C, adenylate cyclase, Rho kinase, transactivation of receptor tyrosine kinases, mitogen activated protein kinase cascades and beta-arrestin) and their likely impact on the development and progression of atherosclerosis.

Inhibitory Activity of Clinical Thiazolidinedione Peroxisome Proliferator Activating Receptor-γ Ligands Toward Internal Mammary Artery, Radial Artery, and Saphenous Vein Smooth Muscle Cell Proliferation

Background—The proliferation of vascular smooth muscle cells (VSMCs) is a known response to arterial injury that is an important part of the process of restenosis and atherosclerosis. People with diabetes have an increased risk of cardiovascular disease resulting from accelerated coronary atherosclerosis. The newest drugs for Type 2 diabetes are thiazolidinediones, which are insulin-sensitizing peroxisome proliferator activating receptor-&ggr; (PPAR&ggr;) ligands. We investigated the antiproliferative effects of troglitazone, rosiglitazone, and pioglitazone on VSMCs derived from the three vascular beds used for coronary artery by-pass grafting: the internal mammary and radial artery and saphenous veins. Methods and Results—The three vessels yielded proliferating cells of slightly differing morphology. Inhibition of cell proliferation was assessed by cell counting and cell cycle studies by Western blotting for phosphorylated retinoblastoma protein. All three thiazolidinediones showed inhibitory potency toward cell proliferation with a potency troglitazone>rosiglitazone≈pioglitazone, and this potency profile was maintained toward the growth factor and insulin-stimulated phosphorylation of the retinoblastoma protein, which controls cell cycle progression. Conclusion—The inhibitory potency of clinical thiazolidinediones toward different vascular sources is dependent on the individual thiazolidinedione and very little on the vascular source.

Thrombin regulates vascular smooth muscle cell proteoglycan synthesis via PAR-1 and multiple downstream signalling pathways

INTRODUCTION Atherosclerosis is the underlying pathological process of most cardiovascular disease. Thrombin is a serine protease which can activate protease activated receptors (PAR) on vascular smooth muscle cells (VSMC) to elicit cellular responses that can contribute to the pathogenesis of atherosclerosis. Human atherosclerosis commences with the binding and retention of lipoproteins by the glycosaminoglycan (GAG) chains of chondroitin/dermatan sulfate proteoglycans. The potential effects of thrombin on the synthesis and structure of CS/DS proteoglycans produced by VSMCs was investigated. MATERIALS AND METHODS VSMCs were derived from human internal mammary arteries. Proteoglycan synthesis was assessed by [(35)S]sulfate and [(3)H]glucosamine incorporation. Proteoglycan size was assessed by SDS-PAGE and size exclusion chromatography. RESULTS AND CONCLUSION Thrombin caused a dose-dependent increase in [(35)S]sulfate and [(3)H]glucosamine incorporation with maximum effects of approximately 150% at the highest doses tested. This increase was associated with increased size of biglycan and decorin assessed by SDS-PAGE. Chemically cleaved glycosaminoglycan (GAG) chains analyzed by SDS-PAGE and size exclusion chromatography were larger for proteoglycans from thrombin treated cells. VSMCs synthesize small GAGs when provided with exogenous xyloside and thrombin treatment also increased the size of the secreted xyloside GAGs. The effect of thrombin was not mimicked by the catalytically inactive FPRCK-HCT and was blocked in a concentration- dependent manner by the PAR-1 antagonist, JNJ5177049. Inhibition of PK C with GF 109203X resulted in concentration dependent but partial inhibition of [(35)S]sulfate incorporation accompanied by a reduction in the size of biglycan and decorin. Epidermal growth factor (EGF) stimulated [(35)S]sulfate incorporation and increased proteoglycan size and this was completely blocked by the EGF receptor tyrosine kinase inhibitor AG1478. AG1478 partially (32%, p<0.01) blocked the effect of thrombin. Thrombin treatment of VSMCs increased the proportion of disaccharides sulfated at the 6 position of the GalNAc residues. Thus, thrombin has actions on VSMCs which increase the length and modify the sulfation pattern of GAG chains on proteoglycans in a manner that would enhance the binding of LDL. If manifest in vivo, this effect on proteoglycan synthesis and structure represents a new biochemical mechanism through which thrombin contributes to the development of atherosclerosis.

Endothelin-1 Actions on Vascular Smooth Muscle Cell Functions As a Target for the Prevention of Atherosclerosis

The formation and progression of atherosclerotic plaques followed by rupture, thrombus formation and vessel blockage leads to ischemic tissue damage and the clinical condition underlying most cardiovascular disease. Therapeutic agents for the prevention of atherosclerosis have all targeted epidemiologically-identified and relatively easily measured risk factors (e.g. lipids and blood pressure). This strategy has proven somewhat effective but is of less than optimal efficacy as rates of cardiovascular disease remain high. Treatment targeting the mechanisms of atherosclerosis in the vessel wall is a conceptually attractive proposition to complement the risk factor directed strategy. Vascular smooth muscle cells (VSMC) are the major cellular component of the vascular media and migration and proliferation leads to the formation of the neointima the development of which renders the vessels particularly sensitive to atherosclerosis. Numerous hormones and growth factors act on VSMC to cause migration, proliferation and the secretion of extracellular matrix and modulation or dysfunction of these processes is the most likely cause of atherosclerosis. Endothelin-1 (ET-1) is a 21 amino acid peptide that acts on 7 transmembrane G protein coupled receptors to elicit a plethora of responses that can modulate the behaviour of VSMCs and thus impact on the development of atherosclerosis. ET-1 is elevated in atherosclerotic plaques. People with diabetes have accelerated atherosclerosis and also show elevated plasma levels of ET-1. This review addresses the actions of ET-1 on VSMC and the signalling pathways through which it mediates its effects as the latter represent potential therapeutic targets for the prevention of atherosclerosis.

Endothelin-1 activates ETA receptors on human vascular smooth muscle cells to yield proteoglycans with increased binding to LDL

OBJECTIVE Lipid retention in the vessel wall by glycosaminoglycan (GAG) chains on chondroitin/dermatan sulfate proteoglycans synthesized by vascular smooth muscle cells (VSMC) have recently been established as an early event in human coronary artery atherosclerosis. GAG structure can be altered by growth factors resulting in enhanced binding to low density lipoprotein (LDL). The aim of this study was to determine if proteoglycans produced by endothelin-1 treated VSMCs had increased binding to human LDL, to examine the effect of endothelin-1 on the synthesis and structure of proteoglycans and to elucidate the signalling pathway. METHODS AND RESULTS Endothelin-1 stimulated an increase in [(35)S]sulfate and [(3)H]glucosamine incorporation into proteoglycans produced by human VSMC. The increase was due to an increase in GAG chain size assessed by SDS-PAGE and size exclusion chromatography. Increased radiolabel incorporation was inhibited by an ET(A) but not an ET(B) receptor antagonist. Endothelin-1 stimulated an increase in the 6:4 position sulfation ratio on the disaccharides of the GAG chains, an effect that was blocked by bosentan. The EGF receptor antagonist AG1478 did not affect the increase in GAG size mediated by endothelin-1. Inhibition of protein kinase C (PKC) with GF109203X or down regulation by PMA pre-treatment attenuated the effect of endothelin-1 on GAG synthesis. CONCLUSION These data demonstrate that endothelin-1 stimulates changes in GAG chain structure that increase binding to LDL. This action of endothelin-1 may represent a new target for the prevention of lipid binding within the vascular wall and the associated complications resulting from this interaction.

The effect of PPAR ligands to modulate glucose metabolism alters the incorporation of metabolic precursors into proteoglycans synthesized by human vascular smooth muscle cells

Abstract PPAR ligands are important effectors of energy metabolism and can modify proteoglycan synthesis by vascular smooth muscle cells (VSMCs). Describing the cell biology of these important clinical agents is important for understanding their full clinical potential, including toxicity. Troglitazone (10 μM) and fenofibrate (30 μM) treatment of VSMCs reduces (35S)-sulphate incorporation into proteoglycans due to a reduction of glycosaminoglycan (GAG) chain length. Conversely, under physiological glucose conditions (5.5 mM), the same treatment increases (3H)-glucosamine incorporation into GAGs. This apparent paradox is the consequence of an increase in the intracellular (3H)-galactosamine specific activity from 48.2 ± 3.2 μCi/ μmol to 90.7 ± 11.0 μCi/ μmol (P < 0.001) and 57.1 ± 2.6 μCi/ μmol (P < 0.05) when VSMCs were treated with troglitazone and fenofibrate, respectively. The increased specific activity observed with troglitazone (10 μM) treatment correlates with a two-fold increase in glucose consumption, while fenofibrate (50 μM) treatment showed a modest (14.6%) increase in glucose consumption. We conclude that the sole use of glucosamine precursors to assess GAG biosynthesis results in misleading conclusions when assessing the effect of PPAR ligands on VSMC proteoglycan biosynthesis.