Narin Osman

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.

Hyperelongated biglycan: the surreptitious initiator of atherosclerosis

Purpose of review To outline a role for the dermatan sulfate proteoglycan biglycan and specifically its growth factor modified form having elongated glycosaminoglycan chains as being a primary initiator of atherosclerosis. Recent findings Antiatherosclerotic therapies have mostly targeted epidemiologically identified, experimentally confirmed risk factors. The efficacy of such therapies is less than optimal, and rates of cardiovascular disease remain stubbornly high. A variety of targets have been actively pursued, but as yet no new therapy has emerged that specifically targets the vessel wall. One area concerns the role of proteoglycans in the trapping of atherogenic lipoproteins as an early and initiating step in atherogenesis. On the basis of studies in human coronary arteries, the prime proteoglycan for lipoprotein retention is biglycan. The glycosaminoglycan chains on biglycan are subject to regulation that yields several structural changes, but most prominently elongation of the chains to form ‘hyperelongated biglycan’. Multiple animal studies and a recent human disorder study have demonstrated the colocalization of atherogenic lipoproteins with biglycan in atherosclerotic lesions. Moreover, in the human atherosclerosis, the deposition of lipid appears to precede the chronic inflammatory response typical of atherosclerotic lesions. Summary The process of biglycan-associated glycosaminoglycan elongation represents a novel potential therapeutic target worthy of full investigation for the prevention of atherosclerosis.

TGF-β stimulates biglycan synthesis via p38 and ERK phosphorylation of the linker region of Smad2

Transforming growth factor (TGF)-β treatment of human vascular smooth-muscle cells increases the expression of biglycan and causes marked elongation of its glycosaminoglycan (GAG) chains. We investigated the role of MAP kinases and Smad transcription factors in this response. TGF-β-stimulated phosphorylation of p38, ERK, and JNK as well as Smad2 at both its carboxy terminal (phospho-Smad2C) and in the linker region (phospho-Smad2L). Pharmacological inhibition of ERK and p38 blocked TGF-β-mediated GAG elongation and expression of biglycan whereas inhibition of JNK had no effect. Inhibition of ERK and p38 but not JNK attenuated the effect of TGF-β to increase phospho-Smad2L. High levels of phospho-Smad2L were detected in a nuclear fraction of TGF-β treated cells. Thus, MAP kinase signaling through ERK and p38 and via phosphorylation of the linker region of Smad2 mediates the effects of TGF-β on biglycan synthesis in vascular smooth-muscle cells.

Smad and p38 MAP Kinase-mediated Signaling of Proteoglycan Synthesis in Vascular Smooth Muscle

Atherosclerosis is the underlying pathological process of most cardiovascular disease. A critical component of the “response to retention” hypothesis of atherogenesis is proteoglycan/low density lipoprotein (LDL) binding. Transforming growth factor β (TGF-β) is present in atherosclerotic lesions, regulates vascular smooth muscle cell (VSMC) proteoglycan synthesis via an unknown signaling pathway, and increases proteoglycan/LDL binding. This pathway was investigated using the activin receptor-like kinase 5 (ALK5) inhibitor SB431542 and inhibitors of p38 MAP kinase as a possible downstream or alternative mediator. TGF-β stimulated and SB431542 inhibited the phosphorylation of Smad2/3. In human VSMC, TGF-β increased [35S]sulfate incorporation into proteoglycans associated with a 19% increase in glycosaminoglycan (GAG) chain size by size exclusion chromatography. SB431542 caused a concentration-dependent decrease in TGF-β-mediated [35S]sulfate incorporation with 92% inhibition at 3 μm. Two different p38 MAP kinase inhibitors, SB203580 and SB202190, but not the inactive analogue SB202474, concentration-dependently blocked TGF-β-mediated [35S]sulfate incorporation. TGF-β increased [3H]glucosamine incorporation into glycosaminoglycans by 180% and [35S]Met/Cys incorporation into proteoglycan core proteins by 35% with both effects completely inhibited by SB431542. Blocking both Smad2/3 and p38 MAP kinase pathways prevented the effect of TGF-β to increase proteoglycan to LDL binding. TGF-β mediates its effects on proteoglycan synthesis in VSMCs via the ALK5/Smad2/3 phosphorylation pathway as well as via the p38 MAP kinase signaling cascade. Further studies of downstream pathways controlling proteoglycan synthesis may identify potential therapeutic targets for the prevention of atherosclerosis and cardiovascular disease.

Transforming growth factor-β regulation of proteoglycan synthesis in vascular smooth muscle: contribution to lipid binding and accelerated atherosclerosis in diabetes

Atherosclerosis is accelerated in the setting of diabetes, but the factors driving this phenomenon remain elusive. Hyperglycemia leads to elevated levels of transforming growth factor (TGF)‐β and TGF‐β has been implicated as a factor in atherosclerosis. Given the established association between hyperglycemia and elevated TGF‐β, it is plausible that elevated TGF‐β levels in diabetes play a pathogenic role in the development of accelerated atherosclerosis. TGF‐β is a potent regulator of extracellular matrix synthesis, including many actions on proteoglycan synthesis that lead to increased binding to low‐density lipoprotein and therefore potentially increased lipid retention in the vessel wall and accelerated atherosclerosis. TGF‐β signals through the canonical TGF‐β receptor I‐mediated phosphorylation of Smad transcription factors and TGF‐β signaling is also known to involve, positively and negatively, interactions with the mitogen‐activated protein kinase pathways. The focus of the present review is on the effects of TGF‐β on proteoglycan synthesis in vascular smooth muscle and particularly the signaling pathways through which TGF‐β exerts its effects, because those pathways may be therapeutic targets for the prevention of pathological modifications in the proteoglycan component of the vessel wall in the vascular diseases of diabetes.

Imatinib inhibits vascular smooth muscle proteoglycan synthesis and reduces LDL binding in vitro and aortic lipid deposition in vivo

The ‘response to retention’ hypothesis of atherogenesis proposes that proteoglycans bind and retain low‐density lipoproteins (LDL) in the vessel wall. Platelet‐derived growth factor (PDGF) is strongly implicated in atherosclerosis and stimulates proteoglycan synthesis. Here we investigated the action of the PDGF receptor inhibitor imatinib on PDGF‐mediated proteoglycan biosynthesis in vitro, lipid deposition in the aortic wall in vivo and the carotid artery ex vivo. In human vSMCs, imatinib inhibited PDGF mediated 35S‐SO4 incorporation into proteoglycans by 31% (P < 0.01) and inhibited PDGF‐mediated size increases in both chemically cleaved and xyloside associated glycosaminoglycan (GAG) chains by 19%, P < 0.05 and 27%, P < 0.05, respectively. Imatinib decreased PDGF stimulation of the 6:4 position sulphation ratio of disaccharides. The half maximal saturation value for LDL binding for proteoglycans from PDGF stimulated cells in the presence of imatinib was approximately 2.5‐fold higher than for PDGF treatment alone. In high fat fed ApoE−/– mice, imatinib reduced total lipid staining area by ∼31% (P < 0.05). Carotid artery lipid accumulation in imatinib treated mice was also reduced. Furthermore, we demonstrate that imatinib inhibits phosphorylation of tyrosine 857, the autophosphorylation site of the PDGF receptor, in vSMCs. Thus imatinib inhibits GAG synthesis on vascular proteoglycans and reduces LDL binding in vitro and in vivo and this effect is mediated via the PDGF receptor. These findings validate a novel mechanism to prevent cardiac disease.

PDGF β-Receptor Kinase Activity and ERK1/2 Mediate Glycosaminoglycan Elongation on Biglycan and Increases Binding to LDL

The initiation of atherosclerosis involves the subendothelial retention of lipoproteins by proteoglycans (PGs). Structural characteristics of glycosaminoglycan (GAG) chains on PGs influence lipoprotein binding and are altered adversely by platelet-derived growth factor (PDGF). The signaling pathway for PDGF-mediated GAG elongation via the PDGF receptor (PDGFR) was investigated. In human vascular smooth muscle cells, PDGF significantly increased (35)S-sulfate incorporation into PGs and GAG chain size. PGs from PDGF-stimulated cells showed increased binding low-density lipoprotein (P < 0.001) in gel mobility shift assays. Knockdown of PDGFRbeta using small interfering RNA demonstrated that PDGF mediated changes in PGs via PDGFRbeta. GAG synthesis and hyperelongation was blocked by inhibition of receptor tyrosine kinase autophosphorylation site Tyr857 activity using Ki11502 or imatinib. Downstream signaling to GAG hyperelongation was mediated through ERK MAPK and not phosphatidylinositol-3 kinase or phospholipase Cgamma. In high-fat-fed apolipoprotein E(-/-) mice, inhibition of PDGFRbeta activity by imatinib reduced aortic total lipid staining area by 35% (P < 0.05). Inhibition of PDGFRbeta tyrosine kinase activity leads to inhibition of GAG synthesis on vascular PGs and aortic lipid area in vivo. PDGFRbeta and its signaling pathways are potential targets for novel therapeutic agents to prevent the earliest stages 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.

Endothelin-1 Stimulation of Proteoglycan Synthesis in Vascular Smooth Muscle is Mediated by Endothelin Receptor Transactivation of the Transforming Growth Factor-β Type I Receptor

We utilized human vascular smooth muscle cells to address the question if a G-protein-coupled receptor, the endothelin (ET) receptor, could transactivate a serine/threonine kinase receptor, specifically the transforming growth factor (TGF)-β receptor, TβRI. Functionality of the interaction was addressed by studying endothelin-1-stimulated proteoglycan synthesis. Signaling molecules were assessed by Western blotting and proteoglycan synthesis by [35S]sulfate and 35S-met/cys incorporation and molecular size by SDS-PAGE. Endothelin-1 treatment led to a time- and concentration-dependent increase in cytosolic phosphoSmad2C, which was inhibited by the mixed endothelin receptor antagonist bosentan and the TβRI antagonist SB431542. Endothelin-1 treatment led to a time-dependent increase in nuclear phosphoSmad2C. Endothelin-1-stimulated proteoglycan synthesis was partially inhibited (40%) by SB431542 and completely blocked by bosentan. The effect of endothelin-1 to stimulate an increase in glycosaminoglycan size on biglycan was also blocked in a concentration-dependent manner by SB431542. These data extend the current paradigm of G-protein coupled receptor signaling to include the transactivation of the serine kinase receptor for TGF-β (TβRI). This response should be considered in the context of response to endothelin-1, and the options for therapeutically targeting endothelin-1 are accordingly broadened to include downstream signaling otherwise associated with TGF-β receptor activation.