Ethanol And Vascular Smooth Muscle Cell Migration

The accumulation of SMC in the intima of arteries is one of the most prominent features of the atherosclerotic plaque and of the intimal hyperplastic lesions which cause restenosis following angioplasty [35,36], It is increasingly recognized that migration of SMC from the media is a key event in progressive intimal thickening leading to atherosclerosis and restenosis [35-37], Consequently, there has been extensive interest in defining both positive and negative regulators of this process and many factors have been identified that may play a role. While ethanol has been shown in vitro to differentially influence the migration of neuronal cells [38] and melanoma cells [39], its effect on SMC migration has not been defined until recently.

The fibrinolytic plasminogen activator system (Figure 9.3) has been hypothesized to play an important role in intimal thickening after various types of vascular injury [40], The inactive proenzyme plasminogen is activated to the proteolytic enzyme plasmin by two plasminogen activators, tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA). The system is regulated by a series of plasminogen activator inhibitors, the most important of which is thought to be plasminogen activator inhibitor type-1 (PAI-1). Several groups have reported the involvement of the fibrinolytic plasminogen system in SMC migration.

Plasminogen[-í> Plasmin

Degrades matrix Actívalos procolfagenaso

PAI-1

Migration

Figure 9.3 The fibrinolytic plasminogen activator system. The inactive proenzyme plasminogen is converted to the proteolytic enzyme plasmin by urokinase-type plasminogen activator (uPA) or tissue-type plasminogen activator (tPA). The system is regulated by a series of plasminogen activator inhibitors (PAI), of which PAI-1 is believed to be the most physiologically important. (* modulated by hemodynamic forces).

Clowes et al. have demonstrated that neointimal thickening post-vascular injury is associated with upregulation of both uPA and tPA [40], Carmeliet et al. demonstrated that uPA null mice do not exhibit exuberant thickening of the intima after vascular injury [41], PAI-1 has been shown to inhibit SMC migration [42] and inactivation of the PAI-1 gene results in abundant neointimal thickening [43], Recently, it has been demonstrated that pulse pressure due to pulsatile flow increases SMC migration in vitro via uPA- and metalloproteinase (MMP)-dependent mechanisms [44], Indeed, uPA gene deletion resulted in blunting of pressure-induced SMC migration despite the endogenous upregulation of MMP [44], A role for the endothelium in protecting the underlying smooth muscle from hemodynamic forces and in playing a pivotal role in hemodynamic force-induced remodelling is underscored by the finding that endothelial cells prevented flow-induced SMC migration [44,45], It appears that, at least in vitro, fluid shear stress induces endothelial cell PAI-1 gene expression and activity and that this in turn inhibits the flow-induced SMC migratory response [45], Modulation of vascular cell uPA and PAI-1 expression by pulse pressure and shear stress may thus represent an important mechanism whereby hemodynamic forces regulate SMC migration and represent potential targets for ethanol to mediate its vascular effects.

With this in mind, the direct effect of ethanol on SMC migration in vitro has been investigated. Hendrickson et al. pretreated cultures of human smooth muscle cells (HuSMC) under static (no flow) or pulsatile flow conditions in the absence or presence of ethanol and then assessed their migration by Transwell assay. Ethanol dose-dependently inhibited migration of SMC from static cultures. Furthermore, ethanol inhibited the flow-induced increase in SMC migration (Figure 9.4) in the absence of any effect on uPA mRNA expression [46], Other mechanisms that are involved in the smooth muscle cell migratory response under basal and flow conditions that ethanol may modulate include the matrix metalloproteinases (MMPs), tissue inhibitors of metalloproteinases (TIMPs), mitogen activated protein kinase (MAPK), and calcium/calmodulin kinase (CaM kinase II). The possible role of these signaling pathways in pulse pressure-induced SMC migration and the effect, if any, of ethanol on their function and activity merits further investigation.

There have been a number of in vitro studies, using cells cultured under static (no flow) conditions, on the molecular regulatory effects of ethanol on endothelial cell-mediated fibrinolysis. Laug demonstrated an ethanol-induced increase in tPA secretion in cultured bovine

Figure 9.4 The effect of ethanol on pulse pressure-induced smooth muscle cell migration. Human smooth muscle cells (HuSMC) in perfused transcapillary cultures were exposed to low or high pulsatile flow conditions, in the absence or presence of ethanol (20 mM). Cells were then harvested and their migration assessed by Transwell assay. Ethanol inhibited the flow-induced increase in HuSMC migration. (Reproduced by permission of Academic Press from Hendrickson et al. (1999), [46].)

Figure 9.4 The effect of ethanol on pulse pressure-induced smooth muscle cell migration. Human smooth muscle cells (HuSMC) in perfused transcapillary cultures were exposed to low or high pulsatile flow conditions, in the absence or presence of ethanol (20 mM). Cells were then harvested and their migration assessed by Transwell assay. Ethanol inhibited the flow-induced increase in HuSMC migration. (Reproduced by permission of Academic Press from Hendrickson et al. (1999), [46].)

aortic endothelial cells [47], An increase in tPA and uPA mRNA and a simultaneous decrease in PAI-1 mRNA in cultured human endothelial cells, concomitant with increased surface-localized EC fibrinolytic activity have been shown [48,49], Miyamoto et al. reported that ethanol enhances agonist-stimulated cAMP-dependent tPA gene transcription in human and bovine EC through differential modulation of a G-protein [50], These in vitro studies may explain the increased fibrinolytic activity found in the plasma of persons consuming moderate amounts of alcohol. As the fibrinolytic plasminogen system is modulated by hemodynamic forces it may be more physiologically relevant to study the effect of ethanol on endothelial fibrinolytic activity in cells exposed to fluid shear stress.

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