Investigating the role of endothelin receptor subtypes in the response to vascular injury

Abstract

Neointimal hyperplasia, the proliferative growth of the innermost layer of the blood vessel wall, is a key process in the response to vascular injury, underlying conditions such as post-interventional restenosis and vein/arterial graft disease. One of the many mediators implicated in this process is endothelin-1 (ET-1), a potent vasoconstrictor with pro-inflammatory and pro-mitogenic actions, which acts through ETA and ETB receptor subtypes. It is well established that ET-1 increases, and ETA blockade reduces, neointima formation following vascular injury. The role of ETB is less clear because these receptors mediate potentially beneficial actions in endothelial cells (EC; such as nitric oxide production, and ET-1 clearance) but detrimental effects elsewhere (such as vascular smooth muscle) and it has been recently reported that non-cell-specific ETB deficiency is associated with increased neointimal lesion size following injury. The work described in this thesis addressed the hypothesis that endogenous ET-1 contributes to neointimal hyperplasia by activation of the ETA receptor, and that this action is moderated by concurrent activation of the ETB receptor expressed in EC. The role of ET receptors in neointimal lesion development was assessed using two models of femoral arterial injury in the mouse: (i) an established method of intraluminal wire-injury, and (ii) adaptation of a model of ligation injury that induces robust neointimal lesion formation without physical damage to the endothelium. Lesion development was assessed using standard histological techniques and this was augmented by development of quantitative optical projection tomography (OPT) to allow three-dimensional analysis of lesions. The role of ETA and ETB receptors in these models was addressed using suitable pharmacological ET receptor antagonists. Following wire-injury, selective ETB blockade (A192621; 30mg.kg-1.day-1; 35 days) increased lesion size and blood pressure without significant altering lesion composition. In contrast, selective ETA blockade (atrasentan; 10mg.kg-1.day-1; 35 days) reduced lesion size and blood pressure. Combined ETA+ETB antagonism had no effect on lesion size, despite reducing blood pressure, and reducing collagen content of the lesions. In the ligation model, neither ETA selective, ETB selective nor ETA+ETB blockade altered lesion size as assessed by standard histology but analysis by OPT indicated that ETA blockade, with or without concurrent ETB blockade, reduced lesion volume. The influence of ETB receptors expressed by ECs on lesion formation was addressed using EC-specific ETB knockout mice. Small vessel myography indicated that endothelium-dependent relaxation was unaltered in femoral arteries from these mice. In addition, no effect on lesion size or rate of development was observed in either wire- or ligation-injury models of neointima formation (although subtle effects on lesion and medial composition were apparent after intra-luminal injury). These results indicate that ETB receptor activation can moderate the detrimental actions of the ETA receptor on neointimal lesion progression, and that this role is dependent on the mode of vascular injury. Furthermore, in this setting, this beneficial action is not primarily mediated by ETB expressed by EC, suggesting that ETB in other cell types can reduce lesion development through another, unidentified mechanism. Therefore, while both ETA selective and non-selective ETA/B antagonists are currently in clinical use, in conditions where similar arterial remodelling processes occur, selective ETA receptor antagonists might be preferred.