The responses of endothelium to insult : does endothelial heterogeneity play a role in in vitro cell models

[摘要] ENGLISH ABSTRACT: Endothelial injury and dysfunction precede the development of cardiovascular diseases. The endothelium may be regarded as the first line of defence against inflammation / obesity-induced vascular injury, therefore gaining more information on the mechanisms of injury and response to injury, as well as modulating endothelial function may be key in the prevention of cardiovascular diseases. Endothelial cells differ in structure and function, therefore endothelial heterogeneity may be relevant when investigating endothelial function and dysfunction. Understanding endothelial heterogeneity in response to pathophysiological stimuli may be of significance in the prevention of cardiovascular diseases. Oleanolic acid (OA), a plant-derived triterpenoid, has been shown to possess endothelio-protective properties; however, its role in reversing endothelial injury is poorly understood.This study investigated endothelial heterogeneity between aortic endothelial cells (AECs) and cardiac microvascular endothelial cells (CMECs) at baseline and in response to an inflammatory insult via the cytokine, tumour necrosis factor-alpha (TNF-α). An in vitro model of endothelial injury was developed by treating AECs and CMECs with 20 ng/ml TNF-α for 24 hours. Endothelial heterogeneity was investigated by comparing intracellular nitric oxide (NO) and reactive oxygen species (ROS) production, protein expression and phosphorylation, and large-scale protein expression and regulation in AECs and CMECs. The experimental techniques included flow cytometry, western blots and proteomic analyses. An ex vivo model of endothelial injury was included to investigate vascular function in aortic rings from lean and high fat diet (HFD) rats. The role of OA in reversing TNF-α-induced injury and modulating vascular function in the ex vivo model was investigated. Although baseline NO-levels were similar between AECs and CMECs, heterogeneity was observed with regards to the NO biosynthetic pathway in terms of increased eNOS expression in CMECs. Baseline ROS levels were heterogeneous between AECs and CMECs, interestingly CMECs possessed higher anti-oxidant capacity. An in vitro model of TNF-α-induced injury was confirmed by decreased NO-levels, increased ROS-levels and necrosis, up-regulation of apoptotic proteins and activation of inflammatory pathways in AECs and CMECs. Here, heterogeneity between AECs and CMECs was also observed: endothelial activation was mediated through different proteins in AECs (CD9 molecule, galectin) and CMECs (ICAM-1 and IL-36α). Apoptosis was mediated by caspase 3 in AECs and Bid in CMECs. AECs appeared to advance to a dysfunctional state shown by up-regulation of endothelin-converting enzyme and angiotensin II-converting enzyme, while CMECs maintained an activated state. OA reversed TNF-α-induced injury through restoring NO-production, decreasing ROS-production in both AECs and CMECs, and inhibiting necrosis in AECs. In the ex vivo model of injury, aortic rings from 16-week HFD rats showed a pro-contractile response to phenylephrine-induced contraction, a response that was reversed by OA. In conclusion, we demonstrated novel findings with regards to endothelial heterogeneity between AECs and CMECs under baseline and TNF-α-treated conditions. Although reduced NO-bioavailability may be the hallmark of endothelial dysfunction, signalling pathways mediating endothelial injury may differ between cell types as was shown in this study. We demonstrated that OA possess protective properties in AECs and CMECS, an observation which was translated to the ex vivo model.

[发布机构] Stellenbosch University