Impinging Flow Mediates Vascular Endothelial Cell Injury through the PKCα/ERK/PPARγ Pathway in vitro

Abstract

Background: This study aims to elucidate the mechanisms underlying endothelial injury in the context of intracranial aneurysm formation and development, which are associated with vascular endothelial injury caused by hemodynamic abnormalities. Specifically, we focus on the involvement of PKCα, an intracellular signaling transmitter closely linked to vascular diseases, and its role in activating MAPK. Additionally, we investigate the protective effects of PPARγ, a vasculoprotective factor known to attenuate vascular injury by mitigating the inflammatory response in the vessel wall. Methods: The study employs a modified T chamber to replicate fluid flow conditions at the artery bifurcation, allowing us to assess wall shear stress effects on human umbilical vein endothelial cells (HUVECs) in vitro. Through experimental manipulations involving PKCα knockdown and Ca2+ and MAPK inhibitors, we evaluate the phosphorylation status of PKCα, NF-κB, ERK5, ERK1/2, JNK1/2/3, and P38, as well as the expression levels of PPARγ, NF-κB, and MMP2 via western blot analysis. The cellular localization of phosphorylated NF-κB was determined using immunofluorescence. Results: Our results showed that impinging flow resulted in the activation of PKCα, followed by the phosphorylation of ERK5, ERK1/2, and JNK1/2/3, leading to a decrease in PPARγ expression, an increase in the expression of NF-κB and MMP2, and the induction of apoptotic injury. Inhibition of PKCα activation or knockdown of PKCα using shRNA leads to a suppression of ERK5, ERK1/2, JNK1/2/3, and P38 phosphorylation, an elevation in PPARγ expression, and a reduction in NF-κB and MMP2 expression, alleviated apoptotic injury. Furthermore, we observe that the regulation of PPARγ, NF-κB, and MMP2 expression is influenced by ERK5 and ERK1/2 phosphorylation, and activation of PPARγ effectively counteracts the elevated expression of NF-κB and MMP2. Conclusion: Our findings suggest that the PKCα/ERK/PPARγ pathway plays a crucial role in mediating endothelial injury under conditions of impinging flow, with potential implications for vascular diseases and intracranial aneurysm development.