Supplementary Material for: Impinging flow mediates vascular endothelial cell injury through the PKCα/ERK/PPARγ pathway in vitro

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.

背景：本研究旨在阐明颅内动脉瘤形成与发展过程中内皮损伤的潜在机制，该过程与血流动力学异常引发的血管内皮损伤密切相关。具体而言，本研究聚焦于与血管疾病紧密关联的细胞内信号转导分子蛋白激酶Cα（PKCα），以及其在激活丝裂原活化蛋白激酶（MAPK）过程中的作用。此外，本研究还探讨了过氧化物酶体增殖物激活受体γ（PPARγ）的保护作用，该因子作为血管保护因子，可通过减轻血管壁炎症反应缓解血管损伤。
方法：本研究采用改良T型腔室模拟动脉分叉处的流体流动条件，以体外评估壁面切应力对人脐静脉内皮细胞（HUVECs）的影响。通过PKCα敲低以及钙、丝裂原活化蛋白激酶抑制剂的实验操作，本研究采用蛋白质印迹法分析了PKCα、核因子κB（NF-κB）、细胞外调节蛋白激酶5（ERK5）、ERK1/2、c-Jun氨基末端激酶1/2/3（JNK1/2/3）及P38的磷酸化水平，同时检测了过氧化物酶体增殖物激活受体γ（PPARγ）、核因子κB（NF-κB）及基质金属蛋白酶2（MMP2）的表达水平。此外，本研究采用免疫荧光法确定了磷酸化NF-κB的细胞定位。
结果：研究结果显示，冲击流可激活PKCα，随后引发ERK5、ERK1/2及JNK1/2/3的磷酸化，进而导致PPARγ表达下调、NF-κB与MMP2表达上调，并诱导细胞凋亡损伤。抑制PKCα激活或通过短发夹RNA（shRNA）敲低PKCα，可抑制ERK5、ERK1/2、JNK1/2/3及P38的磷酸化，提升PPARγ表达，降低NF-κB与MMP2的表达，从而减轻细胞凋亡损伤。进一步研究发现，PPARγ、NF-κB及MMP2的表达调控受ERK5与ERK1/2磷酸化的影响，而PPARγ的激活可有效逆转NF-κB与MMP2的高表达。
结论：本研究结果表明，PKCα/ERK/PPARγ通路在介导冲击流条件下的内皮损伤过程中发挥关键作用，其可能对血管疾病及颅内动脉瘤的发生发展具有潜在的临床意义。