Nuclear Envelope Proteins Nesprin2 And LaminA Regulated Proliferation And Apoptosis Of Endothelial Cells In Response To Shear Stress

The study of shear stress in arterial vessels has important theoretical and clinical significance for our understanding and prevention of cardiovascular disease. Mechanical stress plays a critical role in vascular remodeling, which is an important early component of many cardiovascular diseases, including atherosclerosis. Endothelial cells(ECs) and vascular smooth muscle cells(VSMCs) constitute the main cellular components of the vascular wall. As ECs are exposed directly to blood flow, they are subject to shear stress and, because of their close physical relationship to VSMCs, one might consider that these latter cells are also involved in the process of vascular remodeling.Our previous vascular proteomics study revealed that LaminA might be a protein affected by mechanical stresses; hence, here we have investigated how changes in expression of the nuclear envelope(NE) proteins Nesprin2, SUN1 and LaminA in ECs brought about through shear stress lead to EC proliferation and to apoptosis.ECs of rat aortas were either cultured together with or without VSMCs in a flow chamber system using a parallel plate design, where they were exposed to normal(NSS, 15 dyn/cm2) and low shear stresses(LowSS, 5 dyn/cm2). We found that LowSS depressed the expression of Nesprin2, SUN1 and LaminA in ECs; and both proliferation and apoptosis of ECs were promoted. Co-cultured VSMCs had no effect on NE expressions. By using siRNA to downregulate Nesprin2, SUN1 and LaminA we could also induce EC proliferation and apoptosis.To directly test our hypothesis that Nesprin2 and Lamin A are regulators of EC proliferation and apoptosis, we constructed overexpression plasmids of these proteins, and showed that the plasmids could reverse the proliferaion and apoptosis of ECs brought about by LowSS. The activation of transcription factors, linked specifically with these two NE proteins, was quantified using a Protein/DNA array after ECs were transfected with both their targeted siRNAs and overexpression plasmids. Of specific interest were AP-2 and TFIID for Nesprin2 and Stat-1, 3, 5 and 6 for Lamin A. We also measured how activation of these transcription factors, following application of RNAi or shear stress, was affected. Furthermore, using Ingenuity Pathway Analysis software(IPA) and real-time RT-PCR, the downstream target genes of AP-2, TFIID, Stat-1, 3, 5 and 6 respectively, impacted by Nesprin2 or LaminA were investigated under LowSS application.In general, LowSS significantly depressed the expressions of Nesprin2, SUN1 and LaminA. The downregulation of Nesprin2 induced the activations of its downstream transcription factors AP-2 and TFIID, which subsequently positively mediated AP-2 target gene GEM and TFIID target gene FOS, while negatively regulated TFIID target gene LDLR. On the other hand, LowSS also repressed the expression of LaminA, and then suppressed the phosphorylation of Stat-1, 3, 5, 6. The downregulation of Stat-1, 3, 5, 6 phosphorylation modulated their target genes of BCL2L1, IRF1, IFNG, IL4 and CCND2, and induced the ECs proliferation and apoptosis eventually. These data shed light into the novel aspect for mechanobiological mechanism of EC dysfunction induced by LowSS and potential orientation of atherosclerosis-relevant diseases investigation.