Transcription factor NFAT1 mediates endothelial-to-mesenchymal transition and atherogenesis under disturbed flow conditions

Background Atherosclerosis is a prevalent vascular pathology with a partially understood pathogenesis. Notably, lesions manifest non-uniformly, favoring bifurcations and curvatures characterized by complex hemodynamics. We hypothesize that disturbed flow (DF) plays a pivotal role in disease initiation and progression. Objective Disturbed flow (DF) is known to trigger endothelial-to-mesenchymal transition (EndMT). Through bioinformatics, we identified transcription factor NFAT1 as being closely linked to both EndMT and atherogenesis. This study aims to delineate the mechanistic role of NFAT1 in DF-mediated EndMT, elucidating its impact on endothelial function and inflammation. Methods and Results Atherosclerosis was induced in apoE−/− mice utilizing a partial carotid artery ligation (PCL) model, while DF and pulsatile flow (PF) conditions were recapitulated in primary human umbilical vein endothelial cells (HUVECs) using an orbital shaker system. We observed a robust induction of EndMT alongside the upregulation of NFAT1 within the atherosclerotic lesions of mice; a similar phenotypical shift was corroborated in HUVECs subjected to DF compared with those under PF. Furthermore, chromatin immunoprecipitation (ChIP) and dual-luciferase reporter assays identified NFAT1 as a potent transcriptional activator of the CXCL8 gene. We therefore hypothesized that DF-induced NFAT1 elevation drives EndMT and atherogenesis via transcriptional activation of CXCL8, thereby promoting interleukin-8 (IL-8) secretion. Accordingly, enzyme-linked immunosorbent assay (ELISA) revealed significantly elevated IL-8 levels in the supernatants of DF-treated HUVECs, as well as increased plasma levels of IL-1 (the rodent functional homologue of human IL-8) in atherosclerotic mice. Notably, siRNA-mediated silencing of NFAT1 markedly attenuated DF-induced IL-8 secretion and the EndMT phenotype. Similarly, pharmacological inhibition of the mechanosensitive ion channel Piezo1 with GsMTx4 significantly diminished DF-triggered IL-8 activation and subsequent EndMT. To further validate the role of IL-8, exogenous human IL-8 was administered to HUVECs under static (ST) conditions, which successfully recapitulated a pronounced EndMT profile accompanied by the phosphorylation of the NF-κB p65 subunit. Conclusions In summary, our findings unveil a critical role for the transcription factor NFAT1 in disturbed flow (DF)-driven endothelial-to-mesenchymal transition (EndMT) and the subsequent progression of atherosclerosis. We demonstrate that the Piezo1/NFAT1/CXCL8 axis serves as a quintessential molecular bridge, transducing DF-mediated mechanical stimuli into endothelial inflammation and maladaptive morphological transformation. Collectively, our data suggest that targeting this mechanotransduction pathway may offer a promising therapeutic strategy for the prevention and clinical management of atherosclerotic cardiovascular disease.