Simvastatin rescues vascular health by targeting epigenetic-regulated endothelial-to-mesenchymal transition: a revival of pleiotropic effects?

Comment on the article ‘Statins improve endothelial function via suppression of epigenetic-driven EndMT’ published in Nature/Cardiovascular Research; doi.org/10.1038/s44161-023-00267-1. To investigate the effects of statins on endothelial function, Liu et al.1 generated human endothelial cells (ECs) derived from induced pluripotent stem cells (iPSC-ECs) from three healthy donors without familial hypercholesterolaemia or cardiovascular diseases (CVDs). The study tested the effects of seven statins (pravastatin, atorvastatin, mevastatin, fluvastatin, lovastatin, rosuvastatin, and simvastatin) at three concentrations (0.1 µM, 1 µM and 10 µM) on iPSC-EC functions. Simvastatin upregulated NOS3 expression, a hallmark of functional ECs, in a dose-dependent manner compared to other statins that either had no effect or increased NOS3 expression at a concentration of 10 µM that was found to be cytotoxic. Therefore, 1-µM simvastatin was used for functional assays and transcriptomic and epigenetic profiling in iPSC-ECs. The iPSC-ECs treated with simvastatin showed a higher density of capillary-like tubes and produced a higher level of nitric oxide (NO) than their vehicle-treated counterparts (all P < 0.05). Next-generation sequencing analysis on simvastatin-treated iPSC-ECs showed that genes upregulated by simvastatin are enriched in processes such as angiogenesis, anti-inflammatory response, and extracellular matrix (ECM) organization. Conversely, simvastatin downregulated genes associated with epigenetic regulation of transcription, nucleosome assembly, and chromatin organization. Simvastatin significantly improved iPSC-EC functions in both basal and diabetic or hyperglycaemic (HG) conditions by reducing transcriptional activity of endothelial-to-mesenchymal transition (EndMT)-regulating genes, thus blocking ECs from switching to a mesenchymal phenotype. Yes-associated protein (YAP)-1 was identified as a direct regulator of chromatin accessibility at a previously undescribed enhancer region [SRY box transcription factor 9 (SOX9)] to regulate EndMT genes. Simvastatin blocked nuclear translocation of YAP, as demonstrated by the significant reduction of nuclear/cytoplasmic YAP ratios in simvastatin-treated iPSC-ECs (P < 0.001). The major downstream proteins of YAP signalling, SOX9 and connective tissue growth factor (CTGF), were also downregulated by simvastatin, as was VCAM1, a pro-inflammatory YAP target gene (all P < 0.01). The mevalonate pathway generates squalene for cholesterol biosynthesis or geranylgeranyl pyrophosphate (GGPP), which modulates Rho signalling. Inhibition of geranylgeranyltransferase I (GGTase I), a key intermediate in the mevalonate pathway, has been shown to improve endothelial function by blocking nuclear translocation of YAP1 and repressing EndMT in a RhoA-dependent manner. On the opposite, mevalonic acid (MA) and GGPP abolished simvastatin-mediated improvements on endothelial function. Finally, genetic [using clustered regularly interspaced short palindromic repeats (CRISPR) interference technology] and pharmacological inhibition of any component of the GGTase–RhoA–YAP1–SOX9 signalling axis could improve endothelial function in diabetic conditions, both in vitro (HG medium) and in a db/db diabetic mouse model, effectively repressing EndMT. Statins prevent major cardiovascular (CV) events by lowering serum low-density lipoprotein cholesterol (LDL-C).2 Statins competitively inhibit 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the first and major rate-limiting enzyme of the mevalonate pathway, thereby reducing cholesterol biosynthesis. Since MA is the precursor of numerous metabolites, inhibition of HMG-CoA reductase potentially affects diverse tissue functions and modulate specific signal transduction pathways, resulting in anti-inflammatory and antioxidant activities, improved endothelial function, increased NO bioavailability, and delayed progression of atherosclerotic plaques.3–5 However, apart from in vitro and experimental studies, it is not possible, from the available clinical evidence, to dissect the potential benefits of the pleiotropic effects of statins from those conferred by LDL-C reduction.3 Endothelial dysfunction is a key early step in the development and progression of atherosclerosis. Endothelial-to-mesenchymal transition, a process in which ECs lose their endothelial structural and physiological features and switch to the mesenchymal phenotype, has recently emerged as a potential mechanism that may link endothelial dysfunction to atherosclerosis.6,7 Previous studies have demonstrated that statins cause potent inhibition of EndMT in human cardiac microvascular and glomerular ECs.6,7 However, the precise mechanism by which statins reverse the EndMT process to improve endothelial function has remained unclear. Liu et al.1 investigated the effects of statins in endothelium protection using iPSC-ECs as a model system. Human iPSC-ECs have been shown to accurately recapitulate drug responses in patients and unravel some pathogenic mechanisms of CVDs,8 albeit with several technical limitations, as pointed out by the authors themselves. For example, it is unclear how well the iPS-EC model of HG-induced endothelial dysfunction mirrors the pathophysiology of human endothelial dysfunction in type 2 diabetes (T2D). Finally, iPS-ECs are heterogenous, including arterial, venous, microvascular, and lymphatic ECs. Combining iPS cell technology with CRISPR gene expression editing and knockout systems, the authors identified several tentative therapeutic targets in the epigenetic pathway controlling EndMT. First, they demonstrated that the GGTase–RhoA–YAP1–SOX9 signalling axis has a key role in simvastatin-mediated chromatin remodelling and EndMT inhibition in iPS-ECs. They also found that inhibitors of this pathway, such as a specific GGTase inhibitor and a RhoA inhibitor, were effective in restoring impaired endothelial function in a T2D animal model. These results suggest that inhibition of YAP1–SOX9 or their upstream regulators, such as GGTase I or RhoA, could be a potential therapeutic strategy for the treatment of T2D-associated endothelial dysfunction. Similar mechanisms could be involved in other pathophysiological processes, such as ischaemic myocardial injury.9 However, it remains difficult to demonstrate whether these findings can be translated to clinical applications. First, the observed epigenetic improvement of the endothelial function is not a class effect, as only simvastatin among the seven tested statins improved EC function (as shown by upregulation of NOS3), in a dose-dependent manner. Statins with different lipophilic indices exert distinct effects on different cell types other than liver cells. Predominantly lipophilic statins, such as simvastatin, can easily enter cells and interact with cell membranes, while hydrophilic ones employ transport proteins to gain intracellular access and exhibit higher hepatoselectivity.10,11 Secondly, the authors used 1-µM simvastatin for the in vitro experiments. The mean concentration of statins in human serum (at therapeutic doses) is only 1–15 nmol per L−1, with an even lower pharmacologically active free fraction (because protein binding is very high, 95%–99%). The intracellular concentration of statins in human ECs in vivo is not known. Furthermore, T2D mice were treated with 25-mg simvastatin per kg of body weight. This dose resulted in concentrations much higher than those achievable in patients (approximately 0.1–1 mg per kg of body weight) treated with the dosages selected for lipid-lowering purposes.12 Although the relevance and translational value of these findings need to be handled with caution, this could imply that statins may produce ‘pleiotropic effects’ at different dosages and achieved cellular concentrations. Nevertheless, the study by Liu et al.1 elegantly demonstrates that statins can improve endothelial function through the YAP1–SOX9 signalling axis and that various intermediaries of this pathway, including upstream regulators, could be potential therapeutic targets for the treatment of endothelial dysfunction, particularly in T2D. In conclusion, growing evidence, including the present work of Liu and colleagues, provides provocative insights to support pleiotropic effects of statins and encourage expanded application of statins in non-CV fields, such as systemic inflammatory diseases and cancer.3,5 Further studies with new lipid-lowering agents that inhibit cholesterol synthesis without affecting other cellular pathways will more effectively address unanswered questions about statin pleiotropy. On the other hand, the potential therapeutic targets identified here may deserve further investigation by novel experimental approaches.