Protein Arginine Methylation in Vascular Smooth Muscle Cell Phenotypic Modulation and Calcification

摘要 血管中的血管钙化，动脉硬化，并预测不良心血管死亡率， 发病率。目前还没有开发出直接针对血管钙化的治疗方法。现在有强有力的证据表明 确定了血管平滑肌细胞（VSMC）向“骨样”细胞的成骨分化是至关重要的 导致血管钙化我们和其他人已经证明了SMC衍生的Runx 2（Runt- 相关转录因子2）在调节VSMC的成骨分化中是必不可少的，其诱导 动脉粥样硬化、糖尿病和肾脏疾病中的血管钙化。使用单细胞RNA测序 （scRNA-seq）分析，我们发现了一种新的Runx 2抑制因子及其对SMC表型转换的影响， 动脉粥样硬化具体地说，我们确定蛋白质精氨酸甲基转移酶1（PRMT 1）发挥作用， 在抑制Runx 2和调节SMC表型转换中起关键作用。PRMT 1是一个新兴的监管机构， 然而，在人类的病理过程中，它在血管钙化和动脉粥样硬化中的作用完全是 未知因此，我们有责任提供更多的证据，以证明PRMT 1如何作为一个新的Runx 2 抑制因子在调节血管表型转换和钙化中的作用。被有趣的 观察到Runx 2的上调和PRMT 1的显著下调之间的负相关性， 钙化的动脉粥样硬化病变在人类和小鼠，我们进行了功能研究，使用PRMT 1增益- 和功能丧失的VSMC以及我们的新型SMC特异性PRMT 1转基因小鼠。我们的初步研究 在体外研究中证实PRMT 1在调节Runx 2、SMC表型转换和钙化中起致病作用; SMC特异性转基因PRMT 1抑制主动脉Runx 2和动脉粥样硬化的发展。 ApoE-/-小鼠。进一步的证据表明，PRMT 1介导的Runx 2调节是通过 Runx 2的甲基化。Runx 2相互作用组的无偏蛋白质组学分析揭示了Runx 2的相互作用 与血清反应因子（SRF），一个重要的转录调节收缩SMC标记基因， SRF依赖性SMC标记基因表达失调。基于这些令人兴奋的新发现，我们 假设PRMT 1是一个关键的Runx 2抑制因子，它调节VSMC Runx 2并支配VSMC 表型转换和钙化。利用新的SMC特异性PRMT 1转基因 小鼠模型和全面的多组学方法，该提案将揭示一种新的监管机制， 范式强调PRMT 1/Runx 2信号轴在调节VSMC表型转换和钙化。

Abstract Vascular calcification in blood vessels, stiffens artery and predicts adverse cardiovascular mortality and morbidity. No therapies developed so far directly targeting vascular calcification. Strong evidence has now determined the osteogenic differentiation of vascular smooth muscle cells (VSMC) into “bone-like” cells is critical for the development of vascular calcification. We and others have demonstrated SMC-derived Runx2 (Runt- related transcriptional factor 2) is essential in regulating osteogenic differentiation of VSMC, which induces vascular calcification in atherosclerosis, diabetes and kidney disease. Using single cell RNA sequencing (scRNA-seq) analysis, we discovered a novel Runx2 suppressor and its impact on SMC phenotypic switch in atherosclerosis. Specifically, we determined that the protein arginine methyltransferase 1 (PRMT1) plays a critical role in inhibiting Runx2 and modulating SMC phenotypic switch. PRMT1 is an emerging regulator in human pathological processes, however, its function in vascular calcification and atherosclerosis is entirely unknown. It is thus incumbent upon us to provide additional evidence as to how PRMT1 acts as a new Runx2 suppressor in the modulation of vascular phenotypic switch and calcification. Prompted by the intriguing observations of an inverse correlation between upregulation of Runx2 and marked downregulation of PRMT1 in the calcified atherosclerotic lesions in human and mice, we carried out functional studies using the PRMT1 gain- and loss-of-function VSMC and our novel SMC-specific PRMT1 transgenic mice. Our preliminary studies demonstrated a causative role of PRMT1 in regulating Runx2, SMC phenotypic switch and calcification in vitro; and SMC-specific transgenic PRMT1 inhibited aortic Runx2 and the development of atherosclerosis in vivo in the ApoE-/- mice. Further evidence implicates that PRMT1-directed regulation of Runx2 is mediated through methylation of Runx2. Unbiased proteomics analysis of Runx2 interactome uncovered the interaction of Runx2 with serum response factor (SRF), an essential transcriptional regulator for contractile SMC marker genes, which dysregulates SRF-dependent expression of SMC marker genes. Based on these new and exciting findings, we hypothesize that PRMT1 is a key Runx2 suppressor, which regulates VSMC Runx2 and governs VSMC phenotypic switch and calcification in atherosclerosis. Utilizing the new SMC-specific PRMT1 transgenic mouse model and comprehensive multi-Omics approaches, the proposal will uncover a novel regulatory paradigm highlighting the PRMT1/Runx2 signaling axis in modulating VSMC phenotypic switch and calcification.