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

摘要 血管中的血管钙化，使动脉硬化，并预测不利的心血管死亡率和 发病率。到目前为止，还没有直接针对血管钙化的治疗方法。强有力的证据现在 确定血管平滑肌细胞(VSMC)向“骨样”细胞的成骨分化至关重要 以促进血管钙化的发展。我们和其他人已经演示了SMC派生的Runx2(Run- 相关转录因子2)在调节VSMC成骨分化中起重要作用，从而诱导 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Runx2抑制因子及其对SMC表型转换的影响。 动脉硬化。具体地说，我们确定精氨酸甲基转移酶1(PRMT1)蛋白在 在抑制Runx2和调节SMC表型转换中的关键作用。PRMT1是一种新兴的监管机构 然而，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是完全的 未知。因此，我们有责任就PRMT1如何作为新的Runx2提供更多的证据 抑制因子在调节血管表型转换和钙化中的作用。由耐人寻味的 Runx2的上调与PrMT1的显著下调之间的负相关观察 在人和小鼠的钙化动脉粥样硬化病变中，我们利用PRMT1增益- 和功能丧失的VSMC以及我们的新型SMC特异性PRMT1转基因小鼠。我们的初步研究 证实了PRMT1在调节Runx2、SMC表型转换和体外钙化中的作用； SMC特异性转基因PRMT1在体内抑制Runx2和动脉粥样硬化的发展 载脂蛋白E-/-小鼠。进一步的证据表明，PRMT1对Runx2的调节是通过 Runx2的甲基化。Runx2相互作用组的无偏蛋白质组学分析揭示了Runx2的相互作用 通过血清反应因子(SRF)，一种收缩SMC标记基因的必要转录调节因子， 失调依赖于SRF的SMC标记基因的表达。基于这些令人振奋的新发现，我们 假设PRMT1是一个关键的Runx2抑制因子，它调节VSMC Runx2并控制VSMC 动脉粥样硬化的表型转换和钙化。利用新的SMC特异性PRMT1转基因 鼠标模型和全面的多OMICS方法，该提案将揭示一种新的监管 研究范式强调了PRMT1/Runx2信号轴在调节VSMC表型转换和钙化中的作用。

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.