Novel Interplay of KILN and MKL1 in Vascular Pathophysiology

血管平滑肌细胞（VSMC）表现出显著的表型可塑性， 收缩性VSMC在病理条件下转变为合成状态。合成血管平滑肌细胞表现为 由于它们对炎症激活和收缩性丧失的高度敏感性，在很大程度上导致了各种 血管疾病如血管成形术后再狭窄和动脉瘤。靶向VSMC的早期激活 炎症可能代表阻断血管病理的有吸引力的策略。然而，分子 涉及驱动促炎性VSMC表型的关键调节因子的机制尚不完全清楚， 明白一种尚未被探索的可能性是普遍存在的长非编码RNA （lncRNA）。通过无偏见的RNA-seq研究，我们发现了一种新的人类特异性lncRNA，KILN， 富含促炎性VSMC和患病的人血管（动脉瘤）。RNA-seq显示， KILN在培养的VSMCs中抑制多种炎症基因的表达。值得注意的是，KILN高度 在人源化细菌人工染色体（BAC）转基因小鼠中对炎性损伤的应答 携带KILN（BAC-KILN）或KILN及其邻近基因IL 8（BAC-KILN/IL 8）。都是转基因的 线显示由血管损伤引发的加重的炎症反应。这些结果支持 KILN在促进VSMC炎症和血管疾病中的关键作用。从机械上讲，KILN 与MKL 1一起使用，MKL 1是一种有效的转录辅因子，在反式激活VSMC收缩基因中具有公认的作用。 有趣的是，在培养的VSMC中，Mkl 1缺失小鼠免于动脉瘤形成和MKL 1耗竭 破坏了促炎基因程序这些结果表明，MKL 1和KILN之间的潜在联系， VSMC炎症和血管疾病。事实上，KILN的缺失降低了MKL 1蛋白水平和MKL 1/p65 物理相互作用，后者对促炎基因的反式激活至关重要。这些令人兴奋 初步发现支持了KILN与MKL 1相互作用以稳定MKL 1蛋白的中心假设， 其增强MKL 1/p65激活的VSMC炎症和血管病理。我们提出两个具体的 旨在探索这一假设。目的1阐明KILN在病理过程中的调节和作用 使用BAC转基因小鼠进行血管重塑，并评估KILN在血管和血浆中的表达 动脉瘤患者的外泌体目的2阐明KILN介导血管内皮细胞凋亡的分子机制 涉及KILN破坏MKL 1泛素化以增强MKL 1/p65的炎症和疾病 促炎基因程序的反式激活。成功完成拟议的研究将确定 一种新的分子开关，包括一种新的富含VSMC的lncRNA（KILN），该lncRNA与MKL 1相关， VSMC炎症和病理性血管重塑。这些研究将增进我们对 lncRNA和MKL 1血管病理生理学，可能为治疗策略提供新的见解， 各种炎症性血管疾病。

Vascular smooth muscle cells (VSMCs) exhibit remarkable phenotypic plasticity, whereby differentiated contractile VSMCs switch to a synthetic state under pathological conditions. Synthetic VSMCs are manifested by their high susceptibility to inflammatory activation and loss of contractility, contributing largely to various vascular disorders such as post-angioplasty restenosis and aneurysm. Targeting early activation of VSMC inflammation may represent an attractive strategy to block vascular pathologies. However, molecular mechanisms involving key regulator(s) that drive the proinflammatory VSMC phenotype are incompletely understood. One possibility that has yet to be explored is the pervasive class of long noncoding RNAs (lncRNAs). Through an unbiased RNA-seq study, we discovered a novel human-specific lncRNA, KILN, which is enriched in proinflammatory VSMCs and diseased human vessels (aneurysm). RNA-seq revealed that loss of KILN in cultured VSMCs suppresses expression of multiple inflammatory genes. Notably, KILN is highly responsive to inflammatory insults in humanized Bacterial Artificial Chromosome (BAC) transgenic mice carrying either KILN (BAC-KILN) or both KILN and its neighboring gene IL8 (BAC-KILN/IL8). Both transgenic lines display an exacerbated inflammatory response triggered by vascular injury. These results support a critical role of KILN in promoting VSMC inflammation and vascular disease. Mechanistically, KILN interacts with MKL1, a potent transcriptional cofactor with a recognized role in transactivating VSMC contractile genes. Intriguingly, Mkl1 null mice are protected from aneurysm formation and depletion of MKL1 in cultured VSMCs impairs the proinflammatory gene program. These results suggest a potential link between MKL1 and KILN in VSMC inflammation and vascular disease. Indeed, loss of KILN decreases MKL1 protein levels and MKL1/p65 physical interaction, the latter being critical for transactivation of proinflammatory genes. These exciting preliminary findings support a central hypothesis that KILN interacts with MKL1 to stabilize MKL1 protein, which potentiates MKL1/p65-activated VSMC inflammation and vascular pathologies. We propose two specific aims to probe this hypothesis. Aim 1 will elucidate the regulation and function of KILN during pathological vascular remodeling using BAC transgenic mice, and evaluate KILN expression in vessels and plasma exosomes from aneurysm patients. Aim 2 will elucidate the molecular mechanism of KILN-mediated vascular inflammation and disease involving KILN disruption of MKL1 ubiquitination to potentiate MKL1/p65 transactivation of the proinflammatory gene program. Successful completion of the proposed studies will define a new molecular switch comprising a novel VSMC-enriched lncRNA (KILN) that associates with MKL1 for VSMC inflammation and pathological vascular remodeling. These studies will advance our knowledge of lncRNA and MKL1 vascular pathophysiology, potentially providing novel insights into therapeutic strategies for various inflammatory vascular diseases.