The Role of Vascular MR-Regulated Genes in Vascular Function and Disease

在发达国家，缺血性血管疾病仍然是导致死亡的主要原因。醛固酮(ALDO)是一种类固醇激素，通过与配体激活的转录因子盐皮质激素受体(MR)结合来调节血压(BP)。在临床试验中，Aldo拮抗剂显著降低心血管死亡率和缺血，与全身血压的适度下降不成比例，支持Aldo的直接血管效应有助于Aldo拮抗剂在缺血性心血管疾病中的保护作用。我们最近发现，MR在人血管平滑肌细胞(VSMC)和内皮细胞中表达并调节基因转录程序。在血管损伤和动脉粥样硬化的动物模型中，Aldo输注增加了血管重塑和动脉粥样硬化。我们目前已有的初步数据表明，MR在主动脉中的直接激活诱导了促动脉粥样硬化基因胎盘生长因子(PlGF)的表达，并且小鼠PlGF的遗传缺陷抑制了Aldo刺激的血管损伤，这表明PlGF可能在Aldo介导的血管疾病的机制中发挥作用。PlGF是血管内皮生长因子家族的分泌肽成员，通过与跨膜受体FMS样酪氨酸激酶(Flt-1)结合，促进血管细胞增殖、单核细胞趋化和炎症。在动物模型和人类中，PlGF被认为与动脉粥样硬化和不良缺血事件有关。在这个方案中，我们验证了这样的假设，即VSMC MR的Aldo激活诱导PlGF的表达，PlGF激活Flt-1受体以刺激体内VSMC的增殖、巨噬细胞募集、血管炎症和动脉粥样硬化斑块的形成。我们探索这一假说有三个特定的目的：SA1使用分子方法研究血管MR对PlGF基因的转录调控，SA2通过线缆诱导的小鼠颈动脉损伤模型研究PlGF在体内对Aldo刺激的血管损伤中的作用，SA3在ApoE基因敲除模型中研究PlGF在Aldo刺激的动脉粥样硬化中的体内作用。在这两个小鼠模型中，通过比较PlGF基因缺陷的小鼠、VSMC特异性MR缺失的小鼠和过度表达可溶性Flt-1受体以阻断PlGF信号的小鼠，将探索PlGF介导Aldo刺激的血管疾病的机制。在体内，PlGF转录调节通路在Aldo刺激的损伤和动脉粥样硬化中的作用也将通过染色质免疫沉淀在这些小鼠模型的血管中进行研究。更好地了解血管MR诱导的PlGF在血管重塑和动脉粥样硬化中的作用，将有助于阐明Aldo拮抗剂药物对心血管患者血管保护作用的机制，并将探索PlGF及其信号通路作为预防人类心血管缺血事件的新药物靶点的潜力。

Ischemic vascular disease remains the leading cause of mortality in the developed world. Aldosterone (aldo) is a steroid hormone that acts by binding to the mineralocorticoid receptor (MR), a ligand-activated transcription factor, to regulate blood pressure (bp). In clinical trials, aldo antagonists significantly decrease cardiovascular mortality and ischemia out of proportion to modest decreases in systemic bp, supporting that a direct vascular effect of aldo contributes to the protective role of aldo antagonists in ischemic cardiovascular disease. We recently discovered that MR is expressed and regulates gene transcription programs in human vascular smooth muscle cells (VSMC) and endothelial cells. In animal models of vascular injury and atherosclerosis, aldo infusion increases vascular remodeling and atherosclerosis. We now have preliminary data demonstrating that direct activation of MR in the aorta induces expression of the pro-atherosclerotic gene, placental growth factor (PlGF), and that genetic deficiency of PlGF in mice inhibits aldo-stimulated vascular injury suggesting that PlGF may play a role in the mechanism of aldo-mediated vascular disease. PlGF is a secreted peptide member of the vascular endothelial growth factor family and is known to promote vascular cell proliferation, monocyte chemotaxis, and inflammation by binding to the transmembrane receptor, fms-like tyrosine kinase (Flt-1). PlGF has been implicated in atherosclerosis and adverse ischemic events in animal models and in humans. In this proposal, we test the hypothesis that aldo activation of VSMC MR induces expression of PlGF, which activates Flt-1 receptors to stimulate VSMC proliferation, macrophage recruitment, vascular inflammation, and atherosclerotic plaque formation in vivo. We explore this hypothesis with three specific aims: SA1 uses molecular approaches to investigate the transcriptional regulation of the PlGF gene by vascular MR., SA2 investigates the in vivo role of PlGF in aldo-stimulated vascular injury using a wire-induced carotid injury model in mice, and SA3 investigates the in vivo role of PlGF in aldo-stimulated atherosclerosis in the ApoE knockout model. In these two mouse models, the mechanism by which PlGF mediates aldo-stimulated vascular disease will be explored by comparing mice genetically deficient in PlGF, mice with inducible VSMC-specific MR deletion, and mice overexpressing soluble Flt-1 receptors to block PlGF signaling. The in vivo role of PlGF transcriptional-regulatory pathways in aldo-stimulated injury and atherosclerosis will also be investigated using chromatin immunoprecipitation in vessels from these mouse models. A better understanding of the role of vascular MR induction of PlGF in vascular remodeling and atherosclerosis will help elucidate mechanisms underlying the vascular protective effects of aldo-antagonist drugs in cardiovascular patients and will explore the potential of PlGF and its signaling pathway as new drug targets to prevent cardiovascular ischemic events in humans.