Cellular and Circulating Fortilin in Vascular Diseases

项目摘要 这项题为“血管疾病中的细胞和循环Fortilin”的拨款提案的主要目标是 检验细胞和循环fortilin抑制TGFβ1通路的中心假设， 促进动脉粥样硬化的结合和抑制关键的细胞内和细胞外成分， 道路。这一创新性的假设得到了丰富而令人兴奋的初步数据的充分支持。 动脉粥样硬化是指动脉壁增厚，限制含氧血液向重要血管的输送。 器官，并影响从主动脉和冠状动脉到毛细血管的每一个层次的脉管系统， 微循环它仍然是当今美国最严重的健康问题之一，花费近286美元 每年10亿。单独降低低密度脂蛋白（LDL）水平不能消除动脉粥样硬化， 因为科克伦组织表明，PCSK 9抑制剂对LDL的急剧降低仅是适度的 降低心血管发病率/死亡率。因此，新的分子，其目标将导致停止 动脉粥样硬化性血管病变的进展，必须加以确定。Fortilin是一种172个氨基酸的多 一种功能性蛋白质，不仅在动脉粥样硬化组织中大量表达， 在动脉粥样硬化的存在下，血液循环（如细胞因子和激素）处于较高水平。福尔替林群岛 涉及多种生物学功能但最显著的是调节活性氧（ROS， 氧化还原）、ER应激和细胞凋亡。我们发现，当循环中的福替林被中和时， (HC)小鼠的动脉粥样硬化较少。此外，我们发现巨噬细胞中fortilin的缺失 （M）导致HC小鼠中较少的动脉粥样硬化。探讨Fortilin促进大鼠心肌细胞凋亡的机制 在动脉粥样硬化中，我们进行了一项系统的，无偏倚的NGS RNA-Seq测定，发现缺乏M Fortilin强烈激活抗动脉粥样硬化TGFβ1通路。进一步的研究表明，福替林结合， 抑制该途径的关键分子-细胞外TGFβ1和细胞内Smad 3-，并使M β 1保持在细胞内。 促炎、促动脉粥样硬化表型。在当前的项目中，我们将首先测试假设， 循环中的福替林通过结合和抑制TGFβ1以及使M β 1极化为抗TGFβ1而促进动脉粥样硬化。 炎症表型（Aim 1）。然后，我们将测试的假设，细胞fortilin在M β促进 通过结合和抑制Smad 3以及通过使M β极化为抗炎表型来治疗动脉粥样硬化（Aim 2）。随着所有目标的成功完成，该项目将导致两个不同的和高度创新的战略， 停止动脉粥样硬化：（i）使用α-fortilin mAb破坏循环中fortilin和TGFβ1之间的相互作用， 细胞外空间以从其抑制剂fortilin释放抗动脉粥样硬化TGFβ1，和（ii）产生和 使用小分子量（SMW）化合物破坏福替林-Smad 3相互作用， 被激活。

Project Summary The main goal of this grant proposal titled “Cellular and Circulating Fortilin in Vascular Diseases” is to test the central hypothesis that both cellular and circulating fortilin inhibit the TGFβ1 pathway and promote atherosclerosis by binding and inhibiting the key intracellular and extracellular components of the pathway. The innovative hypothesis is well supported by abundant and exciting preliminary data. Atherosclerosis is the thickening of the wall of arteries that limits transport of oxygen-containing blood to vital organs and affects every level of the vasculature from the aorta and coronary arteries to capillaries and microcirculation. It remains one of the most serious health problems in the U.S. today and costs nearly $286 billion every year. Lowering of the low-density lipoprotein (LDL) levels alone does not eliminate atherosclerosis, as the Cochrane Organization showed that a drastic reduction of LDL by PCSK9 inhibitors only modestly decreased cardiovascular morbidity/mortality. Thus, new molecules, the targeting of which would lead to halting of the progression of atherosclerotic vasculopathy, must be identified. Fortilin is a 172-amino acid multi- functional protein that is not only abundantly expressed in atherosclerotic tissue but is also present in circulation (like cytokines and hormones) at higher levels in the presence of atherosclerosis. Fortilin is implicated in various biological functions but most notably in regulation of reactive oxygen species (ROS, redox), ER stress, and apoptosis. We found that when circulating fortilin was neutralized, hypercholesterolemic (HC) mice developed less atherosclerosis. In addition, we found that the deletion of fortilin in macrophages (MФ) led to less atherosclerosis in HC mice. To explore the mechanism by which fortilin facilitates atherosclerosis, we performed a systematic, unbiased NGS RNA-Seq assay and found that the lack of MФ fortilin robustly activated the anti-atherosclerotic TGFβ1 pathway. Further studies showed that fortilin binds and inhibits the key molecules of the pathway—extracellular TGFβ1 and intracellular Smad3—and kept MФ in the pro-inflammatory, pro-atherosclerotic phenotype. In the current project, we will first test the hypothesis that circulating fortilin facilitates atherosclerosis by binding and inhibiting TGFβ1 and by polarizing MФ to the anti- inflammatory phenotype (Aim 1). We will then test the hypothesis that cellular fortilin in MФ facilitates atherosclerosis by binding and inhibiting Smad3 and by polarizing MФ to the anti-inflammatory phenotype (Aim 2). With all Aims successfully completed, the project will lead to two distinct and highly innovative strategies to halt atherosclerosis: (i) using α-fortilin mAb to disrupt the interaction between circulating fortilin and TGFβ1 in the extracellular space to free up anti-atherosclerotic TGFβ1 from its inhibitor fortilin and (ii) generating and using small molecular weight (SMW) compounds that disrupt the fortilin-Smad3 interaction to allow Smad3 to be activated.