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中的Fortilin”的拨款提案的主要目标是 验证细胞和循环中的Fortilin均抑制转化生长因子β1途径的中心假设 通过结合和抑制关键的细胞内和细胞外成分促进动脉粥样硬化 这条小路。大量令人振奋的初步数据很好地支持了这一创新假说。 动脉粥样硬化是动脉壁的增厚，限制了含氧血液向重要器官的运输。 并影响血管系统的各个层次，从主动脉和冠状动脉到毛细血管 微循环。它仍然是当今美国最严重的健康问题之一，耗资近286美元 每年十亿美元。降低低密度脂蛋白(LDL)水平本身并不能消除动脉粥样硬化， 正如Cochrane组织显示的那样，PCSK9抑制剂大幅降低低密度脂蛋白只能适度地 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情况，必须加以确认。Fortilin是一种由172个氨基酸组成的多氨基酸 一种功能蛋白，不仅在动脉粥样硬化组织中大量表达，而且在 动脉粥样硬化时血液循环(如细胞因子和激素)水平较高。Fortilin是 涉及各种生物学功能，但最显著的是调节活性氧物种(ROS， 氧化还原)、内质网应激和细胞凋亡。我们发现，当循环中和Fortilin时，高胆固醇血症 (HC)小鼠出现较少的动脉粥样硬化。此外，我们还发现巨噬细胞中Fortilin的缺失 (M-Ф)可减轻HC小鼠的动脉粥样硬化。探讨福尔替林的作用机制 对于动脉粥样硬化，我们进行了系统的、无偏倚的NGS RNA-Seq分析，发现缺乏MФ Fortilin有力地激活了抗动脉粥样硬化的转化生长因子β1途径。进一步的研究表明，Fortilin结合和 抑制该途径的关键分子细胞外转化生长因子β1和细胞内Smad3，并使MФ保持在 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Fortilin通过结合和抑制转化生长因子β-1和通过极化M-Ф而促进动脉粥样硬化 炎症表型(目标1)。然后，我们将测试MФ中的细胞Fortilin有助于 通过结合和抑制Smad3以及通过将MФ极化到抗炎表型而导致的动脉粥样硬化(目的 2)。随着所有目标的成功完成，该项目将产生两个不同的和高度创新的战略，以 抗动脉粥样硬化：(1)用α-Fortilin单抗阻断循环Fortilin与转化生长因子β-1的相互作用 从其抑制剂Fortilin中释放抗动脉粥样硬化转化生长因子β1的胞外空间和(Ii)产生和 使用小分子量(SMW)化合物破坏Fortilin-Smad3的相互作用，使Smad3能够 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.