Cellular and Circulating Fortilin in Vascular Diseases

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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.