Downregulation of 24-Dehydrocholesterol Reductase in Monocytes Improves Arteriogenesis in Peripheral Arterial Disease

Abstract Critical limb ischemia (CLI) is the end stage of peripheral artery disease (PAD) and can be an underlying cause of ischemic rest pain, gangrene, and amputation. Primary amputation is often required for the 30% of CLI patients who are not eligible for limb revascularization; thus, an effective therapy to improve neovascularization is urgently needed. During hindlimb ischemia, monocytes are among the first cells to hone in on the ischemia site and contribute to neovascularization. Our recent publication revealed that ischemia training, performed by 24 hours of unilateral femoral artery ligation, led to functional reprogramming of bone marrow-derived monocytes (BM-Mono), enabling them to protect against the outcomes of limb ischemia by increasing perfusion and neovascularization. Mechanistically, this reprogramming resulted in the downregulation of 24-Dehydrocholesterol Reductase (Dhcr24, an important enzyme that converts desmosterol into cholesterol), and a consequent accumulation of desmosterol in those cells. Interestingly, our preliminary data have shown that ischemic-trained monocytes control vascular proliferation, and both vascular cell types, endothelial cells (ECs) and smooth muscle cells (SMCs), have increased expression of IL- 10 and its receptors. Moreover, the macrophages differentiated from the ischemic-trained monocytes possess an anti-inflammatory phenotype. Here our primary scientific goal is to target Dhcr24 in monocytes as a novel and unique strategy to improve neovascularization in the setting of PAD/CLI. First, we will assess whether downregulation of Dhcr24 in BM-Mono improves hindlimb ischemia outcomes, such as perfusion and neovascularization; and second, we will identify the mechanisms by which those BM-Mono with low Dhcr24 expression control vascular proliferation. Our long-term goal is to translate these findings using a Dhcr24 inhibitor into a new therapeutic approach to treat PAD/CLI. We hypothesize that the low expression of Dhcr24 in BM-Mono regulates anti-inflammatory pathways in vascular cells that controls their proliferation, leading to a proper neovascularization in the ischemic limb. We will test our hypothesis in two specific aims. SA1: Determine whether monocyte Dhcr24 regulation alters hindlimb ischemia outcomes. We will determine the dependence of loss- or gain-of-function of monocyte Dhcr24 on limb perfusion, function, and neovascularization using a pre-clinical model of hindlimb ischemia. We expect to demonstrate that low expression of Dhcr24 in BM-Mono plays a beneficial role against hindlimb ischemia. SA2: Identify the mechanism by which monocytes with low Dhcr24 control vascular proliferation We will dissect the underlying mechanisms by which monocytes with low Dhcr24 expression regulate vascular proliferation. We will also inhibit Dhcr24 in human monocytes and assess their inflammatory phenotype. We expect to find that the underlying mechanisms are based on anti-inflammatory pathways.