Reprogrammed Monocytes Improve Neovascularization 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 home 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, the ischemia training process led to a systemic effect that also reprogrammed BM-Mono in the contralateral limb. Here, our primary scientific goal is to expand our data to target Dhcr24 in monocytes as a novel and unique strategy to improve neovascularization in the setting of PAD/CLI. We will first identify whether extracellular vesicles (EVs) are the mechanism underlying the systemic effect of BM-Mono reprogramming; we will then investigate whether loss of Dhcr24 in myeloid cells regulates their crosstalk with endothelial cells (ECs) and consequent endothelium adhesion and transendothelial migration (TEM). We hypothesize that the reprogramming of BM-Mono occurs through systemic EVs that transfer epigenetic cargo to those cells, downregulating Dhcr24 and accumulating desmosterol. These reprogrammed BM-Mono have a lower adhesion rate to ECs and reduced TEM, reducing inflammation and leading to proper arteriogenesis. Our study has three specific aims (SA): SA1: Investigate the systemic effect of ischemia training on monocyte reprogramming We will investigate whether the cargo transfer from EVs released in the circulation during ischemia training is responsible for monocyte reprogramming. SA2: Determine whether myeloid Dhcr24 regulation modifies hindlimb ischemia outcomes We will determine the dependence of loss- or gain-of-function of monocyte Dhcr24 on limb perfusion, limb function, and arteriogenesis, using unique transgenic mouse models that overexpress or delete Dhcr24 specifically in myeloid cells; and then subject these mice to hindlimb ischemia. SA3: Demonstrate whether loss of Dhcr24 in myeloid cells regulates their crosstalk with ECs We will assess whether loss of Dhcr24 in myeloid cells regulates gene expression/phenotype of ECs and myeloid cells in ischemic-limb muscle using single-cell RNA-seq analysis. Further, we will assess if loss of Dhcr24 in monocytes reduces adhesion to the endothelium and TEM.