In contrast to atherogenic factors, mechanisms for protective cell homeostasis and regeneration limiting atherosclerosis with an impact beyond vascular disorders are much underappreciated. Instead of expanding genetic risk variants, PROVASC aimed to (1) characterize novel and inter-dependent cell-specific effects of the crucial CXCR4-CXCL12 axis e.g. in monocyte precursors in depth, (2) to identify and validate a cell-specific role of genetic risk variants affecting this dyad, and (3) to elucidate options for a fine-tuned regulation of CXCL12/CXCR4 through cell-specific miRNA targeting. The three aims were highly inter-related and intricately linked in an overarching context translating findings in-between mice and man. Despite some obstacles in recruiting competent personnel, we have successfully conducted and completed all work packages. In particular, we have dissected the cell-specific roles of CXCR4 in vascular cell types, exerting atheroprotective effects by maintaining endothelial cell (EC) barrier integrity and a contractile smooth muscle cell (SMC) phenotype, and have identified a new cardiovascular risk allele (near CXCR4) with genome-wide significance that correlates with low CXCR4 expression in man (Döring et al., Circulation 2017). In contrast, we have identified rs2802492, an intergenic SNP near CXCL12, to be independently associated with CXCL12 plasma levels and with increased risk for CAD and have found using cell-specific deletion that atherogenic effects of CXCL12 rely on CXCL12 production in arterial ECs, identifying EC-derived CXCL12 as a crucial driver of atherosclerosis and contributor to serum levels (Döring et al., Circulation 2019). As these data indicated a pro-atherogenic role of CXCR4 on circulating leukocytes, additional ongoing studies are interrogating the effects of cell-specific deletion in leukocyte subsets (e.g. in B cells, Döring et al., Circ. Res. 2020) on bone marrow homeostasis and atherosclerosis. In addition, we have generated first constructs for ES injections to obtain humanized CXCL12 mice (and variants), which are underway. Aside from the progress, the pandemic has led to significant delays, in particular concerning the project part for creating mice carrying humanized CXCL12 with a non-coding variant (rs2802492), owing to a lockdown of the mouse facility and emergency freezing of many important strains.
Most importantly, we have screened and validated miRNAs that target and regulate the CXCR4/CXCL12 axis (Cimen et al., under review after revision). Given its crucial role CXCR4 in ECs or VSMCs for vascular integrity, yet atherogenic functions in other cell types, strategies for cell-specifically augmenting CXCR4 function are critical for employing this receptor for therapeutic purposes. Here, we identified miR-206-3p as a vascular-specific CXCR4 repressor and exploited a target-site blocker (CXCR4-TSB) disrupting this interaction to therapeutically increase CXCR4 in the vessel wall. In vitro, CXCR4-TSB enhanced CXCR4 expression in human and murine ECs and VSMCs to promote beneficial effects on cell viability, proliferation, and migration. Systemic administration of CXCR4-TSB in Apoe-deficient mice enhanced Cxcr4 expression in ECs and VSMCs in the vessel wall, reduced vascular permeability and monocyte adhesion to endothelium, and attenuated the development of diet-induced athero-sclerosis. Notably, CXCR4-TSB also affected CXCR4 expression in B cells corroborating its atheroprotective role in this cell type. Analyses of human atherosclerotic plaques revealed a negative correlation between CXCR4 and miR-206-3p, supporting the conservation of this axis in human disease. The disruption of cell-specific microRNA-dependent regulatory pathways, as epitomized by the miR-206-3p-CXCR4 axis, reveals a novel therapeutic approach, and paves the way for a tailored use of TSBs in the treatment of atherosclerosis and other diseases. These data have been submitted in a thoroughly revised manuscript currently under final review for publication.
