Periodic Reporting for period 4 - PROVASC (Cell-specific vascular protection by CXCL12/CXCR4)
Période du rapport: 2021-04-01 au 2023-03-31
Atherosclerosis is the major underlying pathology of cardiovascular diseases, which remain the leading cause of death worldwide and in aging societies such as the endothelial cells (ECs). As a lipid-driven inflammatory disease of arteries atherosclerosis gives rise to vulnerable lesions prone to rupture and thrombotic occlusion. Lesions develop at pre-dilection sites with disturbed flow, where endothelial damage promotes intimal retention of lipoproteins and inflammatory leukocyte recruitment. Past research has largely focused on atherogenic factors and their inhibition but not on boosting a counterbalance by protective mechanisms. Of note, we have recently found that the CXCL12/CXCR4 chemokine-receptor axis protects against atherosclerosis by controlling neutrophil homeostasis and facilitating endothelial regeneration in mice. This is supported by genome-wide association studies, identifying genetic variants near CXCL12 associated with the risk of coronary heart disease. The protective regulation of endothelial repair by microRNAs also involves CXCL12/CXCR4. However, the causal and cell-specific impact of this axis remains unclear.
To balance the ongoing expansion of genetic risk variants, we aim to discover and elucidate new mechanisms for protective cell homeostasis and regeneration counteracting atherosclerosis.
To pursue and achieve this goal in depth, PROVASC will
● dissect cell-specific effects of the CXCR4-CXCL12 axis using an array of mouse lines for conditionnal deletion or bone marrow chimeras to compare resident vs hematopoietic cell compartments.
● validate a role of coding and non-coding genetic risk variants affecting CXCL12 and CXCR4 in different human cell types and humanized mouse models.
● unravel an epigenetic regulation of CXCL12/CXCR4 through cell type-specific microRNAs by identifying relevant microRNAs and targeting sites controlling this axis.
Given the ubiquitous relevance of CXCL12/CXCR4, we expect that the impact of such new mechanisms will extend beyond atherosclerosis to other chronic inflammatory diseases, allowing for tailored strategies of tissue protection and regeneration.
To balance the ongoing expansion of genetic risk variants, we aim to discover and elucidate new mechanisms for protective cell homeostasis and regeneration counteracting atherosclerosis.
To pursue and achieve this goal in depth, PROVASC will
● dissect cell-specific effects of the CXCR4-CXCL12 axis using an array of mouse lines for conditionnal deletion or bone marrow chimeras to compare resident vs hematopoietic cell compartments.
● validate a role of coding and non-coding genetic risk variants affecting CXCL12 and CXCR4 in different human cell types and humanized mouse models.
● unravel an epigenetic regulation of CXCL12/CXCR4 through cell type-specific microRNAs by identifying relevant microRNAs and targeting sites controlling this axis.
Given the ubiquitous relevance of CXCL12/CXCR4, we expect that the impact of such new mechanisms will extend beyond atherosclerosis to other chronic inflammatory diseases, allowing for tailored strategies of tissue protection and regeneration.
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.
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.
In a collaboration with Heiko Runz (Heidelberg), a high-throughput strategy was introduced to systematically study gene function directly in primary CAD-patient cells. The results indicated a function for the CXCR4 signaling pathway (through its ligand MIF), as well as several novel candidate genes impacting lipid uptake into human macrophages. In addition, we anticipate to further verify the effects of relevant variants in CXCL12 in huminazed mice with carrying these variants, and we will elaborate the fine-tuned and cell-specific regulation of the CXCR4/CXCL12 axis by miRNAs.