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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 2 - PROVASC (Cell-specific vascular protection by CXCL12/CXCR4)

Teaser

Summary

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
â— 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.
â— validate a role of coding and non-coding genetic risk variants affecting CXCL12 and CXCR4 in different human cell types and humanized mouse models.
â— 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.

Work performed

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 aims 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 are highly inter-related and intricately linked in an overarching context translating findings in-between mice and man. Despite substantial problems with recruitment of competent personnel we have now managed to initiate and implement all work packages. In particular, we have dissected the cell-specific roles of CXCR4 in vascular cell types (ECs and SMCs), exerting atheroprotective effects by maintaining endothelial barrier integrity and a contractile smooth muscle cell phenotype, and have identified a new cardiovascular risk allele (near CXCR4) with genome-wide significance that correlates with low CXCR4 expression in man (see recent publications, namely DÃ¶ring et al., Circulation 2017). To detail the association between CXCL12 and CAD, we conducted a meta-analysis of genome-wide association studies performed in the EPIC-Norfolk and PROMIS cohorts, and applying conditional analysis, we newly identified rs2802492, an intergenic SNP near CXCL12, to be independently associated with CXCL12 plasma levels and with increased risk for CAD, corroborating CXCL12 as a driver of CAD. Notably, mice with an endohtelial deficiency in CXCL12 displayed a marked reduction of lesion area in the thoracoabdominal aorta and the aortic arch, as well as decreased CXCL12 plasma levels as compared to controls. These data indicate 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 CXCL12 levels (DÃ¶ring et al., Circulation 2019). In addition, we have generated first constructs for humanized CXCL12 mice (and variants) and have started to identify and validate miRNAs that target and regulate the CXCR4/CXCL12 axis.

Final results

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. As this part of the project has been particular laborious and many obstacles remain to be overcome, we also expect to require a no-cost extension of the project for one year.