Sex-specific regulation of the complement C5aR1-CXCL4 axis in revascularization

Henry Nording (Lübeck)1, L. Baron (Lübeck)2, H. Langer (Mannheim)3

1Universitätsklinikum Schleswig-Holstein Medizinische Klinik II / Kardiologie, Angiologie, Intensivmedizin Lübeck, Deutschland; 2Universitätsklinikum Schleswig-Holstein Lübeck, Deutschland; 3Universitätsklinikum Mannheim I. Medizinische Klinik Mannheim, Deutschland


Coronary artery disease (CAD) remains a major cause of death worldwide as well as in Germany. In recent years, sex-specific differences in cardiovascular disease have shifted into focus. There is a growing body of evidence supporting the claim that not only gender (lifestyle and socio-economic factors) but also sex (the genetic-biological and hormonal differences) impact decisively on cardiovascular pathologies (Regitz-Zagrosek and Gebhard 2023). 
In ischemic tissue diseases like CAD, platelets can modulate angiogenesis and thus adaption to vascular occlusion. Previously, we characterized the complement anaphylatoxin C5a-mediated activation of C5a receptor 1 (C5aR1) expressed on platelets as a potent regulator of ischemia-driven revascularization and assessed its role in diseases featuring tissue ischemia in mice as well as cardiovascular patients (Nording et al. 2021, 2023, 2023). 
Interestingly, through these studies we have found evidence for sex-specific differences in the revascularization response of C5aR1 knockout mice compared to WT mice. Male C5aR1-deficient mice display a highly increased revascularization response in the hindlimb ischemia model compared to females. This difference was not significantly apparent in WT mice. Indeed, the effect on revascularization of C5aR1 mice, known to have a pro-revascularization phenotype, was predominantly conveyed by male animals. Female C5aR1-deficient mice did not display a significantly altered revascularization compared to WT. We have used mice deficient for C5aR1 but also the upstream C5 as well as platelet-specific C5aR1 knockout mice to uncover this striking sex-specific difference in revascularization response and found that C5aR1 but not global C5 knockout shows this effect. Indeed, we could show that higher expression of C5aR1 on platelets in male mice impacts on secretion and availability of CXCL4 and appears to be the underlying cause of the observed effect on revascularization. Using C5aR1-/-CXCL4-/- mice we were able to verify this finding. The interdependencies of sex, C5aR1, CXCL4 and revascularization response could be shown by correlations on the level of mRNA using qPCR as well as protein using Western blots and ELISAs. Interestingly, we could show that testosterone but not estrogen or progesterone increased platelet C5aR1 expression, which explains the C5aR1-mediated sex-specific differences. Finally, we were able to verify this finding in human cohorts of CAD as well peripheral artery disease (comparing asymptomatic patients with excellent adaption to vascular occlusion to patients with the same degree of vascular occlusion but with severe symptoms) thus demonstrating translational relevance. The C5aR1-CXCL4 axis may be a mechanism with highly increased relevance in males compared to females. 
In this way, we were able to identify the C5aR1-dependent secretion of CXCL4 to be a mechanism with strong sex-specific regulation. Uncovering such mechanisms is instrumental to overcoming the gender-based inequality in cardiovascular health and is, thus, a highly relevant topic. 
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