Background: Cardiovascular diseases, and in particular coronary artery disease (CAD), are the leading cause of mortality worldwide and represent a high socioeconomic burden (Roth et al. 2020). A growing body of evidence supports an important role of sex-based genetic and hormonal mechanisms on top of gender-based sociocultural or lifestyle-related differences in cardiovascular disease. However, the underlying mechanisms of sex-specific difference in the pathogenesis of cardiovascular diseases are still incompletely understood (Regitz-Zagrosek and Gebhard 2023). Revascularization is an endogenous mechanism of regeneration maintaining tissue perfusion. In previous work, we identified platelet C5aR1 as a potent negative regulator of revascularization in murine hindlimb ischemia model (HLI) because of selective secretion of antiangiogenic protein CXCL4 (Nording et al. 2021).
Methods: The model of hindlimb ischemia (HLI) was performed using 8-12 week old male and female wildtype, C5aR1- (C5aR1-/-) and C5- (C5-/-) deficient mice. Revascularization was monitored by laser-doppler imaging (LDI) for 4 or 14 days followed by organ removal. Complement activation and CXCL4 and C5aR1 expression within ischemic and non-ischemic muscle tissue lysates were measured by ELISA and Western Blot. Murine platelets were isolated and CXCL4 concentration in stimulated supernatants were measured by ELISA.
Results: In HLI experiments sex-specific differences in revascularization in C5aR1-/- mice were observed, that were not present in wildtype controls. In sequential LDI measurements male C5aR1-/- mice showed a significantly enhanced perfusion within the ischemic hindlimb compared to females. Similar results could be observed in C5-/- mice indicating a C5aR1-dependent mechanisms. Ischemia results in a significant increase in complement activation and CXCL4 deposition within ischemic hindlimb muscles in wildtype and C5aR1-/- mice. Furthermore, C3b activity strongly correlates with CXCL4 concentration (r=0.9206) in muscle tissue. In Western Blot analysis of ischemic gastrocnemius muscle tissue male wildtype mice showed a significantly higher expression of C5aR1 compared to female controls. Interestingly, C5a stimulation of isolated platelets resulted in a significant CXCL4 secretion in male but not female animals.
Discussion: The increased expression of C5aR1 in male compared to female mice, which may lead to increased CXCL4 secretion, potentially explains sex specific differences in revascularization in C5aR1-/- mice, where the effect of missing CXCL4 would be larger. Based on the previous identification of the murine C5a-C5aR1-CXCL4 axis as a negative regulator of revascularization these data suggest a significantly increased relevance in male compared to female mice. Furthermore, the knowledge of the underlying mechanisms allows for the introduction of sex-specific therapies.
Sources:
Nording, H. et al. The C5a/C5a receptor 1 axis controls tissue neovascularization through CXCL4 release from platelets. Nat Commun 12, 3352 (2021)
Regitz-Zagrosek, V., Gebhard, C. Gender medicine: effects of sex and gender on cardiovascular disease manifestation and outcomes. Nat Rev Cardiol 20, 236–247 (2023)
Roth, G. et al. Global Burden of Cardiovascular Diseases and Risk Factors, 1990–2019: Update from the GBD 2019 Study. JACC. 76 (25) 2982–3021 (2020)
