Background
Sex-specific differences in cardiovascular disease manifestation remain incompletely understood despite their profound clinical implications (Regitz-Zagrosek & Gebhard, 2023). Previously, we identified platelet complement receptor C5aR1 as a negative regulator of revascularization through selective CXCL4 secretion in hindlimb ischemia (HLI) (Nording et al., 2021). Here, we investigated the molecular mechanisms underlying sex-specific differences in C5aR1-mediated revascularization regulation.
Methods
HLI was induced in 8-12 week-old male and female wild-type, C5aR1^-/-^, C5^-/-^, and platelet-specific C5aR1 knockout (PF4-Cre-C5aR1^fl/fl^) mice. Revascularization was monitored by laser Doppler imaging (LDI) over 14 days. Complement activation, CXCL4 and C5aR1 expression were quantified in ischemic and control muscle tissue by ELISA and Western blot. Isolated platelets were stimulated with C5a, and CXCL4 secretion was measured. Primary megakaryocytes were cultured ex vivo and stimulated with estradiol, testosterone, or progesterone to assess hormone-dependent C5aR1 regulation.
Results
Platelet-specific deletion of C5aR1 resulted in significantly improved revascularization in female mice (days 8-14 post-ischemia), while male C5aR1^fl/fl^PF4-Cre^+^ mice showed no difference compared to controls, revealing sex-specific C5aR1-mediated inhibition of neovascularization. Ischemia induced robust complement activation with C3b deposition strongly correlating with CXCL4 accumulation (r=0.93, p<0.0001). Male mice exhibited 4.5-fold higher platelet C5aR1 expression compared to females, resulting in enhanced C5a-induced CXCL4 secretion and greater CXCL4 deposition in ischemic tissue. Mechanistically, estradiol stimulation of megakaryocytes significantly suppressed C5aR1 expression during proplatelet formation (p<0.05), establishing a hormone-dependent regulatory mechanism. This effect was estradiol-specific and more pronounced in female-derived megakaryocytes.
Discussion
These findings establish an estradiol-C5aR1-CXCL4 axis that mechanistically explains sex-specific revascularization differences: higher estradiol levels in females suppress megakaryocyte C5aR1 expression, reducing platelet C5a responsiveness and subsequent CXCL4 release, thereby permitting enhanced neovascularization. The absent sex differences in platelet-specific C5aR1 knockout mice confirm that platelet C5aR1 is the critical mediator. This hormone-regulated complement-platelet crosstalk represents a novel therapeutic target for sex-tailored interventions in peripheral artery disease and ischemic cardiovascular disorders.
References: Regitz-Zagrosek & Gebhard, Nat Rev Cardiol (2023); Nording et al., Nat Commun (2021)
