Endothelial TLR2/4 signaling in sex dimorphism of pathological angiogenesis and aging - ABSTRACT Numerous studies have demonstrated the male-female health-survival paradox, where females exhibit a higher burden of age-associated disorders yet live longer than males. This paradox arises from a greater burden of chronic diseases in females, particularly those with a strong inflammatory or autoimmune component. A key factor contributing to inflammation-associated pathologies is endothelial cell (EC) dysfunction. This proposal aims to elucidate the molecular basis of sex-dependent differences in EC functions that may shape the outcomes of angiogenesis-driven diseases and aging in general. Our recent findings revealed that female ECs exhibit higher inflammation and reactive oxygen species (ROS) accumulation, with reduced angiogenic potential. These findings suggest biological sexual dimorphism in EC function, although the molecular mechanisms underlying these differences remain unclear. Toll-like receptors (TLRs), key innate proinflammatory mediators, recognize molecular patterns, including both pathogens and endogenous ligands generated by tissue damage and excessive oxidation. Analysis of multiple RNA sequencing datasets revealed that TLR2 and TLR4 pathways, specifically in ECs, are among the most significantly dysregulated in inflammation-associated pathologies and senescence. Moreover, gene expression profiling demonstrates that TLR2/4-related pathways are upregulated in female ECs compared to male ECs in vivo and in vitro. This upregulation seems to be driven by both increased TLR expression and higher levels of endogenous TLR ligands in females, collectively amplifying inflammatory responses. In vivo vascularization models showed a more substantial inflammatory component coupled with impaired angiogenesis and vascular remodeling in females. In EC-specific dual TLR2/4 knockout mice, eliminating these receptors abolished the pronounced differences between females and males in vascular models. This indicates that TLR2/4 pathways dominate sex-related differences in EC functions. While sex- specific differences in vascular diseases can arise from genetic, hormonal, or environmental factors, most research to date has focused on sex hormones. This project aims to shift the focus toward exploring the genetic components, particularly the role of endothelial TLR2 and TLR4 pathways in mediating these differences. Herein, we propose a new hypothesis: TLR2 and TLR4 on the endothelium are critical regulators of vascular responses, and augmented TLR2/4 activation contributes to increased vascular inflammation in females. We propose that endothelial TLR2/4-signaling is required for timely injury responses and tissue regeneration by regulating cytokine production and recruitment of inflammatory cells in females. We will utilize innovative transgenic mouse models, a pharmacological approach using phospholipid-based Reactive Carbonyl Scavengers (RCS) limiting the generation of TLR2 and TLR4, and state-of-the-art proteomics. Our studies will provide insights into the role of innate immune mechanisms in ECs and enhance our understanding of the genetic mechanisms responsible for sex differences in vascular pathologies, leading to more efficient therapeutics for both men and women.
