RESUMO
The pathological mechanisms underlying the sex-dependent presentation of calcific aortic stenosis (AS) remain poorly understood. We aim to analyse sex-specific responses of valve interstitial cells (VICs) to calcific environments and to identify new pathological and potentially druggable targets. First, VICs from stenotic patients were modelled using pro-calcifying media (HP). Both male and female VICs were inflamed upon calcific HP challenge, although the inflammatory response was higher in female VICs. The osteogenic and calcification responses were higher in male VICs. To identify new players involved in the responses to HP, proteomics analyses were performed on additional calcifying VICs. Neuropilin-1 (NRP-1) was significantly up-regulated in male calcifying VICs and that was confirmed in aortic valves (AVs), especially nearby neovessels and calcifications. Regardless of the sex, NRP-1 expression was correlated to inflammation, angiogenesis and osteogenic markers, but with stronger associations in male AVs. To further evidence the role of NRP-1, in vitro experiments of silencing or supplementation with soluble NRP-1 (sNRP-1) were performed. NRP-1 silencing or addition of sNRP-1 reduced/mended the expression of any sex-specific response triggered by HP. Moreover, NRP-1 regulation contributed to significantly diminish the baseline enhanced expression of pro-inflammatory, pro-angiogenic and pro-osteogenic markers mainly in male VICs. Validation studies were conducted in stenotic AVs. In summary, pharmacologic targeting of NRP-1 could be used to target sex-specific phenotypes in AS as well as to exert protective effects by reducing the basal expression of pathogenic markers only in male VICs.
RESUMO
BACKGROUND: Diabetes mellitus (DM) accelerates the progression of aortic stenosis (AS), but how their underlying molecular mechanisms interact is not clear. Moreover, whether DM contributes to clinically relevant sex-differences in AS is unknown. In this work we aim to characterize the sex-specific profile of major pathological mechanisms fundamental to aortic valve (AV) degeneration in AS patients with or without concomitant DM. METHODS: 283 patients with severe AS undergoing surgical valve replacement (27.6% DM, 59.4% men) were recruited. Expression of pathological markers related to AS were thoroughly assessed in AVs and valve interstitial cells (VICs) according to sex and presence of DM. Complementary in vitro experiments in VICs in the presence of high-glucose levels (25 mM) for 24, 48 and 72 h were performed. RESULTS: Oxidative stress and metabolic dysfunction markers were increased in AVs from diabetic AS patients compared to non-diabetic patients in both sexes. However, disbalanced oxidative stress and enhanced inflammation were more predominant in AVs from male AS diabetic patients. Osteogenic markers were exclusively increased in the AVs of diabetic women. Basal characterization of VICs confirmed that oxidative stress, inflammation, calcification, and metabolic alteration profiles were increased in diabetic VICs with sex-specific differences. VICs cultured in hyperglycemic-like conditions triggered inflammatory responses in men, whereas in women rapid and higher production of pro-osteogenic molecules. CONCLUSIONS: DM produces sex-specific pathological phenotypes in AV of AS patients. Importantly, women with diabetes are more prone to develop AV calcification. DM should be considered as a risk factor in AS especially in women.
