ObjectiveWe aim to analyse sex-specific differences in aortic valves (AVs) and valve interstitial cells (VICs) from aortic stenosis (AS) patients.Approach and Results238 patients with severe AS undergoing surgical valve replacement were recruited. Two hundred and two AVs (39.1% women) were used for ex vivo analyses and 36 AVs (33.3% women) for in vitro experiments. AVs from men presented increased levels of the inflammatory molecules interleukin (IL)-1β, IL-6, Rantes, and CD45. Oxidative stress (eNOS, myeloperoxidase, malondialdehyde and nitrotyrosine) was upregulated in male AVs. Concerning fibrosis, similar levels of collagen type I, decreased levels of collagen type III and enhanced fibronectin, active Lox-1 and syndecan-1 expressions were found in AVs from men compared with women. Extracellular matrix (ECM) remodeling was characterized by reduced metalloproteinase-1 and 9 expression and increased tissue inhibitor of metalloproteinase-2 expression in male AVs. Importantly, osteogenic markers (bone morphogenetic protein-9, Rank-L, osteopontin, periostin, osteocalcin and Sox-9) and apoptosis (Bax, Caspase 3, p53, and PARP1) were enhanced in AVs from men as compared to women. Isolated male VICs presented higher myofibroblast-like phenotype than female VICs. Male VICs exhibited increased inflammatory, oxidative stress, fibrotic, apoptosis and osteogenic differentiation markers.ConclusionsOur results suggest that the mechanisms driving the pathogenesis of AS could be different in men and women. Male AVs and isolated VICs presented more inflammation, oxidative stress, ECM remodeling and calcification as compared to those from women. A better knowledge of the pathophysiological pathways in AVs and VICs will allow the development of sex-specific options for the treatment of AS.

Background: Vascular endothelial cells activation and dysfunction mediate inflammation and abnormal coagulation in COVID-19 patients. Mineralocorticoid receptor (MR) signaling and its downstream target Galectin-3 (Gal-3) are known to mediate cardiovascular inflammation and might be involved in the pathogenesis of COVID-19 complications. Accordingly, we aimed to investigate the potential beneficial effects of MR antagonism and Gal-3 inhibition on the inflammatory response induced by SARS-CoV-2 Spike protein in human aortic endothelial cells (HAECs). Methods: HAECs were treated with recombinant SARS-COV2 Spike (S) protein. MR antagonists (namely spironolactone and eplerenone) or the Gal-3 inhibitor G3P-01 were supplemented before and after S protein challenge. HAECs supernatants were assessed by ELISA or Western blotting. Results: HAECs treated with recombinant S protein resulted in enhanced secretion of inflammatory molecules (interleukin-6, monocyte chemoattractant protein-1, interleukin-18, interleukin-27, and interferon-γ) as well as in the thrombosis marker plasminogen activator inhibitor (PAI)-1. This was prevented and reversed by both MR antagonists and G3P-01. Conclusions: These findings indicate that MR/Gal-3 pathway blockade could be a promising option to reduce endothelial inflammation in SARS-CoV-2 infection.

ObjectiveWe aim to analyze sex-related differences in angiogenesis and lymphangiogenesis in aortic valves (AVs) and valve interstitial cells (VICs) from aortic stenosis (AS) patients.Approach and ResultsTotally 230 patients (59% men) with severe AS undergoing surgical valve replacement were recruited. The density of total neovessels was higher in AVs from men as compared to women. Both small and medium neovessels were more abundant in men's AVs. Accordingly, male AVs exhibited higher CD31 and VE-cadherin expressions. The levels of the pro-angiogenic markers, such as vascular endothelial growth factor (VEGF)-A, VEGF receptor (VEGFR)1, VEGFR2, insulin-like growth factor-binding protein-2 (IGFBP-2), interleukin (IL)-8, chemerin, and fibroblast growth factor (FGF)-7, were increased in AVs from men. Transforming growth factor-β expression was higher in male AVs. The expression of antiangiogenic molecules thrombospondin (Tsp)-1, endostatin, and CD36 was upregulated in male AVs, although the levels of Tsp-2, IL-4, IL-12p70, and chondromodulin-1 were similar between both sexes. The number of lymphatic vessels and the expression of the lymphangiogenic markers Lyve-1 and D2-40 was higher in men's AV as well as VEGF-C, VEGF-D, and VEGFR3. Multivariate analyses adjusted for confounders further validated the sex-dependent expression of these targets. VICs isolated from men's AVs secreted higher amounts of the pro-angiogenic factors, VEGF-A, VEGFR1, IGFBP-2, and FGF-7, as well as the pro-lymphangiogenic factors, VEGF-C, VEGF-D, and VEGFR3, than women without changes in antiangiogenic markers.ConclusionOur data show that aberrant angiogenic and lymphangiogenic cues are over-represented in male AVs. Importantly, the VIC is a relevant source of multiple morphogens involved in angiogenesis and lymphangiogenesis likely endowing the AV of men with the predominant calcific AS phenotypes.

Background: There are sex differences in the pathophysiology of aortic valve (AV) calcification in patients with aortic stenosis, although the molecular and cellular mechanisms have not been elucidated. Aldosterone (Aldo) promotes proteoglycan synthesis in valve interstitial cells (VICs) from mitral valves via the mineralocorticoid receptor (MR). We investigated the influence of sex in the role of Aldo/MR pathway in AV alterations in patients with aortic stenosis. METHODS AND RESULTS: MR was expressed by primary aortic VICs and in AVs from patients with aortic stenosis. MR expression positively correlated with VIC activation markers in AVs from both sexes. However, MR expression was positively associated with molecules involved in AV calcification only in AV from men. Aldo enhanced VIC activation markers in cells from men and women. Interestingly, Aldo increased the expression of calcification markers only in VICs isolated from men. In female VICs, Aldo enhanced fibrotic molecules. MR antagonism (spironolactone) blocked all the above effects. Cytokine arrays showed ICAM (intercellular adhesion molecule)-1 and osteopontin to be specifically increased by Aldo in male VICs. In AVs from men, MR expression positively associated with both ICAM-1 (intercellular adhesion molecule-1) and osteopontin. Only in female VICs, estradiol treatment blocked Aldo-induced VICs activation, inflammation, and fibrosis. Conclusions: These findings demonstrate that the Aldo/MR pathway could play a role in early stages of aortic stenosis by promoting VICs activation, fibrosis, and ulterior calcification. Importantly, Aldo/MR pathway is involved in fibrosis in women and in early AV calcification only in men. Accordingly, MR antagonism emerges as a new sex-specific pharmacological treatment to prevent AV alterations.

Mitral valve prolapse (MVP) patients develop myocardial fibrosis that is not solely explained by volume overload, but the pathophysiology has not been defined. Mineralocorticoid receptor antagonists (MRAs) improve cardiac function by decreasing cardiac fibrosis in other heart diseases. We examined the role of MRA in myocardial fibrosis associated with myxomatous degeneration of the mitral valve. Myocardial fibrosis has been analyzed in a mouse model of mitral valve myxomatous degeneration generated by pharmacological treatment with Nordexfenfluramine (NDF) in the presence of the MRA spironolactone. In vitro, adult human cardiac fibroblasts were treated with NDF and spironolactone. In an experimental mouse, MRA treatment reduced interstitial/perivascular fibrosis and collagen type I deposition. MRA administration blunted NDF-induced cardiac expression of vimentin and the profibrotic molecules galectin-3/cardiotrophin-1. In parallel, MRA blocked the increase in cardiac non-fibrillar proteins such as fibronectin, aggrecan, decorin, lumican and syndecan-4. The following effects are blocked by MRA: in vitro, in adult human cardiac fibroblasts, NDF-treatment-induced myofibroblast activation, collagen type I and proteoglycans secretion. Our findings demonstrate, for the first time, the contribution of the mineralocorticoid receptor (MR) to the development of myocardial fibrosis associated with mitral valve myxomatous degeneration. MRA could be a therapeutic approach to reduce myocardial fibrosis associated with MVP.

Mitral valve disease (MVD) is a frequent cause of heart failure and death worldwide, but its etiopathogenesis is not fully understood. Interleukin (IL)-33 regulates inflammation and thrombosis in the vascular endothelium and may play a role in the atherosclerotic process, but its role in mitral valve has not been investigated. We aim to explore IL-33 as a possible inductor of myxomatous degeneration in human mitral valves. We enrolled 103 patients suffering from severe mitral regurgitation due to myxomatous degeneration undergoing mitral valve replacement. Immunohistochemistry of the resected leaflets showed IL-33 and ST2 expression in both valve interstitial cells (VICs) and valve endothelial cells (VECs). Positive correlations were found between the levels of IL-33 and molecules implicated in the development of myxomatous MVD, such as proteoglycans, extracellular matrix remodeling enzymes (matrix metalloproteinases and their tissue inhibitors), inflammatory and fibrotic markers. Stimulation of single cell cultures of VICs and VECs with recombinant human IL-33 induced the expression of activated VIC markers, endothelial–mesenchymal transition of VECs, proteoglycan synthesis, inflammatory molecules and extracellular matrix turnover. Our findings suggest that the IL-33/ST2 system may be involved in the development of myxomatous MVD by enhancing extracellular matrix remodeling.

Aortic stenosis (AS) is a fibrocalcific disease of the aortic valves (AVs). Sex-differences in AS pathophysiology have recently been described. High levels of fatty acid-binding protein 4 (FAPB4) in atherosclerotic plaques have been associated with increased local inflammation, endothelial dysfunction, and plaque vulnerability. FABP4 pharmacological blockade has been shown to be effective for the treatment of atherosclerosis by modulating metabolic and inflammatory pathways. We aimed to analyze the sex-specific expression of FABP4 in AS and its potential role as a therapeutic target. A total of 226 patients (61.5% men) with severe AS undergoing surgical AV replacement were recruited. The FABP4 levels were increased in the AVs of AS patients compared to the control subjects, showing greater expression in the fibrocalcific regions. Male AVs exhibited higher levels of FABP4 compared to females, correlating with markers of inflammation (IL-6, Rantes), apoptosis (Bax, caspase-3, Bcl-2), and calcification (IL-8, BMP-2 and BMP-4). VICs derived from AS patients showed the basal expression of FABP4 in vitro. Osteogenic media induced upregulation of intracellular and secreted FABP4 levels in male VICs after 7 days, along with increased levels of inflammatory, pro-apoptotic, and osteogenic markers. Treatment with BMS309403, a specific inhibitor of FABP4, prevented from all of these changes. Thus, we propose FABP4 as a new sex-specific pharmacological therapeutic target in AS.

Background Accumulating evidence suggest the existence of sex-related differences in the pathogenesis of aortic stenosis (AS) with inflammation, oxidative stress, fibrosis and calcification being over-represented in men. Neutrophil gelatinase-associated lipocalin (NGAL) is expressed in a myriad of tissues and cell types, and it is associated with acute and chronic pathological processes comprising inflammation, fibrosis or calcification. Sex-dependent signatures have been evidenced for NGAL which expression has been associated predominantly in males to metabolic and cardiovascular disorders. We aimed to analyse sex-related differences of NGAL in AS and its role in the inflammatory and fibrocalcific progression of AS. Methods and results 220 (60.45% men) patients with severe AS elective for surgical aortic valve (AV) replacement were recruited. Immunohistochemistry revealed higher expression of NGAL in calcific areas of AVs and that was validated by qPCR in in 65 (60% men) donors. Valve interstitial cells (VICs) were a source of NGAL in these samples. Proteome profiler analyses evidenced higher expression of NGAL in men compared to women, and that was further validated by ELISA. NGAL expression in the AV was correlated with inflammation, oxidative stress, and osteogenic markers, as well as calcium score. The expression of NGAL, both intracellular and secreted (sNGAL), was significantly deregulated only in calcifying male-derived VICs. Depletion of intracellular NGAL in calcifying male-derived VICs was associated with pro-inflammatory profiles, dysbalanced matrix remodelling and pro-osteogenic profiles. Conversely, exogenous NGAL mediated inflammatory and dysbalanced matrix remodelling in calcifying VICs, and all that was prevented by the pharmacological blockade of NGAL. Conclusions Owing to the over-expression of NGAL, the AV from men may be endowed with higher expression of inflammatory, oxidative stress, matrix remodelling and osteogenic markers supporting the progression of calcific AS phenotypes. The expression of NGAL in the VIC emerges as a potential therapeutic checkpoint, with its effects being potentially reverted by the pharmacological blockade of extracellular NGAL.