BackgroundAortic stenosis (AS) is a chronic inflammatory disease, and calcification plays an important role in the progression of the disease. Galectin‐3 (Gal‐3) is a proinflammatory molecule involved in vascular osteogenesis in atherosclerosis. Therefore, we hypothesized that Gal‐3 could mediate valve calcification in AS.Methods and ResultsBlood samples and aortic valves (AVs) from 77 patients undergoing AV replacement were analyzed. As controls, noncalcified human AVs were obtained at autopsy (n=11). Gal‐3 was spontaneously expressed in valvular interstitial cells (VICs) from AVs and increased in AS as compared to control AVs. Positive correlations were found between circulating and valvular Gal‐3 levels. Valvular Gal‐3 colocalized with the VICs markers, alpha‐smooth muscle actin and vimentin, and with the osteogenic markers, osteopontin, bone morphogenetic protein 2, runt‐related transcription factor 2, and SRY (sex‐determining region Y)‐box 9. Gal‐3 also colocalized with the inflammatory markers cd68, cd80 and tumor necrosis factor alpha. In vitro, in VICs isolated from AVs, Gal‐3 induced expression of inflammatory, fibrotic, and osteogenic markers through the extracellular signal‐regulated kinase 1 and 2 pathway. Gal‐3 expression was blocked in VICs undergoing osteoblastic differentiation using its pharmacological inhibitor, modified citrus pectin, or the clustered regularly interspaced short palindromic repeats/Cas9 knockout system. Gal‐3 blockade and knockdown decreased the expression of inflammatory, fibrotic, and osteogenic markers in differentiated VICs.ConclusionsGal‐3, which is overexpressed in AVs from AS patients, appears to play a central role in calcification in AS. Gal‐3 could be a new therapeutic approach to delay the progression of AV calcification in AS.

Objective: Aortic valve (AV) calcification plays an important role in the progression of aortic stenosis (AS). MMP-10 (matrix metalloproteinase-10 or stromelysin-2) is involved in vascular calcification in atherosclerosis. We hypothesize that MMP-10 may play a pathophysiological role in calcific AS. Approach and Results: Blood samples (n=112 AS and n=349 controls) and AVs (n=88) from patients undergoing valve replacement were analyzed. Circulating MMP-10 was higher in patients with AS compared with controls ( P <0.001) and correlated with TNFα (tumor necrosis factor α; r S =0.451; P <0.0001). MMP-10 was detected by immunochemistry in AVs from patients with AS colocalized with aortic valve interstitial cells markers α-SMA (α-smooth muscle actin) and vimentin and with calcification markers Runx2 (Runt-related transcription factor 2) and SRY (sex-determining region Y)-box 9. MMP-10 expression in AVs was further confirmed by RT-qPCR and western blot. Ex vivo, MMP-10 was elevated in the conditioned media of AVs from patients with AS and associated with interleukin-1β (r S =0.5045, P <0.001) and BMP (bone morphogenetic protein)-2 (r S =0.5003, P <0.01). In vitro, recombinant human MMP-10 induced the overexpression of inflammatory, fibrotic, and osteogenic markers (interleukin-1β, α-SMA, vimentin, collagen, BMP-4, Sox9, OPN [osteopontin], BMP-9, and Smad 1/5/8; P <0.05) and cell mineralization in aortic valve interstitial cells isolated from human AVs, in a mechanism involving Akt (protein kinase B) phosphorylation. These effects were prevented by TIMP-1 (tissue inhibitor of metalloproteinases type 1), a physiological MMP inhibitor, or specifically by an anti-MMP-10 antibody. Conclusions: MMP-10, which is overexpressed in aortic valve from patients with AS, seems to play a central role in calcification in AS through Akt phosphorylation. MMP-10 could be a new therapeutic target for delaying the progression of aortic valve calcification in AS.

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.

Rationale: Mitral valve prolapse (MVP) is one of the most common valvular disorders. However, the molecular and cellular mechanisms involved in fibromyxomatous changes in the mitral leaflet tissue have not been elucidated. Aldosterone (Aldo) promotes fibrosis in myocardium, and MR (mineralocorticoid receptor) antagonists (MRAs) improve cardiac function by decreasing cardiac fibrosis. Objective: We investigated the role of the Aldo/MR in the fibromyxomatous modifications associated with MVP. Methods and Results: Aldo enhanced valvular interstitial cell activation markers and induced endothelial-mesenchymal transition in valvular endothelial cells, resulting in increased proteoglycan secretion. MRA blocked all the above effects. Cytokine arrays showed CT-1 (cardiotrophin-1) to be a mediator of Aldo-induced valvular interstitial cell activation and proteoglycan secretion and CD (cluster of differentiation) 14 to be a mediator of Aldo-induced endothelial-mesenchymal transition and proteoglycan secretion in valvular endothelial cells. In an experimental mouse model of MVP generated by nordexfenfluramine administration, MRA treatment reduced mitral valve thickness and proteoglycan content. Endothelial-specific MR deletion prevented fibromyxomatous changes induced by nordexfenfluramine administration. Moreover, proteoglycan expression was slightly lower in the mitral valves of MVP patients treated with MRA. Conclusions: These findings demonstrate, for the first time, that the Aldo/MR pathway regulates the phenotypic, molecular, and histological changes of valvular interstitial cells and valvular endothelial cells associated with MVP development. MRA treatment appears to be a promising option to reduce fibromyxomatous alterations in MVP.

Aldosterone (Aldo) contributes to mitochondrial dysfunction and cardiac oxidative stress. Using a proteomic approach, A-kinase anchor protein (AKAP)-12 has been identified as a down-regulated protein by Aldo in human cardiac fibroblasts. We aim to characterize whether AKAP-12 down-regulation could be a deleterious mechanism which induces mitochondrial dysfunction and oxidative stress in cardiac cells. Aldo down-regulated AKAP-12 via its mineralocorticoid receptor, increased oxidative stress and induced mitochondrial dysfunction characterized by decreased mitochondrial-DNA and Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) expressions in human cardiac fibroblasts. CRISPR/Cas9-mediated knock-down of AKAP-12 produced similar deleterious effects in human cardiac fibroblasts. CRISPR/Cas9-mediated activation of AKAP-12 blunted Aldo effects on mitochondrial dysfunction and oxidative stress in human cardiac fibroblasts. In Aldo-salt-treated rats, cardiac AKAP-12, mitochondrial-DNA and PGC-1α expressions were decreased and paralleled increased oxidative stress. In myocardial biopsies from patients with aortic stenosis (AS, n = 26), AKAP-12, mitochondrial-DNA and PGC-1α expressions were decreased as compared to Controls (n = 13). Circulating Aldo levels inversely correlated with cardiac AKAP-12. PGC-1α positively associated with AKAP-12 and with mitochondrial-DNA. Aldo decreased AKAP-12 expression, impairing mitochondrial biogenesis and increasing cardiac oxidative stress. AKAP-12 down-regulation triggered by Aldo may represent an important event in the development of mitochondrial dysfunction and cardiac oxidative stress.

Background: Soluble ST2 (interleukin 1 receptor-like 1) (sST2) is involved in inflammatory diseases and increased in heart failure (HF). We herein investigated sST2 effects on oxidative stress and inflammation in human cardiac fibroblasts and its pathological role in human aortic stenosis (AS). Methods and results: Using proteomics and immunodetection approaches, we have identified that sST2 down-regulated mitofusin-1 (MFN-1), a protein involved in mitochondrial fusion, in human cardiac fibroblasts. In parallel, sST2 increased nitrotyrosine, protein oxidation and peroxide production. Moreover, sST2 enhanced the secretion of pro-inflammatory cytokines interleukin (IL)-6, IL-1β and monocyte chemoattractant protein-1 (CCL-2). Pharmacological inhibition of transcriptional factor nuclear factor κB (NFκB) restored MFN-1 levels and improved oxidative status and inflammation in cardiac fibroblasts. Mito-Tempo, a mitochondria-specific superoxide scavenger, as well as Resveratrol, a general antioxidant, attenuated oxidative stress and inflammation induced by sST2. In myocardial biopsies from 26 AS patients, sST2 up-regulation paralleled a decrease in MFN-1. Cardiac sST2 inversely correlated with MFN-1 levels and positively associated with IL-6 and CCL-2 in myocardial biopsies from AS patients. Conclusions: sST2 affected mitochondrial fusion in human cardiac fibroblasts, increasing oxidative stress production and inflammatory markers secretion. The blockade of NFκB or mitochondrial reactive oxygen species restored MFN-1 expression, improving oxidative stress status and reducing inflammatory markers secretion. In human AS, cardiac sST2 levels associated with oxidative stress and inflammation. The present study reveals a new pathogenic pathway by which sST2 promotes oxidative stress and inflammation contributing to cardiac damage.

Aortic stenosis (AS) is characterized by pressure overload and causes left ventricular (LV) fibrosis and inflammation, two mechanisms that eventually lead to cardiac dysfunction. Galectin-3 (Gal-3), a β-galactoside-binding lectin, promotes cardiac remodelling. In the present study, we investigated the role of Gal-3 in LV remodelling in patients with AS and the effects of Gal-3 blockade in rats subjected to short-term (6-week) supravalvular aortic banding (AS group). Myocardial biopsies were obtained from 25 patients with severe AS referred for aortic valve replacement and from necropsies of 11 cardiovascular disease-free control individuals. Gal-3 was up-regulated in myocardial biopsies from AS patients compared with controls. Gal-3 directly correlated with parameters assessing myocardial fibrosis and inflammation in AS patients. Normotensive AS animals presented decreased LV diastolic diameter compared with controls. At the histological level, AS rats exhibited a slight increase in LV cross-sectional area and LV wall thickness, and augmented cardiomyocyte width and cross-sectional area. AS animals presented enhanced cardiac Gal-3 expression, which paralleled higher myocardial fibrosis and inflammation. Cardiac Gal-3 was associated with fibrosis and inflammatory markers. Gal-3 pharmacological inhibition prevented the increase in cardiac Gal-3 and normalized histological and molecular alterations in AS rats. In short-term AS, the increase in myocardial Gal-3 expression was associated with cardiac fibrosis and inflammation, alterations that were prevented by Gal-3 blockade. These data suggest that Gal-3 inhibition could be a novel therapeutic approach in the prevention of AS-associated early pathological cardiac remodelling.

Circulating levels of soluble interleukin 1 receptor-like 1 (sST2) are increased in heart failure and associated with poor outcome, likely because of the activation of inflammation and fibrosis. We investigated the pathogenic role of sST2 as an inductor of cardiac fibroblasts activation and collagen synthesis. The effects of sST2 on human cardiac fibroblasts was assessed using proteomics and immunodetection approaches to evidence the upregulation of neuropilin-1 (NRP-1), a regulator of the profibrotic transforming growth factor (TGF)-β1. In parallel, sST2 increased fibroblast activation, collagen and fibrosis mediators. Pharmacological inhibition of nuclear factor-kappa B (NF-κB) restored NRP-1 levels and blocked profibrotic effects induced by sST2. In NRP-1 knockdown cells, sST2 failed to induce fibroblast activation and collagen synthesis. Exogenous NRP-1 enhanced cardiac fibroblast activation and collagen synthesis via NF-κB. In a pressure overload rat model, sST2 was elevated in association with cardiac fibrosis and was positively correlated with NRP-1 expression. Our study shows that sST2 induces human cardiac fibroblasts activation, as well as the synthesis of collagen and profibrotic molecules. These effects are mediated by NRP-1. The blockade of NF-κB restored NRP-1 expression, improving the profibrotic status induced by sST2. These results show a new pathogenic role for sST2 and its mediator, NRP-1, as cardiac fibroblast activators contributing to cardiac fibrosis.