Soluble ST2 levels are related to replacement myocardial fibrosis in severe aortic stenosis.

INTRODUCTION AND OBJECTIVES: Patients with aortic stenosis (AS) exhibit left ventricular (LV) remodeling and replacement myocardial fibrosis (RMF). Whether sST2 is associated with RMF measured by cardiac magnetic resonance and with sex remains unknown. METHODS: We recruited 79 consecutive patients (73.0 [68.0-78.0] years; 61% men) with severe isolated AS underdoing valve replacement. RMF was identified and quantified by late gadolinium enhancement (LGE). Serum sST2 levels were determined. RESULTS: RMF was associated with higher circulating sST2 levels, LV hypertrophy and dilation, and lower LV ejection fraction. All patients with LV dysfunction had RMF. Circulating levels of sST2 ≥ 28.8 ng/mL were associated with RMF and greater LV hypertrophy. LGE mass was correlated with LV remodeling and sST2. Of note, sST2 levels were also associated with the RMF pattern, being higher in midwall than in subendocardial fibrosis. Multivariate analyses showed that only LV ejection fraction and sST2 levels were associated with RMF. Moreover, men had higher levels of sST2 and RMF. RMF was associated with higher LV dilation and hypertrophy only in men and was correlated with LGE mass. CONCLUSIONS: SST2 was an independent factor for RMF in patients with severe isolated AS. The presence of RMF was predicted by sST2 ≥ 28.2 ng/mL, and was associated with greater LV hypertrophy. sST2 expression and clinical associations may be sex-specific.

Sex-dependent expression of neutrophil gelatinase-associated lipocalin in aortic stenosis.

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.

Characterization of the sex-specific pattern of angiogenesis and lymphangiogenesis in aortic stenosis.

Objective: We 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 Results: Totally 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-ß 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. Conclusion: Our 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.

Targeting Fatty Acid-Binding Protein 4 Improves Pathologic Features of Aortic Stenosis.

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.

Sex-Related Signaling of Aldosterone/Mineralocorticoid Receptor Pathway in Calcific Aortic Stenosis.

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.

Sex-Differences in Aortic Stenosis: Mechanistic Insights and Clinical Implications.

Objective: We aim to analyse sex-specific differences in aortic valves (AVs) and valve interstitial cells (VICs) from aortic stenosis (AS) patients. Approach and Results: 238 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)-1ß, 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. Conclusions: Our 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.

Activation of the Interleukin-33/ST2 Pathway Exerts Deleterious Effects in Myxomatous Mitral Valve Disease.

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.

Beneficial Effects of Mineralocorticoid Receptor Antagonism on Myocardial Fibrosis in an Experimental Model of the Myxomatous Degeneration of the Mitral Valve.

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.

Soluble St2 Induces Cardiac Fibroblast Activation and Collagen Synthesis via Neuropilin-1.

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)-ß1. 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.

A New Role for the Aldosterone/Mineralocorticoid Receptor Pathway in the Development of Mitral Valve Prolapse.

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.

A Role for MMP-10 (Matrix Metalloproteinase-10) in Calcific Aortic Valve Stenosis.

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; rS=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-1ß (rS=0.5045, P<0.001) and BMP (bone morphogenetic protein)-2 (rS=0.5003, P<0.01). In vitro, recombinant human MMP-10 induced the overexpression of inflammatory, fibrotic, and osteogenic markers (interleukin-1ß, 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.

Soluble ST2 promotes oxidative stress and inflammation in cardiac fibroblasts: an in vitro and in vivo study in aortic stenosis.

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-1ß 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.

Aldosterone Impairs Mitochondrial Function in Human Cardiac Fibroblasts via A-Kinase Anchor Protein 12.

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.

Galectin-3 down-regulates antioxidant peroxiredoxin-4 in human cardiac fibroblasts: a new pathway to induce cardiac damage.

Galectin-3 (Gal-3) is increased in heart failure (HF) and promotes cardiac fibrosis and inflammation. We investigated whether Gal-3 modulates oxidative stress in human cardiac fibroblasts, in experimental animal models and in human aortic stenosis (AS). Using proteomics and immunodetection approaches, we have identified that Gal-3 down-regulated the antioxidant peroxiredoxin-4 (Prx-4) in cardiac fibroblasts. In parallel, Gal-3 increased peroxide, nitrotyrosine, malondialdehyde, and N-carboxymethyl-lysine levels and decreased total antioxidant capacity. Gal-3 decreased prohibitin-2 expression without modifying other mitochondrial proteins. Prx-4 silencing increased oxidative stress markers. In Gal-3-silenced cells and in heart from Gal-3 knockout mice, Prx-4 was increased and oxidative stress markers were decreased. Pharmacological inhibition of Gal-3 with modified citrus pectin restored cardiac Prx-4 as well as prohibitin-2 levels and improved oxidative status in spontaneously hypertensive rats. In serum from 87 patients with AS, Gal-3 negatively correlated with total antioxidant capacity and positively correlated with peroxide. In myocardial biopsies from 26 AS patients, Gal-3 up-regulation paralleled a decrease in Prx-4 and in prohibitin-2. Cardiac Gal-3 inversely correlated with Prx-4 levels in myocardial biopsies. These data suggest that Gal-3 decreased Prx-4 antioxidant system in cardiac fibroblasts, increasing oxidative stress. In pathological models presenting enhanced cardiac Gal-3, the decrease in Prx-4 expression paralleled increased oxidative stress. Gal-3 blockade restored Prx-4 expression and improved oxidative stress status. In AS, circulating levels of Gal-3 could reflect oxidative stress. The alteration of the balance between antioxidant systems and reactive oxygen species production could be a new pathogenic mechanism by which Gal-3 induces cardiac damage in HF.

A role for fumarate hydratase in mediating oxidative effects of galectin-3 in human cardiac fibroblasts.

AIMS: Galectin-3 (Gal-3), a ß-galactoside-binding lectin involved in cardiac inflammation and fibrosis, could regulate oxidative stress, although the mechanisms have not been elucidated. We herein investigated the changes in oxidative stress-related mediators induced by Gal-3 in human cardiac fibroblasts and in pathological animal and human models of cardiac diseases. RESULTS: Using quantitative proteomics and immunodetection approaches, we have identified that Gal-3 down-regulated fumarate hydratase (FH) in human cardiac fibroblasts. In parallel, Gal-3 increased fumarate production in a time-dependent manner. Gal-3 treatment enhanced carbonylated proteins detected through OxyBlot technique. Interestingly, treatment of cells with fumarate induced oxidative stress, enhanced fibroblast activation markers and increased collagen and interleukin-6 secretion. In Gal-3-silenced cells and in heart from Gal-3 knock-out mice, FH was increased and fumarate was decreased. In myocardial biopsies from patients with aortic stenosis (AS, n=26), FH levels were decreased as compared to Controls (n=13). Cardiac Gal-3 inversely correlated with FH levels in myocardial biopsies. In an experimental model of AS rats, pharmacological inhibition of Gal-3 restored cardiac FH, decreased fumarate concentration and improved oxidative status. CONCLUSION: In human cardiac fibroblasts, Gal-3 decreased FH expression increasing fumarate concentration and promoting oxidative stress. In human AS, cardiac levels of Gal-3 inversely associated with FH. Gal-3 blockade restored FH and improved fumarate and oxidative stress status in AS rats. FH is therefore a key molecule mediating Gal-3-induced oxidative stress in cardiac cells.

Beneficial Effects of Galectin-3 Blockade in Vascular and Aortic Valve Alterations in an Experimental Pressure Overload Model.

Galectin-3 (Gal-3) is involved in cardiovascular fibrosis and aortic valve (AV) calcification. We hypothesized that Gal-3 pharmacological inhibition with modified citrus pectin (MCP) could reduce aortic and AV remodeling in normotensive rats with pressure overload (PO). Six weeks after aortic constriction, vascular Gal-3 expression was up-regulated in male Wistar rats. Gal-3 overexpression was accompanied by an increase in the aortic media layer thickness, enhanced total collagen, and augmented expression of fibrotic mediators. Further, vascular inflammatory markers as well as inflammatory cells content were greater in aorta from PO rats. MCP treatment (100 mg/kg/day) prevented the increase in Gal-3, media thickness, fibrosis, and inflammation in the aorta of PO rats. Gal-3 levels were higher in AVs from PO rats. This paralleled enhanced AV fibrosis, inflammation, as well as greater expression of calcification markers. MCP treatment prevented the increase in Gal-3 as well as fibrosis, inflammation, and calcification in AVs. Overall, Gal-3 is overexpressed in aorta and AVs from PO rats. Gal-3 pharmacological inhibition blocks aortic and AV remodeling in experimental PO. Gal-3 could be a new therapeutic approach to delay the progression and the development of aortic remodeling and AV calcification in PO.

A role for galectin-3 in the development of early molecular alterations in short-term aortic stenosis.

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 ß-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.