Mineralocorticoid Receptors in Vascular Smooth Muscle: Blood Pressure and Beyond.

After half a century of evidence suggesting the existence of mineralocorticoid receptors (MR) in the vasculature, the advent of technology to specifically knockout the MR from smooth muscle cells (SMCs) in mice has elucidated contributions of SMC-MR to cardiovascular function and disease, independent of the kidney. This review summarizes the latest understanding of the molecular mechanisms by which SMC-MR contributes to (1) regulation of vasomotor function and blood pressure to contribute to systemic and pulmonary hypertension; (2) vascular remodeling in response to hypertension, vascular injury, obesity, and aging, and the impact on vascular calcification; and (3) cardiovascular pathologies including aortic aneurysm, heart valve dysfunction, and heart failure. Data are reviewed from in vitro studies using SMCs and in vivo findings from SMC-specific MR-knockout mice that implicate target genes and signaling pathways downstream of SMC-MR. By regulating expression of the L-type calcium channel subunit Cav1.2 and angiotensin II type-1 receptor, SMC-MR contributes to myogenic tone and vasoconstriction, thereby contributing to systemic blood pressure. MR activation also promotes SMC proliferation, migration, production and degradation of extracellular matrix, and osteogenic differentiation by regulating target genes including connective tissue growth factor, osteopontin, bone morphogenetic protein 2, galectin-3, and matrix metallopeptidase-2. By these mechanisms, SMC-MR promotes disease progression in models of aging-associated vascular stiffness, vascular calcification, mitral and aortic valve disease, pulmonary hypertension, and heart failure. While rarely tested, when sexes were compared, the mechanisms of SMC-MR-mediated disease were sexually dimorphic. These advances support targeting SMC-MR-mediated mechanisms to prevent and treat diverse cardiovascular disorders.

Inhibition of the histone methyltransferase EZH2 induces vascular stiffness.

Vascular stiffness increases with aging, obesity and hypertension and predicts cardiovascular risk. The levels of histone H3-lysine-27 methylation (H3K27me) and the histone methyltransferase EZH2 both decrease in aging vessels, driving vascular stiffness. The impact of EZH2 inhibitors on vascular stiffness is unknown. We tested the hypothesis that the EZH2 inhibitor GSK126, currently in development for cancer treatment, increases vascular stiffness and explored underlying molecular mechanisms. Young (3 month) and middle-aged (12 month) male mice were treated with GSK126 for 1-2 months and primary human aortic smooth muscle cells (HASMCs) from young male and female donors were treated with GSK126 for 24-48 h. Stiffness was measured in vivo by pulse wave velocity and in vitro by atomic force microscopy (AFM) and vascular structure was quantified histologically. Extracellular matrix proteins were studied by qRT-PCR, immunoblotting, zymography and chromatin immunoprecipitation. GSK126 treatment decreased H3K27 methylation (H3K27me) and increased acetylation (H3K27ac) in mouse vessels and in HASMCs. In GSK126-treated mice, aortic stiffness increased without changes in vascular fibrosis. EZH2 inhibition enhanced elastin fiber degradation and matrix metalloprotease-2 (MMP2) expression. In HASMCs, GSK126 treatment increased synthetic phenotype markers and intrinsic HASMCs stiffness by AFM with altered cytoskeletal structure and increased nuclear actin staining. GSK126 also increased MMP2 protein expression, activity and enrichment of H3K27ac at the MMP2 promoter in HASMCs. GSK126 causes vascular stiffening, inducing MMP2 activity, elastin degradation, and modulation of SMC phenotype and cytoskeletal stiffness. These findings suggest that EZH2 inhibitors used to treat cancer could negatively impact the vasculature by enhancing stiffness and merits examination in human trials.

The Mineralocorticoid Receptor in the Vasculature: Friend or Foe?

Originally described as the renal aldosterone receptor that regulates sodium homeostasis, it is now clear that mineralocorticoid receptors (MRs) are widely expressed, including in vascular endothelial and smooth muscle cells. Ample data demonstrate that endothelial and smooth muscle cell MRs contribute to cardiovascular disease in response to risk factors (aging, obesity, hypertension, atherosclerosis) by inducing vasoconstriction, vascular remodeling, inflammation, and oxidative stress. Extrapolating from its role in disease, evidence supports beneficial roles of vascular MRs in the context of hypotension by promoting inflammation, wound healing, and vasoconstriction to enhance survival from bleeding or sepsis. Advances in understanding how vascular MRs become activated are also reviewed, describing transcriptional, ligand-dependent, and ligand-independent mechanisms. By synthesizing evidence describing how vascular MRs convert cardiovascular risk factors into disease (the vascular MR as a foe), we postulate that the teleological role of the MR is to coordinate responses to hypotension (the MR as a friend).

PROX1 Inhibits PDGF-B Expression to Prevent Myxomatous Degeneration of Heart Valves.

BACKGROUND: Cardiac valve disease is observed in 2.5% of the general population and 10% of the elderly people. Effective pharmacological treatments are currently not available, and patients with severe cardiac valve disease require surgery. PROX1 (prospero-related homeobox transcription factor 1) and FOXC2 (Forkhead box C2 transcription factor) are transcription factors that are required for the development of lymphatic and venous valves. We found that PROX1 and FOXC2 are expressed in a subset of valvular endothelial cells (VECs) that are located on the downstream (fibrosa) side of cardiac valves. Whether PROX1 and FOXC2 regulate cardiac valve development and disease is not known. METHODS: We used histology, electron microscopy, and echocardiography to investigate the structure and functioning of heart valves from Prox1ΔVEC mice in which Prox1 was conditionally deleted from VECs. Isolated valve endothelial cells and valve interstitial cells were used to identify the molecular mechanisms in vitro, which were tested in vivo by RNAScope, additional mouse models, and pharmacological approaches. The significance of our findings was tested by evaluation of human samples of mitral valve prolapse and aortic valve insufficiency. RESULTS: Histological analysis revealed that the aortic and mitral valves of Prox1ΔVEC mice become progressively thick and myxomatous. Echocardiography revealed that the aortic valves of Prox1ΔVEC mice are stenotic. FOXC2 was downregulated and PDGF-B (platelet-derived growth factor-B) was upregulated in the VECs of Prox1ΔVEC mice. Conditional knockdown of FOXC2 and conditional overexpression of PDGF-B in VECs recapitulated the phenotype of Prox1ΔVEC mice. PDGF-B was also increased in mice lacking FOXC2 and in human mitral valve prolapse and insufficient aortic valve samples. Pharmacological inhibition of PDGF-B signaling with imatinib partially ameliorated the valve defects of Prox1ΔVEC mice. CONCLUSIONS: PROX1 antagonizes PDGF-B signaling partially via FOXC2 to maintain the extracellular matrix composition and prevent myxomatous degeneration of cardiac valves.

Smooth Muscle Mineralocorticoid Receptor Promotes Hypertension After Preeclampsia.

BACKGROUND: Preeclampsia is a syndrome of high blood pressure (BP) with end organ damage in late pregnancy that is associated with high circulating soluble VEGF receptor (sFlt1 [soluble Fms-like tyrosine kinase 1]). Women exposed to preeclampsia have a substantially increased risk of hypertension after pregnancy, but the mechanism remains unknown, leaving a missed interventional opportunity. After preeclampsia, women have enhanced sensitivity to hypertensive stress. Since smooth muscle cell mineralocorticoid receptors (SMC-MR) are activated by hypertensive stimuli, we hypothesized that high sFlt1 exposure in pregnancy induces a postpartum state of enhanced SMC-MR responsiveness. METHODS: Postpartum BP response to high salt intake was studied in women with prior preeclampsia. MR transcriptional activity was assessed in vitro in sFlt1-treated SMC by reporter assays and PCR. Preeclampsia was modeled by transient sFlt1 expression in pregnant mice. Two months post-partum, mice were exposed to high salt and then to AngII (angiotensin II) and BP and vasoconstriction were measured. RESULTS: Women exposed to preeclampsia had significantly enhanced salt sensitivity of BP verses those with a normotensive pregnancy. sFlt1 overexpression during pregnancy in mice induced elevated BP and glomerular endotheliosis, which resolved post-partum. The sFlt1 exposed post-partum mice had significantly increased BP response to 4% salt diet and to AngII infusion. In vitro, SMC-MR transcriptional activity in response to aldosterone or AngII was significantly increased after transient exposure to sFlt1 as was aldosterone-induced expression of AngII type 1 receptor. Post-partum, SMC-MR-KO mice were protected from the enhanced response to hypertensive stimuli after preeclampsia. Mechanistically, preeclampsia mice exposed to postpartum hypertensive stimuli develop enhanced aortic stiffness, microvascular myogenic tone, AngII constriction, and AngII type 1 receptor expression, all of which were prevented in SMC-MR-KO littermates. CONCLUSIONS: These data support that sFlt1-induced vascular injury during preeclampsia produces a persistent state of enhanced sensitivity of SMC-MR to activation. This contributes to postpartum hypertension in response to common stresses and supports testing of MR antagonism to mitigate the increased cardiovascular risk in women after PE.

Mechanism by Which Inflammation and Oxidative Stress Induce Mineralocorticoid Receptor Gene Expression in Aging Vascular Smooth Muscle Cells.

BACKGROUND: Vascular MR (mineralocorticoid receptor) expression increases with age driving aging-associated vascular stiffness and hypertension. MR has two isoforms (1α and 1ß) with distinct 5'-untranslated and promoter sequences (P1 and P2), but the gene regulatory mechanisms remain unknown. We investigated mechanisms driving MR gene transcriptional regulation in aging human smooth muscle cells (SMC). METHODS: MR was quantified in aortic tissue and primary human aortic SMC (HASMC) comparing adult and aged donors and adult HASMC treated with H2O2, to induce aging. Predicted transcription factor (TF) binding sites in the MR gene were validated using chromatin immunoprecipitations and reporter assays. The impact of TF inhibitors on MR isoforms and fibrosis target gene expression was examined. RESULTS: Expression of both MR mRNA isoforms increased with donor age or H2O2 treatment in HASMCs. HIF1α (hypoxia-inducible factor) and the inflammatory TF NFκB (nuclear factor kappa B) both increased with age in HASMCs and are predicted to bind MR promoters. H2O2 induced HIF1α and NFκB expression and DNA binding of HIF1α to the MR P1 promoter and of NFκB to both MR promoters in HASMCs. HIF1α inhibition decreased MR-1α isoform expression while NFκB inhibition decreased both MR isoforms. HIF1α, NFκB, and MR inhibition decreased the expression of a SMC-MR target gene implicated in vascular fibrosis. In human aortic tissues, expression of HIF1α and NFκB each positively correlated with donor age and MR expression (P<0.0001). CONCLUSIONS: These data implicate the inflammatory TF, NFκB, and oxidative stress-induced TF, HIF1α, in regulating SMC MR transcription in aging HASMCs, which drives aging-related vascular stiffness and cardiovascular disease.

Smooth muscle mineralocorticoid receptor as an epigenetic regulator of vascular ageing.

AIMS: Vascular stiffness increases with age and independently predicts cardiovascular disease risk. Epigenetic changes, including histone modifications, accumulate with age but the global pattern has not been elucidated nor are the regulators known. Smooth muscle cell-mineralocorticoid receptor (SMC-MR) contributes to vascular stiffness in ageing mice. Thus, we investigated the regulatory role of SMC-MR in vascular epigenetics and stiffness. METHODS AND RESULTS: Mass spectrometry-based proteomic profiling of all histone modifications completely distinguished 3 from 12-month-old mouse aortas. Histone-H3 lysine-27 (H3K27) methylation (me) significantly decreased in ageing vessels and this was attenuated in SMC-MR-KO littermates. Immunoblotting revealed less H3K27-specific methyltransferase EZH2 with age in MR-intact but not SMC-MR-KO vessels. These ageing changes were examined in primary human aortic (HA)SMC from adult vs. aged donors. MR, H3K27 acetylation (ac), and stiffness gene (connective tissue growth factor, integrin-α5) expression significantly increased, while H3K27me and EZH2 decreased, with age. MR inhibition reversed these ageing changes in HASMC and the decline in stiffness genes was prevented by EZH2 blockade. Atomic force microscopy revealed that MR antagonism decreased intrinsic stiffness and the probability of fibronectin adhesion of aged HASMC. Conversely, ageing induction in young HASMC with H2O2; increased MR, decreased EZH2, enriched H3K27ac and MR at stiffness gene promoters by chromatin immunoprecipitation, and increased stiffness gene expression. In 12-month-old mice, MR antagonism increased aortic EZH2 and H3K27 methylation, increased EZH2 recruitment and decreased H3K27ac at stiffness genes promoters, and prevented ageing-induced vascular stiffness and fibrosis. Finally, in human aortic tissue, age positively correlated with MR and stiffness gene expression and negatively correlated with H3K27me3 while MR and EZH2 are negatively correlated. CONCLUSION: These data support a novel vascular ageing model with rising MR in human SMC suppressing EZH2 expression thereby decreasing H3K27me, promoting MR recruitment and H3K27ac at stiffness gene promoters to induce vascular stiffness and suggests new targets for ameliorating ageing-associated vascular disease.

Neutrophil Gelatinase-Associated Lipocalin From Macrophages Plays a Critical Role in Renal Fibrosis Via the CCL5 (Chemokine Ligand 5)-Th2 Cells-IL4 (Interleukin 4) Pathway.

NGAL (neutrophil gelatinase-associated lipocalin; or lipocalin 2, Lcn2) is a novel mineralocorticoid target in the cardiovascular system. We showed that Lcn2 gene invalidation protects against proteinuria and renal injury upon mineralocorticoid excess and we hypothesized that NGAL produced from macrophages promotes the expression of chemoattractant molecules involved these renal lesions. The role of NGAL was analyzed using myeloid-specific (MΦ KO NGAL) Lcn2 knockout mice challenged with uni-nephrectomy, aldosterone, and salt (NAS) for 6 weeks. The role of the CCL5 (chemokine ligand 5) and IL4 (interleukin 4) in kidney fibrosis was studied by administration of the CCL5 receptor antagonist maraviroc or by injections of an anti-IL4 neutralizing antibody. In CTL mice, NAS increased the renal expression of extracellular matrix proteins, such as collagen I, αSMA, and fibronectin associated with interstitial fibrosis which were blunted in MΦ KO NGAL mice. The expression of CCL5 was blunted in sorted macrophages from MΦ KO NGAL mice challenged by NAS and in macrophages obtained from KO NGAL mice and challenged ex vivo with aldosterone and salt. The pharmacological blockade of the CCL5 receptor reduced renal fibrosis and the CD4+ Th cell infiltration induced by NAS. Neutralization of IL4 in NAS mice blunted kidney fibrosis and the overexpression of profibrotic proteins, such as collagen I, αSMA, and fibronectin. In conclusion, NGAL produced by macrophages plays a critical role in renal fibrosis and modulates the CCL5/IL4 pathway in mice exposed to mineralocorticoid excess.

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.

The Mineralocorticoid Receptor Antagonist Eplerenone Suppresses Interstitial Fibrosis in Subcutaneous Adipose Tissue in Patients With Type 2 Diabetes.

Activation of the mineralocorticoid receptor (MR) may promote dysfunctional adipose tissue in patients with type 2 diabetes, where increased pericellular fibrosis has emerged as a major contributor. The knowledge of the association among the MR, fibrosis, and the effects of an MR antagonist (MRA) in human adipocytes remains very limited. The present substudy, including 30 participants, was prespecified as part of the Mineralocorticoid Receptor Antagonist in Type 2 Diabetes (MIRAD) trial, which randomized patients to either high-dose eplerenone or placebo for 26 weeks. In adipose tissue biopsies, changes in fibrosis were evaluated by immunohistological examination and by the expression of mRNA and protein markers of fibrosis. Treatment with an MRA reduced pericellular fibrosis, synthesis of the major subunits of collagen types I and VI, and the profibrotic factor α-smooth muscle actin compared with placebo in subcutaneous adipose tissue. Furthermore, we found decreased expression of the MR and downstream molecules neutrophil gelatinase-associated lipocalin, galectin-3, and lipocalin-like prostaglandin D2 synthase with an MRA. In conclusion, we present original data demonstrating reduced fibrosis in adipose tissue with inhibition of the MR, which could be a potential therapeutic approach to prevent the extracellular matrix remodeling of adipose tissue in type 2 diabetes.

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.

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.

Arterial Remodeling and Dysfunction in the ZSF1 Rat Model of Heart Failure With Preserved Ejection Fraction.

BACKGROUND: The interplay between the stiffened heart and vessels has long been viewed as a core mechanism in heart failure with preserved ejection fraction, but the incremental vascular molecular remodeling mechanisms from systemic arterial hypertension to heart failure with preserved ejection fraction remain poorly investigated. Our aim was to characterize central arterial remodeling and dysfunction in ZSF1 obese rats and to compare it with hypertensive ZSF1 lean and healthy Wistar-Kyoto controls. METHODS AND RESULTS: Twenty-week-old male ZSF1 obese (n=9), lean (n=9), and Wistar-Kyoto rats (n=9) underwent left ventricular pressure-volume loop evaluation and synchronous acquisition of ascending aortic flow and pressure. Aortic rings underwent functional evaluation, histology, and molecular biology studies. Although mean arterial pressure, characteristic aortic impedance, and reactivity to phenylephrine were similarly increased in hypertensive ZSF1 lean and obese, only ZSF1 obese showed impaired relaxation and upward-shifted end-diastolic pressure-volume relationships despite preserved systolic function indexes, denoting heart failure with preserved ejection fraction. ZSF1 obese phenotype further showed decreased aortic compliance, increased wave reflection, and impaired direct NO donor and endothelial-mediated vasodilation which were accompanied on structural and molecular grounds by aortic media thickening, higher collagen content and collagen/elastin ratio, increased fibronectin and α-5 integrin protein expression and upregulated TGF (transforming growth factor)-ß and CTGF (connective tissue growth factor) levels. CONCLUSIONS: Functional, molecular, and structural disturbances of central vessels and their potentially underlying pathways were newly characterized in experimental heart failure with preserved ejection fraction rendering the ZSF1 obese rat model suitable for preclinical testing.

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.

Myocardial Injury After Ischemia/Reperfusion Is Attenuated By Pharmacological Galectin-3 Inhibition.

Although optimal therapy for myocardial infarction includes reperfusion to restore blood flow to the ischemic region, ischemia/reperfusion (IR) also initiates an inflammatory response likely contributing to adverse left ventricular (LV) extracellular matrix (ECM) remodeling. Galectin-3 (Gal-3), a ß-galactoside-binding-lectin, promotes cardiac remodeling and dysfunction. Our aim is to investigate whether Gal-3 pharmacological inhibition using modified citrus pectin (MCP) improves cardiac remodeling and functional changes associated with IR. Wistar rats were treated with MCP from 1 day before until 8 days after IR (coronary artery ligation) injury. Invasive hemodynamics revealed that both LV contractility and LV compliance were impaired in IR rats. LV compliance was improved by MCP treatment 8 days after IR. Cardiac magnetic resonance imaging showed decreased LV perfusion in IR rats, which was improved with MCP. There was no difference in LV hypertrophy in MCP-treated compared to untreated IR rats. However, MCP treatment decreased the ischemic area as well as Gal-3 expression. Gal-3 blockade paralleled lower myocardial inflammation and reduced fibrosis. These novel data showing the benefits of MCP in compliance and ECM remodeling in IR reinforces previously published data showing the therapeutic potential of Gal-3 inhibition.

CT-1 (Cardiotrophin-1)-Gal-3 (Galectin-3) Axis in Cardiac Fibrosis and Inflammation.

Myocardial fibrosis is a main contributor to the development of heart failure (HF). CT-1 (cardiotrophin-1) and Gal-3 (galectin-3) are increased in HF and associated with myocardial fibrosis. The aim of this study is to analyze whether CT-1 regulates Gal-3. Proteomic analysis revealed that Gal-3 was upregulated by CT-1 in human cardiac fibroblasts in parallel with other profibrotic and proinflammatory markers. CT-1 upregulation of Gal-3 was mediated by ERK (extracellular signal-regulated kinase) 1/2 and Stat-3 (signal transducer and activator of transcription 3) pathways. Male Wistar rats and B6CBAF1 mice treated with CT-1 (20 µg/kg per day) presented higher cardiac Gal-3 levels and myocardial fibrosis. In CT-1-treated rats, direct correlations were found between cardiac CT-1 and Gal-3 levels, as well as between Gal-3 and perivascular fibrosis. Gal-3 genetic disruption in human cardiac fibroblasts and pharmacological Gal-3 inhibition in mice prevented the profibrotic and proinflammatory effects of CT-1. Dahl salt-sensitive hypertensive rats with diastolic dysfunction showed increased cardiac CT-1 and Gal-3 expression together with cardiac fibrosis and inflammation. CT-1 and Gal-3 directly correlated with myocardial fibrosis. In HF patients, myocardial and plasma CT-1 and Gal-3 were increased and directly correlated. In addition, HF patients with high CT-1 and Gal-3 plasma levels presented an increased risk of cardiovascular death. Our data suggest that CT-1 upregulates Gal-3 which, in turn, mediates the proinflammatory and profibrotic myocardial effects of CT-1. The elevation of both molecules in HF patients identifies a subgroup of patients with a higher risk of cardiovascular mortality. The CT-1/Gal-3 axis emerges as a candidate therapeutic target and a potential prognostic biomarker in HF.

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.