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BACKGROUND: Calcific aortic valve disease (CAVD), a major cause for surgical aortic valve replacement, currently lacks available pharmacological treatments. Cadherin-11 (Cad11), a promising therapeutic target, promotes aortic valve calcification in vivo, but direct Cad11 inhibition in clinical trials has been unsuccessful. Targeting of downstream Cad11 effectors instead may be clinically useful; however, the downstream effectors that mediate Cad11-induced aortic valve cellular pathogenesis have not been investigated. APPROACH AND RESULTS: Immunofluorescence of calcified human aortic valves revealed that GTP-Rac1 is highly upregulated in calcified leaflets and is 2.15 times more co-localized with Cad11 in calcified valves than GTP-RhoA. Using dominant negative mutants in porcine aortic valve interstitial cells (PAVICs), we show that Cad11 predominantly regulates Runx2 nuclear localization via Rac1. Rac1-GEF inhibition via NSC23766 effectively reduces calcification in ex vivo porcine aortic valve leaflets treated with osteogenic media by 2.8-fold and also prevents Cad11-induced cell migration, compaction, and calcification in PAVICs. GTP-Rac1 and Trio, a known Cad11 binding partner and Rac1-GEF, are significantly upregulated in Nfatc1Cre; R26-Cad11Tg/Tg (Cad11 OX) mice that conditionally overexpress Cad11 in the heart valves by 3.1-fold and 6.3-fold, respectively. Finally, we found that the Trio-specific Rac1-GEF inhibitor, ITX3, effectively prevents Cad11-induced calcification and Runx2 induction in osteogenic conditions. CONCLUSION: Here we show that Cad11 induces many cellular pathogenic processes via Rac1 and that Rac1 inhibition effectively prevents many Cad11-induced aortic disease phenotypes. These findings highlight the therapeutic potential of blocking Rac1-GEFs in CAVD.
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Estenose da Valva Aórtica , Calcinose , Animais , Valva Aórtica/patologia , Estenose da Valva Aórtica/patologia , Caderinas , Calcinose/etiologia , Células Cultivadas , Camundongos , SuínosRESUMO
Cell plasticity plays a key role in embryos by maintaining the differentiation potential of progenitors. Whether postnatal somatic cells revert to an embryonic-like naïve state regaining plasticity and redifferentiate into a cell type leading to a disease remains intriguing. Using genetic lineage tracing and single-cell RNA sequencing, we reveal that Oct4 is induced by nuclear factor κB (NFκB) at embyronic day 9.5 in a subset of mouse endocardial cells originating from the anterior heart forming field at the onset of endocardial-to-mesenchymal transition. These cells acquired a chondro-osteogenic fate. OCT4 in adult valvular aortic cells leads to calcification of mouse and human valves. These calcifying cells originate from the Oct4 embryonic lineage. Genetic deletion of Pou5f1 (Pit-Oct-Unc, OCT4) in the endocardial cell lineage prevents aortic stenosis and calcification of ApoE−/− mouse valve. We established previously unidentified self-cell reprogramming NFκB- and OCT4-mediated inflammatory pathway triggering a dose-dependent mechanism of valve calcification.
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OBJECTIVE: Calcific aortic valve disease (CAVD) is an actively regulated degenerative disease process. Clinical lesions exhibit marked 3D complexity not represented in current in vitro systems. We here present a unique mechanically stressed 3D culture system that recapitulates valve interstitial cell (VIC) induced matrix calcification through myofibroblastic activation and osteoblastic differentiation. We test the hypothesis that valve endothelial (VEC) - interstitial collaborative interactions modulate the risk and complexity of calcific pathogenesis within mechanically stressed and pro-inflammatory environments. APPROACH AND RESULTS: Porcine aortic valve endothelial and interstitial cells (VEC and VIC) were seeded in a mechanically constrained collagen hydrogels alone or in co-culture configurations. Raised 3D VIC-filled lesions formed within 7 days when cultured in osteogenic media (OGM), and surprisingly exacerbated by endothelial coculture. We identified a spatially coordinated pro-endochondral vs. pro-osteogenic signaling program within the lesion. VEC underwent Endothelial-to-Mesenchymal Transformation (EndMT) and populated the lesion center. The spatial complexity of molecular and cellular signatures of this 3D in vitro CAVD system were consistent with human diseased aortic valve histology. SNAI1 was highly expressed in the VEC and subendothelial direct VIC corroborates with human CAVD lesions. Spatial distribution of Sox9 vs. Runx2 expression within the developed lesions (Sox9 peri-lesion vs. Runx2 predominantly within lesions) mirrored their expression in heavily calcified human aortic valves. Finally, we demonstrate the applicability of this platform for screening potential pharmacologic therapies through blocking the canonical NFκB pathway via BAY 11-7082. CONCLUSIONS: Our results establish that VEC actively induce VIC pathological remodeling and calcification via EndMT and paracrine signaling. This mechanically constrained culture platform enables the interrogation of accelerated cell-mediated matrix remodeling behavior underpinned by this cellular feedback circuit. The high fidelity of this complex 3D model system to human CAVD mechanisms supports its use to test mechanisms of intercellular communication in valves and their pharmacological control.
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Estenose da Valva Aórtica , Calcinose , Animais , Valva Aórtica , Diferenciação Celular , Células Cultivadas , Humanos , SuínosRESUMO
INTRODUCTION: Structural valve degeneration (SVD) leads to the failure of aortic valve bioprostheses. It is suspected that lipid-derived factors could play a role in SVD. We hypothesised that oxidised low-density lipoprotein (OxLDL), OxLDL/LDL, OxLDL/high-density lipoprotein (OxLDL/HDL) and proprotein convertase subtilisin/kexin 9 (PCSK9) may be associated with SVD. METHODS: We included 199 patients who underwent an aortic valve replacement with a bioprosthesis and had an echocardiography follow-up to evaluate the function of the prosthesis. SVD was defined as an increase in mean transprosthetic gradient (≥10â mmâ Hg) or a worsening of transprosthetic regurgitation (≥1/3) during the follow-up. RESULTS: After a mean follow-up of 8±3.5â years, 41(21%) patients developed SVD. The univariate predictors of SVD were LDL (p=0.03), apolipoprotein B (p=0.01), OxLDL (p=0.02), OxLDL/HDL (p=0.009) and LDL associated with small, dense particles (LDL-C<255Å) (p=0.02). In a model adjusted for covariates, only OxLDL/HDL (OR 1.49, 95%CI 1.08 to 2.07 per 10 units, p=0.01) remained associated with SVD. There was a significant interaction between OxLDL/HDL and PCSK9 on SVD (p=0.05). After adjustment, compared with patients with low OxLDL/HDL (median, <25.4) and low PCSK9 (median, <298â ng/mL) (referent), patients with both an elevated OxLDL/HDL ratio and PCSK9 had a higher risk of SVD (OR 2.93, 95% CI 1.02 to 9.29, p=0.04). CONCLUSIONS: OxLDL/HDL ratio is independently associated with SVD.
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Insuficiência da Valva Aórtica/etiologia , Valva Aórtica/cirurgia , Bioprótese , Implante de Prótese de Valva Cardíaca/efeitos adversos , Implante de Prótese de Valva Cardíaca/instrumentação , Próteses Valvulares Cardíacas , Lipoproteínas HDL/sangue , Lipoproteínas LDL/sangue , Falha de Prótese , Idoso , Idoso de 80 Anos ou mais , Valva Aórtica/diagnóstico por imagem , Insuficiência da Valva Aórtica/sangue , Insuficiência da Valva Aórtica/diagnóstico por imagem , Biomarcadores/sangue , Distribuição de Qui-Quadrado , Estudos Transversais , Ecocardiografia Doppler , Feminino , Humanos , Modelos Lineares , Modelos Logísticos , Masculino , Análise Multivariada , Razão de Chances , Pró-Proteína Convertase 9/sangue , Estudos Prospectivos , Desenho de Prótese , Medição de Risco , Fatores de Risco , Fatores de TempoRESUMO
BACKGROUND: We previously reported that plasma Lp-PLA2 was associated with aortic valve disease progression and degeneration of bioprostheses. Low systemic arterial compliance and high valvuloarterial impedance (Z(va)) are predictors of poor survival in patients with aortic valve disease. However, the prevalence of high Z(va) after AVR is largely unknown and whether Lp-PLA2 could predict Z(va) has not been documented. We investigated the relationships between plasma lipoprotein-associated phospholipase A2 (Lp-PLA2) mass and activity and valvuloarterial impedance (Z(va)), an index of global LV hemodynamic load, in patients that underwent aortic valve replacement (AVR). METHODS: A total of 195 patients with aortic bioprostheses underwent echocardiographic assessment of the prosthetic aortic valve function 8±3.4 years after AVR. Lp-PLA2 mass and activity were measured. RESULTS: In this group of patients, the mean Z(va) was elevated (5.73±1.21 mm Hg·ml(-1)·m(2)). In univariate analyses, Lp-PLA2 mass (p=0.003) and Lp-PLA2 activity (p=0.046) were associated with Z(va). After adjustment for covariates including age, gender, clinical risk factors, anti-hypertensive medications, body mass index and prosthesis size, Lp-PLA2 mass was associated with high Z(va) (≥4.5 mm Hg·ml(-1)·m(2)) (OR: 1.29, 95%CI: 1.10-1.53; p=0.005) and was inversely related with the systemic arterial compliance (ß=-0.01, SEM=0.003; p=0.003). CONCLUSIONS: An increased Z(va), an index of excessive hemodynamic load, was highly prevalent 8-year post-AVR and was independently related to circulating Lp-PLA2.
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1-Alquil-2-acetilglicerofosfocolina Esterase/sangue , Valva Aórtica/cirurgia , Artérias/fisiopatologia , Complacência (Medida de Distensibilidade) , Próteses Valvulares Cardíacas , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , MasculinoRESUMO
BACKGROUND: Elevated lipoprotein(a) (Lp[a]) is associated with aortic stenosis (AS). Oxidized phospholipids (OxPL) are key mediators of calcification in valvular cells and are carried by Lp(a). OBJECTIVES: This study sought to determine whether Lp(a) and OxPL are associated with hemodynamic progression of AS and AS-related events. METHODS: OxPL on apolipoprotein B-100 (OxPL-apoB), which reflects the biological activity of Lp(a), and Lp(a) levels were measured in 220 patients with mild-to-moderate AS. The primary endpoint was the progression rate of AS, measured by the annualized increase in peak aortic jet velocity in m/s/year by Doppler echocardiography; the secondary endpoint was need for aortic valve replacement and cardiac death during 3.5 ± 1.2 years of follow-up. RESULTS: AS progression was faster in patients in the top tertiles of Lp(a) (peak aortic jet velocity: +0.26 ± 0.26 vs. +0.17 ± 0.21 m/s/year; p = 0.005) and OxPL-apoB (+0.26 ± 0.26 m/s/year vs. +0.17 ± 0.21 m/s/year; p = 0.01). After multivariable adjustment, elevated Lp(a) or OxPL-apoB levels remained independent predictors of faster AS progression. After adjustment for age, sex, and baseline AS severity, patients in the top tertile of Lp(a) or OxPL-apoB had increased risk of aortic valve replacement and cardiac death. CONCLUSIONS: Elevated Lp(a) and OxPL-apoB levels are associated with faster AS progression and need for aortic valve replacement. These findings support the hypothesis that Lp(a) mediates AS progression through its associated OxPL and provide a rationale for randomized trials of Lp(a)-lowering and OxPL-apoB-lowering therapies in AS. (Aortic Stenosis Progression Observation: Measuring Effects of Rosuvastatin [ASTRONOMER]; NCT00800800).
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Estenose da Valva Aórtica/sangue , Estenose da Valva Aórtica/diagnóstico por imagem , Valva Aórtica/patologia , Calcinose/sangue , Calcinose/diagnóstico por imagem , Progressão da Doença , Lipoproteína(a)/sangue , Fosfolipídeos/sangue , Adulto , Idoso , Valva Aórtica/diagnóstico por imagem , Estenose da Valva Aórtica/mortalidade , Biomarcadores/sangue , Calcinose/mortalidade , Morte , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Oxirredução , UltrassonografiaRESUMO
BACKGROUND: Mendelian randomization studies have highlighted that lipoprotein(a) [Lp(a)] was associated with calcific aortic valve disease (CAVD). Lp(a) transports oxidized phospholipids with a high content in lysophosphatidylcholine. Autotaxin (ATX) transforms lysophosphatidylcholine into lysophosphatidic acid. We hypothesized that ATX-lysophosphatidic acid could promote inflammation/mineralization of the aortic valve. METHODS AND RESULTS: We have documented the expression of ATX in control and mineralized aortic valves. By using different approaches, we have also investigated the role of ATX-lysophosphatidic acid in the mineralization of isolated valve interstitial cells and in a mouse model of CAVD. Enzyme-specific ATX activity was elevated by 60% in mineralized aortic valves in comparison with control valves. Immunohistochemistry studies showed a high level of ATX in mineralized aortic valves, which colocalized with oxidized phospholipids and apolipoprotein(a). We detected a high level of ATX activity in the Lp(a) fraction in circulation. Interaction between ATX and Lp(a) was confirmed by in situ proximity ligation assay. Moreover, we documented that valve interstitial cells also expressed ATX in CAVD. We showed that ATX-lysophosphatidic acid promotes the mineralization of the aortic valve through a nuclear factor κB/interleukin 6/bone morphogenetic protein pathway. In LDLR(-/-)/ApoB(100/100)/IGFII mice, ATX is overexpressed and lysophosphatidic acid promotes a strong deposition of hydroxyapatite of calcium in aortic valve leaflets and accelerates the development of CAVD. CONCLUSIONS: ATX is transported in the aortic valve by Lp(a) and is also secreted by valve interstitial cells. ATX-lysophosphatidic acid promotes inflammation and mineralization of the aortic valve and thus could represent a novel therapeutic target in CAVD.
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Estenose da Valva Aórtica/metabolismo , Valva Aórtica/metabolismo , Lipoproteína(a)/biossíntese , Diester Fosfórico Hidrolases/biossíntese , Idoso , Animais , Valva Aórtica/patologia , Estenose da Valva Aórtica/patologia , Feminino , Humanos , Inflamação/metabolismo , Inflamação/patologia , Lipoproteína(a)/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-IdadeRESUMO
BACKGROUND AND PURPOSE: Ectonucleotide pyrophosphatase/PDE1 (NPP1) is an ectoenzyme, which plays a role in several disorders including calcific aortic valve disease (CAVD). So far, compounds that have been developed as inhibitors of NPP1 lack potency and specificity. Quinazoline-4-piperidine sulfamides (QPS) have been described as potent inhibitors of NPP1. However, their mode of inhibition as well as their selectivity and capacity to modify biological processes have not been investigated. EXPERIMENTAL APPROACH: In the present series of experiments, we have evaluated the efficacy of two derivatives, QPS1-2, in inhibiting human NPP1, and we have evaluated the effect of the most potent derivative (QPS1) on other ectonucleotidases as well as on the ability of this compound to prevent phosphate-induced mineralization of human primary aortic valve interstitial cells (VICs). KEY RESULTS: The QPS1 derivative is a potent (Ki 59.3 ± 5.4 nM) and selective non-competitive inhibitor of human NPP1. Moreover, QPS1 also significantly inhibited the K121Q NPP1 gene variant (Ki 59.2 ± 14.5 nM), which is prevalent in the general population. QPS1 did not significantly alter the activity of other nucleotide metabolizing ectoenzymes expressed at the cell surface, namely NPP3, NTPDases (1-3), ecto-5'-nucleotidase and ALP. Importantly, QPS1 in the low micromolar range (≤10 µM) prevented phosphate-induced mineralization of VICs and lowered the rise of osteogenic genes as expected for NPP1 inhibition. CONCLUSIONS AND IMPLICATIONS: We have provided evidence that QPS1 is a potent and selective non-competitive inhibitor of NPP1 and that it prevented pathological mineralization in a cellular model.
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Pirofosfatases/antagonistas & inibidores , Quinazolinas/farmacologia , Adulto , Animais , Valva Aórtica/citologia , Apoptose/efeitos dos fármacos , Células COS , Chlorocebus aethiops , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Diester Fosfórico HidrolasesRESUMO
AIMS: Calcific aortic valve stenosis (CAVS) is the most common heart valve disease. In the present work we sought to determine the reversibility of mineralization in the aortic valve. METHODS AND RESULTS: By using in vitro analyses we found that valve interstitial cells (VICs) have the ability to resorb minerals. We documented that agonist of P2Y2 receptor (P2Y2R) promoted the expression of carbonic anhydrase XII (CAXII) at the cell membrane of VICs, whereby minerals are resorbed. P2Y2R-mediated mineral resorption was corroborated by using mouse VICs isolated from wild type and P2Y2R(-/-) mice. Measurements of extracellular pH (pHe) by using core-shell nanosensors revealed that P2Y2R-mediated CAXII export to the cell membrane led to an acidification of extracellular space, whereby minerals are resorbed. In vivo, we next treated LDLR(-/-)/ApoB(100/100)/IGF2 mice, which had developed CAVS under a high-fat/high-sucrose diet for 8 months, with 2-thioUTP (a P2Y2R agonist) or saline for the next 2 months. The administration of 2-thioUTP (2mg/kg/day i.p.) reduced the mineral volume in the aortic valve measured with serial microCT analyses, which improved hemodynamics and reduced left ventricular hypertrophy (LVH). Examination of leaflets at necropsy confirmed a lower level of mineralization and fibrosis along with higher levels of CAXII in mice under 2-thioUTP. In another series of experiment, the administration of acetazolamide (a CA inhibitor) prevented the acidification of leaflets and the regression of CAVS induced by 2-thioUTP in LDLR(-/-)/ApoB(100/100)/IGF2 mice. CONCLUSION: P2Y2R-mediated expression of CAXII by VICs acidifies the extracellular space and promotes the regression of CAVS.
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Estenose da Valva Aórtica/etiologia , Estenose da Valva Aórtica/metabolismo , Calcinose/complicações , Calcinose/metabolismo , Anidrases Carbônicas/metabolismo , Valvas Cardíacas/metabolismo , Animais , Estenose da Valva Aórtica/diagnóstico , Estenose da Valva Aórtica/tratamento farmacológico , Calcinose/patologia , Modelos Animais de Doenças , Espaço Extracelular/metabolismo , Valvas Cardíacas/patologia , Masculino , Camundongos , Camundongos Transgênicos , Minerais/metabolismo , Agonistas do Receptor Purinérgico P2Y/farmacologia , Receptores Purinérgicos P2Y2/metabolismoRESUMO
AIMS: In this study, we sought to determine the role of ecto-nucleotidases and adenosine receptors in calcific aortic valve disease (CAVD). The expression of ecto-nucleotidases, which modify the levels of extracellular nucleotides/nucleosides, may control the mineralization of valve interstitial cells (VICs). We hypothesized that expression of ectonucleotide pyrophosphatase/phosphodiesterase 1 (NPP1), which generates AMP, and 5'-nucleotidase (CD73), an enzyme using AMP as a substrate to produce adenosine, may co-regulate the mineralization of the aortic valve. METHODS AND RESULTS: We have investigated the expression of NPP1 and 5'-nucleotidase in CAVD tissues and determined the role of these ecto-nucleotidases on the mineralization of isolated VICs. In CAVD tissues (stenotic and sclerotic), we documented that NPP1 and 5'-nucleotidase were overexpressed by VICs. In isolated VICs, we found that mineralization induced by adenosine triphosphate was decreased by silencing NPP1 and 5'-nucleotidase, suggesting a role for adenosine. Adenosine and specific A2a adenosine receptor (A2aR) agonist increased the mineralization of VICs. Silencing of A2aR in human VICs and the use of A2aR(-/-) mouse VICs confirmed that A2aR promotes the mineralization of cells. Also, A2aR-mediated mineralization was negated by the transfection of a mutant dominant-negative Gαs vector. Through several lines of evidence, we next documented that adenosine stimulated the mineralization of VICs through a cAMP/protein kinase A (PKA)/cAMP response element-binding protein (CREB) pathway, and found that CREB positively regulated the expression of NPP1 in a positive feedback loop by physically interacting with the promoter. CONCLUSION: Expression of NPP1 and 5'-nucleotidase by VICs promotes the mineralization of the aortic valve through A2aR and a cAMP/PKA/CREB pathway.
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Adenosina Trifosfatases/fisiologia , Adenosina/fisiologia , Estenose da Valva Aórtica/fisiopatologia , Valva Aórtica/patologia , Valva Aórtica/fisiopatologia , Calcinose/fisiopatologia , Cardiopatias Congênitas/fisiopatologia , Doenças das Valvas Cardíacas/fisiopatologia , Receptor A2A de Adenosina/fisiologia , 5'-Nucleotidase/fisiologia , Idoso , Animais , Doença da Válvula Aórtica Bicúspide , AMP Cíclico/fisiologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/fisiologia , Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Diester Fosfórico Hidrolases/fisiologia , Pirofosfatases/fisiologia , Receptor A2A de Adenosina/deficiência , Receptor A2A de Adenosina/genética , Transdução de Sinais/fisiologiaRESUMO
OBJECTIVES: The purpose of this prospective study was to examine the relationship between plasma lipoprotein-associated phospholipase A2 (Lp-PLA2) activity and the progression rate of aortic stenosis (AS). BACKGROUND: We recently reported that Lp-PLA2 is highly expressed in stenotic aortic valves where it may contribute to the mineralization of valvular interstitial cells. METHODS: Patients with AS were prospectively recruited in the PROGRESSA (Metabolic Determinants of the Progression of Aortic Stenosis) study. AS progression rate was assessed by annualized increase in peak aortic jet velocity (Vpeak), mean gradient (MG), and aortic valve area index (AVAi). Circulating Lp-PLA2 activity was measured and dichotomized based on the median value. RESULTS: Of 183 patients included in this subanalysis of the PROGRESSA study, 70% were men and the mean age was 66 ± 13 years. Over the 2.5 ± 1.4 years of follow up, the AS progression rate tended to be higher in patients with high versus low Lp-PLA2 activity (annualized Vpeak = 0.17 ± 0.23 m/s vs. 0.12 ± 0.18 m/s; p = 0.14). There was a significant interaction (p < 0.05) between baseline AS severity and Lp-PLA2 activity with respect to impact on AS progression rate. In patients with mild AS (i.e., Vpeak <3 m/s; n = 123), increased Lp-PLA2 activity was associated with a significantly faster AS progression rate (Vpeak 0.16 ± 0.18 m/s vs. 0.09 ± 0.14 m/s; p = 0.01) but not in patients with moderate or severe AS (p = 0.99). After adjustment for other risk factors, increased Lp-PLA2 activity remained independently associated with faster AS progression rate (p = 0.005) in the former subset. CONCLUSIONS: There was no significant association between plasma Lp-PLA2 activity or mass and stenosis progression in the whole cohort. However, increased Lp-PLA2 activity was associated with a faster stenosis progression rate in the subset of patients with mild AS. These findings provide an impetus for the elaboration of a randomized trial targeting Lp-PLA2 activity in patients with early stages of calcific aortic valve disease.
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1-Alquil-2-acetilglicerofosfocolina Esterase/sangue , Estenose da Valva Aórtica/sangue , Estenose da Valva Aórtica/fisiopatologia , Idoso , Valva Aórtica/patologia , Valva Aórtica/fisiopatologia , Progressão da Doença , Ecocardiografia Doppler , Feminino , Humanos , Masculino , Estudos Prospectivos , Função Ventricular EsquerdaRESUMO
AIMS: LV hypertrophy (LVH) is frequent after aortic valve replacement (AVR) and is often associated with comorbidities, including hypertension, obesity, renal failure and prosthesis-patient mismatch (PPM). However, whether other biological mechanism(s) may participate to LVH after AVR is still unknown. Parathyroid hormone (PTH) may play a role in LVH. However, it is presently unknown whether PTH is associated with LVH in patients that have undergone an AVR. METHODS: In this cross-sectional study, 195 patients have been investigated at a mean of 8 ± 3.5 years following AVR. LV function and mass were evaluated by Doppler echocardiography. The plasma levels of PTH, 25-hydroxyvitamin D (25-OHD), calcium and phosphate were measured. RESULTS: There were 102 (52%) patients with LVH after AVR. In univariate analyses, PTH blood level was associated with LV mass (LVMi) and LVH. After adjustment for other risk factors, elevated PTH remained associated with LVMi (p=0.003) and LVH (p=0.02). In turn, the blood levels of 25-OHD and the renal function (GFR) were independently and inversely related to the blood level of PTH. CONCLUSIONS: After AVR, the level of PTH is independently associated with LVH. In turn, the level of PTH is related with the renal function and the level of 25-OHD.
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Valva Aórtica/cirurgia , Implante de Prótese de Valva Cardíaca/efeitos adversos , Hipertrofia Ventricular Esquerda/etiologia , Hormônio Paratireóideo/sangue , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/sangue , Cálcio/sangue , Distribuição de Qui-Quadrado , Estudos Transversais , Ecocardiografia Doppler , Feminino , Taxa de Filtração Glomerular , Humanos , Hipertrofia Ventricular Esquerda/sangue , Hipertrofia Ventricular Esquerda/diagnóstico , Hipertrofia Ventricular Esquerda/fisiopatologia , Rim/fisiopatologia , Modelos Lineares , Modelos Logísticos , Masculino , Análise Multivariada , Fosfatos/sangue , Valor Preditivo dos Testes , Estudos Prospectivos , Fatores de Risco , Fatores de Tempo , Resultado do Tratamento , Função Ventricular Esquerda , Vitamina D/análogos & derivados , Vitamina D/sangueRESUMO
Calcific aortic valve disease (CAVD) is a disorder characterized by an abnormal mineralization, which may have intricate links with inflammation. Interleukin-6 (IL-6) and its cognate cytokines are widely expressed and exert pleiotropic effects on different tissues. In this study, we examined the expression of the IL-6 family of cytokines in human CAVD by using a transcriptomic approach and we performed in-depth functional assays with valve interstitial cells (VICs) to unravel the process regulating IL-6 expression and its role during the mineralization of the aortic valve. We documented by both microarray and q-PCR analyses an elevated expression of IL-6 in human CAVD, which was correlated with the remodeling process. IL-6 was highly expressed by VICs. We found that following treatment with a phosphate-containing medium the level of IL-6 expressed by VICs increased by several-fold. Phosphate-induced expression of IL-6 relied on reduced PI3K/Akt signaling downstream of the P2Y2 receptor (P2Y2R). In this regard, we found by using transfection experiments that Akt-1 is a negative regulator of the NF-κB pathway. In addition, by using a siRNA targeting IL-6 we found that phosphate-induced mineralization was largely dependent on IL-6 expression. A transfection of Akt-1 rescued the hypermineralizing phenotype of P2Y2R(-/-) mouse VICS (MVICs). Hence, we documented a novel mechanism whereby P2Y2R and Akt modulate the NF-κB pathway and its downstream target IL-6, which is a strong promoter of the mineralization of VICs.
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Estenose da Valva Aórtica/genética , Valva Aórtica/metabolismo , Valva Aórtica/patologia , Calcinose/genética , Interleucina-6/genética , Fosfatos/farmacologia , Proteínas Proto-Oncogênicas c-akt/genética , Receptores Purinérgicos P2Y2/genética , Animais , Valva Aórtica/efeitos dos fármacos , Estenose da Valva Aórtica/induzido quimicamente , Estenose da Valva Aórtica/metabolismo , Estenose da Valva Aórtica/patologia , Calcinose/induzido quimicamente , Calcinose/metabolismo , Calcinose/patologia , Células Cultivadas , Meios de Cultura/química , Meios de Cultura/farmacologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Interleucina-6/antagonistas & inibidores , Interleucina-6/metabolismo , Camundongos , Camundongos Knockout , NF-kappa B/genética , NF-kappa B/metabolismo , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptores Purinérgicos P2Y2/metabolismo , Transdução de SinaisRESUMO
OBJECTIVES: In this study, we sought to determine the metabolic markers associated with structural valve degeneration (SVD). BACKGROUND: Structural valve degeneration (SVD) is the major cause of bioprosthetic valve failure leading to bioprostheses (BPs) stenosis or regurgitation. We hypothesized that lipoprotein-associated phospholipase A2 (Lp-PLA2) is involved in the SVD of BPs. METHODS: We included 197 patients who underwent aortic valve replacement with a bioprosthetic valve and had echocardiographic follow-up to evaluate valve function. Moreover, explanted BPs (n = 39) were analysed by immunohistochemistry for the expression of Lp-PLA2. RESULTS: After a mean follow-up of 7·9 ±0·2 years, forty-one patients (21%) were identified as developing SVD. Patients with SVD had significantly higher plasma level of Lp-PLA2 mass (151·8 ± 9·2 ng/mL vs. 133·2 ± 3·4 ng/mL, P = 0·03) and activity (27·6 ± 0·9 nmol/min/mL vs. 25·0 ± 0·4 nmol/min/mL, P = 0·005). Multivariate analysis revealed that Lp-PLA2 activity (OR: 1·09, 95% CI: 1·01-1·18; P = 0·03) was the strongest independent predictor of SVD. Immunohistochemistry studies of explanted BP showed that 77% of explanted BPs had the expression of Lp-PLA2, which correlated with the density of macrophages (CD68), and ox-LDL levels in bioprosthetic tissues. CONCLUSIONS: Increased blood plasma activity of Lp-PLA2 is associated with higher prevalence of SVD. These findings open new avenues for the identification of patients at risk for SVD and for the development of pharmacotherapy aiming at the prevention of SVD.
Assuntos
1-Alquil-2-acetilglicerofosfocolina Esterase/fisiologia , Bioprótese , Oclusão de Enxerto Vascular/etiologia , Próteses Valvulares Cardíacas , Falha de Prótese/etiologia , Idoso , Insuficiência da Valva Aórtica/etiologia , Insuficiência da Valva Aórtica/cirurgia , Estenose da Valva Aórtica/etiologia , Estenose da Valva Aórtica/cirurgia , Estudos Transversais , Ecocardiografia Doppler , Feminino , Humanos , Masculino , Estudos ProspectivosRESUMO
OBJECTIVES: This study sought to document the presence and role of lipoprotein-associated phospholipase A2 (Lp-PLA2) in calcific aortic valve disease (CAVD). BACKGROUND: CAVD is a chronic disorder characterized by pathological mineralization and remodeling. Studies have indicated that human CAVD tissues are infiltrated by lipids and that inflammation may play a role in the pathobiology. We hypothesized that Lp-PLA2 (encoded by the PLA2G7 gene) is expressed in CAVD and may play a role in the mineralization of valve interstitial cells. METHODS: We have documented the expression of the phospholipase A2 family of genes in aortic valves by using a transcriptomic assay. Messenger ribonucleic acid and protein expression were confirmed in aortic valves explanted from 60 patients by quantitative polymerase chain reaction and immunohistochemistry, respectively. The effect of lysophosphatidylcholine, the product of Lp-PLA2 activity, was documented on the mineralization of valve interstitial cell cultures. RESULTS: Transcriptomic analyses of CAVD and control nonmineralized aortic valves revealed that Lp-PLA2 was increased by 4.2-fold in mineralized aortic valves. Higher expression of Lp-PLA2 in stenotic aortic valves was confirmed by quantitative polymerase chain reaction, immunohistochemistry, and enzymatic Lp-PLA2 activity. The number of Lp-PLA2 transcripts correlated with several indexes of tissue remodeling. In vitro, lysophosphatidylcholine increased the expression of alkaline phosphatase, the ectonucleotide pyrophosphatase/phosphodiesterase 1 enzyme, sodium-dependent phosphate cotransporter 1 (encoded by the SLC20A1 gene), and osteopontin. We then showed that lysophosphatidylcholine-induced mineralization involved ectonucleotidase enzyme as well as apoptosis through a protein-kinase-A-dependent pathway. CONCLUSIONS: Together, these results demonstrated that Lp-PLA2 is highly expressed in CAVD, and it plays a role in the mineralization of valve interstitial cells. Further work is necessary to document whether Lp-PLA2 could be considered as a novel target in CAVD.
Assuntos
1-Alquil-2-acetilglicerofosfocolina Esterase/genética , Valva Aórtica/enzimologia , Calcinose/genética , Regulação da Expressão Gênica , Cardiopatias Congênitas/genética , Doenças das Valvas Cardíacas/genética , RNA Mensageiro/genética , 1-Alquil-2-acetilglicerofosfocolina Esterase/biossíntese , Idoso , Valva Aórtica/diagnóstico por imagem , Valva Aórtica/patologia , Doença da Válvula Aórtica Bicúspide , Calcinose/diagnóstico , Calcinose/enzimologia , Ecocardiografia Doppler , Feminino , Seguimentos , Cardiopatias Congênitas/diagnóstico , Cardiopatias Congênitas/enzimologia , Doenças das Valvas Cardíacas/diagnóstico , Doenças das Valvas Cardíacas/enzimologia , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Prognóstico , Índice de Gravidade de DoençaRESUMO
BACKGROUND: Calcific aortic valve disease (CAVD) is a chronic disorder characterized by the mineralization of the aortic valve and involving fibrosis. OBJECTIVES: In this work we sought to determine if the fibrotic component of the remodeling process of CAVD was related to the use of angiotensin-converting enzyme inhibitors (ACEi) and/or angiotensin receptor blockers (ARBs). METHODS: In 477 patients with CAVD, the aortic valve was examined by histology. A semiquantitative score of fibrosis was generated and associations with clinical/cardiometabolic variables examined. In a subset of 103 patients the aortic valve was available to study the infiltration by inflammatory cells and expression of interleukin-6 (IL-6) by quantitative real-time PCR. RESULTS: The fibrosis score of the aortic valve was independently related to the hemodynamic severity of CAVD measured by echocardiography. The fibrotic score of the aortic valve was also related to the expression of IL-6. The use of ARBs but not of ACEi was associated with a lower fibrosis score of the aortic valve even after correction for covariates. In addition, patients under ARBs had lower aortic valve inflammation and expression of IL-6. CONCLUSIONS: These findings suggest that ARBs may alter the fibrotic process of the aortic valve in CAVD, possibly by lowering tissue inflammation.
Assuntos
Antagonistas de Receptores de Angiotensina/uso terapêutico , Calcinose/tratamento farmacológico , Calcinose/patologia , Cardiopatias Congênitas/tratamento farmacológico , Cardiopatias Congênitas/patologia , Doenças das Valvas Cardíacas/tratamento farmacológico , Doenças das Valvas Cardíacas/patologia , Interleucina-6/biossíntese , Idoso , Valva Aórtica/imunologia , Valva Aórtica/patologia , Doença da Válvula Aórtica Bicúspide , Calcinose/imunologia , Feminino , Fibrose , Cardiopatias Congênitas/imunologia , Doenças das Valvas Cardíacas/imunologia , Humanos , Imuno-Histoquímica , Interleucina-6/análise , Masculino , Reação em Cadeia da Polimerase em Tempo RealRESUMO
BACKGROUND: Calcific aortic valve disease (CAVD) is a chronic disorder characterized by a fibrocalcific remodelling. It is suspected that lipid retention within the aortic valve may be one important mechanism participating to aortic valve remodelling. Lipoprotein lipase (LPL) is implicated in lipid metabolism and may play a role in lipid retention within the aortic valve. METHODS: In 57 patients, CAVD were analysed for the expression of LPL by q-PCR and immunohistochemistry. Expression of oxidized-LDL (ox-LDL) and decorin was also documented. In addition, a complete blood profile, including the size of LDL and high-density lipoprotein (HDL) particles, were performed to find associations between the blood lipid profile and expression of ox-LDL and LPL within CAVD. RESULTS: Immunohistochemistry studies revealed that LPL was expressed in stenotic aortic valves as a diffuse staining and also in dense cellular areas where macrophages were abundant. Expression of LPL co-localized with decorin and ox-LDL. In turn, valves with higher amount of ox-LDL had elevated number of LPL transcripts. In addition, we documented that the small, dense HDL phenotype was associated with an elevated amount of ox-LDL and LPL transcripts within CAVD. Furthermore, expression of LPL was associated with several indices of fibrocalcific remodelling of the aortic valve. CONCLUSION: Expression of LPL within CAVD is related to the amount of ox-LDL, which is, in turn, associated with the small, dense HDL phenotype. Lipid retention associated with smaller HDL particles may participate in the expression of LPL, whereby a fibrocalcific remodelling of the aortic valve is promoted.
Assuntos
Estenose da Valva Aórtica/enzimologia , Valva Aórtica/patologia , Lipase Lipoproteica/metabolismo , Lipoproteínas HDL/metabolismo , Lipoproteínas LDL/metabolismo , Calcificação Vascular/enzimologia , Idoso , Valva Aórtica/enzimologia , Estenose da Valva Aórtica/patologia , Feminino , Fibrose/enzimologia , Humanos , Imuno-Histoquímica , Masculino , Calcificação Vascular/patologiaRESUMO
The regulation of phosphate (Pi) handling is crucial during calcification of the aortic valve. Gene profiling of Pi transporters revealed that VIC culture expresses SLC201A1/Pit1 and SLC20A2/Pit2. On exposure to a mineralizing medium (2 mM Pi), the expression of Pi transporters in VIC culture is increased several folds, with the highest magnitude for SLC20A1. By using siRNAs, we established that silencing SLC20A1 significantly reduced Pi-induced mineralization of VICs. In human pathological specimens, we found that the expression of SCL20A1 was increased in CAVD tissues compared to control non-mineralized aortic valves. Treatment of VIC culture with Pi promoted the loss of mitochondrial membrane potential (ΔΨm) and cytochrome c release within the cytosol, leading to apoptosis. Inhibition of Pi transporters with phosphonoformic acid (PFA) prevented Pi-mediated apoptosis of VICs. Moreover, we discovered that the level of the Akt-1 transcript is diminished in CAVD tissues compared with control valves. Accordingly, treatment with Pi caused a reduction of the Akt-1 transcript in VIC culture, and treatment with PFA or siRNA against SLC20A1 restored the level of Akt-1. Overexpression of Akt-1 (pCMVAkt-1) prevented both Pi-induced apoptosis and mineralization of VIC culture. These results strongly suggest that overexpression of SLC20A1 promotes apoptosis and mineralization by altering the level of Akt-1.
Assuntos
Calcinose/genética , Expressão Gênica/efeitos dos fármacos , Cardiopatias Congênitas/genética , Doenças das Valvas Cardíacas/genética , Fosfatos/farmacologia , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Cotransportadoras de Sódio-Fosfato Tipo III/genética , Idoso , Idoso de 80 Anos ou mais , Valva Aórtica/efeitos dos fármacos , Valva Aórtica/metabolismo , Valva Aórtica/patologia , Apoptose/efeitos dos fármacos , Doença da Válvula Aórtica Bicúspide , Calcinose/metabolismo , Calcinose/patologia , Células Cultivadas , Citocromos c/metabolismo , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Feminino , Cardiopatias Congênitas/metabolismo , Cardiopatias Congênitas/patologia , Doenças das Valvas Cardíacas/metabolismo , Doenças das Valvas Cardíacas/patologia , Humanos , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Pessoa de Meia-Idade , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Fosfatos/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Interferente Pequeno/genética , Transdução de Sinais/efeitos dos fármacos , Proteínas Cotransportadoras de Sódio-Fosfato Tipo III/antagonistas & inibidores , Proteínas Cotransportadoras de Sódio-Fosfato Tipo III/metabolismoRESUMO
Calcific aortic valve disease (CAVD) is the most frequent heart valve disorder. Studies indicate that mineralization of the aortic valve may be related to the inflammatory process. However, no clear evidence has been given regarding clinical evolution of aortic stenosis and the inflammatory process within the aortic valve. Aortic valves excised from 285 patients with CAVD undergoing aortic valve replacement were analyzed for the presence of chronic inflammatory infiltrates, and those findings were related to the hemodynamic severity of aortic stenosis. In a subset of 57 patients, in whom additional valvular tissue and the clinical progression rate of aortic stenosis were available, the density of leukocytes was determined as well as the number of TNF-α transcripts. Histological analyses revealed that in 81 (28.4 %) patients, the presence of chronic inflammatory infiltrates was documented within CAVD tissue, which was characterized by the existence of a cluster of cells as well as the presence of neovascularisation and osseous metaplasia. The presence of an inflammatory process within the CAVD tissue was independently related to the remodeling process and the peak transaortic gradient. In addition, the density of leukocytes within CAVD tended to correlate (r = 0.25, p = 0.05) with the progression rate of aortic stenosis. Dense inflammatory infiltrate within CAVD is associated with an active remodeling process, the severity of aortic stenosis, and the hemodynamic progression rate.