Rihab Bouchareb

Altered DNA methylation of long noncoding RNA H19 in calcific aortic valve disease promotes mineralization by silencing NOTCH1

Background: Calcific aortic valve disease is characterized by an abnormal mineralization of the aortic valve. Osteogenic activity in the aortic valve is under the control of NOTCH1, which regulates the expression of key pro-osteogenic genes such as RUNX2 and BMP2. Long noncoding RNAs (lncRNAs) may reprogram cells by altering the gene expression pattern. Methods: Multidimensional genomic profiling was performed in human aortic valves to document the expression of lncRNAs and the DNA methylation pattern in calcific aortic valve disease. In-depth functional assays were carried out to document the impact of lncRNA on the mineralization of the aortic valve. Results: We documented that lncRNA H19 (H19) was increased in calcific aortic valve disease. Hypomethylation of the promoter region was observed in mineralized aortic valves and was inversely associated with H19 expression. Knockdown and overexpression experiments showed that H19 induces a strong osteogenic phenotype by altering the NOTCH1 pathway. Gene promoter analyses showed that H19 silenced NOTCH1 by preventing the recruitment of p53 to its promoter. A knockdown of H19 in valve interstitial cells (VICs) increased the expression of NOTCH1 and decreased the level of RUNX2 and BMP2, 2 downstream targets repressed by NOTCH1. In rescue experiments, the transfection of a vector encoding for the active Notch intracellular domain prevented H19-induced mineralization of valve interstitial cells. Conclusions: These findings indicate that a dysregulation of DNA methylation in the promoter of H19 during calcific aortic valve disease is associated with a higher expression of this lncRNA, which promotes an osteogenic program by interfering with the expression of NOTCH1.

P2Y2 receptor represses IL-6 expression by valve interstitial cells through Akt: implication for calcific aortic valve disease.

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.

Mechanical strain induces the production of spheroid mineralized microparticles in the aortic valve through a RhoA/ROCK-dependent mechanism

Calcific aortic valve disease (CAVD) is a chronic disorder characterized by an abnormal mineralization of the leaflets, which is accelerated in bicuspid aortic valve (BAV). It is suspected that mechanical strain may promote/enhance mineralization of the aortic valve. However, the effect of mechanical strain and the involved pathways during mineralization of the aortic valve remains largely unknown. Valve interstitial cells (VICs) were isolated and studied under strain conditions. Human bicuspid aortic valves were examined as a model relevant to increase mechanical strain. Cyclic strain increased mineralization of VICs by several-fold. Scanning electron microscope (SEM) and energy dispersive X-ray (EDX) analyses revealed that mechanical strain promoted the formation of mineralized spheroid microparticles, which coalesced into larger structure at the surface of apoptotic VICs. Apoptosis and mineralization were closely associated with expression of ENPP1. Inhibition of ENPP1 greatly reduced mineralization of VIC cultures. Through several lines of evidence we showed that mechanical strain promoted the export of ENPP1-containing vesicles to the plasma membrane through a RhoA/ROCK pathway. Studies conducted in human BAV revealed the presence of spheroid mineralized structures along with the expression of ENPP1 in areas of high mechanical strain. Mechanical strain promotes the production and accumulation of spheroid mineralized microparticles by VICs, which may represent one important underlying mechanism involved in aortic valve mineralization. RhoA/ROCK-mediated export of ENPP1 to the plasma membrane promotes strain-induced mineralization of VICs.

Innate and Adaptive Immunity in Calcific Aortic Valve Disease

Calcific aortic valve disease (CAVD) is the most common heart valve disorder. CAVD is a chronic process characterized by a pathologic mineralization of valve leaflets. Ectopic mineralization of the aortic valve involves complex relationships with immunity. Studies have highlighted that both innate and adaptive immunity play a role in the development of CAVD. In this regard, accumulating evidence indicates that fibrocalcific remodelling of the aortic valve is associated with activation of the NF-κB pathway. The expression of TNF-α and IL-6 is increased in human mineralized aortic valves and promotes an osteogenic program as well as the mineralization of valve interstitial cells (VICs), the main cellular component of the aortic valve. Different factors, including oxidized lipid species, activate the innate immune response through the Toll-like receptors. Moreover, VICs express 5-lipoxygenase and therefore produce leukotrienes, which may amplify the inflammatory response in the aortic valve. More recently, studies have emphasized that an adaptive immune response is triggered during CAVD. Herein, we are reviewing the link between the immune response and the development of CAVD and we have tried, whenever possible, to keep a translational approach.

Molecular biology of calcific aortic valve disease: towards new pharmacological therapies.

Calcific aortic valve disease (CAVD) is a chronic process leading to fibrosis and mineralization of the aortic valve. Investigations in the last several years have emphasized that key underlying molecular processes are involved in the pathogenesis of CAVD. In this regard, the processing of lipids and their retention has been underlined as an important mechanism that triggers inflammation. In turn, inflammation promotes/enhances the mineralization of valve interstitial cells, the main cellular component of the aortic valve. On the other hand, transformation of valve interstitial cells into myofibroblasts and osteoblast-like cells is determined by several signaling pathways having reciprocal cross-talks. In addition, the mineralization of the aortic valve has been shown to rely on ectonucleotidase and purinergic signaling. In this review, the authors have highlighted key molecular underpinnings of CAVD that may have significant relevance for the development of novel pharmaceutical therapies.

Early Development of Calcific Aortic Valve Disease and Left Ventricular Hypertrophy in a Mouse Model of Combined Dyslipidemia and Type 2 Diabetes Mellitus

Objective— This study aimed to determine the potential impact of type 2 diabetes mellitus on left ventricular dysfunction and the development of calcified aortic valve disease using a dyslipidemic mouse model prone to developing type 2 diabetes mellitus. Approach and Results— When compared with nondiabetic LDLr−/−/ApoB100/100, diabetic LDLr−/−/ApoB100/100/IGF-II mice exhibited similar dyslipidemia and obesity but developed type 2 diabetes mellitus when fed a high-fat/sucrose/cholesterol diet for 6 months. LDLr−/−/ApoB100/100/IGF-II mice showed left ventricular hypertrophy versus C57BL6 but not LDLr−/−/ApoB100/100 mice. Transthoracic echocardiography revealed significant reductions in both left ventricular systolic fractional shortening and diastolic function in high-fat/sucrose/cholesterol fed LDLr−/−/ApoB100/100/IGF-II mice when compared with LDLr−/−/ApoB100/100. Importantly, we found that peak aortic jet velocity was significantly increased in LDLr−/−/ApoB100/100/IGF-II mice versus LDLr−/−/ApoB100/100 animals on the high-fat/sucrose/cholesterol diet. Microtomography scans and Alizarin red staining indicated calcification in the aortic valves, whereas electron microscopy and energy dispersive x-ray spectroscopy further revealed mineralization of the aortic leaflets and the presence of inflammatory infiltrates in diabetic mice. Studies showed upregulation of hypertrophic genes (anp, bnp, b-mhc) in myocardial tissues and of osteogenic genes (spp1, bglap, runx2) in aortic tissues of diabetic mice. Conclusions— We have established the diabetes mellitus –prone LDLr−/−/ApoB100/100/IGF-II mouse as a new model of calcified aortic valve disease. Our results are consistent with the growing body of clinical evidence that the dysmetabolic state of type 2 diabetes mellitus contributes to early mineralization of the aortic valve and calcified aortic valve disease pathogenesis.

OxLDL-derived lysophosphatidic acid promotes the progression of aortic valve stenosis through a LPAR1-RhoA–NF-κB pathway

Aims Oxidatively modified lipoproteins may promote the development/progression of calcific aortic valve stenosis (CAVS). Oxidative transformation of low-density lipoprotein (OxLDL) generates lysophosphatidic acid (LPA), a lipid mediator that accumulates in mineralized aortic valves. LPA activates at least six different G protein-coupled receptors, which may play a role in the pathophysiology of CAVS. We hypothesized that LPA derived from OxLDL may promote a NF-κB signature that drives osteogenesis in the aortic valve. Methods and results The role of OxLDL-LPA was examined in isolated valve interstitial cells (VICs) and the molecular pathway was validated in human explanted aortic valves and in a mouse model of CAVS. We found that OxLDL-LPA promoted the mineralization and osteogenic transition of VICs through LPAR1 and the activation of a RhoA-NF-κB pathway. Specifically, we identified that RhoA/ROCK activated IκB kinase alpha, which promoted the phosphorylation of p65 on serine 536 (p65 pS536). p65 pS536 was recruited to the BMP2 promoter and directed an osteogenic program not responsive to the control exerted by the inhibitor of kappa B. In LDLR-/-/ApoB100/100/IGFII transgenic mice (IGFII), which develop CAVS under a high-fat and high-sucrose diet the administration of Ki16425, a Lpar1 blocker, reduced by three-fold the progression rate of CAVS and also decreased the osteogenic activity as measured with a near-infrared fluorescent probe that recognizes hydroxyapatite of calcium. Conclusions OxLDL-LPA promotes an osteogenic program in the aortic valve through a LPAR1-RhoA/ROCK-p65 pS536 pathway. LPAR1 may represent a suitable target to prevent the progression of CAVS.

Association between plasma lipoprotein levels and bioprosthetic valve structural degeneration

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 (≥10 mm Hg) or a worsening of transprosthetic regurgitation (≥1/3) during the follow-up. Results After a mean follow-up of 8±3.5 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, <298 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.

Carbonic anhydrase XII in valve interstitial cells promotes the regression of calcific aortic valve stenosis

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.

Soluble CD14 is associated with the structural failure of bioprostheses

INTRODUCTION Aortic valve bioprostheses, which do not mandate chronic anticoagulation, are prone to structural valve degeneration (SVD). The processes involved in SVD are likely multifactorial. We hypothesized that inflammation and macrophage activation could be involved in SVD. METHODS In 203 patients with an aortic valve bioprosthesis, we evaluated the association between the macrophage activation marker soluble CD14 (sCD14) and SVD. RESULTS After a mean follow-up of 8 ± 3 years, 42 (21%) patients developed SVD. Patients with SVD had higher peak (44 ± 13 mmHg vs. 25 ± 12 mmHg, p < .0001) and mean (24 ± 7 mmHg vs. 12 ± 5 mmHg, p < .0001) transprosthetic gradients. On univariable analysis, low-density lipoprotein cholesterol (LDL) and sCD14 were associated with SVD. After correction for covariates, sCD14 (OR: 1.12, 95%CI: 1.02-1.23, p = .01) remained independently associated with SVD. In turn, sCD14 was associated with the HOMA index and high-density lipoprotein (HDL) level. Patients with a metabolic syndrome (MetS) had higher level of sCD14. In a model corrected for age, sex, HOMA and HDL, the MetS remained independently associated with sCD14 levels (β = 0.65, SE = 0.30, p = .03). CONCLUSION Circulating level of sCD14 is an independent predictor of SVD. In turn, patients with MetS have higher sCD14 levels.