Microcalcification and Thoracic Aortopathy: A Window Into Disease Severity.

BACKGROUND: Patients with thoracic aortopathy are at increased risk of catastrophic aortic dissection, carrying with it substantial mortality and morbidity. Although granular medial calcinosis (medial microcalcification) has been associated with thoracic aortopathy, its relationship to disease severity has yet to be established. METHODS: One hundred one thoracic aortic specimens were collected from 57 patients with thoracic aortopathy and 18 control subjects. Standardized histopathologic scores, immunohistochemistry, and nanoindentation (tissue elastic modulus) were compared with the extent of microcalcification on von Kossa histology and 18F-sodium fluoride autoradiography. RESULTS: Microcalcification content was higher in thoracic aortopathy samples with mild (n=28; 6.17 [2.71-10.39]; P≤0.00010) or moderate histopathologic degeneration (n=30; 3.74 [0.87-11.80]; P<0.042) compared with control samples (n=18; 0.79 [0.36-1.90]). Alkaline phosphatase (n=26; P=0.0019) and OPN (osteopontin; n=26; P=0.0045) staining were increased in tissue with early aortopathy. Increasingly severe histopathologic degeneration was related to reduced microcalcification (n=82; Spearman ρ, -0.51; P<0.0001)-a process closely linked with elastin loss (n=82; Spearman ρ, -0.43; P<0.0001) and lower tissue elastic modulus (n=28; Spearman ρ, 0.43; P=0.026).18F-sodium fluoride autoradiography demonstrated good correlation with histologically quantified microcalcification (n=66; r=0.76; P<0.001) and identified areas of focal weakness in vivo. CONCLUSIONS: Medial microcalcification is a marker of aortopathy, although progression to severe aortopathy is associated with loss of both elastin fibers and microcalcification.18F-sodium fluoride positron emission tomography quantifies medial microcalcification and is a feasible noninvasive imaging modality for identifying aortic wall disruption with major translational promise.

Podocyte as the link between sterile inflammation and diabetic kidney disease.

Shahzad et al. examined the underlying mechanisms of sterile inflammation in diabetic kidney disease, specifically the role of NLRP3 inflammasome activation in podocytes. Using mouse models with gain-of-function and loss-of-function mutations in podocyte Nlrp3, or caspase-1 loss-of-function mutations in podocytes, they identified that Nlrp3 activation in these cells is central for development of diabetic kidney disease but not solely dependent on canonical mechanisms and caspase-1. These findings position podocyte-mediated immune cell-like functions as potential therapeutic targets for diabetic kidney disease.

Resistin induces cardiac fibroblast-myofibroblast differentiation through JAK/STAT3 and JNK/c-Jun signaling.

Cardiac fibrosis is characterized by excessive deposition of extracellular matrix proteins and myofibroblast differentiation. Our previous findings have implicated resistin in cardiac fibrosis; however, the molecular mechanisms underlying this process are still unclear. Here we investigated the role of resistin in fibroblast-to-myofibroblast differentiation and elucidated the pathways involved in this process. Fibroblast-to-myofibroblast transdifferentiation was induced with resistin or TGFß1 in NIH-3T3 and adult cardiac fibroblasts. mRNA and protein expression of fibrotic markers were analyzed by qPCR and immunoblotting. Resistin-knockout mice, challenged with a high-fat diet (HFD) for 20 weeks to stimulate cardiac impairment, were analyzed for cardiac function and fibrosis using histologic and molecular methods. Cardiac fibroblasts stimulated with resistin displayed increased fibroblast-to-myofibroblast conversion, with increased levels of αSma, col1a1, Fn, Ccn2 and Mmp9, with remarkable differences in the actin network appearance. Mechanistically, resistin promotes fibroblast-to-myofibroblast transdifferentiation and fibrogenesis via JAK2/STAT3 and JNK/c-Jun signaling pathways, independent of TGFß1. Resistin-null mice challenged with HFD showed an improvement in cardiac function and a decrease in tissue fibrosis and reduced mRNA levels of fibrogenic markers. These findings are the first to delineate the role of resistin in the process of cardiac fibroblast-to-myofibroblast differentiation via JAK/STAT3 and JNK/c-Jun pathways, potentially leading to stimulation of cardiac fibrosis.

FTO-Dependent N6-Methyladenosine Regulates Cardiac Function During Remodeling and Repair.

BACKGROUND: Despite its functional importance in various fundamental bioprocesses, studies of N6-methyladenosine (m6A) in the heart are lacking. Here, we show that the FTO (fat mass and obesity-associated protein), an m6A demethylase, plays a critical role in cardiac contractile function during homeostasis, remodeling, and regeneration. METHODS: We used clinical human samples, preclinical pig and mouse models, and primary cardiomyocyte cell cultures to study the functional role of m6A and FTO in the heart and in cardiomyocytes. We modulated expression of FTO by using adeno-associated virus serotype 9 (in vivo), adenovirus (both in vivo and in vitro), and small interfering RNAs (in vitro) to study its function in regulating cardiomyocyte m6A, calcium dynamics and contractility, and cardiac function postischemia. We performed methylated (m6A) RNA immunoprecipitation sequencing to map transcriptome-wide m6A, and methylated (m6A) RNA immunoprecipitation quantitative polymerase chain reaction assays to map and validate m6A in individual transcripts, in healthy and failing hearts, and in myocytes. RESULTS: We discovered that FTO has decreased expression in failing mammalian hearts and hypoxic cardiomyocytes, thereby increasing m6A in RNA and decreasing cardiomyocyte contractile function. Improving expression of FTO in failing mouse hearts attenuated the ischemia-induced increase in m6A and decrease in cardiac contractile function. This is performed by the demethylation activity of FTO, which selectively demethylates cardiac contractile transcripts, thus preventing their degradation and improving their protein expression under ischemia. In addition, we demonstrate that FTO overexpression in mouse models of myocardial infarction decreased fibrosis and enhanced angiogenesis. CONCLUSIONS: Collectively, our study demonstrates the functional importance of the FTO-dependent cardiac m6A methylome in cardiac contraction during heart failure and provides a novel mechanistic insight into the therapeutic mechanisms of FTO.

Activated platelets promote an osteogenic programme and the progression of calcific aortic valve stenosis.

AIMS: Calcific aortic valve stenosis (CAVS) is characterized by a fibrocalcific process. Studies have shown an association between CAVS and the activation of platelets. It is believed that shear stress associated with CAVS promotes the activation of platelets. However, whether platelets actively participate to the mineralization of the aortic valve (AV) and the progression of CAVS is presently unknown. To identify the role of platelets into the pathobiology of CAVS. METHODS AND RESULTS: Explanted control non-mineralized and mineralized AVs were examined by scanning electron microscope (SEM) for the presence of activated platelets. In-depth functional assays were carried out with isolated human valve interstitial cells (VICs) and platelets as well as in LDLR-/- apoB100/100 IGFII (IGFII) mice. Scanning electron microscope and immunogold markings for glycoprotein IIb/IIIa (GPIIb/IIIa) revealed the presence of platelet aggregates with fibrin in endothelium-denuded areas of CAVS. In isolated VICs, collagen-activated platelets induced an osteogenic programme. Platelet-derived adenosine diphosphate induced the release of autotaxin (ATX) by VICs. The binding of ATX to GPIIb/IIIa of platelets generated lysophosphatidic acid (LysoPA) with pro-osteogenic properties. In IGFII mice with CAVS, platelet aggregates were found at the surface of AVs. Administration of activated platelets to IGFII mice accelerated the development of CAVS by 2.1-fold, whereas a treatment with Ki16425, an antagonist of LysoPA receptors, prevented platelet-induced mineralization of the AV and the progression of CAVS. CONCLUSIONS: These findings suggest a novel role for platelets in the progression of CAVS.

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.

Autotaxin Derived From Lipoprotein(a) and Valve Interstitial Cells Promotes Inflammation and Mineralization of the Aortic Valve.

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.

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.

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(-/-)/ApoB(100/100), diabetic LDLr(-/-)/ApoB(100/100)/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(-/-)/ApoB(100/100)/IGF-II mice showed left ventricular hypertrophy versus C57BL6 but not LDLr(-/-)/ApoB(100/100) mice. Transthoracic echocardiography revealed significant reductions in both left ventricular systolic fractional shortening and diastolic function in high-fat/sucrose/cholesterol fed LDLr(-/-)/ApoB(100/100)/IGF-II mice when compared with LDLr(-/-)/ApoB(100/100). Importantly, we found that peak aortic jet velocity was significantly increased in LDLr(-/-)/ApoB(100/100)/IGF-II mice versus LDLr(-/-)/ApoB(100/100) 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(-/-)/ApoB(100/100)/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.

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.

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.

Genetic susceptibility to diabetic kidney disease is linked to promoter variants of XOR.

The lifetime risk of kidney disease in people with diabetes is 10-30%, implicating genetic predisposition in the cause of diabetic kidney disease (DKD). Here we identify an expression quantitative trait loci (QTLs) in the cis-acting regulatory region of the xanthine dehydrogenase, or xanthine oxidoreductase (Xor), a binding site for C/EBPß, to be associated with diabetes-induced podocyte loss in DKD in male mice. We examine mouse inbred strains that are susceptible (DBA/2J) and resistant (C57BL/6J) to DKD, as well as a panel of recombinant inbred BXD mice, to map QTLs. We also uncover promoter XOR orthologue variants in humans associated with high risk of DKD. We introduced the risk variant into the 5'-regulatory region of XOR in DKD-resistant mice, which resulted in increased Xor activity associated with podocyte depletion, albuminuria, oxidative stress and damage restricted to the glomerular endothelium, which increase further with type 1 diabetes, high-fat diet and ageing. Therefore, differential regulation of Xor contributes to phenotypic consequences with diabetes and ageing.

Timing of bioprosthetic valve fracture in transcatheter valve-in-valve intervention: impact on valve durability and leaflet integrity.

BACKGROUND: Bioprosthetic valve fracture (BVF) can be used to improve transcatheter heart valve (THV) haemodynamics following a valve-in-valve (ViV) intervention. However, whether BVF should be performed before or after THV deployment and the implications on durability are unknown.  Aims: We sought to assess the impact of BVF timing on long-term THV durability. METHODS: The impact of BVF timing was assessed using small ACURATE neo (ACn) or 23 mm SAPIEN 3 (S3) THV deployed in 21 mm Mitroflow valves compared to no-BVF controls. Valves underwent accelerated wear testing up to 200 million (M) cycles (equivalent to 5 years). At 200M cycles, THV were evaluated by hydrodynamic testing, second-harmonic generation (SHG) microscopy, scanning electron microscopy (SEM) and histology. RESULTS: At 200M cycles, the regurgitant fraction (RF) and effective orifice area (EOA) for the ACn were 8.03±0.30%/1.74±0.01 cm2 (no BVF), 12.48±0.70%/1.97±0.02 cm2 (BVF before ViV) and 9.29±0.38%/2.21±0.0 cm2 (BVF after ViV), respectively. For the S3 these values were 2.63±0.51%/1.26±0.01 cm2, 2.03±0.42%/1.65±0.01 cm2, and 1.62±0.38%/2.22±0.01 cm2, respectively. Further, SHG and SEM revealed a higher degree of superficial leaflet damage when BVF was performed after ViV for the ACn and S3. However, the histological analysis revealed significantly less damage, as determined by matrix density analysis, through the entire leaflet thickness when BVF was performed after ViV with the S3 and a similar but non-significant trend with the ACn.  Conclusions: BVF performed after ViV appears to offer superior long-term EOA without increased RF. Ultrastructure leaflet analysis reveals that the timing of BVF can differentially impact leaflets, with more superficial damage but greater preservation of overall leaflet structure when BVF is performed after ViV.

Resistin deletion protects against heart failure injury by targeting DNA damage response.

AIMS: Increased resistin (Retn) levels are associated with development of cardiovascular diseases. However, the role of Retn in heart failure (HF) is still unclear. Here we probed the functional and molecular mechanism underlying the beneficial effect of Retn deletion in HF. METHODS AND RESULTS: Wild-type (WT) and adipose tissue-specific Retn-knockout (RKO) mice were subjected to transverse aortic constriction (TAC)-induced HF. Cardiac function and haemodynamic changes were measured by echocardiography and left ventricular catheterization. Adipose tissue Retn deletion attenuated while Retn cardiac-selective overexpression, via a recombinant adeno-associated virus-9 vector, exacerbated TAC-induced hypertrophy, cardiac dysfunction, and myocardial fibrosis in WT and RKO mice. Mechanistically, we showed that Gadd45α was significantly increased in RKO HF mice while cardiac overexpression of Retn led to its downregulation. miR148b-3p directly targets Gadd45α and inhibits its expression. Retn overexpression upregulated miR148b-3p expression and triggered DNA damage response (DDR) in RKO-HF mice. Inhibition of miR148b-3p in vivo normalized Gadd45α expression, decreased DDR, and reversed cardiac dysfunction and fibrosis. In vitro Retn overexpression in adult mouse cardiomyocytes activated miR148b-3p and reduced Gadd45α expression. Gadd45α overexpression in H9C2-cardiomyoblasts protected against hydrogen peroxide- and Retn-induced DDR. CONCLUSION: These findings reveal that diminution in circulating Retn reduced myocardial fibrosis and apoptosis, and improved heart function in a mouse model of HF, at least in part, through attenuation of miR148b-3p and DDR. The results of this study indicate that controlling Retn levels may provide a potential therapeutic approach for treating pressure overload-induced HF.

Isolation of Conditionally Immortalized Mouse Glomerular Endothelial Cells with Fluorescent Mitochondria.

Glomerular endothelial cell (GEC) dysfunction can initiate and contribute to glomerular filtration barrier breakdown. Increased mitochondrial oxidative stress has been suggested as a mechanism resulting in GEC dysfunction in the pathogenesis of some glomerular diseases. Historically the isolation of GECs from in vivo models has been notoriously challenging due to difficulties in isolating pure cultures from glomeruli. GECs have complex growth requirements in vitro and a very limited lifespan. Here, we describe the procedure for isolating and culturing conditionally immortalized GECs with fluorescent mitochondria, enabling the tracking of mitochondrial fission and fusion events. GECs were isolated from the kidneys of a double transgenic mouse expressing the thermolabile SV40 TAg (from the Immortomouse), conditionally promoting proliferation and suppressing cell differentiation, and a photo-convertible fluorescent protein (Dendra2) in all mitochondria (from the photo-activatable mitochondria [PhAMexcised] mouse). The stable cell line generated allows for cell differentiation after inactivation of the immortalizing SV40 TAg gene and photo-activation of a subset of mitochondria causing a switch in fluorescence from green to red. The use of mitoDendra2-GECs allows for live imaging of fluorescent mitochondria's distribution, fusion, and fission events without staining the cells.

Increased Ca2+ influx through CaV1.2 drives aortic valve calcification.

Calcific aortic valve disease (CAVD) is heritable, as revealed by recent GWAS. While polymorphisms linked to increased expression of CACNA1C - encoding the CaV1.2 L-type voltage-gated Ca2+ channel - and increased Ca2+ signaling are associated with CAVD, whether increased Ca2+ influx through the druggable CaV1.2 causes CAVD is unknown. We confirmed the association between increased CaV1.2 expression and CAVD in surgically removed aortic valves from patients. We extended our studies with a transgenic mouse model that mimics increased CaV1.2 expression within aortic valve interstitial cells (VICs). In young mice maintained on normal chow, we observed dystrophic valve lesions that mimic changes found in presymptomatic CAVD and showed activation of chondrogenic and osteogenic transcriptional regulators within these valve lesions. Chronic administration of verapamil, a CaV1.2 antagonist used clinically, slowed the progression of lesion development in vivo. Exploiting VIC cultures, we demonstrated that increased Ca2+ influx through CaV1.2 drives signaling programs that lead to myofibroblast activation of VICs and upregulation of genes associated with aortic valve calcification. Our data support a causal role for Ca2+ influx through CaV1.2 in CAVD and suggest that early treatment with Ca2+ channel blockers is an effective therapeutic strategy.

Implication of Lipids in Calcified Aortic Valve Pathogenesis: Why Did Statins Fail?

Calcific Aortic Valve Disease (CAVD) is a fibrocalcific disease. Lipoproteins and oxidized phospholipids play a substantial role in CAVD; the level of Lp(a) has been shown to accelerate the progression of valve calcification. Indeed, oxidized phospholipids carried by Lp(a) into the aortic valve stimulate endothelial dysfunction and promote inflammation. Inflammation and growth factors actively promote the synthesis of the extracellular matrix (ECM) and trigger an osteogenic program. The accumulation of ECM proteins promotes lipid adhesion to valve tissue, which could initiate the osteogenic program in interstitial valve cells. Statin treatment has been shown to have the ability to diminish the death rate in subjects with atherosclerotic impediments by decreasing the serum LDL cholesterol levels. However, the use of HMG-CoA inhibitors (statins) as cholesterol-lowering therapy did not significantly reduce the progression or the severity of aortic valve calcification. However, new clinical trials targeting Lp(a) or PCSK9 are showing promising results in reducing the severity of aortic stenosis. In this review, we discuss the implication of lipids in aortic valve calcification and the current findings on the effect of lipid-lowering therapy in aortic stenosis.

Isolation of Mouse Interstitial Valve Cells to Study the Calcification of the Aortic Valve In Vitro.

The calcification of aortic valve cells is the hallmark of aortic stenosis and is associated with valve cusp fibrosis. Valve interstitial cells (VICs) play an important role in the calcification process in aortic stenosis through the activation of their dedifferentiation program to osteoblast-like cells. Mouse VICs are a good in vitro tool for the elucidation of the signaling pathways driving the mineralization of the aortic valve cell. The method described herein, successfully used by these authors, explains how to obtain freshly isolated cells. A two-step collagenase procedure was performed with 1 mg/mL and 4.5 mg/mL. The first step is crucial to remove the endothelial cell layer and avoid any contamination. The second collagenase incubation is to facilitate the migration of VICs from the tissue to the plate. In addition, an immunofluorescence staining procedure for the phenotype characterization of the isolated mouse valve cells is discussed. Furthermore, the calcification assay was performed in vitro by using the calcium reagent measurement procedure and alizarin red staining. The use of mouse valve cell primary culture is essential for testing new pharmacological targets to inhibit cell mineralization in vitro.

Proteomic Architecture of Valvular Extracellular Matrix: FNDC1 and MXRA5 Are New Biomarkers of Aortic Stenosis.

This study analyzed the expression of extracellular matrix (ECM) proteins during aortic valve calcification with mass spectrometry, and further validated in an independent human cohort using RNAseq data. The study reveals that valve calcification is associated with significant disruption in ECM and metabolic pathways, and highlights a strong connection between metabolic markers and ECM remodeling. It also identifies FNDC1 and MXRA5 as novel ECM biomarkers in calcified valves, electing them as potential targets in the development and progression of aortic stenosis.

Platelets: Implications in Aortic Valve Stenosis and Bioprosthetic Valve Dysfunction From Pathophysiology to Clinical Care.

Aortic stenosis (AS) is the most common heart valve disease requiring surgery in developed countries, with a rising global burden associated with aging populations. The predominant cause of AS is believed to be driven by calcific degeneration of the aortic valve and a growing body of evidence suggests that platelets play a major role in this disease pathophysiology. Furthermore, platelets are a player in bioprosthetic valve dysfunction caused by their role in leaflet thrombosis and thickening. This review presents the molecular function of platelets in the context of recent and rapidly evolving understanding the role of platelets in AS, both of the native aortic valve and bioprosthetic valves, where there remain concerns about the effects of subclinical leaflet thrombosis on long-term prosthesis durability. This review also presents the role of antiplatelet and anticoagulation therapies on modulating the impact of platelets on native and bioprosthetic aortic valves, highlighting the need for further studies to determine whether these therapies are protective and may increase the life span of surgical and transcatheter aortic valve implants. By linking molecular mechanisms through which platelets drive disease of native and bioprosthetic aortic valves with studies evaluating the clinical impact of antiplatelet and antithrombotic therapies, we aim to bridge the gaps between our basic science understanding of platelet biology and their role in patients with AS and ensuing preventive and therapeutic implications.