Zinc ameliorates human aortic valve calcification through GPR39 mediated ERK1/2 signalling pathway.

AIMS: Calcific aortic valve disease (CAVD) is the most common heart valve disease in the Western world. It has been reported that zinc is accumulated in calcified human aortic valves. However, whether zinc directly regulates CAVD is yet to be elucidated. The present study sought to determine the potential role of zinc in the pathogenesis of CAVD. METHODS AND RESULTS: Using a combination of a human valve interstitial cell (hVIC) calcification model, human aortic valve tissues, and blood samples, we report that 20 µM zinc supplementation attenuates hVIC in vitro calcification, and that this is mediated through inhibition of apoptosis and osteogenic differentiation via the zinc-sensing receptor GPR39-dependent ERK1/2 signalling pathway. Furthermore, we report that GPR39 protein expression is dramatically reduced in calcified human aortic valves, and there is a significant reduction in zinc serum levels in patients with CAVD. Moreover, we reveal that 20 µM zinc treatment prevents the reduction of GPR39 observed in calcified hVICs. We also show that the zinc transporter ZIP13 and ZIP14 are significantly increased in hVICs in response to zinc treatment. Knockdown of ZIP13 or ZIP14 significantly inhibited hVIC in vitro calcification and osteogenic differentiation. CONCLUSIONS: Together, these findings suggest that zinc is a novel inhibitor of CAVD, and report that zinc transporter ZIP13 and ZIP14 are important regulators of hVIC in vitro calcification and osteogenic differentiation. Zinc supplementation may offer a potential therapeutic strategy for CAVD.

Aldo-keto reductase family 1 member B induces aortic valve calcification by activating hippo signaling in valvular interstitial cells.

AIMS: Calcific aortic valve disease (CAVD) is a primary cause of cardiovascular mortality; however, its mechanisms are unknown. Currently, no effective pharmacotherapy is available for CAVD. Aldo-keto reductase family 1 member B (Akr1B1) has been identified as a potential therapeutic target for valve interstitial cell calcification. Herein, we hypothesized that inhibition of Akr1B1 can attenuate aortic valve calcification. METHODS AND RESULTS: Normal and degenerative tricuspid calcific valves from human samples were analyzed by immunoblotting and immunohistochemistry. The results showed significant upregulation of Akr1B1 in CAVD leaflets. Akr1B1 inhibition attenuated calcification of aortic valve interstitial cells in osteogenic medium. In contrast, overexpression of Akr1B1 aggravated calcification in osteogenic medium. Mechanistically, using RNA sequencing (RNAseq), we revealed that Hippo-YAP signaling functions downstream of Akr1B1. Furthermore, we established that the protein level of the Hippo-YAP signaling effector active-YAP had a positive correlation with Akr1B1. Suppression of YAP reversed Akr1B1 overexpression-induced Runx2 upregulation. Moreover, YAP activated the Runx2 promoter through TEAD1 in a manner mediated by ChIP and luciferase reporter systems. Animal experiments showed that the Akr1B1 inhibitor epalrestat attenuated aortic valve calcification induced by a Western diet in LDLR-/- mice. CONCLUSION: This study demonstrates that inhibition of Akr1B1 can attenuate the degree of calcification both in vitro and in vivo. The Akr1B1 inhibitor epalrestat may be a potential treatment option for CAVD.

Lipoprotein-associated phospholipase A2 activity, genetics and calcific aortic valve stenosis in humans.

BACKGROUND: Lipoprotein-associated phospholipase A2 (Lp-PLA2) activity has been shown to predict calcific aortic valve stenosis (CAVS) outcomes. Our objective was to test the association between plasma Lp-PLA2 activity and genetically elevated Lp-PLA2 mass/activity with CAVS in humans. METHODS AND RESULTS: Lp-PLA2 activity was measured in 890 patients undergoing cardiac surgery, including 476 patients undergoing aortic valve replacement for CAVS and 414 control patients undergoing coronary artery bypass grafting. After multivariable adjustment, Lp-PLA2 activity was positively associated with the presence of CAVS (OR=1.21 (95% CI 1.04 to 1.41) per SD increment). We selected four single nucleotide polymorphisms (SNPs) at the PLA2G7 locus associated with either Lp-PLA2 mass or activity (rs7756935, rs1421368, rs1805017 and rs4498351). Genetic association studies were performed in eight cohorts: Quebec-CAVS (1009 cases/1017 controls), UK Biobank (1350 cases/349 043 controls), European Prospective Investigation into Cancer and Nutrition-Norfolk (504 cases/20 307 controls), Genetic Epidemiology Research on Aging (3469 cases/51 723 controls), Malmö Diet and Cancer Study (682 cases/5963 controls) and three French cohorts (3123 cases/6532 controls), totalling 10 137 CAVS cases and 434 585 controls. A fixed-effect meta-analysis using the inverse-variance weighted method revealed that none of the four SNPs was associated with CAVS (OR=0.99 (95% CI 0.96 to 1.02, p=0.55) for rs7756935, 0.97 (95% CI 0.93 to 1.01, p=0.11) for rs1421368, 1.00 (95% CI 1.00 to 1.01, p=0.29) for rs1805017, and 1.00 (95% CI 0.97 to 1.04, p=0.87) for rs4498351). CONCLUSIONS: Higher Lp-PLA2 activity is significantly associated with the presence of CAVS and might represent a biomarker of CAVS in patients with heart disease. Results of our genetic association study suggest that Lp-PLA2 is however unlikely to represent a causal risk factor or therapeutic target for CAVS.

Semicarbazide-Sensitive Amine Oxidase Increases in Calcific Aortic Valve Stenosis and Contributes to Valvular Interstitial Cell Calcification.

INTRODUCTION: Calcific aortic valve stenosis (CAVS) is a common disease associated with aging. Oxidative stress participates in the valve calcification process in CAVS. Semicarbazide-sensitive amine oxidase (SSAO), also referred to as vascular adhesion protein 1 (VAP-1), transforms primary amines into aldehydes, generating hydrogen peroxide and ammonia. SSAO is expressed in calcified aortic valves, but its role in valve calcification has remained largely unexplored. The aims of this study were to characterize the expression and the activity of SSAO during aortic valve calcification and to establish the effects of SSAO inhibition on human valvular interstitial cell (VIC) calcification. METHODS: Human aortic valves from n = 80 patients were used for mRNA extraction and expression analysis, Western blot, SSAO activity determination, immunohistochemistry, and the isolation of primary VIC cultures. RESULTS: SSAO mRNA, protein, and activity were increased with increasing calcification within human aortic valves and localized in the vicinity of the calcified zones. The valvular SSAO upregulation was consistent after stratification of the subjects according to cardiovascular and CAVS risk factors associated with increased oxidative stress: body mass index, diabetes, and smoking. SSAO mRNA levels were significantly associated with poly(ADP-ribose) polymerase 1 (PARP1) in calcified tissue. Calcification of VIC was inhibited in the presence of the specific SSAO inhibitor LJP1586. CONCLUSION: The association of SSAO expression and activity with valvular calcification and oxidative stress as well as the decreased VIC calcification by SSAO inhibition points to SSAO as a possible marker and therapeutic target to be further explored in CAVS.

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.

Nucleotide ecto-enzyme metabolic pattern and spatial distribution in calcific aortic valve disease; its relation to pathological changes and clinical presentation.

BACKGROUND: Extracellular nucleotide metabolism contributes to chronic inflammation, cell differentiation, and tissue mineralization by controlling nucleotide and adenosine concentrations and hence its purinergic effects. This study investigated location-specific changes of extracellular nucleotide metabolism in aortic valves of patients with calcific aortic valve disease (CAVD). Individual ecto-enzymes and adenosine receptors involved were analyzed together with correlation with CAVD severity and risk factors. RESULTS: Nucleotide and adenosine degradation rates were adversely modified on the aortic surface of stenotic valve as compared to ventricular side, including decreased ATP removal (1.25 ± 0.35 vs. 2.24 ± 0.61 nmol/min/cm2) and adenosine production (1.32 ± 0.12 vs. 2.49 ± 0.28 nmol/min/cm2) as well as increased adenosine deamination (1.28 ± 0.31 vs. 0.67 ± 0.11 nmol/min/cm2). The rates of nucleotide to adenosine conversions were lower, while adenosine deamination was higher on the aortic sides of stenotic vs. non-stenotic valve. There were no differences in extracellular nucleotide metabolism between aortic and ventricular sides of non-stenotic valves. Furthermore, nucleotide degradation rates, measured on aortic side in CAVD (n = 62), negatively correlated with echocardiographic and biochemical parameters of disease severity (aortic jet velocity vs. ATP hydrolysis: r = - 0.30, p < 0.05; vs. AMP hydrolysis: r = - 0.44, p < 0.001; valvular phosphate concentration vs. ATP hydrolysis: r = - 0.26, p < 0.05; vs. AMP hydrolysis: r = - 0.25, p = 0.05) while adenosine deamination showed positive correlation trend with valvular phosphate deposits (r = 0.23, p = 0.07). Nucleotide and adenosine conversion rates also correlated with CAVD risk factors, including hyperlipidemia (AMP hydrolysis vs. serum LDL cholesterol: r = - 0.28, p = 0.05; adenosine deamination vs. total cholesterol: r = 0.25, p = 0.05; LDL cholesterol: r = 0.28, p < 0.05; triglycerides: r = 0.32, p < 0.05), hypertension (adenosine deamination vs. systolic blood pressure: r = 0.28, p < 0.05) and thrombosis (ATP hydrolysis vs. prothrombin time: r = - 0.35, p < 0.01). Functional assays as well as histological and immunofluorescence, flow cytometry and RT-PCR studies identified all major ecto-enzymes engaged in nucleotide metabolism in aortic valves that included ecto-nucleotidases, alkaline phosphatase, and ecto-adenosine deaminase. We have shown that changes in nucleotide-converting ecto-enzymes were derived from their altered activities on valve cells and immune cell infiltrate. We have also demonstrated a presence of A1, A2a and A2b adenosine receptors with diminished expression of A2a and A2b in stenotic vs. non-stenotic valves. Finally, we revealed that augmenting adenosine effects by blocking adenosine deamination with deoxycoformycin decreased aortic valve thickness and reduced markers of calcification via adenosine-dependent pathways in a mouse model of CAVD. CONCLUSIONS: This work highlights profound changes in extracellular nucleotide and adenosine metabolism in CAVD. Altered extracellular nucleotide hydrolysis and degradation of adenosine in stenotic valves may affect purinergic responses to support a pro-stenotic milieu and valve calcification. This emphasizes a potential mechanism and target for prevention and therapy. .

Valve Interstitial Cell-Specific Cyclooxygenase-1 Associated With Calcification of Aortic Valves.

BACKGROUND: The molecular mechanisms underlying aortic valve calcification are poorly understood. Here, we aimed to identify the master regulators of calcification by comparison of genes in valve interstitial cells (VICs) with calcified and noncalcified aortic valves. METHODS: Calcified aortic valves were surgically excised from patients with aortic valve stenosis who required aortic valve replacements. Noncalcified and calcified sections were obtained from aortic valve leaflets. Collagenase-digested tissues were seeded into dishes, and VICs adhering to the dishes were cultured for 3 weeks, followed by comprehensive gene expression analysis. Functional analyses of identified proteins were performed by in vitro calcification assays. Tissue localization was determined by immunohistochemical staining for normal (n = 11) and stenotic valves (n = 30). RESULTS: We found 87 genes showing greater than a twofold change in calcified tissues. Among these genes, 68 were downregulated and 19 were upregulated. Cyclooxygenase-1 (COX1) messenger RNA and protein levels were upregulated in VICs from calcified tissues. The COX1 messenger RNA and protein levels in VICs were also strongly increased by stimulation with osteoblast differentiation medium. These were VIC-specific phenotypes and were not observed in other cell types. Immunohistochemical staining revealed that COX1-positive VICs were specifically localized in the calcified area of aortic valve tissues. CONCLUSIONS: The VIC-specific COX1 overexpression played a crucial role in calcification by promoting osteoblast differentiation in aortic valve tissues.

Transcatheter Aortic Heart Valves: Histological Analysis Providing Insight to Leaflet Thickening and Structural Valve Degeneration.

OBJECTIVES: This study investigated processes causing leaflet thickening and structural valve degeneration (SVD). BACKGROUND: Although transcatheter aortic valve replacement (TAVR) has changed the treatment of aortic stenosis, concerns remain regarding SVD, potentially related to valve thrombosis and thickening, based on studies using computed tomography (CT). Detailed histological analyses are provided to help attain insights into these processes. METHODS: Explanted transcatheter heart valves (THVs) were evaluated for thrombosis, fibrosis, and calcification for quantification of leaflet thickness. Immunohistochemical and microscopy approaches were used to investigate SVD-associated mechanisms. RESULTS: THVs (n = 23) were obtained from 22 patients (median 81 years of age; 50% male) from 0 to 2,583 days post TAVR. Maximal leaflet thickness increased relative to implant duration (ρ = 0.427; p = 0.027). THVs explanted after >2 years were thicker than those explanted after <2 years (p = 0.007). All THVs had adherent thrombus on both aortic and ventricular sides, which beyond 60 days was seen in combination with fibrosis and beyond 4 years had calcification. Early thrombus formation (<60 days) occurred despite rapid endothelialization with an abnormal hyperplastic phenotype. Fibrosis was observed in 6 patients on both the aortic and the ventricular THV surfaces, remodeled over time, and was associated with matrix metalloproteinase-1 expression. Five THVs showed overt calcification associated with adherent thrombus and fibrosis. CONCLUSIONS: There is a time-dependent degeneration of THVs consisting of thrombus formation, endothelial hyperplasia, fibrosis, tissue remodeling, proteinase expression, and calcification. Future investigation is needed to further understand these mechanisms contributing to leaflet thickening and SVD.

Histone deacetylase 6 reduction promotes aortic valve calcification via an endoplasmic reticulum stress-mediated osteogenic pathway.

OBJECTIVE: Aortic valve (AoV) calcification occurs via a pathophysiologic process that includes osteoblastic differentiation of valvular interstitial cells (VICs). Histone deacetylases (HDACs) have been shown to be involved in the pathogenesis of vascular diseases. Here, we investigated the role of HDAC6 in AoV calcification. METHODS: AoV cusps from patients with aortic stenosis (n = 7) and normal controls (n = 7) were subjected to determination of calcified nodules and HDAC6 expression. Human VICs were cultured in osteogenic media and treated with 10 uM tubacin or HDAC6 small interfering RNA silencing to inhibit HDAC6. Treatment with 100 uM tauroursodeoxycholic acid was used to suppress endoplasmic reticulum stress. Activating transcription factor 4 (ATF4) small interfering RNA was used to knock down ATF4. Alizarin red staining was used to evaluate calcified nodules formation of VICs cultured with osteogenic media for 14 days. RESULTS: HDAC6 expression was significantly reduced in AoV tissue of patients with aortic stenosis compared with controls. Tubacin treatment or HDAC6 silencing markedly promoted osteoblastic differentiation accompanied by endoplasmic reticulum stress activation in VICs. The HDAC6 inhibition-induced osteogenic pathway was mediated by endoplasmic reticulum stress/ATF4 pathway as indicated by tauroursodeoxycholic acid pretreatment or ATF4 silencing. Finally, alizarin red staining showed that HDAC6 inhibition promoted osteoblastic differentiation of VICs, which could be suppressed by tauroursodeoxycholic acid. CONCLUSIONS: HDAC6 inhibition promotes AoV calcification via an endoplasmic reticulum stress/ATF4-mediated osteogenic pathway. HDAC6 may be a novel target for AoV calcification prevention and treatment.

CD39 and CD73 in the aortic valve-biochemical and immunohistochemical analysis in valve cell populations and its changes in valve mineralization.

BACKGROUND: The calcific aortic valve disease (CAVD) is a common heart pathology that involves inflammation, fibrosis, and calcification of aortic valve leaflets. All these processes could be affected by changes in the extracellular purinergic signaling that depend on the activity of ectonucleotidases, mainly ectonucleoside triphosphate diphosphohydrolase 1 (CD39, eNTPD1) and ecto-5'nucleotidase (CD73, e5NT). OBJECTIVE AND METHODS: We investigated the localization of CD39 and CD73 proteins in human noncalcified and calcified aortic valves using immunohistochemistry together with analysis of NTPDases and e5NT activities in aortic valve homogenates by analysis of substrate into product conversion by high-performance liquid chromatography. We also measured the rates of extracellular nucleotide catabolism on the surface of isolated cultured aortic valve endothelial (hAVECs) and interstitial cells (hAVICs) as well as characterized cellular CD39 and CD73 distribution. RESULTS: In noncalcified valves, CD39 and CD73 were expressed in both endothelial and interstitial cells, while in calcified valves, the expressions of CD39 and CD73 were significantly down-regulated with the exception of calcified regions where the expression of CD73 was maintained. This correlated with activities in valve homogenates. NTPDase was reduced by 35% and e5NT activity by 50% in calcified vs. noncalcified valve. CD39 and CD73 were present mainly in the cell membrane of hAVECs, but in hAVICs, these proteins were also present intracellularly. The rates of extracellular adenosine triphosphate and adenosine monophosphate hydrolysis in isolated hAVECs and hAVICs were comparable. CONCLUSION: The presence of ectonucleotidases in valves and especially in aortic valve interstitial cells highlights important local role of purinergic signaling and metabolism. Changes in the local expression and hence the activity of CD39 and CD73 in calcified valves suggest their potential role in CAVD.

DNA methylation of a PLPP3 MIR transposon-based enhancer promotes an osteogenic programme in calcific aortic valve disease.

Aims: Calcific aortic valve disease (CAVD) is characterized by the osteogenic transition of valve interstitial cells (VICs). In CAVD, lysophosphatidic acid (LysoPA), a lipid mediator with potent osteogenic activity, is produced in the aortic valve (AV) and is degraded by membrane-associated phospholipid phosphatases (PLPPs). We thus hypothesized that a dysregulation of PLPPs could participate to the osteogenic reprograming of VICs during CAVD. Methods and results: The expression of PLPPs was examined in human control and mineralized AVs and comprehensive analyses were performed to document the gene regulation and impact of PLPPs on the osteogenic transition of VICs. We found that PLPP3 gene and enzymatic activity were downregulated in mineralized AVs. Multidimensional gene profiling in 21 human AVs showed that expression of PLPP3 was inversely correlated with the level of 5-methylcytosine (5meC) located in an intronic mammalian interspersed repeat (MIR) element. Bisulphite pyrosequencing in a larger series of 67 AVs confirmed that 5meC in intron 1 was increased by 2.2-fold in CAVD compared with control AVs. In isolated cells, epigenome editing with clustered regularly interspersed short palindromic repeats-Cas9 system containing a deficient Cas9 fused with DNA methyltransferase (dCas9-DNMT) was used to increase 5meC in the intronic enhancer and showed that it reduced significantly the expression of PLPP3. Knockdown experiments showed that lower expression of PLPP3 in VICs promotes an osteogenic programme. Conclusions: DNA methylation of a MIR-based enhancer downregulates the expression of PLPP3 and promotes the mineralization of the AV.

Interleukin-32 plays an essential role in human calcified aortic valve cells.

Interleukin-32 (IL-32) is an inflammatory cytokine produced mainly by T, natural killer, and epithelial cells. Previous studies on IL-32 have primarily investigated its proinflammatory properties. The IL-32 also has been described as an activator of the p38 mitogen-activated protein kinase (MAPK) and NF-κB, and induces several cytokines. In this study, we hypothesized that the inflammatory regulators NF-κB, MAP kinase, STAT1, and STAT3 are associated with the expression of the IL-32 protein in human calcified aortic valve cells. This study comprised aortic valve sclerotic patients and control group patients without calcified aortic valve. Increased IL-32 expression in calcified aortic valvular tissue was shown by immunohistochemical staining and western blotting. There was an increase in NF-κB p65 level, p-ERK, p-JNK, and p-p38 MAPK activation underlying IL-32 expression in the study. The level of p-STAT3 but not p-STAT1 was found to be increased in calcified aortic valve tissue. In cultured primary human aortic valve interstitial cells, inhibition of NF-κB or MAPK kinase pathways results in a decrease of IL-32 expression. Treatment of recombinant IL-32 induced the levels of TNF-α, IL-6, IL-1ß, and IL-8. Our findings demonstrate that IL-32 may be an important pro-inflammatory molecule involved in calcific aortic valve disease.

ADAMTS5 Deficiency in Calcified Aortic Valves Is Associated With Elevated Pro-Osteogenic Activity in Valvular Interstitial Cells.

OBJECTIVE: Extracellular matrix proteinases are implicated in the pathogenesis of calcific aortic valve disease. The ADAMTS5 (a disintegrin and metalloproteinase with thrombospondin motifs 5) enzyme is secreted, matrix-associated metalloendopeptidase, capable of degrading extracellular matrix proteins, particularly matrilin 2. We sought to determine the role of the ADAMTS5/matrilin 2 axis in mediating the phenotype transition of valvular interstitial cells (VICs) associated with calcific aortic valve disease. APPROACH AND RESULTS: Levels of ADAMTS5, matrilin 2, and α-SMA (α-smooth muscle actin) were evaluated in calcified and normal human aortic valve tissues and VICs. Calcified aortic valves have reduced levels of ADAMTS5 and higher levels of matrilin 2 and α-SMA. Treatment of normal VICs with soluble matrilin 2 caused an increase in α-SMA level through Toll-like receptors 2 and 4, which was accompanied by upregulation of runt-related transcription factor 2 and alkaline phosphatase. In addition, ADAMTS5 knockdown in normal VICs enhanced the effect of matrilin 2. Matrilin 2 activated nuclear factor (NF) κB and NF of activated T cells complex 1 and induced the interaction of these 2 NFs. Inhibition of either NF-κB or NF of activated T cells complex 1 suppressed matrilin 2's effect on VIC phenotype change. Knockdown of α-SMA reduced and overexpression of α-SMA enhanced the expression of pro-osteogenic factors and calcium deposit formation in human VICs. CONCLUSIONS: Matrilin 2 induces myofibroblastic transition and elevates pro-osteogenic activity in human VICs via activation of NF-κB and NF of activated T cells complex 1. Myofibroblastic transition in human VICs is an important mechanism of elevating the pro-osteogenic activity. Matrilin 2 accumulation associated with relative ADAMTS5 deficiency may contribute to the mechanism underlying calcific aortic valve disease progression.

Effects of Raloxifene on the Proliferation and Apoptosis of Human Aortic Valve Interstitial Cells.

We aimed to explore the effects of raloxifene (RAL) on the proliferation and apoptosis of human aortic valve interstitial cells (AVICs). Different concentrations of RAL were used to act on AVICs. MTS kit is used to test the effects of different concentrations of RAL on the proliferation of AVICs. Cell cycle and apoptosis test used flow cytometry after seven-day treatment. The relative expression levels of caspase-3 and caspase-8 are tested with RT-qPCR and Western blot. The results of MTS testing revealed that the absorbance value (OD value) of the cells in the concentration groups of 10 and 100 nmol/L RAL at a wavelength of 490 nm at five, seven, and nine days significantly decreased compared with that in the control group. Meanwhile, the results of flow cytometry of the cells collected after seven days showed that the ratio of the S stage and the cell apoptosis rate of AVICs can be significantly reduced by RAL in the concentration groups of 10 and 100 nmol/L. The mRNA and protein expressions of caspase-3 and caspase-8 were significantly decreased compared with those in the control group. This study laid the foundation for further treatment of aortic valve disease by using RAL.

Matrix metalloproteinase inhibitor, doxycycline and progression of calcific aortic valve disease in hyperlipidemic mice.

Calcific aortic valve disease (CAVD) is the most common cause of aortic stenosis. Currently, there is no non-invasive medical therapy for CAVD. Matrix metalloproteinases (MMPs) are upregulated in CAVD and play a role in its pathogenesis. Here, we evaluated the effect of doxycycline, a nonselective MMP inhibitor on CAVD progression in the mouse. Apolipoprotein (apo)E(-/-) mice (n = 20) were fed a Western diet (WD) to induce CAVD. After 3 months, half of the animals was treated with doxycycline, while the others continued WD alone. After 6 months, we evaluated the effect of doxycycline on CAVD progression by echocardiography, MMP-targeted micro single photon emission computed tomography (SPECT)/computed tomography (CT), and tissue analysis. Despite therapeutic blood levels, doxycycline had no significant effect on MMP activation, aortic valve leaflet separation or flow velocity. This lack of effect on in vivo images was confirmed on tissue analysis which showed a similar level of aortic valve gelatinase activity, and inflammation between the two groups of animals. In conclusion, doxycycline (100 mg/kg/day) had no effect on CAVD progression in apoE(-/-) mice with early disease. Studies with more potent and specific inhibitors are needed to establish any potential role of MMP inhibition in CAVD development and progression.

Deficiency in the anti-aging gene Klotho promotes aortic valve fibrosis through AMPKα-mediated activation of RUNX2.

Fibrotic aortic valve disease (FAVD) is an important cause of aortic stenosis, yet currently there is no effective treatment for FAVD due to its unknown etiology. The purpose of this study was to investigate whether deficiency in the anti-aging Klotho gene (KL) promotes high-fat-diet-induced FAVD and to explore the underlying molecular mechanism. Heterozygous Klotho-deficient (KL(+/-) ) mice and WT littermates were fed with a high-fat diet (HFD) or normal diet for 13 weeks, followed by treatment with the AMPKα activator (AICAR) for an additional 2 weeks. A HFD caused a greater increase in collagen levels in the aortic valves of KL(+/-) mice than of WT mice, indicating that Klotho deficiency promotes HFD-induced aortic valve fibrosis (AVF). AMPKα activity (pAMPKα) was decreased, while protein expression of collagen I and RUNX2 was increased in the aortic valves of KL(+/-) mice fed with a HFD. Treatment with AICAR markedly attenuated HFD-induced AVF in KL(+/-) mice. AICAR not only abolished the downregulation of pAMPKα but also eliminated the upregulation of collagen I and RUNX2 in the aortic valves of KL(+/-) mice fed with HFD. In cultured porcine aortic valve interstitial cells, Klotho-deficient serum plus cholesterol increased RUNX2 and collagen I protein expression, which were attenuated by activation of AMPKα by AICAR. Interestingly, silencing of RUNX2 abolished the stimulatory effect of Klotho deficiency on cholesterol-induced upregulation of matrix proteins, including collagen I and osteocalcin. In conclusion, Klotho gene deficiency promotes HFD-induced fibrosis in aortic valves, likely through the AMPKα-RUNX2 pathway.

Challenges in developing a reseeded, tissue-engineered aortic valve prosthesis.

OBJECTIVES: Biological heart valve prostheses are characterized by a limited durability due to the degenerative processes after implantation. Tissue engineering may provide new approaches in the development of optimized valvular grafts. While re-endothelialization of decellularized heart valves has already been successfully implemented, interstitial repopulation still remains an unaccomplished objective although it is essential for valvular functionality and regeneration potential. The aim of this study was to compare different concepts for an improved in vitro interstitial repopulation of decellularized heart valves. A novel 3D heart valve model has been developed to investigate the cell behaviour of valvular interstitial cells (VIC) in their physiological environment and to evaluate the potential of in vitro repopulation of acellular heart valves. METHODS: Ovine aortic heart valves were decellularized by detergent solutions and additionally treated with trypsin or laser perforation. Subsequently, the decellularized extracellular matrices (dECM) were reseeded with ovine VIC using reseeding devices to provide a repopulation of the matrix on a defined area under controlled conditions. After an initial attachment of the VIC, reseeded dECM were transferred into a transwell system to improve the nutrient supply inside the valvular matrix. Cell migration and expression of cell markers were analysed histologically. The results were compared with VIC cultivation in a biological scaffold. RESULTS: VIC did not migrate into the matrix of untreated dECM and reseeding in laser perforated dECM showed inconsistent results. However, trypsinization increased the susceptibility of the valvular cusps to VIC penetration and repopulation of superficial areas. Additionally, the cultivation of reseeded dECM in a transwell system significantly increased the total number of cells repopulating the valvular matrix and their mean migration distance, representing the best repopulation results. Immunohistological analysis and in situ zymography revealed a low activation status of repopulating VIC after 7 days of culture. CONCLUSIONS: A comparison of different techniques for increasing interstitial repopulation of detergent dECM revealed that an additional limited trypsin treatment was most effective. Nevertheless, a complete interstitial repopulation of decellularized heart valves remains a challenging endeavour. Additional experimental fine-tuning may improve the in vitro results of heart valve tissue engineering.

Nucleotide Catabolism on the Surface of Aortic Valve Xenografts; Effects of Different Decellularization Strategies.

Extracellular nucleotide metabolism controls thrombosis and inflammation and may affect degeneration and calcification of aortic valve prostheses. We evaluated the effect of different decellularization strategies on enzyme activities involved in extracellular nucleotide metabolism. Porcine valves were tested intact or decellularized either by detergent treatment or hypotonic lysis and nuclease digestion. The rates of ATP hydrolysis, AMP hydrolysis, and adenosine deamination were estimated by incubation of aorta or valve leaflet sections with substrates followed by HPLC analysis. We demonstrated relatively high activities of ecto-enzymes on porcine valve as compared to the aortic wall. Hypotonic lysis/nuclease digestion preserved >80 % of ATP and AMP hydrolytic activity but reduced adenosine deamination to <10 %. Detergent decellularization completely removed (<5 %) all these activities. These results demonstrate high intensity of extracellular nucleotide metabolism on valve surface and indicate that various valve decellularization techniques differently affect ecto-enzyme activities that could be important in the development of improved valve prostheses.

Enzymatically Modified Low-Density Lipoprotein Is Present in All Stages of Aortic Valve Sclerosis: Implications for Pathogenesis of the Disease.

BACKGROUND: We have demonstrated previously that enzymatically degraded low-density lipoprotein (eLDL) is an essential causative component for the initiation of atherosclerosis. Here, we investigated the different stages of human aortic valve sclerosis for the presence of eLDL and effectors of the innate immune system, as well as the interaction of eLDL with isolated valvular interstitial cells/myofibroblasts to discover possible pathways leading to aortic valve sclerosis. METHODS AND RESULTS: Human aortic valvular tissue was obtained from 68 patients undergoing valve replacement surgery. Patients were classified into 3 groups (mild, moderate, or severe aortic valve sclerosis), and clinical data for statistical analysis were gathered from all patients. Immunohistochemical staining demonstrated extensive extracellular deposits of eLDL throughout all grades of aortic valve sclerosis. Complementary analysis of lipid composition revealed higher concentrations of the decisive components of eLDL (ie, unesterified cholesterol and linoleic acid) compared with internal control tissues. Further, the complement component C3d and terminal complement complexes colocalized with eLDL compatible with the proposal that subendothelially deposited eLDL is enzymatically transformed into a complement activator at early stages of valvular cusp lesion development. Gene expression profiles of proteases and complement components corroborated by immunohistochemistry demonstrated an upregulation of the protease cathepsin D (a possible candidate for LDL degradation to eLDL) and the complement inhibitor CD55. Surprisingly, substantial C-reactive protein expression was not observed before grade 2 aortic valve sclerosis as investigated with microarray analysis, reverse transcription-polymerase chain reaction analysis, and immunohistochemistry. Finally, we demonstrated cellular uptake of eLDL by valvular interstitial cells/myofibroblasts. CONCLUSIONS: The present study is a startup of a hypothesis on the pathogenesis of aortic valve sclerosis declaring extracellular lipoprotein modification, subsequent complement activation, and cellular uptake by valvular interstitial cells/myofibroblasts as integral players.

Multimodality and molecular imaging of matrix metalloproteinase activation in calcific aortic valve disease.

UNLABELLED: Calcific aortic valve disease (CAVD) is the most common cause of aortic stenosis. Matrix metalloproteinases (MMPs) are upregulated in CAVD and contribute to valvular remodeling and calcification. We investigated the feasibility and correlates of MMP-targeted molecular imaging for detection of valvular biology in CAVD. METHODS: Apolipoprotein E-deficient (apoE(-/-)) mice were fed a Western diet (WD) for 3, 6, and 9 mo (n = 108) to induce CAVD. Wild-type mice served as the control group (n = 24). The development of CAVD was tracked with CT, echocardiography, MMP-targeted small-animal SPECT imaging using (99m)Tc-RP805, and histologic analysis. RESULTS: Key features of CAVD­leaflet thickening and valvular calcification­were noted after 6 mo of WD and were more pronounced after 9 mo. These findings were associated with a significant reduction in aortic valve leaflet separation and a significant increase in transaortic valve flow velocity. On in vivo SPECT/CT images, MMP signal in the aortic valve area was significantly higher at 6 mo in WD mice than in control mice and decreased thereafter. The specificity of the signal was demonstrated by blocking, using an excess of nonlabeled precursor. Similar to MMP signal, MMP activity as determined by in situ zymography and valvular inflammation by CD68 staining were maximal at 6 mo. In vivo (99m)Tc-RP805 uptake correlated significantly with MMP activity (R(2) = 0.94, P < 0.05) and CD68 expression (R(2) = 0.98, P < 0.01) in CAVD. CONCLUSION: MMP-targeted imaging detected valvular inflammation and remodeling in a murine model of CAVD. If this ability is confirmed in humans, the technique may provide a tool for tracking the effect of emerging medical therapeutic interventions and for predicting outcome in CAVD.