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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., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Currently, aortic valve replacement is the only treatment capable of relieving left ventricle pressure overload in patients with severe aortic stenosis. It aims to improve cardiac function and revert hypertrophy, by triggering myocardial reverse remodeling. Despite immediately relieving afterload, reverse remodeling turns out to be extremely variable. Among other factors, the extent of reverse remodeling may depend on how well ubiquitin-proteasome system tackle hypertrophy. Therefore, we assessed tagged ubiquitin and ubiquitin ligases in the left ventricle collected from patients undergoing valve replacement and tested their association to the degree of reverse remodeling. Patients were classified according to the regression of left ventricle mass (ΔLVM) and assigned to complete (ΔLVM≥15%) or incomplete (ΔLVM≤5%) reverse remodeling groups. No direct inter-group differences were observed. Nevertheless, correlation analysis supports a fundamental role of the ubiquitin-proteasome system during reverse remodeling. Indeed, total protein ubiquitination was associated to hypertrophic indexes such as interventricular septal thickness (r = 0.55, p = 0.03) and posterior wall thickness (r = 0.65, p = 0.009). No significant correlations were observed for Muscle Ring Finger 3. Surprisingly, though, higher levels of atrogin-1 were associated to postoperative interventricular septal thickness (r = 0.71, p = 0.005). In turn, Muscle Ring Finger 1 correlated negatively with this postoperative hypertrophy marker (r = -0.68, p = 0.005), suggesting a cardioprotective role during reverse remodeling. No significant correlations were found with left ventricle mass regression, although a trend for a negative association between the ligase Murine Double Minute 2 and mass regression (r = -0.44, p = 0.10) was found. Animal studies will be necessary to understand whether this ligase is protective or detrimental. Herein, we show, for the first time, an association between the preoperative myocardial levels of ubiquitin ligases and postoperative hypertrophy, highlighting the therapeutic potential of targeting ubiquitin ligases in incomplete reverse remodeling., Competing Interests: The authors have declared that no competing interests exist.

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., Competing Interests: Competing interests: BA has received research funding from Pfizer, Merck and Ionis Pharmaceuticals and is a consultant for Novartis. PM is a consultant for Casebia Therapeutics., (© Author(s) (or their employer(s)) 2020. No commercial re-use. See rights and permissions. Published by BMJ.)

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., (Copyright © 2020 The Society of Thoracic Surgeons. Published by Elsevier Inc. All rights reserved.)

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; r S =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-1β (r S =0.5045, P <0.001) and BMP (bone morphogenetic protein)-2 (r S =0.5003, P <0.01). In vitro, recombinant human MMP-10 induced the overexpression of inflammatory, fibrotic, and osteogenic markers (interleukin-1β, 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.

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/cm 2 ) and adenosine production (1.32 ± 0.12 vs. 2.49 ± 0.28 nmol/min/cm 2 ) as well as increased adenosine deamination (1.28 ± 0.31 vs. 0.67 ± 0.11 nmol/min/cm 2 ). 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. .

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., Competing Interests: The authors declare that there are no conflicts of interest regarding the publication of this paper., (Copyright © 2020 Nathalie Mercier et al.)

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., (Copyright © 2018 The American Association for Thoracic Surgery. Published by Elsevier Inc. All rights reserved.)

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.

The beneficial effects of exercise training (EX) on cardiac pathology are well recognized. Previously, we found that the effects of EX on cardiac dysfunction in mice critically depend on the underlying etiology. EX exerted beneficial effects after myocardial infarction (MI); however, cardiac pathology following pressure overload produced by transverse aortic constriction (TAC) was aggravated by EX. In the presented study, we investigated whether the contrasting effects of EX on cardiac dysfunction can be explained by an etiology-specific response of endothelial nitric oxide (NO) synthase (eNOS) to EX, which divergently affects the balance between nitric oxide and superoxide. For this purpose, mice were exposed to eight weeks of voluntary wheel running or sedentary housing (SED), immediately after sham, MI, or TAC surgery. Left ventricular (LV) function was assessed using echocardiography and hemodynamic measurements. EX ameliorated LV dysfunction and remodeling after MI, but not following TAC, in which EX even aggravated fibrosis. Strikingly, EX attenuated superoxide levels after MI, but exacerbated NOS-dependent superoxide levels following TAC. Similarly, elevated eNOS S-glutathionylation and eNOS monomerization, which were observed in both MI and TAC, were corrected by EX in MI, but aggravated by EX after TAC. Additionally, EX reduced antioxidant activity in TAC, while it was maintained following EX in MI. In conclusion, the present study shows that EX mitigates cardiac dysfunction after MI, likely by attenuating eNOS uncoupling-mediated oxidative stress, whereas EX tends to aggravate cardiac dysfunction following TAC, likely due to exacerbating eNOS-mediated oxidative stress.

Aortic valve stenosis (AVS) is the most common valvular heart disease in the Western world. Therapy based on apolipoprotein A-I (apoA-I), the major protein component of high-density lipoproteins, results in AVS regression in experimental models. Nevertheless, apoA-I degradation by proteases might lead to suboptimal efficacy of such therapy. An activatable probe using a quenched fluorescently labeled full-length apoA-I protein was generated to assess apoA-I-degrading protease activity in plasma derived from 44 men and 20 women with severe AVS (age 65.0 ± 10.4 years) as well as from a rabbit model of AVS. In human and rabbit AVS plasma, apoA-I-degrading protease activity was significantly higher than in controls (humans: 0.038 ± 0.009 vs 0.022 ± 0.005 RFU/s, p < 0.0001; rabbits: 0.033 ± 0.016 vs 0.017 ± 0.005 RFU/s, p = 0.041). Through the use of protease inhibitors, we identified metalloproteinases (MMP) as exerting the most potent proteolytic effect on apoA-I in AVS rabbits (67%, p < 0.05 vs control), while the cysteine protease cathepsin S accounted for 54.2% of apoA-I degradation in human plasma (p < 0.05 vs control) with the maximum effect seen in women (68.8%, p < 0.05 vs men). Accordingly, cathepsin S activity correlated significantly with mean transaortic pressure gradient in women (r = 0.5, p = 0.04) but not in men (r = - 0.09, p = 0.60), and was a significant independent predictor of disease severity in women (standardized beta coefficient 0.832, p < 0.001) when tested in a linear regression analysis. ApoA-I proteolysis is increased in AVS. Targeting circulating cathepsin S may lead to new therapies for human aortic valve disease.

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-1β, and IL-8. Our findings demonstrate that IL-32 may be an important pro-inflammatory molecule involved in calcific aortic valve disease.

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., (© 2017 American Heart Association, Inc.)

Histoenzymological methods were used to study metabolism of smooth muscle cells of intramural myocardial arteries during experimental aortic or pulmonary artery stenosis. Aortic stenosis was accompanied by changes in smooth muscles of the left ventricle manifested by deceleration of tricarboxylic acid cycle, inhibition of oxidation of free fatty acids and their metabolites, flux redistribution in the glycolytic cascade, and inhibition of shuttle systems and biosynthetic processes. Similar metabolic alterations were observed in vessels of the ventricular septum, but they were not revealed in vessels of the right ventricle (except glycolysis stimulation). Under conditions of pulmonary artery stenosis, histoenzymological alterations in vascular smooth muscle of both ventricles and ventricular septum were similar, which attested to acceleration of tricarboxylic acid cycle, stimulation of oxidation of the free fatty acids with their metabolites, acceleration of glycolysis, and activation of the shuttle systems and biosynthetic processes. Comparative analysis of histoenzymological alterations revealed substantial differences in the character of metabolic changes under conditions of increased left and right ventricular afterload, which can be caused by peculiarities in myocardial blood flow, severity of circulatory disorders, severity of hypoxia, and intensity of processes maintaining ionic homeostasis in vascular smooth muscles and transport across the histohematic barriers. The data attest to important metabolic role of glycolysis in vascular smooth muscles of the myocardium, especially under conditions of enhanced afterload of the right ventricle.

Background and Aim of the Study: Calcified aortic valve disease (CAVD) is an actively regulated disease that shares pathophysiological hallmarks with atherosclerosis. One of these common features is extracellular matrix (ECM) remodeling, which consists of a dynamic degradation and deposition of the ECM composition. Granzymes (Grs) are ECM- degrading and pro-apoptotic proteases that have been detected in atherosclerotic lesions, but their role in CAVD remains unknown., Methods: The expression of granzymes and perforin was characterized in heavily stenotic valves (n = 20) and control valves (n = 6) using quantitative RT-PCR and immunohistochemistry., Results: Quantitative RT-PCR revealed that levels of granzymes A, B, H, K and M mRNA were 4.9-fold (p < 0.001), 7.1-fold (p < 0.001), 4.6-fold (p < 0.001), 4.7-fold (p < 0.001) and 2.8-fold (p = 0.069) higher, respectively, in stenotic aortic valves than in control valves. Perforin mRNA levels were 3.6-fold (p < 0.001) higher in stenotic valves than in control valves. Granzyme A immunohistochemical positivity was observed in mast cells and lymphocytes, granzyme H in mast cells but not in lymphocytes, and granzyme K in lymphocytes but not in mast cells. A statistical analysis was also performed to investigate the effect of statin treatment on granzyme expression, but no differences were found when compared to non-statin-treated patients., Conclusions: The data acquired showed that CAVD is characterized by an increased expression of granzymes A, B, H, K, and perforin.

Histoenzymological changes, indicating inhibition of the main metabolic processes, were found in the conduction cardiomyocytes of the left ventricle and ventricular septum in experimental stenosis of the aorta. The histoenzymological changes in the conduction system of both ventricles and ventricular septum were similar in experimental stenosis of the pulmonary artery and indicated primarily activation of glycolysis. The histoenzymological profile of conduction cardiomyocytes differed little in cases when the increase of the pressure load was complicated or not complicated by the development of heart failure, particularly in pulmonary artery stenosis. The histoenzymological changes in the conduction system in response to increased afterload differed significantly from those in the contractile myocardium and correlated with the level of cellular functional activity and sensitivity to the regulatory and alterative exposure. These data attest to minor role of metabolic shifts in conduction cell injuries with increasing afterload, primarily, of the right ventricle.

The NADPH oxidases are a key family of ROS (reactive oxygen species)-producing enzymes which may differentially contribute to cardiac pathophysiology. Animal studies show uncertain results regarding the regulation of cardiac Nox4 by pressure overload and no data are available on human myocardial Nox4. In the present study, we evaluated Nox4 expression and its relationship with myocardial remodelling and LV (left ventricular) function in patients with severe AS (aortic valve stenosis). Endomyocardial biopsies from 34 patients with AS were obtained during aortic valve replacement surgery. LV morphology and function were assessed by echocardiography. Myocardial samples from subjects deceased of non-CVDs (cardiovascular diseases) were analysed as controls. Nox4 localization was evaluated by immunohistochemistry and quantified by Western blot. Myocardial capillary density, fibrosis and cardiomyocyte dimensions and apoptosis were assessed histologically to evaluate myocardial remodelling. Nox4 was present in samples from all subjects and expressed in cardiomyocytes, VSMCs (vascular smooth muscle cells), endothelium and fibroblasts. Nox4 levels were reduced 5-fold in AS patients compared with controls (P<0.01). Nox4 levels directly correlated with cardiomyocyte cross-sectional area (r=0.299, P<0.05) and diameter (r=0.406, P<0.05) and capillary density (r=0.389, P<0.05), and inversely with cardiomyocyte apoptosis (r=-0.316, P<0.05) in AS patients. In addition, Nox4 levels correlated with echocardiographic parameters (LV ejection fraction: r=0.353, P<0.05; midwall fractional shortening: r=0.355, P<0.05; deceleration time: r=-0.345, P<0.05) in AS patients. Nox4 is expressed in human myocardium and reduced in AS patients. The observed associations of Nox4 with cardiomyocyte parameters and capillary density in AS patients suggest a potential role of Nox4 deficiency in the myocardial remodelling present in the human pressure-overloaded heart.

Background and Aim of the Study: Currently, there is an increased incidence of aortic valve stenosis (AS) in patients undergoing hemodialysis (HD), though the exact mechanisms are not fully understood. Myeloperoxidase (MPO) is a leukocyte-derived enzyme that catalyzes the formation of reactive oxygen species and is an index of oxidative stress. The study aim was to examine, immunohistochemically, the expression of MPO, using surgically resected aortic valve specimens from AS patients undergoing HD., Methods: The study population consisted of 15 HD patients and 19 non-HD patients with severe AS undergoing aortic valve replacement. Frozen aortic valve samples obtained surgically from AS patients were stained immunohistochemically with antibodies against smooth muscle cells, neutrophils, macrophages, T lymphocytes, CD31, MPO and 4-hydroxy-2-nonenal (4-HNE)., Results: Quantitative analyses showed that the macrophage-positive area, and numbers of T lymphocytes, neutrophils, CD31-positive microvessels and MPO-positive cells in HD patients were significantly higher than in non-HD patients (macrophages, p < 0.0001; T lymphocytes, p < 0.0001; neutrophils, p < 0.0001; CD31, p < 0.0001; MPO, p < 0.0001). Moreover, the number of MPO-positive cells was positively correlated with CD31-positive microvessels and the 4-HNE-positive macrophage score (CD31, R = 0.73, p < 0.0001; 4-HNE, R = 0.49; p < 0.005)., Conclusion: These findings suggest that MPO is highly expressed in the aortic valves of AS patients undergoing HD. Furthermore, MPO is positively associated with neovascularization and oxidative stress, which contribute to a rapid progression of AS in HD patients.

Maladaptive cardiac hypertrophy predisposes one to arrhythmia and sudden death. Cytochrome P450 (CYP)-derived epoxyeicosatrienoic acids (EETs) promote anti-inflammatory and antiapoptotic mechanisms, and are involved in the regulation of cardiac Ca(2+)-, K(+)- and Na(+)-channels. To test the hypothesis that enhanced cardiac EET biosynthesis counteracts hypertrophy-induced electrical remodeling, male transgenic mice with cardiomyocyte-specific overexpression of the human epoxygenase CYP2J2 (CYP2J2-TG) and wildtype littermates (WT) were subjected to chronic pressure overload (transverse aortic constriction, TAC) or β-adrenergic stimulation (isoproterenol infusion, ISO). TAC caused progressive mortality that was higher in WT (42% over 8 weeks after TAC), compared to CYP2J2-TG mice (6%). In vivo electrophysiological studies, 4 weeks after TAC, revealed high ventricular tachyarrhythmia inducibility in WT (47% of the stimulation protocols), but not in CYP2J2-TG mice (0%). CYP2J2 overexpression also enhanced ventricular refractoriness and protected against TAC-induced QRS prolongation and delocalization of left ventricular connexin-43. ISO for 14 days induced high vulnerability for atrial fibrillation in WT mice (54%) that was reduced in CYP-TG mice (17%). CYP2J2 overexpression also protected against ISO-induced reduction of atrial refractoriness and development of atrial fibrosis. In contrast to these profound effects on electrical remodeling, CYP2J2 overexpression only moderately reduced TAC-induced cardiac hypertrophy and did not affect the hypertrophic response to β-adrenergic stimulation. These results demonstrate that enhanced cardiac EET biosynthesis protects against electrical remodeling, ventricular tachyarrhythmia, and atrial fibrillation susceptibility during maladaptive cardiac hypertrophy.

Objective: Calcific aortic valve disease is a leading cardiovascular disease in the elderly, and progressive calcification results in the failure of valvular function. Aortic valve interstitial cells (AVICs) from stenotic valves express higher levels of bone morphogenetic protein-2 in response to Toll-like receptor 4 stimulation. We recently found that Toll-like receptor 4 interacts with Notch1 in human AVICs. This study tests the hypothesis that Notch1 promotes the pro-osteogenic response of human AVICs., Approach and Results: AVICs isolated from diseased human valves expressed higher levels of bone morphogenetic protein-2 and alkaline phosphatase after lipopolysaccharide stimulation. The augmented pro-osteogenic response is associated with elevated cellular levels of Notch1 and enhanced Notch1 cleavage in response to lipopolysaccharide stimulation. Inhibition or silencing of Notch1 suppressed the pro-osteogenic response in diseased cells, and the Notch 1 ligand, Jagged1, enhanced the response in AVICs isolated from normal human valves. Interestingly, extracellular signal-regulated protein kinases 1/2 (ERK1/2) and nuclear factor-κB phosphorylation induced by lipopolysaccharide was markedly reduced by inhibition or silencing of Notch1 and enhanced by Jagged1. Inhibition of ERK1/2 or nuclear factor-κB also reduced bone morphogenetic protein-2 and alkaline phosphatase expression induced by lipopolysaccharide., Conclusions: Notch1 mediates the pro-osteogenic response to Toll-like receptor 4 stimulation in human AVICs. Elevated Notch1 levels and enhanced Notch1 activation play a major role in augmentation of the pro-osteogenic response of AVICs of stenotic valves through modulation of ERK1/2 and nuclear factor-κB activation. These pathways could be potential therapeutic targets for prevention of the progression of calcific aortic valve disease.

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., (© 2013 Stichting European Society for Clinical Investigation Journal Foundation. Published by John Wiley & Sons Ltd.)

Arterial endothelial cells maintain vascular homeostasis and vessel tone in part through the secretion of nitric oxide (NO). In this study, we determined how aortic valve endothelial cells (VEC) regulate aortic valve interstitial cell (VIC) phenotype and matrix calcification through NO. Using an anchored in vitro collagen hydrogel culture system, we demonstrate that three-dimensionally cultured porcine VIC do not calcify in osteogenic medium unless under mechanical stress. Co-culture with porcine VEC, however, significantly attenuated VIC calcification through inhibition of myofibroblastic activation, osteogenic differentiation, and calcium deposition. Incubation with the NO donor DETA-NO inhibited VIC osteogenic differentiation and matrix calcification, whereas incubation with the NO blocker l-NAME augmented calcification even in 3D VIC-VEC co-culture. Aortic VEC, but not VIC, expressed endothelial NO synthase (eNOS) in both porcine and human valves, which was reduced in osteogenic medium. eNOS expression was reduced in calcified human aortic valves in a side-specific manner. Porcine leaflets exposed to the soluble guanylyl cyclase inhibitor ODQ increased osteocalcin and α-smooth muscle actin expression. Finally, side-specific shear stress applied to porcine aortic valve leaflet endothelial surfaces increased cGMP production in VEC. Valve endothelial-derived NO is a natural inhibitor of the early phases of valve calcification and therefore may be an important regulator of valve homeostasis and pathology., (Copyright © 2013 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Extracellular signal-regulated kinases 1 and 2 (ERK1/2) are central mediators of cardiac hypertrophy and are discussed as potential therapeutic targets. However, direct inhibition of ERK1/2 leads to exacerbated cardiomyocyte death and impaired heart function. We have previously identified ERK(Thr188) autophosphorylation as a regulatory phosphorylation of ERK1/2 that is a key factor in cardiac hypertrophy. Here, we investigated whether interference with ERK(Thr188) phosphorylation permits the impairment of ERK1/2-mediated cardiac hypertrophy without increasing cardiomyocyte death. The impact of ERK(Thr188) phosphorylation on cardiomyocyte hypertrophy and cell survival was analyzed in isolated cells and in mice using the mutant ERK2(T188A), which is dominant-negative for ERK(Thr188) signaling. ERK2(T188A) efficiently attenuated cardiomyocyte hypertrophic responses to phenylephrine and to chronic pressure overload, but it affected neither antiapoptotic ERK1/2 signaling nor overall physiological cardiac function. In contrast to its inhibition of pathological hypertrophy, ERK2(T188A) did not interfere with physiological cardiac growth occurring with age or upon voluntary exercise. A preferential role of ERK(Thr188) phosphorylation in pathological types of hypertrophy was also seen in patients with aortic valve stenosis: ERK(Thr188) phosphorylation was increased 8.5 ± 1.3-fold in high-gradient, rapidly progressing cases (≥40 mmHg gradient), whereas in low-gradient, slowly progressing cases, the increase was not significant. Because interference with ERK(Thr188) phosphorylation (i) inhibits pathological hypertrophy and (ii) does not impair antiapoptotic ERK1/2 signaling and because ERK(Thr188) phosphorylation shows strong prevalence for aortic stenosis patients with rapidly progressing course, we conclude that interference with ERK(Thr188) phosphorylation offers the possibility to selectively address pathological types of cardiac hypertrophy.

On the basis of the role of small, leucine-rich proteoglycans (SLRPs) in fibrogenesis and inflammation, we hypothesized that they could be involved in cardiac remodeling and reverse remodeling as occurs during aortic stenosis and after aortic valve replacement. Thus, in a well-characterized aortic banding-debanding mouse model, we examined the SLRPs decorin and lumican and enzymes responsible for synthesis of their glycosaminoglycan (GAG) chains. Four weeks after banding of the ascending aorta, mice were subjected to a debanding operation (DB) and were subsequently followed for 3 or 14 days. Sham-operated mice served as controls. Western blotting revealed a 2.5-fold increase in the protein levels of glycosylated decorin in mice with left ventricular pressure overload after aortic banding (AB) with a gradual decrease after DB. Interestingly, protein levels of three key enzymes responsible for decorin GAG chain synthesis were also increased after AB, two of them gradually declining after DB. The inflammatory chemokine (C-X-C motif) ligand 16 (CXCL16) was increased after AB but was not significantly altered following DB. In cardiac fibroblasts CXCL16 increased the expression of the GAG-synthesizing enzyme chondroitin polymerizing factor (CHPF). The protein levels of lumican core protein with N-linked oligosaccharides increased by sevenfold after AB and decreased again 14 days after DB. Lumican with keratan sulfate chains was not regulated. In conclusion, this study shows alterations in glycosylated decorin and lumican core protein that might be implicated in myocardial remodeling and reverse remodeling, with a potential important role for CS/DS GAG chain-synthesizing enzymes.

Background: Lipoprotein-associated phospholipase A2 (Lp-PLA2) is an inflammatory mediator involved in atherosclerosis. Since aortic valve stenosis (AVS) is regarded as an atherosclerosis-like inflammatory disease, we sought to investigate whether AVS is associated with elevated Lp-PLA2., Methods: Plasma Lp-PLA2 levels were determined in 48 consecutive patients with severe AVS without atherosclerotic vascular disease and compared with the values obtained in 48 controls matched for age, sex and cardiovascular risk factors., Results: Lp-PLA2 was higher in AVS than in controls (242.3±50.4 vs. 151.9±28.1 ng/mL, p<0.0001). Lp-PLA2 correlated inversely with aortic valve area (AVA) (r=-0.53; p=0.0001) and positively with mean pressure gradient (PG) (r=0.32; p=0.029). In multivariable analysis C-reactive protein (CRP) (OR=1.42; 95% CI 0.95-2.1; p=0.09) and AVA (OR=0.003; 95% CI 0.00004-0.23; p<0.01) were independently associated with Lp-PLA2 above a mean of 242 ng/mL. After adjustment for CRP, AVA was the only independent predictor of Lp-PLA2 in AVS patients (p<0.001)., Conclusions: This study is the first to show that AVS is characterized by increased plasma Lp-PLA2 levels associated with the severity of AVS, which suggests active involvement of Lp-PLA2 in the pathogenesis of AVS.

Calcific aortic valve disease is the most common heart valve disorder. So far, there is no medical treatment for calcific aortic valve disease. The expression of ectonucleotidases, which metabolize nucleotides into phosphate products, may influence the calcification of the aortic valve. In this study, we investigated if the administration of an ectonucleotidase inhibitor, ARL67156 (6-N,N-Diethyl-D-β,γ-dibromomethyleneATP trisodium salt), may prevent the calcification of the aortic valve in the warfarin-induced mineralization rat model. Male Wistar rats were treated with warfarin or warfarin+ARL67156 for 28 days. All rats had comprehensive Doppler-echocardiographic studies at 28 day. A gene profiling of ectonucleotidases expressed in aortas of rats was documented by quantitative real-time PCR. The amount of calcium was determined by quantitative method and von Kossa staining. Ex vivo cultures of rat aortas were also used to further assess the effect of ARL67156 on the calcifying process and Akt signaling. Mineralization of the aorta/aortic valve was documented in warfarin-treated rats and was accompanied by the development of aortic stenosis. These changes were paralleled by an increased of ectonucleotide pyrophosphatase/phosphodiesterase-1 (ENPP1). Administration of the ectonucleotidase inhibitor, ARL67156 prevented the development of aortic stenosis by lowering the level of apoptosis and mineralization of the aortic valve/aorta. In addition, ARL67156 normalized the level of pAkt, an important kinase involved in the survival pathway. Inhibition of ectonucleotidase activity prevented the development of calcific aortic valve disease in a rat model. On that account, ectonucleotidase may represent a novel target in the treatment of calcific aortic valve disease., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Background: Prominent features of myocardial remodeling in heart failure with preserved ejection fraction (HFPEF) are high cardiomyocyte resting tension (F(passive)) and cardiomyocyte hypertrophy. In experimental models, both reacted favorably to raised protein kinase G (PKG) activity. The present study assessed myocardial PKG activity, its downstream effects on cardiomyocyte F(passive) and cardiomyocyte diameter, and its upstream control by cyclic guanosine monophosphate (cGMP), nitrosative/oxidative stress, and brain natriuretic peptide (BNP). To discern altered control of myocardial remodeling by PKG, HFPEF was compared with aortic stenosis and HF with reduced EF (HFREF)., Methods and Results: Patients with HFPEF (n=36), AS (n=67), and HFREF (n=43) were free of coronary artery disease. More HFPEF patients were obese (P<0.05) or had diabetes mellitus (P<0.05). Left ventricular myocardial biopsies were procured transvascularly in HFPEF and HFREF and perioperatively in aortic stenosis. F(passive) was measured in cardiomyocytes before and after PKG administration. Myocardial homogenates were used for assessment of PKG activity, cGMP concentration, proBNP-108 expression, and nitrotyrosine expression, a measure of nitrosative/oxidative stress. Additional quantitative immunohistochemical analysis was performed for PKG activity and nitrotyrosine expression. Lower PKG activity in HFPEF than in aortic stenosis (P<0.01) or HFREF (P<0.001) was associated with higher cardiomyocyte F(passive) (P<0.001) and related to lower cGMP concentration (P<0.001) and higher nitrosative/oxidative stress (P<0.05). Higher F(passive) in HFPEF was corrected by in vitro PKG administration., Conclusions: Low myocardial PKG activity in HFPEF was associated with raised cardiomyocyte F(passive) and was related to increased myocardial nitrosative/oxidative stress. The latter was probably induced by the high prevalence in HFPEF of metabolic comorbidities. Correction of myocardial PKG activity could be a target for specific HFPEF treatment.

Oxidative stress may contribute to the hemodynamic progression of aortic valve stenosis, and is associated with activation of the nuclear enzyme poly(ADP-ribose) polymerase (PARP) 1. The aim of the present study was to assess the transcriptional profile and the topological distribution of PARP-1 in human aortic valves, and its relation to the stenosis severity. Human stenotic aortic valves were obtained from 46 patients undergoing aortic valve replacement surgery and used for mRNA extraction followed by quantitative real-time PCR to correlate the PARP-1 expression levels with the non invasive hemodynamic parameters quantifying the stenosis severity. Primary isolated valvular interstitial cells (VICs) were used to explore the effects of cytokines and leukotriene C(4) (LTC(4)) on valvular PARP-1 expression. The thickened areas of stenotic valves with tricuspid morphology expressed significantly higher levels of PARP-1 mRNA compared with the corresponding part of bicuspid valves (0.501 vs 0.243, P=0.01). Furthermore, the quantitative gene expression levels of PARP-1 were inversely correlated with the aortic valve area (AVA) (r=-0.46, P=0.0469) and AVA indexed for body surface area (BSA) (r=-0.498; P=0.0298) only in tricuspid aortic valves. LTC(4) (1nM) significantly elevated the mRNA levels of PARP-1 by 2.38-fold in VICs. Taken together, these data suggest that valvular DNA-damage pathways may be associated with inflammation and the stenosis severity in tricuspid aortic valves., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Degenerative aortic stenosis is the most common worldwide cause of valve replacement. While it shares certain risk factors with coronary artery disease, it is not delayed or reversed by reducing exposure to risk factors (e.g., therapies that lower lipids). Therefore, it is necessary to better understand its pathophysiology for preventive measures to be taken. In this work, aortic valve samples were collected from 20 patients that underwent aortic valve replacement (55% males, mean age of 74 years) and 20 normal control valves were obtained from necropsies (40% males, mean age of 69 years). The proteome of the samples was analyzed by quantitative differential electrophoresis (2D-DIGE) and mass spectrometry, and 35 protein species were clearly increased in aortic valves, including apolipoprotein AI, alpha-1-antitrypsin, serum albumin, lumican, alfa-1-glycoprotein, vimentin, superoxide dismutase Cu-Zn, serum amyloid P-component, glutathione S-transferase-P, fatty acid-binding protein, transthyretin, and fibrinogen gamma. By contrast, 8 protein species were decreased (transgelin, haptoglobin, glutathione peroxidase 3, HSP27, and calreticulin). All of the proteins identified play a significant role in cardiovascular processes, such as fibrosis, homeostasis, and coagulation. The significant changes observed in the abundance of key cardiovascular proteins strongly suggest that they can be involved in the pathogenesis of degenerative aortic stenosis. Further studies are warranted to better understand this process before we can attempt to modulate it.

Valvular heart diseases represent an important public health burden. With the decrease in the incidence of rheumatic heart disease, calcific aortic stenosis has now become the most common valvular disease in Western countries. Its prevalence increases with age, such that its affects about 4% of the elderly population and it is the most common motive for valve replacement. Several tissue abnormalities were observed in aortic valves from patients suffering from aortic stenosis: presence of large calcium deposits, inflammatory cells, lipids, and neocapillaries as well as extracellular matrix remodeling. The aortic valves show three characteristic layers: the fibrosa composed mainly of collagen bundles, the spongiosa which consists of a proteoglycan matrix, and the ventricularis which contains several elastic lamellae. The components of the extracellular matrix are synthesized by valvular mesenchymal cells. The turn-over of collagen and elastic fibers is low; the other macromolecules are more rapidly synthesized and hydrolysed. Serine proteases such as enzymes of the fibrinolytic system and matrix metalloproteinases play a role in the remodeling of the extracellular matrix. The hydrolysis of adhesive proteins, such as fibronectin, by plasmin triggers the apoptosis of valvular (myo)fibroblasts, a biological process named anoikis. Cellular events and extracellular matrix remodeling thus participate to the evolution of aortic valves towards aortic stenosis., (© Société de Biologie, 2012.)

Contractile cardiomyocyte metabolism was studied by histochemical methods in experimental stenosis of the aorta complicated and not by heart failure. Acceleration of the citric acid cycle, more intense oxidation of free fatty acids and their metabolites, glycolysis intensification, and higher activity of shuttle mechanisms were found in the contractile cardiomyocytes in stenosis of the aorta not complicated by heart failure. The presence of these metabolic shifts in the myocardium of all studied compartments suggests their association with not only more intense heart work, but also with the effects of total systems neurohumoral factors. Comparative study of myocardial metabolism in two variants of experimental stenosis of the aorta has revealed changes prognostically unfavorable for the development of heart failure. These changes include exhaustion of glycogen reserve, glycolysis inhibition, and metabolism shift towards biosynthetic processes. These data indicate an important role of glycolysis in support of myocardial contractile function during the acute phase of pressure overloading of the heart.

Objective: Matrix metalloproteinases participate in remodeling of extracellular matrix, which is central to the development of aortic stenosis. Synthesis of certain matrix metalloproteinases is induced by the glycoprotein extracellular matrix metalloproteinase inducer. We investigated whether extracellular matrix metalloproteinase inducer and membrane-type 1 matrix metalloproteinase are abundant in calcific aortic valve and their role in the pathogenesis of this condition., Methods: Sixteen patients who underwent surgery for aortic stenosis (n = 12) or heart transplantation for ischemic cardiomyopathy (n = 4) were reviewed. Expression of extracellular matrix metalloproteinase inducer and membrane-type 1 matrix metalloproteinase proteins was assessed by Western blot (n = 4 per group), immunohistochemistry for aortic stenosis (n = 12) and ischemic cardiomyopathy (n = 2), and in situ zymography (n = 3 per group). Functional relevance was investigated using an artificial valve model., Results: Extracellular matrix metalloproteinase inducer and membrane-type 1 matrix metalloproteinase were abundant in all stenotic valves. Control valves did not stain for either protein. Double immunofluorescence colocalized extracellular matrix metalloproteinase inducer and membrane-type 1 matrix metalloproteinase to macrophages. On Western blotting, both proteins were more abundant in stenotic valves than in control valves. In situ zymography demonstrated greater gelatinolytic activity in stenotic valves than in control valves. Silencing of the extracellular matrix metalloproteinase inducer gene using small interfering RNA reduced migration of monocytes in an artificial valve model., Conclusions: Extracellular matrix metalloproteinase inducer and membrane-type 1 matrix metalloproteinase were demonstrated on macrophages in stenotic aortic valves, into which extracellular matrix metalloproteinase inducer may promote monocyte immigration. The latter protein may therefore represent a potential target to reduce the development of aortic stenosis., (Copyright © 2011 The American Association for Thoracic Surgery. Published by Mosby, Inc. All rights reserved.)

Calcific aortic valve stenosis (CAS) is the most frequent heart valve disease in the elderly, accompanied by valve calcification. Tumor necrosis factor-α (TNF-α), a pleiotropic cytokine secreted mainly from macrophages, has been detected in human calcified valves. However, the role of TNF-α in valve calcification remains unclear. To clarify whether TNF-α accelerates the calcification of aortic valves, we investigated the effect of TNF-α on human aortic valve interstitial cells (HAVICs) obtained from patients with CAS (CAS group) and with aortic regurgitation or aortic dissection having a noncalcified aortic valve (control group). HAVICs (2 × 10(4)) were cultured in a 12-well dish in Dulbecco's modified Eagle's medium with 10% fetal bovine serum. The medium containing TNF-α (30 ng/ml) was replenished every 3 days after the cells reached confluence. TNF-α significantly accelerated the calcification and alkaline phosphatase (ALP) activity of HAVICs from CAS but not the control group after 12 days of culture. Furthermore, gene expression of calcigenic markers, ALP, bone morphogenetic protein 2 (BMP2), and distal-less homeobox 5 (Dlx5) were significantly increased after 6 days of TNF-α treatment in the CAS group but not the control group. Dorsomorphin, an inhibitor of mothers against decapentaplegic homologs (Smads) 1/5/8 phosphorylation, significantly inhibited the enhancement of TNF-α-induced calcification, ALP activity, Smad phosphorylation, and Dlx5 gene expression of HAVICs from the CAS group. These results suggest that HAVICs from the CAS group have greater sensitivity to TNF-α, which accelerates the calcification of aortic valves via the BMP2-Dlx5 pathway.

Background and Aim of the Study: Aortic valve stenosis (AS) is the most common valvular heart disease in the western world, and in adults is invariably caused by the calcification of a normal tricuspid or congenital bicuspid valve. Calcific AS, as an active disease process, is characterized by lipid accumulation, inflammation and calcification that mimic atherosclerosis. Paraoxonase-1 (PON-1) is a high-density lipoprotein (HDL)-bound enzyme that exerts antiatherogenic properties by protecting low-density lipoprotein (LDL)-cholesterol from oxidative modification. The study aim was to examine the association between PON-1 activity and AS., Methods: A total of 93 patients with angiographically normal coronary arteries was enrolled into the study. Transthoracic echocardiography was used to diagnose and grade the AS before the patients underwent selective coronary angiography. The patients were allocated to three groups of mild AS (n = 34), moderate AS (n = 31) and severe AS (n = 28). Paraoxonase activity was measured using a spectrophotometric technique., Results: The mean PON-1 activity in patients with severe AS (64.4 +/- 29.8 U/l) was significantly lower than that in patients with mild and moderate AS (97.1 +/- 72.6 and 146.8 +/- 133.9 U/l; p = 0.03 and p = 0.002, respectively). Typically, PON-1 activity tended to be lower in moderate AS than in mild AS (p = 0.07). The serum PON-1 activity correlated positively with the aortic valve area, but negatively with the aortic mean and maximum gradients., Conclusion: The study results showed that PON-1 activity is lower in patients with calcific AS. In addition, PON-1 activity was inversely correlated with the severity of AS.

Rationale: Multiple cyclic nucleotide phosphodiesterases (PDEs) degrade cAMP in cardiomyocytes but the role of PDEs in controlling cAMP signaling during pathological cardiac hypertrophy is poorly defined., Objective: Evaluate the beta-adrenergic regulation of cardiac contractility and characterize the changes in cardiomyocyte cAMP signals and cAMP-PDE expression and activity following cardiac hypertrophy., Methods and Results: Cardiac hypertrophy was induced in rats by thoracic aortic banding over a time period of 5 weeks and was confirmed by anatomic measurements and echocardiography. Ex vivo myocardial function was evaluated in Langendorff-perfused hearts. Engineered cyclic nucleotide-gated (CNG) channels were expressed in single cardiomyocytes to monitor subsarcolemmal cAMP using whole-cell patch-clamp recordings of the associated CNG current (I(CNG)). PDE variant activity and protein level were determined in purified cardiomyocytes. Aortic stenosis rats exhibited a 67% increase in heart weight compared to sham-operated animals. The inotropic response to maximal beta-adrenergic stimulation was reduced by approximately 54% in isolated hypertrophied hearts, along with a approximately 32% decrease in subsarcolemmal cAMP levels in hypertrophied myocytes. Total cAMP hydrolytic activity as well as PDE3 and PDE4 activities were reduced in hypertrophied myocytes, because of a reduction of PDE3A, PDE4A, and PDE4B, whereas PDE4D was unchanged. Regulation of beta-adrenergic cAMP signals by PDEs was blunted in hypertrophied myocytes, as demonstrated by the diminished effects of IBMX (100 micromol/L) and of both the PDE3 inhibitor cilostamide (1 micromol/L) and the PDE4 inhibitor Ro 201724 (10 micromol/L)., Conclusions: Beta-adrenergic desensitization is accompanied by a reduction in cAMP-PDE and an altered modulation of beta-adrenergic cAMP signals in cardiac hypertrophy.

Objectives: The aim of this study was to determine whether oxidative stress is increased in calcified, stenotic aortic valves and to examine mechanisms that might contribute to increased oxidative stress., Background: Oxidative stress is increased in atherosclerotic lesions and might play an important role in plaque progression and calcification. The role of oxidative stress in valve disease is not clear., Methods: Superoxide (dihydroethidium fluorescence and lucigenin-enhanced chemiluminescence), hydrogen peroxide H2O2 (dichlorofluorescein fluorescence), and expression and activity of pro- and anti-oxidant enzymes were measured in normal valves from hearts not suitable for transplantation and stenotic aortic valves that were removed during surgical replacement of the valve., Results: In normal valves, superoxide levels were relatively low and distributed homogeneously throughout the valve. In stenotic valves, superoxide levels were increased 2-fold near the calcified regions of the valve (p < 0.05); noncalcified regions did not differ significantly from normal valves. Hydrogen peroxide levels were also markedly elevated in calcified regions of stenotic valves. Nicotinamide adenine dinucleotide phosphate oxidase activity was not increased in calcified regions of stenotic valves. Superoxide levels in stenotic valves were significantly reduced by inhibition of nitric oxide synthases (NOS), which suggests uncoupling of the enzyme. Antioxidant mechanisms were reduced in calcified regions of the aortic valve, because total superoxide dismutase (SOD) activity and expression of all 3 SOD isoforms was significantly decreased. Catalase expression also was reduced in pericalcific regions., Conclusions: This study provides the first evidence that oxidative stress is increased in calcified regions of stenotic aortic valves from humans. Increased oxidative stress is due at least in part to reduction in expression and activity of antioxidant enzymes and perhaps to uncoupled NOS activity. Thus, mechanisms of oxidative stress differ greatly between stenotic aortic valves and atherosclerotic arteries.

Background and Aim of the Study: A growing body of data indicates an independent association between serum gamma-glutamyltransferase (GGT) activity, a marker of increased oxidative stress, and cardiovascular diseases. The process of calcific aortic valve disease has been shown to present characteristics of atherosclerosis. The study aim was to evaluate the possible role of serum GGT in patients with calcific aortic valve disease., Methods: The results of patients' echocardiography studies from 2005 for the presence of calcific aortic valve disease in the forms of aortic stenosis (AS) and aortic valve calcification (AVC) without significant valve stenosis, were retrospectively evaluated. Age-and gender-matched patients with normal aortic valve morphology were selected at random as a control group. A total of 383 patients was enrolled into the study (126 with AS, 133 with AVC, 124 controls). Serum GGT activity, along with other liver enzyme analyses and laboratory results, were determined and compared among the groups., Results: Age, gender and clinical and laboratory results were similar among the three groups. Median serum GGT levels in the AS, AVC and control groups were 23.0 U/1 (mean 31.5 +/- 24.9 U/1), 22.0 U/1 (mean 27.6 +/- 18.6 U/) and 18.0 U/l (mean 22.4 +/- 16.4 U/l), respectively. Compared to controls, AS patients had significantly higher serum GGT and C-reactive protein levels, while the differences between AVC patients and controls for these parameters were not significant., Conclusion: The study results suggest that serum GGT activity is increased in patients with calcific AS. These increases seem to occur in advanced rather than milder forms of calcific aortic valve disease.

Objective: We hypothesized that reactive oxygen species (ROS) contribute to progression of aortic valve (AV) calcification/stenosis., Methods and Results: We investigated ROS production and effects of antioxidants tempol and lipoic acid (LA) in calcification progression in rabbits given 0.5% cholesterol diet +10(4) IU/d Vit.D2 for 12 weeks. Superoxide and H2O2 microfluorotopography and 3-nitrotyrosine immunoreactivity showed increased signals not only in macrophages but preferentially around calcifying foci, in cells expressing osteoblast/osteoclast, but not macrophage markers. Such cells also showed increased expression of NAD(P)H oxidase subunits Nox2, p22phox, and protein disulfide isomerase. Nox4, but not Nox1 mRNA, was increased. Tempol augmented whereas LA decreased H2O2 signals. Importantly, AV calcification, assessed by echocardiography and histomorphometry, decreased 43% to 70% with LA, but increased with tempol (P < or = 0.05). Tempol further enhanced apoptosis and Nox4 expression. In human sclerotic or stenotic AV, we found analogous increases in ROS production and NAD(P)H oxidase expression around calcifying foci. An in vitro vascular smooth muscle cell (VSMC) calcification model also exhibited increased, catalase-inhibitable, calcium deposit with tempol, but not with LA., Conclusions: Our data provide evidence that ROS, particularly hydrogen peroxide, potentiate AV calcification progression. However, tempol exhibited a paradoxical effect, exacerbating AV/vascular calcification, likely because of its induced increase in peroxide generation.

Objective: To observe the effects of Xiongshao Capsule (XSC) on blood vessel collagenase gene expression in experimental rabbits with arterial restenosis, and to probe its mechanisms for preventing restenosis., Methods: Restenosis rabbit model was established by injuring endothelium of abdominal aorta by balloon dilation and feeding with high fatty diet for 6 weeks. Eighty rabbits were randomly allocated into 8 groups, Group A, normal rabbit for control; Group B, rabbit with simple injured arterial endothelium; Group C, model rabbits at different times after modeling (3 days for Group C1, 2 weeks for Group C2, and 6 weeks for Group C3); Group D, model rabbit treated with Probucol for 6 weeks; Group E and F, model rabbit treated with small and large dose of XSC respectively. The effect of XSC on collagenase gene expression during the course of restenosis was observed adopting RT-PCR method and computer image analyzer, and its mechanisms in preventing RS were probed by combined analyzing the change of collagen and patho-morphological examination., Results: Compensatory dilation of lumens appeared at the end of the 2nd week; while 6 weeks after modeling, the diameters of lumens obviously diminished with an apparently increased proliferation index. The cell proliferation inhibiting effect in Group D and F was significant. The total amount of collagen increased and reached the peak at the 2nd week but without conspicuous accumulation on intima, which increased gradually and reached its peak at the 6th week. In Group D-F, especially in Group F, the amount of collagen in vascular wall (intima, media and externa) was lesser than that in Groups C. MMP-1 mRNA showed weak expression in Group A and Group C1-C3; significant difference only existed in comparing Group F with C3 (P < 0.05)., Conclusion: XSC could markedly increase the MMP-1 mRNA expression in injured portion of vessels, suggesting that its action in preventing RS might be related with the up-regulating of MMP-1 mRNA expression, increasing collagen degradation and reducing collagen deposition in vascular wall.

Objectives: Protein kinase C (PKC) is a central enzyme in the regulation of growth and hypertrophy. Little was known on PKC isoform regulation in human heart. Goal of this study was to characterize the isoforms of protein kinase C in human heart, their changes during ontogenesis, and their regulation in myocardial hypertrophy and heart failure., Methods: In left ventricular and atrial samples from adults with end-stage dilated cardiomyopathy (DCM), from adults with severe aortic stenosis (AS), from small infants undergoing repair of ventricular septal defects, and from healthy organ donors (CO), activity of protein kinase C and the expression of its isozymes were examined., Results: In the adult human heart, the isoforms PKC-alpha, PCK-beta, PKC-delta, PKC-epsilon, PKC-lambda/-iota, and PKC-zeta were detected both on protein and on mRNA level. All isozymes are subjected to downregulation during ontogenesis. No evidence, however, exists for an isoform shift from infancy to adulthood. DCM leads to a pronounced upregulation of PKC-beta. Severe left ventricular hypertrophy in AS, however, recruits a distinct isoform pattern, i.e., isoforms PKC-alpha, PKC-delta, PKC-epsilon, PKC-lambda/-iota, and PKC-zeta are upregulated, whereas PKC-beta is not changed under this condition., Conclusion: This work gives evidence for a differential recruitment of human PKC isoforms in various forms of myocardial hypertrophy and heart failure.

Background: Aortic valve stenosis is the most frequent indication for valve replacement surgery, and is commonly associated with pathologic calcification. Previous investigations by our group have shown a strong association of transforming growth factor-beta1 (TGF-beta1)-related mechanisms with calcific aortic stenosis in both cell culture and clinical pathology studies., Methods: In the present investigations we sought to investigate the sequence of events involved in TGF-beta1-initiated aortic valve interstitial cell calcification in cell culture, and to study related gene expression pattern differences comparing calcific aortic stenosis surgical specimens with normal aortic valve leaflets., Results: Sheep aortic valve interstitial cells (SAVIC) in culture progressively calcified over 14 days after the addition of TGF-beta1 to a significantly greater extent than non-TGF-beta1 controls. The TGF-beta1-induced SAVIC calcification was associated with maximal levels of alkaline phosphatase by 72 hours. Annexin V positive apoptosis was increased in TGF-beta1-treated SAVIC cultures at 14 days compared with controls. Matrix metalloproteinase 9 per gel zymography was detectable only in SAVIC cultures treated with TGF-beta1 from seven days on. Matrix metalloproteinase 2 was present in all SAVIC cultures per gel zymograms, either with or without TGF-beta1, but the active form of matrix metalloproteinase 2 significantly increased over 14 days in response to TGF-beta1. Quantitative gene expression studies (re: RNA levels) of human aortic valve cusps obtained at cardiac surgery demonstrated a number of related trends, including upregulation of the expression of TGF-beta1, alkaline phosphatase, and matrix metalloproteinase 9 in calcified human aortic valves., Conclusions: Transforming growth factor-beta1 causes SAVIC to calcify due to an early maximal increase in alkaline phosphatase activity with associated apoptotic events and increased matrix metalloproteinase 9. These TGF-beta1-related mechanistic events may be of clinical relevance based upon the gene expression pattern changes observed in calcific aortic stenosis valve cusps.

Aims: Aortic stenosis (AS) is characterized by extensive remodelling of the valves, including infiltration of inflammatory cells, extracellular matrix degradation, and fibrosis. The molecular mechanisms behind this adverse remodelling have remained obscure. In this article, we study whether cathepsin G, an angiotensin II (Ang II)-forming elastolytic enzyme, contributes to progression of AS., Methods and Results: Stenotic aortic valves (n = 86) and control valves (n = 17) were analysed for cathepsin G, transforming growth factor-beta1 (TGF-beta1), and collagens I and III with RT-PCR and immunohistochemistry. Valvular collagen/elastin ratio was quantified by histochemistry. In stenotic valves, cathepsin G was present in mast cells and showed increased expression (P < 0.001), which correlated positively (P < 0.001) with the expression levels of TGF-beta1 and collagens I and III. TGF-beta1 was also present in mast cell-rich areas and cathepsin G induced losartan-sensitive TGF-beta1 expression in cultured fibroblasts. Collagen/elastin ratio was increased in stenotic valves (P < 0.001) and correlated positively with smoking (P = 0.02). Nicotine in cigarette smoke activated mast cells and induced TGF-beta1 expression in cultured fibroblasts. Fragmented elastin was observed in stenotic valves containing activated cathepsin G-secreting mast cells and in normal valves treated with cathepsin G., Conclusion: In stenotic aortic valves, mast cell-derived cathepsin G may cause adverse valve remodelling and AS progression.

Objectives: Aortic surgery for progressive aortic valve disease or aortic aneurysm after previous coronary artery bypass grafting (CABG) is a challenging procedure. We report the outcome of aortic reoperation after previous CABG and evaluate our management of patent grafts and our methods for obtaining myocardial protection., Methods: From February 2001 to July 2003, 6 patients with progressive aortic valve disease and aneurysm of the thoracic aorta were operated on. The group comprised 3 men and 3 women with a mean age of 67.6 years. There were 4 patients with an aneurysm of the aortic arch, 1 with chronic ascending aortic dissection, and 1 with progressive aortic valve stenosis. The interval between previous CABG and aortic surgery was 74.0 +/- 44.2 months. All reoperations were performed via median resternotomy. Myocardial protection was obtained by hypothermic perfusion of patent in-situ arterial grafts following cold-blood cardioplegia administration via the aortic root under aortic cross clamping., Results: The operative procedure was aortic arch replacement in 4 patients, ascending aortic replacement with double CABG in 1, and aortic valve replacement in 1. All patients survived the reoperation. Postoperative maximum creatine kinase-MB was 49.2 +/- 29.8 and no new Q-waves occurred in the electrocardiogram nor were any new wall motion abnormalities recognized on echocardiography. There were no late deaths during a follow-up of 30.7 months., Conclusion: Reoperative aortic procedures after CABG can be performed safely with myocardial protection via hypothermic perfusion of a patent in-situ arterial graft.

Aims: Aortic valve diseases are characterized by pathological remodelling of valvular tissue but the cellular and molecular effectors involved in these processes are not well known. The role of matrix metalloproteinase (MMP)-2, MMP-9, MMP-3, MMP-7, and tissue inhibitor of matrix metalloproteinase (TIMP)-1 and TIMP-2 are investigated here., Methods and Results: Histological analysis of pathological valves [aortic stenosis (AS) (n=49), aortic regurgitation (AR) (n=23)] and control valves (n=8) was performed. The main tissue abnormalities (calcification, inflammatory cells, and capillaries) observed in AS were less severe or absent in AR. However, both groups of pathological valves displayed similar histological signs of extracellular matrix (ECM) remodelling. Biochemical analysis of MMPs and TIMPs (gelatin and casein zymography and ELISA) was performed on valve extracts. MMP-2 activity was not significantly different in control and pathological valves. Increases in MMP-9 and MMP-3 in AS demonstrated an inflammatory state. Finally, there was a four- to seven-fold increase of TIMP-1 in pathological valves. TIMP-1, TIMP-2, and MMP-2 were synthesized by the valvular interstitial cells in primary culture., Conclusion: This study demonstrates the involvement of the MMP/TIMP system in ECM remodelling of both AS and AR. These findings provide evidence of inflammatory injury more severe in AS than in AR and involvement of mesenchymal cell response.

Calcific aortic valve stenosis, the main heart valve disease in the elderly, is based on progressive calcification and fibrous thickening of the valve. Several reports addressed the pathogenesis of tissue calcification in this disorder, but few data exist on the molecular mechanisms of the fibrosis and remodeling of the extracellular matrix. The cytokine "receptor activator of nuclear factor kappa B ligand" (RANKL), is expressed in stenotic aortic valves and involved in valvular calcification during calcific aortic valve stenosis. The present study aimed to assess the influence of RANKL on the molecular mechanisms of connective tissue remodeling. In an established cell culture model of primary human aortic valve myofibroblasts, stimulation with RANKL increased cell proliferation as compared to medium alone. Matrix metalloproteinase (MMP)-1 was detectable time-dependently in conditioned media from RANKL-stimulated cells, but absent in media from control cells. MMP-1 activity was increased by RANKL, as measured by collagenase activity assay. Zymography showed an increase in active MMP-2 in RANKL-stimulated cells. These results support the concept that MMPs are involved in the connective tissue remodeling during calcific aortic valve stenosis. RANKL might regulate this process by promoting cell proliferation and MMP expression and activation.

Aortic stenosis (AS) results in myocyte and extracellular matrix remodeling in the human left ventricle (LV). The myocardial renin-angiotensin system is activated and collagens I and III and fibronectin accumulate. We determined the yet unknown regulation of enzymes that control collagen turnover, i.e., LV matrix metalloproteinases (MMP) and their tissue inhibitors (TIMPs) in human AS. We compared LV samples from AS patients undergoing elective aortic valve replacement (n=19) with nonused donor hearts with normal LV function (controls, n=12). MMP-2, MMP-9, MT1-MMP, and extracellular matrix metalloproteinase inducer (EMMPRIN), TIMP-1, TIMP-2, TIMP-3, and TIMP-4 mRNA were quantitated by real-time RCR. MMP-1, MMP-2, MMP-3, TIMP-3, TIMP-4, and EMMPRIN protein were measured by immunoblotting and MMP-9 and TIMP-1 protein by ELISA. Gelatinolytic MMP-2 and MMP-9 activity was measured by zymography. MMP-2 was increased in AS at mRNA, protein, and activity levels (131%, 193%, and 138% of controls). MMP-3 protein (308%) and EMMPRIN mRNA and protein were also upregulated (171% and 200%). In contrast, MMP-1 (37%) and MMP-9 mRNA, protein, and activity (26%, 21%, and 52%) were downregulated. MMP-9 activity was inversely correlated with LV size. TIMP-1 mRNA and protein were decreased (55% and 73%). In contrast, TIMP-2 mRNA (358%), TIMP-3 mRNA and protein (145% and 249%) were increased. TIMP-4 mRNA was not altered, but TIMP-4 protein was upregulated to 350%. Changes were similar in AS patients with normal and impaired LV ejection fraction. The dysregulation of myocardial MMPs and TIMPs in human AS starts at an early disease stage when LV function is still normal. In spite of upregulation of some MMPs the balance between MMP and TIMP is shifted towards MMP inhibition in human AS and may contribute to collagen accumulation.

Objective: The degeneration of bicuspid aortic valve and its frequent association with ascending aortic pathology, point to a still unidentified genetic tissue defect with unknown mediators. Metalloproteinases (MMPs) are lytic enzymes that have been strongly implicated in aneurysm formation. The purpose of this study was to detect the presence of these enzymes in aortic valvular tissue in healthy and diseased aortic valves with or without the presence of synchronous ascending aortic pathology., Methods: Aortic valve specimens from 26 aortic valve replacement patients as well as 4 healthy control tricuspid aortic valves were included. 10 patients had bicuspid aortic valves, and 16 had tricuspid aortic valves. Half of our patient population had a concomitant aortic procedure for aortic pathology. The study detected MMPs 1,2 and 9 as well as their Tissue inhibitors (TIMPs) 1 and 2. MMP and TIMP detection was accomplished with the construction of a tissue micro array and immunohistochemistry., Conclusions: MMP-9 expression was significantly higher in bicuspid aortic valves compared to normal valves (P<0.05). When compared to the tricuspid valve group, MMP-9 mean value was significantly higher in bicuspid valves (P<0.05). When the entire rest of the valve group (n=4+16, i.e. control and tricuspid valve groups) was compared to the bicuspid valve group, bicuspid valves had significantly higher MMP-2, and MMP-9 (P<0.01) expression. TIMP expression also changed in diseased valves, among different patient groups. This increased proteolytic presence in bicuspid aortic valves may attribute to the observed decreased elastin and collagen content, and their resultant functional failure.

Calcific aortic valve stenosis (AS), the main heart valve disease in the elderly, is characterized by extensive remodeling of the extracellular matrix. Matrix metalloproteinases (MMPs) are upregulated in calcific AS and might modulate matrix remodeling. The regulatory mechanisms are unclear. As recent studies have suggested that calcific AS might result from an inflammatory process involving leukocyte invasion and activation, the present study aimed to elucidate the role of the pro-inflammatory cytokine interleukin (IL)-1 beta on MMP expression and cell proliferation in human aortic valves. Immunohistochemistry for leukocytes, IL-1 beta and MMP-1 was performed on aortic valves with (n=6) and without (n=6) calcification obtained at valve replacement or autopsy. Stenotic valves showed marked leukocyte infiltration and associated expression of IL-1 beta and MMP-1. In control valves only scattered leukocytes, low staining for MMP-1 and no staining for IL-1 beta were present. Double-label immunostaining localized IL-1 beta expression mainly to leukocytes and MMP-1 expression to myofibroblasts. Stimulation of cultured human aortic valve myofibroblasts with IL-1 beta lead to a time-dependently increased expression of MMP-1 and MMP-2 by Western blotting and zymography, whereas MMP-9 remained unchanged. Cell proliferation was increased by IL-1 beta as determined by bromodesoxyuridine incorporation. Thus, IL-1 beta may regulate remodeling of the extracellular matrix in calcific AS.