Safety of Phosphodiesterase-5 Inhibitors in Valvular Heart Disease.

ABSTRACT: Erectile dysfunction is a common entity in clinical practice. Primary erectile dysfunction, not related to vasculopathy or psychiatric disorder, can be readily treated with phosphodiesterase inhibitors. These drugs have many physiologic effects that can alter a patient's hemodynamic profile considerably, especially in the presence of concomitant structural heart disease, specifically valvular heart disease. Although some contraindications to the use of PDE5 inhibitors in patients with cardiovascular disease are defined, the effect of these drugs in the presence of valvular heart disease is not well documented. The purpose of this review is to analyze the data regarding the safety of PDE5 inhibitors in patients with valvular heart disease.

Biallelic loss-of-function variants in PLD1 cause congenital right-sided cardiac valve defects and neonatal cardiomyopathy.

Congenital heart disease is the most common type of birth defect, accounting for one-third of all congenital anomalies. Using whole-exome sequencing of 2718 patients with congenital heart disease and a search in GeneMatcher, we identified 30 patients from 21 unrelated families of different ancestries with biallelic phospholipase D1 (PLD1) variants who presented predominantly with congenital cardiac valve defects. We also associated recessive PLD1 variants with isolated neonatal cardiomyopathy. Furthermore, we established that p.I668F is a founder variant among Ashkenazi Jews (allele frequency of ~2%) and describe the phenotypic spectrum of PLD1-associated congenital heart defects. PLD1 missense variants were overrepresented in regions of the protein critical for catalytic activity, and, correspondingly, we observed a strong reduction in enzymatic activity for most of the mutant proteins in an enzymatic assay. Finally, we demonstrate that PLD1 inhibition decreased endothelial-mesenchymal transition, an established pivotal early step in valvulogenesis. In conclusion, our study provides a more detailed understanding of disease mechanisms and phenotypic expression associated with PLD1 loss of function.

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.

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.

Hyaluronidase 2 Deficiency Causes Increased Mesenchymal Cells, Congenital Heart Defects, and Heart Failure.

BACKGROUND: Hyaluronan (HA) is required for endothelial-to-mesenchymal transition and normal heart development in the mouse. Heart abnormalities in hyaluronidase 2 (HYAL2)-deficient (Hyal2-/- ) mice and humans suggested removal of HA is also important for normal heart development. We have performed longitudinal studies of heart structure and function in Hyal2-/- mice to determine when, and how, HYAL2 deficiency leads to these abnormalities. METHODS AND RESULTS: Echocardiography revealed atrial enlargement, atrial tissue masses, and valvular thickening at 4 weeks of age, as well as diastolic dysfunction that progressed with age, in Hyal2-/- mice. These abnormalities were associated with increased HA, vimentin-positive cells, and fibrosis in Hyal2-/- compared with control mice. Based on the severity of heart dysfunction, acute and chronic groups of Hyal2-/- mice that died at an average of 12 and 25 weeks respectively, were defined. Increased HA levels and mesenchymal cells, but not vascular endothelial growth factor in Hyal2-/- embryonic hearts, suggest that HYAL2 is important to inhibit endothelial-to-mesenchymal transition. Consistent with this, in wild-type embryos, HYAL2 and HA were readily detected, and HA levels decreased with age. CONCLUSIONS: These data demonstrate that disruption of normal HA catabolism in Hyal2-/- mice causes increased HA, which may promote endothelial-to-mesenchymal transition and proliferation of mesenchymal cells. Excess endothelial-to-mesenchymal transition, resulting in increased mesenchymal cells, is the likely cause of morphological heart abnormalities in both humans and mice. In mice, these abnormalities result in progressive and severe diastolic dysfunction, culminating in heart failure.

Whole Exome Sequencing, Familial Genomic Triangulation, and Systems Biology Converge to Identify a Novel Nonsense Mutation in TAB2-encoded TGF-beta Activated Kinase 1 in a Child with Polyvalvular Syndrome.

OBJECTIVE: To use whole exome sequencing (WES) of a family trio to identify a genetic cause for polyvalvular syndrome. METHODS AND RESULTS: A male child was born with mild pulmonary valve stenosis and mild aortic root dilatation, and an atrial septal defect, ventricular septal defect, and patent ductus arteriosus that were closed surgically. Subsequently, the phenotype of polyvalvular syndrome with involvement of both semilunar and both atrioventricular valves emerged. His family history was negative for congenital heart disease. Because of hypotonia, myopia, soft pale skin, joint hypermobility, and mild facial dysmorphism, either Noonan syndrome- or William syndrome-spectrum disorders were suspected clinically. However, chromosomal analysis was normal and commercially available Noonan syndrome and William syndrome genetic tests were negative. Whole exome sequencing of the patient and both parents was performed. Variants were analyzed by sporadic and autosomal recessive inheritance models. A sporadic mutation, annotated as c.1491 T > A, in TAB2, resulting in a nonsense mutation, p.Y497X, in the TAB2-encoded TGF-beta activated kinase 1 (TAK1) was identified as the most likely disease-susceptibility gene. This mutation results in elimination of the terminal 197 amino acids, including the C-terminal binding motif critical for interactions with TRAF6 and TAK1. CONCLUSIONS: The combination of WES, genomic triangulation, and systems biology has uncovered perturbations in TGF-beta activated kinase 1 signaling as a novel pathogenic substrate for polyvalvular syndrome.

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.

Calpain-Calcineurin-Nuclear Factor Signaling and the Development of Atrial Fibrillation in Patients with Valvular Heart Disease and Diabetes.

Calpain, calcineurin (CaN), and nuclear factor of activated T cell (NFAT) play a key role in the development of atrial fibrillation. Patients with valvular heart disease (VHD) are prone to develop atrial fibrillation (AF). Thus, our current study was aimed at investigating whether activation of calpain-CaN-NFAT pathway is associated with the incidence of AF in the patients with VHD and diabetes. The expressions of calpain 2 and alpha- and beta-isoforms of CaN catalytic subunit (CnA) as well as NFAT-c3 and NFAT-c4 were quantified by quantitative reverse transcription-polymerase chain reaction in atrial tissues from 77 hospitalized patients with VHD and diabetes. The relevant protein content was measured by Western blot and calpain 2 in human atrium was localized by immunohistochemistry. We found that the expressions of calpain 2, CnA alpha and CnA beta, and NFAT-c3 but not NFAT-c4 were significantly elevated in the samples from patients with AF compared to those with sinus rhythm (SR). Elevated protein levels of calpain 2 and CnA were observed in patients with AF, and so was the enhanced localization of calpain 2. We thereby concluded that CaN together with its upstream molecule, calpain 2, and its downstream effector, NFAT-c3, might contribute to the development of AF in patients with VHD and diabetes.

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.

COX2 inhibition reduces aortic valve calcification in vivo.

OBJECTIVE: Calcific aortic valve disease (CAVD) is a significant cause of morbidity and mortality, which affects ≈1% of the US population and is characterized by calcific nodule formation and stenosis of the valve. Klotho-deficient mice were used to study the molecular mechanisms of CAVD as they develop robust aortic valve (AoV) calcification. Through microarray analysis of AoV tissues from klotho-deficient and wild-type mice, increased expression of the gene encoding cyclooxygenase 2 (COX2; Ptgs2) was found. COX2 activity contributes to bone differentiation and homeostasis, thus the contribution of COX2 activity to AoV calcification was assessed. APPROACH AND RESULTS: In klotho-deficient mice, COX2 expression is increased throughout regions of valve calcification and is induced in the valvular interstitial cells before calcification formation. Similarly, COX2 expression is increased in human diseased AoVs. Treatment of cultured porcine aortic valvular interstitial cells with osteogenic media induces bone marker gene expression and calcification in vitro, which is blocked by inhibition of COX2 activity. In vivo, genetic loss of function of COX2 cyclooxygenase activity partially rescues AoV calcification in klotho-deficient mice. Moreover, pharmacological inhibition of COX2 activity in klotho-deficient mice via celecoxib-containing diet reduces AoV calcification and blocks osteogenic gene expression. CONCLUSIONS: COX2 expression is upregulated in CAVD, and its activity contributes to osteogenic gene induction and valve calcification in vitro and in vivo.

Differential expression of MMP-2, MMP-9 and TIMP proteins in thoracic aortic aneurysm - comparison with and without bicuspid aortic valve: a meta-analysis.

BACKGROUND: It is believed that the balance of matrix metalloproteinases (MMPs) and the tissue inhibitors of metalloproteinases (TIMPs), in the aorta, play a critical role in aneurysm formation. The objective of this study was to perform a meta-analysis of studies reporting protein expression of MMPs and TIMPs in the ascending aorta of thoracic aortic aneurysms (TAA) cases and to examine this expression in persons with TAA and bicuspid aortic valves (BAV). METHODS: OvidSP Medline and EMbase were systematically searched for studies that were: human ascending TAA cases with measurement of MMP or TIMP protein expression in the aorta and a control group. A similar search was conducted for BAV compared to those with a normal or trileaflet aortic valve (TAV). RESULTS: Eight studies fulfilled the inclusion criteria. There was a significant increase in MMP-9 and no change in MMP-2, in the aorta from persons with TAA (N = 106) compared to control (N = 30). There was also a highly significant reduction in TIMP-1 and TIMP-2 in TAA (N = 93) compared to control (N = 24) resulting in a MMP-9 to TIMP-1 or TIMP-2 ratio over 3.5 fold greater than controls. There was a highly significant increase in MMP-2 but not MMP-9 in TAA with BAV (N = 112) compared to TAV (N = 53). There was a significant reduction for TIMP-1 in BAV compared to TAV but no change in TIMP-2, TIMP-3 or TIMP-4. CONCLUSIONS: These data suggest that MMP may be implicated in the pathogenesis of TAA and there is a differential expression with MMP-9 increased and TIMP-1 and -2 reduced in the most common forms of TAA. MMP-2 is increased and only TIMP-1 decreased in TAA with BAV compared to TAV.

Analysis of extracellular superoxide dismutase and Akt in ascending aortic aneurysm with tricuspid or bicuspid aortic valve.

Ascending aortic aneurysm (AsAA) is a consequence of medial degeneration (MD), deriving from apoptotic loss of smooth muscle cells (SMC) and fragmentation of elastin and collagen fibers. Alterations of extracellular matrix structure and protein composition, typical of medial degeneration, can modulate intracellular pathways. In this study we examined the relevance of superoxide dismutase (SOD3) and Akt in AsAA pathogenesis, evaluating their tissue distribution and protein levels in ascending aortic tissues from controls (n=6), patients affected by AsAA associated to tricuspid aortic valve (TAV, n=9) or bicuspid aortic valve (BAV, n=9). The results showed a significant reduction of SOD3, phospho-Akt and Akt protein levels in AsAA tissues from patients with BAV, compared to controls, whereas the differences observed between controls and patients with TAV  were not significant. The decreased levels of SOD3 and Akt in BAV aortic tissues are associated with decreased Erk1/Erk2 phosphorylation and MMP-9 levels increase. The authors suggest a role of decreased SOD3 protein levels in the progression of AsAA with BAV and a link between ECM modifications of aortic media layer and impaired Erk1/Erk2 and Akt signaling in the late stages of the aortopathy associated with BAV.

Elevated expression of lipoprotein-associated phospholipase A2 in calcific aortic valve disease: implications for valve mineralization.

OBJECTIVES: This study sought to document the presence and role of lipoprotein-associated phospholipase A2 (Lp-PLA2) in calcific aortic valve disease (CAVD). BACKGROUND: CAVD is a chronic disorder characterized by pathological mineralization and remodeling. Studies have indicated that human CAVD tissues are infiltrated by lipids and that inflammation may play a role in the pathobiology. We hypothesized that Lp-PLA2 (encoded by the PLA2G7 gene) is expressed in CAVD and may play a role in the mineralization of valve interstitial cells. METHODS: We have documented the expression of the phospholipase A2 family of genes in aortic valves by using a transcriptomic assay. Messenger ribonucleic acid and protein expression were confirmed in aortic valves explanted from 60 patients by quantitative polymerase chain reaction and immunohistochemistry, respectively. The effect of lysophosphatidylcholine, the product of Lp-PLA2 activity, was documented on the mineralization of valve interstitial cell cultures. RESULTS: Transcriptomic analyses of CAVD and control nonmineralized aortic valves revealed that Lp-PLA2 was increased by 4.2-fold in mineralized aortic valves. Higher expression of Lp-PLA2 in stenotic aortic valves was confirmed by quantitative polymerase chain reaction, immunohistochemistry, and enzymatic Lp-PLA2 activity. The number of Lp-PLA2 transcripts correlated with several indexes of tissue remodeling. In vitro, lysophosphatidylcholine increased the expression of alkaline phosphatase, the ectonucleotide pyrophosphatase/phosphodiesterase 1 enzyme, sodium-dependent phosphate cotransporter 1 (encoded by the SLC20A1 gene), and osteopontin. We then showed that lysophosphatidylcholine-induced mineralization involved ectonucleotidase enzyme as well as apoptosis through a protein-kinase-A-dependent pathway. CONCLUSIONS: Together, these results demonstrated that Lp-PLA2 is highly expressed in CAVD, and it plays a role in the mineralization of valve interstitial cells. Further work is necessary to document whether Lp-PLA2 could be considered as a novel target in CAVD.

Serum levels of matrix metalloproteinases 2 and 9 and TGFBR2 gene screening in patients with ascending aortic dilatation.

Development of ascending aortic dilatation (AAD) in about 10 % of patients operated for aortic valve disease (AVD) is probably based on intrinsic pathology of the aortic wall. This may involve an abnormality in the process of extracellular matrix remodelling. The present study evaluated the serum levels of specific metalloproteinases (MMP-2 and MMP-9) and investigated the gene for transforming growth factor receptor 2 (TGFBR2) in 28 patients with AVD associated with AAD (mean age 60.6 years), in 29 patients (68.9 years) with AVD without AAD, and in 30 healthy controls (45.3 years). The serum levels of MMPs were determined by ELISA. Further, we focused on genetic screening of the TGFBR2 gene. Plasma MMP-2 concentrations were significantly higher in the groups of patients compared to the controls: median 1315.0 (mean 1265.2 ± SD 391.3) in AVD with AAD, 1240.0 (1327.8 ± 352.5) in AVD without AAD versus 902.5 (872.3 ± 166.2) ng/ml in the healthy controls, in both cases P < 0.001. The serum levels of MMP-9 were significantly higher in AVD with AAD patients [107.0 (202.3 ± 313.0)] and in AVD without AAD patients [107.0 (185.8 ± 264.3)] compared to the healthy controls [14.5 (21.2 ± 24.8) ng/ml], in both cases P < 0.001. No significant correlation was observed between plasma MMP-2 and MMP-9 and ascending aorta diameter. Genetic screening did not reveal any variation in the TGFBR2 gene in the patients. Measurement of MMP levels is a simple and relatively rapid laboratory test that could be used as a biochemical indicator when evaluated in combination with imaging techniques.

Association of angiotensin I-converting enzyme gene insertion/deletion polymorphism with rheumatic heart disease in Indian population and meta-analysis.

Rheumatic heart disease (RHD) is one of the most severe consequences of rheumatic fever. It has been suggested that angiotensin I-converting enzyme (ACE) may be involved in the increased valvular fibrosis and calcification in the pathogenesis of RHD. We conducted a case-control study to look for association of ACE I/D polymorphism with RHD in Indian population. The study incorporated 300 patients (170 males and 130 females) with RHD, and 200 controls (118 males and 82 females). We also subgrouped RHD patients into mitral valve lesion (MVL) and combined valve lesion (CVL). ACE I/D polymorphism was identified using polymerase chain reaction method. We also performed a meta-analysis of three published studies and the present study (636 RHD cases and 533 controls) to evaluate the association between the ACE I/D polymorphisms and RHD risk. A significant difference in ACE ID and DD genotypes distribution between RHD cases (OR = 1.62, 95% CI = 1.11-2.36 and OR = 2.08, 95% CI = 1.02-4.15, respectively) and corresponding controls was observed. On comparing the ACE genotypes of MVL and CVL subgroups with controls, ID and DD genotypes were also significantly associated with CVL (FDR Pcorr = 0.009, OR = 2.19 and FDR Pcorr = 0.014, OR = 3.29, respectively). Meta-analysis also suggested association of the ACE D allele (FDR Pcorr = 0.036, OR-1.22, 95% CI 1.02-1.45) with RHD. In conclusion, ACE ID and DD genotypes are associated with an increased risk of RHD, particularly CVL. This suggests that the ACE I/D gene polymorphism may play an important role in the pathogenesis of RHD.

Low transcriptional activity haplotype of matrix metalloproteinase 1 is less frequent in bicuspid aortic valve patients.

PURPOSE: Bicuspid aortic valve (BAV) is the most common congenital cardiac anomaly which affects 0.5-2% of the population. It can be associated with other cardiac congenital lesions such as aortic aneurysms or aortic coarctation. Some genetic abnormalities have been suggested as the underlying cause of BAV and aortic root dilatation, but no clear genetic substrate and no specific pathogenic gene variant have already been identified. Increased matrix metalloproteinase activity has been described in the aortic wall of thoracic aortic aneurysms (TAA). METHODS: 87 patients with BAV and 77 controls with normal tricuspid aortic valve were prospectively assessed. We analysed three functional polymorphisms (-1607 1G/2G, -519 A/G, and -340 T/C) in the matrix metalloproteinase (MMP)-1 gene using polymerase chain reaction and restriction fragment length analysis. RESULTS: We found a haplotype composed of the lower activity allele from each polymorphism (-1607 1G/-519 A/-340 C) significantly less frequent in BAV group (p=0.016; OR [95% CI]=0.37 [0.16-0.85]), association even more clear when we consider only men (p=0.0005, OR [95% CI]=0.24 [0.10-0.56]). We also found a borderline statistical significance in the distribution of the -1607 alleles, being 2G allele more frequent in patients with TAA (p=0.053). This association was stronger and statistically significant when we consider only men (p=0.013; OR [95% CI]=2.0 [1.16-3.50]). In addition, genotype -1607 2G2G, theoretically the more active transcriptionally, was also significantly more frequent in TAA group, independently of aortic valve morphology. CONCLUSIONS: Our study suggests that specific genotypes of MMP1 gene could be in part responsible of the complications of BAV pathology, like TAA.

Anti-correlation between longevity gene SirT1 and Notch signaling in ascending aorta biopsies from patients with bicuspid aortic valve disease.

About 1-2% of the population present with bicuspid aortic valves (BAV), a defect of the aortic valve resulting in the formation of two leaflets instead of three. This disease leads to an abnormal aorta, altered in strength and size, which in turn is a high risk factor for potentially lethal events such as aortic dissection and aneurysm formation. BAV is inheritable, with a demonstrated association with Notch1, a member of the Notch intercellular signaling pathway that is implicated in various cardiovascular disorders. Sirtuin 1 (SirT1) is a protein deacetylase of the sirtuin family, whose activation appears beneficial for cardiac diseases. A recent study has shown that SirT1 can limit Notch signaling in model systems of vascular growth. If a concomitant dysregulation in Notch and SirT1 signaling pathways can cause the phenotypic form of human BAV is unknown. To address this issue, we analyzed human ascending aorta biopsies from BAV and control patients obtained at the time of cardiac surgery. RNA and proteins were extracted from formalin-fixed and paraffin-embedded specimens, and quantitative real-time PCR and immunoblotting were used to determine the expression of sirtuins and members of the Notch family of proteins. We found a significant increase in SirT1 expression that correlates with a decreased expression of the Notch signaling effectors detected. We put forward the idea that an altered interaction between SirT1 and Notch signaling could participate in BAV pathogenesis and that these molecules could be used as potential clinical markers.

Matrix metalloproteinase-2 and -9 levels in patients with dilated ascending aorta and bicuspid aortic valve.

BACKGROUND: Predictors of aortic dilatation are not well-described in patients with bicuspid aortic valve (BAV). Changes in extracellular matrix composition in the aortic wall may play an important role. Our study aimed to examine the relationship between ascending aortic dilatation and biochemical markers for collagen metabolism, such as matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) levels in patients with BAV. METHODS: All patients underwent cardiac echocardiography using a standard protocol, and aortic measurements were made in end-diastole. One hundred twelve BAV patients with no or mild valvular impairment were recruited and grouped according to the aortic dimensions corrected for body surface area (BSA) and age. There were 54 patients with dilated ascending aorta (Group 1) and 58 patients with nondilated ascending aorta (group 2). The plasma levels of MMP-2 and MMP-9 were determined by ELISA. RESULTS: The mean ascending aorta diameter was 4.49 ± 0.49 mm in group 1 and 3.51 ± 0.46 mm in group 2 (P < 0.001). There were no significant difference in gender, BSA, presence of hypertension, diabetes mellitus, hyperlipidemia, and smoking between the 2 groups. Nevertheless, no significant difference was observed in the levels of MMP-2 and MMP-9 between the 2 groups. The ascending aorta diameter correlated significantly with age (r = 0.438 P < 0.001). No significant correlation was observed between plasma MMP-2 and MMP-9 concentration and ascending aorta diameter, respectively (r = -0.005 P = 0.58, r = -0.106 P = 0.07). Multivariate analysis showed that age was independent predictor of aortic dilatation (P ≤ 0.001). CONCLUSION: Age was an independent predictor of aortic dilatation in patients with BAV, whereas MMP-2 and 9 levels were not relevant by aortic dilatation.

Antioxidant enzymes reduce DNA damage and early activation of valvular interstitial cells in aortic valve sclerosis.

OBJECTIVE: Accumulation of reactive oxygen species (ROS) and remodeling of the microstructure of the cusp characterize aortic valve sclerosis, the early phase of calcific aortic valve disease. These events are associated with activation of valvular interstitial cells (VICs) toward an osteogenic-like phenotype. Because ROS cause DNA damage and transcriptional activation we investigated the relationship between ROS, DNA damage response, and transdifferentiation of VICs. METHODS AND RESULTS: Human aortic valve cusps and patient-matched VICs were collected from 39 patients both with and without calcific aortic valve disease. VICs were exposed to hydrogen peroxide (0.1-1 mmol/L) after cell transduction with extracellular superoxide dismutase/catalase adenoviruses and characterized for DNA-damage response, osteogenic transdifferentiation, and calcification. ROS induce relocalization of phosphorylated γH2AX, MRE11, and XRCC1 proteins with expression of osteogenic signaling molecule RUNX2 via AKT. We report a sustained activation of γH2AX in aortic valve sclerosis-derived VICs suggesting their impaired ability to repair DNA damage. Adenovirus superoxide dismutase/catalase transduction decreases ROS-induced DNA damage and VIC transdifferentiation in aortic valve sclerosis-derived cells. Finally, adenoviral transduction with catalase reverts ROS-mediated calcification and cellular transdifferentiation. CONCLUSIONS: We conclude that the ROS-induced DNA damage response is dysfunctional in early asymptomatic stages of calcific aortic valve disease. We unveiled an association among ROS, DNA-damage response, and cellular transdifferentiation, reversible by antioxidant enzymes delivery.