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

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

A Role for MMP-10 (Matrix Metalloproteinase-10) in Calcific Aortic Valve Stenosis.

OBJECTIVE: Aortic valve (AV) calcification plays an important role in the progression of aortic stenosis (AS). MMP-10 (matrix metalloproteinase-10 or stromelysin-2) is involved in vascular calcification in atherosclerosis. We hypothesize that MMP-10 may play a pathophysiological role in calcific AS. Approach and Results: Blood samples (n=112 AS and n=349 controls) and AVs (n=88) from patients undergoing valve replacement were analyzed. Circulating MMP-10 was higher in patients with AS compared with controls (P<0.001) and correlated with TNFα (tumor necrosis factor α; rS=0.451; P<0.0001). MMP-10 was detected by immunochemistry in AVs from patients with AS colocalized with aortic valve interstitial cells markers α-SMA (α-smooth muscle actin) and vimentin and with calcification markers Runx2 (Runt-related transcription factor 2) and SRY (sex-determining region Y)-box 9. MMP-10 expression in AVs was further confirmed by RT-qPCR and western blot. Ex vivo, MMP-10 was elevated in the conditioned media of AVs from patients with AS and associated with interleukin-1ß (rS=0.5045, P<0.001) and BMP (bone morphogenetic protein)-2 (rS=0.5003, P<0.01). In vitro, recombinant human MMP-10 induced the overexpression of inflammatory, fibrotic, and osteogenic markers (interleukin-1ß, α-SMA, vimentin, collagen, BMP-4, Sox9, OPN [osteopontin], BMP-9, and Smad 1/5/8; P<0.05) and cell mineralization in aortic valve interstitial cells isolated from human AVs, in a mechanism involving Akt (protein kinase B) phosphorylation. These effects were prevented by TIMP-1 (tissue inhibitor of metalloproteinases type 1), a physiological MMP inhibitor, or specifically by an anti-MMP-10 antibody. CONCLUSIONS: MMP-10, which is overexpressed in aortic valve from patients with AS, seems to play a central role in calcification in AS through Akt phosphorylation. MMP-10 could be a new therapeutic target for delaying the progression of aortic valve calcification in AS.

Apolipoprotein A-I proteolysis in aortic valve stenosis: role of cathepsin S.

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.

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

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

Exercise Training Has Contrasting Effects in Myocardial Infarction and Pressure Overload Due to Divergent Endothelial Nitric Oxide Synthase Regulation.

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.

Interference with ERK(Thr188) phosphorylation impairs pathological but not physiological cardiac hypertrophy.

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.

Histoenzymological Characteristics of Smooth Muscle Cells in Myocardial Vessels: A Comparative Study under Conditions of Increased Left or Right Ventricular Afterload.

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.

Expression and Localization of Granzymes and Perforin in Human Calcific Aortic Valve Disease.

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.

Side-specific endothelial-dependent regulation of aortic valve calcification: interplay of hemodynamics and nitric oxide signaling.

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.

Notch1 promotes the pro-osteogenic response of human aortic valve interstitial cells via modulation of ERK1/2 and nuclear factor-κB activation.

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.

Low myocardial protein kinase G activity in heart failure with preserved ejection fraction.

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.

Lipoprotein lipase in aortic valve stenosis is associated with lipid retention and remodelling.

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.

Decreased Nox4 levels in the myocardium of patients with aortic valve stenosis.

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.

Myeloperoxidase and progression of aortic valve stenosis in patients undergoing hemodialysis.

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.

Histoenzymological characteristics of the heart conduction system: comparative study with left or right ventricle afterload.

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.

Proteomic profile of human aortic stenosis: insights into the degenerative process.

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.

Increased transcript level of poly(ADP-ribose) polymerase (PARP-1) in human tricuspid compared with bicuspid aortic valves correlates with the stenosis severity.

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.

Lipoprotein-associated phospholipase A2 is elevated in patients with severe aortic valve stenosis without clinically overt atherosclerosis.

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.

Tumor necrosis factor-α accelerates the calcification of human aortic valve interstitial cells obtained from patients with calcific aortic valve stenosis via the BMP2-Dlx5 pathway.

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.

Decreased expression and activity of cAMP phosphodiesterases in cardiac hypertrophy and its impact on beta-adrenergic cAMP signals.

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.