Fibrosis and expression of extracellular matrix proteins in human interventricular septum in aortic valve stenosis and regurgitation.

Valvular heart disease leads to ventricular pressure and/or volume overload. Pressure overload leads to fibrosis, which might regress with its resolution, but the limits and details of this reverse remodeling are not known. To gain more insight into the extent and nature of cardiac fibrosis in valve disease, we analyzed needle biopsies taken from the interventricular septum of patients undergoing surgery for valve replacement focusing on the expression and distribution of major extracellular matrix protein involved in this process. Proteomic analysis performed using mass spectrometry revealed an excellent correlation between the expression of collagen type I and III, but there was little correlation with the immunohistochemical staining performed on sister sections, which included antibodies against collagen I, III, fibronectin, sarcomeric actin, and histochemistry for wheat germ agglutinin. Surprisingly, the immunofluorescence intensity did not correlate significantly with the gold standard for fibrosis quantification, which was performed using Picrosirius Red (PSR) staining, unless multiplexed on the same tissue section. There was also little correlation between the immunohistochemical markers and pressure gradient severity. It appears that at least in humans, the immunohistochemical pattern of fibrosis is not clearly correlated with standard Picrosirius Red staining on sister sections or quantitative proteomic data, possibly due to tissue heterogeneity at microscale, comorbidities, or other patient-specific factors. For precise correlation of different types of staining, multiplexing on the same section is the best approach.

Hypoxic Culture Maintains Cell Growth of the Primary Human Valve Interstitial Cells with Stemness.

The characterization of aortic valve interstitial cells (VICs) cultured under optimal conditions is essential for understanding the molecular mechanisms underlying aortic valve stenosis. Here, we propose 2% hypoxia as an optimum VIC culture condition. Leaflets harvested from patients with aortic valve regurgitation were digested using collagenase and VICs were cultured under the 2% hypoxic condition. A significant increase in VIC growth was observed in 2% hypoxia (hypo-VICs), compared to normoxia (normo-VICs). RNA-sequencing revealed that downregulation of oxidative stress-marker genes (such as superoxide dismutase) and upregulation of cell cycle accelerators (such as cyclins) occurred in hypo-VICs. Accumulation of reactive oxygen species was observed in normo-VICs, indicating that low oxygen tension can avoid oxidative stress with cell-cycle arrest. Further mRNA quantifications revealed significant upregulation of several mesenchymal and hematopoietic progenitor markers, including CD34, in hypo-VICs. The stemness of hypo-VICs was confirmed using osteoblast differentiation assays, indicating that hypoxic culture is beneficial for maintaining growth and stemness, as well as for avoiding senescence via oxidative stress. The availability of hypoxic culture was also demonstrated in the molecular screening using proteomics. Therefore, hypoxic culture can be helpful for the identification of therapeutic targets and the evaluation of VIC molecular functions in vitro.

A Role for Peroxisome Proliferator-Activated Receptor Gamma Agonists in Counteracting the Degeneration of Cardiovascular Grafts.

ABSTRACT: Aortic valve replacement for severe stenosis is a standard procedure in cardiovascular medicine. However, the use of biological prostheses has limitations especially in young patients because of calcifying degeneration, resulting in implant failure. Pioglitazone, a peroxisome proliferator-activated receptor gamma (PPAR-gamma) agonist, was shown to decrease the degeneration of native aortic valves. In this study, we aim to examine the impact of pioglitazone on inflammation and calcification of aortic valve conduits (AoC) in a rat model. Cryopreserved AoC (n = 40) were infrarenally implanted into Wistar rats treated with pioglitazone (75 mg/kg chow; n = 20, PIO) or untreated (n = 20, controls). After 4 or 12 weeks, AoC were explanted and analyzed by histology, immunohistology, and polymerase chain reaction. Pioglitazone significantly decreased the expression of inflammatory markers and reduced the macrophage-mediated inflammation in PIO compared with controls after 4 (P = 0.03) and 12 weeks (P = 0.012). Chondrogenic transformation was significantly decreased in PIO after 12 weeks (P = 0.001). Calcification of the intima and media was significantly reduced after 12 weeks in PIO versus controls (intima: P = 0.008; media: P = 0.025). Moreover, echocardiography revealed significantly better functional outcome of the AoC in PIO after 12 weeks compared with control. Interestingly, significantly increased intima hyperplasia could be observed in PIO compared with controls after 12 weeks (P = 0.017). Systemic PPAR-gamma activation prevents inflammation as well as intima and media calcification in AoC and seems to inhibit functional impairment of the implanted aortic valve. To further elucidate the therapeutic role of PPAR-gamma regulation for graft durability, translational studies and long-term follow-up data should be striven for.

Aortic Regurgitation Is Associated With Ascending Aortic Remodeling in the Nondilated Aorta.

OBJECTIVE: The probability of aortic complications in patients with bicuspid aortic valve is higher in association with aortic regurgitation (AR) compared with aortic stenosis (AS) or normally functioning valves. The objective of this study was to determine whether this is related to the specific characteristics of aneurysmatic dilatation that includes AR or whether AR itself has a negative impact on the aortic wall, independent of aneurysmatic dilatation. Approach and Results: Nondilated aortic specimens were harvested intraoperatively from individuals with tricuspid aortic valves and either AS (n=10) or AR (n=16). For controls, nondilated aortas were harvested during autopsies from individuals with tricuspid aortic valves and no evidence of aortic valve disease (n=10). Histological and immunohistochemical analyses revealed that compared with control aortas, overall medial degeneration was more severe in AR-aortas (P=0.005) but not AS-aortas (P=0.23). This pathological remodeling included mucoid extracellular matrix accumulation (P=0.005), elastin loss (P=0.003), elastin fragmentation (P=0.008), and decreased expression of fibrillin (P=0.003) and collagen (P=0.008). Furthermore, eNOS (endothelial nitric oxide synthase) expression was decreased in the intima (P=0.0008) and in vasa vasorum (P=0.004) of AR-aortas but not AS-aortas (all P>0.05). Likewise, subendothelial apoptosis was increased in AR-aortas (P=0.03) but not AS-aortas (P=0.50). CONCLUSIONS: AR has a negative effect on the nondilated ascending aortic wall. Accordingly, our results support the need for more detailed studies of the aortic wall in relation to aortic valve disease and may ultimately lead to more aggressive clinical monitoring and/or surgical criteria for patients with relevant AR. Graphic Abstract: A graphic abstract is available for this article.

Molecular recognition of aortic valve stenosis and regurgitation.

OBJECTIVE: Aortic valve stenosis (AS) presents a disease during which there are changes of the aortic valve structure that modify the blood structure of patients. The aim of this study was to improve the effectiveness of differential diagnostics of aortic stenosis and aortic regurgitation using molecular techniques on both mRNA (RT-PCR) and protein (biochip protein). PATIENTS AND METHODS: An experimental group (n = 58) consisting of patients with aortic valve stenosis (n = 26) and aortic regurgitation (AR, n = 32) was compared with a control group (n = 35). Both blood serum and valve tissue samples were used for the determination of gene expression specific genes related to inflammatory response (CRP, IL6, IL2R, IL6R, TNFR1, and 2) as well as genes and proteins involved in remodeling of the extracellular matrix (MMP9, TIMP, Emilin-1). RESULTS: We found that hsCRP and IL6 plasma levels of patients with AS were higher than both controls and patients with AR (mean 5.6 ng/ml). The differences between AS and AR were detected only in mRNA levels of MMP9 and TIMP where increased levels characteristic for AS were found (about 74%, p < 0.01 and 87%, p < 0.001 higher than AR). CONCLUSIONS: The achieved results could contribute to the improvement of early diagnosis of selected cardiovascular disease in the future and improve the quality of patient's life.

Interaction Between Sacubitril and Valsartan in Preventing Heart Failure Induced by Aortic Valve Insufficiency in Rats.

BACKGROUND: Synergistic interactions between neprilysin inhibition (NEPi) with sacubitril and angiotensin receptor type1 blockade (ARB) with valsartan have been implicated in improvement of left ventricular (LV) contractility, relaxation, exercise tolerance, and fibrosis in preexisting heart failure (HF) induced by aortic valve insufficiency (AVI). It is not known whether this pharmacologic synergy can prevent cardiovascular pathology in a similar AVI model. Our aim was to investigate the pharmacology of sacubitril/valsartan in an experimental setting with therapy beginning immediately after creation of AVI. METHODS: HF was induced through partial disruption of the aortic valve in rats. Therapy began 3 hours after valve disruption and lasted 8 weeks. Sacubitril/valsartan (68 mg/kg), valsartan (31 mg/kg), sacubitril (31 mg/kg), or vehicle were administered daily via oral gavage (N=8 in each group). Hemodynamic assessments were conducted using Millar technology, and an exercise tolerance test was conducted using a rodent treadmill. RESULTS: Only sacubitril/valsartan increased total arterial compliance and ejection fraction (EF). Therapies with sacubitril/valsartan and valsartan similarly improved load-dependent (dP/dtmax) and load independent indices (Ees) of LV contractility, and exercise tolerance, whereas sacubitril did not. None of the therapies improved LV relaxation (dP/dtmin), whereas all reduced myocardial fibrosis. CONCLUSIONS: 1) The synergistic interaction between NEPi and ARB in early therapy with sacubitril/valsartan leads to increased total arterial compliance and EF. 2) Improvement in indices of LV contractility, and exercise tolerance with sacubitril/valsartan is likely because of ARB effect of valsartan. 3) All three therapies provided antifibrotic effects, suggesting both ARB and NEPi are capable of reducing myocardial fibrosis.

Increased mesenchymal podoplanin expression is associated with calcification in aortic valves.

BACKGROUND AND AIM OF THE STUDY: Calcific aortic valve disease (CAVD) is a progressive disease starting from mild valvular sclerosis and progressing to severe aortic stenosis (AS) with calcified valves. The origin of the calcification is proposed to be mesenchymal cells which have differentiated towards an osteoblastic phenotype. Podoplanin is a glycoprotein expressed in the endothelium of lymphatic vessels and in osteoblasts and osteocytes, mesenchymal cells, as well as in many carcinomas and aortic atherosclerotic lesions. In CAVD, its expression has been evaluated only as a marker of the lymphatic vasculature. MATERIALS AND METHODS: We determined podoplanin expression in human aortic valves in four patient groups: control (C, n=7), aortic regurgitation (AR, n=8), aortic regurgitation and fibrosis (AR + f, n=15) and AS (n=49) by immunohistochemistry and quantitative real-time PCR (RT-PCR). RESULTS: Immunohistochemically, podoplanin expression was significantly increased in AR + f and AS groups when compared with the control and AR groups and the level of expression positively correlated with the extent of calcification and vascularity. Podoplanin mRNA levels were 1.7-fold higher in the AS group as compared with the control group (P=.05). Podoplanin-positivity was present not only in lymphatic vessel endothelium but also in osteoblasts, osteocytes, chondrocytes, macrophages and extracellular matrix. The majority of the podoplanin-positivity was in spindle cells with a myofibroblastic phenotype, often associated with calcifications. Tricuspid valves had more calcification-associated podoplanin than bi/unicuspid valves (median 1.52 vs 1.16, P<.001). CONCLUSIONS: CAVD is characterized by an increased expression of podoplanin; this is associated with the differentiation of valvular interstitial cells into calcium-producing, myofibroblast-like cells. In addition, tricuspid valves express relatively more podoplanin than bi/unicuspid valves.

Degenerative aortic valve disease and diabetes: Implications for a link between proteoglycans and diabetic disorders in the aortic valve.

Degenerative aortic valve disease in combination with diabetes is an increasing burden worldwide. There is growing evidence that particularly small leucine-rich proteoglycans are involved in the development of degenerative aortic valve disease. Nevertheless, the role of these molecules in this disease in the course of diabetes has not been elucidated in detail and previous studies remain controversial. Therefore, the aim of this study is to broaden the knowledge about small leucine-rich proteoglycans in degenerative aortic valve disease and the influence of diabetes and hyperglycaemia on aortic valves and valvular interstitial cells is examined. Analyses were performed using reverse-transcription polymerase chain reaction, Western blot, enzyme-linked immunosorbent assay, (immuno)histology and colorimetric assays. We could show that biglycan, but not decorin and lumican, is upregulated in degenerated human aortic valve cusps. Subgroup analysis reveals that upregulation of biglycan is stage-dependent. In vivo, loss of biglycan leads to stage-dependent calcification and also to migratory effects on interstitial cells within the extracellular matrix. In late stages of degenerative aortic valve disease, diabetes increases the expression of biglycan in aortic valves. In vitro, the combinations of hyperglycaemic with pro-degenerative conditions lead to an upregulation of biglycan. In conclusion, biglycan represents a potential link between degenerative aortic valve disease and diabetes.

Effects of the cardiac myosin activator Omecamtiv-mecarbil on severe chronic aortic regurgitation in Wistar rats.

BACKGROUND: Aortic regurgitation (AR) is a valvular disease that can lead to systolic heart failure. Treatment options besides cardiac surgery are limited and consequently severe AR is associated with higher mortality and morbidity when not operated. In this investigation, we examined the effects of a novel cardiac myosin activator, Omecamtiv-mecarbil (OM), in rats with chronic severe AR. METHODS: AR was created by retrograde puncture of the aortic valve leaflets in 20 adults Wistar rats. 12 animals survived the acute AR phase and were randomized 2 months thereafter into OM (n = 7) or placebo groups (n = 5). Two rats underwent a sham operation and served as controls. Equal volumes of OM or placebo (NaCl 0.9%) were perfused in the femoral vein by continuous infusion (1.2 mg/kg/hour) during 30 min. Doppler-echocardiography was performed before and at the end of the infusion periods. RESULTS: OM increased indices of global cardiac function (cardiac output, stroke volume), and increased systolic performance (fractional shortening, ejection fraction, left ventricular end systolic diameter) (all p < 0.05). These effects concurred with decreases in indices of LV preload (left atrial size, left ventricular end diastolic diameter) as well in the aortic pre-ejection period / left ventricular ejection time ratio (all p < 0.05). The severity score of the regurgitant AR jet did not change. Placebo infusion did not affect these parameters. CONCLUSION: The cardiac myosin activator OM exerts favorable hemodynamic effects in rats with experimental chronic AR.

Aortic stenosis and aortic regurgitation express different titin isoforms: Differences and relationships with functional and geometric characteristics.

Background-Titin represents an important biomechanical sensor which determines compliance and diastolic/systolic function of the left ventricle (LV). To assess the different titin-isoform expression and the relationships with functional and geometric patterns, we analyzed titin-isoform expression and cardiomyocytes contractile function in myocardial biopsy samples of patients undergoing aortic valve replacement (AVR) for aortic stenosis (AS) and for aortic regurgitation (AR). Method -Specimens, collected from the LV of 35 with AS and 35 with AR undergoing AVR were analyzed for titin-isoform expression and cardiomyocytes force measurement. Ten donor hearts were analyzed as controls for normal values. Results were implemented with preoperative geometry and function assessed by Doppler echocardiography. Results-Compared to controls, N2BA/N2B titin-isoforms ratio was reduced to 0.24 in AS (p < 0.001) but increased to 0.51 in AR (p < 0.001). N2BA/N2B titin-isoforms ratio was further reduced in 8 patients with severe (restrictive) diastolic dysfunction (0.17 ±â€¯0.03, p < 0.001) but was increased in patients with severe systolic dysfunction (0.58 ±â€¯0.07, p < 0.001). As compared to controls, Fpasive was higher in AS (6.7 ±â€¯0.2 vs 4.4 ± 0.4 kN/m2, p < 0.001) but was lower in AR (3.7 ±â€¯0.2 vs 4.4 ±â€¯0.4 kN/m2, p < 0.001). Total force was comparable. Fpassive was significantly higher in AS patients with severe than with moderate LV diastolic dysfunction (7.1 ± 0.5 vs 6.6. ±â€¯0.6, p = 0.004). Conclusions-titin-isoform expression differs in AS and AR as adaptive response to different pathophysiologic scenarios. Co-expressing isoforms at varying ratios results in modulation of the passive mechanical behavior of the LV at different degree of dysfunction and allows for compensative adjustment of the diastolic/systolic properties of the myocardium.

Differential cardiac hypertrophy and signaling pathways in pressure versus volume overload.

Mechanical overload can be classified into pressure overload and volume overload, causing concentric and eccentric cardiac hypertrophy, respectively. Here, we aimed to differentiate the load-mediated signaling pathways involved in pressure versus volume overload cardiac hypertrophy. Pressure or volume overload was imposed on C57BL/6J mice by transverse aortic constriction (TAC) or aortic regurgitation (AR), respectively. After surgery (2 wk), left ventricular structure and function were evaluated by echocardiographic, hemodynamic, and histological analyses. Signaling pathways related to hypertrophy, fibrosis, angiogenesis, and apoptosis were studied by histological analysis, RT-PCR, and Western blot analysis. Although mean wall stress was similar in both TAC and AR mice, systolic wall stress was significantly increased in TAC and diastolic wall stress was mainly elevated in AR. TAC or AR induced concentric or eccentric compensated hypertrophy, respectively. TAC was associated with more significant fibrosis and apoptosis, whereas AR was associated with more significant angiogenesis. MAPK kinase family, ß-arrestin-2, Akt, and Ca2+-related signaling pathways were markedly activated in TAC but mildly upregulated or unchanged in AR. Pressure overload and volume overload induce different phenotypic and molecular adaptations in cardiac hypertrophy. Most load-related signaling pathways assessed in this study predominate in pressure but not volume overload. The stimulus-specific heterogeneity in the signaling pathways requires distinct manipulations for further mechanistic and pharmacological studies. NEW & NOTEWORTHY Using the transverse aortic constriction mouse model and the newly developed aortic regurgitation mouse model, we delineated the prominent differences between concentric and eccentric cardiac hypertrophy on morphological, functional, and molecular levels. Our findings are important for the precise diagnosis and treatment of these two types of cardiac hypertrophy. Listen to this article's corresponding podcast at http://ajpheart.podbean.com/e/chinese-english-language-podcast-on-differential-cardiac-remodeling-in-tac-vs-ar/ .

Early Activation of Growth Pathways in Mitral Leaflets Exposed to Aortic Regurgitation: New Insights from an Animal Model.

BACKGROUND AND AIM OF THE STUDY: Mitral leaflet enlargement in patients with chronic aortic regurgitation (AR) has been identified as an adaptive mechanism potentially able to prevent functional mitral regurgitation (FMR) in response to left ventricular (LV) dilatation. The timing of valve enlargement is not known, and the related mechanisms are largely unexplored. METHODS: AR was induced in 58 rats, and another 54 were used as sham controls. Animals were euthanized at different time points after AR creation (48 h, one week, and three months), and AR severity, FMR and LV dilatation were assessed using echocardiography. Mitral valves were harvested to document the reactivation of embryonic growth pathways. RESULTS: AR animals had increased LV dimensions and mitral annulus size. No animal developed FMR. No change in leaflet length or thickness was seen at 48 h; however, anterior mitral leaflets were longer and thicker in AR animals at one week and three months. Molecular changes were present early (at 48 h and at one week), with positive staining for transforming growth factor-b1 (TGF-b1), Alpha-smooth muscle actin (α-SMA) and matrix metalloproteinase-2 (MMP-2), which suggested active matrix remodeling. Increased gene expression for collagen 1, TGF-ß1, α-SMA and MMP-2 was found in the mitral valve at 48 h and at one week, but after three months their expression had returned to normal. CONCLUSIONS: This model of AR induces active expansion and thickening of the mitral leaflets. Growth signals are expressed acutely, but not at three months, which suggests that most of this enlargement occurs at an early stage. The stimulation of valvular growth could represent a new strategy for the prevention of FMR.

Interplay of mitochondria apoptosis regulatory factors and microRNAs in valvular heart disease.

Valvular heart disease (VHD) is an active process involving a wide range of pathological changes. The major complications of VHD are stenosis and regurgitation, which are macroscopic phenomena, induced in part through cellular changes. Altered expression of mitochondria associated genes causes membrane potential depolarization, leading to the increased levels of apoptosis observed in cardiac dysfunction. Objective of this study is to find molecular medicine candidates that can control expression of the key mitochondria apoptosis regulatory genes. Present study aims to assess the way microRNA are involved in regulating mitochondrial apoptosis regulatory genes and observation of their expression in the heart valve dysfunction. Apoptotic genes PUMA and DRP1 were found to be highly expressed, whereas anti-apoptotic gene ARC was down regulated. The expression level of GATA-4 transcription factor was also reduced in cardiac valve tissues. MicroRNAs miR-15a and miR-29a were repressed, while miR-214 was up regulated. Furthermore, study showed that PUMA, DRP1 and ARC expression might be attenuated by their respective miRNAs. Our results indicate that mitochondria regulatory genes might be controlled by miR-15a, miR-29a and miR-214, in VHD patients. Present study may provide platform for future research regarding potential therapeutic role of miRNAs in CVDs.

Calcification and Oxidative Modifications Are Associated With Progressive Bioprosthetic Heart Valve Dysfunction.

BACKGROUND: Bioprosthetic heart valves (BHVs), fabricated from glutaraldehyde-pretreated bovine pericardium or porcine aortic valves, are widely used for the surgical or interventional treatment of heart valve disease. Reoperation becomes increasingly necessary over time because of BHV dysfunction. METHODS AND RESULTS: Forty-seven explanted BHV aortic valve replacements were retrieved at reoperation for clinically severe BHV dysfunction over the period 2010-2016. Clinical explant analyses of BHV leaflets for calcium (atomic absorption spectroscopy) and oxidized amino acids, per mass spectroscopy, were primary end points. Comorbidities for earlier BHV explant included diabetes mellitus and coronary artery bypass grafting. Mean calcium levels in BHV leaflets were significantly increased compared with unimplanted BHV (P<0.001); however, time to reoperation did not differ comparing calcified and noncalcified BHV. BHV dityrosine, an oxidized amino acid cross-link, was significantly increased in the explants (227.55±33.27 µmol/mol [dityrosine/tyrosine]) but was undetectable in unimplanted leaflets (P<0.001). BHV regional analyses revealed that dityrosine, ranging from 57.5 to 227.8 µmol/mol (dityrosine/tyrosine), was detectable only in the midleaflet samples, indicating the site-specific nature of dityrosine formation. 3-Chlorotyrosine, an oxidized amino acid formed by myeloperoxidase-catalyzed chlorinating oxidants, correlated with BHV calcium content in leaflet explant analyses from coronary artery bypass graft patients (r=0.62, P=0.01) but was not significantly correlated with calcification in non-coronary artery bypass graft explanted BHV. CONCLUSIONS: Both increased BHV leaflet calcium levels and elevated oxidized amino acids were associated with bioprosthesis dysfunction necessitating reoperation; however, BHV calcium levels were not a determinant of implant duration, indicating a potentially important role for oxidized amino acid formation in BHV dysfunction.

The influence of pump rotation speed on hemodynamics and myocardial oxygen metabolism in left ventricular assist device support with aortic valve regurgitation.

Aortic valve regurgitation (AR) is a serious complication under left ventricular assist device (LVAD) support. AR causes LVAD-left ventricular (LV) recirculation, which makes it difficult to continue LVAD support. However, the hemodynamics and myocardial oxygen metabolism of LVAD support with AR have not been clarified, especially, how pump rotation speed influences them. An animal model of LVAD with AR was newly developed, and how pump rotation speed influences hemodynamics and myocardial oxygen metabolism was examined in acute animal experiments. Five goats (55 ± 9.3 kg) underwent centrifugal type LVAD, EVAHEART implantation. The AR model was established by placing a vena cava filter in the aortic valve. Hemodynamic values and the myocardial oxygen consumption, delivery, and oxygen extraction ratio (O2ER) were evaluated with changing pump rotation speeds with or without AR (AR+, AR-). AR+ was defined as Sellers classification 3 or greater. AR was successfully induced in five goats. Diastolic aortic pressure was significantly lower in AR+ than AR- (p = 0.026). Central venous pressure, mean left atrial pressure, and diastolic left ventricular pressure were significantly higher in AR+ than AR- (p = 0.010, 0.047, and 0.0083, respectively). Although systemic flow did not improve with increasing pump rotation speed, LVAD pump flow increased over systemic flow in AR+, which meant increasing pump rotation speed increased LVAD-LV recirculation and did not contribute to effective systemic circulation. O2ER in AR- decreased with increasing pump rotation speed, but O2ER in AR+ was hard to decrease. The O2ER in AR+ correlated positively with the flow rate of LVAD-LV recirculation (p = 0.012). AR caused LVAD-LV recirculation that interfered with the cardiac assistance of LVAD support and made it ineffective to manage with high pump rotation speed.

Loss of Axin2 results in impaired heart valve maturation and subsequent myxomatous valve disease.

AIMS: Myxomatous valve disease (MVD) is the most common aetiology of primary mitral regurgitation. Recent studies suggest that defects in heart valve development can lead to heart valve disease in adults. Wnt/ß-catenin signalling is active during heart valve development and has been reported in human MVD. The consequences of increased Wnt/ß-catenin signalling due to Axin2 deficiency in postnatal valve remodelling and pathogenesis of MVD were determined. METHODS AND RESULTS: To investigate the role of Wnt/ß-catenin signalling, we analysed heart valves from mice deficient in Axin2 (KO), a negative regulator of Wnt/ß-catenin signalling. Axin2 KO mice display enlarged mitral and aortic valves (AoV) after birth with increased Wnt/ß-catenin signalling and cell proliferation, whereas Sox9 expression and collagen deposition are decreased. At 2 months in Axin2 KO mice, the valve extracellular matrix (ECM) is stratified but distal AoV leaflets remain thickened and develop aortic insufficiency. Progressive myxomatous degeneration is apparent at 4 months with extensive ECM remodelling and focal aggrecan-rich areas, along with increased BMP signalling. Infiltration of inflammatory cells is also observed in Axin2 KO AoV prior to ECM remodelling. Overall, these features are consistent with the progression of human MVD. Finally, Axin2 expression is decreased and Wnt/ß-catenin signalling is increased in myxomatous mitral valves in a murine model of Marfan syndrome, supporting the importance of Wnt/ß-catenin signalling in the development of MVD. CONCLUSIONS: Altogether, these data indicate that Axin2 limits Wnt/ß-catenin signalling after birth and allows proper heart valve maturation. Moreover, dysregulation of Wnt/ß-catenin signalling resulting from loss of Axin2 leads to progressive MVD.

Krox20 heterozygous mice: A model of aortic regurgitation associated with decreased expression of fibrillar collagen genes.

BACKGROUND: The mechanism involved in the onset of aortic valve (AoV) disease remains unclear despite its poor prognosis and frequency. Recently, we reported that Krox20 (EGR2 in humans) is involved in AoV development and dysfunction. AIM: Analyze Krox20 heterozygous mice (Krox20(+/-)) to discover whether incomplete expression of Krox20 can cause valvular diseases. METHODS: Transcriptional levels of Col1a2/COL1A2 and Krox20/EGR2 in AoVs from Krox20(+/-) mice and human patients operated on for severe aortic regurgitation were evaluated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Human control valves were obtained from three transplanted patients without AoV disease. Twenty-one heterozygous Krox20(+/-) mice were compared with 35 controls at different ages. Three independent measurements of valve thickness were performed on magnified tissue sections using Image J software. In vivo valve structure and function were evaluated using the high-frequency Vevo(®) 2100 echocardiogram. RESULTS: qRT-PCR analysis using AoVs from patients with severe aortic regurgitation showed a decrease in EGR2 expression associated with significant downregulation of COL1A2 expression (P<0.05). Similar results were observed in the AoVs of Krox20(+/-) mice. Anatomical examination revealed that incomplete invalidation of Krox20 caused significant thickening of the aortic leaflet compared with controls (145±22 vs. 75±24µm; P=0.01). Within the mutant group, this thickening worsened significantly over time (Krox20(+/-) mice aged>7 vs.<7months: 136±48 vs. 102±41µm; P<0.001). Moreover, the aortic leaflets of embryonic day 18.5 Krox20(+/-) embryos were significantly more thickened than those from controls, suggesting that this disease begins during embryonic development. Echo-Doppler analysis showed a significant increase in AoV dysfunction in heterozygous versus control mice (53% vs. 17%; P<0.001), suggesting a tight relationship between valve architecture and function. Morphometric analysis revealed that the most severe AoV dysfunction was always associated with the most thickened valves. Classic histological analysis revealed that mutant AoVs had extracellular matrix disorganization, with features of human myxomatous degeneration, including excess of proteoglycan deposition in spongiosa and reduction of collagen fibre in fibrosa, but no calcification. CONCLUSION: Decreased expression of Krox20 in mice causes degeneration of the aortic leaflets and disorganization of the extracellular matrix, causing valvular dysfunction.

Endurance training or beta-blockade can partially block the energy metabolism remodeling taking place in experimental chronic left ventricle volume overload.

BACKGROUND: Patients with chronic aortic valve regurgitation (AR) causing left ventricular (LV) volume overload can remain asymptomatic for many years despite having a severely dilated heart. The sudden development of heart failure is not well understood but alterations of myocardial energy metabolism may be contributive. We studied the evolution of LV energy metabolism in experimental AR. METHODS: LV glucose utilization was evaluated in vivo by positron emission tomography (microPET) scanning of 6-month AR rats. Sham-operated or AR rats (n = 10-30 animals/group) were evaluated 3, 6 or 9 months post-surgery. We also tested treatment intervention in order to evaluate their impact on metabolism. AR rats (20 animals) were trained on a treadmill 5 times a week for 9 months and another group of rats received a beta-blockade treatment (carvedilol) for 6 months. RESULTS: MicroPET revealed an abnormal increase in glucose consumption in the LV free wall of AR rats at 6 months. On the other hand, fatty acid beta-oxidation was significantly reduced compared to sham control rats 6 months post AR induction. A significant decrease in citrate synthase and complex 1 activity suggested that mitochondrial oxidative phosphorylation was also affected maybe as soon as 3 months post-AR.Moderate intensity endurance training starting 2 weeks post-AR was able to partially normalize the activity of various myocardial enzymes implicated in energy metabolism. The same was true for the AR rats treated with carvedilol (30 mg/kg/d). Responses to these interventions were different at the level of gene expression. We measured mRNA levels of a number of genes implicated in the transport of energy substrates and we observed that training did not reverse the general down-regulation of these genes in AR rats whereas carvedilol normalized the expression of most of them. CONCLUSION: This study shows that myocardial energy metabolism remodeling taking place in the dilated left ventricle submitted to severe volume overload from AR can be partially avoided by exercise or beta-blockade in rats.

Loss of Krox20 results in aortic valve regurgitation and impaired transcriptional activation of fibrillar collagen genes.

AIMS: Heart valve maturation is achieved by the organization of extracellular matrix (ECM) and the distribution of valvular interstitial cells. However, the factors that regulate matrix components required for valvular structure and function are unknown. Based on the discovery of its specific expression in cardiac valves, we aimed to uncover the role of Krox20 (Egr-2) during valve development and disease. METHODS AND RESULTS: Using series of mouse genetic tools, we demonstrated that loss of function of Krox20 caused significant hyperplasia of the semilunar valves, while atrioventricular valves appeared normal. This defect was associated with an increase in valvular interstitial cell number and ECM volume. Echo Doppler analysis revealed that adult mutant mice had aortic insufficiency. Defective aortic valves (AoVs) in Krox20(-/-) mice had features of human AoV disease, including excess of proteoglycan deposition and reduction of collagen fibres. Furthermore, examination of diseased human AoVs revealed decreased expression of KROX20. To identify downstream targets of Krox20, we examined expression of fibrillar collagens in the AoV leaflets at different stages in the mouse. We found significant down-regulation of Col1a1, Col1a2, and Col3a1 in the semilunar valves of Krox20 mutant mice. Utilizing in vitro and in vivo experiments, we demonstrated that Col1a1 and Col3a1 are direct targets of Krox20 activation in interstitial cells of the AoV. CONCLUSION: This study identifies a previously unknown function of Krox20 during heart valve development. These results indicate that Krox20-mediated activation of fibrillar Col1a1 and Col3a1 genes is crucial to avoid postnatal degeneration of the AoV leaflets.