Nancy Côté

Characterization of Novel Phosphodiesterases in the Bovine Ovarian Follicle

Abstract The phosphodiesterase (PDE) family is a group of enzymes that catalyzes the transformation of cyclic nucleotides into 5′ nucleotides. Based on rodents, the current mammalian model of PDE distribution in the ovarian follicle predicts Pde3a in the oocyte and Pde4d in the somatic cells. Using bovine as an experimental model, the present results showed that PDE3 was the predominant PDE activity in oocytes. However, cumulus cell cAMP-PDE activity was predominantly resistant to inhibition by 3-isobutyl-methylxantine, indicating PDE8 activity (60% of total PDE activity) and a minor role for PDE4 (<5%). A total of 20% of total oocyte PDE activity was also attributed to PDE8. The PDE activity measurements in mural granulosa cells from 2 to 6 mm in diameter suggest the presence of PDE4 and PDE8. In granulosa cells from follicles >10 mm, total PDE and PDE8 activities along with PDE8A protein level were increased compared with smaller follicles. The RT-PCR experiments showed that cumulus cells expressed PDE8A, PDE8B, and PDE10A. Western blot experiments showed PDE8A, PDE8B, and PDE4D proteins in mural granulosa cells and cumulus-oocyte complexes. PDE8 inhibition using dipyridamole in a dose-dependent manner increased cAMP levels in the cumulus-oocyte complexes and delayed oocyte nuclear maturation. These results are the first to demonstrate the functional presence of PDE8 in the mammalian ovarian follicle. This challenges the recently described cell-specific expression of cAMP-PDEs in the ovarian follicle and the notion that PDE4 is the predominant granulosa/cumulus cell PDE. These findings have implications for our understanding of hormonal regulation of folliculogenesis and the potential application of PDE inhibitors as novel contraceptives.

Impact of Metabolic Syndrome on Progression of Aortic Stenosis Influence of Age and Statin Therapy

OBJECTIVES The aims of this study were to examine prospectively the relationship between metabolic syndrome (MetS) and aortic stenosis (AS) progression and to evaluate the effect of age and statin therapy on AS progression in patients with or without MetS. BACKGROUND Despite the clear benefits of statin therapy in primary and secondary coronary heart disease prevention, several recent randomized trials have failed to demonstrate any significant effect of this class of drugs on the progression of AS. Previous retrospective studies have reported an association between MetS and faster AS progression. METHODS This predefined substudy included 243 of the 269 patients enrolled in the ASTRONOMER (AS Progression Observation: Measuring Effects of Rosuvastatin) trial. Follow-up was 3.4 ± 1.3 years. AS progression rate was measured by calculating the annualized increase in peak aortic jet velocity measured by Doppler echocardiography. RESULTS Patients with MetS (27%) had faster stenosis progression (+0.25 ± 0.21 m/s/year vs. +0.19 ± 0.19 m/s/year, p = 0.03). Predictors of faster AS progression in multivariate analysis were older age (p = 0.01), higher degree of valve calcification (p = 0.01), higher peak aortic jet velocity at baseline (p = 0.007), and MetS (p = 0.005). Impact of MetS on AS progression was most significant in younger (< 57 years) patients (MetS: +0.24 ± 0.19 m/s/year vs. no MetS: +0.13 ± 0.18 m/s/year, p = 0.008) and among patients receiving statin therapy (+0.27 ± 0.23 m/s/year vs. +0.19 ± 0.18 m/s/year, p = 0.045). In multivariate analysis, the MetS-age interaction was significant (p = 0.01), but the MetS-statin use interaction was not. CONCLUSIONS MetS was found to be a powerful and independent predictor of faster AS progression, with more pronounced impact in younger patients. These findings emphasize the importance of routinely identifying and treating MetS in AS patients. The apparent faster stenosis progression in the subset of normocholesterolemic patients with MetS receiving the statin will need to be confirmed by future studies.

Increased Biglycan in Aortic Valve Stenosis Leads to the Overexpression of Phospholipid Transfer Protein via Toll-Like Receptor 2

Aortic stenosis (AS) is the most common valvular heart disease, and it is suspected that atherosclerotic mechanisms are involved in the development of this disorder. Therefore, the retention of lipids within the aortic valve may play a role in the pathobiology of AS. In this study, a gene expression microarray experiment was conducted on human aortic valves with and without AS. The expression levels of transcripts encoding proteoglycans and enzymes involved in lipid retention were compared between the two groups. The microarray results were subsequently replicated in a cohort of 87 AS valves and 36 control valves. In addition, the interaction between proteoglycan and lipid-modifying enzyme was documented in isolated valve interstitial cells (VICs). The microarray results indicated that only biglycan (BGN) and phospholipid transfer protein (PLTP) were overexpressed in the AS valves. These results were then confirmed by quantitative PCR. The immunohistochemical analysis revealed a colocalization of BGN, PLTP, and Toll-like receptor-2 (TLR 2) in AS valves. In vitro, we showed that BGN induces the production of PLTP in VICs via the stimulation of TLR 2. Thus, increased accumulation of BGN in AS valves contributes to the production of PLTP via TLR 2. These results suggest that intricate links between valve matrix proteins, inflammation, and lipid retention are involved in the pathobiology of AS.

Clinical ResearchValve DiseaseImpact of Metabolic Syndrome on Progression of Aortic Stenosis: Influence of Age and Statin Therapy

OBJECTIVES The aims of this study were to examine prospectively the relationship between metabolic syndrome (MetS) and aortic stenosis (AS) progression and to evaluate the effect of age and statin therapy on AS progression in patients with or without MetS. BACKGROUND Despite the clear benefits of statin therapy in primary and secondary coronary heart disease prevention, several recent randomized trials have failed to demonstrate any significant effect of this class of drugs on the progression of AS. Previous retrospective studies have reported an association between MetS and faster AS progression. METHODS This predefined substudy included 243 of the 269 patients enrolled in the ASTRONOMER (AS Progression Observation: Measuring Effects of Rosuvastatin) trial. Follow-up was 3.4 ± 1.3 years. AS progression rate was measured by calculating the annualized increase in peak aortic jet velocity measured by Doppler echocardiography. RESULTS Patients with MetS (27%) had faster stenosis progression (+0.25 ± 0.21 m/s/year vs. +0.19 ± 0.19 m/s/year, p = 0.03). Predictors of faster AS progression in multivariate analysis were older age (p = 0.01), higher degree of valve calcification (p = 0.01), higher peak aortic jet velocity at baseline (p = 0.007), and MetS (p = 0.005). Impact of MetS on AS progression was most significant in younger (< 57 years) patients (MetS: +0.24 ± 0.19 m/s/year vs. no MetS: +0.13 ± 0.18 m/s/year, p = 0.008) and among patients receiving statin therapy (+0.27 ± 0.23 m/s/year vs. +0.19 ± 0.18 m/s/year, p = 0.045). In multivariate analysis, the MetS-age interaction was significant (p = 0.01), but the MetS-statin use interaction was not. CONCLUSIONS MetS was found to be a powerful and independent predictor of faster AS progression, with more pronounced impact in younger patients. These findings emphasize the importance of routinely identifying and treating MetS in AS patients. The apparent faster stenosis progression in the subset of normocholesterolemic patients with MetS receiving the statin will need to be confirmed by future studies.

New insight into the role of phosphodiesterase 3A in porcine oocyte maturation

BackgroundThe ovulatory surge of gonadotropins triggers oocyte maturation and rupture of the ovarian follicle. The resumption of nuclear maturation in the oocyte from the prophase stage is characterized by germinal vesicle breakdown (GVBD). It has previously been shown that specific inhibition of cAMP degradation by PDE3 prevents the resumption of oocyte meiosis. However, no report has characterized the activity of PDE3 in the porcine oocyte, or the implication of the cAMP-PDE3 pathway in the entire nuclear maturation process. In this study, PDE3 activity in the oocyte was assessed during in vitro maturation (IVM) and the possible roles of the cAMP-PDE3 pathway in the resumption and progression of meiosis were investigated in terms of different models of oocyte maturation.ResultsCyclic AMP-degrading PDE activity was detected in the cumulus-oocyte complex (COC) and was partially inhibited by a specific PDE3 inhibitor, cilostamide. When measured only in the denuded oocyte, PDE activity was almost completely inhibited by cilostamide, suggesting that cAMP-PDE3 activity is the major cAMP-PDE in porcine oocytes. PDE3A mRNA was detected by RT-PCR. PDE3 activity did not vary significantly during the early hours of IVM, but a maximum was observed at 13 hours. In cumulus-oocyte complexes, meiosis resumed after 20.81 hours of culture. PDE3 inhibition no longer maintained meiotic arrest if sustained beyond 17.65 hours of IVM, 3 hours prior to resumption of meiosis. Thereafter, PDE3 inhibition progressively lost its efficacy in GVBD. When the protein phosphatase 1 and 2A inhibitor okadaic acid was continuously or transiently (3 hours) present during IVM, meiosis resumed prematurely; PDE3 inhibition was unable to prevent GVBD. However, PDE3 inhibition in COC treated with OA for 3 hours significantly delayed meiosis at the intermediate stage.ConclusionThe present investigation has demonstrated that PDE3A is the major cAMP-degrading PDE in the oocyte. It regulates the resumption of meiosis until 3 hours prior to GVBD and transiently affects meiotic progression.

ATP acts as a survival signal and prevents the mineralization of aortic valve

Calcific aortic valve disease (CAVD) is a disorder related to progressive mineralization of valvular tissue that is a leading cause of heart disease. Thus far, there is no medical treatment to prevent the mineralization of aortic valves. It is generally thought that pathologic mineralization is linked to apoptosis of vascular cells. However, the role of apoptosis during mineralization as well as the survival signals for valvular interstitial cells (VICs), the main cellular component of aortic valves, remains to be identified. Here, through several lines of evidence, we show that bioavailability of extracellular ATP is a signal which determines survival or apoptosis of VICs and, in doing so, plays a major role in the development of CAVD. Specifically, in CAVD and in VIC cultures undergoing mineralization, we found a high level of the ectonucleotidase ENPP1. In addition, a genetic polymorphism in the intron 9 of the ENPP1 gene was associated with CAVD in a case-control cohort as well as with mRNA expression levels of ENPP1 in aortic valves. A high level of ENPP1 in CAVD promoted apoptosis-mediated mineralization of VICs by depleting the extracellular pool of ATP. We then documented that release of ATP by VICs promoted cell survival via the P2Y(2) receptor and the PI3K/Akt signaling pathway. Hence, our results show that level of ENPP1 modulates extracellular concentration of ATP, which is an important survival signal for VICs. These findings may help to develop novel pharmacological treatment for CAVD.

Angiotensin receptor blockers are associated with a lower remodelling score of stenotic aortic valves

Eur J Clin Invest 2011; 41 (11): 1172–1179

Inhibition of ectonucleotidase with ARL67156 prevents the development of calcific aortic valve disease in warfarin-treated rats

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

Impact of hypertension and renin-angiotensin system inhibitors in aortic stenosis.

Experimental studies revealed that renin–angiotensin system (RAS) could play a crucial role in the pathophysiology of aortic stenosis (AS). The objectives of this study were to examine (i) the impact of hypertension on AS progression and clinical events and (ii) the effect of angiotensin‐converting enzyme inhibitors (ACEIs) and angiotensin‐receptor blockers (ARBs).

Insulin Resistance and LVH Progression in Patients With Calcific Aortic Stenosis: A Substudy of the ASTRONOMER Trial

OBJECTIVES The objective of this substudy of the ASTRONOMER (Aortic Stenosis Progression Observation: Measuring Effects of Rosuvastatin) trial was to examine the association between insulin resistance and progression of left ventricular hypertrophy (LVH) in patients with aortic stenosis (AS). BACKGROUND In a recent cross-sectional study, the authors reported that the metabolic syndrome was associated with an increased prevalence of concentric LVH in patients with AS. As a central feature of the metabolic syndrome, insulin resistance could be an important mediator of this association. METHODS This substudy included 250 of 269 patients enrolled in ASTRONOMER. Follow-up was 3.4 ± 1.3 years. Insulin resistance was evaluated using the homeostatic assessment model (HOMA) index, and patients were dichotomized using the median HOMA index value (1.24). The rate of LVH progression was estimated by calculating the annualized change in LV mass index (LVMi), measured on echocardiography. The presence of LVH was defined as an LVMi >47 g/m(2.7) in women and >49 g/m(2.7) in men. RESULTS There was a significant progression of LVH among the patients without LVH at baseline (n = 134; p < 0.0001) but not in those with it (n = 116; p = NS). In those without LVH at baseline, the annualized progression rate of LVMi was significantly faster in the subset with HOMA >1.24 compared to that in the subset with HOMA <1.24 (2.49 ± 4.38 g/m(2.7)/year vs. -0.03 ± 3.90 g/m(2.7)/year; p = 0.001). During follow-up, LVH developed in 46% of patients with HOMA >1.24 compared to 11% of those with HOMA <1.24 (p = 0.0005). Independent predictors of faster LVH progression identified on multivariate analysis were history of hypertension (p = 0.048), degree of aortic valve calcification (p = 0.035), and HOMA index (p = 0.02). CONCLUSIONS In this ASTRONOMER substudy, insulin resistance was a powerful independent predictor of progression to LVH in patients with AS. Visceral obesity and ensuing insulin resistance may thus present novel therapeutic targets in AS patients.