Autotaxin and Lipoprotein Metabolism in Calcific Aortic Valve Disease

Calcific aortic valve disease (CAVD) is a complex trait disorder characterized by calcific remodeling of leaflets. Genome-wide association (GWA) study and Mendelian randomization (MR) have highlighted that LPA, which encodes for apolipoprotein(a) [apo(a)], is causally associated with CAVD. Apo(a) is the protein component of Lp(a), a LDL-like particle, which transports oxidized phospholipids (OxPLs). Autotaxin (ATX), which is encoded by ENPP2, is a member of the ecto-nucleotidase family of enzymes, which is, however, a lysophospholipase. As such, ATX converts phospholipids into lysophosphatidic acid (LysoPA), a metabolite with potent and diverse biological properties. Studies have recently underlined that ATX is enriched in the Lp(a) lipid fraction. Functional experiments and data obtained in mouse models suggest that ATX mediates inflammation and mineralization of the aortic valve. Recent findings also indicate that epigenetically-driven processes lower the expression of phospholipid phosphatase 3 (PLPP3) and increased LysoPA signaling and inflammation in the aortic valve during CAVD. These recent data thus provide novel insights about how lipoproteins mediate the development of CAVD. Herein, we review the implication of lipoproteins in CAVD and examine the role of ATX in promoting the osteogenic transition of valve interstitial cells (VICs)

High expression of the Pi-transporter SLC20A1/Pit1 in calcific aortic valve disease promotes mineralization through regulation of Akt-1

The regulation of phosphate (Pi) handling is crucial during calcification of the aortic valve. Gene profiling of Pi transporters revealed that VIC culture expresses SLC201A1/Pit1 and SLC20A2/Pit2. On exposure to a mineralizing medium (2 mM Pi), the expression of Pi transporters in VIC culture is increased several folds, with the highest magnitude for SLC20A1. By using siRNAs, we established that silencing SLC20A1 significantly reduced Pi-induced mineralization of VICs. In human pathological specimens, we found that the expression of SCL20A1 was increased in CAVD tissues compared to control nonmineralized aortic valves. Treatment of VIC culture with Pi promoted the loss of mitochondrial membrane potential (DYm) and cytochrome c release within the cytosol, leading to apoptosis. Inhibition of Pi transporters with phosphonoformic acid (PFA) prevented Pi-mediated apoptosis of VICs. Moreover, we discovered that the level of the Akt-1 transcript is diminished in CAVD tissues compared with control valves. Accordingly, treatment with Pi caused a reduction of the Akt-1 transcript in VIC culture, and treatment with PFA or siRNA against SLC20A1 restored the level of Akt-1. Overexpression of Akt-1 (pCMVAkt-1) prevented both Pi-induced apoptosis and mineralization of VIC culture. These results strongly suggest that overexpression of SLC20A1 promotes apoptosis and mineralization by altering the level of Akt-1

Arginine methylation, the characterization of a post-translational modification

Proteins are known to be post-translationally modified. This thesis will discuss arginine methylation, one of the many post-translational modifications that occur within the cell. The enzymes that catalyze this post-translational modification are called arginine methyltransferases. The three main types of methylated arginines include monomethylated arginine (MMA), asymmetric dimethylated arginine (aDMA) and symmetric dimethylated arginines (sDMA). Type I arginine methyltransferases catalyze the formation of MMA and aDMA; Type II enzyme catalyze the formation of MMA and sDMA. Protein arginine methylation has been implicated in the regulation of many different cellular processes, including transcription, cellular localization, protein-protein interaction and signal transduction.The purpose of this work was to further characterize arginine methylation by identifying new members of the arginine methyltransferase enzyme family in Drosophila melanogaster and to study the effects of protein arginine methylation on novel substrates. I identified and characterized nine homologues of arginine methyltransferases in Drosophila that were named DART1 to DART9, for drosophila arginine methyltransferases 1-9. All nine enzymes are expressed at various developmental stages. I discovered that a substrate of mammalian enzyme protein arginine methyltransferase 1 (PRMT1) can also be methylated by PRMT5. I also identified HIV-1 Tat protein as the first substrate of the novel enzyme PRMT6

Mechanical strain induces the production of spheroid mineralized microparticles in the aortic valve through a RhoA/ROCK-dependent mechanism.

Calcific aortic valve disease (CAVD) is a chronic disorder characterized by an abnormal mineralization of the leaflets, which is accelerated in bicuspid aortic valve (BAV). It is suspected that mechanical strain may promote/enhance mineralization of the aortic valve. However, the effect of mechanical strain and the involved pathways during mineralization of the aortic valve remains largely unknown. Valve interstitial cells (VICs) were isolated and studied under strain conditions. Human bicuspid aortic valves were examined as a model relevant to increase mechanical strain. Cyclic strain increased mineralization of VICs by several-fold. Scanning electron microscope (SEM) and energy dispersive X-ray (EDX) analyses revealed that mechanical strain promoted the formation of mineralized spheroid microparticles, which coalesced into larger structure at the surface of apoptotic VICs. Apoptosis and mineralization were closely associated with expression of ENPP1. Inhibition of ENPP1 greatly reduced mineralization of VIC cultures. Through several lines of evidence we showed that mechanical strain promoted the export of ENPP1-containing vesicles to the plasma membrane through a RhoA/ROCK pathway. Studies conducted in human BAV revealed the presence of spheroid mineralized structures along with the expression of ENPP1 in areas of high mechanical strain. Mechanical strain promotes the production and accumulation of spheroid mineralized microparticles by VICs, which may represent one important underlying mechanism involved in aortic valve mineralization. RhoA/ROCK-mediated export of ENPP1 to the plasma membrane promotes strain-induced mineralization of VICs

Parathyroid hormone is associated with the LV mass after aortic valve replacement

Aims : LV hypertrophy (LVH) is frequent after aortic valve replacement (AVR) and is often associated with comorbidities, including hypertension, obesity, renal failure and prosthesis-patient mismatch (PPM). However, whether other biological mechanism(s) may participate to LVH after AVR is still unknown. Parathyroid hormone (PTH) may play a role in LVH. However, it is presently unknown whether PTH is associated with LVH in patients that have undergone an AVR. Methods : In this cross-sectional study, 195 patients have been investigated at a mean of 8±3.5 years following AVR. LV function and mass were evaluated by Doppler echocardiography. The plasma levels of PTH, 25-hydroxyvitamin D (25-OHD), calcium and phosphate were measured. Results : There were 102 (52%) patients with LVH after AVR. In univariate analyses, PTH blood level was associated with LV mass (LVMi) and LVH. After adjustment for other risk factors, elevated PTH remained associated with LVMi (p=0.003) and LVH (p=0.02). In turn, the blood levels of 25-OHD and the renal function (GFR) were independently and inversely related to the blood level of PTH. Conclusions : After AVR, the level of PTH is independently associated with LVH. In turn, the level of PTH is related with the renal function and the level of 25-OHD

Lp-PLA2 is associated with structural valve degeneration of bioprostheses

Objectives: In this study, we sought to determine the metabolic markers associated with structural valve degeneration (SVD). Background: Structural valve degeneration (SVD) is the major cause of bioprosthetic valve failure leading to bioprostheses (BPs) stenosis or regurgitation. We hypothesized that lipoprotein-associated phospholipase A2 (Lp-PLA2) is involved in the SVD of BPs. Methods: We included 197 patients who underwent aortic valve replacement with a bioprosthetic valve and had echocardiographic follow-up to evaluate valve function. Moreover, explanted BPs (n = 39) were analysed by immunohistochemistry for the expression of Lp-PLA2. Results: After a mean follow-up of 7·9 ±0·2 years, forty-one patients (21%) were identified as developing SVD. Patients with SVD had significantly higher plasma level of Lp-PLA2 mass (151·8 ± 9·2 ng/mL vs. 133·2 ± 3·4 ng/mL, P = 0·03) and activity (27·6 ± 0·9 nmol/min/mL vs. 25·0 ± 0·4 nmol/min/mL, P = 0·005). Multivariate analysis revealed that Lp-PLA2 activity (OR: 1·09, 95% CI: 1·01–1·18; P = 0·03) was the strongest independent predictor of SVD. Immunohistochemistry studies of explanted BP showed that 77% of explanted BPs had the expression of Lp-PLA2, which correlated with the density of macrophages (CD68), and ox-LDL levels in bioprosthetic tissues. Conclusions: Increased blood plasma activity of Lp-PLA2 is associated with higher prevalence of SVD. These findings open new avenues for the identification of patients at risk for SVD and for the development of pharmacotherapy aiming at the prevention of SVD

Characterization of the Drosophila protein arginine methyltransferases DART1 and DART4.

The role of arginine methylation in Drosophila melanogaster is unknown. We identified a family of nine PRMTs (protein arginine methyltransferases) by sequence homology with mammalian arginine methyltransferases, which we have named DART1 to DART9 ( Drosophila arginine methyltransferases 1-9). In keeping with the mammalian PRMT nomenclature, DART1, DART4, DART5 and DART7 are the putative homologues of PRMT1, PRMT4, PRMT5 and PRMT7. Other DART family members have a closer resemblance to PRMT1, but do not have identifiable homologues. All nine genes are expressed in Drosophila at various developmental stages. DART1 and DART4 have arginine methyltransferase activity towards substrates, including histones and RNA-binding proteins. Amino acid analysis of the methylated arginine residues confirmed that both DART1 and DART4 catalyse the formation of asymmetrical dimethylated arginine residues and they are type I arginine methyltransferases. The presence of PRMTs in D. melanogaster suggest that flies are a suitable genetic system to study arginine methylation

P2Y2 receptor represses IL-6 expression by valve interstitial cells through Akt : implication for calcific aortic valve disease

Calcific aortic valve disease (CAVD) is a disorder characterized by an abnormal mineralization, which may have intricate links with inflammation. Interleukin-6 (IL-6) and its cognate cytokines are widely expressed and exert pleiotropic effects on different tissues. In this study, we examined the expression of the IL-6 family of cytokines in human CAVD by using a transcriptomic approach and we performed in-depth functional assays with valve interstitial cells (VICs) to unravel the process regulating IL-6 expression and its role during the mineralization of the aortic valve. We documented by both microarray and q-PCR analyses an elevated expression of IL-6 in human CAVD, which was correlated with the remodeling process. IL-6 was highly expressed by VICs. We found that following treatment with a phosphate-containing medium the level of IL-6 expressed by VICs increased by several-fold. Phosphate-induced expression of IL-6 relied on reduced PI3K/Akt signaling downstream of the P2Y2 receptor (P2Y2R). In this regard, we found by using transfection experiments that Akt-1 is a negative regulator of the NF-¿B pathway. In addition, by using a siRNA targeting IL-6 we found that phosphate-induced mineralization was largely dependent on IL-6 expression. A transfection of Akt-1 rescued the hypermineralizing phenotype of P2Y2R-/- mouse VICS (MVICs). Hence, we documented a novel mechanism whereby P2Y2R and Akt modulate the NF-¿B pathway and its downstream target IL-6, which is a strong promoter of the mineralization of VIC