Effet des fibrates sur la synthèse périphérique et le métabolisme hépatique de la bilirubine, un endobiotique aux propriétés anti-oxydantes et athéro-protectrice

La bilirubine, metabolite de Thème, est un antioxydant puissant exerçant des fonctions athéro-protectrices à des concentrations modérées; cependant elle est neurotoxique à des concentrations élevées. Synthétisée lors du métabolisme de l'hème par l'action successive des enzymes, hème-oxygénase (HO-1) et biliverdine-réductase (BVR), la bilirubine est métabolisée par l'UDP-glucuronosyltransférase (UGT1A1) dans le foie, afin d'être éliminée sous forme glucurono-conjuguée par le transporteur, Multidrug resistance Related Protein (MRP)2. Dans le cadre de la présente étude, nous avons validé une explication pharmacologique supplémentaire du mode d'action des fibrates, médication actuellement sur le marché dans le traitement de la dyslipidémie (prévenant l'athérosclérose in vivo). Les données obtenues démontrent que les fibrates modulent positivement les enzymes de la synthèse et du métabolisme de la bilirubine, favorisant ainsi ses propriétés antioxydantes et athéro-protectrice dans la paroi vasculaire et protègent l'organisme contre ses accumulations toxiques au niveau hépatique. Donc, un mode d'action additionnel des fibrates, pourrait être pris en considération, résultant en une élévation modérée de la concentration locale de la bilirubine et profitant de ses capacités antioxydantes et par la suite athéro-protectrices

PPAR α

Hypolipidemic fibrates activate the peroxisome proliferator-activated receptor (PPAR) α to modulate lipid oxidation and metabolism. The present study aimed at evaluating how 3 PPARα agonists, namely, fenofibrate, gemfibrozil, and Wy14,643, affect bilirubin synthesis and metabolism. Human umbilical vein epithelial cells (HUVEC) and coronary artery smooth muscle cells (CASMC) were cultured in the absence or presence of the 3 activators, and mRNA, protein, and/or activity levels of the bilirubin synthesizing heme oxygenase- (HO-) 1 and biliverdin reductase (BVR) enzymes were determined. Human hepatocytes (HH) and HepG2 cells sustained similar treatments, except that the expression of the bilirubin conjugating UDP-glucuronosyltransferase (UGT) 1A1 enzyme and multidrug resistance-associated protein (MRP) 2 transporter was analyzed. In HUVECs, gemfibrozil, fenofibrate, and Wy14,643 upregulated HO-1 mRNA expression without affecting BVR. Wy14,643 and fenofibrate also caused HO-1 protein accumulation, while gemfibrozil and fenofibrate favored the secretion of bilirubin in cell media. Similar positive regulations were also observed with the 3 PPARα ligands in CASMCs where HO-1 mRNA and protein levels were increased. In HH and HepG2 cells, both UGT1A1 and MRP2 transcripts were also accumulating. These observations indicate that PPARα ligands activate bilirubin synthesis in vascular cells and metabolism in liver cells. The clinical implications of these regulatory events are discussed

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

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

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

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-ß, [gamma]-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

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

Pi induces apoptosis of valve interstitial cells through the mitochondrial pathway.

<p><b>A</b>. The percentage of apoptotic cells, measured by TUNEL assay, increased significantly during VIC mineralization, whereas treatment with PFA blocked this response. <b>B</b>. The mitochondrial membrane potential (ΔΨm) decreased following treatment with Pi, but addition of PFA protected the ΔΨm. <b>C.</b> Epifluorescence images of VICs with the MitoPT TRME fluorescent dye in different conditions. In control cells, the mitochondrial uptake of MitoPT TRME gives a clear and distinct fluorescent pattern, indicating a normal ΔΨm. Following treatment with Pi, however, the fluorescent pattern is diffuse and accompanied by an abnormal mitochondrial morphology indicating a loss in the ΔΨm. The addition of PFA prevented Pi-mediated loss in the ΔΨm. <b>D.</b> This protection with PFA was also confirmed with an immunofluorescence assay measuring cytochrome <i>c</i> release in VICs under mineralizing condition. <b>E</b>. Cyclosporin A which is an inhibitor of MTP, prevented Pi-mediated apoptosis of VIC cultures as detected with TUNEL assay. <b>F.</b> The effect of cyclosporine A on Pi-mediated apoptosis was confirmed by the APOPercentage assay, which relies on changes in membrane asymmetry during apoptosis. <b>G</b>. Cyclosporin A prevented Pi-induced mineralization of VIC cultures. (For in vitro experiments n = 3); PFA: Phosphonoformic acid; MTP: mitochondrial permeability transition pore; * p<0.0001 compared to negative control (Ctn); # p<0.0001 compared to mineralizing medium (PO₄).</p

Akt-1 a regulator of Pi-induced mineralization.

<p>Transfection of VICs with a pCMVAkt-1 resulted in higher expression of Akt-1 transcripts. <b>B</b>. The transfection of Akt-1 (pCMVAkt-1) prevented Pi-induced apoptosis of VICs (measured with the TUNEL assay). <b>C</b>. Transfection of Akt-1 reduced significantly Pi-induced mineralization of VIC cultures. <b>D.</b> Inhibition of PI3K, a kinase acting upstream of Akt, with Ly294002 increased mineralization of VIC cultures by several-folds. <b>E and F.</b> Similarly, the knockdown of Akt-1 (F) resulted in higher mineralization of VIC cultures (E). (For in vitro experiments n = 3); pCMV empty is the control (ctn) in panels A–C * p<0.0001 compared to negative control (Ctn); # p<0.0001 compared to mineralizing medium (PO₄).</p

Expression of SCL20A1 in human calcific aortic valve disease.

<p><b>A</b>. In human calcific aortic valve disease (CAVD) tissues, expression of SLC20A1 was increased, both in tricuspid and bicuspid aortic valves, when compared to control non-mineralized aortic valves. <b>B, C and D</b>. Immunostaining for SLC20A1 revealed faint expression in control aortic valves (B), whereas in CAVD tissues we observed a strong immunostaining in areas of tissue remodelling in the vicinity of calcific nodules (C) (100×) (in panel D magnification 200× of inset in C). * p<0.005 compared to control (Ctn).</p

Pi transporters in human valve interstitial cells (VICs).

<p><b>A</b>. In isolated VICs, only transcripts of SLC20A1 and SL20A2 were expressed. <b>B.</b> Following exposure to Pi, VICs have increased expression of SLC20A1 and SLC20A2 transcripts by several folds, with the highest magnitude for SLC20A1 (results expressed in function of SLC20A2 as the referent) (SLC20A1 increased by 5.3-folds when compared to SLC20A2) <b>C</b>. Treatment with PFA, a Pi transporter inhibitor, blocked the rise of SLC20A1 and SLC20A2 transcripts induced by the mineralizing medium (Pi). <b>D</b>. PFA prevented the mineralization of VIC cultures induced by Pi. <b>E</b>. PFA prevented the Pi-induced rise of osteopontin, osteonectin, osteocalcin, Runx2 and alkaline phosphatase transcripts. <b>F and G</b>. In isolated VICs, siRNA-mediated knockdown of SLC20A1 (F) resulted in a decreased Pi-induced mineralization (G). When compared with the knockdown of SLC20A2, the siRNA against SLC20A1 provided a greater reduction of mineralization of VIC cultures (G). (For in vitro experiments n = 3); PFA: Phosphonoformic acid; * p<0.0001 compared to negative control (Ctn); # p<0.005 compared to mineralizing medium (PO₄).</p