Direct protective effects of poly-unsaturated fatty acids, DHA and EPA, against activation of cardiac late sodium current: a mechanism for ischemia selectivity.

Polyunsaturated fatty acids (PUFAs) such as docosahexaenoic and eicosapentaenoic acids (DHA, EPA) exert ischemic anti-arrhythmic effects. However, their mechanism of action remains unknown. The present study was designed to investigate their potential effect on the regulation of the late sodium current as the basis for their ischemic anti-arrhythmic activity. Human isoforms of wild-type SCN5A and DeltaKPQ-mutated cardiac sodium channels were stably transfected in HEK 293 cells and, the resulting currents were recorded using the patch clamp technique in whole cell configuration. In addition to their effect to inhibit peak I(Na), acute application of DHA and EPA blocked veratridine-induced late sodium current (late I(Na-Verat)) in a concentration--dependent manner with IC(50) values of 2.1 +/- 0.5 microM and 5.2 +/- 0.8 microM,for DHA and EPA, respectively. Channels availability was reduced, resulting in a significant leftward shift of the steadystate inactivation curve by -10.0 +/- 2.1 mV and -8.5 +/- 0.2 mV for DHA and EPA, respectively. Similar inhibitory effects of DHA and EPA were also observed on late I(Na-KPQ). In addition to their role as blocking agents of peak I(Na), DHA and EPA reduced human late I(Na). These results could explain the antiarrhythmic properties of DHA and EPA during ischemia or following ischemia-reperfusion.

Direct effect of F12511, a systemic inhibitor of Acyl-CoA cholesterol acyltransferase on bovine aortic endothelial cells.

F12511(S)-2',3',5'-trimethyl-4'-hydroxy-alpha-dodecylthio-alpha-phenylacetanilide (F12511) is a new Acyl-CoA cholesterol acyltransferase (ACAT) inhibitor that not only reduces the plasma cholesterol levels but also has anti-atherosclerotic actions in animals models. The study's aim was to analyze if F12511 may directly modify the ability of tumor necrosis factor--alpha (TNF-alpha)-incubated bovine aortic endothelial cells (BAEC) to express endothelial nitric oxide synthase (eNOS) protein and inflammatory-related proteins such as platelet endothelial cell adhesion molecule (PECAM) and CD40 ligand (CD40L). The addition of increasing concentrations of F12511 (10 to 10 mol/L) failed to modify the level of eNOS protein expressed in control BAEC. TNF-alpha (10 ng/mL) reduced the expression of eNOS protein. In TNF-alpha--incubated BAEC, F12511 protected eNOS expression in a concentration-dependent manner. TNF-alpha stimulated the expression of both CD40L and PECAM in cultured BAEC. F12511 (10 mol/L) failed to modify the expression of CD40L and PECAM in control and TNF-alpha-incubated BAEC. Reverse transcriptase polymerase chain reaction showed a marked expression of the ACAT-2 isoform and absent of expression of the ACAT-1 isoform in BAEC. The presence of ACAT-2 isoform in BAEC was further confirmed by Western blot. F12511 failed to modify the expression of the proinflammatory associated proteins PECAM and CD40L in the endothelium but protected eNOS expression in the endothelial cells exposed to inflammatory conditions.

Regulation of the scavenger receptor BI and the LDL receptor by activators of aldosterone production, angiotensin II and PMA, in the human NCI-H295R adrenocortical cell line.

In human adrenal cells, cholesterol for steroidogenesis is derived from both high-density lipoproteins (HDL) via the Scavenger Receptor Class B Type I (SR-BI) and low-density lipoproteins (LDL) via the LDL receptor pathway. We have previously shown that, in the human adrenocortical carcinoma cell line, NCI-H295R, SR-BI and LDL receptor expression and steroidogenesis are coordinately regulated by activators of protein kinase A (PKA) leading to glucocorticoid synthesis. In the present study, we studied whether SR-BI and LDL receptor expression are regulated by activators of the protein kinase C (PKC) signaling pathway, such as angiotensin II, which stimulate mineralocorticoid synthesis. First, it is shown that, in NCI-H295R cells, aldosterone synthesis is stimulated by a phorbol ester (phorbol-12-myristate-13 acetate, PMA), a potent PKC activator. Northern blot analysis indicated that both angiotensin II and PMA stimulated SR-BI expression in a time-dependent manner. LDL receptor expression is slightly stimulated by PMA. The induction of SR-BI gene expression occurs at the transcriptional level, via an activation of the human SR-BI promoter, as shown by transient transfection experiments. Finally, SR-BI protein level was increased in angiotensin II- and PMA-stimulated cells, resulting in higher lipoprotein binding and specific cholesteryl ester (CE) uptake from HDL, as well from LDL after angiotensin II and PMA stimulation.

PPARalpha and PPARdelta activators inhibit cytokine-induced nuclear translocation of NF-kappaB and expression of VCAM-1 in EAhy926 endothelial cells.

Endothelium injury is a primary event in atherogenesis, which is followed by monocyte infiltration, macrophage differentiation, and smooth muscle cell migration. Peroxisome proliferator-activated receptors (PPARs) are transcription factors now recognized as important mediators in the inflammatory response. The aim of this study was to develop a human endothelial model to evaluate anti-inflammatory properties of PPAR activators. PPAR proteins (alpha, delta and gamma) are expressed in EAhy926 endothelial cells (ECs). Pirinixic acid (Wy-14643), fenofibrate, fenofibric acid, the Merck ligand PPARdelta activator L-165041, 15-deoxy-Delta(12,14)-prostaglandin J2, but not rosiglitazone (BRL-49653) inhibited the induced expression of vascular cell adhesion molecule-1 (VCAM-1), as measured by enzyme linked immunosorbent assay (ELISA), and monocyte binding to activated-EAhy926 cells. The PPARdelta activator L-165041 had the greatest potency to reduce cytokine-induced monocyte chemotactic protein-1 (MCP-1) secretion. All PPAR activators tested which impaired VCAM-1 expression reduced significantly nuclear p65 amount. These results show that EAhy926 endothelial cells are an adequate tool to substantiate and characterize inflammatory impacts of PPAR activators.

Anti-atherosclerotic properties of the acyl-coenzyme A:cholesterol acyltransferase inhibitor F 12511 in casein-fed New Zealand rabbits.

SUMMARY: The anti-atherosclerotic properties of F 12511, a novel acyl-coenzyme A:cholesterol acyltransferase (ACAT) inhibitor, were studied in rabbits that were fed a cholesterol-free casein-rich diet and developed endogenous hypercholesterolemia and fibrofatty preatheroma lesions. After 6 weeks of casein feeding, an endothelial abrasion was performed in the abdominal aorta; at week 8, a control group was maintained on this diet while F 12511 (8 mg/kg/d) was administered as a diet admixture for the subsequent 24 weeks. Total plasma cholesterol level rose to 250-300 mg/dl in both groups before starting the treatment; F 12511 time-dependently reduced total plasma cholesterol by 50%, and also decreased by 50% the incidence of lesions and macrophage accumulation in uninjured aorta (thoracic arch, celiac bifurcation). Residual lesions in the treated group were characterized by few macrophages, essentially under the endothelium, and by a larger content of smooth muscle cells. Quantitative image analysis of serial sections of mechanically injured abdominal aorta revealed a 20% surface covered by preatheroma lesions in the placebo group; F 12511 significantly reduced this surface. These data suggest that the combination of endogenous hypercholesterolemia with endothelial injury in the rabbit may offer a useful model to study atherosclerosis; lipid lowering by F 12511 reduces the incidence of vascular lesions and macrophage infiltration and may reinforce the fibrous skeleton of the atheroma.