Nitric Oxide Mediates the Effect of Fluvastatin on Intercellular Adhesion Molecule-1 and Platelet Endothelial Cell Adhesion Molecule-1 Expression on Human Endothelial Cells

Abstract

Leukocyte and platelet adhesion to endothelial cells, an early step in the pathogenesis of atherosclerosis, is mediated through adhesion molecules. It has been shown that statins decrease adhesion molecule expression. We examined the hypothesis that fluvastatin decreased intercellular adhesion molecule-1 (ICAM-1) and platelet endothelial cell adhesion molecule-1 (PECAM-1) expression through a nitric oxide-mediated pathway. Human iliac artery endothelial cells were exposed to fluvastatin in the presence or absence of 2 mM N-monomethyl-L-arginine (L-NMMA). Flow cytometry analysis was used to measure ICAM-1 and PECAM-1 expression. In a separate experiment, confluent cell cultures were exposed in a serum-free medium to fluvastatin 20 microM, and the supernatant was collected for nitrate/nitrite determination after 6 and 48 hr of incubation. Protein was isolated and processed for immunoblotting with monoclonal antibodies specific for endothelial nitric oxide synthase (eNOS), Ser(1177)-phosphorylated eNOS, and AMP kinase. Relative band intensity was assessed with densitometry. Results are presented as the mean +/- standard deviation (SD), and p < 0.05 was considered significant. ICAM-1 and PECAM-1 were expressed constitutively. Human iliac artery endothelial cells (HIAECS) treated with 5 microM fluvastatin did not exhibit reduced expression of PECAM-1 or ICAM-1. Incubation with 10 microM fluvastatin reduced basal expression of both ICAM-1 and PECAM-1. Fluorescence intensity (FI) for these substance was as follows: 3638 +/- 1671, p = 0.01 and PECAM-1 vs. control FI 276 +/- 52 vs. 522 +/- 78, p = 0.02. In the presence of 2 mM L-NMMA, fluvastatin failed to decrease the expression of ICAM-1 (fluvastatin 10 microM + L-NMMA: FI was 3042 +/- 1378 vs. 3638 +/- 1671 for the control p = 0.01) or PECAM-1 (fluvastatin 10 microM + L-NMMA: FI was 415 +/- 188 vs. 522 +/- 78 for the control, p = 0.1). Incubation with 20 microM fluvastatin similarly reduced ICAM-1 expression (FI was 2014 +/- 1595 vs. 3638 +/- 1671 for the control, p = 0.02) and PECAM-1 expression (FI was 196 +/- 109 vs. 522 +/- 78 for the control, p = 0.02). This reduction was prevented in the presence of 2 mM L-NMMA. L-NMMA in a concentration of 2 mM had no significant effect on adhesion molecule expression (p > 0.05 for all comparisons of the control FI versus 2 mM L-NMMA mean FI). After a 48 hr incubation with 20 microM fluvastatin there was a 219 +/- 35% increase in the cell eNOS protein content (p = 0.01) and a 170 +/- 26% increase in the cell AMPK protein content (p = 0.02). Ser(1177)-phosphorylated eNOS protein levels were increased by 41 +/- 8% (p = 0.03). The nitric oxide concentration in the medium of the HIAEC treated with 20 microM fluvastatin for 48 hr was significantly higher than that in the control (p = 0.0004), pointing to increased production during the incubation period. Fluvastatin thus decreases basal expression of ICAM-1 and PECAM-1. Competitive inhibition of eNOS with L-NMMA abolishes the effect of fluvastatin on ICAM-1 and PECAM-1 expression. The statin up-regulates eNOS and AMP kinase, one of the enzymes that activates eNOS via phosphorylation at Ser(1177). We have shown that after a 48-hr exposure to fluvastatin there is an increased amount of the phosphorylated enzyme in the endothelial cells.