Nitric oxide mediates the effect of fluvastatin on intercellular adhesion molecule-1 and platelet endothelialcell adhesion molecule-1 expressionon human endothelial cells

Eleftherios S. Xenos, Scott Stevens, Michael Freeman, David C. Cassada, Mitchell Goldman

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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 μM, 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), Ser1177- 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 μM fluvastatin did not exhibit reduced expression of PECAM-1 or ICAM-1. Incubation with 10 μM 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 μM + L-NMMA: FI was 3042 ± 1378 vs. 3638 ± 1671 for the control p = 0.01) or PECAM-1 (fluvastatin 10 μM + L-NMMA: FI was 415 ± 188 vs. 522 ± 78 for the control, p = 0.1). Incubation with 20 μM 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 μM 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). Ser1177-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 μM 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 Ser1177. We have shown that after a 48-hr exposure to fluvastatin there is an increased amount of the phosphorylated enzyme in the endothelial cells.

Original languageEnglish (US)
Pages (from-to)386-392
Number of pages7
JournalAnnals of Vascular Surgery
Volume19
Issue number3
DOIs
StatePublished - Jan 1 2005

Fingerprint

fluvastatin
Intercellular Adhesion Molecule-1
Nitric Oxide Synthase Type III
Nitric Oxide
Blood Platelets
Endothelial Cells
CD31 Antigens
omega-N-Methylarginine
Hydroxymethylglutaryl-CoA Reductase Inhibitors
Adenylate Kinase
Proteins
AMP-Activated Protein Kinases
Densitometry
Iliac Artery
Serum-Free Culture Media
Enzymes
Nitrites
Immunoblotting
Nitrates

All Science Journal Classification (ASJC) codes

  • Surgery
  • Cardiology and Cardiovascular Medicine

Cite this

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title = "Nitric oxide mediates the effect of fluvastatin on intercellular adhesion molecule-1 and platelet endothelialcell adhesion molecule-1 expressionon 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 μM, 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), Ser1177- 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 μM fluvastatin did not exhibit reduced expression of PECAM-1 or ICAM-1. Incubation with 10 μM 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 μM + L-NMMA: FI was 3042 ± 1378 vs. 3638 ± 1671 for the control p = 0.01) or PECAM-1 (fluvastatin 10 μM + L-NMMA: FI was 415 ± 188 vs. 522 ± 78 for the control, p = 0.1). Incubation with 20 μM 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 μM 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). Ser1177-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 μM 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 Ser1177. We have shown that after a 48-hr exposure to fluvastatin there is an increased amount of the phosphorylated enzyme in the endothelial cells.",
author = "Xenos, {Eleftherios S.} and Scott Stevens and Michael Freeman and Cassada, {David C.} and Mitchell Goldman",
year = "2005",
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language = "English (US)",
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journal = "Annals of Vascular Surgery",
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TY - JOUR

T1 - Nitric oxide mediates the effect of fluvastatin on intercellular adhesion molecule-1 and platelet endothelialcell adhesion molecule-1 expressionon human endothelial cells

AU - Xenos, Eleftherios S.

AU - Stevens, Scott

AU - Freeman, Michael

AU - Cassada, David C.

AU - Goldman, Mitchell

PY - 2005/1/1

Y1 - 2005/1/1

N2 - 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 μM, 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), Ser1177- 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 μM fluvastatin did not exhibit reduced expression of PECAM-1 or ICAM-1. Incubation with 10 μM 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 μM + L-NMMA: FI was 3042 ± 1378 vs. 3638 ± 1671 for the control p = 0.01) or PECAM-1 (fluvastatin 10 μM + L-NMMA: FI was 415 ± 188 vs. 522 ± 78 for the control, p = 0.1). Incubation with 20 μM 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 μM 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). Ser1177-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 μM 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 Ser1177. We have shown that after a 48-hr exposure to fluvastatin there is an increased amount of the phosphorylated enzyme in the endothelial cells.

AB - 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 μM, 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), Ser1177- 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 μM fluvastatin did not exhibit reduced expression of PECAM-1 or ICAM-1. Incubation with 10 μM 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 μM + L-NMMA: FI was 3042 ± 1378 vs. 3638 ± 1671 for the control p = 0.01) or PECAM-1 (fluvastatin 10 μM + L-NMMA: FI was 415 ± 188 vs. 522 ± 78 for the control, p = 0.1). Incubation with 20 μM 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 μM 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). Ser1177-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 μM 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 Ser1177. We have shown that after a 48-hr exposure to fluvastatin there is an increased amount of the phosphorylated enzyme in the endothelial cells.

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