Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2013 May;345(2):170-9.
doi: 10.1124/jpet.112.201442. Epub 2013 Feb 22.

Attenuation by statins of membrane raft-redox signaling in coronary arterial endothelium

Yu-Miao Wei  1 , Xiang Li, Jing Xiong, Justine M Abais, Min Xia, Krishna M Boini, Yang Zhang, Pin-Lan Li
Affiliations

Attenuation by statins of membrane raft-redox signaling in coronary arterial endothelium

Yu-Miao Wei et al. J Pharmacol Exp Ther. 2013 May.

Abstract

Membrane raft (MR)-redox signaling platforms associated with NADPH oxidase are involved in coronary endothelial dysfunction. Here, we studied whether statins interfere with the formation of MR-redox signaling platforms to protect the coronary arterial endothelium from oxidized low-density lipoprotein (OxLDL)-induced injury and from acute hypercholesterolemia. In cultured human coronary arterial endothelial cells, confocal microscopy detected the formation of an MRs clustering when they were exposed to OxLDL, and such MR platform formation was inhibited markedly by statins, including pravastatin and simvastatin. In these MR clusters, NADPH oxidase subunits gp91(phox) and p47(phox) were aggregated and were markedly blocked by both statins. In addition, colocalization of acid sphingomyelinase (ASM) and ceramide was induced by OxLDL, which was blocked by statins. Electron spin resonance spectrometry showed that OxLDL-induced superoxide (O2(.-)) production in the MR fractions was substantially reduced by statins. In coronary artery intima of mice with acute hypercholesterolemia, confocal microscopy revealed a colocalization of gp91(phox), p47(phox), ASM, or ceramide in MR clusters. Such colocalization was rarely observed in the arteries of normal mice or significantly reduced by pretreatment of hypercholesterolemic mice with statins. Furthermore, O2(.-) production in situ was 3-fold higher in the coronary arteries from hypercholesterolemic mice than in those from normal mice, and such increase was inhibited by statins. Our results indicate that blockade of MR-redox signaling platform formation in endothelial cell membrane may be another important therapeutic mechanism of statins in preventing endothelial injury and atherosclerosis and may be associated with their direct action on membrane cholesterol structure and function.

PubMed Disclaimer

Figures

Fig. 1.
Fig. 1.
OxLDL-induced MR clustering in HCAECs in the absence or presence of statins. HCAECs were stimulated with control, OxLDL (100 µg/ml, 30 minutes) without or with pravastatin (Prava, 10 µM)/simvastatin (Simva, 5 µM) pretreatment, and stained with a MR probe, Alexa 488-cholera toxin B (Al488-CTXB). (A) Representative images of HCAECs with Al488-CTXB staining. (B) Summarized dose effects of OxLDL (25–150 µg/ml, 30 minutes) on the MR clustering, as indicated by percentage of positive cells with CTXB staining (n = 4). (C) Summarized effects of statins on OxLDL-induced MR clustering (n = 4). Some groups of cells were pretreated with mevalonate (Mev, 10 µM), farnesol (Far, 10 µM), or geranylgeraniol (Ger, 10 µM) or transfected with oncogenic Rac1 cDNA. Shown is the percentage of cells with MR clustering. *P < 0.05, versus control; #P < 0.05, versus vehicle; $P < 0.05, versus statin.
Fig. 2.
Fig. 2.
Effects of statins on OxLDL-induced colocalization of ASM and ceramide in HCAECs. Cells were stained with Alexa488-conjugated anti-ASM and TR-conjugated anti-ceramide antibodies. (A) Representative images show the colocalization (yellow) of ASM (green, Alexa488-anti-ASM) and ceramide (red, TR-anti-ceramide) under the stimulation of OxLDL (100 µg/ml) with or without pretreatment of statins. (B) Summarized data show the colocalization coefficiency indicating the relative ratio of cells with colocalized yellow spots (n = 5). *P < 0.05, versus control; #P < 0.05, versus vehicle.
Fig. 3.
Fig. 3.
Effects of statins on OxLDL-induced colocalization of MR clusters and gp91phox or p47phox in HCAECs. Cells were stained with MR marker, Alexa488-CTXB, and TR-conjugated anti-gp91phox (TR-anti-gp91) or p47phox (TR-anti-p47) antibody. Representative images show the colocalization (yellow) of MR marker CTXB (green, Alexa488-CTXB) and gp91phox (red, TR-anti-gp91) (A) or p47 (red, TR-anti-p47) (B) under the stimulation of OxLDL (100 µg/ml, 30 minutes) with or without pretreatment of statins. (C) Summarized data show the colocalization coefficiency indicating the relative ratio of cells with colocalized yellow spots (n = 5). *P < 0.05, versus control; #P < 0.05, versus vehicle.
Fig. 4.
Fig. 4.
ESR spectrometric analysis of O2.− production in HCAECs stimulated by OxLDL in the absence or presence of statins. (A) Cells were pretreated with vehicle (Vehl), pravastatin (Prava, 10 µM), or simvastatin (Simva, 5 µM) and stimulated with OxLDL. Representative ESR spectrographs of O2.− trapped by CMH with NADPH as substrates. (B) Summarized data show relative O2.− production induced by OxLDL, compared with control (n = 5). (C) Summarized data show relative O2.− production induced by sphingomyelinase (SMase), compared with control (n = 5). Some groups of cells were pretreated with MR disruptors: MCD or filipin. *P < 0.05, versus control; #P < 0.05, versus vehicle.
Fig. 5.
Fig. 5.
Effect of statins on plasma cholesterol levels in mice with acute hypercholesterolemia. Mice were injected intraperitoneally with vehicle (Control) or poloxamer 407 (P407, 0.5 g/kg) to induce acute hypercholesterolemia (P407). In case of pretreatment with statins, mice were intragastrically fed statins (pravastatin, 140 mg/kg/day; simvastatin, 70 mg/kg/day) for a week and then treated with vehicle (Statin) or P407 (Statin+P407). Summarized data show the plasma cholesterol concentrations in mice (n = 5). **P < 0.01, versus control; ##P < 0.01, versus P407.
Fig. 6.
Fig. 6.
Statins block the NADPH oxidase subunits clustering in MR clusters in coronary arteries of mice with acute hypercholesterolemia. Frozen sections of mouse hearts were stained with Al488-anti-flotillin-1 and TR-anti gp91phox or TR-anti-p47phox. Representative merged images displayed yellow dots or patches indicating the colocalization of MR marker protein flotillin-1 with NADPH oxidase subunit gp91phox (A) or p47phox (B). Each image includes an enlarged view of region of interest at the lower right corner. Scale bar, 50 µm. (C) Summarized data show the colocalization coefficiency (n = 5). *P < 0.05, versus control; #P < 0.05, versus vehicle.
Fig. 7.
Fig. 7.
Statins block ceramide or ASM clustering in MR clusters in coronary arteries of mice with acute hypercholesterolemia. Frozen sections of mouse hearts were stained with Al488-anti-flotillin-1 and TR-anti-ASM or TR-anti-ceramide. Representative merged images displayed yellow dots or patches indicating the colocalization of MR marker protein flotillin-1 with ASM (A) or ceramide (B). Each image includes an enlarged view of region of interest at the lower right corner. Scale bar, 50 µm. (C) Summarized data show the colocalization coefficiency (n = 5). *P < 0.05, versus control; #P < 0.05, versus vehicle.
Fig. 8.
Fig. 8.
Measurement of O2.− production in situ in mouse coronary arteries. (A) Frozen sections of hearts without fixation were incubated with DHE. Representative merged images (DHE red fluorescence merged with transmission light) show increases in DHE-O2.− fluorescence in situ in mouse coronary arteries. Scale bar, 50 µm. (B) Summarized data show the DHE fluorescence intensity (n = 5). **P < 0.01, versus control; ##P < 0.01, versus vehicle.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. (1994) Randomised trial of cholesterol lowering in 4444 patients with coronary heart disease: the Scandinavian Simvastatin Survival Study (4S). Lancet 344:1383–1389 - PubMed
    1. (1998) Prevention of cardiovascular events and death with pravastatin in patients with coronary heart disease and a broad range of initial cholesterol levels. The Long-Term Intervention with Pravastatin in Ischaemic Disease (LIPID) Study Group. N Engl J Med 339:1349–1357 - PubMed
    1. Alvarez E, Rodiño-Janeiro BK, Ucieda-Somoza R, González-Juanatey JR. (2010) Pravastatin counteracts angiotensin II-induced upregulation and activation of NADPH oxidase at plasma membrane of human endothelial cells. J Cardiovasc Pharmacol 55:203–212 - PubMed
    1. Araki K, Masaki T, Katsuragi I, Kakuma T, Yoshimatsu H. (2008) Effects of pravastatin on obesity, diabetes, and adiponectin in diet-induced obese mice. Obesity (Silver Spring) 16:2068–2073 - PubMed
    1. Bao JX, Jin S, Zhang F, Wang ZC, Li N, Li PL. (2010a) Activation of membrane NADPH oxidase associated with lysosome-targeted acid sphingomyelinase in coronary endothelial cells. Antioxid Redox Signal 12:703–712 - PMC - PubMed

Publication types

MeSH terms