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COX-2 Inhibition and Inhibition of Cytosolic Phospholipase A2 Increase CD36 Expression and Foam Cell Formation in THP-1 Cells

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Lipids

Abstract

Cardiovascular safety of cyclooxygenase (COX)-2-selective inhibitors and nonselective nonsteroidal anti-inflammatory drugs (NSAIDs) is of worldwide concern. COX-2 inhibitors and NSAIDs act by inhibiting arachidonic acid metabolism to prostaglandins. They confer a cardiovascular hazard manifested as an elevated risk of myocardial infarction. Mechanisms underlying these cardiovascular effects are uncertain. Here we determine whether interference with cytosolic phospholipase A2 (cPLA-2) or COX-2 through pharmacologic blockade or silencing RNA impacts expression of scavenger receptor CD36 and scavenger receptor A, both involved in cholesterol uptake in monocytes and macrophages. THP-1 human monocytes and human peripheral blood mononuclear cells were exposed to celecoxib, a COX-2 selective inhibitor currently in clinical use, and to arachidonyl trifluoromethyl ketone (AACOCF3), an arachidonic acid analog that selectively inhibits cPLA-2. Celecoxib and AACOCF3 each upregulated expression of CD36, but not scavenger receptor A, as determined by quantitative PCR and immunoblotting. Silencing of cPLA-2 or COX-2 had comparable effects to pharmacologic treatments. Oil red O staining revealed a profound increase in foam cell transformation of THP-1 macrophages exposed to either celecoxib or AACOCF3 (both 25 μM), supporting a role for the COX pathway in maintaining macrophage cholesterol homeostasis. Demonstration of disrupted cholesterol balance by AACOCF3 and celecoxib provides further evidence of the possible mechanism by which COX inhibition may promote lipid overload leading to atheromatous lesion formation and increased cardiovascular events.

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Abbreviations

AACOCF3:

Arachidonyl trifluoromethyl ketone

COX:

Cyclooxygenase

cPLA-2:

Cytosolic phospholipase A2

FCS:

Fetal calf serum

GAPDH:

Glyceraldehyde-3-phosphate dehydrogenase

IFN:

Interferon

HRP:

Horseradish peroxidase

LDL:

Low density lipoprotein

NSAIDs:

Nonsteroidal anti-inflammatory drugs

PBMC:

Peripheral blood mononuclear cells

PMA:

Phorbol 12-myristate 13-acetate

RCT:

Reverse cholesterol transport

ScR-A:

Scavenger receptor A

TGF:

Transforming growth factor

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Acknowledgments

This work was supported by an Innovative Research Grant from the Arthritis Foundation and by a Winthrop University Hospital Pilot and Feasibility Grant.

Conflict of interest

None declared.

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Authors and Affiliations

  1. Department of Medicine, Winthrop-University Hospital, 222 Station Plaza North, Suite 502, Mineola, NY, 11501, USA

    Kamran Anwar, Iryna Voloshyna, Michael J. Littlefield, Steven E. Carsons, Peter A. Wirkowski, Nadia L. Jaber, Andrew Sohn, Sajan Eapen & Allison B. Reiss

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  1. Kamran Anwar
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  2. Iryna Voloshyna
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Correspondence to Allison B. Reiss.

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Fig. 1.

Dose-response curve for celecoxib effect on CD36, ScR-A1 and COX-2 expression in THP-1 monocytes. THP-1 monocytes were incubated with increasing concentrations of celecoxib for 18h. As demonstrated, increasing celecoxib (up to 50 μM) results in a decrease in COX-2 message and upregulation of CD36 message. However, ScR-A1 expression did not change significantly with increasing concentrations of celecoxib (n=3). **- P<0.01 vs. control cells in media alone, ***- P<0.001 vs. control cells. (PDF 14 kb)

Fig. 2.

Effect of COX-2 inhibition by celecoxib and cPLA-2 inhibition by AACOCF3 on CD36 and ScR-A1 mRNA expression in THP-1 monocytes. THP-1 monocytes were incubated for 18 hours under the following four conditions: 1) media alone, 2) IFN-γ [500u/ml], 3) AACOCF3 [25 μM], 4) celecoxib [25 μM]; 5) TGF-β (120 pg/ml). Following incubation, total RNA was isolated from cells exposed to each condition. RNA was reverse transcribed, CD36 and ScR-A1 messages were analyzed by QRT-PCR. The expressions of target genes were normalized to that of GAPDH. A. CD36 gene expression graphed as relative mRNA expression and compared with results obtained from untreated THP-1 monocytes. The data represent the mean and SEM of three independent experiments (n=3), **- P<0.01 vs. control cells in media alone, ## - P<0.01 vs. IFN – γ, AACOCF3, celecoxib. B. ScR-A1 gene expression graphed as relative mRNA expression and compared with results obtained from untreated THP-1 monocytes. The data represent the mean and SEM of three independent experiments (n=3), NS, ## - P<0.01 vs. control, IFN – γ, AACOCF3, celecoxib. (PDF 23 kb)

Fig. 3.

Effect of celecoxib and AACOCF3 on foam cell transformation in THP-1 macrophages under cholesterol loading conditions using oxidized LDL. THP-1 macrophages were treated with oxidized LDL (50 µg/ml, 48 hours). Cells were then exposed for 48 hours to the following five conditions: 1) untreated control; 2) IFN-γ [500 U/ml]; 3) celecoxib [25 µM]; 4) AACOCF3 [25 µM]; 5) TGF-β [120 pg/ml]. A. Cells were stained with Oil red O to detect foam cells. B. Graphic representation of percentage foam cells shows a significant increase in foam cell transformation with celecoxib, AACOCF3 or IFN-γ (n=4). ** - P<0.01 vs. control cells, *** - P<0.001 vs. control cells, # - P<0.05 vs. celecoxib, AACOCF3. (PDF 293 kb)

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Anwar, K., Voloshyna, I., Littlefield, M.J. et al. COX-2 Inhibition and Inhibition of Cytosolic Phospholipase A2 Increase CD36 Expression and Foam Cell Formation in THP-1 Cells. Lipids 46, 131–142 (2011). https://doi.org/10.1007/s11745-010-3502-4

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