Effect of cyclooxygenase inhibition on cholesterol efflux proteins and atheromatous foam cell transformation in THP-1 human macrophages: a possible mechanism for increased cardiovascular risk

Abstract

Both selective cyclooxygenase (COX)-2 inhibitors and non-steroidal anti-inflammatory drugs (NSAIDs) have been beneficial pharmacological agents for many patients suffering from arthritis pain and inflammation. However, selective COX-2 inhibitors and traditional NSAIDs are both associated with heightened risk of myocardial infarction. Possible pro-atherogenic mechanisms of these inhibitors have been suggested, including an imbalance in prostanoid production leaving the pro-aggregatory prostaglandins unopposed, but the precise mechanisms involved have not been elucidated. We explored the possibility that downregulation of proteins involved in reverse cholesterol transport away from atheromatous plaques contributes to increased atherogenesis associated with COX inhibition. The reverse cholesterol transport proteins cholesterol 27-hydroxylase and ATP-binding cassette transporter A1 (ABCA1) export cholesterol from macrophages. When mechanisms to process lipid load are inadequate, uncontrolled cholesterol deposition in macrophages transforms them into foam cells, a key element of atheromatous plaques. We showed that in cultured THP-1 human monocytes/macrophages, inhibition of COX-1, COX-2, or both reduced expression of 27-hydroxylase and ABCA1 message (real-time reverse transcription-polymerase chain reaction) and protein (immunoblot). The selective COX-2 inhibitor N-(2-cyclohexyloxy-4-nitrophenyl)methanesulfonamide (NS398) significantly reduced 27-hydroxylase and ABCA1 message (to 62.4% +/- 2.2% and 71.1% +/- 3.9% of control, respectively). Incubation with prostaglandin (PG) E2 or PGD2 reversed reductions in both of these cholesterol transport proteins induced by NS398. Cholesterol-loaded THP-1 macrophages showed significantly increased foam cell transformation in the presence of NS398 versus control (42.7% +/- 6.6% versus 20.1% +/- 3.4%, p = 0.04) as determined by oil red O staining. Pharmacological inhibition of COX in monocytes is involved in downregulation of two proteins that mediate cholesterol efflux: cholesterol 27-hydroxylase and ABCA1. Because these proteins are anti-atherogenic, their downregulation may contribute to increased incidence of cardiac events in patients treated with COX inhibitors. Reversal of inhibitory effects on 27-hydroxylase and ABCA1 expression by PGD2 and PGE2 suggests involvement of their respective signaling pathways. NS398-treated THP-1 macrophages show greater vulnerability to form foam cells. Increased cardiovascular risk with COX inhibition may be ascribed at least in part to altered cholesterol metabolism.

Figure 1

Detection and quantitation of cholesterol 27-hydroxylase in THP-1 cells exposed to NS398. (a) Dose-dependent decrease in 27-hydroxylase mRNA expression in THP-1 monocytes treated with the COX-2 inhibitor NS398. Cultured THP-1 monocytic cells were untreated or exposed to NS398 for 18 hours. After isolation of total RNA, the RNA was reverse-transcribed and the cDNA amplified by quantitative real-time polymerase chain reaction as described. Signals obtained from the amplification of GAPDH message were used as internal controls. (b) Dose-dependent decrease in 27-hydroxylase protein expression in THP-1 monocytes treated with the COX-2 inhibitor NS398. Cultured THP-1 monocytic cells were untreated or exposed to NS398 for 18 hours. Total cell protein was isolated and 27-hydroxylase detected with specific rabbit polyclonal anti-human 27-hydroxylase antibody. Western blotting was performed with an anti-β-actin antibody to confirm equal protein loading. At 100 mM NS398 concentration, cell death was statistically significant (14.8% ± 6.3%). COX, cyclooxygenase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NS398, N-(2-cyclohexyloxy-4-nitrophenyl)methanesulfonamide. ** p < 0.01.

Figure 2

Quantitation of ABCA1 message in THP-1 cells exposed to NS398. Dose-dependent decrease in ABCA1 mRNA expression in THP-1 monocytes treated with the COX-2 inhibitor NS398. Cultured THP-1 monocytic cells were untreated or exposed to NS398 for 18 hours. After isolation of total RNA, the RNA was reverse-transcribed and the cDNA amplified by quantitative real-time polymerase chain reaction as described. Signals obtained from the amplification of GAPDH message were used as internal controls. At 100 mM NS398 concentration, cell death was statistically significant (14.8% ± 6.3%). ABCA1, ATP-binding cassette transporter A1; COX, cyclooxygenase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NS398, N-(2-cyclohexyloxy-4-nitrophenyl)methanesulfonamide. ** p < 0.01.

Figure 3

QRT-PCR for 27-hydroxylase and ABCA1 message in indomethacin-treated THP-1 cells. (a) 27-Hydroxylase mRNA expression is decreased by the non-specific COX inhibitor indomethacin in a dose-dependent fashion in THP-1 monocytes. Cultured THP-1 monocytic cells were untreated or exposed to increasing doses of indomethacin for 18 hours. After isolation of total RNA, the RNA was reverse-transcribed and the cDNA amplified by QRT-PCR as described. Signals obtained from the amplification of GAPDH message were used as internal controls. (b) ABCA1 mRNA expression is decreased by the non-specific COX inhibitor indomethacin in a dose-dependent fashion in THP-1 monocytes. Cultured THP-1 monocytic cells were untreated or exposed to increasing doses of indomethacin for 18 hours. After isolation of total RNA, the RNA was reverse-transcribed and the cDNA amplified by QRT-PCR as described. Signals obtained from the amplification of GAPDH message were used as internal controls. At 50 mM indomethacin concentration, cell death was statistically significant (16.8% ± 1.0%). ABCA1, ATP-binding cassette transporter A1; COX, cyclooxygenase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; QRT-PCR, quantitative real-time polymerase chain reaction. * p < 0.05, ** p < 0.01.

Figure 4

Detection and quantitation of cholesterol 27-hydroxylase in THP-1 cells exposed to SC560. (a) 27-Hydroxylase mRNA expression in THP-1 monocytes is decreased by the specific COX-1 inhibitor SC560. Cultured THP-1 monocytic cells were untreated or exposed to increasing doses of SC560 for 24 hours. After isolation of total RNA, the RNA was reverse-transcribed and the cDNA amplified by quantitative real-time polymerase chain reaction as described. Signals obtained from the amplification of GAPDH message were used as internal controls. (b) Decrease in 27-hydroxylase protein expression in THP-1 monocytes treated with the COX-1 inhibitor SC560. Cultured THP-1 human monocytes were untreated or exposed to SC560 for 24 hours. Total cell protein was isolated and 27-hydroxylase detected with specific rabbit polyclonal anti-human 27-hydroxylase antibody. Western blotting was performed with an anti-β-actin antibody to confirm equal protein loading. COX, cyclooxygenase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; SC560, 5-(4-Chlorophenyl)-1-(4-methoxyphenyl)-3-trifluoromethylpyrazol. ** p < 0.01.

Figure 5

QRT-PCR for 27-hydroxylase and ABCA1 message in NS398-treated THP-1 cells exposed to prostaglandins. (a) 27-Hydroxylase message is decreased by the COX-2 inhibitor NS398 and this decrease is reversed by prostaglandins E₁, E₂, and D₂. THP-1 human monocytes were exposed to the following conditions represented by the six bars (from left to right): (1) RPMI 1640, (2) NS398 (50 μM), (3) PGE₁ (0.1 μM) + NS398 (50 μM), (4) PGE₂ (0.1 μM) + NS398 (50 μM), (5) PGE₁ (0.1 μM) + PGE₂ (0.1 μM) + NS398 (50 μM), and (6) PGD₂ (14 μM) + NS398 (50 μM) (all 18-hour exposures). Cells were extracted for total RNA and were evaluated for 27-hydroxylase mRNA expression by QRT-PCR. Signals obtained from the amplification of GAPDH message were used as internal controls. (b) ABCA1 message is decreased by the COX-2 inhibitor NS398 and this decrease is reversed by prostaglandins E₁, E₂, and D₂. THP-1 human monocytes were exposed to the following conditions represented by the six bars (from left to right): (1) RPMI 1640, (2) NS398 (50 μM), (3) PGE₁ (0.1 μM) + NS398 (50 μM), (4) PGE₂ (0.1 μM) + NS398 (50 μM), (5) PGE₁ (0.1 μM) + PGE₂ (0.1 μM) + NS398 (50 μM), and (6) PGD₂ (14 μM) + NS398 (50 μM) (all 18-hour exposures). Cells were extracted for total RNA and were evaluated for ABCA1 mRNA expression by QRT-PCR. Signals obtained from the amplification of GAPDH message were used as internal controls. **p < 0.01 compared to NS398 (n = 5). ABCA1, ATP-binding cassette transporter A1; COX, cyclooxygenase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NS398, N-(2-cyclohexyloxy-4-nitrophenyl)methanesulfonamide; PG, prostaglandin; QRT-PCR, quantitative real-time polymerase chain reaction.

Figure 6

QRT-PCR for 27-hydroxylase and ABCA1 message in NS398-treated THP-1 macrophages exposed to prostaglandins or TXA₂. (a) 27-Hydroxylase message in THP-1 macrophages is decreased by the COX-2 inhibitor NS398 and this decrease is reversed by prostaglandins E₂ and D₂, but not TXA₂. THP-1 human macrophages were exposed to the following conditions represented by the five bars (from left to right): (1) RPMI 1640, (2) NS398 (50 μM), (3) PGE₂ (0.1 μM) + NS398 (50 μM), (4) PGD₂ (14 μM) + NS398 (50 μM), and (5) TXA₂ (3 μM) + NS398 (50 μM) (24-hour exposures to NS398 alone followed by addition of indicated PG or TXA₂ for a further 24 hours). Cells were extracted for total RNA and were evaluated for 27-hydroxylase mRNA expression by QRT-PCR. Signals obtained from the amplification of GAPDH message were used as internal controls. (b) ABCA1 message is decreased by the COX-2 inhibitor NS398 in THP-1 macrophages and this decrease is reversed by prostaglandins E₂ and D₂, but not TXA₂. THP-1 human macrophages were exposed to the following conditions represented by the five bars (from left to right): (1) RPMI 1640, (2) NS398 (50 μM), (3) PGE₂ (0.1 μM) + NS398 (50 μM), (4) PGD₂ (14 μM) + NS398 (50 μM), and (5) TXA₂ (3 μM) + NS398 (50 μM) (24-hour exposures to NS398 alone followed by addition of indicated PG or TXA₂ for a further 24 hours). Cells were extracted for total RNA and were evaluated for ABCA1 mRNA expression by QRT-PCR. Signals obtained from the amplification of GAPDH message were used as internal controls. *p < 0.05, **p < 0.01 compared to NS398 (n = 3). ABCA1, ATP-binding cassette transporter A1; COX, cyclooxygenase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NS398, N-(2-cyclohexyloxy-4-nitrophenyl)methanesulfonamide; PG, prostaglandin; QRT-PCR, quantitative real-time polymerase chain reaction; TXA₂, thromboxane A₂.

Figure 7

Decrease in ABCA1 protein in THP-1 macrophages exposed to NS398. Cultured THP-1 human macrophages were untreated or exposed to increasing concentrations of NS398 for 18 hours. Total cell protein was isolated and ABCA1 detected with specific rabbit polyclonal anti-ABCA1 antibody. Western blotting was performed with an anti-β-actin antibody to confirm equal protein loading. ABCA1, ATP-binding cassette transporter A1; NS398, N-(2-cyclohexyloxy-4-nitrophenyl)methanesulfonamide.

Figure 8

NS398 increases foam cell formation in THP-1 macrophages. THP-1 differentiated macrophages were treated with acetylated low-density lipoprotein (50 μg/ml, 48 hours) and further incubated alone or with the addition of NS398 (10 μM, 18 hours). Representative photomicrographs of oil red O staining to detect foam cells. NS398, N-(2-cyclohexyloxy-4-nitrophenyl)methanesulfonamide.

Figure 9

COX inhibition impairs reverse cholesterol transport. COX-1/2 inhibition downregulates 27-hydroxylase and ABCA1, thereby decreasing cholesterol efflux, in turn promoting the accumulation of cholesterol in macrophages that transform into foam cells. This effect is restored by the addition of prostaglandins. AA, arachidonic acid; ABCA1, ATP-binding cassette transporter A1; COX, cyclooxygenase; PG, prostaglandin; TXA, thromboxane A.