A novel anti-atherogenic role for COX-2--potential mechanism for the cardiovascular side effects of COX-2 inhibitors

Abstract

Atherosclerosis, the underlying cause of cardiovascular disease, is characterized by lipid accumulation, lipoprotein oxidation, and inflammation. Products of the cyclooxygenase (COX) pathway participate in acute and chronic inflammation. The inducible form of COX, COX-2, generates lipid mediators of inflammation that are pro-inflammatory and COX-2-selective inhibitors are potent anti-inflammatory agents. However, clinical data suggest an increased risk of cardiovascular side effects in patients using COX-2-selective inhibitors. In this paper, we sought to determine the effect of COX-2 deficiency on atherosclerosis-related lipoprotein metabolism in mice. We demonstrate that COX-2 deficiency resulted in (i) accumulation of lipids in circulation and liver, (ii) pro-inflammatory properties of HDL as measured by HDL's increased reactive oxygen species (ROS) content, decreased paraoxonase 1 (PON1) activity, decreased serum apoA-1, reduced ability to efflux cholesterol and to prevent LDL oxidizability, and (iii) increased TXB(2) in circulation. Moreover, when placed on an atherogenic diet, COX-2 deficiency resulted in (i) increased lipid deposition in the aorta, (ii) a further dramatic imbalance in circulating eicosanoids, i.e. decreased serum PGI(2) coupled with increased PGE(2) and TXB(2), and (iii) a marked elevation of pro-inflammatory cytokines, TNF and IL-6. Our results suggest, for the first time, that COX-2 deficiency contributes to the pro-atherogenic properties of HDL in mice.

Fig. 1

Absence of COX-2 results in increased lipid deposition in aorta and liver. (A) Lipid accumulation in the aorta (n=5 per group) and (B) liver cholesterol content (n=8–12 per group) were determined as described in Materials and Methods. Aortic lipid and liver cholesterol content are represented individually (open circles) and as averages (black bar) of each group. P-values were calculated by T-test for statistical analysis. WT = COX-2^+/+ wild-type mice, KO = COX-2^−/− mice, (C) = chow diet, (A) = atherogenic diet.

Fig. 2

ROS accumulation on HDL from COX-2^−/− mice and C57BL6/J wild-type mice treated with rofecoxib. Individual FPLC fractions from pooled serum samples from COX-2^+/+, COX-2^−/− mice (A and B) and C57BL6/J mice treated with either vehicle or rofecoxib (C and D) were assayed for cholesterol (A and C) and ROS (B and D). WT = COX-2^+/+ wild-type mice, KO = COX-2^−/− mice, (C) = chow diet, (A) = atherogenic diet.

Fig. 3

HDL from COX-2^−/− mice is dysfunctional and pro-inflammatory. (A) Individual FPLC fractions from pooled serum samples from COX-2^+/+ and COX-2^−/− mice were tested for PON activity (represented as units/mL). (B) Levels of apoA-1 in serum (20 μg/mL) were determined by ELISA. Concentrations are represented individually (open circles) and as averages (black bar) for each group (n=8–12). (C) HDL (25 μg/mL) isolated from COX-2^+/+ and COX-2^−/− mice was incubated for 6 hours with RAW cells preloaded with ³H-cholesterol. Percentage efflux is represented individually (open circles) and as averages (black bar) for each group (n=8–12). (D) HDL from COX-2^+/+ and COX-2^−/− mice was used in a monocyte chemotaxis assay as described in Materials and Methods. Data represented as average with one standard deviation of number of migrated monocytes in 9 fields for each HDL. Data are representative of three experiments. P-values were calculated by T-test for statistical analysis. WT = COX-2^+/+ wild-type mice, KO = COX-2^−/− mice. (C) = chow diet, (A) = atherogenic diet, hLDL = human LDL, hHDL = human HDL, none = no addition control.

Fig. 4

Absence of COX-2 alters the inflammatory balance of circulating eicosanoids. Concentrations of TXB₂ (A), PGE₂ (B), 6-keto PGF_1α (C), and LTB₄ (D) in serum from COX-2^+/+ and COX-2^−/− mice were determined by EIA kits. Eicosanoid levels are represented as individual concentrations (open circles) and averages (black bar) for each group (n=8–12). P-values were calculated by T-test for statistical analysis. WT = COX-2^+/+ wild-type mice, KO = COX-2^−/− mice, (C) = chow diet, (A) = atherogenic diet.

Fig. 5

Absence of COX-2 increases pro-inflammatory cytokines in circulation when challenged with an atherogenic diet. Concentrations of MCP-1 (A), TNF (B), IFN-γ (C), IL-6 (D), and IL-12 (E) in serum (50 μL) from COX-2^+/+ and COX-2^−/− mice were determined by cytometric bead array. Cytokine levels are represented as individual concentrations (open circles) and averages (black bar) for each group (n=8–12). P-values were calculated by T-test for statistical analysis. P-value denoted by (*) was determined by Mann-Whitney U test. WT = COX-2^+/+ wild-type mice, KO = COX-2^−/− mice, (C) = chow, (A) = atherogenic diet.