Cyclooxygenase-2 in endothelial and vascular smooth muscle cells restrains atherogenesis in hyperlipidemic mice

Abstract

Background: Placebo-controlled trials of nonsteroidal anti-inflammatory drugs selective for inhibition of cyclooxygenase-2 (COX-2) reveal an emergent cardiovascular hazard in patients selected for low risk of heart disease. Postnatal global deletion of COX-2 accelerates atherogenesis in hyperlipidemic mice, a process delayed by selective enzyme deletion in macrophages.

Methods and results: In the present study, selective depletion of COX-2 in vascular smooth muscle cells and endothelial cells depressed biosynthesis of prostaglandin I2 and prostaglandin E2, elevated blood pressure, and accelerated atherogenesis in Ldlr knockout mice. Deletion of COX-2 in vascular smooth muscle cells and endothelial cells coincided with an increase in COX-2 expression in lesional macrophages and increased biosynthesis of thromboxane. Increased accumulation of less organized intimal collagen, laminin, α-smooth muscle actin, and matrix-rich fibrosis was also apparent in lesions of the mutants.

Conclusions: Although atherogenesis is accelerated in global COX-2 knockouts, consistent with evidence of risk transformation during chronic nonsteroidal anti-inflammatory drug administration, this masks the contrasting effects of enzyme depletion in macrophages versus vascular smooth muscle cells and endothelial cells. Targeting delivery of COX-2 inhibitors to macrophages may conserve their efficacy while limiting cardiovascular risk.

Keywords: atherogenesis; cyclooxygenase; endothelial cell; prostaglandin; vascular smooth muscle.

Figure 1

Impact of COX-2 deletion on prostaglandin biosynthesis in mice on high fat diet. Overnight (5pm to 10am) fasting urine from WT and vascular COX-2 mutants were collected at the end of HFD feeding, and prostanoid metabolites were analyzed by liquid chromatography-mass spectrometry as described in the Methods. Urinary PGI-M (A and E) and PGE-M (B and F) were depressed in male and female COX-2 KOs after 3 and 6 months on HFD, respectively. Tx-M was increased in female KOs fed 6 months HFD. One-way ANOVA showed a significant effect of genotype on urinary prostanoid levels in both genders fed a HFD. Holm Sidak’s multiple comparison tests were used to test significant differences between WT and COX-2 mutants. Data are means ± SEMs. *p< 0.05, ***p<0.001, ****p<0.0001; n=18-22 per genotype.

Figure 2

Vascular specific COX-2 deletion elevates systolic blood pressure. Systolic blood pressure (SBP) was measured in conscious mice using the computerized non-invasive tail-cuff system as described in the Methods. SBP of male mice was elevated after 3 months on a HFD in E/V DKO compared to WT mice (A). This hypertensive phenotype was further augmented in all mutants after 6 months on a HFD, with the exception of female EC KOs (B). One-way ANOVA showed a significant effect of genotype on SBP in both genders. Holm Sidak’s multiple comparison tests were used to test significant differences between WT and COX-2 mutants. Data are means ± SEMs. **p< 0.01, ***p< 0.001; n=18-22 per genotype.

Figure 3

Vascular COX-2 restrains atherogenesis. Aortic atherosclerotic lesion burden, represented by the percentage of lesion area to total aortic area, was quantified by en face analysis of aortas from male mice fed a HFD for 3 and of female mice at 6 months. Representative en face preparations are shown (Lower panels). Lesion area tended to increase in male or female COX-2 mutants fed a HFD for 3 or 6 months respectively (A and B). One-way ANOVA revealed a significant effect of genotype (male, P= 0.0001 and female, P= 0.003) on lesion progression. Holm Sidak’s multiple comparison tests were used to test significant differences between WT and COX-2 KOs. Data are means ± SEMs. **p< 0.01, ****p<0.0001; n=18-22 per genotype.

Figure 4

Vascular COX-2 restrains aortic root lesion burden and orientation of structural collagen in lesions. A. Quantification of cross-sectional analysis of aortic root samples from female mice fed 6 months HFD, was performed by measuring total lesion area across the aortic root as detailed in Methods. One-way ANOVA (Kruskal-Wallis test) revealed a significant effect of genotype (P=0.048) on lesion progression. Dunnett’s multiple comparison tests were used to test significant differences between WT and COX-2 KOs. *p< 0.05. Data are means ± SEMs. n=5 per group and 7-9 sections from each animal were averaged. B. Second harmonic generation (SHG) detection of structural collagen in WT (n=22) and E/V DKO (n= 21) lesions from female mice on a HFD for 6 months. Fast Fourier transform (FFT)-based analyses revealed a more random orientation of collagen fibers in E/V DKO lesions. Data are means ± SEMs. **p < 0.01 (Mann-Whitney test, two-tails). C and D. Representative WT and E/V DKO SHG images are shown.

Figure 5

Morphometric consequences of vascular COX-2 deletion on lesion development. Lesion morphology in aortic roots from female mice fed HFD for 6 months were analyzed. Quantification of immunohistochemical staining of collagen (A), laminin (B), α-SMA (C) and VCAM-1 (D) from each genotype are shown in parallel with their representative aortic root sections. One-way ANOVA (Kruskal-Wallis test) revealed a significant effect of genotype (P<0.05) on lesion morphology. Dunnett’s multiple comparison tests were used to test significant differences between WT and COX-2 KOs. Data are means ± SEMs. n=4-5 per genotype. L- lumen, m- media, nc- necrotic core, fc- foam cell.

Figure 6

Vascular COX-2 deletion increases COX-2 expression in lesional macrophages. Lesion morphology in aortic roots from female mice fed a HFD for 6 months were analyzed. Quantification of immunohistochemistry staining of COX-2 (A) and CD11b (B) from WT and E/V DKO are shown. A Mann-Whitney test (two-tailed) revealed a significant increase in COX-2 positive cells in DKO compared to WT, **p< 0.01; n=8-12 per genotype. Data are means ± SEMs. C. Representative aortic root sections from COX-2 staining (left panel) and CD11b (right panel) from E/V DKO are shown. Arrows indicate lesional cells that are stained positive for both COX-2 and CD11b. L- lumen, m- media.