P2Y receptors and atherosclerosis in apolipoprotein E-deficient mice

Abstract

Background and purpose: P2Y nucleotide receptors are involved in the regulation of vascular tone, smooth muscle cell (SMC) proliferation and inflammatory responses. The present study investigated whether they are involved in atherosclerosis.

Experimental approach: mRNA of P2Y receptors was quantified (RT-PCR) in atherosclerotic and plaque-free aorta segments of apolipoprotein E-deficient (apoE(-/-)) mice. Macrophage activation was assessed in J774 macrophages, and effects of non-selective purinoceptor antagonists on atherosclerosis were evaluated in cholesterol-fed apoE(-/-) mice.

Key results: P2Y(6) receptor mRNA was consistently elevated in segments with atherosclerosis, whereas P2Y(2) receptor expression remained unchanged. Expression of P2Y(1) or P2Y(4) receptor mRNA was low or undetectable, and not influenced by atherosclerosis. P2Y(6) mRNA expression was higher in cultured J774 macrophages than in cultured aortic SMCs. Furthermore, immunohistochemical staining of plaques demonstrated P2Y(6)-positive macrophages, but few SMCs, suggesting that macrophage recruitment accounted for the increase in P2Y(6) receptor mRNA during atherosclerosis. In contrast to ATP, the P2Y(6)-selective agonist UDP increased mRNA expression and activity of inducible nitric oxide synthase and interleukin-6 in J774 macrophages; this effect was blocked by suramin (100-300 microM) or pyridoxal-phosphate-6-azophenyl-2'-4'-disulphonic acid (PPADS, 10-30 microM). Finally, 4-week treatment of cholesterol-fed apoE(-/-) mice with suramin or PPADS (50 and 25 mg.kg(-1).day(-1) respectively) reduced plaque size, without changing plaque composition (relative SMC and macrophage content) or cell replication.

Conclusions and implications: These results suggest involvement of nucleotide receptors, particularly P2Y(6) receptors, during atherosclerosis, and warrant further research with selective purinoceptor antagonists or P2Y(6) receptor-deficient mice.

Figure 1

Comparative expression of mRNA for P2Y₂ receptors (A), P2Y₆ receptors (B) and the ratio P2Y₆/P2Y₂ (C) in aortic arches of old (18 months) atherosclerotic apoE^–/– and plaque-free WT mice (18 months) on regular chow. The expression of the P2Y₆ receptor was significantly increased in arteries with atherosclerotic lesions. β-Actin was used as an endogenous reference gene and the mean value of the WT was used as a calibrator and set to one. Results show mean and SEM, n= 6 (apoE^–/–) or n= 4 (WT). *P < 0.05, ***P < 0.001. WT, wild-type.

Figure 2

Relative expression of mRNA for P2Y₂ receptors, P2Y₆ receptors and the ratio P2Y₆/P2Y₂ in endothelial cell-denuded aortae of young (A) and old (B) apoE^–/– mice on regular chow. Young apoE^–/– mice (4 months) that had not developed atherosclerotic plaques yet, showed no regional differences in the expression of P2Y₂ or P2Y₆ receptors between aortic arch (AA) and thoracic aorta (TA). In contrast, old apoE^–/– (18 months) with established plaques in the aortic arch had more P2Y₆ receptor transcripts in the atherosclerotic region. β-Actin was used as an endogenous reference gene and the mean value of the TA was used as calibrator and set to one. Results show mean and SEM, n= 5. *P < 0.05, **P < 0.01.

Figure 3

Relative expression of mRNA for P2Y₂ receptors (A), P2Y₆ receptors (B) and the ratio P2Y₆/P2Y₂ (C) in naïve murine aortic SMCs (SMC aorta), in a primary culture of aortic SMCs (cultured SMC) and in J774 macrophages (J774). The expression of P2Y₆ receptors decreased upon cultivation of SMCs, whereas J774 macrophages displayed a relatively high expression. ΔC_T= C_Tβ-actin – C_T P2Y_{2 or 6}. Results show mean and SEM, n= 5; *P < 0.05, **P < 0.01, anova, Bonferroni post hoc test. SMC, smooth muscle cell.

Figure 4

Staining of α-smooth muscle actin (A, C, brown), of P2Y₆ receptors (B, D, G, H, I, red) and of the macrophage marker mac-3 (E, red) in consecutive transverse sections of a plaque-free (A, B) and an atherosclerotic (C, D, E, F, G, H, I) segment of the aorta of an apoE^–/– mouse (18 months old). α-Smooth muscle actin-positive staining was observed in the media of both segments, but also in SMCs of the fibrous cap. Mac-3-positive staining was found in the plaque, and occasionally in the inner media. P2Y₆-positive staining was most abundant in SMCs of the media (arrow heads) in segments with (D, G) or without (B) plaques. However, in the plaque P2Y₆ receptors were mostly present in macrophages (D, I, arrows) and in a few SMCs in the fibrous cap (D, H, arrow head). Staining of P2Y₆ receptors was completely abolished after incubation of the antibody with the polypeptide that had been used to immunize the rabbits, illustrating the selectivity of the antibody (F). The dashed line shows the border between media and plaque. Magnification: 200× (A, B); 100× (C, D, E, F); 1000× (G, H, I). FC, fibrous cap; m, media; SMC, smooth muscle cell.

Figure 6

iNOS (A) and IL-6 (B) mRNA expression in J774 macrophages after stimulation (6 h) with 1 mM UDP, with and without suramin (100 and 300 µM) or PPADS (10 and 30 µM). The UDP-induced up-regulation of iNOS and IL-6 was antagonized by both non-selective purinoceptor antagonists. Results show mean and SEM of four independent experiments. *P < 0.05, **P < 0.01 different from 1 mM UDP, anova and Dunnett's test. IL-6, interleukin 6; iNOS, inducible nitric oxide synthase; PPADS, pyridoxal-phosphate-6-azophenyl-2′-4′-disulphonic acid; UDP, uridine 5′ diphosphate.

Figure 5

Concentrations of nitrite (A, B) and IL-6 (C, D) in supernatants of J774 macrophages after a 42-h incubation with increasing concentrations of ATP (A, C) or UDP (B, D). Results show mean and SEM of four independent experiments. *P < 0.05, **P < 0.01, anova, Dunnett's post hoc test. ATP, adenosine 5′ triphosphate; IL-6, interleukin 6; UDP, uridine 5′ diphosphate.

Figure 7

Cross-sectional plaque area at the level of the aortic root in control, suramin (50 mg·kg⁻¹·day⁻¹), and PPADS (25 mg·kg⁻¹·day⁻¹)-treated apoE^–/– mice (4 months) on a Western-type diet (A). Plaque size was significantly lower in mice treated with suramin or PPADS. Relative areas of macrophages (mac-3, B) and SMCs (α-SMC actin, C), and cell proliferation (percentage PCNA-positive nuclei, D) were not influenced by suramin or PPADS. Results show mean and SEM, n= 6. *P < 0.05, **P < 0.01, Kruskal Wallis anova. PCNA, proliferating cell nuclear antigen; PPADS, pyridoxal-phosphate-6-azophenyl-2′-4′-disulphonic acid; SMC, smooth muscle cell.

Figure 8

mRNA expression of P2Y₂ receptors (A), P2Y₆ receptors (B), the macrophage marker CD68 (C), α-SMC actin (D), iNOS (E) and IL-6 (F) in the atherosclerotic aortic arch (AA) and in virtually plaque-free thoracic aorta (TA) segments of control mice and apoE^–/– mice (4 months), on a Western-type diet, treated with suramin or PPADS. Atherosclerotic AA segments showed increased mRNA expression of P2Y₆ receptors, CD68 and iNOS (tendency), unaltered expression of P2Y₂ receptors and IL-6, and slightly decreased α-SMC actin mRNA. Treatment with suramin or PPADS increased mRNA of P2Y₂ receptors and showed a tendency to decrease iNOS activation, whereas the expression of P2Y₆ receptors, CD68, α-SMC actin and IL-6 were not influenced by the purinoceptor antagonists. Results show mean and SEM, n= 6. Significances of a factorial anova with segment (AA or TA) and drug treatment as factors are shown; the interaction term was never significant. IL-6, interleukin 6; iNOS, inducible nitric oxide synthase; PPADS, pyridoxal-phosphate-6-azophenyl-2′-4′-disulphonic acid; SMC, smooth muscle cell.