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. 2016 Oct:85:11-20.
doi: 10.1016/j.vph.2016.06.001. Epub 2016 Jun 26.

Deletion of P2Y2 receptor reveals a role for lymphotoxin-α in fatty streak formation

Shaomin Qian  1 , April Hoggatt  1 , Yava L Jones-Hall  2 , Carl F Ware  3 , Paul Herring  1 , Cheikh I Seye  4
Affiliations

Deletion of P2Y2 receptor reveals a role for lymphotoxin-α in fatty streak formation

Shaomin Qian et al. Vascul Pharmacol. 2016 Oct.

Abstract

Background: Lymphotoxin alpha (LTα) is expressed in human atherosclerotic lesions and genetic variations in the LTα pathway have been linked to myocardial infarction. Activation of the P2Y2 nucleotide receptor (P2Y2R) regulates the production of LTα. in vitro. We aimed to uncover a potential pathway linking purinergic receptor to LTα-mediated inflammatory processes pivotal to the early stages of atherosclerosis in apolipoprotein E (ApoE(-)(/)(-)) deficient mice.

Methods and results: En face immunostaining revealed that P2Y2R and VCAM-1 are preferentially expressed in the atherosclerosis prone site of the mouse aortic sinus. Deletion of the P2Y2R gene suppresses VCAM-1 expression. Compared with ApoE(-)(/)(-) mice, ApoE(-)(/)(-) mice lacking the P2Y2R gene (ApoE(-)(/)(-)/P2Y2R(-)(/)(-)) did not develop fatty streak lesions when fed a standard chow diet for 15weeks. Systemic and CD4(+) T cell production of the pro-inflammatory cytokine lymphotoxin-alpha (LTα) were specifically inhibited in ApoE(-)(/)(-)/P2Y2R(-)(/)(-)mice. Anti-LTα preventive treatment was initiated in ApoE(-)(/)(-)mice with intraperitoneal administration of recombinant human tumor necrosis factor receptor 1 fusion protein (TNFR1-Fc) on 5 consecutive days before the disease onset. Remarkably, none of the TNFR1:Fc-treated ApoE(-)(/)(-)mice exhibited atherosclerotic lesions at any developmental stage.

Significance: ApoE(-)(/)(-) mice deficient in P2Y2R exhibit low endothelial cell VCAM-1 levels, decreased production of LTα and delayed onset of atherosclerosis. These data suggest that targeting this nucleotide receptor could be an effective therapeutic approach in atherosclerosis.

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Figures

Fig. 1
Fig. 1
Differential expression of VCAM-1 in regions of the mouse aorta. A, representative en face immunostaining for VCAM-1 expression in the aorta of wild-type mice. Endothelial cell nuclei and VCAM-1 are in blue and green, respectively. Scale bar, 20 μm. P < 0.05. n = 15 visual fields. B, Flow cytometry analysis of EC surface expression of VCAM-1 in the aortic sinus and descending thoracic aorta of wild-type mice. Graphs show that the number of cells expressing VCAM-1 on the cell surface was greater in the aortic sinus as compared to the descending aorta. Endothelial cells incubated with only a second antibody were used as controls.
Fig. 2
Fig. 2
High P2Y2R expression in the inner curvature of the aortic arch in wild-type mice. A–B, representative en face images and quantitative histograms of P2Y2R-positive cells in different areas of the aortic sinus and descending thoracic aorta represented in the schematic. Endothelial cell nuclei and P2Y2R are in blue and green, respectively. Scale bar, 20 μm. P < 0.05. n = 15 visual fields from 4 different mice.
Fig. 3
Fig. 3
Effect of P2Y2R gene deletion on VCAM-1 in different regions of the mouse aorta. A, Real-time PCR analysis of VCAM-1 mRNA levels in the aortic sinus of wild-type (n = 4) and P2Y2R−/− (n = 3) mice. P < 0.05. B, representative en face immunostaining for VCAM-1 expression in the arch and descending thoracic aorta in P2Y2R−/− mice. No VCAM-1-positive cell was detected in either part of the aorta. Endothelial cell nuclei are in shown blue. Scale bar, 20 μm. P < 0.05. n = 15 visual fields from 4 different mice.
Fig. 4
Fig. 4
VCAM-1 promoter reporter assay. Luciferase activity in endothelial cells transfected with a pGL3 vector containing murine VCAM-1 promoter and stimulated with UTP-γ-S. T Error bars represent standard error of mean from triplicates. P < 0.05.
Fig. 5
Fig. 5
Analysis of atherosclerotic lesions in the aortic sinus of male ApoE−/− and ApoE−/−/P2Y2R−/− littermate mice fed standard chow diet for 15 weeks. A, Aortic sinus cross sections stained with Masson’s trichrome for gross morphological analysis and classification of lesions as shown in Table 2. B–C, Cross sections were stained with Oil Red O and the relative lesion area was calculated by dividing the lesion area by the total cross-sectional area. D, Representative images of immunohistological staining of atherosclerotic lesions in the aortic sinus. Adjacent sections were stained with, Mac-3 antibody, VCAM-1 and smooth muscle α-actin antibodies, respectively. Each scale bar represents 100 μm.
Fig. 6
Fig. 6
Time course of LTα, and TNF-α production in activated CD4+ T cells. A, CD4+ T cells were stimulated with anti-CD3 mAb, and the culture supernatants were collected after 1, 2, and 3 days for cytokine measurement. Data shown are mean ± SEM of 4 experiments, P < 0.05. B, CD4+ T cells were stimulated with anti-CD3 mAb and collected at the indicated time periods. Total RNA was extracted and subjected to Real-time PCR for the detection of LTα, and TNF-α mRNAs. Cytokine mRNA levels were quantified by densitometric analysis.
Fig. 7
Fig. 7
Effect of TNFR1:Fc on the development of atherosclerosis in ApoE−/− mice. TNFR1:Fc or human IgG1 was administered (100 μg/mouse for 5 consecutive days) to 5 week-old ApoE−/− mice fed a standard chow diet. Mice were sacrificed at 15 weeks of age. A, Aortic sinus cross sections were stained with Masson’s trichrome for gross morphological evaluation. B–C, Cross sections were stained with Oil Red O and the relative lesion area was then quantified. P < 0.05 vs IgG-injected controls.
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