β-Carotene Attenuates Angiotensin II-Induced Aortic Aneurysm by Alleviating Macrophage Recruitment in Apoe(-/-) Mice

Abstract

Abdominal aortic aneurysm (AAA) is a common chronic degenerative disease characterized by progressive aortic dilation and rupture. The mechanisms underlying the role of α-tocopherol and β-carotene on AAA have not been comprehensively assessed. We investigated if α-tocopherol and β-carotene supplementation could attenuate AAA, and studied the underlying mechanisms utilized by the antioxidants to alleviate AAA. Four-months-old Apoe(-/-) mice were used in the induction of aneurysm by infusion of angiotensin II (Ang II), and were orally administered with α-tocopherol and β-carotene enriched diet for 60 days. Significant increase of LDL, cholesterol, triglycerides and circulating inflammatory cells was observed in the Ang II-treated animals, and gene expression studies showed that ICAM-1, VCAM-1, MCP-1, M-CSF, MMP-2, MMP-9 and MMP-12 were upregulated in the aorta of aneurysm-induced mice. Extensive plaques, aneurysm and diffusion of inflammatory cells into the tunica intima were also noticed. The size of aorta was significantly (P = 0.0002) increased (2.24±0.20 mm) in the aneurysm-induced animals as compared to control mice (1.17±0.06 mm). Interestingly, β-carotene dramatically controlled the diffusion of macrophages into the aortic tunica intima, and circulation. It also dissolved the formation of atheromatous plaque. Further, β-carotene significantly decreased the aortic diameter (1.33±0.12 mm) in the aneurysm-induced mice (β-carotene, P = 0.0002). It also downregulated ICAM-1, VCAM-1, MCP-1, M-CSF, MMP-2, MMP-9, MMP-12, PPAR-α and PPAR-γ following treatment. Hence, dietary supplementation of β-carotene may have a protective function against Ang II-induced AAA by ameliorating macrophage recruitment in Apoe(-/-) mice.

Figure 1. Induction of abdominal aneurysm in Apoe−/− mice.

(A) Abdominal aneurysm observed in the Ang II-treated Apoe−/− mice; (B) Plaque reduction in the entire aorta and pseudoaneurysm observed in the abdominal and thoracic aorta of β-carotene- treated animals; (C) Abdominal aneurysm resembles with Ang II-treated mice but plaque formation was reduced in the α-tocopherol-treated animals.

Figure 2. Biochemical analysis of the Apoe−/− experimental groups.

Serum TC, triglycerides and LDL levels were analysed in the different study groups. (Ang II-treated (n = 6), β-carotene-treated (n = 6) and control (n = 6). P values of less than 0.05 were considered significant.

Figure 3. Histopathological analysis of abdominal aortic tissues.

(A–C)Ang II treatment. Multiple foci of adhesive atheroma formation seen in the tunica intima. Severe vacuolar and fatty degeneration and accumulation, and invasion of modified macrophages (foam cells) are also seen; (D–F) β-carotene treatment. Mild to moderate dissolution of atheromatous plaque in the tunica intima. Foci of calcified region (box) in which sheet like structure composed of chondrocytelike cells; collagen and inflammatory cells in the intimal layer of aorta (arrow) are seen; (G–I) α-tocopherol treatment. Complete atheromatous plaque dissolution is visible in the intimal part of aorta. (J–L) Combined antioxidant treatment. Mild disintegration or dissolution of atheromatous plaques and thrombus (arrow) are seen.

Figure 4. Confocal microscopy of CD45.2 expression in aortic tissue sections of Apoe−/− mice.

(A–C CD45.2 Ang II- treated group) 45.2 over-expression of CD45.2 protein seen in the atheromatous plaques in the tunica media of Ang II- treated Apoe−/− mice; (D–F CD45.2 α-tocopherol-treated) Mild levels of expression are seen in α-tocopherol-treated group; (G–I CD45.2 β-carotene-treated) CD45.2 is not expressed in the β-carotene-treated group; (J–L CD45.2 α-tocopherol+β-carotene-treated) Combined treatment shows mild expressions of CD45.2 (M–O Control).

Figure 5. (A) FACS analysis of peripheral blood against Mac3.

Significant increase in the Mac3 positive cells (R2) in Ang II-treated animals compared with control (R1); (B) FACS analysis of peripheral blood against CD45.2. Slight increase in the percentage of CD45.2 cells is seen in the Ang II-treated (R1) animals compared with control (R1); (C–E) FACS analysis of peripheral blood against Mac3. α-tocopherol-treatment slightly decreased Mac3 expression whereas no marked difference is seen in the β-carotene-treated group. Combined treatment shows downregulation of Mac3 on macrophages; (F–H) FACS analysis of peripheral blood against CD45.2. No marked difference is seen between the control and antioxidant-treated groups with CD45.2 expression.

Figure 6. Confocal microscopic images of expression of Mac3 in the aorta of Apoe−/− mice in the different treatment groups.

(A–C Mac3- Ang II-treated) Mac3 is over-expressed in the atheromatous plaques of aortic tunica media of Ang II-treated mice; (D–F Mac3-α-tocopheroltreated & G- I Mac3- β-carotene- treated) Dissoluted or loosely cohesive plaques seen in the tunica media in the antioxidant-treated group; (J–L Control).

Figure 7. Confocal microscopic images of expression of ICAM-1 (CD54) in the aorta of Apoe−/− mice.

(A–C ICAM-1 Ang II-treated) Over-expression of ICAM-1 protein is seen in the atheromatous plaques of tunica intima of Ang II-treated animals. (D–F ICAM-1 α -tocopheroltreated) ICAM-1 expression is not seen in the α-tocopherol- treated group; (G–L ICAM-1 β-carotene-treated) Less expression of ICAM-1 is seen in the dissoluted or loosely cohesive plaques of tunica intima of both β-carotene-treated and combined antioxidant-treated animals. (MO) Control.

Figure 8. Confocal microscopic images of expression of VEGF in the aorta of Apoe−/− mice.

(A–C VEGF Ang II-treated) Upregulation of VEGF protein is seen in the atheromatous plaques of tunica intima of Ang II-treated animals. (D–F VEGF α-tocopherol-treated) VEGF expression is not seen in the α-tocopherol-treated group. (G–L) Lesser expression of VEGF is seen in the dissolved or loosely cohesive plaques of tunica intima of both β-carotene-treated and combined antioxidant-treated animals. (M–O) Control.

Figure 9. Confocal microscopic images of expression of RAR in the aorta of Apoe−/− mice.

(A–C RAR Ang II-treated) RAR expression is not seen in the Ang II-treated group; (D–I) Over-expression of RAR- α is observed in the dissoluted and loosely cohesive atheromatous plaques of tunica intima of both groups treated with α-tocopherol and β-carotene antioxidants.