Tongxinluo mitigates atherogenesis by regulating angiogenic factors and inhibiting vasa vasorum neovascularization in apolipoprotein E-deficient mice

Abstract

Vasa vasorum (VV) neovascularization contributes to atherogenesis and its expansion and distribution is correlated with intraplaque expression of angiogenic factors. The present study investigated the roles of Tongxinluo (TXL), a traditional Chinese medication, on VV proliferation and atherogenesis. In vitro, TXL pre-treatment reversed the tumor necrosis factor-a (TNF-a) induced expression of vascular endothelial growth factor A (VEGF-A) and angiopoietin-1 (ANGPT-1) but not ANGPT-2, leading to increased ratio of ANGPT-1 to ANGPT-2. Consistently, TXL treatment (at a dosage of 0.38, 0.75, 1.5 g/kg/d, respectively) decreased the expression of VEGF-A while increased that of ANGPT-1 in early atherosclerotic lesions of apolipoprotein E deficient (apoE-/-) mice. On aortic ring assay, microvessels sprouting from aortas were significantly inhibited in TXL-treated mice. Moreover, VV neovascularization in plaques was markedly reduced with TXL treatment. Histological and morphological analysis demonstrated that TXL treatment reduced plaque burden, plaque size and changed the plaque composition. These data suggest that TXL inhibits early atherogenesis through regulating angiogenic factor expression and inhibiting VV proliferation in atherosclerotic plaque. Our study shed new light on the anti-atherosclerotic effect of TXL.

Keywords: Tongxinluo; atherogenesis; neovascularization; vasa vasorum.

Figure 1. TXL solution regulates VEGF-A, ANGPT-1 and ANGPT-2 expression in TNF-α-stimulated RAW 264.7 cells

A. qRT-PCR analysis of mRNA level of VEGF-A in TNF-α-stimulated RAW 264.7 cells pretreated with or without TXL solution (100, 200, 500 μg/ml). B. Representative western blot image and quantification of protein level of VEGF-A in TNF-α-stimulated RAW 264.7 cells pretreated with or without TXL solution. C. qRT-PCR analysis of relative mRNA level of ANGPT-1 in TNF-α-stimulated RAW 264.7 cells pretreated with or without TXL solution. D. Representative western blot image and quantification of protein level of ANGPT-1 in TNF-α-stimulated RAW 264.7 cells pretreated with or without TXL solution. E. qRT-PCR analysis of relative mRNA level of ANGPT-2 in TNF-α-stimulated RAW 264.7 cells pretreated with or without TXL solution. F. Representative western blot image and quantification of protein level of ANGPT-2 in TNF-α-stimulated RAW 264.7 cells pretreated with or without TXL solution. G. Quantification of the protein ratio of ANGPT-1/ANGPT-2 in TNF-α-stimulated RAW 264.7 cells pretreated with or without TXL solution. Bar 1: TNF-α only; bar 2: TNF-α+TXL 100 μg/ml; bar 3: TNF-α+TXL 200 μg/ml; bar 4: TNF-α+TXL 500 μg/ml; Data are mean ±SEM. * P<0.05 vs TNF-α only; # P<0.05 vs TNF-α+TXL 100 μg/ml.

Figure 2. TXL regulates angiogenic factor expression in apoE−/− mice

Representative immunohistochemical images and quantification of A. VEGF-A, B. ANGPT-1, C. ANGPT-2 levels in atherosclerotic plaques of apoE−/− mice. Representative western blot images and quantification of D. VEGF-A, E. ANGPT-1 and F. ANGPT-2 expression in treated mice aortas. G. Quantification of the protein ratio of ANGPT-1/ANGPT-2 in four groups of treated mice. Bar 1: control; bar 2: TXL-L; bar 3: TXL-M; bar 4: TXL-H; Data are mean ±SEM. * P <0.05 vs Control; ^# P <0.05 vs TXL-L.

Figure 3. TXL inhibits plaque-associated angiogenesis ex vivo and in vivo

A. Representative images of mouse aortic ring sprouts with four treatment groups. B. Quantification of sprouting number in aortic rings. C. Quantification of sprouting length in aortic rings. D. Representative images of VV proliferation by Tomato lectin staining in aortic root plaques. E. Quantitative analysis of VV number in four treatments. Data are mean ±SEM. * P <0.05 vs Control; ^# P <0.05 vs TXL-L.

Figure 4. TXL suppresses early atherogenesis in apoE−/− mice

A. Representative images of en face Oil-Red O staining of aortas in four groups of mice. B. Quantitative analysis of en face aorta lesions expressed as percentage lesion area relative to total aorta area. C. Representative images of cross-sectional aortic root lesions by H&E staining. D. Quantitative analysis of cross-sectional plaque area in aortic roots. Data are mean ± SEM. * P <0.05 vs Control; ^# P <0.05 vs TXL-L.

Figure 5. TXL stabilizes atherosclerotic plaque in apoE−/− mice

A. Representative image and quantification of lipid deposition in four treatment groups by Oil-Red O staining. B. Representative immunohistochemical MOMA-2 staining image and quantification of plaque macrophage content. C. Representative immunohistochemical α-SMA staining and quantification of plaque smooth muscle cell content. D. Representative image and quantification of plaque collagen content by Picrosirius Red staining. E. Quantitative analysis of fibrous cap thickness by Picrosirius Red staining. Data are mean ±SEM.* P <0.05 vs Control; ^# P <0.05 vs TXL-L.