Down-regulation of microRNA-342-5p or Up-regulation of Wnt3a Inhibits Angiogenesis and Maintains Atherosclerotic Plaque Stability in Atherosclerosis Mice

Abstract

Evidence has demonstrated that microRNA-342-5p (miR-342-5p) is implicated in atherosclerosis (AS), but little is known regarding its intrinsic regulatory mechanisms. Here, we aimed to explore the effect of miR-342-5p targeting Wnt3a on formation of vulnerable plaques and angiogenesis of AS. ApoE^-/- mice were fed with high-fat feed for 16 w to replicate the AS vulnerable plaque model. miR-342-5p and Wnt3a expression in aortic tissues of AS were detected. The target relationship between miR-342-5p and Wnt3a was verified. Moreover, ApoE^-/- mice were injected with miR-342-5p antagomir and overexpression-Wnt3a vector to test their functions in serum lipid levels, inflammatory and oxidative stress-related cytokines, aortic plaque stability and angiogenesis in plaque of AS mice. miR-342-5p expression was enhanced and Wnt3a expression was degraded in aortic tissues of AS mice and miR-342-5p directly targeted Wnt3a. Up-regulating Wnt3a or down-regulating miR-342-5p reduced blood lipid content, inflammatory and oxidative stress levels, the vulnerability of aortic tissue plaque and inhibited angiogenesis in aortic plaque of AS mice. Functional studies show that depleting miR-342-5p can stabilize aortic tissue plaque and reduce angiogenesis in plaque in AS mice via restoring Wnt3a.

Keywords: Atherosclerosis; MicroRNA-342-5p; Microvessel density; Vulnerability index; Vulnerable plaque; Wnt3a.

Fig. 1

miR-342-5p increases and Wnt3a decreases in aortic tissues of ApoE^−/− mice and miR-342-5p directly targets Wnt3a. A Expression of miR-342-5p in aortic tissue of mice detected by RT-qPCR. B Expression of Wnt3a mRNA in aortic tissue of mice detected by RT-qPCR (n = 12). C, D Wnt3a and β-catenin protein expression in aortic tissues of mice tested by Western blot analysis (n = 12). E Binding site of miR-342-5p within Wnt3a 3′-UTR. F miR-342-5p agomir dose-dependently decreased the relative activity in cells transfected with Wnt3a 3′-UTR (N = 3). G Relative activity of luciferase in cells with wild-type and mutant Wnt3a 3′-UTR after transfected with miR-342-5p agomir or scramble (N = 3). *P < 0.05 vs. the normal group, ^#P < 0.05 vs. the NC group. & P < 0.05 vs. the miR-342-5p agomir group. Measurement data were indicated as mean ± standard deviation. Comparisons between two groups were formulated by t-test, while comparisons among multiple groups were assessed by one-way ANOVA followed by Tukey’s multiple comparisons test. AS, atherosclerosis; NC, negative control

Fig. 2

Effects of up-regulated Wnt3a or down-regulated miR-342-5p on the lipid levels in ApoE^−/− mice. A Comparison of HDL-C contents in serum of mice group. B Comparison of LDL-C contents in serum of mice group. C Comparison of TG contents in serum of mice group. D Comparison of TC contents in serum of mice group. n = 12. *P < 0.05 vs. the normal group, ^#P < 0.05 vs. the NC group. & P < 0.05 vs. the miR-342-5p agomir group. Measurement data were indicated as mean ± standard deviation. Comparisons among multiple groups were assessed by one-way ANOVA followed by Tukey’s multiple comparisons test. AS, atherosclerosis; NC, negative control

Fig. 3

Effects of overexpression of Wnt3a or low expression of miR-342-5p on inflammatory and oxidative stress-related cytokines in serum of ApoE^−/− mice. A Comparison of IL-5 contents in serum of mice group. B Comparison of IL-12p70 contents in serum of mice group. C Comparison of IFN-γ contents in serum of mice group. D Comparison of TNF-α contents in serum of mice group. E, Comparison of MDA content in serum of mice group. F Comparison of SOD activity in serum of mice group. n = 12. *P < 0.05 vs. the normal group, ^#P < 0.05 vs. the NC group. & P < 0.05 vs. the miR-342-5p agomir group. Measurement data were indicated as mean ± standard deviation. Comparisons among multiple groups were assessed by one-way ANOVA followed by Tukey’s multiple comparisons test. AS, atherosclerosis; NC, negative control

Fig. 4

Effects of depletion of miR-342-5p or restoration of Wnt3a on lipid and collagen content in aortic plaque of ApoE^−/− mice. A Results of aortic HE staining in mice (scale bar: 50 μm). B Comparison of aortic plaque area in each group of mice. C Results of aortic Oil Red O staining in all groups of mice (scale bar: 100 μm). D Comparison of lipid content in aortic tissue of mice. E Results of aortic Sirius red staining in each group of mice (scale bar: 50 μm). F Comparison of collagen contents in aortic tissues of mice. n = 12. ^#P < 0.05 vs. the NC group. & P < 0.05 vs. the miR-342-5p agomir group. Measurement data were indicated as mean ± standard deviation. Comparisons among multiple groups were assessed by one-way ANOVA followed by Tukey’s multiple comparisons test. AS, atherosclerosis; NC, negative control

Fig. 5

Effects of high expression of Wnt3a or poor expression of miR-342-5p on aortic plaque vulnerability of ApoE^−/− mice. A Immunohistochemical staining of MOMA-2 in each group of mice (scale bar: 50 μm). B Quantitative analysis of figure A. C Immunohistochemical staining of α-SMA in each group of mice (scale bar: 50 μm). D Quantitative analysis of figure C. E Comparison of plaque vulnerability index in aortic tissues of AS mice. n = 12. ^#P < 0.05 vs. the NC group. & P < 0.05 vs. the miR-342-5p agomir group. Measurement data were indicated as mean ± standard deviation. Comparisons among multiple groups were assessed by one-way ANOVA followed by Tukey’s multiple comparisons test. AS, atherosclerosis; NC, negative control

Fig. 6

Effects of low expression of miR-342-5p or overexpression of Wnt3a on MVD in aortic plaque of ApoE^−/− mice. A Immunohistochemical staining of CD34 in each group of ApoE^−/− mice (scale bar: 50 μm). B Comparison of MVD in aortic plaque in ApoE^−/− mice. C Comparison of CD34 protein expression in ApoE^−/− mice. n = 12. ^#P < 0.05 vs. the NC group. & P < 0.05 vs. the miR-342-5p agomir group. Measurement data were indicated as mean ± standard deviation. Comparisons among multiple groups were assessed by one-way ANOVA followed by Tukey’s multiple comparisons test. AS, atherosclerosis; NC, negative control