Figure 5. Effect of CXCR4 deletion on cholesterol efflux, vascular tone and SMC phenotype.

(A, B) Quantification of cholesterol efflux from lipid-laden macrophages using HDL or pooled serum from TaglnCre⁺ vs TaglnCre^-Cxcr4^fl/flApoe^−/− mice (A, n=6) or from SmmhcCre⁺ vs SmmhcCre^-Cxcr4^fl/flApoe^−/− mice (B, n=4) after 12 weeks of HFD as acceptor. Data represent mean±SD (Mann-Whitney test for comparing Cre- vs Cre+; 2-way ANOVA with Bonferroni post-test for comparing without vs with cAMP). (C) ApoA1 protein levels in serum of TaglnCre⁺ vs TaglnCre^-Cxcr4^fl/flApoe^−/− mice after 12 weeks of HFD, as measured by Western blot analysis A representative blot and densitometry quantification is shown (n=6; mean±SD; Mann-Whitney test). (D,E) Acetylcholine-induced endothelium-dependent relaxation (D, n=20 rings with endothelium or n=9-10 rings without endothelium from 8-9 mice) and DETA-NONOate-induced endothelium-independent relaxation (E, n=16-18 rings with endothelium or n=11-16 rings without endothelium from 8-9 mice) of aortic rings from TaglnCre⁺ vs TaglnCre^-Cxcr4^fl/flApoe^−/− mice after 12 weeks of HFD, with or without removal of the endothelium, as indicated. Data represent mean±SEM (2-way repeated measures ANOVA with Bonferroni post-test for comparing Cre^- vs Cre⁺). (F) Ratio of L-NAME-induced contraction (300 µM L-NAME) to basal pre-contraction without L-NAME in aortic rings from TaglnCre⁺ vs TaglnCre^-Cxcr4^fl/flApoe^−/− mice after 12 weeks of HFD (n=20-21 rings from 8-9 mice). Data represent mean±SEM (Mann-Whitney test). (G) Relative quantification of transgrelin (Tagln), smooth muscle-myosin heavy chain (Smmhc), vimentin and CD248 (endosialin) mRNA expression in thoracic aortas of TaglnCre⁺ vs TaglnCre^-Cxcr4^fl/flApoe^−/− and SmmhcCre⁺ vs SmmhcCre^-Cxcr4^fl/flApoe^−/− mice after normalization to 18S rRNA (n=3-9). Data represent mean±SD, Student’s t-test with Welch correction or Mann-Whitney test, as appropriate. (H) Representative images and quantification of protein expression of CD248 (endosialin), vimentin and CD68 and lipid deposition (Nile red) in atherosclerotic lesions (plaque cap) in mice with SMC-specific Cxcr4 deficiency, as compared to controls (n=6-10). Data represent mean±SD and were analyzed by Student’s t-test with Welch correction or Mann-Whitney test, as appropriate (G,H). (I) Relative quantification of Tagln, smoothelin, CD248 and vimentin mRNA expression in hAoSMCs, pretreated with endoIWR1 (1 µM) and stimulated with WNT3a (200 ng/ml), as indicated. Data are normalized to Gapdh expression (mean±SD, data combined from 3-7 experiments, each performed in triplicate; 1-way ANOVA with Sidak’s multiple comparison test or Kruskal-Wallis test with Dunn’s multiple comparison test, as appropriate). (J) Relative quantification of Tagln, smoothelin, CD248 and vimentin mRNA expression in hAoSMCs stimulated with (protease-resistant) CXCL12 (100-300 ng/ml), TGF-β (2.5 ng/ml) or PDGF-BB (20 ng/ml) as indicated. Data are normalized to Gapdh expression (mean±SD, data combined from 3-5 experiments, each performed in triplicate; 1-way ANOVA with Sidak’s multiple comparison test or Kruskal-Wallis test with Dunn’s multiple comparison test, as appropriate). (A-J) *P<0.05; **P<0.01; ***P<0.001.