Figure 2.

Ccn2 deficiency causes de-differentiation of SMCs. (A) Experiment design and aorta segments used for RNA, protein, and histological analyses. (B, C) Ccn2 mRNA (B) and Ccn2 protein (C) level in descending thoracic aortas. (D) Peptides of Ccn2 and Gapdh quantified to obtain results in C. Peptide positions are indicated. (E) Cross-sections of descending thoracic aortas stained by Masson’s trichrome (mtc). Scale bars (overview) = 100 µm; scale bars (magnification) = 20 µm. (F) Medial cross-sectional area of descending thoracic aorta. We found no difference in the relative effect of genotype on this measure between female and male mice. (G) GSEA based on microarray data obtained from primary Ccn2^Δ/Δ (n = 9) and Ccn2^fl/fl (n = 6) SMCs. The 15 most down- and up-regulated gene ontology biological process terms (based on significance) are shown. Dot sizes indicate the number of genes regulated in Ccn2^Δ/Δ SMCs for each term. The ratio shows coverage of a given term by genes regulated in Ccn2^Δ/Δ SMCs, and dot colours indicate the level of significance. (H) Volcano plot showing regulation of genes in primary Ccn2^Δ/Δ SMCs compared with Ccn2^fl/fl SMCs. Two thousand one hundred seventy-five genes were up-regulated and 1366 genes were down-regulated (unadjusted P < 0.05) in Ccn2^Δ/Δ SMCs. Only Hlx displayed significant up-regulation after correction for multiple testing (see supplementary material online, Table S1). Myocd and genes encoding pre-specified markers for contractile SMCs are shown in magenta, and Lgals3 is shown in red. (I) Validation of selected genes by qPCR of descending thoracic aortas. Data in B, C, F, and I were analysed by unpaired t-test with Welch’s correction or Mann–Whitney U test. Data in G were analysed using clusterProfiler 4.0 package⁴¹ in R.