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. 2023 Aug 9;65:102838. doi: 10.1016/j.redox.2023.102838

Fig. 1.

Fig. 1

Global deletion of cytoglobin in the mouse accelerates the decrease in smooth muscle contractile genes and activation of DNA repair pathways in the carotid artery ligation model. (a) Representative immunofluorescence staining of the right (uninjured) and left (injured) common carotid arteries of cytoglobin wildtype (WT) and knockout (KO) littermate mice 3-days post-ligation, harvested and analyzed for cytoglobin content (red – CYGB, blue – DAPI, green – autofluorescence marking elastic lamina). Cytoglobin immunostaining was evident in the media and adventitia (top) and lost in the KO mice; scale bar, 40 μm. (b) Results from bulk RNA-Seq analysis of the entire left (injured) and right (uninjured) common carotid arteries from mice that underwent left common artery ligation for 3 days. 2993 genes differentially changed in the injured cytoglobin knockout mice (n = 5) were isolated from differential gene expression analysis of wildtype injured (Left) and uninjured (Right) vessels (n = 4) and cytoglobin knockout injured and uninjured (n = 5; 1.5 - fold change FDR <0.05). (c) Top down- and up-regulated pathways from gene ontology analysis of the 2993 genes altered in the differential gene expression analysis. (d) Heat map comparing smooth muscle cell contractile genes in CYGB-WT and CYGB-KO mice 3 days after left common carotid artery ligation. Each individual square represents the z-score value of the count obtained for the selected gene from one individual mouse using the combined bulk RNA-seq results from the right (uninjured) and left (injured) common carotid arteries of cytoglobin wildtype (WT, n = 4) and knockout (KO, n = 5) littermate mice 3-days post-ligation. (e) Representative immunofluorescence images and fluorescence quantitation from CYGB-WT and CYGB-KO carotids probing for ACTA2, 3 days after ligation; scale bar, 50 μm. (f) Gene set enrichment analysis based on a curated set of 197 DNA damage factors associated with DNA repair pathways; The number for each bar represents the nominal P value for each pathway. (g) Indirect immunofluorescence staining for the marker of DNA repair activation γH2AX (phosphorylated histone 2AX) of uninjured (right common) and injured (left common) mouse carotid arteries 3-days post ligation (red – γH2AX, blue – DAPI, green – autofluorescence marking elastic lamina); scale bar, 100 μm. Right panel, quantitation of results. For statistical analysis throughout the figure, results were analyzed by two-way ANOVA, followed by Tukey's post hoc test. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)