Figure 4.

Loss of OCT4 in EC results in increased EndoMT, neovascularization, and the number of VCAM1⁺ cells in plaques and decreased mitochondrial respiration in vitro. (A and C) Representative immunostaining on serially sectioned BCAs collected from Oct4^+/+ or Oct4^Δ/Δ mice fed WD for 18 weeks; fibrous cap area (A) or lesion area (C). (A) White arrows indicating YFP⁺ACTA2⁺ EC undergoing EndoMT. Scale bar = 20 µm. (B) Quantification of the percentage of YFP⁺ACTA2⁺ cells within the total YFP⁺DAPI⁺ cell population in the 30 µm protective fibrous cap area of the male and female lesions. Data were analysed by unpaired t-test (males) or non-parametric Mann–Whitney test (females); *P < 0.05 (n = 12 males; 12 females) vs. Oct4^Δ/Δ (n = 12 males; 11 females) mice. (C) White arrows indicating capillary-like YFP⁺ neovessels. Scale bar = 50 µm. (D) Quantification of the number of YFP⁺ intraplaque capillary-like neovessels. Values = mean ± S.E.M.; data were analysed by non-parametric Mann–Whitney test; *P < 0.05 Oct4^Δ/Δ (n = 9 males; 12 females) vs. Oct4^+/+ (n = 9 males; 11 females) mice. (E) Representative immunostaining serially sectioned BCAs collected from Oct4^+/+ or Oct4^Δ/Δ male fed WD for 10 weeks. Scale bar = 10 µm. (F) Quantification of the percentage of YFP⁺VCAM1 ⁺ DAPI⁺ cells within the total YFP⁺ cell population at the luminal surface of the male and female vessels. Values = mean ± S.E.M.; *P < 0.05 by unpaired t-test (females) or unpaired t-test with Welch’s correction (males). Oct4^+/+ (n = 8 males; 11 females) and Oct4^+/+ (n = 8 males; 11 females) mice after 10 weeks of WD feeding. (G) Graphical representation of the Seahorse XF24 Cell Mito Stress Test assays measuring the oxygen consumption rates (OCR) in OCT4^WT and OCT4^ex1_KO HUVECs with arrows indicating treatments with specific stressors: oligomycin, carbonyl cyanite-4 (trifluoromethoxy) phenylhydrazone (FCCP), and Rotenone/Antimycin A. (H) Quantification of OCR in OCT4^WT and OCT4^ex1_KO HUVECs revealed a significant difference in basal mitochondrial respiration, maximum respiration, spare respiratory capacity (SRC), and ATP production. Values = mean ± S.E.M.; *P < 0.05 by two-way ANOVA, n = 3 independent experiments.