Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Endothelial Intracellular ANG (Angiogenin) Protects Against Atherosclerosis by Decreasing Endoplasmic Reticulum Stress

doi: 10.1161/atvbaha.121.317339

Figure Lengend Snippet: Figure 4. Sodium 4-phenylbutyrate (PBA) inhibited the activation of endoplasmic reticulum (ER) stress to attenuate atherosclerosis in endothelial ANG (angiogenin)- and apolipoprotein E-deficient (ANGfl/flTie2cre/+/ApoE−/−) mice. ANGfl/flTie2cre/+/ApoE−/− mice and control littermates (ANGfl/fl/ApoE−/− mice) on a Western diet (WD) for 13 wk underwent an intraperitoneal injection of PBA (100 mg/kg every other day) and phosphate-buffered saline (PBS) during the final 4 wk. A, Hematoxylin and eosin (HE) staining of aortic plaques. Scale bars indicate 500 µm. B, Quantification of the average area of atherosclerotic lesions. n=10 per group. C, Representative en face aorta obtained by Oil Red O staining of the whole aorta. D, Quantitative data of the aortic plaque area are shown as the percentage of the total surface area of the aorta. n=10 per group. E, EGF-like module-containing mucin-like hormone receptor-like 1 (F4/80+; red) and 4′-6-diamidino-2-phenylindole (DAPI; blue) immunostaining in aortas, and representative images are shown. Scale bars indicate 500 µm. F, Quantitative analysis of F4/80+ staining. n=10 per group. G and H, Protein expression levels of ICAM-1 (intercellular cell adhesion molecule-1), VCAM-1 (vascular cell adhesion molecule-1), MCP-1 (monocyte chemotactic protein-1) (n=6 per group; G), GRP78 (glucose- regulated protein 78), IRE1 (inositol-requiring enzyme-1), phosphorylated IRE1 (p-IRE1), sXBP1 (spliced X-box-binding protein 1), and CHOP (C/EBP-homologous protein) (n=6 per group; H) in whole aortas were evaluated by Western blots, and the corresponding quantitative analysis was performed. Data are expressed as the means±SEM. The data were analyzed by 2-way ANOVA with Bonferroni post hoc tests.

Article Snippet: Our study strictly abided by the guidelines as described by the NIH and in the Arteriosclerosis, Thrombosis, and Vascular Biology Council Statement for considering sex as a biological variable.25,26 We used only male mice because estrogen is reported to affect the progression of atherosclerosis and male mice are less likely to generate a null Tie2-Cre allele compared with female mice.22,27,28 Our study also adhered to the guidelines for experimental atherosclerosis studies described in the American Heart Association Statement.29 Five-week-old ApoE−/−, ANG−/−/ApoE−/−, ANGfl/ fl/ApoE−/−, ANGfl/flTie2cre/+/ApoE−/−, and ANGfl/flLyz2cre/+/ ApoE−/− mice were fed a WD (21% fat, 0.15% cholesterol, SLAC, Shanghai, China) for 13 weeks.30 An intraperitoneal injection of sodium 4-phenylbutyrate (PBA, 100 mg/kg every other day; No. HY-15654, MCE, Monmouth Junction) and phosphate-buffered saline (No. G4202, Servicebio, Wuhan, China) was performed during the final 4 weeks in ANGfl/fl/ ApoE−/− mice and ANGfl/flTie2cre/+/ApoE−/− mice.14 For identification of the floxed allele, the primers were as follows: forward primer ATCTTGCCTCAGGAACCCC and reverse primer AGGAGTGGTCGGCTGTAGAA.

Techniques: Activation Assay, Control, Western Blot, Injection, Saline, Staining, Immunostaining, Expressing, Binding Assay