Nidogen 2 Overexpression Promotes Hepatosteatosis and Atherosclerosis

Figures

• 

  Figure 1. Expression of NID2 protein is elevated in human atherosclerotic arteries and murine steatotic livers. (A) Representative western blot images for NID2 and β-tubulin protein expression in human atherosclerotic inner curvature (IC) and non-atherosclerotic descending aorta (DA) vascular tissue. The bar diagram shows mean protein levels expressed as a ratio of NID2 to β-tubulin. (B) Representative Western blot images for NID2 (red arrowhead points to the correct band) and GAPDH in the livers of control diet (CD)- and calorie-matched high-fat diet (HFD, 12 weeks)-fed C57BL/6J mice. The bar diagram represents the mean NID2 protein expression (n = 4). Statistical analyses were performed using a two-tailed unpaired t-test (A,B). Data represent mean ± SEM. * p < 0.05, and **** p < 0.0001.

• 

  Figure 2. NID2 overexpression enhances liver and epididymal white adipose tissue mass in male mice. (A) The schematic diagram illustrates the experimental plan. Apoe−/− mice were injected with control (Ctrl) and NID2-AAV intraperitoneally, fed a Western diet for 12 weeks, and analyzed. (B–E) Male control and NID2-AAV-injected Apoe−/− mice were utilized to measure NID2 mRNA levels in various organs by qRT-PCR at least in duplicate. Bar diagrams represent mRNA expression in the liver (B, n = 10), kidney (C, n = 6), epididymal white adipose tissue (EpiWAT, D, n = 7–10), and heart (E, n = 10). Bar diagrams show body weight gain (F), plasma total cholesterol (G), fasting blood glucose (H), whole-body fat/lean mass (I), liver weight (J), adipose tissue weight (K), and spleen weight (L) (n = 5–6). A two-tailed unpaired t-test (C,G–K), two-tailed unpaired Mann–Whitney test (B,D,E,L), and two-way ANOVA followed by Sidak post hoc test for multiple comparisons (F) were utilized for statistical analyses. Data represent mean ± SEM. ns: non-significant. * p < 0.05, *** p < 0.001 and **** p < 0.0001.

• 

  Figure 3. NID2 overexpression in mice promotes hepatic lipid accumulation and fibrosis. Male Apoe−/− mice were injected with control and NID2-AAV intraperitoneally, fed a Western diet for 12 weeks, and analyzed. (A) Representative Western blot images for NID2 and GAPDH protein expression in the livers of control and NID2-overexpressing mice (n = 3). (B) Representative images of liver sections stained with H & E (lipid droplets), ORO (neutral lipid accumulation), and Sirius red (fibrosis); scale bar 100 μm. (C–H) Bar diagrams represent lipid accumulation (C, n = 6), fibrosis area (D, n = 5), hepatic triglyceride (E, n = 3–4), NEFA levels (F, n = 3–4), plasma triglyceride (G, n = 5) and NEFA levels (H, n = 5), in control and NID2-AAV-injected mice. Statistical analyses were performed using a two-tailed unpaired t-test (C–H). Data represent mean ± SEM. ns: non-significant. * p < 0.05, and ** p < 0.01.

• 

  Figure 4. NID2 overexpression augments atherosclerosis in male hypercholesterolemic mice. Male Apoe−/− mice were injected with control (Ctrl) and NID2-AAV intraperitoneally, fed a Western diet for 12 weeks and analyzed. (A) Representative in situ images of the aortic arch (red arrowheads point to atherosclerotic lesions). (B) Representative ORO staining of whole aortas; scale bar 5 mm. The bar diagram represents ORO-positive areas in whole aortas (n = 6). (C) Representative images of aortic root cross-sections stained with H & E (lesion area and necrotic core), ORO (lipid accumulation), and Masson’s trichrome (collagen content); scale bar 200 μm. (D–G) Bar diagrams show lesion area (D), lipid deposition (E), collagen content (F), and necrotic core area (G) (n = 5–6). Statistical analyses were performed using a two-tailed unpaired t-test (B,D–G). Data represent mean ± SEM. * p < 0.05, and ** p < 0.01.

• 

  Figure 5. NID2 overexpression inhibits the activation of the lipid metabolism-related protein AMPK. (A) Representative Western blot images for lipid metabolism and pro-inflammatory proteins utilizing liver lysates from control and NID2-AAV-injected mice. Bar diagrams represent mean protein expression (B,C) as the ratios of phospho-total proteins ACC (B) and AMPK (C), and protein levels of IL-6 (D) and TNFα (E) (n = 5). Statistical analyses were performed using a two-tailed unpaired t-test. Data represent mean ± SEM. ns: non-significant. * p < 0.05.