Combined metabolic stress and nitric oxide synthase inhibition induces tissue-specific remodeling across cardiovascular and hematopoietic compartments during obesity

Figures

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  Figure 1. Combined metabolic stress and NOS inhibition differentially affects organ remodeling. (a) Experimental design. C57BL/6J mice were fed chow, HFD, or HFD plus L–NAME for 15 weeks. Created with BioRender.com. (b) Body weight measured after 15 weeks of diet treatment in control, HFD, and HFD + L-NAME groups. (total N = 23 from 4 cohorts of 4–5 animals per group) (c) Liver weights (total N = 5–10 animals from 2 cohorts of 2–5 animals per group), (d) spleen weights (N = 18–23 animals from 4 cohorts), (e) total heart weights (total N = 22−35 animals from 6 cohorts of 3–6 animals per group), (f) and femur length (total N = 9–10 animals from 2 cohorts of 4–5 animals per group) (g) Correlation between spleen and heart weight (total N = 13–19 from 4 cohorts of 3–5 animals per group). Each point represents one biological replicate. Statistical significance was determined by Kruskal-Wallis with Dunn's multiple comparisons test for (b,d,e,f). For data passing Shapiro-Wilk test for normality, One-Way ANOVA with Tukey's multiple comparisons test was used.

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  Figure 2. Cardiac fibrosis and collagen composition are comparable between HFD and HFD + L-NAME mice. (a) Representative picrosirius red-stained transverse heart sections and quantification of fibrotic area. Scale bar = 200 µm. (b) Polarized light images of picrosirius red stained heart sections were quantified for the ratio of collagen type I (red) to collagen type III (green). Scale bar = 25μm. Data was quantified from N = 4–5 sections per group obtained from individual mice. Assumption of normality was tested by Shapiro–Wilk test and statistical significance was determined by One-Way ANOVA with Tukey's multiple comparisons test.

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  Figure 3. Combined metabolic stress and NOS inhibition alters obesity-induced circulating myeloid-cell expansion. Frequencies of (a) CD11b+ myeloid cells, (b) Ly6G+ neutrophils, (c) monocytes, and (d) Ly6Chi inflammatory monocytes in peripheral blood collected from mice after 15 weeks of dietary intervention. N = 3–4 individual mice per group with each point representing one individual biological replicate. Normality was determined by Shapiro–Wilk test and statistical significance was determined by One-Way ANOVA with Tukey's multiple comparisons test.

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  Figure 4. Bone marrow remodeling is selectively enhanced by combined metabolic stress and NOS inhibition. (a) Distal femur sections stained with hematoxylin and eosin (H&E); scale bar=500um. (b) Adipocyte numbers were quantified in the bone marrow across the region extending from the growth plate to 1600 μm. Data was quantified from N = 9–10 sections per group obtained from two cohorts of 4–5 individual mice per group. (c) Quantification of trabecular bone volume (BV) expressed as a percentage of the total volume (TV) measured within a 1.5 × 1.5 mm region above the growth plate. Normality was determined by Shapiro–Wilk test and statistical significance was determined by One-Way ANOVA with Tukey's multiple comparisons test.