Perivascular adipose tissue promotes vascular dysfunction in murine lupus

Figures

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  Figure 1. MRL/lpr mice with active disease exhibited partial lipodystrophy. (A) Body weights were monitored weekly. (B) Total weight, fat mass and fat percentage at baseline (7 weeks) and with active disease (14 weeks) measured by NMR. (C-G) Representative images and weights of adipose tissue depots. Subcutaneous (SQAT, (C), gonadal (gAT, (D), retroperitoneal (E), brown (BAT, (F), thoracic aortic PVAT (tPVAT, red arrows, (G) and abdominal aortic PVAT (aPVAT, yellow arrows, (G) and mesenteric arterial PVAT (mPVAT, yellow arrows, (H). In all images, MRL tissues are depicted on the left and MRL/lpr on the right. n=10-15. ****p<0.0001 vs MRL (control). ns, not significant.

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  Figure 2. MRL/lpr mice were hypermetabolic. Comprehensive laboratory animal monitoring system (CLAMS) was performed at age of 14 weeks. VO2 (A), CO2 (B), heat production (D), activity (E), and food consumption (F) were measured, and respiratory exchange ratio (C) was calculated. n=4. *p<0.05, ***p<0.001, ****p<0.0001 vs MRL (control). ns, not significant.

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  Figure 3. MRL/lpr mice with active disease displayed normal blood pressure and metabolic health. Systolic blood pressure (A) was measured using tail cuff (n=8). Fasting blood glucose (B) and insulin (C) levels were measured, and estimated insulin sensitivity as insulin resistance index (HOMA-IR) (D) was calculated using the University of Oxford HOMA calculator software (n=15). Glucose tolerance tests (GTT, (E) were performed and the area under the curve (AUC, (F) between MRL/lpr and MRL was compared (n=5). (G) and (H) representative plasma lipoprotein profiles measured via FPLC using pooled plasma (0.22 ml) from 4 MRL/lpr mice and 5 MRL mice. Plasma levels of triglyceride (I) and cholesterol (J) were also measured using biochemical assays (n=10). VLDL, very low-density lipoprotein; IDL, intermediate-density lipoprotein; LDL, low-density lipoprotein; HDL, high-density lipoprotein. ns, not significant.

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  Figure 4. MRL/lpr mice exhibited impaired endothelium-dependent relaxation, which was exacerbated by PVAT. (A) Concentration-response curves to Ach in all groups. (B) Concentration-response curves to Ach in the absence of PVAT. (C) Concentration-response curves to Ach in the presence of PVAT. Gray shaded areas indicate reductions in Ach-induced relaxation in MRL/lpr mice. (D) Maximum responses (Emax) to Ach from all concentration-response curves. (E) Negative logarithm of EC50 (pEC50) for all concentration-response curves. (F) Area under the curve (AUC) for all concentration-response curves. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Ach, acetylcholine; PVAT, perivascular adipose tissue. ns, not significant.

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  Figure 5. PVAT exhibited whitening and vascular remodeling in MRL/lpr mice. (A) Hematoxylin and eosin (H&E), scale bar=100 μm. (B) Quantitative analysis of average adipocyte size (area per adipocyte, μm2); (C) the relative distribution of adipocytes calculated as frequency of adipocyte area (%) over total area. (D) Masson Trichrome (MT) staining for collagen, scale bar=100 μm; (E) Verhoeff-Van Gieson (VVG) staining for elastin, scale bar=200 μm; (F) UCP1 staining for beige adipocytes, scale bar=100 μm; (G) CD45 staining for leukocytes, scale bar=20 μm; and (H-L) quantification of adventitial areas, adventitia-media ratio, media-lumen ratio, percentage of positive UCP1 and CD45 areas. n=10. ****p < 0.0001 vs MRL (control). ns, not significant.

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  Figure 6. PVAT from MRL/lpr mice exhibited decreased adipogenic and thermogenic marker genes (A), and increased proinflammatory genes including cytokines (B), chemokines (C), and inflammatory marker (D) genes. n=6. *p<0.05, **p < 0.01, vs MRL (control). ns, not significant.