Thoracic perivascular adipose tissue inhibits VSMC apoptosis and aortic aneurysm formation in mice via the secretome of browning adipocytes

Abstract

Abdominal aortic aneurysm (AAA) is a dangerous vascular disease without any effective drug therapies so far. Emerging evidence suggests the phenotypic differences in perivascular adipose tissue (PVAT) between regions of the aorta are implicated in the development of atherosclerosis evidenced by the abdominal aorta more vulnerable to atherosclerosis than the thoracic aorta in large animals and humans. The prevalence of thoracic aortic aneurysms (TAA) is much less than that of abdominal aortic aneurysms (AAA). In this study we investigated the effect of thoracic PVAT (T-PVAT) transplantation on aortic aneurysm formation and the impact of T-PVAT on vascular smooth muscle cells. Calcium phosphate-induced mouse AAA model was established. T-PVAT (20 mg) was implanted around the abdominal aorta of recipient mice after removal of endogenous abdominal PVAT (A-PVAT) and calcium phosphate treatment. Mice were sacrificed two weeks after the surgery and the maximum external diameter of infrarenal aorta was measured. We found that T-PVAT displayed a more BAT-like phenotype than A-PVAT; transplantation of T-PVAT significantly attenuated calcium phosphate-induced abdominal aortic dilation and elastic degradation as compared to sham control or A-PVAT transplantation. In addition, T-PVAT transplantation largely preserved smooth muscle cell content in the abdominal aortic wall. Co-culture of T-PVAT with vascular smooth muscle cells (VSMCs) significantly inhibited H₂O₂- or TNFα plus cycloheximide-induced VSMC apoptosis. RNA sequencing analysis showed that T-PVAT was enriched by browning adipocytes and anti-apoptotic secretory proteins. We further verified that the secretome of mature adipocytes isolated from T-PVAT significantly inhibited H₂O₂- or TNFα plus cycloheximide-induced VSMC apoptosis. Using proteomic and bioinformatic analyses we identified cartilage oligomeric matrix protein (COMP) as a secreted protein significantly increased in T-PVAT. Recombinant COMP protein significantly inhibited VSMC apoptosis. We conclude that T-PVAT exerts anti-apoptosis effect on VSMCs and attenuates AAA formation, which is possibly attributed to the secretome of browning adipocytes.

Keywords: abdominal aortic aneurysm; apoptosis; browning; perivascular adipose tissue; secretome; vascular smooth muscle cells.

Fig. 1. Thoracic PVAT (T-PVAT) exhibits a more BAT-like phenotype than abdominal PVAT (A-PVAT).

a Hematoxylin and eosin (H&E) staining of A-PVAT and T-PVAT from C57/BL6 mice (Scale bar = 200 μm). b Average of adipocyte size. c Western blotting analysis of Ucp1. d Immunohistochemical staining of Ucp1. e Quantitative RT-PCR of browning markers (n = 5, **P < 0.01, ***P < 0.001).

Fig. 2. Transplantation of T-PVAT attenuates AAA formation.

a Experimental scheme. 8-week-old male C57BL/6 mice were transplanted with isolated T-PVAT and A-PVAT at the infrarenal region of the abdominal aorta, and then undergone calcium phosphate treatment to induce AAA. b Representative photographs of abdominal aorta 14 days after CaPO₄ treatment with sham control and transplantation of T-PVAT or A-PVAT. c Maximal diameter of abdominal aorta. d Cross-section lesion area. e H&E staining of abdominal aorta. (n = 6–10, *P < 0.05; **P < 0.01; ***P < 0.001) (Scale bar = 200 μm).

Fig. 3. Transplantation of T-PVAT attenuates elastin fragmentation and increases collagen deposition of abdominal aortic wall.

a Representative image of Elastica Van Gieson (EVG) staining. b Representative images of Sirius red staining. c Assessment of medial elastica fragmentation. d Quantification of Sirius red positive area (n = 5–9; *P < 0.05; ***P < 0.001) (Scale bar = 200 μm).

Fig. 4. Transplantation of T-PVAT preserves medial smooth muscle cell content in the aortic wall.

a Immunofluorescence staining of alpha smooth muscle actin (SMA). b Immunohistochemical staining of cleaved Caspase 3. c TUNEL staining. d Quantification of SMA positive area. e Quantification of cleaved-Caspase 3 positive area. f Quantification of TUNEL staining (n = 5–9; **P < 0.01; ***P < 0.001) (Scale bar = 200 μm).

Fig. 5. Co-culture of T-PVAT inhibits VSMC apoptosis.

Primary VSMCs were co-cultured with/without A-TVAT or T-PVAT for 24 h and then treated with 400 μM H₂O₂ for 24 h (a–e) or 50 ng/mL TNFα plus 5 μg/mL cycloheximide for 8 h (f–j). a, f Representative images of Western blotting analysis. b, g Quantification of cleaved-Caspase 3. c, h Quantification of cleaved-Parp. d, i Annexin V/PI staining and flow cytometry analysis. e, j Percentage of apoptotic cells (Annexin V⁺ ) (n = 4–10; *P < 0.05; **P < 0.01; ***P < 0.001; NS not significant).

Fig. 6. T-PVAT is enriched in browning adipocytes and anti-apoptotic secretory proteins.

a Heatmap of differentially expressed genes (DEGs) from RNA-seq data. b Volcano plot from RNA-seq data. c GO enrichment analysis for the DEGs. d Top 20 biological processes for the upregulated secreted proteins. The circle size represents the gene number. The color gradient shows the adjusted P value (n = 3).

Fig. 7. The secretome of adipocytes isolated from T-PVAT inhibits VSMC apoptosis.

Primary VSMCs were cultured with conditioned medium collected from adipocytes and SVF that were isolated from T-PVAT or A-PVAT, and then cells were treated with 400 μM H₂O₂ for 24 h (a–h) or 50 ng/mL TNFα plus 5 μg/mL cycloheximide for 8 h (i–p). a, d, i, l Representative images of Western blotting analysis. b, e, j, m Quantification of cleaved-Parp. c, f, k, n Quantification of cleaved-Caspase 3. g, o Annexin V/PI staining and flow cytometry analysis. h, p Percentage of apoptotic cells (Annexin V⁺ ) (n = 3–6; *P < 0.05; ***P < 0.001; NS not significant).

Fig. 8. Secreted COMP from T-PVAT inhibits VSMC apoptosis.

a Screening scheme. b Western blotting analysis of COMP expression in the lysates and culture medium of adipocytes derived from A-PVAT and T-PVAT (three independent repeated experiments were performed). c Immunohistochemical staining of COMP. d Annexin V/PI staining and flow cytometry analysis. e Percentage of apoptotic cells (Annexin V+). f Representative images of Western blotting analysis. g Quantification of cleaved-Parp. h Quantification of cleaved-Caspase 3. i Quantification of survivin. j Quantification of Bcl-2 (n = 3–6; *P < 0.05; **P < 0.01; ***P < 0.001; NS not significant).