Vasodilator-Stimulated Phosphoprotein: Regulators of Adipokines Resistin and Phenotype Conversion of Epicardial Adipocytes

Abstract

BACKGROUND Endothelial dysfunction plays a central part in the pathogenesis of coronary atherosclerosis. The adipokine resistin is one of the key players in endothelial cell dysfunction. In addition, the role of epicardial fat in coronary artery endothelial dysfunction is also emphasized. We investigated whether vasodilator-stimulated phosphoprotein (VASP) is involved in resistin-related endothelial dysfunction and the phenotype conversion of epicardial adipocytes. MATERIAL AND METHODS Cell proliferation and migration were evaluated by MTT and Transwell chamber assay, respectively. Next, we took epicardial fat samples from patients with valvular heart disease and non- coronary artery disease. Gene expression was determined by reverse transcription- quantitative polymerase chain reaction and relative abundance of the protein by Western blotting. RESULTS Resistin induced endothelial proliferation and migration in a dose-dependent manner. Both resistin-induced cell proliferation and migration were effectively blocked by ablation of VASP. The brown adipose tissue-specific genes for uncoupling protein 1 (UCP-1) and PR-domain-missing16 (PRDM16) decreased, but the white adipose tissue-specific genes for resistin and RIP140 increased in VASP-deficient adipocytes compared with the LV-sicntr group. However, disruption of the Ras homolog gene family member A (RhoA) /Rho-associated kinase (ROCK) in VASP-deficient adipocytes with specific inhibitors inverted the adipocyte phenotype existing in VASP-deficient adipocytes. Furthermore, the expressions of proinflammatory cytokines interleukin-6 (IL-6), interleukin-8 (IL-8), and monocyte chemoattractantprotein-1 (MCP-1) in VASP-deficient adipocytes were markedly upregulated compared with the LV-sicntr group. CONCLUSIONS These results suggest a physiological role for VASP in coronary atherosclerosis through regulating adipokine resistin and phenotype conversion of epicardial adipose tissue.

Figure 1

Knockdown of VASP by siRNA. Both gene and protein expressions were obviously lower after knockdown of VASP by siRNA compared with LV-sicntr group in HCAECs. * p<0.05.

Figure 2

Effect of VASP on proliferation of resistin. (A) After 24h incubation, resistin induced endothelial proliferation in a dose-dependent manner (0–200 ng/ml) with MTT assay. (B) Resistin-induced cell proliferation could be effectively blocked by ablation of VASP with or without 100 ng/ml of resistin. * p<0.05.

Figure 3

Effect of VASP on migration of resistin. (A) HCAEC migration in different doses of resistin(0–200 ng/ml) was studied by transwell chamber assay. The results showed that resistin promoted migration of HCAECs after 12 h incubation. (B) Ablation of VASP disrupted the function of resistin on cell migration with or without 100 ng/ml of resistin. * p<0.05.

Figure 4

(A) IL-6 induced the differentiation of brown adipocytes into white adipocytes. (B) VASP contents were decreased during brown-to-white adipocyte trans-differentiation phenotype conversion. * p<0.05.

Figure 5

Ablation of VASP promoted brown-to-white phenotype conversion of epicardial adipocytes. (A) Actin cytoskeletal staining with or without VASP. (B)The abundance of of UCP-1 and PRDM16 decreased, but the resistin and RIP140 expressions increased in VASP-deficient adipocytes compared with LV-sicntr group. * p<0.05.

Figure 6

(A–D) Ablation of VASP increased expressions of inflammatory adipocytokines in epicardial adipose tissue. The mRNA expressions of IL-6, IL-8, TNF-α and MCP-1 increased in VASP-deficient adipocytes compared with LV-sicntr group. * p<0.05.

Figure 7

RhoA/ROCK signal was involved in VASP-mediated phenotype conversion. After treatment with RhoA/ROCK specific blocker Y-27632 2HCL in VASP-deficient adipocytes, the expressions of UCP-1 and PRDM16 were significantly increased in protein levels compared with LV-sicntr group. * p<0.05.