Nox2 mediates high fat high sucrose diet-induced nitric oxide dysfunction and inflammation in aortic smooth muscle cells

Abstract

Diet-induced obesity and metabolic syndrome are important contributors to cardiovascular diseases. The decreased nitric oxide (NO) bioactivity in endothelium and the impaired response of smooth muscle cell (SMC) to NO significantly contribute to vascular pathologies, including atherosclerosis and arterial restenosis after angioplasty. Sarco/endoplasmic reticulum Ca(2+) ATPase (SERCA) is an important mediator of NO function in both endothelial cells and SMCs, and its irreversible oxidation impairs its stimulation by NO. We used C57BL/6J mice fed a high fat high sucrose diet (HFHSD) to study the role of SMC SERCA in diet-induced obesity and metabolic syndrome. We found that HFHSD upregulated Nox2 based NADPH oxidase, induced inflammation, increased irreversible SERCA oxidation, and suppressed the response of aortic SERCA to NO. Cultured aortic SMCs from mice fed HFHSD showed increased reactive oxygen species production, Nox2 upregulation, irreversible SERCA oxidation, inflammation, and a decreased ability of NO to inhibit SMC migration. Overexpression of wild type SERCA2b or downregulation of Nox2 restored NO-mediated inhibition of migration in SMCs isolated from HFHSD-fed mice. In addition, tumor necrosis factor alpha (TNFα) increased Nox2 which induced SERCA oxidation and inflammation. Taken together, Nox2 induced by HFHSD plays significant roles in controlling SMC responses to NO and TNFα-mediated inflammation, which may contribute to the development of cardiovascular diseases in diet-induced obesity and metabolic syndrome.

Keywords: High fat high sucrose diet; Nitric oxide; Nox2; Sarco/endoplasmic reticulum Ca(2+) ATPase; Smooth muscle cell.

Figure 1. High fat, high sucrose diet (HFHSD) increases Nox2, SERCA oxidation, inflammation and decreases NO bioactivity and NO-stimulated SERCA activity in intact aorta

A. Representative immunostainings of aortas from mice fed HFHSD or normal diet (ND) for 16 weeks. Quantitation in graph. Pictures were taken at 40×. *p < 0.05 vs. ND, n=5–10. B. HFHSD inhibits aortic eNOS phosphorylation. *p < 0.05 vs. ND, n=3. C. HFHSD inhibits NO-stimulated aortic SERCA activity. *p < 0.05 vs. ND, n=5–8.

Figure 2. NADPH oxidase, SERCA oxidation and VCAM1 are increased in smooth muscle cells (SMCs) from HFHSD-fed mice

A. mRNA levels of indicated genes in SMCs isolated from aortas of HFHSD- or ND-fed mice and cultured in 0.2% FBS DMEM overnight. *p < 0.05 vs. ND, n=5. B. Representative Western blots of homogenates of aortic SMCs isolated from HFHSD- or ND-fed mice and cultured in 0.2% FBS DMEM overnight. Nox2 (58 kDa), Rac1 (21 kDa), Nox4 (66 kDa), SERCA and SERCA C674-SO₃H (110 kDa), p65NFκB and p-p65NFκB (65 kDa), VCAM1 (100 kDa), TNFα (17 kDa), GAPDH (36 kDa). Quantitation of band intensities in graph. *p < 0.05 vs. ND, n=3–10.

Figure 3. Smooth muscle cells from HFHSD-fed mice have increased ROS and macrophage adhesion, and impaired NO-inhibited migration

A. ROS production in aortic SMCs is increased after 16 week HFHSD. Quantitation of fluorescence intensities in graph. *p < 0.05 vs. ND, n=3. B. NO donor fails to inhibit migration of cultured aortic SMCs isolated from HFHSD-fed mice. *p < 0.05 vs. serum, n=12. C. Overexpression of SERCA2b restores the inhibition of cell migration by NO donor in HFHSD SMCs. *p < 0.05 vs. LacZ (vector control), n=5. D. HFHSD enhances macrophage adhesion to cultured SMCs, quantified in graph. *p < 0.05 vs. ND, n=6.

Figure 4. Downregulation of Nox2 restores NO function and inhibits inflammation in HFHSD SMCs

A. Nox2 knockdown by siRNA decreases irreversible SERCA oxidation and the expression of inflammatory molecules. *p < 0.05 vs. control siRNA, n=3–7. B. Nox2 siRNA has no effects on other NADPH oxidase components mRNA. *p < 0.05 vs. control siRNA, n=3–6. C. Nox2 siRNA restored the inhibition of SMC migration by NO donor DETA NONOate. *p < 0.05 vs. control siRNA, n=6. D. Nox2 knockdown reduces macrophage adhesion to cultured SMCs isolated from HFHSD-fed mice. *p < 0.05 vs. control siRNA, n=4.

Figure 5. Nox2 mediates TNFα-induced SERCA oxidation and inflammation in SMCs

A. TNFα (10 ng/ml) increases Nox2 expression, SERCA oxidation, and activates NFκB and VCAM1 in SMCs. SMCs were isolated from aortas of mice fed ND. Band intensities normalized by GADPH and expressed as percentage of PBS-control. *p < 0.05 vs. PBS, n=3–6. B. Nox2 siRNA blocks the effects of TNFα on SERCA oxidation and inflammation. Quantitation of band intensities indicated in graph. *p < 0.05 vs. control siRNA, n=3–6.

Figure 6. Scheme of the potential mechanisms by which HFHSD contributes to cardiovascular diseases

HFHSD-induced TNFα stimulates Nox2 and inflammation, leading to SERCA oxidation, decreased NO bioactivity, and increased SMC migration and macrophage adhesion to SMC, which can all contribute to vessel restenosis and atherosclerosis in settings of obesity.