Panagiotis Kougias, MD, Hong Chai, MD, PhD, Peter H. Lin, MD, Alan B

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Adipocyte-derived cytokine resistin causes endothelial dysfunction of porcine coronary arteries Panagiotis Kougias, MD, Hong Chai, MD, PhD, Peter H. Lin, MD, Alan B. Lumsden, MD, Qizhi Yao, MD, PhD, Changyi Chen, MD, PhD Journal of Vascular Surgery Volume 41, Issue 4, Pages 691-698 (April 2005) DOI: 10.1016/j.jvs.2004.12.046 Copyright © 2005 The Society for Vascular Surgery Terms and Conditions

Fig 1 Effect of resistin on the vasomotor function of porcine coronary arteries. Pig right coronary arteries (n = 10) were cultured with medium-dimethyl sulfoxide (as control) or treated with resistin (10 or 40 ng/mL) for 24 hours. A, Maximal contraction of the vessel rings in response to thromboxane A2 analogue U46619 (10−7 M). Contractility was increased in the rings treated with resistin; however, this did not reach statistical significance (P > .05, t test and analysis of variance [ANOVA]) compared with controls. B, Precontracted vessels were tested for endothelium-dependent relaxation by addition of bradykinin (10−9 to 10−5 M). We observed a statistically significant reduction in the resistin-treated samples compared with controls (*P < .05, ANOVA). C, Endothelium-independent relaxation in response to sodium nitroprusside (10−6 M) was significantly reduced in the vessels treated with 40 ng/mL of resistin (*P < .05, t test and ANOVA). Data shown are means ± SEM. Journal of Vascular Surgery 2005 41, 691-698DOI: (10.1016/j.jvs.2004.12.046) Copyright © 2005 The Society for Vascular Surgery Terms and Conditions

Fig 2 Effect of resistin on the O2− production in porcine coronary arteries. O2− levels in porcine coronary arteries were tested with lucigenin-enhanced chemiluminescence assay. The data were normalized with area (mm2) of the ring and are expressed as relative light units (RLU/sec/mm2). Resistin (40 ng/mL) significantly increased the O2− levels of vessel rings (*P < .05, n = 3). The antioxidant seleno-L-methionine (SeMet) blocked the resistin-induced O2− production. Journal of Vascular Surgery 2005 41, 691-698DOI: (10.1016/j.jvs.2004.12.046) Copyright © 2005 The Society for Vascular Surgery Terms and Conditions

Fig 3 Effect of antioxidant seleno-L-methionine (SeMet) on the resistin-induced vasomotor dysfunction of porcine coronary arteries. Vessel rings were cultured with SeMet (100 μM), resistin (40 ng/mL), or both for 24 hours (n = 5). A, Maximal contraction of the vessel rings in response to U46619 (10−7 M). There was no statistically significant difference among the rings treated with resistin or SeMet, or both, compared with controls (t test and analysis of variance [ANOVA]). B, Endothelium-dependent vasorelaxation of the precontracted vessel rings in response to bradykinin (10−9 to 10−5 M). Resistin significantly inhibited bradykinin-induced vasorelaxation (*P < .05, ANOVA). SeMet blocked this effect. C, Endothelium-independent vasorelaxation in response to SNP (10−6 M). Resistin 40 ng/mL interfered with the sodium nitroprusside-induced vasorelaxation (*P < 0.05, t-test, and ANOVA), and SeMet reversed this effect. Journal of Vascular Surgery 2005 41, 691-698DOI: (10.1016/j.jvs.2004.12.046) Copyright © 2005 The Society for Vascular Surgery Terms and Conditions

Fig 4 Effect of resistin on the endothelial nitric oxide synthase (eNOS) mRNA level of porcine coronary arteries. Porcine coronary rings were cultured for 24 hours in medium (dimethyl sulfoxide [control]) with resistin at concentrations of 10 and 40 ng/mL, with seleno-L-methionine (SeMet), or with resistin (40 ng/mL) plus SeMet. The total mRNA was purified from endothelial layers of the rings. The eNOS mRNA expression was quantified with real-time PCR. The eNOS mRNA level in each sample was normalized to that of glyceraldehyde phosphate dehydrogenase (GAPDH). Relative eNOS mRNA level was presented as [2^(Ct[GAPDH] − Ct[eNOS])]. Treatment with resistin (40 ng/mL) significantly decreased the eNOS mRNA expression compared with controls (*P < .05, n = 6, t test). This effect was reversed with the addition of SeMet. Journal of Vascular Surgery 2005 41, 691-698DOI: (10.1016/j.jvs.2004.12.046) Copyright © 2005 The Society for Vascular Surgery Terms and Conditions

Fig 5 Effect of resistin on the endothelial nitric oxide synthase (eNOS) immunoreactivity of porcine coronary arteries. Avidin-biotin complex immunoperoxidase procedure was used to detect eNOS protein levels in both control and resistin-treated vessels. A, Control. B, SeMet (seleno-L-methionine) alone (control). C, Resistin (10 ng/mL). D, Resistin (10 ng/mL) plus SeMet. E, Resistin (40 ng/mL). F, Resistin (40 ng/mL) plus SeMet. High dose resistin (40 ng/mL) treatment substantially reduced eNOS immunoreactivity compared with controls, but the antioxidant SeMet reversed this effect (n = 3). Arrows indicate endothelial cells. Journal of Vascular Surgery 2005 41, 691-698DOI: (10.1016/j.jvs.2004.12.046) Copyright © 2005 The Society for Vascular Surgery Terms and Conditions

Fig 6 Effect of resistin on the endothelial nitric oxide synthase (eNOS) mRNA levels of porcine coronary artery endothelial cells in culture. The cells were incubated with resistin (10 or 40 ng/mL) for 24 hours. Real-time polymerase chain reaction was used to assess eNOS mRNA expression. The eNOS mRNA level in each sample was normalized to that of glyceraldehyde-s-phosphate dehydrogenase (GAPDH). Relative eNOS mRNA level was presented as 2^[Ct(GAPDH) - Ct(eNOS)]. Addition of resistin (10 or 40 ng/mL) substantially decreased the eNOS mRNA expression (* P < 0.05, n = 6, t test). Data shown are means ± SEM. Journal of Vascular Surgery 2005 41, 691-698DOI: (10.1016/j.jvs.2004.12.046) Copyright © 2005 The Society for Vascular Surgery Terms and Conditions