Growth hormone–releasing peptide ghrelin inhibits homocysteine-induced endothelial dysfunction in porcine coronary arteries and human endothelial cells

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Growth hormone–releasing peptide ghrelin inhibits homocysteine-induced endothelial dysfunction in porcine coronary arteries and human endothelial cells Nasim Hedayati, MD, Suman Annambhotla, MD, Jun Jiang, MD, PhD, Xinwen Wang, MD, PhD, Hong Chai, MD, PhD, Peter H. Lin, MD, Qizhi Yao, MD, PhD, Changyi Chen, MD, PhD Journal of Vascular Surgery Volume 49, Issue 1, Pages 199-207 (January 2009) DOI: 10.1016/j.jvs.2008.08.065 Copyright © 2009 The Society for Vascular Surgery Terms and Conditions

Fig. 1 Effects of ghrelin and homocysteine (Hcy) on vasomotor functions in porcine coronary arteries. The vessel rings (n = 8) were treated with or without ghrelin, Hcy, or Hcy plus ghrelin, for 24 hours. Vasomotor reactivity was determined by the myograph system in response to vasoactive drugs. Error bars represent the standard error of the mean. A, Vessel contraction in response to the thromboxane A2 analog U46619 (10−7 M). B, Endothelium-dependent vasorelaxation in response to accumulative concentrations of bradykinin (10−9 to 10−5 M). C. Endothelium-independent vasorelaxation in response to sodium nitroprusside (SNP, 10−5 M). D. Effects of deacylated ghrelin (D-ghrelin) and Hcy on vasomotor functions in porcine coronary arteries. The vessel rings were treated with or without D-ghrelin, Hcy or Hcy plus D-ghrelin for 24 hours. The vessel rings were precontracted with the thromboxane A2 analog U46619 (10−7 M), and endothelium-dependent vasorelaxation was recorded in response to accumulative concentrations of bradykinin (10−9 to 10−5 M). B and D, *P < .05 by t test and analysis of variance. B, C, and D, EC, Endothelial cell. Relative unit to GAPDH. Journal of Vascular Surgery 2009 49, 199-207DOI: (10.1016/j.jvs.2008.08.065) Copyright © 2009 The Society for Vascular Surgery Terms and Conditions

Fig 2 Effects of ghrelin, deacylated ghrelin (D-ghrelin), and homocysteine (Hcy) on endothelial nitric oxide synthase (eNOS) messenger RNA (mRNA) levels in porcine coronary arteries. The vessel rings (n = 3) were treated with or without ghrelin, D-ghrelin, Hcy, or Hcy plus ghrelin or D-ghrelin for 24 hours. The mRNA levels of porcine eNOS and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were determined by real time polymerase chain reaction. A. Effects of ghrelin and Hcy. B. Effects of D-ghrelin and Hcy. Relative eNOS mRNA levels were calculated as 2 [Ct(GAPDH)-Ct(eNOS)]; *P < .05. Both t test and analysis of variance were performed. Error bars show the standard error of the mean. Journal of Vascular Surgery 2009 49, 199-207DOI: (10.1016/j.jvs.2008.08.065) Copyright © 2009 The Society for Vascular Surgery Terms and Conditions

Fig 3 Effects of ghrelin and homocysteine (Hcy) on endothelial nitric oxide synthase (NOS) immunoreactivity in porcine coronary arteries. The vessel rings were treated with or without ghrelin, Hcy, or Hcy plus ghrelin for 24 hours. Porcine eNOS protein levels (arrows) were determined with immunohistochemical staining. A, Control. B, Homocysteine (Hcy). C, Ghrelin (Ghr). D, Homocysteine plus ghrelin. Original magnification ×400. Journal of Vascular Surgery 2009 49, 199-207DOI: (10.1016/j.jvs.2008.08.065) Copyright © 2009 The Society for Vascular Surgery Terms and Conditions

Fig 4 Effects of ghrelin and homocysteine (Hcy) on superoxide anion production in porcine coronary arteries. The vessel rings (n = 8) were treated with or without ghrelin, Hcy, or Hcy plus ghrelin for 24 hours. Superoxide anion production from the endothelial surface of porcine coronary rings was detected by lucigenin-enhanced chemiluminescence. Relative light units per second (RLU/s) were recorded and the measurements between 5 and 10 minutes were averaged and normalized to the area of each arterial segment as RLU/s/mm2. Error bars show the standard error of the mean. *P < .05; both t test and analysis of variance were performed. Journal of Vascular Surgery 2009 49, 199-207DOI: (10.1016/j.jvs.2008.08.065) Copyright © 2009 The Society for Vascular Surgery Terms and Conditions

Fig 5 Effects of ghrelin and homocysteine (Hcy) on endothelial nitric oxide synthase (eNOS) protein levels in human coronary artery endothelial cells (HCAECs). The cells were treated with or without ghrelin, Hcy (50 μM), or Hcy plus ghrelin (100 ng/mL) for 24 hours. Human eNOS protein levels were determined with Western blot and band intensity quantitation. Error bars show the standard error of the mean. A, Ghrelin concentration-dependent study (0, 0.5, 5, 50, and 100 ng/mL). B, Effect of D-ghrelin (100 ng/mL). C, Blocking effect of antighrelin receptor antibody (anti-GHS-R1a Ab, 1:500). Journal of Vascular Surgery 2009 49, 199-207DOI: (10.1016/j.jvs.2008.08.065) Copyright © 2009 The Society for Vascular Surgery Terms and Conditions