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. 2009 Jan;49(1):199-207.
doi: 10.1016/j.jvs.2008.08.065. Epub 2008 Nov 22.

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

Nasim Hedayati  1 , Suman Annambhotla, Jun Jiang, Xinwen Wang, Hong Chai, Peter H Lin, Qizhi Yao, Changyi Chen
Affiliations

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

Nasim Hedayati et al. J Vasc Surg. 2009 Jan.

Abstract

Objective: Ghrelin, a novel growth hormone-releasing peptide, is implicated to play a protective role in cardiovascular tissues. However, it is not clear whether ghrelin protects vascular tissues from injury secondary to risk factors such as homocysteine (Hcy). This study investigated the effect and potential mechanisms of ghrelin on Hcy-induced endothelial dysfunction.

Methods: Porcine coronary artery rings were incubated for 24 hours with ghrelin (100 ng/mL), Hcy (50 microM), or ghrelin plus Hcy. Endothelial vasomotor function was evaluated using the myograph tension model. The response to the thromboxane A(2)analog U46619, bradykinin, and sodium nitroprusside was analyzed. Endothelial nitric oxide synthase (eNOS) expression was determined using real-time polymerase chain reaction and immunohistochemistry staining, and superoxide anion production was documented lucigenin-enhanced chemiluminescence analysis. Human coronary artery endothelial cells (HCAECs) were treated with different concentrations of Hcy, ghrelin, or antighrelin receptor antibody for 24 hours, and eNOS protein levels were determined by Western blot analysis.

Results: Maximal contraction with U46619 and endothelium-independent vasorelaxation with sodium nitroprusside were not different among the four groups. However, endothelium-dependent vasorelaxation with bradykinin (10(-6) M) was significantly reduced by 34% with Hcy compared with controls (P < .05). The addition of ghrelin to Hcy had a protective effect, with 61.6% relaxation, which was similar to controls (64.7%). Homocysteine significantly reduced eNOS expression, whereas ghrelin cotreatment effectively restored eNOS expression to the control levels. Superoxide anion levels, which were increased by 100% with Hcy, returned to control levels with ghrelin cotreatment. Ghrelin also effectively blocked the Hcy-induced decrease of eNOS protein levels in HCAECs in a concentration-dependent manner. Antighrelin receptor antibody effectively inhibited the effect of ghrelin.

Conclusion: Ghrelin has a protective effect in the porcine coronary artery by blocking Hcy-induced endothelial dysfunction, improving eNOS expression, and reducing oxidative stress. Ghrelin also shows a protective effect on HCACEs from the Hcy-induced decrease in eNOS protein levels. The effect of ghrelin is receptor-dependent. Thus, ghrelin administration may have beneficial effects in the treatment of vascular disease in patients with hyperhomocysteinemia.

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Figures

Fig 1
Fig 1
Effects of ghrelin and homocysteine (Hcy) on vasomotor functions in porcine coronary arteries. The vessel rings 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. A. Vessel contraction in response to thromboxane A2 analog U46619 (10-7M). B. Endothelium-dependent vasorelaxation in response to accumulative concentrations of bradykinin (10-9 to 10-5M). C. Endothelium-independent vasorelaxation in response to sodium nitroprusside (SNP, 10-5M). n = 8. *P<0.05 (t-test and ANOVA test). D. Effects of deacylated ghrelin (D-ghrelin) and homocysteine (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 thromboxane A2 analog U46619 (10-7M), and endothelium-dependent vasorelaxation was recorded in response to accumulative concentrations of bradykinin (10-9 to 10-5M). n = 8. *P<0.05 (t-test and ANOVA test).
Fig 1
Fig 1
Effects of ghrelin and homocysteine (Hcy) on vasomotor functions in porcine coronary arteries. The vessel rings 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. A. Vessel contraction in response to thromboxane A2 analog U46619 (10-7M). B. Endothelium-dependent vasorelaxation in response to accumulative concentrations of bradykinin (10-9 to 10-5M). C. Endothelium-independent vasorelaxation in response to sodium nitroprusside (SNP, 10-5M). n = 8. *P<0.05 (t-test and ANOVA test). D. Effects of deacylated ghrelin (D-ghrelin) and homocysteine (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 thromboxane A2 analog U46619 (10-7M), and endothelium-dependent vasorelaxation was recorded in response to accumulative concentrations of bradykinin (10-9 to 10-5M). n = 8. *P<0.05 (t-test and ANOVA test).
Fig 1
Fig 1
Effects of ghrelin and homocysteine (Hcy) on vasomotor functions in porcine coronary arteries. The vessel rings 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. A. Vessel contraction in response to thromboxane A2 analog U46619 (10-7M). B. Endothelium-dependent vasorelaxation in response to accumulative concentrations of bradykinin (10-9 to 10-5M). C. Endothelium-independent vasorelaxation in response to sodium nitroprusside (SNP, 10-5M). n = 8. *P<0.05 (t-test and ANOVA test). D. Effects of deacylated ghrelin (D-ghrelin) and homocysteine (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 thromboxane A2 analog U46619 (10-7M), and endothelium-dependent vasorelaxation was recorded in response to accumulative concentrations of bradykinin (10-9 to 10-5M). n = 8. *P<0.05 (t-test and ANOVA test).
Fig 1
Fig 1
Effects of ghrelin and homocysteine (Hcy) on vasomotor functions in porcine coronary arteries. The vessel rings 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. A. Vessel contraction in response to thromboxane A2 analog U46619 (10-7M). B. Endothelium-dependent vasorelaxation in response to accumulative concentrations of bradykinin (10-9 to 10-5M). C. Endothelium-independent vasorelaxation in response to sodium nitroprusside (SNP, 10-5M). n = 8. *P<0.05 (t-test and ANOVA test). D. Effects of deacylated ghrelin (D-ghrelin) and homocysteine (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 thromboxane A2 analog U46619 (10-7M), and endothelium-dependent vasorelaxation was recorded in response to accumulative concentrations of bradykinin (10-9 to 10-5M). n = 8. *P<0.05 (t-test and ANOVA test).
Fig 2
Fig 2
Effects of ghrelin, deacylated ghrelin (D-ghrelin) and homocysteine (Hcy) on eNOS mRNA levels in porcine coronary arteries. The vessel rings 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 GAPDH were determined by real time PCR. A. Effects of ghrelin and Hcy. B. Effects of D-ghrelin and Hcy. Relative eNOS mRNA levels were calculated as 2ˆ[Ct (GAPDH)-Ct(eNOS)]. n = 3. *P<0.05. Both t-test and ANOVA test were performed.
Fig 2
Fig 2
Effects of ghrelin, deacylated ghrelin (D-ghrelin) and homocysteine (Hcy) on eNOS mRNA levels in porcine coronary arteries. The vessel rings 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 GAPDH were determined by real time PCR. A. Effects of ghrelin and Hcy. B. Effects of D-ghrelin and Hcy. Relative eNOS mRNA levels were calculated as 2ˆ[Ct (GAPDH)-Ct(eNOS)]. n = 3. *P<0.05. Both t-test and ANOVA test were performed.
Fig 3
Fig 3
Effects of ghrelin and homocysteine (Hcy) on eNOS 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 were determined with immunohistochemistical staining. A. Control. B. Homocysteine (Hcy). C. Ghrelin. D. Hcy plus ghrelin.
Fig 4
Fig 4
Effects of ghrelin and homocysteine (Hcy) on superoxide anion production in porcine coronary arteries. The vessel rings 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. n = 8. *P<0.05. Both t-test and ANOVA test were performed.
Fig 5
Fig 5
Effects of ghrelin and homocysteine (Hcy) on eNOS protein levels in HCAECs. The cells were treated with or without ghrelin, Hcy (50 μM) or plus ghrelin (100 ng/mL) for 24 hours. Human eNOS protein levels were determined with western blot and band intensity quantitation. 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 anti-ghrelin receptor antibody (anti-GHS-R1a Ab, 1:500).
Fig 5
Fig 5
Effects of ghrelin and homocysteine (Hcy) on eNOS protein levels in HCAECs. The cells were treated with or without ghrelin, Hcy (50 μM) or plus ghrelin (100 ng/mL) for 24 hours. Human eNOS protein levels were determined with western blot and band intensity quantitation. 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 anti-ghrelin receptor antibody (anti-GHS-R1a Ab, 1:500).
Fig 5
Fig 5
Effects of ghrelin and homocysteine (Hcy) on eNOS protein levels in HCAECs. The cells were treated with or without ghrelin, Hcy (50 μM) or plus ghrelin (100 ng/mL) for 24 hours. Human eNOS protein levels were determined with western blot and band intensity quantitation. 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 anti-ghrelin receptor antibody (anti-GHS-R1a Ab, 1:500).
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