[Frontiers in Bioscience E4, 2525-2535, June 1, 2012]

Homocysteine-impaired angiogenesis is associated with VEGF/VEGFR inhibition

Quan Zhang1, Qian Li1, Yu Chen2, Xiao Huang3,4, Irene Hwa Yang5, Lu Cao1, Wei-Kang Wu1, Hong-Mei Tan1

1Department of Pathophysiology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510080, China, 2Department of Anesthesiology, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou 510080, China, 3Department of Pharmacology, Cardiovascular Research Center, Temple University School of Medicine, Philadelphia, PA, USA, 4Department of Cardiology, Second Hospital of NanChang University, Jiang Xi, China, 5Yale affiliation Primary Care Internal Medicine Residency Program, Yale University School of Medicine, CT, USA

TABLE OF CONTENTS

1. Abstract
2. Introduction
3. Materials and methods
3.1. Cell culture
3.2. Cell proliferation assay
3.3. Cell viability assay
3.4. Cell cycle analysis
3.5. Cell migration assay
3.6. Matrigel assay
3.7. Zebrafish embryos angiogenesis assay
3.8. Quantitative real time PCR for mRNA expression analyses
3.9. Western blot analysis
3.10. Statistical methods
4. Results
4.1. Hcy inhibited HUVEC proliferation and viability
4.2. Hcy induced HUVEC cell cycle G1/S arrest
4.3. Hcy inhibited HUVEC migration
4.4. Hcy suppressed tube formation
4.5. Hcy inhibited the vessel formation in zebrafish embryo
4.6. Hcy inhibited expression of VEGF, VEGFR1 and VEGFR2
4.7. Hcy decreased ERK1/2 and Akt expression in HUVEC
5. Discussion
6. Acknowledgment
7. References

1. ABSTRACT

This study investigated the effects of homocysteine (Hcy) on angiogenesis in cultured human umbilical vein endothelial cells (HUVEC) and zebrafish embryos. We found that Hcy (50 micro mol/L) significantly decreased cell numbers, viability, and induced a G1/S arrest in HUVEC in the presence of adenosine (Ade, 50 micro mol/L). Hcy, in combination with Ade, reduced migration and suppressed tube-like formation on Matrigel in HUVEC. Further, Hcy reduced subintestinal vessel formation in zebrafish embryos. Interestingly, Hcy-induced inhibitory effects on cell growth, migration, tube-like formation, and vessel formation in HUVEC and zebra fish embryos were abolished by the supplement of recombinant VEGF (10ng/ml). Finally, Hcy in combination with Ade reduced the mRNA levels of VEGF, VEGFR-1, VEGFR-2, and attenuated protein levels of VEGF, ERK1/2 and Akt. The present study suggests that Hcy inhibits angiogenesis, and that the mechanism anti-angiogenic effects of Hcy may be through VEGF/VEGFR, Akt, and ERK1/2 inhibition.