DNA methylation is a potent regulator of gene expression. The influence of beta-carotene (BC) and arachidonic acid (AA) on angiogenesis--a new blood vessel formation, was reported. The tyrosine kinase VEGFR-2 receptor (KDR) activation by vascular endothelial growth factor is one of the main angiogenic mechanisms. This study was aimed to investigate a possible role of CpG island methylation on regulation of the pro-angiogenic KDR gene expression after incubation of human endothelial cells with BC and/or AA.
Methods: Human umbilical vein endothelial cells (HUVEC) were incubated with BC (1-10 microM) and/or 3 microM AA for 24 hours. The CpG island methylation was quantified using the COBRA method and restriction enzymes' digestion (NewEngland BioLabs). Intracellular protein concentrations were determined by Western blot analysis using the specific antibodies (Santa Cruz).
Results: Incubation with BC and AA, decreased methylation of the KDR promoter region. These results well-correlated with the detected, by qRT-PCR, up-regulation of KDR gene expression by BC (p=0.035) as well as by AA. Incubation with BC (p=0.02) and AA (p=0.0014) increased the KDR protein levels in HUVECs.
Conclusion: The changes in CpG island methylation of the KDR the pro-angiogenic gene promoter, represents one of the mechanisms involved in regulation of angiogenic response by BC and AA.