DNA methylation is a mechanism regulating gene expression. Beta-carotene (BC), potent pro-vitamin A/retinoic acid source in human, was shown to have pro-chemotactic activity and stimulate expression of pro-angiogenic genes in endothelium. Angiogenesis is an important mechanism in tumour malignancy. Fatty acids stimulate BC uptake. The arachidonic acid (AA) metabolites were shown the procancerogenic activity. This study was undertaken to define the possible changes in DNA methylation in endothelial cells and its progenitors after incubation with BC and AA.

Human umbilical vein endothelial cells (HUVEC) and isolated from cord blood endothelial progenitors (EPC) were incubated with BC (1-10mM) and 3mM arachidonic acid (AA) for 24 hours. The CpG island methylation was quantified using the Combined Bisulphite Restriction Analysis (COBRA) method (HotStarTaq Master Mix Kit, Qiagen) and the PCR products were digested by restriction enzymes (NewEngland BioLabs). Global DNA methylation was analysed with cytosine extension assay using methyl-sensitive restriction enzyme HpaII (New England Biolabs), which allows [³H]dCTP to be incorporated into the DNA strands..

The global DNA methylation analysis pointed to the tendency to down-regulation of DNA methylation in HUVEC and EPC, after incubation with AA (p=0.919) or BC (p=0.227). Of the 18 investigated genes connected with the endothelial cell proangiogenic activity, DNA methylation was regulated in the promoter regions of: integrinb3, connexin 43, CXCR4, KDR, MMP-2, laminin, Notch4 and VCAM1 genes .

Conclusion: The CpG island methylation might be an important mechanism of changes in the expression of pro-angiogenic genes after stimulation with beta-carotene and arachidonic acid. The presented results are from a pilot study and need further observation.

Project supported by Polish Committee of Science Grant No: 0143/P01/2006/31 and The European Nutrigenomics Organisation NUGO Exchange Grand

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