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Nymph Chan, Shikun He, Stephen J. Ryan, Jr., David R. Hinton; Epigenetic Regulation of Choroidal Neovascularization. Invest. Ophthalmol. Vis. Sci. 2012;53(14):3019.
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Choroidal neovascularization (CNV) is a blinding complication of age-related macular degeneration. The pathogenesis of CNV is still under investigation, and epigenetic regulation can be a new target for a therapeutic approach in treating CNV. The aim was to examine the role of histone deacetylation inhibitors (HDACi) in the suppression of angiogenesis in vitro, and its regulation of pro- and anti-angiogenic genes in new blood vessel formation in CNV.
Cultured early passage human fetal retinal pigment epithelial (RPE) cells and bovine choroidal endothelial cells (BCEC) were used in the study. The role of HDACs in the regulation of expression of HIF-1α, PEDF, VEGF and VEGFR2 was analyzed by ChIP assay. 5×105 of RPE cells were treated with or without 0.5 μM of trichostatin A (TSA) for 24 h, and fragmented chromatin was immunoprecipitated with normal mouse IgG, anti-RNA Polymerase II or anti-Acetyl-Histone H3 antibodies. Pulled down DNA was amplified by PCR for the promoter regions of the genes of interest and the DNA products were resolved on a 1% agarose gel. The effect of TSA on the formation of tube-like structures was tested by treating BCECs with or without 0.7 μM of TSA for 24 h. 1 x 104 of BCECs per condition were transferred onto 50 μL of reconstituted basement membrane matrix and incubated with or without 25 ng/mL of VEGF at 37°C for 6 h. The effect of another HDACi, MS-275, on the expression of the four genes was examined by treating RPE cells with 0 - 0.3 μM of MS-275 for 18 to 24 h, with or without 150 μM of CoCl2 for 6 h. Western blots were performed to analyze expression of the four genes.
Results from the ChIP assay showed that in TSA-treated RPE cells, less RNA Polymerase II and Acetyl-Histone H3 were associated with the promoter regions of HIF-1α, VEGF and VEGFR2, but more RNA Polymerase II and Acetyl-Histone H3 were associated with the PEDF promoter. This suggests that under the effect of TSA, the PEDF gene was actively being transcribed, while there was less transcriptional activity at the HIF-1α, VEGF and VEGFR2 promoters. Another HDACi, MS-275, also reduced the expression of HIF-1α, VEGF and VEGFR2, and up-regulated PEDF, indicating that the expression of these genes was likely regulated by epigenetics. Further, TSA inhibited the formation of capillary-like structures in BCECs, which showed that TSA could inhibit angiogenesis.
The down-regulation of HIF-1α, VEGF and VEGFR2, and up-regulation of PEDF after TSA treatment is likely affected by epigenetics. Moreover, TSA also inhibits tube formation in BCECs. It is conceivable that HDACi inhibits angiogenesis in CNV by modulating genes involved in regulating new blood vessel formation. These data illustrate that epigenetic agents can emerge as a new type of treatment for CNV.
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