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Bharani Krishna Mynampati Arunadithya, sandeep Grover, K V Chalam; Bevacizumab complements inhibitory effects of proton beam radiation on VEGF enriched proliferative choroidal endothelial cells. Invest. Ophthalmol. Vis. Sci. 2017;58(8):882.
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© ARVO (1962-2015); The Authors (2016-present)
To investigate the effects of escalating doses of proton beam radiation with and without bevacizumab on vascular endothelial growth factor (VEGF)-enriched choroidal vascular endothelial cells (CVECs)
The CVECs were treated with VEGF to induce cellular proliferation to mimic subretinal neovascular membrane in macular degeneration. Six well plates (n=5) were prepared for 5 different proton dosage levels - 0,2,4,8 and 12 cobalt grey equivalent (CGE). Before proton beam exposure, CVECs were treated with bevacizumab at 2mg/ml. Three wells in each plate were treated with bevacizumab and the remaining 3 wells were not treated (control). On day 5, cell proliferation was measured with WST-1 assay and trypan blue exclusion assay with automated Vi cell XR analyzer. Reactive oxygen species (ROS) levels were also measured using dihydrorhodamine 123.
In controls, the cell proliferation rates (WST-1 assay) with exposure to various proton beam dosages were 87.6% (p=0.29), 84% (p=0.12), 51.6% (p<0.0001) and 39% (p<0.0001) with 2,4,8 and 12 CGE, respectively. In the presence of bevacizumab, cell proliferation rates were reduced even more, 92.9 % (p=0.95), 70.6% (p=0.08), 38.5% (p=0.0008) and 20.1% (p<0.0001) with 2, 4, 8 and 12 CGE, respectively. Similarly, the cell count (T.blue assay) at various dosages were 88.0% (p=0.44), 70.6% (p=0.015), 46.2% (p=0.0002) and 36% (p<0.0001) with 2, 4, 8 and 12 CGE, compared to control cells. However, in the presence of bevacizumab, these cell counts were again much more reduced 53.03% (p=0.0032), 43.9 % (p=0.0009), 27.5% (p<0.0001), and 27.5% (p<0.0001) with 2,4,8 and 12 CGE, respectively. ROS levels were found to be increased at higher doses of proton beam exposure and they measured 100% (p=0.95), 100.6% (p>0.99), 111.9% (p=0.17) and 118.1% (p=0.02) with dosages of 2, 4, 8 and 12 CGE, respectively, as compared to controls. Similarly, in the presence of bevacizumab, ROS levels further increased, measuring at 100% (p=0.9), 101.36% (p= 0.9), 114.3% (p=0.6) and 135.5% (p=0.02) with dosages of 2, 4, 8 and 12 CGE, respectively
There was significant inhibition of the proliferation of VEGF-enriched CVECs with escalating doses of proton beam exposure in a dose-dependent manner. Treatment of these cells with bevacizumab further complemented the inhibitory effect of proton beam radiation treatment.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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