Abstract
Purpose: :
Previous work from our laboratory has shown that activation of Phospholipase Cγ1(PLCγ1) activation is required for tubulogenesis of endothelial cells and distruption of PLCγ1 binding with VEGFR–2/FLK–1 impairs the ability of VEGFR–2 to promote tubulogenesis in cell culture system. In our current study using chorioallantoic membrane (CAM) model, we studied the role of activation of PLCγ1 in angiogenesis.
Methods: :
9 days old fertilized chicken eggs were used in CAM assay. Baseline vessel formation was photographed in 12 CAM by using S6D microscope with Leica DC 300F camera, and IM50 system. m–3M3FBS (50 µM) a potent Phospholipase C activator was placed on 8 CAM and control (DMSO with DMEM ) was placed on 4 CAM. These CAM were photographed 24 hours after incubation. The vascular patterns and density were analysed using the digital Kodak ID 3.5 image analysis system. The difference (delta) between the baseline and 24 hours after treatment or control was measured for each CAM. The statistical significance between the two groups was evaluated using t–test for independent samples on the two sample calculator.
Results: :
The mean of the change in measurement 24 hours after incubation in the control group was 18, and the mean of change in measurement 24 hours after incubation with the PLCγ1 activator group was 30.75 ( P=0.07). In our work with PLCγ 1 inhibitor (U–73132) the mean of the change in measurement 24 hours after incubation in the control group was 43, and the mean of change in measurement 24 hours after incubation of the treatment group was –17( P=0.00039). The mean baseline measurement in the treatment group with the activator was 40 and the mean baseline measurement in the control group was 39, these two groups were not statisctically significant ( P=0.96). This data shows that the vascular density was higher in the drug (PLCγ1 activator) group as compared to the control group.
Conclusions: :
The data shows that activation of PLCγ1 increases the growth of blood vessel in an in vivo model of angiogenesis, corraborating the previous results on cell culture system. Although this data is preliminary, however it shows a trend towards significance. Further work is underway to refine the drug dosage. Comparing the PLCγ1 activator data to the study on CAM with PLCγ1 inhibitor there is definite increase in vascularity in the group treated with PLCγ1 activator. This work indicates that PLCγ1 plays a vital role in angiogenesis. Therefore the targeting of PLCγ1 may offer a potential novel therapeutic modality for treatment of ocular neovascularization.
Keywords: proliferation • neovascularization