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K. S. Edgar, R. van Haperen, R. de Crom, T. Gardiner, D. M. Mc Donald; Overexpression of Endothelial Nitric Oxide Synthase (eNOS) Increases Vascular Branch Formation in vitro and Modulates Neovascularisation in Oxygen Induced Retinopathy (OIR). Invest. Ophthalmol. Vis. Sci. 2009;50(13):3130.
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Neovascularisation occurs in response to tissue ischemia and vascular endothelial growth factor (VEGF) stimulation. In the retina, new vessels fail to recover the ischemic tissue and instead infiltrate the transparent vitreous by a poorly understood mechanism. Nitric oxide produced by eNOS is a downstream mediator of VEGF function and plays a crucial role in vascular formation and maturation. Here, we investigated the extent to which overexpression of eNOS induces branch formation in vitro and promotes vascular recovery of the ischemic retina. In order to achieve this, we used a transgenic mouse model expressing eNOS fused to green fluorescent protein (GFP) under the control of an endogenous eNOS promotor.
Wild type (WT) and eNOSGFP mice were exposed to 75% oxygen for 5 days from P7 to P12 and returned to room air. Eyes were collected at P12 and P17, flat mounted and the vasculature stained with B simplicifolia isolectin. Avascular, normal vascular and neovascular areas were quantified using image analysis software. eNOS is known to play an important role in capillary closure in this model therefore all results were expressed relative to P12. Aortas were removed and cut into 1mm rings, embedded in Matrigel and after 7 days the number of sprouts produced quantified.
The formation of sprouts from aortic explants was significantly greater in eNOSGFP mice compared to WT (fold change 1.2±0.039; p<0.001). In the OIR treated retinas at P12 there was a significant increase in the vaso-obliterated area in the eNOSGFP eyes (57.31±1.3 vs 48.38±1.7; p<0.01) compared to WT littermate controls. The extent of revascularisation from P12 to P17 was significantly greater in eNOS overexpressing animals (1.523±0.04 vs 1.377±0.03; p<0.05), in addition, there was also an increase in neovascular formation in this group (16.07±0.78 vs 11.77±0.76; p<0.01).
The increased sprouting seen in explants of aortic tissue from eNOSGFP mice demonstrates an important role for eNOS in vascular branch formation. In the ischemic retina however, while there is an increase in normal vasculature, there is also an increase in neovascular tuft formation, suggesting that ischemia partially dysregulates the role of eNOS in forming a viable vasculature.
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