Neutralizing VEGF decreases tortuosity and alters endothelial cell division orientation in arterioles and veins in a rat model of ROP: relevance to plus disease.

Plus disease, manifest by dilation and tortuosity of retinal vessels, is an important feature of severe retinopathy of prematurity (ROP) and is predictive of poor outcome and vision loss in preterm infants.1 However, the causes of plus disease are incompletely understood. Better understanding of the mechanisms of plus disease may lead to earlier treatments or prevention of severe ROP. Early investigators proposed that retinal vascular dilation and tortuosity in plus disease were a result of midperipheral mesenchymal shunting and increased retinal blood flow.2 However, studies using color Doppler imaging to measure blood flow in the central retinal artery were in disagreement. In one study, there were no significant flow differences between preterm infants with and without ROP, and in infants with ROP, there were no flow differences between those with and without plus disease.3 In another study, there was reduced blood flow in infants with plus disease.4 Neither study showed increased blood flow as initially hypothesized. However, measurements of blood flow within the central retinal artery are difficult in infant eyes and may not reflect that in the mesenchymal shunt or peripheral vessels. Increased blood flow increases shear stress, the in-plane frictional force, on endothelial cells within blood vessels. In tortuous vessels, the acutely curved part of the vessel is believed to have greater shear stress and the opposite side reduced shear stress. Shear stress can activate endothelial nitric oxide synthetase (eNOS) to produce nitric oxide (NO), which is important in vessel homeostasis.5-7 One outcome of NO is vascular muscle relaxation and vessel dilatation, which is a feature of plus disease in ROP. Besides shear stress, other stimuli, including hypoxia and growth factors like vascular endothelial growth factor (VEGF) can increase eNOS expression.8 Hypoxia has been associated with arterial tortuosity after middle cerebral artery occlusion,9 and when hypoxia is chronic, tortuosity is believed to be a form of angiogenesis through vessel lengthening. VEGF is mechanistically involved in the intravitreous neovascularization that occurs in animal models of ROP,10,11 and VEGF RNA was increased in the retina in a human infant with ROP.12 VEGF induces blood flow to ischemic myocardium by increasing collateral vessel formation,13 and VEGF also increases the size of capillaries during remodeling.14 VEGF is ineffective at improving angiogenesis in enos-/- mice in a murine ischemic limb model, suggesting that eNOS may be a downstream mediator of VEGF.15 Finally, eNOS was shown to be involved in hyperoxia-induced vaso-obliteration and angiogenesis in an enos-/- mouse model.16 In this last study, VEGF was not found to be downstream of eNOS. Therefore, eNOS can be activated by increased blood flow and hypoxia and may be a downstream mediator of VEGF-induced angiogenesis in some tissues. We asked whether plus disease is in part related to increased VEGF bioactivity and, if so, whether eNOS is involved. We used the rat 50/10 oxygen-induced retinopathy (50/10 OIR) model, which develops peripheral avascular retina, intravitreous neovascularization, and arteriolar tortuosity similar to human ROP.17,18 We also measured the orientation of the cleavage of mitotic endothelial cells during anaphase19 within newly formed intraretinal arterioles and veins of pups in the 50/10 OIR model after treatment with a neutralizing antibody to VEGF as a method of assessing the effect of VEGF on dilation and tortuosity of large retinal vessels.