Abstract
Purpose: :
To determine the effect of inhibiting VEGF bioactivity with a neutralizing antibody to VEGF or a VEGF receptor 2 inhibitor on retinal endothelial tip cell filopodia in the 50/10 rat oxygen induced retinopathy (OIR) model.
Methods: :
: Litters with 12-14 newborn pups and mothers were exposed to 24 hours of 50% oxygen followed by 10% oxygen repeated for seven cycles in an Oxycycler incubator. At postnatal day p12, the right eyes of pups in one litter were given intravitreous injections of either 1 µl of a neutralizing VEGF 164 antibody (50 ng/µl) or an IgG control. Similarly, pups of another litter were injected with SU5416 (10µM), a non-specific VEGF receptor 2 tyrosine kinase inhibitor or DMSO as a control. The doses of SU5416 and VEGF 164 antibody were chosen because we found their dose significantly reduced IVNV in the rat OIR model at p18. The left eyes remained non-injected. At p14, the retinas were removed, flat-mounted, and stained with Alexa Fluor 568 conjugated Grifonnia isolectin B4 (5 µg/ml). Flat-mounts were then imaged with a Leica SP2 laser scanning confocal microscope. Approximately 12-15 tip cells per retina were analyzed along the leading edge of vascular development. The number of filopodia in each tip cell was counted and their lengths were measured with ImageTool. The filopodia lengths for each tip cell was added together and expressed as a total length.
Results: :
There was no significant difference in the number of filopodia per tip cell between eyes injected with the VEGF 164 antibody and the IgG control (ttest, n=5, p=.07) or between those injected with SU5416 and DMSO (ttest, n=5, p=.5). With eyes injected with SU5416, there was a significant 1.5 fold reduction in total filopodia length per tip cell compared to DMSO injected eyes (ttest, n=5, p=.002).
Conclusions: :
Using a VEGF receptor 2 inhibitor, SU5416, filopodial length, but not number of filopodia in endothelial tip cells was reduced. Since the dose of SU5416 used caused reduction in IVNV, our data support that inhibiting the receptor tyrosine kinase activity of angiogenic receptors (e.g. VEGF R2) reduces angiogenesis by reducing the extension but not the number of filopodia in individual endothelial tip cells.
Keywords: retinopathy of prematurity • vascular endothelial growth factor • growth factors/growth factor receptors