Reduction in endothelial tip cell filopodia corresponds to reduced intravitreous but not intraretinal vascularization in a model of ROP.
To determine the effect of a vascular endothelial growth factor receptor 2 tyrosine kinase (VEGFR2) inhibitor on intravitreous neovascularization (IVNV), endothelial tip cell filopodia, and intraretinal vascularization in a rat model of retinopathy of prematurity (ROP). Within 4h of birth, newborn Sprague-Dawley rat pups and their mothers were cycled between 50% and 10% oxygen daily until postnatal day (p)12. Pups were given intravitreous injections of VEGFR2 inhibitor, SU5416, or control (dimethyl sulfoxide, DMSO) and returned to oxygen cycling until p14, then placed into room air. Intravitreous neovascularization (IVNV), avascular/total retinal areas, and endothelial tip cell filopodial number and length were determined in lectin-labeled neurosensory retinal flat mounts. Cryosections or fresh tissue were analyzed for phospho-VEGFR1, phospho-VEGFR2, activated caspase-3, or phospho-beta3 integrin. Human umbilical venous (HUVECs) and human choroidal endothelial cells (ECs) were treated with VEGFR2 inhibitor to determine effect on VEGFR2 phosphorylation and on directed EC migration toward a VEGF gradient. Filopodial length and number of migrated ECs were also measured. Compared to control, the VEGFR2 inhibitor reduced VEGFR2 phosphorylation in HUVECs in vitro and clock hours and areas of IVNV but not percent avascular retina in vivo. Filopodial length and number of filopodia/EC tip cell were reduced in retinal flat mounts at doses that inhibited IVNV, whereas at lower doses, only a reduction in filopodial length/EC tip cell was found. There was no difference in phosphorylated beta3 integrin and cleaved caspase-3 labeling in VEGFR2 inhibitor-treated compared to control in vivo. Doses of the VEGFR2 inhibitor that reduced filopodial length and number of filopodia/migrating EC corresponded to reduced EC migration in in vitro models. VEGFR2 inhibitor reduced IVNV and filopodial number and length/EC tip cell without interfering with intraretinal vascularization. Reducing the number and length of filopodia/endothelial tip cell may reduce guidance cues for endothelial cells to migrate into the vitreous without interfering with migration into the retina toward a VEGF gradient.
Budd
Steven
S
Department of Ophthalmology, University of North Carolina Chapel Hill, 103 Mason Farm Road, CB # 7041, 6135 NSRB, Chapel Hill, NC 27599, USA. budd@med.unc.edu
Byfield
Grace
G
Martiniuk
David
D
Geisen
Pete
P
Hartnett
Mary Elizabeth
ME
D020533:Angiogenesis Inhibitors / Q000494:pharmacology*; D000818:Animals; D000831:Animals, Newborn; D053148:Caspase 3 / Q000378:metabolism; D002465:Cell Movement / Q000187:drug effects; D002478:Cells, Cultured; D004195:Disease Models, Animal; D004305:Dose-Response Relationship, Drug; D042783:Endothelial Cells / Q000187:drug effects* / Q000201:enzymology; D006801:Humans; D007211:Indoles / Q000494:pharmacology*; D007231:Infant, Newborn; D039661:Integrin beta3 / Q000378:metabolism; D009389:Neovascularization, Pathologic / Q000188:drug therapy* / Q000201:enzymology / Q000503:physiopathology; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D011554:Pseudopodia / Q000187:drug effects* / Q000201:enzymology; D011758:Pyrroles / Q000494:pharmacology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012171:Retinal Vessels / Q000187:drug effects* / Q000201:enzymology / Q000503:physiopathology; D012178:Retinopathy of Prematurity / Q000188:drug therapy* / Q000201:enzymology / Q000503:physiopathology; D013997:Time Factors; D040281:Vascular Endothelial Growth Factor Receptor-1 / Q000378:metabolism; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000037:antagonists & inhibitors* / Q000378:metabolism; D014822:Vitreous Body / Q000098:blood supply*
D020533:Angiogenesis Inhibitors; D007211:Indoles; D039661:Integrin beta3; D047428:Protein Kinase Inhibitors; D011758:Pyrroles; C116890:Semaxinib; D040281:Vascular Endothelial Growth Factor Receptor-1; D040301:Vascular Endothelial Growth Factor Receptor-2; C505410:Casp3 protein, rat; D053148:Caspase 3