ORIGINAL ARTICLE Lipid hydroperoxide-induced tumor necrosis factor (TNF)-a, vascular endothelial growth factor and neovascularization in the rabbit cornea: effect of TNF inhibition Toshihiko Ueda, Takako Ueda, Shohei Fukuda, Richard Browne, Edwin Jenis, 1 Robert Spengler, 1 Richard Chou, 1 Peter Buch, 2 Ahmad Aljada, Paresh Dandona, 3 R. Sasisekharan, 4 C. Kathleen Dorey 5 and Donald Armstrong Departments of Clinical Laboratory Science, 1 Pathology, 2 Ophthalmology and 3 Medicine, State University of New York at Buffalo, Buffalo, NY 14214, USA. 4 Division of Health Sciences and Technology, Harvard University±Mas- sachusetts Institute of Technology, Cambridge, MA 02139, USA. 5 Schepens Eye Research Institute, Harvard Medical School, Boston, MA 02114, USA Lipid hydroperoxides (LHP) at high concentrations are cytotoxic, but at sublethal concentration, they induce synthesis of cytokine vascular growth factors. Intra- corneal injections of 30 lg LHP placed 5 mm from the superior limbus stimulated early vasodilation of limbal vasculature and a rapidly developing, sustained neo- vascularization. Under these conditions, vessels grew at the rate of 0.3 mm/day to a total length of 7.5 mm, 25 days after injection. Cholesterol peroxides were less effective. Developing vessels were oriented towards the stimulus. Around the developing vessel there was dissolution of the stromal extracellular matrix. The most distal endothelial cells displayed prominent endoplasmic reticulum, a lack of basement membrane or tight junction complexes and leakage of ¯uorescein dye. Both the injection site and superior quadrant showed increased levels of tumor necrosis factor (TNF)-a and vascular endothelial growth factor after exposure to LHP. The neovascular response was inhibited by simultaneous administration of TNF-a antibody or pentoxifylline, an inhibitor of TNF-a syn- thesis. This corneal model of peroxide-induced neo- vascularization should prove useful for temporal studies of events in the initiation and propagation of signals leading to neovascularization, and for evaluat- ing effects of treatment on neovascular growth. Key words : Cornea, lipid hydroperoxide, neovasculariza- tion, rabbit, tumor necrosis factor-a, vascular endothelial growth factor. (Received 24 March 1997; accepted 13 October 1997) Introduction Angiogenesis is a developmental process that is essential for homeostasis. 1 The plasticity of ex- isting blood vessels to respond to appropriate stimuli by chemotaxis, proliferation and matura- tion into new vessels is an important feature of wound healing. 2 On the other hand, ocular neo- vascularization is a key event in pathological processes such as in¯ammation and diabetic retinopathy. Neovascularization is especially rel- evant in cancer where it promotes expansion of the tumor and metastasis. 3 Free radicals have also been implicated in the morbidity of diabetic retinopathy. In this disorder, hyperglycemia generates free radicals 4 and exacerbates lipid hydroperoxide (LHP) production in vascular en- dothelial cells. 5 Serum LHP is elevated in diabetes and is increased further in those patients with Correspondence to D. Armstrong, Department of Clinical Lab- oratory Science, State University of New York at Buffalo, 26 Cary Hall, 3435 Street, Buffalo, NY 14214, USA. Tel: (+1) 716 829-3630; Fax: (+1) 716 829-3601. Preliminary report: Ueda T, Ueda T, Jenis E, et al. Lipid hydroperoxide induced corneal neovascularization. Invest Ophthalmol Vis Sci 1995; 36, S31 (abstr). Supported by grants 150-4581 (D.A.) and 91058G (R.S.) from the American Heart Association, NY Af®liate, NO1 WH32122 (D.A.), and an in- stitutional award to T.U. and S.F. from Showa University School of Medicine. Angiogenesis 1997; 1; 174±184 174 Angiogenesis á Vol 1 á No 2 á 1997 Ó 1997 Rapid Science Publishers