Endogenous TNFα suppression of neovascularization in corneal stroma in mice

PURPOSE. To examine the role of tumor necrosis factor alpha (TNF alpha) in stromal neovascularization in injured cornea in vivo and in cytokine-enhanced vessel-like endothelial cell tube formation in vitro. METHODS. An in vitro model of angiogenesis was used to examine the roles of TNF alpha on tube formation by human umbilical vein endothelial cells (HUVECs) cocultured with fibroblasts on induction by transforming growth factor beta 1 (TGF beta 1) and vascular endothelial growth factor (VEGF). Central cauterization was used to induce stromal neovascularization in corneas of wild-type (WT) and TNF alpha-null (Tnf alpha(-/-)) mice. At 7, 14, or 21 days of injury, experimental mice were killed, and the eyes were enucleated and subjected to histologic and immunohistochemical examination and real-time reverse transcription polymerase chain reaction. RESULTS. HUVECs formed a vessel-like tube structure on the fibroblast feeder layer. Adding TGF beta 1, VEGF, or both augmented vessel-like tube formation by HUVECs cocultured with fibroblasts. Adding TNF alpha (5 ng/mL) completely abolished the formation of tube-like structures despite the presence or absence of TGF beta 1 or VEGF in coculture. In vivo, cauterization of the central cornea induced the formation of CD31(+) new vessels surrounding the limbus in WT mice. More prominent central stromal neovascularization accompanied by increased expression of TGF beta 1 and VEGF was found in Tnf alpha(-/-) mice compared with WT mice. CONCLUSIONS. In addition to inhibiting TGF beta 1 and VEGF expression by fibroblasts, endogenous TNF alpha may counter the induction effects of TGF beta 1 and VEGF on vascular endothelial cells and may block neovascularization.