Endogenous TNFalpha suppression of neovascularization in corneal stroma in mice.
OBJECTIVE
To examine the role of tumor necrosis factor alpha (TNFalpha) 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 TNFalpha on tube formation by human umbilical vein endothelial cells (HUVECs) cocultured with fibroblasts on induction by transforming growth factor beta1 (TGFbeta1) and vascular endothelial growth factor (VEGF). Central cauterization was used to induce stromal neovascularization in corneas of wild-type (WT) and TNFalpha-null (Tnfalpha(-/-)) 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 TGFbeta1, VEGF, or both augmented vessel-like tube formation by HUVECs cocultured with fibroblasts. Adding TNFalpha (5 ng/mL) completely abolished the formation of tube-like structures despite the presence or absence of TGFbeta1 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 TGFbeta1 and VEGF was found in Tnfalpha(-/-) mice compared with WT mice.


CONCLUSIONS
In addition to inhibiting TGFbeta1 and VEGF expression by fibroblasts, endogenous TNFalpha may counter the induction effects of TGFbeta1 and VEGF on vascular endothelial cells and may block neovascularization.
Fujita
Shuko
S
Department of Ophthalmology, Wakayama Medical University, Kimiidera, Wakayama, Japan. shuko@wakayama-med.ac.jp
Saika
Shizuya
S
Kao
Winston Whei-Yang
WW
Fujita
Kyoko
K
Miyamoto
Takeshi
T
Ikeda
Kazuo
K
Nakajima
Yuji
Y
Ohnishi
Yoshitaka
Y
D000818:Animals; D018920:Coculture Techniques; D016510:Corneal Neovascularization / Q000378:metabolism / Q000517:prevention & control*; D003319:Corneal Stroma / Q000098:blood supply*; D004195:Disease Models, Animal; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism; D016326:Extracellular Matrix Proteins / Q000096:biosynthesis / Q000235:genetics; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016212:Transforming Growth Factor beta / Q000096:biosynthesis / Q000235:genetics; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology / Q000502:physiology*; D014471:Umbilical Veins / Q000166:cytology; D042461:Vascular Endothelial Growth Factor A / Q000096:biosynthesis / Q000235:genetics; D000099222:betaIG-H3 Protein
D016326:Extracellular Matrix Proteins; D012333:RNA, Messenger; D016212:Transforming Growth Factor beta; D014409:Tumor Necrosis Factor-alpha; D042461:Vascular Endothelial Growth Factor A; D000099222:betaIG-H3 Protein; C467480:vascular endothelial growth factor A, mouse