Regulation of matrix metalloproteinase expression by tumor necrosis factor in a murine model of retinal neovascularization.
OBJECTIVE
Hypoxia and growth factors are postulated to be involved in the development of retinal neovascularization through the regulation of extracellular proteinase production. It has been shown that matrix metalloproteinases (MMPs) are elevated in the retina during the neovascularization process. However, the factors and mechanisms that regulate the expression of these enzymes are not well characterized. The present study examines the potential role of tumor necrosis factor (TNF)-alpha as a regulator of MMPs in the retinal neovascularization process.


METHODS
C57/Bl6 mice were treated with 75% oxygen (experimental) or room air (control) from postnatal days (P)7 through P12, followed by room air until P17. Retinas were collected at P13, P15, or P17 and total RNA analyzed for the relative level of TNFalpha, TNF receptor (p55), and TNFalpha-converting enzyme (TACE). Immunostaining was used to identify changes in TNF protein expression as well as to localize TNFalpha within specific retinal cell types. The role of TNFalpha in stimulating retinal microvascular endothelial cell (RMVEC) proteinase production was evaluated using isolated murine RMVECs grown in normoxic or hypoxic conditions. Message expression was analyzed by RT-PCR and protein expression by zymographic analysis.


RESULTS
TNFalpha mRNA was increased in the retinas of experimental animals on P13 and P15, during the early stages of retinal neovascularization. In addition to being expressed by Müller glial cells and the inner nuclear layer, additional expression was noted in the outer nuclear layer of experimental animals. No significant level of apoptosis was detected in the retina of experimental animals with retinal neovascularization. Isolated RMVECs did not significantly increase MMP production directly in response to a hypoxic stimulus, but required the presence of exogenous TNFalpha. TNFalpha increased the expression of MT1-MMP, MMP-3, and MMP-9 in these cells. The levels of TACE and p55, proteins important in mediating the response of cells to TNFalpha, were found to be increased by the angiogenic protein, vascular endothelial growth factor (VEGF), which was also elevated in the experimental retinas.


CONCLUSIONS
TNFalpha levels increase in experimental mouse retinas exposed to hypoxic stimuli. Increased production of MMPs by RMVECs does not occur directly in response to a hypoxic stimulus. These cells are responsive, however, to stimulation by TNFalpha, which enhances the production of specific members of the MMP family. VEGF also plays a role in this process through its regulation of TACE and p55 mRNA in the vascular endothelial cells. These findings support the hypothesis that these two growth factors have a role in the regulation of extracellular proteinase expression during retinal neovascularization.
Majka
Susan
S
Division of Ophthalmology, University of New Mexico School of Medicine, Albuquerque, New Mexico 87131, USA.
McGuire
Paul G
PG
Das
Arup
A
D051722:ADAM Proteins; D000072198:ADAM17 Protein; D000818:Animals; D000831:Animals, Newborn; D017209:Apoptosis; D007124:Immunoenzyme Techniques; D020287:In Situ Nick-End Labeling; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism; D020780:Matrix Metalloproteinase 9 / Q000378:metabolism; D020782:Matrix Metalloproteinases / Q000378:metabolism*; D008666:Metalloendopeptidases / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D023421:Models, Animal; D012313:RNA / Q000302:isolation & purification; D012333:RNA, Messenger / Q000378:metabolism; D018124:Receptors, Tumor Necrosis Factor / Q000235:genetics / Q000378:metabolism; D015861:Retinal Neovascularization / Q000201:enzymology* / Q000473:pathology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014409:Tumor Necrosis Factor-alpha / Q000502:physiology*
D012333:RNA, Messenger; D018124:Receptors, Tumor Necrosis Factor; D014409:Tumor Necrosis Factor-alpha; D012313:RNA; D051722:ADAM Proteins; D020782:Matrix Metalloproteinases; D008666:Metalloendopeptidases; D020778:Matrix Metalloproteinase 2; D020780:Matrix Metalloproteinase 9; D000072198:ADAM17 Protein; C000606463:Adam17 protein, mouse