Abnormal vessel formation in the choroid of mice lacking tissue inhibitor of metalloprotease-3.
OBJECTIVE
Tissue inhibitor of metalloprotease (TIMP)-3 is an inhibitor of matrix metalloprotease (MMP) and regulates angiogenesis. In the eye, TIMP3 is tightly associated with Bruch's membrane. In this study, the authors analyzed mice lacking TIMP3 for retinal abnormalities.


METHODS
Mice with targeted disruption of the Timp3 gene were generated (Timp3(-/-)) and bred into C57/Bl6 and CD1 backgrounds. Eyes were analyzed by light and electron microscopy. Vasculature was examined by scanning laser ophthalmoscopy, corrosion casts, and whole mount preparations. MMP activity was assessed by in situ zymography, angiogenic potential was evaluated by tube formation, and aortic ring assays and signaling pathways were studied by immunoblotting.


RESULTS
TIMP3-deficient mice develop abnormal vessels with dilated capillaries throughout the choroid. Enhanced MMP activity in the choroid region of Timp3(-/-) eyes was detected when compared with controls. Timp3(-/-)-derived tissue showed an increased angiogenic activity over wild-type, an effect that could specifically be inhibited by recombinant TIMP3. Moreover, the antiangiogenic property of TIMP3 was demonstrated to reside within the C-terminal domain. When VEGFR2 inhibitor was added to Timp3(-/-) aortic explants, endothelial sprout formation was markedly reduced, which provided evidence for an unbalanced VEGF-mediated angiogenesis in Timp3(-/-) animals. Finally, angiogenic signaling pathways are activated in Timp3(-/-)-derived cells.


CONCLUSIONS
These findings suggest that the distinct choroidal phenotype in mice lacking TIMP3 may be the result of a local disruption of extracellular matrix and angiogenic homeostasis, and they support an important role of TIMP3 in the regulation of choroidal vascularization.
Janssen
Andreas
A
Institute of Human Genetics, University of Regensburg, Regensburg, Germany.
Hoellenriegel
Julia
J
Fogarasi
Marton
M
Schrewe
Heinrich
H
Seeliger
Mathias
M
Tamm
Ernst
E
Ohlmann
Andreas
A
May
Christian Albrecht
CA
Weber
Bernhard H F
BH
Stöhr
Heidi
H
D000818:Animals; D001013:Aorta, Thoracic / Q000187:drug effects / Q000503:physiopathology; D001808:Blood Vessels / Q000002:abnormalities / Q000473:pathology; D016570:Bruch Membrane / Q000473:pathology; D002196:Capillaries / Q000254:growth & development; D002829:Choroid / Q000098:blood supply* / Q000201:enzymology; D005123:Eye / Q000378:metabolism; D066298:In Vitro Techniques; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism; D020780:Matrix Metalloproteinase 9 / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D009389:Neovascularization, Pathologic / Q000209:etiology; D018919:Neovascularization, Physiologic / Q000187:drug effects / Q000502:physiology*; D010641:Phenotype; D010857:Pigment Epithelium of Eye / Q000473:pathology; D017434:Protein Structure, Tertiary / Q000502:physiology; D011994:Recombinant Proteins / Q000494:pharmacology; D012160:Retina / Q000473:pathology; D015398:Signal Transduction; D019717:Tissue Inhibitor of Metalloproteinase-3 / Q000172:deficiency / Q000235:genetics / Q000494:pharmacology / Q000502:physiology*; D042461:Vascular Endothelial Growth Factor A / Q000494:pharmacology; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000037:antagonists & inhibitors
D011994:Recombinant Proteins; D019717:Tissue Inhibitor of Metalloproteinase-3; D042461:Vascular Endothelial Growth Factor A; D040301:Vascular Endothelial Growth Factor Receptor-2; D020778:Matrix Metalloproteinase 2; D020780:Matrix Metalloproteinase 9