Tissue inhibitor of metalloproteases-3 (TIMP3) deficiency leads to abnormal vessel formation in the choroid
Andreas Janssen 1,
Julia Hoellenriegel 1,
Marton Fogarasi 1,
Heinrich Schrewe 2,
Mathias Seeliger 3,
Ernst R. Tamm 4,
Andreas Ohlmann 4,
Chr. Albrecht May 5,
Bernhard HF Weber 6,
and
Heidi B. Stoehr 1*
1 Human Genetics, University of Regensburg, Regensburg, Germany
2 Max-Planck Institute for Molecular Genetics, Berlin, Germany
3 Department of Pathophysiology of Vision and Neuroophthalmology, University of Tubingen, Tubingen, Germany
4 Institute of Human Anatomy and Embryology, University Regensburg, Regensburg, Germany
5 Anatomy, Carl Gustav Carus Medical Faculty, TU Dresden, Dresden, Germany
6 Human Genetics, University Regensburg, Regensburg, Germany
* To whom correspondence should be addressed. E-mail: heidi.stoehr{at}klinik.uni-regensburg.de.
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Abstract |
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Purpose. Tissue inhibitor of metalloproteases-3 (TIMP3) is an inhibitor of matrix metalloproteases (MMPs) and regulates angiogenesis. In the eye, TIMP3 is tightly associated with Bruch's membrane. In this study we analyzed mice lacking TIMP3 for retinal abnormalities.
Methods. Mice with a 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 signalling 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 to controls. Timp3-/--derived tissue showed an increased angiogenic activity over wild-type, an effect that could specifically be inhibited by recombinant TIMP3. Moreover, the anti-angiogenic 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 provides evidence for an unbalanced VEGF-mediated angiogenesis in Timp3-/- animals. Finally, angiogenic signalling pathways are activated in Timp3-/--derived cells.
Conclusions. Our findings suggest that the distinct choroidal phenotype in mice lacking TIMP3 may be the result of a local disruption of both ECM and angiogenic homeostasis and supports an important role of TIMP3 in the regulation of choroidal vascularisation.
Key Words:
choroid, metalloproteinases, angiogenesis, tissue inhibitor of metalloproteinases-3, vascular endothelial growth factor
