gms | German Medical Science

25th Annual Meeting of the German Retina Society

German Retina Society

01.06. - 02.06.2012, Münster

Inhibition or genetic knockout of more than one cathepsin reduces angiogenic sprout formation in vitro and Laser-CNV formation in mice

Meeting Abstract

  • Anima Bühler - Universitäts-Augenklinik Freiburg i. B.
  • S. Berger - Universitäts-Augenklinik Freiburg i. B.
  • F. Werner - Institut für molekulare Medizin und Zellforschung der Universität Freiburg
  • G. Martin - Universitäts-Augenklinik Freiburg i. B.
  • H.T. Agostini - Universitäts-Augenklinik Freiburg i. B.
  • T. Reinheckel - Institut für molekulare Medizin und Zellforschung der Universität Freiburg
  • A. Stahl - Universitäts-Augenklinik Freiburg i. B.

German Retina Society. 25th Annual Conference of the German Retina Society. Münster, 01.-02.06.2012. Düsseldorf: German Medical Science GMS Publishing House; 2012. Doc12rg06

doi: 10.3205/12rg06, urn:nbn:de:0183-12rg064

This is the translated version of the article.
The original version can be found at: http://www.egms.de/de/meetings/rg2012/12rg06.shtml

Published: May 30, 2012

© 2012 Bühler et al.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en). You are free: to Share – to copy, distribute and transmit the work, provided the original author and source are credited.


Outline

Text

Purpose: Cathepsins are a family of lysosomal and secreted proteases that are involved in

intracellular protein turnover and matrix invasion. Cathepsin B (Ctsb) and Z (Ctsz) are two cystein cathepsin family members with possible roles in angiogenesis. We therefore investigated the angiomodulatory function of these cathepsins in vitro as well as in a mouse model of laser-induced choroidal neovascularization (Laser-CNV).

Methods: Ctsb-/- mice, Ctsz-/- mice, Ctsb/Ctsz double knockouts (Ctsb/z DKO) and C57BL/6 wildtype controls underwent argon laser treatment to induce choroidal neovascularisation (CNV). 10 days after laser treatment, animals were perfused with FITC dextran, the eyes were collected and choroidal flatmounts prepared. The CNV area was quantified individually for each lesion. For in vitro analysis, endothelial cell (EC) sprouting was analyzed using a spheroidal sprouting assay in collagen matrix under VEGF stimulation. A pan cathepsin inhibitor was added to the matrix. VEGF 165 stimulation alone served as positive control.

Results: In the Laser CNV model, neither Ctsb-/- nor Ctsz-/- mice show a significant difference in CNV area compared to wildtyp controls (p=0.7 and p=0.1 respectively). Ctsb/z DKO, however display a significantly reduced area of CNV formation compared to wild type controls (p=0.001). Similarly, VEGF-induced EC spheroid sprouting in vitro was significantly suppressed using the cell-permeable broad-spectrum cysteine cathepsin inhibitor E64d (p=0.0001).

Conclusion: Our results show that the knockout of either Ctsb or Ctsz alone does not significantly alter Laser-induced CNV formation in mice. However, Laser-CNV formation is significantly reduced in Ctsb/z DKO mice suggesting a compensatory mechanism between different members of the cathepsin family. In accordance EC sprout formation is significantly reduced with broad-spectrum cathepsin inhibitor. The EC spheroid assay also gives a strong hint that the anti-angiogenic effect of cathepsin inhibition is mediated directly via an endothelial mechanism without the involvement of other cell types.