March 2012
Volume 53, Issue 14
Free
ARVO Annual Meeting Abstract  |   March 2012
Inhibition Or Genetic Knockout Of More Than One Cathepsin Reduces Angiogenic Sprout Formation In Vitro And Laser-CNV Formation In Mice
Author Affiliations & Notes
  • Anima D. Buehler
    Ophthalmology, University Eye Hospital Freiburg, Freiburg, Germany
  • Stefanie Berger
    Ophthalmology, University Eye Hospital Freiburg, Freiburg, Germany
  • Fee Werner
    Institute for Molecular Medicine and Cell Research, Faculty of Biology, Albert-Ludwigs-University Freiburg, Freiburg, Germany
  • Gottfried Martin
    Ophthalmology, University Eye Hospital Freiburg, Freiburg, Germany
  • Hansjuergen Agostini
    Ophthalmology, University Eye Hospital Freiburg, Freiburg, Germany
  • Thomas Reinheckel
    Institute for Molecular Medicine and Cell Research, Faculty of Biology, Albert-Ludwigs-University Freiburg, Freiburg, Germany
  • Andreas Stahl
    Ophthalmology, University Eye Hospital Freiburg, Freiburg, Germany
  • Footnotes
    Commercial Relationships  Anima D. Buehler, None; Stefanie Berger, None; Fee Werner, None; Gottfried Martin, None; Hansjuergen Agostini, None; Thomas Reinheckel, None; Andreas Stahl, None
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science March 2012, Vol.53, 3014. doi:
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      Anima D. Buehler, Stefanie Berger, Fee Werner, Gottfried Martin, Hansjuergen Agostini, Thomas Reinheckel, Andreas Stahl; Inhibition Or Genetic Knockout Of More Than One Cathepsin Reduces Angiogenic Sprout Formation In Vitro And Laser-CNV Formation In Mice. Invest. Ophthalmol. Vis. Sci. 2012;53(14):3014.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: : Cathepsins are a family of lysosomal and secreted proteases that are involved in intracellular protein turnover and matrix invasion, respectively. Cathepsin B (Ctsb) and Z (Ctsz) are two cysteine 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 neovascularization (CNV). 10 days after laser treatment, animals were perfused with FITC dextran, the eyes were collected and choroidal flatmounts were 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-cysteine 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 wildtype 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).

Conclusions: : Our results show that 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 in vitro is significantly reduced with broad-spectrum cathepsin inhibition. 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.

Keywords: choroid: neovascularization 
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