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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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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).
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.
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).
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.
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