Cathepsin L in bone marrow-derived cells is required for retinal and choroidal neovascularization

Abstract

Many vision-threatening diseases are characterized by intraocular neovascularization, (e.g., proliferative diabetic retinopathy and age-related macular degeneration). Although a new therapy with anti-VEGF antibodies is being used to treat these intraocular neovascular disorders, the visual recovery is limited, mainly because of the remnants of fibrovascular tissues. The ideal goal of the treatment is to prevent the invasion of new vessels into the avascular tissue through a matrix barrier. The purpose of this study was to determine the role played by cathepsin L, a matrix degrading enzyme, on intraocular angiogenesis. Used established animal models of retinal and choroidal neovascularization, we demonstrated that an inhibition of cathepsin L by specific inhibitors resulted in a significant decrease of intraocular neovascularization. A similar decrease of neovascularization was found in cathepsin L-deficient mice. Transplantation of bone marrow from cathepsin L-deficient mice into wild-type mice significantly reduced the degree of intraocular neovascularization. In addition, immunocytochemical analyses demonstrated that VE cadherin-positive endothelial progenitor cells, but not CD43-positive or Iba-1-positive cells, were the major cells contributing to the production of cathepsin L. These data indicate that cathepsin L expressed in endothelial progenitor cells plays a critical role in intraocular angiogenesis and suggest a potential therapeutic approach of targeting cathepsin L for neovascular ocular diseases.

Figure 1

Effects of periocular injections of Z-FF-FMK, a cathepsin L–specific inhibitor, on a mouse model of retinal neovascularizaion. A–H: Photomicrographs of retinal sections histochemically stained with the endothelial cell–selective lectin from Griffonia simplicifolia. Retinal blood vessels within the retina and neovascularization on the surface of the retinas are stained with the reaction product. Scale bars; 100 μm. A and B: The retina of P17 mice raised in room air (no oxygen exposure). No extraretinal neovascularization is observed. B: Higher magnification image of A. C and D: The retina of P17 mice that were exposed to 75 ± 3% oxygen from P5 to P12 and received daily intraperitoneal injection of DMSO (40 μmol/L) from P12 to P17. Extraretinal neovascularization is observed as brown clumps on the surface of the retina. D: Higher magnification image of C. Arrowheads point to the extraretinal neovascularization. E and F: The retina of P17 mice that were exposed to 75 ± 3% oxygen from P5 to P12 and received daily intraperitoneal injection of Z-FF-FMK (40 μmol/L) from P12 to P17. Extraretinal neovascularization is observed as brown clumps on the surface of the retina. F: Higher magnification image of E. Arrowheads point to the extraretinal neovascularization. G and H: The retina of P17 mice that were exposed to 75 ± 3% oxygen from P5 to P12 and received daily intraperitoneal injection of Z-FF-FMK (400 μmol/L) from P12 to P17. Extraretinal neovascularization appears to be reduced. H: Higher magnification image of G. Arrowheads point to the extraretinal neovascularization. I: Quantification of the total area of endothelial cell staining in retinal sections of mouse with oxygen-induced ischemic retinopathy. Only preretinal vascular cells were counted. Neovascularization in mice that had a periocular injection of Z-FF-FMK (n = 8 both at 40 μmol/L and 400 μmol/L) was significantly reduced compared with mice that had vehicle (DMSO) injection only (n = 8 both at 40 μmol/L and 400 μmol/L). *P < 0.01.

Figure 2

Effects of periocular injections of Z-FF-FMK, a cathepsin L–specific inhibitor, on laser-induced choroidal neovascularization (CNV) in a mouse. A–D: Representative photomicrographs of retinal sections stained with hematoxylin and eosin (HE). Arrowheads show the site of the CNV. Scale bars = 20 μm. A: The retinal section of the mice that received daily periocular injections of DMSO for 14 days after laser irradiation. B: The retinal section of the mice that received daily periocular injections of Z-Phe-Leu-COCHO (800 nmol/L) for 14 days after laser irradiation. C: The retinal section of the mice that received daily periocular injections of Z-FF-FMK (40 μmol/L) for 14 days after laser irradiation. D: The retinal section of the mice that received daily periocular injections of Z-FF-FMK (400 μmol/L) for 14 days after laser irradiation. The CNV is barely detectable at the site of laser injury. E: Quantification of the total volume of CNV at 14 days after laser irradiation of mouse retina. The neovascularizations in mice that had received periocular injections of Z-FF-FMK (n = 9 at 40 μmol/L and n = 8 at 400 μmol/L) are significantly smaller than that of mice that received vehicle (DMSO) injection alone (n = 9 at 40 μmol/L and n = 8 at 400 μmol/L). Injection of cathepsin S–specific inhibitor (Z-Phe-Leu-COCHO; n = 8 both at 80 nmol/L and 800 nmol/L) does not lead to an inhibition of the CNV compared with the mice that received vehicle injection alone. *P < 0.001. F–H: Representative figures of CNV as choroidal flatmounts. Scale bar = 200 μm. F: The vehicle (DMSO)-injected eye. G: The cathepsin S–specific inhibitor (Z-Phe-Leu-COCHO; 800 nmol/L)-injected eye. H: The cathepsin L–specific inhibitor (Z-FF-FMK; 400 μmol/L)-injected eye. I: CNV areas in the choroidal flatmounts were measured in the vehicle (DMSO)-injected eyes (n = 8), cathepsin S–specific inhibitor (Z-Phe-Leu-COCHO; 800 nmol/L)-injected eyes (n = 8), and cathepsin L–specific inhibitor (Z-FF-FMK; 400 μmol/L)-injected eyes (n = 8), and the results of the quantitative analyses are shown in the graph. CNV area is significantly smaller in cathepsin L inhibitor-injected eyes than cathepsin S inhibitor–injected eyes or vehicle-treated eyes. *P < 0.01.

Figure 3

Effects of periocular injections of Z-FF-FMK on the fluorescein dye intensity of laser-induced choroidal neovascularization (CNV). A and B: Representative fundus angiograms at four minutes after fluorescein dye injection. Arrowheads show the site of CNV. A: The vehicle (DMSO)-injected eye. B: The eye that received cathepsin L–specific inhibitor (Z-FF-FMK; 400 μmol/L). C: Quantification of the grades of fluorescein dye intensity of the CNV in mice at 14 days after laser irradiation. The fluorescein intensity of CNV in mice that received periocular injection of Z-FF-FMK (n = 6) was significantly lower than that of mice that received vehicle (DMSO) injection alone (n = 6). *P = 0.03.

Figure 4

Reduced retinal neovascularization in cathepsin L−/− mice with oxygen-induced ischemic retinopathy. A–D: Representative photomicrographs of retinal section histochemically stained with the endothelial cell–selective lectin from Griffonia simplicifolia. Retinal blood vessels within the retina and neovascularization on the surface of the retina are stained positively. Scale bars = 100 μm. A: The retina of P17 wild-type mice that were exposed to 75 ± 3% oxygen from P5 to P12. Extraretinal neovascularization is observed as brown clumps on the surface of the retina. B: Higher magnification image of A. Arrowheads point to the extraretinal neovascularization. C: The retina of P17 cathepsin L−/− mice that were exposed to 75 ± 3% oxygen from P5 to P12. Extraretinal neovascularization is barely observed. D: Higher magnification image of C. E: Quantification of the total area of endothelial cell staining in retinal sections of mice with oxygen-induced ischemic retinopathy. Neovascularization after hyperoxia is significantly reduced in cathepsin L−/− mice (n = 8) compared with that in wild-type mice (n = 8). *P < 0.001.

Figure 5

Reduction of the fluorescein dye intensity of laser-induced choroidal neovascularization (CNV) in cathepsin L−/− mice. A and B: Representative fundus angiogram at four minutes after fluorescein dye injection. A: The fluorescein angiogram of wild-type mouse at 14 days after laser irradiation. Intense dye leakage from the CNV is observed (arrow). B: The fluorescein angiogram of cathepsin L−/− mouse at 14 days after laser irradiation. Dye leakage at the site of laser irradiation is reduced remarkably (arrow). C: Quantification of the grades of fluorescein dye intensity of the CNV in a mouse retina 14 days after laser irradiation. The fluorescein intensity of the CNV in the cathepsin L−/− mice (n = 7) is significantly weaker than that of wild-type mice (n = 7). *P < 0.01.

Figure 6

Inhibition of laser-induced choroidal neovascularization (CNV) by the transplantation of bone marrow cells derived from a cathepsin L−/− mice into a wild-type mouse. A–D: Representative photomicrographs of a retinal section stained with hematoxylin and eosin (H&E). Laser treatment was performed 30 days after bone marrow transplantation. Scale bars = 20 μm. A: The retina of wild-type mouse without bone marrow transplantation at 14 days after laser irradiation. Arrowheads point to the CNV. B: The retina of cathepsin L−/− mouse without bone marrow transplantation at 14 days after laser irradiation. CNV is barely detectable at the laser site. C: The retina of wild-type mouse that received bone marrow transplantation from a wild-type mouse and at 14 days after laser irradiation. Arrowheads point to the CNV. D: The retina of wild-type mouse that received bone marrow transplantation from a cathepsin L−/− mouse and at 14 days after laser irradiation. Neovascularization is barely detectable. E: Quantification of the total volume of CNV in a mouse retina at 14 days after laser irradiation. Neovascularization in wild-type mice that received transplantation of bone marrow cells from cathepsin L−/− mice (n = 7) is significantly reduced compared with the wild-type mice that received a transplantation of bone marrow derived from wild-type mice (n = 8). Neovascularization in wild-type mice that received transplantation of bone marrow derived from cathepsin L−/− mice is comparable with that of cathepsin L−/− mice (n = 8). *P < 0.01. F–I: Representative photographs of CNV in choroidal flatmounts at 14 days after laser irradiation. Arrowheads show the site of the CNV. Scale bars = 100 μm. F: Wild-type mouse without bone marrow transplantation. G: Cathepsin L−/− mouse without bone marrow transplantation. H: Wild-type mouse that received bone marrow transplantation from wild-type mouse. I: Wild-type mouse that received bone marrow transplantation from a cathepsin L−/− mouse. Neovascularization appears smaller than that of the wild-type mouse that received bone marrow transplantation from wild-type mouse. J: CNV areas in the choroidal flatmounts were measured in wild-type mice (n = 7), cathepsin L−/− mice (n = 8), wild-type mice that received transplantation of bone marrow cells from wild-type mice (n = 8), and wild-type mice that received transplantation of bone marrow cells from cathepsin L−/− mice (n = 8). CNV area in wild-type mice that received transplantation of bone marrow cells from cathepsin L−/− mice is significantly reduced compared with the wild-type mice that received a transplantation of bone marrow derived from wild-type mice. *P < 0.01.

Figure 7

Immunohistochemical examination of the expression of cathepsin L and various cell markers at the site of the choroidal neovascularization (CNV) in the mouse retina with laser-induced CNV at 2 days after laser irradiation. Cell nuclei are counterstained with Topro 3. Scale bars = 10 μm A: Immunostaining of cathepsin L (green) and VE cadherin (red). At 2 days after laser irradiation, staining of cathepsin L (green) can be seen at the laser site. The staining of cathepsin L is observed at the corresponding area of VE cadherin-positive cells (red). A higher magnification image confirms the colocalization of cathepsin L–positive cells and VE cadherin–positive cells. B: Immunostaining of cathepsin L (green) and CD43 (red). Only a few CD43-positive cells are observed at the laser irradiation site, and these cells do not show cathepsin L expression. C: Immunostaining of cathepsin L (green) and Iba1 (red). Only a few Iba1-positive cells are observed at the laser irradiation site, and these cells do not show cathepsin L expression.