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• W2085021432 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. 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. The eye as an optical instrument must maintain a clear optical pathway. As such, it contains different transparent avascular tissues (e.g., cornea, crystalline lens, vitreous body, and outer retina), but an invasion of blood vessels into the avascular tissue can lead to hemorrhage and exudates, which significantly impairs their transparency and hence vision. In fact, the majority of diseases that lead to vision depression in industrialized countries are disorders that are characterized by intraocular neovascularization, (e.g., proliferative diabetic retinopathy, retinal vein occlusion, retinopathy of prematurity, and age-related macular degeneration). Among these diseases, the proliferative diabetic retinopathy, retinal vein occlusion, and retinopathy of prematurity are characterized by the development of new vessels in the retina that proliferate into the vitreal cavity. Age-related macular degeneration is characterized by the formation of new vessels from the choroidal vessels, which invade the outer layers of the neural retina. This formation of new blood vessels is called a choroidal neovascularization (CNV). For the cells of the new vessels to invade the avascular tissue within the eye, the cells must penetrate a matrix barrier separating the vascular tissue from the avascular tissue. In retinal neovascularization, retinal vascular endothelial cells need to degrade their own basement membrane and also the basement membrane of the Mueller cells forming the internal limiting membrane to migrate and proliferate into the avascular vitreous. In a CNV, the choroidal neovessels need to breach the Bruch membrane, an extracellular matrix composed mainly of elastin and collagen laminae, and grow into the neural retina. Although the alterations of the matrix composing the Bruch membrane have been investigated in detail,1Chong NH Keonin J Luthert PJ Frennesson CI Weingeist DM Wolf RL Mullins RF Hageman GS Decreased thickness and integrity of the macular elastic layer of Bruch's membrane correspond to the distribution of lesions associated with age-related macular degeneration.Am J Pathol. 2005; 166: 241-251Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar the mechanism of the degradation and invasion through the matrix barrier within the eye has not been fully explored. Until recently, it was assumed that the neovascularization develops from the activation, migration, and proliferation of resident endothelial cells. This idea was changed when Asahara et al2Asahara T Murohara T Sullivan A Silver M van der Zee R Li T Witzenbichler B Schatteman G Isner JM Isolation of putative projenitor endothelial cells for angiogenesis.Science. 1997; 275: 964-967Crossref PubMed Scopus (7691) Google Scholar reported that peripheral blood contains a population of bone marrow–derived endothelial progenitor cells (EPCs) that differentiate into endothelial cells at the sites of postnatal vasculogenesis and pathological neovascularization. The results of studies on animal models of retinal neovascularization3Friedlander M Dorrell MI Ritter MR Marchetti V Moreno SK El-Kalay M Bird AC Banin E Aguilar E Progenitor cells and retinal angiogenesis.Angiogenesis. 2007; 10: 89-101Crossref PubMed Scopus (52) Google Scholar and CNV4Csaky KG Baffi JZ Byrnes GA Wolfe JD Hilmer SC Flippin J Cousins SW Recruitment of bone marrow–derived endothelial cells to experimental choroidal neovascularization by local expression of vascular endothelial growth factor.Exp Eye Res. 2004; 78: 1107-1116Crossref PubMed Scopus (72) Google Scholar, 5Espinosa-Heidmann DG Caicedo A Hernandez EP Csaky KG Cousins SW Bone marrow-derived progenitor cells contribute to experimental choroidal neovascularization.Invest Ophthalmol Vis Sci. 2003; 44: 4914-4919Crossref PubMed Scopus (126) Google Scholar, 6Espinosa-Heidmann DG Reinoso MA Pina Y Csaky KG Caicedo A Cousins SW Quantitative enumeration of vascular smooth muscle cells and endothelial cells derived from bone marrow precursors in experimental choroidal neovascularization.Exp Eye Res. 2005; 80: 369-378Crossref PubMed Scopus (76) Google Scholar, 7Sengupta N Caballero S Mames RN Butler JM Scott EW Grant MB The role of adult bone marrow–derived stem cells in choroidal neovascularization.Invest Ophthalmol Vis Sci. 2003; 44: 4908-4913Crossref PubMed Scopus (141) Google Scholar, 8Tomita M Yamada H Adachi Y Cui Y Yamada E Higuchi A Minamino K Suzuki Y Matsumura M Ikehara S Choroidal neovascularization is provided by bone marrow cells.Stem Cells. 2004; 22: 21-26Crossref PubMed Scopus (50) Google Scholar have provided evidence that EPCs may be major contributors to intraocular angiogenic disorders. For example, experiments on laser-induced CNV in chimeric mice (viz., C57BL/6 mice with bone marrow transplantation from green fluorescent protein [GFP]-transgenic mice) showed that 50% to 60% of the endothelial cells of a CNV were GFP-positive.6Espinosa-Heidmann DG Reinoso MA Pina Y Csaky KG Caicedo A Cousins SW Quantitative enumeration of vascular smooth muscle cells and endothelial cells derived from bone marrow precursors in experimental choroidal neovascularization.Exp Eye Res. 2005; 80: 369-378Crossref PubMed Scopus (76) Google Scholar In addition, cells expressing the EPC marker AC133 were identified in the specimens of surgically excised CNVs of human patients.9Sheridan CM Rice D Hiscott PS Wong D Kent DL The presence of AC133-positive cells suggests a possible role of endothelial progenitor cells in the formation of choroidal neovascularization.Invest Ophthalmol Vis Sci. 2006; 47: 1642-1645Crossref PubMed Scopus (52) Google Scholar An important property of EPCs is their ability to invade the extracellular matrix.10Bagley RG Walter-Yohrling J Cao X Weber W Simons B Cook BP Chartrand SD Wang C Madden SL Teicher BA Endothelial precursor cells as a model of tumor endothelium: characterization and comparison with mature endothelial cells.Cancer Res. 2003; 63: 5866-5873PubMed Google Scholar Thus, Bagley and coworkers10Bagley RG Walter-Yohrling J Cao X Weber W Simons B Cook BP Chartrand SD Wang C Madden SL Teicher BA Endothelial precursor cells as a model of tumor endothelium: characterization and comparison with mature endothelial cells.Cancer Res. 2003; 63: 5866-5873PubMed Google Scholar studied AC133+/CD34+ bone marrow progenitor cells in a coculture assay using human SKOV3 ovarian cancer cell clusters in collagen as a stimulus for the invasion of EPCs. They showed that EPCs were able to invade the malignant cell cluster through a matrigel barrier, whereas human microvascular endothelial cells were not able to invade the malignant cell cluster. These results suggested that the EPCs have a greater proliferative and invasive capacity than mature vascular endothelial cells. It has recently been shown that the major factor responsible for the greater angiogenic activity of EPCs was their high expression of cathepsin L.11Urbich C Heeschen C Aicher A Sasaki K Bruhl T Farhadi MR Vajkoczy P Hofmann WK Peters C Pennacchio LA Abolmaali ND Chavakis E Reinheckel T Zeiher AM Dimmeler S Cathepsin L is required for endothelial progenitor cell-induced neovascularization.Nat Med. 2005; 11: 206-213Crossref PubMed Scopus (265) Google Scholar Thus, Urbich and associates11Urbich C Heeschen C Aicher A Sasaki K Bruhl T Farhadi MR Vajkoczy P Hofmann WK Peters C Pennacchio LA Abolmaali ND Chavakis E Reinheckel T Zeiher AM Dimmeler S Cathepsin L is required for endothelial progenitor cell-induced neovascularization.Nat Med. 2005; 11: 206-213Crossref PubMed Scopus (265) Google Scholar demonstrated that the protease cathepsin L was essential for the degradation and invasion of the matrix in vitro by EPCs using a mouse hind limb ischemia model. They concluded that cathepsin L plays a critical role in the EPC-mediated neovascularization. The cathepsins include the catalytic classes of serine, asaparate, and cysteine peptidases exhibiting endo- or exopeptidase activities.12Turk V Turk B Turk D Lysosomal cysteine proteases: facts and opportunities.EMBO J. 2001; 20: 4629-4633Crossref PubMed Scopus (633) Google Scholar Anti-VEGF therapy is being used to treat intraocular neovascular disorders, and some improvement of vision has been obtained.13Ciulla TA Rosenfels PJ Antivascular endothelial growth factor therapy for neovascular age-related macular degeneration.Curr Opin Ophthalmol. 2009; 20: 158-163Crossref PubMed Scopus (111) Google Scholar, 14Ciulla TA Rosenfeld PJ Anti-vascular endothelial growth factor therapy for neovascular ocular diseases other than age-related macular degeneration.Curr Opin Ophthalmol. 2009; 20: 166-174Crossref PubMed Scopus (83) Google Scholar Nevertheless, it is still difficult to regain a complete visual recovery because of the remnants of fibrovascular scar tissue and the concomitant damage of the neural retina. Therefore, a goal of an ideal treatment against intraocular neovascular disorders is to prevent the development and progression of new vessels into the avascular tissue. Although the critical roles of cathepsin L and EPCs have been demonstrated in the angiogenesis in other organs, a PubMed search did not identify any studies investigating the role of cathepsin L in ocular angiogenesis. Thus, the purpose of this study was to investigate the role played by cathepsin L in ocular neovascularization. To accomplish this, we used established animal models of retinal and choroidal neovascularization. We shall show that an inhibition of cathepsin L by specific inhibitors resulted in a significant decrease in the size of the intraocular neovascularization. Similar findings were made in cathepsin L gene–deficient mice (cathepsin L−/− mice). Immunocytochemical analyses demonstrated that VE cadherin–positive cells, highly likely EPCs, were the major cells that express cathepsin L. Cathepsin L inhibtor (Z-FF-FMK) and cathepsin S inhibitor (Z-Phe-Leu-COCHO) were obtained from Calbiochem (Darmstadt, Germany); polyclonal antiserum against mouse VE cadherin from R&D Systems (Minneapolis, MN) and from Abcam (Cambridge, MA); polyclonal antiserum against mouse CD43 from Becton Dickinson (Franklin Lakes, NJ); polyclonal antiserum against mouse Iba1 from Wako (Osaka, Japan); polyclonal antiserum against mouse cathepsin L from R&D Systems (Minneapolis, MN); biotinylated Griffonia simplicifolia lectin B4 (GSA) from Vector Laboratories (Burlingame, CA); streproavidin-phosphatase from Dako (Glostrup, Denmark); FITC-conjugated secondary antibody from Invitrogen (Carlsbad, CA); PE-conjugated secondary antibody from Invitrogen (Carlsbad, CA); and Topro 3 from Invitrogen (Carlsbad, CA). All animals were treated in accordance with the principles described in the Guiding Principles in the Care and Use of Animals in Tokyo Medical and Dental University, and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. The research protocol was approved by the Ethics Committee for Animal Experimentation at Tokyo Medical and Dental University. C57BL/6 mice were obtained from CLEA Japan Inc. (Tokyo, Japan) and housed in our institutional animal care facilities. C57BL/6 mice with targeted disruption of the cathepsin L gene, cathepsin L−/− mice, were generated as described.15Koike M Shibata M Waguri S Yoshimura K Tanida I Kominami E Gotow T Peters C von Figura K Mizushima N Saftig P Uchiyama Y Participation of autophagy in storage of lysosomes in neurons from mouse models of neuronal ceroid-lipofuscinoses (Batten disease).Am J Pathol. 2005; 167: 1713-1728Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar Genotyping to verify the absence of the cathepsin L gene of the targeting vector was accomplished by polymerase chain reaction of the DNA from tail biopsy specimens. Ischemic retinopathy was produced in C57BL/6 mice or cathepsin L−/− mice by the method described by Smith et al.16Smith LEH Wesolowski E McLellan A Kostyk SK D'Amato R Sullivan R D'amore PA Oxygen-induced retinopathy in the mouse.Invest Ophthalmol Vis Sci. 1994; 35: 101-111PubMed Google Scholar On postnatal day (P) 7, pups and their mothers were placed in airtight incubators and exposed to an atmosphere of 75 ± 3% oxygen for 5 days. Oxygen was continuously monitored with a PROOX model 110 oxygen controller (Reming Bioinstruments, Redfield, NY). On P12, the mice were returned to room air. On P17, the pups were sacrificed with an overdose of pentobarbital sodium, and their eyes were rapidly removed and frozen in optimal cutting temperature embedding compound (Miles, Elkhart, IN) and 10-μm-thick sections were cut. To investigate the effect of specific pharmacological inhibition of cathepsin L, cathepsin L inhibitor (Z-FF-FMK) and cathepsin S inhibitor (Z-Phe-Leu-COCHO) were used. Periocular injections of 1 μl of the inhibitor were performed using a microinjector (CellTram, Eppendorf, Germany). Two independent experiments were performed to investigate the effect of periocular injections of cathepsin inhibitors. In the first experiment, mice received daily periocular injections of 40 μmol/L (8 mice) or 400 μmol/L (8 mice) of Z-FF-FMK and vehicle of the same concentration (DMSO) in the fellow eye from P12 to P17. In the second set of experiments, mice received daily periocular injections of 80 nmol/L (10 mice) or 800 nmol/L (10 mice) of Z-Phe-Leu-COCHO and vehicle in the fellow eye from P12 to P17. Retinal flatmounts were prepared by a modification of a described technique.17Ohno-Matsui K Uetama T Yoshida T Hayano M Itoh T Morita I Mochizuki M Reduced retinal angiogenesis in MMP-2-deficient mice.Invest Ophthalmol Vis Sci. 2003; 44: 5370-5375Crossref PubMed Scopus (79) Google Scholar After the mice were anesthetized, the descending aorta was clamped, the right atrium was cut, and the animal was perfused through the right ventricle with 1 ml phosphate buffered saline (PBS) containing 50 mg/ml fluorescein-labeled dextran (2 × 106 average MW; Sigma, St. Louis, MO). The eyes were removed and fixed for 1 hour in 10% phosphate-buffered formalin. The cornea and lens were removed, and the entire retina was then carefully dissected from the eye cup. Then radial cuts were made from the edge of the retina to the equator in all four quadrants. The retinas were flatmounted on a microscope slide in an aqueous medium (Aquamount; BDH, Poole, UK) with the photoreceptors facing downward. Flatmounts were carefully examined by fluorescence microscopy (BX51; Olympus), and images were taken with a CCD camera and imported to a computer system. The Bruch membrane was ruptured by laser photocoagulation to generate a CNV.18Tobe T Ortega S Luna JD Ozaki H Okamoto N Derevjanik NL Vinores SA Basilico C Campochiaro PA Targeted disruption of the FGF2 gene does not prevent choroidal neovascularization in a murine model.Am J Pathol. 1998; 153: 1641-1646Abstract Full Text Full Text PDF PubMed Scopus (301) Google Scholar Briefly, 4- to 5-week-old C57BL/6J mice or cathepsin L−/− mice were anesthetized with ketamine hydrochloride (100 mg/kg body weight), and the pupils were dilated with 1% tropicamide (Santen, Tokyo, Japan). Two diode laser photocoagulation spots (100-μm size, 0.1-second duration, 120 mW) were made on each retina with a slit-lamp delivery system (Ultima 2000SE, Coherent, Santa Clara, CA) and a handheld coverslip as a contact lens. Burns were made at the 11 to 1 and 5 to 7 o'clock positions around the optic nerve head. A production of a bubble at the time of laser irradiation, which indicated a rupture of the Bruch membrane, was an important end point that induced a CNV. Thus, only burns in which a bubble was produced were analyzed. Fourteen days after the laser irradiation, fluorescein angiography was performed using an intraperitoneal injection of 0.2 ml of 2.5% sodium fluorescein (Alcon Laboratories, Inc., Fort Worth, TX). Fourteen days after the laser irradiation, the mice were sacrificed by transcardiac perfusion of 5 ml 4% paraformaldehyde in PBS. The eyes were enucleated, fixed in paraformaldehyde, embedded in paraffin, and cut into 5-μm sections. Three independent experiments were performed to investigate the effect of periocular injections of cathepsin inhibitors. In the first experiment, mice received periocular injections of 40 μmol/L (8 mice) or 400 μmol/L (8 mice) of Z-FF-FMK, and vehicle in the fellow every two days for 2 weeks after the laser irradiation. In the second experiment, mice received 3 μl periocular injections of 80 nmol/L (10 mice) or 800 nmol/L (10 mice) of Z-Phe-Leu-COCHO, and vehicle in the fellow eye every two days for 2 weeks after the laser irradiation. And in the third experiment, eight mice with laser-induced CNV and without injections were analyzed as non-treated controls. The size of CNV lesions was measured in choroidal flatmounts. Mice were anesthetized and perfused with 1 ml PBS containing 50 mg/ml fluorescein-labeled dextran (Sigma, St. Louis, MO). After the eyes were enucleated and briefly fixed in 4% PFA, the anterior segment was removed and the retinas were carefully dissected from the eyecup. Four radial cuts were made from the edge to the equator, and the eyecup was flatmounted with the photoreceptors facing downward. The flatmounts were examined by fluorescence microscopy (BX51; Olympus) at ×40 magnification. Photographs of the retina were taken with a CCD camera and imported to a computer system. Four to six minutes after injecting fluorescein into the intraperitoneal cavity, fluorescein angiograms were taken with a fundus camera (TRC-50IX; Topcon, Tokyo, Japan) with a built-in filter for fluorescein. The images were downloaded into a computer with a Windows operating system using a 3-color CCD color video camera (640 × 480 pixels; DXC-970MD; Sony, Tokyo, Japan). The intensity of the fluorescein staining of the CNV was determined as reported by Takehana and associates.19Takehana Y Kurokawa T Kitamura T Tsukahara Y Akahane S Kitazawa M Yoshimura N Suppression of laser-induced choroidal neovascularization by oral tranilast in the rat.Invest Ophthalmol Vis Sci. 1999; 40: 459-466PubMed Google Scholar Briefly, late-phase angiograms taken 100 to 140 seconds after the dye injection were graded by two examiners in a masked fashion. The angiograms were graded as: score 0, no staining; score 1, slightly stained; score 2, moderately stained; and score 3, strongly stained. The differences in the scores were evaluated by Wilcoxon signed rank tests. Serial sections (10 μm) were cut through the entire eye, and the sections were histochemically stained with biotinylated GSA B4 as described.20Marshall J Hussain A Starita C Moore D Patmore A Aging and Bruch's membrane.in: Marmor M Wolfensberger T The Retinal Pigment Epithelium. Oxford University Press, New York1988: 669-692Google Scholar GSAB4 binds selectively to vascular cells. Slides were incubated in 4% paraformaldehyde for 30 minutes, washed with 0.05 M Tris buffer (TB; pH 7.4), incubated in methanol-H2O2 for 10 minutes at 4°C, washed with 0.05 M TB, and incubated for 30 minutes in 10% normal swine serum. Slides were rinsed with 0.05 M TB, and incubated 2 hours at 37°C with 1:20 GSA, rinsed again with 0.05 M TB, and incubated with undiluted streptoavidin-phosphatase for 30 minutes at room temperature. After a 10-minute wash in 0.05 M TB (pH 7.6), the slides were developed with diaminobenzidine (Dako). For quantification, lectin-stained sections were examined at ×400 magnification with a model Q600 HR Leica microscope (Heidelberg, Germany). The images were digitized with a three-color CCD video camera and a frame grabber. The accompanying software (Quantimet; Leica) was used to delineate lectin-stained cells, and the area of neovascularization was measured. The area of the neovascularization was set to the area covered by the proliferated retinal pigmented epithelium, and the volume of CNV was determined with volume-calculating software (TRY/3D-SUFII, RATOC System Engineering Co Ltd, Tokyo, Japan). Mann–Whitney U tests were used to determine whether significant differences existed between experimental mean values. A P < 0.05 was considered significant. All statistical analyses were done using StatView software (SAS Institute, Cary, NC). The area of CNV in the choroidal flatmounts was outlined with a cursor moved by the computer mouse, and the area was determined with the public domain NIH image program (developed at the NIH and available on the Internet at http://rsb.info.nih.gov/nih-image/). Image analysis was performed with the observer masked as to treatment or the types of mice used for the experiments. To determine the contribution of cathepsin L to the development of a CNV, bone marrow cells that contain EPCs that synthesize cathepsin L were transplanted into wild-type mice. The bone marrow cells were obtained from three cathepsin L −/− mice. To isolate the bone marrow cells, the femur and tibia were dissected and placed in RPMI 1640 culture medium (Invitrogen-Gibco, Grand Island, NY) containing 2.5% HEPES (1 M) and 1% gentamicin at 4°C. Bone marrow was obtained by slowly flushing the medium inside a diaphyseal channel with a syringe through a 27-gauge needle. The bone marrow was homogenized by passing the fluid through an 18-gauge needle and filtered through a nylon filter (70 μm; Spectrum, Houston, TX). The fluid was centrifuged and the pellets were resuspended in a medium described earlier. Recipient mice (C57BL/6J) were lethally irradiated (950 cGy) and given 107 nonpurified bone marrow cells intravenously (200 μl). The blood components were allowed to reconstitute for 1 month. The survival rate of mice transplanted with exogenous bone marrow was 100%. In contrast, irradiated mice without exogenous bone marrow died approximately 10 to 14 days after the irradiation. One month after the bone marrow transplantation, CNVs were induced in recipient mice by making four separate choroidal burns in each eye with a red diode laser. Two weeks later, the animals were sacrificed and the eyes removed for histological examinations. Preliminary experiments using GFP transgenic mice, transgenic for the chicken β-actin promoter-GFP and cytomegalovirus enhancer (stock no. 003291; The Jackson Laboratory, Bar Harbor, ME), revealed that the chimerism of this bone marrow with this transplantation protocol was more than 90% (data not shown). To determine the contribution of cathepsin L to the development of a CNV and to identify the cells that expressed cathepsin L in the process of CNV development, mice were sacrificed at 2 and 6 days after laser irradiation of the retina with an overdose of pentobarbital sodium. The eyes were enucleated, fixed in 4% paraformaldehyde, embedded in optimal cutting temperature embedding compound, and cut into 10-μm-thick frozen sections. The sections were incubated overnight in a humidified chamber at room temperature with rabbit polyclonal antibody against mouse cathepsin L (dilution 1:10; 10 μg/ml, R&D Systems, MN), or with VE-cadherin, which selectively binds to endothelial cells and mononuclear cells including monocytes and macrophages (dilution 1:50, BD Biosciences, NJ), or with CD43, which selectively binds to monocytes, granulocytes, and lymphocytes, (dilution 1:50, BD Biosciences, NJ), or with Iba1, which selectively binds to microglia/macrophages (dilution 1:50; 2 μg/ml, Wako, Osaka, Japan). Immunoreactive cells were identified by incubating the sections in FITC-conjugated secondary antibody or PE-conjugated secondary antibody. The nuclei were counterstained with Topro3 for fluorescence staining, and the sections were observed with a confocal optical microscopy (Carl Zeiss Inc., Germany). Daily periocular injection of 40 μmol/L or 400 μmol/L of Z-FF-FMK, a specific cathepsin L inhibitor, from P12 to P17 was well-tolerated and caused a statistically significant reduction in the area of the retinal neovascularization from 619.5 ± 48.0 μm2 with 40 μmol/L DMSO to 307.1 ± 108.0 μm2 with 40 μmol/L Z-FF-FMK, and from 578.4 ± 263.7 μm2 with 400 μmol/L DMSO to 150.4 ± 93.7 μm2 with 400 μmol/L Z-FF-FMK (Figure 1, A–I). The volume of the CNV in eyes that received periocular injections of 40 μmol/L or 400 μmol/L of Z-FF-FMK were 4.34 × 105 ± 3.37 × 105 μm3 and 2.38 × 105 ± 1.70 × 105 μm3, respectively, which were significantly smaller than the 10.65 × 105 ± 3.90 × 105 μm3 with 40 μmol/L DMSO and 11.29 × 105 ± 3.90 × 105 μm3 with 400 μmol/L DMSO in eyes that received periocular injections of vehicle. The volumes of the CNV in eyes that received daily periocular injections of Z-Phe-Leu-COCHO, a specific inhibitor of cathepsin S, were 10.23 × 105 ± 3.33 × 105 μm3 with 80 nmol/L and 10.66 × 105 ± 4.06 × 105 μm3 with 800 nmol/L on day 14 after the laser irradiation. These volumes were not significantly different from that in the vehicle injected eyes (Figure 2, A–E). Choroidal flatmount analyses was used to quantify the CNV area, and the results demonstrated that the CNV area was smaller in the mice treated with 400 μmol/L Z-FF FMK (5.28 × 103 ± 1.63 × 103 μm2) than in the mice treated with 800 nmol/L Z-Phe-Leu-COCHO (9.12 × 103 ± 2.82 × 103 μm2) or DMSO-treated controls (8.64 × 103 ± 2.18 × 103 μm2; Figure 2, F–I). The intensity of the CNV in the fluorescein angiograms was significantly weaker in eyes that received Z-FF-FMK (1.17 ± 0.75) than in eyes that received vehicle injections (2.50 ± 0.84) (Figure 3, A–C). To determine the contribution of cathepsin L to the development of retinal neovascularization and CNV, the degree of ocular neovascularization in cathepsin L−/− mice was compared with that in wild-type mice. The results demonstrated that cathepsin L−/− mice had significantly smaller areas of retinal neovascularization: 130.9 ± 88.3 μm2 in cathepsin L−/− and 599.2 ± 226.1 μm2 in wild-type mice (Figure 4, A–E). No differences were detected in the development of retinal vasculature in P12 wild-type mice without any treatment, in P12 wild-type that had received periocular injections of 400 μmol/L of Z-FF-FMK, and in P12 cathepsin L−/− mice (see supplemental Figure S1 at http://ajp.amjpathol.org). In addition, the intensity of the CNV in the fluorescein angiograms was significantly weaker in eyes of cathepsin L−/− mice (0.33 ± 0.52) than in the eyes of wild-type mice (2.57 ± 0.79; Figure 5, A–C). The volume of the CNV in cathepsin L−/− mice (1.86 × 105 ± 1.68 × 105 μm3) was significantly smaller than that in wild-type mice (10.14 × 105 ± 4.14 × 105 μm3; Figure 6, A, B, and E).Figure 5Reduction 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−/− mo" @default.
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• W2085021432 date "2010-05-01" @default.
• W2085021432 modified "2023-09-27" @default.
• W2085021432 title "Cathepsin L in Bone Marrow-Derived Cells Is Required for Retinal and Choroidal Neovascularization" @default.
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• W2085021432 doi "https://doi.org/10.2353/ajpath.2010.091027" @default.
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