Cathepsin L in Bone Marrow-Derived Cells Is Required for Retinal and Choroidal Neovascularization

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,¹ 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 al² 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 neovascularization³ and CNV^4,5,6,7,8 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.⁶ In addition, cells expressing the EPC marker AC133 were identified in the specimens of surgically excised CNVs of human patients.⁹An important property of EPCs is their ability to invade the extracellular matrix.¹⁰ Thus, Bagley and coworkers¹⁰ 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.¹¹ Thus, Urbich and associates¹¹ 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.¹²Anti-VEGF therapy is being used to treat intraocular neovascular disorders, and some improvement of vision has been obtained.^13,14 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.