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.     

EML4-ALK mutations in lung cancer that confer resistance to ALK inhibitors

The EML4 (echinoderm microtubule-associated protein-like 4)-ALK (anaplastic lymphoma kinase) fusion-type tyrosine kinase is an oncoprotein found in 4 to 5% of non-small-cell lung cancers, and clinical trials of specific inhibitors of ALK for the treatment of such tumors are currently under way. Here, we report the discovery of two secondary mutations within the kinase domain of EML4-ALK in tumor cells isolated from a patient during the relapse phase of treatment with an ALK inhibitor. Each mutation developed independently in subclones of the tumor and conferred marked resistance to two different ALK inhibitors. (Funded by the Ministry of Health, Labor, and Welfare of Japan, and others.).     

Inhibitory role of CD19 in the progression of experimental autoimmune encephalomyelitis by regulating cytokine response

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory demyelinating disease of the central nerve system that is considered a T helper type 1 (Th1)-mediated autoimmune disease. EAE currently serves as an experimental animal model for multiple sclerosis in human. Cytokines, such as interferon-gamma and interleukin-10, play a key role in the development and remission of EAE. Recent studies have also shown a role for B cells in the pathogenesis of EAE. Therefore, we examined the role of CD19, a B cell-specific surface molecule that defines signaling thresholds critical for B-cell responses and autoimmunity, on the development of EAE. Following immunization with myelin oligodendrocyte glycoprotein (MOG) peptide, CD19-deficient (CD19(-/-)) mice exhibited higher clinical and pathological severity scores of EAE than wild-type mice. The increased severity of EAE in CD19(-/-) mice was associated with polarized Th1 cytokines in the inflamed central nerve system but not with anti-MOG antibodies in the serum. MOG-primed CD19(-/-) B cells produced high levels of interferon-gamma, and transfer of MOG-primed CD19(-/-) B cells to wild-type mice worsened the disease. Thus, CD19 modulates the Th1/Th2 cytokine balance in B cells and plays a critical role as a suppressive molecule in the development of EAE.     

Oncogenic MAP2K1 mutations in human epithelial tumors

The scirrhous subtype of gastric cancer is a highly infiltrative tumor with a poor outcome. To identify a transforming gene in this intractable disorder, we constructed a retroviral complementary DNA (cDNA) expression library from a cell line (OCUM-1) of scirrhous gastric cancer. A focus formation assay with the library and mouse 3T3 fibroblasts led to the discovery of a transforming cDNA, encoding for MAP2K1 with a glutamine-to-proline substitution at amino acid position 56. Interestingly, treatment with a MAP2K1-specific inhibitor clearly induced cell death of OCUM-1 but not of other two cells lines of scirrhous gastric cancer that do not carry MAP2K1 mutations, revealing the essential role of MAP2K1(Q56P) in the transformation mechanism of OCUM-1 cells. By using a next-generation sequencer, we further conducted deep sequencing of the MAP2K1 cDNA among 171 human cancer specimens or cell lines, resulting in the identification of one known (D67N) and four novel (R47Q, R49L, I204T and P306H) mutations within MAP2K1. The latter four changes were further shown to confer transforming potential to MAP2K1. In our experiments, a total of six (3.5%) activating mutations in MAP2K1 were thus identified among 172 of specimens or cell lines for human epithelial tumors. Given the addiction of cancer cells to the elevated MAP2K1 activity for proliferation, human cancers with such MAP2K1 mutations are suitable targets for the treatment with MAP2K1 inhibitors.     

Photodynamic therapy as salvage treatment for local failure after chemoradiotherapy in patients with esophageal squamous cell carcinoma: a phase II study

Local failure at the primary site is a major problem after chemoradiotherapy (CRT) in patients with esophageal squamous cell carcinoma (ESCC). Salvage surgery is the only treatment option with curative intent, but it is associated with high morbidity and mortality. The aim of this study was to evaluate the efficacy and safety of salvage photodynamic therapy (PDT) after CRT. Patients with histologically proven local failure limited to the submucosal layer, and without any metastasis after definitive CRT (≥50 Gy) for ESCC were enrolled in the study. PDT began with intravenous administration of 2 mg/kg of porfimer sodium followed 48-72 hr later by excimer dye laser irradiation with a fluence of 75 J/cm2. The primary endpoint was a complete response (CR) to treatment with PDT, and the secondary endpoints were toxicity related to PDT, progression-free survival (PFS) and overall survival (OS). Twenty-five patients were enrolled in the study. A CR was attained in 19 of 25 patients treated with PDT (CR rate, 76%; 95% CI, 55-91%). One treatment-related death (4%) caused by gastrointestinal hemorrhage at the irradiated site occurred 33 days after PDT. No adverse events greater than grade 3 were related to PDT in the other patients. After a median follow-up of 48 months after PDT, the PFS and OS at 3 years were 40% (95% CI, 21-59%) and 38% (95% CI, 17-60%), respectively. PDT is a potentially curative and tolerable salvage treatment after CRT for carefully selected patients with local failure without any metastasis.     

Transient elevation of serum cystatin C concentrations during perioperative cisplatin-based chemotherapy in esophageal cancer patients

Purpose

In the present study, the time–concentration profile of platinum (Pt) in plasma was compared to that of serum cystatin C (Cys C) in Japanese esophageal cancer patients receiving perioperative cisplatin-based chemotherapy.

Methods

Five male and one female patients receiving 2 successive cycles of cisplatin-based chemotherapy combined with 5-fluorouracil, the treatment for esophageal squamous cell carcinoma, participated in this study. The pharmacokinetic parameters in each patient were calculated from the individual plasma Pt concentration–time curve after intravenous infusion of cisplatin using the one-compartment model.

Results

Within a week of starting the first cycle of chemotherapy, serum Cys C concentrations increased in all patients from 122.6 to 143.0?%, subsequently returning to baseline levels in approximately 10?days. A similar increase in serum Cys C levels also occurred during the second treatment cycle. However, no increase in serum creatinine levels was observed during either treatment cycle. In addition, the concentration of plasma Pt 2?days after treatment in the first and second cycles did not correlate with those of either serum Cys C or creatinine. Finally, the half-life of Pt in plasma during the first treatment cycle was not significantly different from that in the second cycle.

Conclusions

These findings suggest that concentration fluctuations in serum Cys C are unlikely to correlate with Pt elimination from the plasma and that renal function estimates based on serum Cys C concentration might be underestimated during perioperative cisplatin-based chemotherapy for esophageal cancer.     

Diethyldithiocarbamate induces apoptosis in HHV-8-infected primary effusion lymphoma cells via inhibition of the NF-κB pathway

Primary effusion lymphoma (PEL) is a subtype of B-cell lymphoma caused by human herpes virus 8/Kaposi sarcoma-associated herpes virus (HHV-8/KSHV), which is mostly found in patients with AIDS and has poor prognosis. Nuclear factor (NF)-κB pathway is constitutively activated in HHV-8-infected PEL cells and plays a crucial role in tumorigenesis. Recently, it has been shown that diethyldithiocarbamate (DDTC), an active metabolite of disulfiram, has apoptotic activity in cancer cells. Here, we investigated the effect of DDTC on PEL using a PEL mouse model generated by intraperitoneal injection of BC-3 cells, a PEL cell line. DDTC ameliorated the symptoms of PEL in these mice, such as development of ascites, splenomegaly and increase of body weight, in comparison with PBS-treated controls. Moreover, we determined in vitro that DDTC suppressed the constitutively activated NF-κB pathway in BC-3 cells. Methylthiotetrazole assay revealed that the cell proliferation of various PEL cell lines was significantly suppressed by the treatment of DDTC. DDTC also induced the expression of cleaved caspase-3, an apoptosis marker, whereas the addition of Q-VD-OPh, a pan-caspase inhibitor, inhibited cell apoptosis induced by DDTC treatment. Together, our results indicated that DDTC induces apoptosis via inhibition of the NF-κB signaling pathway in HHV-8-infected PEL cells. This study suggests the potential use of DDTC as a therapeutic approach for PEL.     

Effects of hyaluronic acid and CD44 interaction on the proliferation and invasiveness of malignant pleural mesothelioma

Hyaluronic acid (HA) has been proposed as a biochemical marker of malignant pleural mesothelioma (MPM). The present study focused on the implications of HA and CD44 interaction in the proliferation and invasiveness of MPM. The proliferation and invasive activity was evaluated in two human mesothelioma cell lines, ACC-MESO-1 and K921MSO, by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and the transwell chamber model. The knockdown of CD44 gene expression was accomplished by transfection of the cells with small interfering RNA. Flow cytometry revealed that both the ACC-MESO-1 and K921MSO cell lines highly expressed CD44. Treatment with HA enhanced the proliferation in both mesothelioma cell lines in comparison to cells without HA treatment. The treatment with HA (25???g/ml) also significantly upregulated the invasion of both types of cells. The silencing of CD44 significantly abrogated the effect of HA treatment on the proliferation of ACC-MESO-1 cells and significantly suppressed the proliferation of K921MSO cells. HA?CCD44 binding is important for the migration and proliferation of mesothelioma cells. Therefore, the HA?CCD44 interaction is a potentially useful therapeutic target in MPM.