Inhibition of vascular adhesion protein-1 enhances the anti-tumor effects of immune checkpoint inhibitors.

Modulation of the immunosuppressive tumor microenvironment (TME) is essential for enhancing the anti-tumor effects of immune checkpoint inhibitors (ICIs). Adhesion molecules and enzymes such as vascular adhesion protein-1 (VAP-1), which are expressed in some cancers and tumor vascular endothelial cells, may be involved in the generation of an immunosuppressive TME. In this study, the role of VAP-1 in TME was investigated in 2 murine colon cancer models and human cancer cells. Intraperitoneal administration of the VAP-1-specific inhibitor U-V296 inhibited murine tumor growth by enhancing IFN-γ-producing tumor antigen-specific CD8+ T cells. U-V296 exhibited significant synergistic anti-tumor effects with ICIs. In the TME of mice treated with U-V296, the expression of genes associated with M2-like macrophages, Th2 cells (Il4, Retnla, and Irf4), angiogenesis (Pecam1), and fibrosis (Acta2, Loxl2) were significantly decreased, and the Th1/Th2 balance was increased. H2 O2 , an enzymatic product of VAP-1, which promoted the production of IL-4 by mouse Th2 and inhibited IFN-γ by mouse Th1 and human tumor-infiltrating lymphocytes, was decreased in tumors and CD31+ tumor vascular endothelial cells in the TMEs of mice treated with VAP-1 inhibitor. TCGA database analysis showed that VAP-1 expression was a negative prognostic factor in human cancers, exhibiting a significant positive correlation with IL-4, IL4R, and IL-13 expression and a negative correlation with IFN-γ expression. These results indicated that VAP-1 is involved in the immunosuppressive TMEs through H2 O2 -associated Th2/M2 conditions and may be an attractive target for the development of combination cancer immunotherapy with ICIs.

Increased Microcirculation on Optic Nerve Head by Laser Speckle Flowgraphy at Early Stage of Leber Hereditary Optic Neuropathy.

Leber hereditary optic neuropathy (LHON) is a mitochondrial disorder predominantly affecting young men. Characteristic features of an early stage of LHON are peripapillary telangiectatic microangiopathy with optic disc hyperaemia and swelling of the retinal nerve fibre layers. We evaluated the microcirculation of the optic nerve head (ONH) by laser speckle flowgraphy (LSFG) in a 79-year-old man and a 36-year-old woman with LHON. The ONH microcirculation of the tissue area was markedly increased in the early stage in both patients. LSFG may be a useful noninvasive method to suspect individuals to have an early stage of LHON.

Inhibition of Vascular Adhesion Protein-1 for Treatment of Graft-Versus-Host Disease in Mice.

Graft-versus-host disease (GVHD) is a potentially lethal complication of hematopoietic stem cell transplantation (HSCT). GVHD comprises acute and chronic forms. To date, several approaches to treat acute GVHD or chronic GVHD have been reported. However, there is no literature precedent regarding all-in-one methods to address the 2 GVHD types. Severe inflammation in organs affected by GVHD is highly problematic, and vascular adhesion protein-1 (VAP-1) is known to be detrimentally involved in various inflammatory diseases. Based on the previous reports, we envisaged that there would be a link between GVHD and VAP-1, and we strived to create effective therapies for the 2 types of GVHD using a mouse model of GVHD. Our investigation indicated that expression of VAP-1 was elevated in organs disordered by GVHD. Hence, we subsequently attempted to block VAP-1 by using a novel inhibitor. Our results indicate that systemic injection of the inhibitor prevented aberrant influx of inflammatory cells into tissues and thereby mitigate GVHD-elicited inflammation and fibrosis. Collectively, our study suggests that the increased expression of VAP-1 is detrimentally associated with the development of GVHD and that the blockade of VAP-1 could be a promising medical modality to combat the acute and chronic variants.-Mukai, S., Ogawa, Y., Kawakami, Y., Mashima, Y., Tsubota, K. Inhibition of vascular adhesion protein-1 for treatment of graft-versus-host disease in mice.

Visual prognosis better in eyes with less severe reduction of visual acuity one year after onset of Leber hereditary optic neuropathy caused by the 11,778 mutation.

BACKGROUND: Patients with Leber hereditary optic neuropathy (LHON) have a progressive decrease of their visual acuity which can deteriorate to <0.1. Some patients can have a partial recovery of their vision in one or both eyes. One prognostic factor associated with a recovery of vision is an early-age onset. The purpose of this study was to determine other clinical factors that are predictive of a good visual recovery. METHODS: Sixty-one Japanese LHON patients, with the 11,778 mutation and a mean age of 23.1 ± 12.1 years at the onset, were studied. All patients were initially examined at an acute stage of LHON and were followed for 3 to 10 years. At 1 year after the onset, the lowest visual acuity was <0.1 in all eyes. We studied the following parameters of patients with/without a final visual acuity of ≥ 0.2: sex; heavy consumption of cigarettes and alcohol; taking idebenone; mean age at onset; mean lowest visual acuity; and distribution of the lowest and the final visual acuity. RESULTS: Fifteen (24.6%) of the 61 patients or 25 (20.5%) of the 122 eyes had a recovery of their visual acuity to ≥ 0.2. The mean age at onset of these 15 patients with visual recovery to ≥ 0.2 was 17.5 ± 7.7 years, and that of the 46 patients without visual recovery to ≥ 0.2 was 25.0 ± 12.8 years (P = 0.02, Mann-Whitney U test). The mean lowest visual acuity of the 25 eyes with visual recovery ≥ 0.2 was 0.04, and that of the 97 eyes without visual recovery to ≥ 0.2 was 0.015 (P < 0.001, Mann-Whitney U test). Fifty percent (15/30) of the eyes whose lowest visual acuity was ≥ 0.04 during 1 year after the onset had a visual recovery to ≥ 0.2, while 11% (10/92) of the eyes whose the lowest visual acuity was ≤ 0.03 had a visual recovery to ≥ 0.2 (P < 0.001, χ 2 test). There were no significant differences in the other clinical factors. CONCLUSION: A final visual acuity of ≥ 0.2 was associated with a less severe reduction of the visual acuity at 1 year after the onset. Our findings can be used to predict the visual prognosis in LHON patients.

Vascular Adhesion Protein-1 Blockade Suppresses Ocular Inflammation After Retinal Laser Photocoagulation in Mice.

Purpose: To investigate the effect of the vascular adhesion protein-1 (VAP-1) inhibitor RTU-1096 on retinal morphologic changes and ocular inflammation after retinal laser photocoagulation in mice. Methods: C57BL/6JJcl mice were fed a diet containing RTU-1096, a specific inhibitor for VAP-1, or a control diet ad libitum for 7 days. Laser photocoagulation was performed on the peripheral retina of the animals. The semicarbazide sensitive amine oxidase (SSAO) activities in plasma and chorioretinal tissues were measured. Optical coherence tomography (OCT) images were acquired before and at 1, 3, and 7 days after laser photocoagulation, and thickness of the individual retinal layers was measured. Intravitreal leukocyte infiltration was assessed by histologic analysis. The expression level of intercellular adhesion molecule-1 (ICAM-1) in retinal tissues were examined by quantitative real-time PCR. Results: One day after laser photocoagulation, the thickness of the outer nuclear layer (ONL) increased in the laser group compared with in the control group, and RTU-1096 administration abrogated the ONL thickening. Histologic analysis and OCT observation revealed that laser photocoagulation caused infiltration of inflammatory cells and the appearance of hyperreflective foci at the vitreoretinal surface, both of which were suppressed by RTU-1096 administration. In addition, systemic administration of RTU-1096 reduced upregulation of the leukocyte adhesion molecules ICAM-1 in the retina. Conclusions: The current data indicate that VAP-1/SSAO inhibition may be a potential therapeutic strategy for the prevention of macular edema secondary to scatter laser photocoagulation in patients with ischemic retinal diseases such as diabetic retinopathy.

Long-Term Protection of Genetically Ablated Rabbit Retinal Degeneration by Sustained Transscleral Unoprostone Delivery.

Purpose: To evaluate the long-term protective effects of transscleral unoprostone (UNO) against retinal degeneration in transgenic (Tg) rabbits (Pro347Leu rhodopsin mutation). Methods: The UNO release devices (URDs) were implanted into the sclerae of Tg rabbits and ERG, optical coherence tomography (OCT), and ophthalmic examinations were conducted for 40 weeks. Unoprostone metabolites in retina, choroid/RPE, aqueous humor, and plasma from wild-type (Wt) rabbits were measured using liquid chromatography-tandem mass spectrometry. In situ hybridization and immunohistochemistry evaluated the retinal distribution of big potassium (BK) channels, and RT-PCR evaluated the expressions of BK channels and m-opsin at 1 week after URD treatment. Results: The URD released UNO at a rate of 10.2 ±1.0 µg/d, and the release rate and amount of UNO decreased during 32 weeks. Higher ERG amplitudes were observed in the URD-treated Tg rabbits compared with the placebo-URD, or nontreated controls. At 24 weeks after implantation into the URD-treated Tg rabbits, OCT images showed preservation of retinal thickness, and histologic examinations (44 weeks) showed greater thickness of outer nuclear layers. Unoprostone was detected in the retina, choroid, and plasma of Wt rabbits. Retina/plasma ratio of UNO levels were 38.0 vs. 0.68 ng UNO*hour/mL in the URD-treated group versus control (topical UNO), respectively. Big potassium channels were observed in cone, cone ON-bipolar, and rod bipolar cells. Reverse-transcriptase PCR demonstrated BK channels and m-opsins increased in URD-treated eyes. Conclusions: In Tg rabbits, URD use slowed the decline of retinal function for more than 32 weeks, and therefore provides a promising tool for long-term treatment of RP.

Protective effects of sustained transscleral unoprostone delivery against retinal degeneration in S334ter rhodopsin mutant rats.

It has been suggested that unoprostone isopropyl (UNO) has potent neuroprotective activity in the retina. The effect of sustained transscleral UNO delivery to the posterior segment of the eye on photoreceptor degeneration was evaluated. UNO was loaded into a device made of poly(ethyleneglycol) dimethacrylate by polydimethylsiloxane mold-based UV-curing. The amount of UNO diffusing from these devices was measured using high-performance liquid chromatography. The polymeric devices that released UNO at 1.8 µg/day were implanted on the sclerae of S334ter rats at postnatal 21 days, and electroretinograms (ERGs) were compared with those of topical application and placebo devices. Retinal thickness was evaluated by histological examination. Western blots of specimens 4 weeks after implantation were performed. ERGs showed that the UNO-loaded device prevented the reduction of ERG amplitudes 2 and 4 weeks after implantation, compared with results using a placebo device or topical application. Histological examination showed that the UNO-loaded device prevented the reduction of retinal thickness, and Western blots of specimens indicated that the UNO-loaded device decreased expression of ERK1/2, phosphorylated ERK1/2, and caspase-3. A device that provided sustained UNO administration protected against retinal degeneration in rhodopsin mutant rats, and thus, may have translational potential as a sustainable method to administer drugs to treat retinitis pigmentosa. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 104B: 1730-1737, 2016.

A platform for controlled dual-drug delivery to the retina: protective effects against light-induced retinal damage in rats.

Controlled transscleral co-delivery of two drugs, edaravone (EDV) and unoprostone (UNO), using a platform that comprises a microfabricated reservoir, controlled-release cover, and drug formulations, which are made of photopolymerized poly(ethyleneglycol) dimethacrylates, shows synergistic retinal neuroprotection against light injury in rats when compared with single-drug-loaded devices. The device would offer a safer therapeutic method than intravitreal injections for retinal disease treatments.

Association of HK2 and NCK2 with normal tension glaucoma in the Japanese population.

Although family studies and genome-wide association studies have shown that genetic factors play a role in glaucoma, it has been difficult to identify the specific genetic variants involved. We tested 669 single nucleotide polymorphisms (SNPs) from the region of chromosome 2 that includes the GLC1B glaucoma locus for association with primary open-angle glaucoma (POAG) and normal tension glaucoma (NTG) in the Japanese population. We performed a two-stage case-control study. The first cohort consisted of 123 POAG cases, 121 NTG cases and 120 controls: the second cohort consisted of 187 POAG cases, 286 NTG cases, and 271 controls. Out of six SNPs showing significant association with POAG in the first round screening, seven SNPs were tested in the second round. Rs678350 in the HK2 gene coding sequence showed significant allelic (p=0.0027 in Stage Two, 2.7XE-4 in meta-analysis) association with POAG, and significant allelic (p=4.7XE-4 in Stage Two, 1.0XE-5 in meta-analysis) association with NTG. Although alleles in the TMEM182 gene did not show significant association with glaucoma in the second round, subjects with the A/A allele in TMEM182 rs869833 showed worse visual field mean deviation (p=0.01). Even though rs2033008 in the NCK2 gene coding sequence did not show significant association in the first round, it had previously shown association with NTG so it was tested for association with NTG in round 2 (p=0.0053 in Stage Two). Immunohistochemistry showed that both HK2 and NCK2 are expressed in the retinal ganglion cell layer. Once multi-testing was taken into account, only HK2 showed significant association with POAG and NTG in Stage Two. Our data also support previous reports of NCK2 association with NTG, and raise questions about what role TMEM182 might play in phenotypic variability. Our data suggest that HK2 may play an important role in NTG in the Japanese population.

Serum vascular adhesion protein-1 correlates with vascular endothelial growth factor in patients with type II diabetes.

AIMS: To study serum levels of soluble vascular adhesion protein (sVAP)-1 in type II diabetic patients with retinopathy. METHODS: Serum samples were obtained from 53 consecutive patients, including 14 cases with non-angiogenic ocular diseases, i.e., epiretinal membrane (ERM) and idiopathic macular hole (MH), 19 cases with age-related macular degeneration (AMD), and 20 cases with diabetic retinopathy (DR). Protein levels of sVAP-1, intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1, and vascular endothelial growth factor (VEGF) were determined by enzyme-linked immunosorbent assay. Enzymatic activity of semicarbazide-sensitive amine oxidase (SSAO) was also measured. RESULTS: Serum level of sVAP-1 showed a moderate correlation with SSAO activity in all cases. Patients with DR had higher levels of serum sVAP-1 than subjects with ERM and MH, or those with AMD; however, severity of DR is not related to the serum levels of sVAP-1. Serum sVAP-1 correlated positively with VEGF in patients with DR, but not in those with ERM and MH, or those with AMD. Neither soluble ICAM-1 nor VCAM-1 correlated with VEGF, even in subjects with DR. CONCLUSION: The current data demonstrate the elevated serum levels of sVAP-1 and correlation between sVAP-1 and VEGF in patients with type II diabetes.

Association of Toll-like receptor 4 gene polymorphisms in Japanese subjects with primary open-angle, normal-tension, and exfoliation glaucoma.

PURPOSE: To determine whether polymorphisms in the Toll-like receptor 4 (TLR4) gene are associated with primary open-angle glaucoma (POAG), normal-tension glaucoma (NTG), and exfoliation glaucoma (XFG) in Japanese individuals. DESIGN: Genetic association study. SETTING: Multicenter study. STUDY POPULATION: One hundred eighty-four unrelated Japanese patients with POAG, 365 unrelated patients with NTG, and 109 unrelated patients with XFG from 5 hospitals. PROCEDURES: Genomic DNA was extracted from leukocytes of the peripheral blood, and 8 polymorphisms in the TLR4 genes were amplified by polymerase chain reaction (PCR) and directly sequenced. Allele and genotype frequencies and the inferred haplotypes were estimated. MAIN OUTCOME MEASURES: Differences in allele and genotype frequencies and haplotypes between subjects with POAG, NTG, and XFG. RESULTS: The allele frequency of rs2149356 of the TLR4 gene in the POAG, NTG, and XFG groups was the most significantly different from that of the control group (minor allele frequency 0.446, 0.395, 0.404, vs 0.308; P = .000058, P = .0030, and P = .015). The allele frequencies of the 5 TLR4 SNPs were higher in all of the glaucoma groups than that in the control group. The statistics of genotypes of TLR4 were approximately the same for all allele frequencies. The haplotypic frequencies with Tag SNPs studied earlier showed that only POAG was statistically significant. Other haplotypes, such as rs10759930, rs1927914, rs1927911, and rs2149356, had higher statistical significance (overall P = .00078 in POAG, overall P = .018 in NTG, and overall P = .014 in XFG). CONCLUSIONS: This study demonstrated that TLR4 polymorphisms are associated with NTG in the Japanese, and they also play a role in the pathogenesis of POAG and XFG.

Soluble vascular adhesion protein-1 accumulates in proliferative diabetic retinopathy.

PURPOSE: Vascular adhesion protein (VAP)-1, a multifunctional molecule with adhesive and enzymatic properties, is expressed at the surface of vascular endothelial cells of mammals. It also exists as a soluble form (sVAP-1), which is implicated in oxidative stress via its enzymatic activity. This study explores a link between increased level of sVAP-1 and oxidative stress in proliferative diabetic retinopathy (PDR) with a focus on mechanistic components to form sVAP-1 by shedding from retinal endothelial cells. METHODS: Protein levels of sVAP-1 and N epsilon-(hexanoyl)lysine (HEL), an oxidative stress marker, in the vitreous samples from patients with PDR or non-PDR were measured by ELISA. The mechanism of VAP-1 shedding under diabetic condition, exposure to high glucose and/or inflammatory cytokines, was explored using cultured retinal capillary endothelial cells. RESULTS: Protein level of sVAP-1 was increased and correlated with HEL in the vitreous fluid of patients with PDR. Retinal capillary endothelial cells released sVAP-1 when stimulated with high glucose or inflammatory cytokines, such as TNF-α and IL-1ß in vitro. Furthermore, matrix metalloproteinase-2 and -9, type IV collagenases, were the key molecules to mediate the protein cleavage of VAP-1 from retinal capillary endothelial cells. CONCLUSIONS: Our data for the first time provide evidence on the link between sVAP-1 and type IV collagenases in the pathogenesis of PDR.

Blockade of vascular adhesion protein-1 attenuates choroidal neovascularization.

PURPOSE: Vascular adhesion protein (VAP)-1 is an adhesion molecule elucidated as a mediator of the leukocyte recruitment cascade. The purpose of this study was to investigate the role of VAP-1 in ocular inflammatory neovascularization using a mouse laser-induced choroidal neovascularization (CNV) model. METHODS: CNV was induced with 532 nm laser irradiation in C57BL/6 mice, and production of VAP-1 protein in the retinal pigment epithelium (RPE) choroid during CNV formation was examined. CNV animals were treated with the specific VAP-1 inhibitor U-V002 or vehicle solution, and the volume of CNV tissue was evaluated with volumetric measurements. Macrophage infiltration into the CNV lesions was evaluated using two different techniques, flatmount staining and real-time polymerase chain reaction (PCR) for F4/80. The protein levels of intercellular adhesion molecule (ICAM)-1, monocyte chemoattractant protein (MCP)-1, P-selectin, and vascular endothelial growth factor (VEGF) in the RPE-choroid were measured with enzyme-linked immunosorbent assay (ELISA). RESULTS: VAP-1 inhibition significantly suppressed CNV formation in a dose-dependent manner and reduced macrophage infiltration into CNV lesions. Furthermore, VAP-1 blockade decreased the expression of ICAM-1 and MCP-1, both of which play a pivotal role in macrophage recruitment. CONCLUSIONS: Our data suggest VAP-1 has an important role during ocular inflammatory neovascularization through leukocyte recruitment. VAP-1 inhibition may be a novel and potent therapeutic strategy in treating CNV formation.

Topical isopropyl unoprostone for retinitis pigmentosa: microperimetric results of the phase 2 clinical study.

INTRODUCTION: The purpose of this study was to determine whether topical 0.15% isopropyl unoprostone (IU), a BK-channel activator, could improve or maintain the central retinal sensitivity in patients with middle- to late-stage retinitis pigmentosa (RP). IU was approved for glaucoma and ocular hypertension in 1994. The drug re-profiling strategy is one of the effective ways to develop safe drugs for patients with RP. METHODS: A randomized, double-blind, and placebo-controlled phase II safety/efficacy trial was conducted. One hundred and nine patients with middle- to late-stage RP having a visual acuity of ≥0.5 were studied at six ophthalmological centers in Japan. The treatments of IU/day were divided into three groups: placebo group; two-drop group; and four-drop group for 24 weeks. The primary outcome measure was changes in the retinal sensitivity from baseline in the central 2° determined by MP-1 microperimetry (MP-1, Nidek, Japan). The secondary outcomes were changes in best-correct visual acuity, contrast sensitivity, retinal sensitivity of the central 10° by MP-1, mean deviation (MD) by a Humphrey field analyzer (HFA; Carl Zeiss Meditec, Dublin, CA, USA) 10-2, and the Visual Functioning Questionnaire 25 (VFQ-25) questionnaire scores. RESULTS: There was a tendency for a dose-dependent responsiveness in retinal sensitivity in the central 2°, MD, and total VFQ-25 score after 24 weeks of IU instillation by a simple linear regression analysis. A stratified analysis showed a significant dose-dependent responsiveness of the 2° central retinal sensitivity in more advanced patients (P = 0.028). The number of patients having a ≥4 dB decrease in the primary outcome measure was significantly fewer in the four-drop group than in the placebo group (P = 0.02). No adverse reactions were observed. CONCLUSIONS: A higher dose of IU can delay progression of the central retinal sensitivity decrease through an improvement of retinal sensitivity.

VAP-1-mediated M2 macrophage infiltration underlies IL-1ß- but not VEGF-A-induced lymph- and angiogenesis.

Vascular adhesion protein-1 (VAP-1) contributes to inflammatory and angiogenic diseases, including cancer and age-related macular degeneration. It is expressed in blood vessels and contributes to inflammatory leukocyte recruitment. The cytokines IL-1ß and vascular endothelial growth factor A (VEGF-A) modulate angiogenesis, lymphangiogenesis, and leukocyte infiltration. The lymphatic endothelium expresses intercellular adhesion molecule-1 and vascular adhesion molecule-1, which facilitate leukocyte transmigration into the lymphatic vessels. However, whether lymphatics express VAP-1 and whether they contribute to cytokine-dependent lymph- and angiogenesis are unknown. We investigated the role of VAP-1 in IL-1ß- and VEGF-A-induced lymph- and angiogenesis using the established corneal micropocket assay. IL-1ß increased VAP-1 expression in the inflamed cornea. Our in vivo molecular imaging revealed significantly higher VAP-1 expression in neovasculature than in the preexisting vessels. VAP-1 was expressed in blood but not lymphatic vessels in vivo. IL-1ß-induced M2 macrophage infiltration and lymph- and angiogenesis were blocked by VAP-1 inhibition. In contrast, VEGF-A-induced lymph- and angiogenesis were unaffected by VAP-1 inhibition. Our results indicate a key role for VAP-1 in lymph- and angiogenesis-related macrophage recruitment. VAP-1 might become a new target for treatment of inflammatory lymph- and angiogenic diseases, including cancer.

Unoprostone reduces oxidative stress- and light-induced retinal cell death, and phagocytotic dysfunction, by activating BK channels.

PURPOSE: Unoprostone isopropyl (unoprostone) is a docosanoid currently used as an antiglaucoma agent. Unoprostone is known to have neuroprotective effects and to activate large conductance Ca²âº-activated K⁺ (BK) channels. Recently, unoprostone has been tested in clinical studies as a therapeutic agent for retinitis pigmentosa (RP) and studies have demonstrated an improvement in retinal sensitivity and in the protection of central retinal sensitivity with its use. However, the mechanism of action underlying unoprostone's protective effect in RP is not fully known. It is well known that the pathogenesis of RP can be accelerated by oxidative stress or light irradiation. Therefore, the current study investigated the effects and the underlying mechanism of action of unoprostone on oxidative stress- and light irradiation-induced damage in photoreceptor and retinal pigment epithelial cultures. METHODS: The study used the mouse retinal cone-cell line 661W to investigate the effects of unoprostone and its major metabolite, unoprostone-free acid (M1), on oxidative stress- or light irradiation-induced cell death, and a human retinal pigment epithelial cell line (ARPE-19), was used to investigate the effects on light-induced disruption of phagocytotic function in a latex bead assay. Additionally, we examined whether the effects of unoprostone and M1 were mediated by BK channels using iberiotoxin, a selective inhibitor of BK channels. RESULTS: Unoprostone and M1 protected against light- or H2O2-induced cell death in 661W cells, and against light-induced phagocytotic dysfunction in ARPE-19 cells. Additionally, iberiotoxin inhibited the protective effects of unoprostone and M1. CONCLUSIONS: These findings indicate that unoprostone has protective effects on oxidative stress- and light irradiation-induced damage in vitro and that these effects are mediated by activation of BK channels. This confirms that unoprostone represents a promising therapeutic agent for the treatment of RP and other retinal diseases.

Low levels of plasma endothelin-1 in patients with retinitis pigmentosa.

PURPOSE: The aim of this study was to elucidate the role of endothelin-1 (ET-1) in the pathophysiology of retinitis pigmentosa (RP). METHODS: Plasma ET-1 levels and ophthalmic features in 50 RP patients were compared with those in 20 healthy-eye control subjects. Plasma ET-1 concentrations were determined using a commercially available enzyme-linked immunosorbent assay kit. RESULTS: Mean plasma ET-1 levels of RP patients (1.88 +/- 0.56 pg/mL) were significantly lower than those of control subjects (2.30 +/- 0.30 pg/mL, Mann-Whitney's U test; P < 0.01). However, ET-1 concentrations varied markedly in each patient. Among RP patients, a significant correlation of ET-1 concentrations was not observed in terms of its hereditary forms or other clinical factors. CONCLUSION: ET-1 may be important in the pathogenesis of RP, and measurement of its plasma concentrations may also contribute to additional insights into the retinal hemodynamics of RP.

Vascular adhesion protein-1 regulates leukocyte transmigration rate in the retina during diabetes.

Vascular adhesion protein-1 (VAP-1) is an endothelial adhesion molecule that possesses semicarbazide-sensitive amine oxidase (SSAO) activity and is involved in leukocyte recruitment. Leukocyte adhesion to retinal vessels is a predominant feature of experimentally induced diabetic retinopathy (DR). However, the role of VAP-1 in this process is unknown. Diabetes was induced by i.p. injection of Streptozotocin in Long-Evans rats. The specific inhibitor of VAP-1, UV-002, was administered by daily i.p. injections. The expression of VAP-1 mRNA in the retinal extracts of normal and diabetic animals was measured by real-time quantitative polymerase chain reaction (PCR). Firm leukocyte adhesion was quantified in retinal flatmounts after intravascular staining with concanavalin A (ConA). Leukocyte transmigration rate was quantified by in vivo acridine orange leukocyte staining (AOLS). In diabetic rats, the rate of leukocyte transmigration into the retinal tissues of live animals was significantly increased, as determined by AOLS. When diabetic animals were treated with daily injections of the VAP-1 inhibitor (0.3 mg/kg), leukocyte transmigration rate was significantly reduced (P < 0.05). However, firm adhesion of leukocytes in diabetic animals treated with the inhibitor did not differ significantly from vehicle-treated diabetic controls. This work provides evidence for an important role of VAP-1 in the recruitment of leukocyte to the retina in experimental DR. Our results reveal the critical contribution of VAP-1 to leukocyte transmigration, with little impact on firm leukocyte adhesion in the retinas of diabetic animals. VAP-1 inhibition might be beneficial in the treatment of DR.