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.

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.

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.

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.

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.

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.

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.

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.

Inhibition of vascular adhesion protein-1 suppresses endotoxin-induced uveitis.

Inflammatory leukocyte accumulation is a common feature of major ocular diseases, such as uveitis, diabetic retinopathy, and age-related macular degeneration. Vascular adhesion protein-1 (VAP-1), a cell surface and soluble molecule that possesses semicarbazide-sensitive amine oxidase (SSAO) activity, is involved in leukocyte recruitment. However, the expression of VAP-1 in the eye and its contribution to ocular inflammation are unknown. Here, we investigated the role of VAP-1 in an established model of ocular inflammation, the endotoxin-induced uveitis (EIU), using a novel and specific inhibitor. Our inhibitor has a half-maximal inhibitory concentration (IC(50)) of 0.007 microM against human and 0.008 microM against rat SSAO, while its IC(50) against the functionally related monoamine oxidase (MAO) -A and MAO-B is >10 microM. In the retina, VAP-1 was exclusively expressed in the vasculature, and its expression level was elevated during EIU. VAP-1 inhibition in EIU animals significantly suppressed leukocyte recruitment to the anterior chamber, vitreous, and retina, as well as retinal endothelial P-selectin expression. Our data suggest an important role for VAP-1 in the recruitment of leukocytes to the immune-privileged ocular tissues during acute inflammation. VAP-1 inhibition may become a novel strategy in the treatment of ocular inflammatory diseases.

Vascular adhesion protein-1 blockade suppresses choroidal neovascularization.

Vascular adhesion protein-1 (VAP-1) is an endothelial cell adhesion molecule involved in leukocyte recruitment. Leukocytes and, in particular, macrophages play an important role in the development of choroidal neovascularization (CNV), an integral component of age-related macular degeneration (AMD). Previously, we showed a role for VAP-1 in ocular inflammation. Here, we investigate the expression of VAP-1 in the choroid and its role in CNV development. VAP-1 was expressed in the choroid, exclusively in the vessels, and colocalized in the vessels of the CNV lesions. VAP-1 blockade with a novel and specific inhibitor significantly decreased CNV size, fluorescent angiographic leakage, and the accumulation of macrophages in the CNV lesions. Furthermore, VAP-1 blockade significantly reduced the expression of inflammation-associated molecules such as tumor necrosis factor (TNF) -alpha, monocyte chemoattractant protein (MCP) -1, and intercellular adhesion molecule (ICAM) -1. This work provides evidence for an important role of VAP-1 in the recruitment of macrophages to CNV lesions, establishing a novel link between VAP-1 and angiogenesis. Inhibition of VAP-1 may become a new therapeutic strategy in the treatment of AMD.

Discordance between subjective perimetric visual fields and objective multifocal visual evoked potential-determined visual fields in patients with hemianopsia.

PURPOSE: To investigate the concordance between subjectively and objectively acquired visual fields in patients with subjectively determined hemianopsia. DESIGN: Retrospective observational study. METHODS: Ten patients, six men and four women, ranging in age from 28 to 68 years, were studied. Goldmann or Humphrey perimeters were used to obtain the subjectively determined visual fields for up to 25 degrees of eccentricity, and the VERIS Scientific System (Electro-Diagnostic Imaging, San Francisco, California, USA) was used to record multifocal visual evoked potential [VEPs] (mfVEPs) to obtain the objective visual fields. Each of the 60 black-and-white segments of the checkerboard stimulus was alternated according to a binary m sequence. The first slices of the second-order kernels were extracted and analyzed. RESULTS: In five cases, the visual field loci where the mfVEPs were within normal limits corresponded to the scotomatous areas obtained by conventional perimetry. In these discordant cases, the lesions (e.g., arteriovenous malformation) were located in the occipital lobe. Two of these cases had a complete recovery of the subjective visual field. The lesions of the concordant cases were located outside the occipital lobe (e.g., pituitary adenoma). In these cases, no visual field improvement was seen. The temporal crescent syndrome was ruled out in patients with posterior lesions by computed tomography (CT) or magnetic resonance imaging (MRI) findings. CONCLUSIONS: In some patients with occipital lesions, the subjective and objective visual field results are discordant, and some of them will show a recovery of the visual field deficits.

Quantitative evaluation of tear meniscus height from fluorescein photographs.

PURPOSE: To describe a method of quantifying the tear meniscus height (TMH) and using these values to diagnose patients with dry eye. METHODS: Thirty-eight eyes of 19 healthy subjects and 14 patients diagnosed with dry eye were studied. Each eye received 2 microL of 1% fluorescein-sodium solution in the conjunctival sac, and digital photographs were taken of the external surface of the eye with a fundus camera. The tear meniscus was extracted from the digitalized image and used to measure the TMH with NIH image software. The correlation between the mean TMH and Schirmer test values or cotton-thread test values was evaluated. RESULTS: The means of the upper and lower TMH in healthy subjects were 0.22 +/- 0.06 and 0.24 +/- 0.08 mm, respectively. The comparable values in patients with dry eye were 0.17 +/- 0.04 and 0.17 +/- 0.07 mm (upper TMS, P = 0.01; lower TMH, P = 0.04; unpaired t test). A significant correlation was found between the TMH and Schirmer test values (r = 0.72, P = 0.01) but not with cotton-thread test values. CONCLUSIONS: Fluorescein photographs of the eye can be used to quantify the upper and lower TMH separately and simultaneously without specialized equipment. This technique can be used for the diagnosis of dry eye.

Woman with atypical unilateral Leber's hereditary optic neuropathy with visual improvement.

We describe a patient with Leber's hereditary optic neuropathy (LHON) who had a unilateral involvement and a gradual recovery of vision. A 50-year-old woman was referred to our clinic in December 2004 for the treatment of left optic neuritis. The visual acuity was 0.01 in her left eye and 1.5 in her right eye. The left eye had a central scotoma and a relative afferent pupillary defect. Ophthalmoscopy revealed a hyperaemic optic disc with indistinct margins in the left eye. Fluorescein angiography showed circumpapillary microangiopathy in both eyes and staining of the left optic disc. An nt 11778 mutation was identified and she was diagnosed with LHON. The central scotoma gradually improved, and the visual acuity had recovered to 0.3 in August 2007. LHON should still be considered even in older female patients presenting with unilateral acute visual loss when microangiopathy is seen. In such cases, molecular testing is effective in confirming a diagnosis of LHON.

Association between open-angle glaucoma and gene polymorphism for heat-shock protein 70-1.

PURPOSE: Heat-shock proteins (HSPs) or antibodies against them may contribute to glaucomatous optic neuropathy. We investigated the associations of HSP70-1 polymorphisms with open-angle glaucoma (OAG) in a Japanese population. METHODS: In 241 normal Japanese controls and 501 Japanese OAG patients, including 211 with primary open-angle glaucoma (POAG) and 290 with normal-tension glaucoma (NTG), two single-nucleotide polymorphisms, A-110C and G+190C, of HSP70-1 were identified by using an Invader assay and polymerase chain reaction-restriction fragment length polymorphism, respectively. Genotype distributions were compared between controls and OAG patients. Age at diagnosis, untreated maximum intraocular pressure, and visual field defects at diagnosis were examined for associations with the polymorphisms. RESULTS: Distribution of the A-110C genotype (AA versus AC+CC) differed significantly between controls and OAG patients (P = 0.007), POAG patients (P = 0.007), or NTG patients (P = 0.032). The genotype distribution of the G+190C polymorphism did not differ significantly between the controls and any patient group. No significant differences in the clinical characteristics of the patients were detected between genotype-defined groups by logistic regression analysis. CONCLUSION: The A-110C polymorphism of HSP70-1 may be associated with OAG pathogenesis in Japanese 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.

Involvement of insulin-like growth factor-I and insulin-like growth factor binding protein-3 in corneal fibroblasts during corneal wound healing.

PURPOSE: The involvement of downstream messengers of transforming growth factor (TGF)-beta in the differentiation of corneal fibroblasts into myofibroblasts was investigated. The effects of insulin-like growth factor (IGF)-I and insulin-like growth factor binding protein (IGFBP)-3 upregulated by TGF-beta were examined in human corneal fibroblasts, and the possible involvement of IGF axis components in corneal wound healing was assessed in a mouse model. METHODS: Human corneal fibroblasts were incubated with TGF-beta2 or IGF-I, to investigate IGF-I, IGF-II, IGFBP-3, type I collagen, and alpha-smooth muscle actin (alpha-SMA) mRNA, as well as IGFBP-3 protein expression, during myofibroblast differentiation. DNA synthesis was evaluated with a 5-bromo-2'-deoxyuridine (BrdU) incorporation assay. IGFBP-3 mRNA expression, protein expression, and immunolocalization were investigated in mouse corneas after photorefractive keratectomy (PRK). RESULTS: TGF-beta2 treatment induced expression of IGF-I and IGFBP-3 mRNA and of IGFBP-3 protein in human corneal fibroblasts. TGF-beta2 and IGF-I both stimulated expression of type I collagen. TGF-beta2 but not IGF-I potently stimulated alpha-SMA mRNA expression. IGF-I potently stimulated basal DNA synthesis, whereas IGFBP-3 inhibited it. IGF-I potently stimulated proliferation of TGF-beta2-activated myofibroblasts without reversing the activated fibrogenic phenotype, whereas IGFBP-3 suppressed IGF-I-induced proliferation of corneal fibroblasts. IGFBP-3 mRNA and protein increased in mouse corneas soon after PRK, when in vivo immunostaining of the corneas showed expression of IGFBP-3 in the deep layer of the corneal stroma. CONCLUSIONS: These results suggest that during corneal wound healing, TGF-beta stimulates IGF axis components, whereas IGFBP-3 may modulate IGF-I-induced myofibroblast proliferation to suppress corneal mesenchymal overgrowth.

SNPs and interaction analyses of noelin 2, myocilin, and optineurin genes in Japanese patients with open-angle glaucoma.

PURPOSE: To evaluate the noelin 2 gene as a disease-causing factor for open-angle glaucoma (OAG) and the interactions between the noelin 2 (OLFM2), optineurin (OPTN), and myocilin (MYOC) genes. METHODS: OLFM2 was analyzed in 770 Japanese subjects including 215 patients with elevated intraocular pressure (IOP), 277 with normal IOP, 38 with juvenile open-angle glaucoma, and 240 control subjects. Two single-nucleotide polymorphisms (SNPs) in OPTN (c.412G-->A and c.603T-->A) and one SNP in MYOC (c.227G-->A) were examined. Single genes were investigated by univariate analysis and the gene-gene interactions by logistic regression analysis. Associations between genotypes and clinical characteristics at the time of diagnosis were examined. RESULTS: In OLFM2, 12 sequence variants were identified in 770 Japanese subjects. Arg144Gln (exon 4) was identified in two (0.3%) of the patients and in none of the control subjects. Combinations of OLFM2/317A and OPTN/412A or OLFM2/1281T and OPTN/412A were associated with patients with elevated IOP (P = 0.018 or P = 0.012, respectively). The combination of OLFM2/317G and OPTN/603A was significantly associated with elevated IOP (P = 0.018). No significant association was detected between SNPs in OLFM2 and in MYOC. Patients with normal IOP and with OLFM2/678A+OPTN/412G or OLFM2/1281C+OPTN/412G had significantly worse visual field scores (P = 0.022 or 0.030, respectively). CONCLUSIONS: The Arg144Gln mutation in OLFM2 is a possible disease-causing mutation in Japanese patients with OAG. Common polymorphisms in OLFM2 and OPTN may interactively contribute to the development of OAG, indicating a polygenic etiology.

Polymorphism of beta-adrenergic receptors and susceptibility to open-angle glaucoma.

PURPOSE: The human trabecular meshwork and ciliary body, which express beta-adrenergic receptors (ADRB1 and ADRB2), control aqueous humor dynamics. We investigated associations of ADRB polymorphisms with open-angle glaucoma (OAG), because ADRB gene polymorphisms alter receptor function. METHODS: We studied 240 Japanese controls and 505 Japanese OAG patients including 211 with primary open-angle glaucoma (POAG), and 294 with normal-tension glaucoma (NTG). Associations of four polymorphisms (Ser49Gly and Arg389Gly in the ADRB1 gene; Arg16Gly and Gln27Glu in the ADRB2 gene) were compared between patients and controls. Age, intraocular pressure (IOP), and visual field defects at diagnosis were examined for associations with polymorphisms. RESULTS: The Arg389Gly polymorphism in the ADRB1 gene showed significantly different allele and genotype frequencies in patients with NTG than in controls (p = 0.004 and 0.006, respectively). Other polymorphisms did not show a significant frequency difference. In POAG patients, carriers of Gly16 in the ADRB2 gene were significantly younger at diagnosis than noncarriers (p<0.001). The IOP at diagnosis was significantly higher in OAG patients carrying 27Glu in the ADRB2 gene than in patients without this allele (p<0.001). Clinical characteristics of OAG patients did not differ significantly in relation to other polymorphisms. CONCLUSIONS: Certain polymorphisms of the ADRB1 and ADRB2 genes influence the pathophysiology of OAG in Japanese patients.

Mitochondrial DNA mutations with Leber's hereditary optic neuropathy in Japanese patients with open-angle glaucoma.

PURPOSE: Abnormal optic disc excavations are found in patients with Leber's hereditary optic neuropathy (LHON). The purpose of this study was to determine whether heteroplasmy for the major three LHON mutations or for the rare LHON mutations are risk factors for open-angle glaucoma. METHODS: Blood samples from 835 Japanese subjects were screened with the Invader assay for ten LHON-associated mutations: three major mutations (G3460A, G11778A, T14484C) and seven rare mutations (T9101C, G9804A, C14482A, C14482G, G14459A, T14498C, and A14510G). Of the 835 subjects, 241 were patients with primary open-angle glaucoma (POAG), 310 were patients with normal-tension glaucoma (NTG), and 284 were healthy controls. RESULTS: Five POAG patients and three NTG patients had one of five mutations, C9099A, T9101G, T9101C, G9804A, or G11778A, but none of these patients had LHON. The C9099A (Ile191Met) and T9101G (Ile192Ser) mutations were novel and identified within the probes by lack of signal in the assay. Two patients with the G11778A mutation showed heteroplasmy, with 15% mutant mtDNA in the male patient and 80% in the female patient. The remaining LHON-associated mutations were not detected in any of the subjects. A case-control study did not show a significant difference (P = 0.099): eight potentially disease-associated variants in 551 patients versus zero variants in the 284 controls. CONCLUSIONS: Rare LHON-associated mitochondrial DNA mutations were found in Japanese patients with open-angle glaucoma (OAG). However, whether mitochondrial DNA mutations are risk factors for OAG is still open to question.

Relaxation of encircling buckle improved choroidal blood flow in a patient with visual field defect following encircling procedure.

BACKGROUND: We report a patient with a visual field defect after retinal reattachment by the encircling procedure for rhegmatogenous retinal detachment. We confirmed improved ocular blood flow after relaxation of the buckle. CASE: A 24-year-old woman with a visual field defect appearing after an encircling procedure for rhegmatogenous retinal detachment. OBSERVATIONS: Before and after relaxing the encircling buckle, we measured tissue blood flow in the fundus of each eye of the patient using a Heidelberg retina flow meter. Preoperative measurements showed a reduction of blood flow at the disc rim in the diseased fundus, while retinal blood flow was not reduced (P = 0.026, disc rim area versus retinal area, one-factor analysis of variance, ANOVA). Indocyanine green angiography showed extensive peripheral filling delay. Electroretinography showed low a-wave and b-wave amplitudes, but normal oscillatory potential. The base value of the electro-oculogram was severely reduced in the right eye. The blood flow values after surgery indicated a significant improvement of blood flow (P = 0.01, one-factor ANOVA). No further progression in the visual field defect was observed, and visual acuity of the right eye improved from 0.8 to more than 1.0. CONCLUSIONS: These results suggest that the choroidal circulation disturbance, which was found after the encircling procedure, had a plausible role in the development of the visual field defect.