Yoshihisa Oguchi

VEGF164-mediated Inflammation Is Required for Pathological, but Not Physiological, Ischemia-induced Retinal Neovascularization

Hypoxia-induced VEGF governs both physiological retinal vascular development and pathological retinal neovascularization. In the current paper, the mechanisms of physiological and pathological neovascularization are compared and contrasted. During pathological neovascularization, both the absolute and relative expression levels for VEGF164 increased to a greater degree than during physiological neovascularization. Furthermore, extensive leukocyte adhesion was observed at the leading edge of pathological, but not physiological, neovascularization. When a VEGF164-specific neutralizing aptamer was administered, it potently suppressed the leukocyte adhesion and pathological neovascularization, whereas it had little or no effect on physiological neovascularization. In parallel experiments, genetically altered VEGF164-deficient (VEGF120/188) mice exhibited no difference in physiological neovascularization when compared with wild-type (VEGF+/+) controls. In contrast, administration of a VEGFR-1/Fc fusion protein, which blocks all VEGF isoforms, led to significant suppression of both pathological and physiological neovascularization. In addition, the targeted inactivation of monocyte lineage cells with clodronate-liposomes led to the suppression of pathological neovascularization. Conversely, the blockade of T lymphocyte–mediated immune responses with an anti-CD2 antibody exacerbated pathological neovascularization. These data highlight important molecular and cellular differences between physiological and pathological retinal neovascularization. During pathological neovascularization, VEGF164 selectively induces inflammation and cellular immunity. These processes provide positive and negative angiogenic regulation, respectively. Together, new therapeutic approaches for selectively targeting pathological, but not physiological, retinal neovascularization are outlined.

Leukocytes mediate retinal vascular remodeling during development and vaso-obliteration in disease

Retinal ischemia can cause vision-threatening pathological neovascularization. The mechanisms of retinal ischemia are not fully understood, however. Here we have shown that leukocytes prune the retinal vasculature during normal development and obliterate it in disease. Beginning at postnatal day 5 (P5) in the normal rat, vascular pruning began centrally and extended peripherally, leaving behind a less dense, smaller-caliber vasculature. The pruning was correlated with retinal vascular expression of intercellular adhesion molecule-1 (ICAM-1) and coincided with an outward-moving wave of adherent leukocytes composed in part of cytotoxic T lymphocytes. The leukocytes adhered to the vasculature through CD18 and remodeled it through Fas ligand (FasL)-mediated endothelial cell apoptosis. In a model of oxygen-induced ischemic retinopathy, this process was exaggerated. Leukocytes used CD18 and FasL to obliterate the retinal vasculature, leaving behind large areas of ischemic retina. In vitro, T lymphocytes isolated from oxygen-exposed neonates induced a FasL-mediated apoptosis of hyperoxygenated endothelial cells. Targeting these pathways may prove useful in the treatment of retinal ischemia, a leading cause of vision loss and blindness.

Autologous serum eye drops for the treatment of severe dry eye in patients with chronic graft-versus-host disease

Summary:We investigated the efficacy and safety of autologous serum eye drops for the treatment of severe dry eye after allogeneic haematopoietic stem cell transplantation (SCT). A total of 14 patients (four males and 10 females; median age, 31.0 years) with severe dry eye associated with chronic graft-versus-host disease (cGVHD) were enrolled in this study. All patients were refractory to treatment with conventional artificial tears. Autologous serum eye drops, a solution made of 20% autologous serum in sterile saline, were applied 10 times per eye per day. The patients were evaluated every 4 weeks according to visual acuity, corneal sensitivity, vital staining of the ocular surface, tear dynamics, and subjective assessments of symptoms (complaints scores). The median follow-up period was 19.4 months (range: 4–41 months). After 4 weeks of treatment, significant improvement was observed in both complaint scores (from 33.7±12.3 to 23.6±10.6 points; P<0.01) and fluorescein scores (from 5.8±2.0 to 2.4±0.9 points; P<0.005). Significant improvements were observed also in rose-bengal staining and tear break-up time. In seven of the 14 patients, the responses were maintained for 6–41 months (median:19.4±8.3 months), while six of the other seven patients required treatment with punctal plugs in addition to autologous serum eye drops. One of these other seven patients developed eczema around the eyelids, after which the treatment was discontinued. No serious adverse events were observed. We conclude that autologous serum eye drops are safe and effective for treating severe dry eye associated with cGVHD and that more efficient control of dry eye may be achieved by the combined use of autologous serum eye drops with punctal plugs.

Dry eye after haematopoietic stem cell transplantation

AIMS To determine the incidence, natural course, and severity of dry eye occurring or worsening after haematopoietic stem cell transplantation (SCT). METHODS At a tertiary care hospital, 53 patients undergoing allogeneic or autologous SCT followed by at least 180 days of follow up were studied prospectively. Examination included grading of symptoms of dry eye, evaluation of ocular surface, tear break up time, and Schirmer tests with and without nasal stimulation. Meibomian gland secretion was also examined using a slit lamp while applying steady digital pressure. RESULTS Of the 53 patients, 44 received allografts. Half of these patients (22) developed dry eye or their pre-existing dry eye worsened after SCT, while none of nine autograft recipients did. Onset of dry eye was 171 (SD 59) days after SCT. Two types of dry eye occurred. One (n=10) was severe with ocular surface findings resembling Sjögren’s syndrome and reduction of reflex tearing soon after onset. A mild type (n=12) had unimpaired reflex tearing. Meibomian gland dysfunction (MGD) was more frequent and severe in patients with dry eye and chronic graft versus host disease (GVHD), and overall severity of dry eye was greater in patients with MGD and chronic GVHD. CONCLUSIONS Dry eye after SCT occurred only in allograft recipients, and was not evident in autograft recipients. The severe form of dry eye had a tendency to develop rapidly. Further study on the prediction and treatment of severe dry eye after SCT is necessary.

Do idebenone and vitamin therapy shorten the time to achieve visual recovery in Leber hereditary optic neuropathy

Objectives: The authors investigated the effectiveness of idebenone combined with vitamin B2 and vitamin C in the treatment of patients with Leber hereditary optic neuropathy (LHON) in an early stage as compared with untreated patients with LHON. These agents may stimulate the formation of ATP. Materials and Methods: For this retrospective study, the authors selected 28 outpatients with LHON from the Keio University Hospital. These patients were followed for 2 to 19 years from disease onset. They were divided into two groups: 14 untreated patients (11778 mutation in 10 patients, 3460 mutation in 2 patients, and 14484 mutation in 2 two patients); and 14 treated patients (11778 mutation in 11 patients, 3460 mutation in 1 patient, and 14484 mutation in 2 patients). The treated patients were administered medical treatment with idebenone, vitamin B2, and vitamin C for at least 1 year. The current study evaluated the following: 1) number of eyes with visual recovery ≥0.3; 2) interval between the onset of LHON and the beginning of visual recovery; 3) interval between the onset of LHON and visual recovery to 0.3; and 4) interval between the beginning of medical treatment and the beginning of visual recovery in the treated subjects. Results: There was no significant difference in the number of eyes with visual recovery ≥0.3 in the two groups with the 3460, 11778, or 14484 mutation. Patients with visual recovery showed a fenestrated scotoma or a clearing of central vision. The mean interval between the onset of LHON and the beginning of visual recovery was significantly shorter in the treated group (11.1 months) than in the untreated group (17.4 months) (P = 0.03). The mean interval between the onset of LHON and visual recovery to 0.3 was significantly shorter in the treated group (17.6 months) than in the untreated group (34.4 months) (P = 0.01). The mean interval between the initiation of medical treatment to the beginning of visual recovery was 5.4 months. Conclusions: Results suggest that the administration of idebenone, vitamin B2, and vitamin C sped the recovery of vision in patients with LHON.

Spectrum of pathogenic mitochondrial DNA mutations and clinical features in Japanese families with Leber's hereditary optic neuropathy

PURPOSE To investigate the incidence and clinical significance of primary or proposed secondary mitochondrial DNA (mtDNA) mutations in Japanese patients with Lebers hereditary optic neuropathy (LHON). METHODS Blood samples from the 80 unrelated Japanese patients with bilateral optic atrophy were screened for primary LHON mutations. Patients found to have a primary LHON mutation were then tested for 9 proposed secondary LHON mutations. We investigated the association between these mutations and clinical characteristics. RESULTS Primary mtDNA mutations were identified in 68 patients: at np 3460 in 3 (4%) of 68 patients, at np 11,778 in 59 patients (87%), and at np 14,484 in 6 patients (9%). We identified 5 secondary mtDNA mutations (at np 3394, 4216, 7444, 9438 or 13,708) in 10 (15%) of 68 LHON patients and 3 mutations (at np 3394, 4216 or 3708) in 6 (7%) of 90 healthy Japanese individuals. No patient was positive for more than one secondary mutation. The frequency of secondary mutations was similar in the 68 LHON patients and 90 controls. The clinical features of the Japanese patients with any of the 3 primary LHON mutations were similar to those of Caucasian patients, despite different mtDNA backgrounds in these populations. The percentage of patients with familial LHON harboring the 3460 or 14,484 mutations was lower in the Japanese population. CONCLUSIONS Japanese patients with LHON exhibited a very high incidence (87%) of the 11,778 primary mutation. Most of the proposed secondary LHON mutations were rare in the Japanese population and they, except the 7444 mutation, may not influence the clinical features of LHON.

Use of High-speed, High-resolution Thermography to Evaluate the Tear Film Layer

PURPOSE To evaluate the tear film layer in patients with dry eye and in normal subjects by measuring the corneal temperature with infrared radiation thermography. METHODS One eye of each of 13 patients with dry eye and one eye of each of seven normal subjects were evaluated randomly. The corneal temperature was measured continuously with a recently improved infrared radiation thermography technique. We calculated the k value, which reflected the steepness of the corneal temperature change. The bigger the k value was, the more rapid was the decrease in corneal temperature, and this was directly related to increased evaporation. RESULTS With normal blinking, the mean k value for patients with dry eye (5.6 +/- 2.9 per second) was significantly less than that in the control subjects (9.3 +/- 5.0 per second; P < .05). Keeping the eyes open after closing the eyes significantly decreased the k values compared with normal blinking in both groups (P < .05). CONCLUSIONS Our findings demonstrate the usefulness of this method of measuring corneal temperature to evaluate the tear film layer. High-speed, high-resolution thermography detected subtle changes in corneal temperature with enhanced sensitivity and spatial and temporal resolution. We found that the mean k value, and therefore the rate of decline in corneal temperature in patients with dry eye, was significantly less than that in normal subjects. The k value may therefore reflect tear film layer stability. The measurement of the changes in the corneal temperature can thus give us valuable information on the tear film layer.

Detection of human papillomavirus infection in squamous tumours of the conjunctiva and lacrimal sac by immunohistochemistry, in situ hybridisation, and polymerase chain reaction

BACKGROUND Squamous tumours of the ocular surface, including the lacrimal pathway, range from benign lesions to invasive carcinomas. Some of these tumours are associated with human papillomavirus (HPV) infection, with the types of HPV differing among papillomas and dysplastic or malignant lesions. METHODS The relation between squamous tumours of the conjunctiva and lacrimal sac and HPV infection was investigated in 17 individuals with such tumours. Nine of the 17 tumours were benign, four were dysplastic lesions, and four were carcinomas. RESULTS Eight specimens showed positive immunohistochemical staining with antibodies to HPV; four of these eight were papillomas, three were dysplastic lesions, and one was a carcinoma. Koilocytosis was detected in seven of these eight tumours. Five of the eight specimens positive for immunohistochemical staining were also positive for HPV DNA by in situ hybridisation, and all eight were positive for HPV DNA by the polymerase chain reaction (PCR) method. CONCLUSION Approximately 50% of squamous tumours of the ocular surface and lacrimal sac were associated with HPV infection. This is the first report, to our knowledge, of the detection of HPV in the field of ophthalmology by a combination of immunohistochemistry, in situ hybridisation, and PCR.

Macular hole retinal detachment in highly myopic eyes: ultrastructure of surgically removed epiretinal membrane and clinicopathologic correlation.

Purpose: To elucidate the pathogenesis of macular hole retinal detachment (RD) in highly myopic eyes by investigating the ultrastructure of surgically removed epiretinal membranes (ERM). Methods: Five consecutive Japanese patients with macular hole RD in highly myopic eyes underwent vitrectomy with attempted removal of the ERM around the hole. The surgical specimens were examined by light microscopy and by scanning and transmission electron microscopy. Results: Extremely thin, translucent sheets of epiretinal tissue were harvested from all patients during surgery, resulting in successful retinal reattachment. Ultrastructural examination revealed that the ERM consisted of cortical vitreous and various cellular components. Fibrous astrocytes were the major cell population and extended cytoplasmic processes with membrane‐associated vesicles onto the cortical vitreous. Gap junctions were observed between the interdigitating processes of fibrous astrocytes. The cortical vitreous contained abundant newly formed collagen, including fibrous long‐spacing collagen, surrounded by sparsely distributed native vitreous collagen. Conclusions: Active synthesis of new collagen may be regulated by fibrous astrocytes by means of transmission of metabolic substances through gap junctions and cytoplasmic vesicles. The frequent occurrence of newly formed collagen aggregates may subsequently lead to a diffusely condensed posterior cortical vitreous that exerts tangential traction on the posterior retina, causing macular hole RD.

Interleukin-8 concentrations in conjunctival epithelium brush cytology samples correlate with neutrophil, eosinophil infiltration, and corneal damage.

Purpose. To evaluate whether interleukin-8 (IL-8) and RANTES (regulated on activation, normal T-cell expressed and secreted) concentrations in the supernatants of conjunctival epithelial samples from patients with vernal keratoconjunctivitis (VKC) correlate with the number of infiltrating eosinophils or neutrophils and with the severity of corneal lesions. Methods. Thirty-four patients with VKC, 5 patients with seasonal allergic conjunctivitis, and 10 volunteers without allergic diseases were enrolled in this study. Conjunctival epithelial cells were collected by brush cytology and the number of inflammatory cells was counted. The chemokine expression in the cells was investigated by immunocytochemistry and the chemokine concentrations of the cell suspensions were measured by enzyme-linked immunosorbent assay. Results. The percentages of eosinophils and neutrophils in cell suspensions from VKC patients with corneal erosion or ulcer were higher than those from subjects with clear corneas or superficial punctate keratopathy. IL-8 concentrations in the supernatant of samples correlated significantly with the percentages of neutrophils and eosinophils in paired cell suspensions. No correlation was observed between RANTES and the percentages of eosinophils. Positive staining for IL-8 was observed in the cytosol of conjunctival epithelial cells. Conclusion. IL-8 in the extracellular space of the conjunctival epithelium may play a role in the recruitment of neutrophils and possibly eosinophils and in the pathogenesis of corneal damage in severe allergic diseases.