Miyo Matsumura

Hypoxia and Vascular Endothelial Growth Factor Selectively Up-regulate Angiopoietin-2 in Bovine Microvascular Endothelial Cells

Recent studies have shown that the angiopoietin-Tie2 system is a predominant regulator of vascular integrity. In this study, we investigated the effect of two known angiogenic stimuli, hypoxia and vascular endothelial growth factor (VEGF), on these molecules. VEGF induced both a time- and concentration-dependent increase in angiopoietin-2 (Ang2) mRNA expression in bovine microvascular endothelial cells. This up-regulation was derived primarily from an increased transcription rate as evidenced by nuclear run-on assay and mRNA decay study. The increased Ang2 expression upon VEGF treatment was almost totally abolished by inhibition of tyrosine kinase or mitogen-activated protein kinase and partially by suppression of protein kinase C. Hypoxia also directly increased Ang2 mRNA expression. In contrast, Ang1 and Tie2 responded to neither of these stimuli. The enhanced Ang2 expression following VEGF stimulation and hypoxia was accompanied by de novo protein synthesis as detected by immunoprecipitation. In a mouse model of ischemia-induced retinal neovascularization, Ang2 mRNA was up-regulated in the ischemic inner retinal layer, and remarkable expression was observed in neovascular vessels. These data suggest that both hypoxia- and VEGF-induced neovascularization might be facilitated by selective induction of Ang2, which deteriorates the integrity of preexisting vasculature.

Retinal Nerve Fiber Layer Defect as an Early Manifestation of Diabetic Retinopathy

PURPOSE An incidence of and risk factors for retinal nerve fiber layer defect were investigated in patients with type II diabetes mellitus and compared with that of age-matched control subjects. METHODS The authors photographed the retinal nerve fiber layer of the right eye in each of 137 patients with diabetes and 144 healthy control subjects. The level of diabetic retinopathy ranged from levels 1 (no microaneurysm) to 4 (eyes with localized intra-retinal microvascular abnormalities or venous beading). Risk factors for the nerve fiber layer defect analyzed were age of patients, visual acuity, axial length, optic disc size, glycosylated hemoglobin, systolic blood pressure, and level of diabetic retinopathy. RESULTS Defects of the retinal nerve fiber layer were found in 6/30 (20%) eyes with level 1 retinopathy, 8/14 (57%) eyes with level 2 retinopathy, 24/47 (51%) eyes with level 3 retinopathy, and 36/46 (78%) eyes with level 4 retinopathy. These defect incidences were significantly higher than that of the control group, which had 5/144 (3.5%) defects (P < or = 0.001). Risk factors for this nerve defect were level of diabetic retinopathy (P = 0.002), high systolic blood pressure (P = 0.0232), and patients age (P = 0.0478). Not correlated with the incidence of the retinal nerve fiber layer defect were visual acuity, disc size, axial length, and glycosylated hemoglobin level at the time of examination. CONCLUSION These findings suggest that the retinal nerve fiber layer defect is common in patients with early diabetic retinopathy. Risk factors for this defect were higher level of diabetic retinopathy, systemic hypertension, and advanced age.

Pigment epithelium derived factor as a neuroprotective agent against ischemic retinal injury.

Purpose. Pigment epithelium-derived factor (PEDF) is a protein shown to have neurotrophic activity. The purpose of this study was to determine whether PEDF is neuroprotective of retinal neurons that are exposed to transient ischemia-reperfusion. Methods. Transient retinal ischemia was produced by increasing the intraocular pressure for 45 min in albino rats eyes. Immediately after reperfusion, PEDF was injected intravitreally into the experimental eyes. Injury was evaluated morphologically and by measuring the thickness of the inner retinal layers (IRL) and by counting the number of retinal ganglion cells (RGC) in epon embedded sections. Results. Morphologic and morphometric analysis of the thickness of the IRL and the counting of RGC demonstrated that PEDF injected immediately after reperfusion protected the eyes partially but significantly from the ischemic injury. Cocnlusions. Intravitreal injection of PEDF even after the ischemia can ameliorate retinal injury. PEDF may be useful in preventing neuronal degeneration in the inner retina resulting from ischemia.

Upregulation of pigment epithelium-derived factor after laser photocoagulation

PURPOSE To determine the changes in the expression of pigment epithelium-derived factor in cultured human retinal pigment epithelial cells and rat retinas after laser photocoagulation. METHODS Experimental study of laser photocoagulation on human retinal pigment epithelial cells in culture and on adult rats. Reverse transcription-polymerase chain reaction and semiquantitative polymerase chain reaction analysis were used. RESULTS After photocoagulation, the mRNA expression of pigment epithelium-derived factor was upregulated in human retinal pigment epithelial cells at 6 hours and then gradually decreased. Compared with controls, significantly higher levels of pigment epithelium-derived factor were observed in rat retinas from 6 to 24 hours after laser photocoagulation (P <.005), and they were still higher than before photocoagulation at 2 weeks. CONCLUSION An upregulation of pigment epithelium-derived factor in retinal pigment epithelial cells and in the retina after photocoagulation suggests that pigment epithelium-derived factor plays a role in inhibiting neovascularization by its antiangiogenic activity.

Müller cells in detached human retina express glial fibrillary acidic protein and vimentin

We investigated the expression of vimentin and glial fibrillary acidic protein (GFAP) within Müller cells in normal human retinas and in detached human retinas of proliferative vitreoretinopathy (PVR) cases using the immunogold method. Muller cells in normal retinas showed vimentin immunoreactivity and faint GFAP immunoreactivity; however, in detached retinas they showed distinct GFAP immunoreactivity as well as vimentin immunoreactivity. Immunoelectron microscopic observation revealed that intermediate filaments (IF) within Müller cells in normal retinas showed vimentin immunoreactivity and that those within Müller cells in detached retinas showed both vimentin and GFAP immunoreactivity. Double staining for vimentin and GFAP showed that in detached retinas, these two protein immunoreactivities were observed in the same filaments. These results indicate that IF of human Müller cells consist of vimentin under normal conditions and that Müller cells in detached retinas contain different IF, which consist of vimentin and GFAP.

Phagocytic Activity of Cultured Retinal Pigment Epithelial Cells from Chick Embryo: Inhibition by Melatonin and Cyclic AMP, and Its Reversal by Taurine and Cyclic GMP

Cultured chick retinal pigment epithelial cells phagocytosed polystyrene latex particles. The phagocytosis was inhibited very specifically by melatonin, which attained 50% inhibition at about 10(-16) M. Other indole compounds such as 5-methoxytryptophol, 5-hydroxytryptophol, 6-hydroxymelatonin, N-acetylserotonin, 5-methoxytryptamine and serotonin were also inhibitory although their effects were less than 1/10,000 that of melatonin. Possible retinal neurotransmitters, acetylcholine, gamma-aminobutyric acid, glycine, dopamine, aspartic acid and glutamic acid, had no or only a minimum inhibitory effect, and was also the case for prostaglandin D2, E2, F2 alpha, and I2. Taurine was not inhibitory at all. Among nucleotides, cyclic AMP specifically inhibited phagocytosis, giving 50% inhibition at about 10(-11) M. Melatonin inhibition was increased by copresence of isobutylmethylxanthine. Inhibition by either melatonin or cyclic AMP was reversed by dibutyryl cyclic GMP. The reversal was observed also with compounds which were expected to increase intracellular cyclic GMP. Prostaglandin D2 reversed inhibition in both cases, but its effect was incomplete and per se it had an inhibitory effect. Melatonin derivatives reversed inhibition by melatonin alone but not inhibition by cyclic AMP. Taurine efficiently reversed both kinds of inhibition. Other possible neurotransmitters were ineffective. Taurine was thus the most effective of these compounds. We suggest the following hypothetic control mechanism of phagocytic activity of the pigment epithelial cells: melatonin and cyclic AMP are intercellular and intracellular signals, respectively, of stopping phagocytosis, while taurine and cyclic GMP are intercellular and intracellular signals, respectively of cancelling this stop signal. Phagocytic activity of chick retinal pigment epithelial cells might be regulated by the concentration ratio of melatonin to taurine in the interphotoreceptor space.

Epiretinal Membrane of Proliferative Diabetic Retinopathy: An Immunohistochemical Study

Nineteen cases of epiretinal membrane (ERM) in proliferative diabetic retinopathy were studied using immunohistochemical methods. Antibodies against type I-IV collagen, fibronectin (FN), glial fibrillary acidic protein (GFAP), vimentin and the monoclonal antibody (MAB) against human Müller cells were used to examine the membranes. Type II collagen was found on one side of the ERMs in 95% of the cases. The other types of collagen, FN and vimentin were also identified in most cases. Müller cells (GFAP- and MAB-positive) were found in 2/19 cases (11%), and astrocytes (GFAP-positive but MAB-negative) were found in 10/19 cases (53%). These results suggest that type II collagen (vitreal collagen) may act as a scaffolding in the formation of ERMs and that glial elements of the ERMs consist mainly of astrocytes.

HISTOLOGICAL AND IMMUNOHISTOCHEMICAL STUDY OF IDIOPATHIC EPIRETINAL MEMBRANE

We used electron microscopy and light-microscopic immunohistochemistry of cellular and extracellular markers to characterize the cellular and extracellular components of 15 surgically resected idiopathic epiretinal membranes (IEMs). Ten specimens from the eyes with posterior vitreous detachment (PVD) consisted of inner limiting membrane, collagen layer and a flattened cell layer. Six out of the 10 specimens were also examined immunohistochemically, and fibronectin and type I, II, III and IV collagens were identified in a characteristic lamellar construct in the IEMs. On the other hand, 5 specimens obtained from the eyes without PVD consisted mainly of a thick layer of collagen fibrils with or without a flattened cell layer. Two of the 5 specimens were also examined immunohistochemically, and the collagen fibrils in the specimens were identified as type II collagen. Glial cells (glial fibrillary acidic protein-immunoreactive cells) were also identified in 3 specimens. These results indicate that there are some variations in the IEMs.

Multicenter genetic study of retinitis pigmentosa in Japan: I. Genetic heterogeneity in typical retinitis pigmentosa☆

A nationwide, multicenter study of typical retinitis pigmentosa (RP) was carried out in collaboration with 18 hospitals throughout Japan to obtain current information for genetic counseling. We analyzed the genetic heterogeneity of RP based on the parental consanguinity of 434 probands registered during a 6-month period in 1990. A gradual decline in the frequency of consanguineous marriage was recognized among the normal parents of RP patients. The relative frequencies of inheritance patterns were estimated as: autosomal recessive, 25.2%; autosomal dominant, 16.9%; X-linked, 1.6%; and simplex, 56.3%. A comparison of these results with previous reports in Japan revealed a decline in the relative frequency of autosomal recessive cases and an increase in simplex cases. This suggests a decrease in the incidence of autosomal recessive retinitis pigmentosa in Japan, as well as the necessity for exhaustive investigations aimed at identifying inheritance patterns for RP patients seeking genetic counseling.

Multicenter genetic study of retinitis pigmentosa in Japan: II. Prevalence of autosomal recessive retinitis pigmentosa

Retinitis pigmentosa (RP) is a group of genetically heterogeneous diseases with autosomal recessive (AR), autosomal dominant, and X-linked modes of inheritance. Autosomal recessive retinitis pigmentosa (ARRP) is the most common form in Japan. A genetic analysis was done to determine the prevalence of ARRP indirectly, to provide an estimation of changing trends in the overall prevalence of RP. Data on the frequency of consanguinity and marriage year of normal parents of 59 ARRP patients were obtained from a nationwide multicenter survey of typical retinitis pigmentosa conducted in 1990. The gene frequency of ARRP was 0.01145 (Dahlbergs formula). In 1990, the number of young symptomatic ARRP patients decreased, while the number of patients aged 40 years and older increased. The total number of symptomatic ARRP patients in 1990 was nearly 21% higher than in 1970. Despite a dramatic decline in consanguinity in recent decades in Japan, the number of ARRP patients has increased. This increase is attributed to greater longevity and overall population growth. Our results suggest that the total number of RP patients has not decreased, and may even have increased.