Hajime Inomata

Expression of vascular endothelial growth factor in experimental choroidal neovascularization

Abstract• Background: Although the choroidal neovascularization (CNV) is a common pathologic feature of a number of different eye diseases, its pathological mechanisms have not been fully elucidated. We investigated the expression of vascular endothelial growth factor (VEGF) in CNV using an experimental primate model. • Method: CNV was induced by intense laser photocoagulation in four monkey eyes. Single eyes were enucleated at 1, 3, 7 or 14 days after photocoagulation and examined immunohistochemically for VEGF, macrophage antigen, von Willebrand factor and glial fibrillary acidic protein (GFAP). Expression of VEGF mRNA was examined byin situ hybridization. • Results: One day after photocoagulation, the normal structure of the outer portion of the retina and the inner portion of the choroid was destroyed. Three days after photocoagulation, choroidal vascular endothelial cells migrated into the subretinal space through the defect in Bruchs membrane. Increased expression of VEGF was detected in the accumulating macrophages, migrating retinal pigment epithelial (RPE) cells and Muller cells. Maximal expression of VEGF was observed between 3 and 7 days after wounding, and many newly formed vessels extended into the subretinal space 7–14 days after photocoagulation. • Conclusion: VEGF derived from RPE cells, macrophages and Müller cells may play a role in the formation of CNV.

Vascular Endothelial Growth Factor Plays a Role in Hyperpermeability of Diabetic Retinal Vessels

In rat diabetic retinas, we immunohistochemically looked for vascular endothelial growth factor (VEGF) which is also known as vascular permeability factor (VPF). In nondiabetic retinas, VEGF immunoreactivity was weak and restricted to the nerve fiber and ganglion cell layers. On the other hand, in diabetic retinas, VEGF immunoreactivity was markedly increased and was observed in all layers of the retina, especially in the perivascular area. Hyperpermeability of these vessels was confirmed by immunohistochemically detecting extravasation of albumin. These findings indicate that vascular endothelial growth factor plays an important role in blood-retinal barrier breakdown in diabetic retinopathy.

The relationship between accumulation of advanced glycation end products and expression of vascular endothelial growth factor in human diabetic retinas

Summary Both advanced glycation end products and vascular endothelial growth factor are believed to play a role in the pathogenesis of diabetic retinopathy. It is known that vascular endothelial growth factor causes retinal neovascularization and a breakdown of the blood-retinal barrier; how advanced glycation end products affect the retina, however, remains largely unclear. The substance Ne-(carboxymethyl)lysine is a major immunologic epitope, i. e. a dominant advanced glycation end products antigen. We generated an anti-Ne-(carboxymethyl)lysine antibody to investigate the relationship between the localization of advanced glycation end products and that of vascular endothelial growth factor in 27 human diabetic retinas by immunohistochemistry. Nine control retinas were also examined. In all 27 diabetic retinas, Ne-(carboxymethyl)lysine was located in the thickened vascular wall. In 19 of the 27 retinas, strand-shaped Ne-(carboxymethyl)lysine immunoreactivity was also observed around the vessels. In all 27 diabetic retinas, vascular endothelial growth factor revealed a distribution pattern similar to that of Ne-(carboxymethyl)lysine. Vascular endothelial growth factor was also located in the vascular wall and in the perivascular area. Neither Ne-(carboxymethyl)lysine nor vascular endothelial growth factor immunoreactivity was detected in the 9 control retinas. Vessels with positive immunoreactivity for Ne-(carboxymethyl)lysine and/or vascular endothelial growth factor were counted. A general association was noted between accumulation of Ne-(carboxymethyl)lysine and expression of vascular endothelial growth factor in the eyes with non-proliferative diabetic retinopathy (p < 0.01) and proliferative diabetic retinopathy (p < 0.05). [Diabetologia (1997) 40: 764–769]

Relocalization of Apoptosis-Inducing Factor in Photoreceptor Apoptosis Induced by Retinal Detachment in Vivo

Apoptosis-inducing factor (AIF) is a novel mediator in apoptosis. AIF is a flavoprotein that is normally confined to the mitochondrial intermembrane space, yet translocates to the nucleus in several in vitro models of apoptosis. To investigate the role of AIF in the apoptotic process in vivo, we induced retinal detachment (RD) by subretinal injection of sodium hyaluronate, either in Brown Norway rats or in C3H mice. Apoptotic DNA fragmentation, as determined by terminal nick-end labeling, was most prominent 3 days after RD. The subcellular localization of AIF was examined by immunohistochemistry and immunoelectron microscopy. In normal photoreceptor cells, AIF was present in the mitochondrion-rich inner segment. However, AIF was found in the nucleus after RD. Photoreceptor apoptosis developed similarly in C3H control mice, and in mice bearing the gld or lpr mutations, indicating that cell death occurs independently from the CD95/CD95 ligand system. Both the mitochondrio-nuclear transition of AIF localization and the nuclear DNA fragmentation were inhibited by subretinal application of brain-derived neurotrophic factor. To our knowledge, this is the first description of AIF relocalization occurring in a clinically relevant, in vivo model of apoptosis.

Targeted gene transfer to corneal endothelium in vivo by electric pulse

A novel method of in vivo targeted gene transfer to intentionally selected areas of the corneal endothelium was developed. Plasmid DNA with the lacZ gene coding for β-galactosidase was injected into the anterior chamber of adult Wistar rats, and eight pulses of electricity at intensities ranging from 5 to 40 V/cm were delivered for 50 ms to the cornea with a specially designed electric probe in order to determine the effect of gene transfer on the corneal endothelial cells. Gene expression was visualized by enzymatic color reaction using X-gal in enucleated eyes on days 1, 3, 7, 14 and 21 after gene transfer. The treated eyes were then photographed and the X-gal-positive areas were evaluated by an image analyzer. The ratios of the areas (X-gal-positive area/area of entire corneal endothelium × 100%) were then calculated to determine gene transfection efficiency. The expression of β-galactosidase was clearly detected in the cytoplasm of the corneal endo- thelial cells as early as day 1 and lasted until day 21. The most intense gene expression was observed on days 1 and 3 (5.21% on day 1 and 6.45% on day 3). The expression of β-galactosidase on day 3 was most evident following delivery of 20 V electric pulses (0.09% at 5 V, 0.03% at 10 V, 6.45% at 20 V). β-Galactosidase expression was limited to the corneal endothelial cells in highly selected areas and no β-galactosidase expression was detected in any other intra- or extraocular tissues. In addition, no cell damage was apparent in the cornea and no inflammation was detected in any other intraocular tissues. Thus, low-voltage electric pulses successfully transferred the gene of interest to highly selective areas of the corneal endothelium without inducing any pathological changes. This targeted gene transfer method appears to have great potential for use in gene therapy for ocular diseases.

Formation of Drusen in the Human Eye

Light and electron microscopy of drusen formation in the human eye showed yellow-white spots in the fundus with two morphologic patterns: that of typical drusen and a nodular accumulation of cellular components beneath the retinal pigment epithelial cells. By electron microscopy, the progression of drusen formation could be classified into four stages. Stage I showed budding or evagination of retinal pigment epithelial cells into the subpigment epithelial space. This evaginated portion was connected to the retinal pigment epithelial cell cytoplasm and was surrounded by its basement membrane. In Stage II the evaginated portion of the cell was completely separate from the cytoplasm of its parent retinal pigment epithelial cell. In Stage III, the evaginated portion showed degeneration and disintegration. Finally, in Stage IV, an accumulation of vesicular, granular, tubular, and linear material was seen free within the nodular space beneath the retinal pigment epithelial cell.

Critical role of photoreceptor apoptosis in functional damage after retinal detachment

Purpose. Although apoptosis is assumed to play a pivotal role in retinal function loss, its mechanism and real influence on retinal function are still unclear. To investigate the relation between retinal function and apoptosis, we studied photoreceptor apoptosis in experimental retinal detachment (RD). Methods. We induced RD by subretinal injection of sodium hyaluronate in Brown Norway rats. Apoptotic photoreceptors were detected by TdT-dUTP Terminal Nick-End Labeling (TUNEL). To evaluate the function of the detached retina, electroretinograms (ERGs) were taken on day 1, 3 with corneal electrodes and full-field stimulation. Results. Apoptotic DNA fragmentation appeared 12 hours after RD, was most prominent on day 3, and decreased thereafter. The ERGs showed that the amplitudes of dark-adapted a-waves and light adapted 2Hz b-waves decreased immediately after RD and continued to decrease over time. The administration of Fas/Fc chimera recombinant protein or a caspase inhibitor, Z-VAD.fmk, failed to prevent either photoreceptor apoptosis or retinal functional damage. In contrast, brain derived neurotrophic factor (BDNF) and basic fibroblast growth factor (bFGF) significantly impeded both apoptosis and dysfunction. The ERGs recognized the functional changes sensitively, and these ERG changes correlated well to the amount of photoreceptor apoptosis. Immunohistochemical study showed that apoptosis-inducing factor (AIF), a novel caspase-independent apoptotic factor, was relocalized from mitochondria to the nucleus in this process. Conclusions. The present results showed that apoptosis was a key phenomenon in the retinal dysfunction in RD and that this process was transmitted mainly by mitochondria-dependent pathways rather than Fas/Fas-L or downstream caspase dependent pathways.

Experimental subretinal neovascularization is inhibited by adenovirus-mediated soluble VEGF/flt-1 receptor gene transfection: a role of VEGF and possible treatment for SRN in age-related macular degeneration

Accumulating evidence has shown the importance of vascular endothelial growth factor (VEGF) in chorioretinal angiogenesis. However, whether or not VEGF is indeed critical for the pathogenesis of subretinal neovascularization (SRN) in adulthood, which is a serious complication of age-related macular degeneration, has to be further investigated. We constructed an adenovirus expressing an entire ectodomain of the human VEGF receptor/flt-1 fused to Fc portion of human IgG (Adflt-ExR): this soluble receptor is secreted from Adflt-ExR-transfected cells. We studied the effect of Adflt-ExR on the formation of experimental SRN. Experimental SRN was induced by intense photocoagulation on the retina in pigmented rats and Adflt-ExR was injected into the femoral muscle. The formation of SRN assessed by fluorescein angiography was more significantly inhibited for 7 days in the Adflt-ExR-treated rats than in the control rats who received either an adenovirus vector encoding LacZ gene or balanced salt solution (BSS). The serum concentration of this soluble receptor increased for 7 days and thereafter gradually decreased. An immunohistochemical study disclosed the fibroblast cell proliferation and inflammatory cell infiltration to be reduced in the photocoagulation spot of Adflt-ExR-treated rats. VEGF plays a crucial role in the formation of SRN and VEGF soluble receptor gene transfection can inhibit SRN. This method will contribute to future gene therapy for age-related macular degeneration.

Hypoxia-induced expression of vascular endothelial growth factor by retinal glial cells promotes in vitro angiogenesis

To determine whether retinal glial cells (RGCs) participate in the paracrine regulation of retinal neovascularization, we investigated whether cultured RGCs synthesize and release vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) under normoxic or hypoxic conditions. Northern blot analysis demonstrated that cultured RGCs transcribed both VEGF mRNA with two molecular bands approximately 3.9 and 4.3 kilobases (kb), and bFGF mRNA with approximately 3.7 and 6.0 kb. The expression of VEGF mRNA was greatly enhanced by hypoxic cultivation (2% oxygen) when compared with normoxic cultivation (20% oxygen), while the expression of bFGF mRNA by RGCs was not significantly affected by hypoxia. The effects of RGCs-conditioned media (CM) on tritiated-thymidine incorporation and in vitro angiogenesis by retinal capillary endothelial cells (RECs) in producing the formation of capillary-like tubes in type I collagen gels, were evident in the observation that RGCs-CM harvested after hypoxic cultivation significantly enhanced tritiated-thymidine incorporation (1.9 times, P<0.01) and in vitro angiogenesis (2.4 times, P<0.01) compared with the normoxic RGCs-CM. These enhancing effects of RGCs-CM at hypoxia were suppressed by anti-VEGF neutralizing antibody. Furthermore, RECs were shown to express mRNA encoding the VEGF receptor flt-1 by northern blot analysis. These results suggest that VEGF expressed by RGCs under hypoxic conditions plays an integral role in the initiation and progression of retinal neovascularization in a paracrine manner.

Histologic Findings and Prognosis of Uveal Malignant Melanoma in Japanese Patients

PURPOSE To determine factors of uveal malignant melanoma that would help to predict prognosis in Japanese patients. METHODS From 1969 to 1994, 23 eyes with uveal malignant melanoma were enucleated from 23 Japanese patients, and 16 of these patients were studied. In the 16 patients, enucleated eyes were evaluated for tumor characteristics, including cell type and microvascular architecture. Patient records were reviewed for follow-up therapy and outcome. Mortality rate was evaluated four years after enucleation. RESULTS In Japanese patients with uveal malignant melanoma, the average tumor size was larger (average largest diameter, 11.36 mm; average increase, 6.25 mm) and the average patient age was younger (55.2 years old) than previously reported for white populations. Patients with spindle cell type melanoma tended to have a better prognosis than those with epithelioid cell type (four-year mortality rates: spindle cell type, 0%; mixed cell type, 43%; epithelioid cell type, 66%). Microvascular architecture patterns interpreted as either network or closed loop patterns were associated with a poorer prognosis (network pattern, P = .03; closed loop pattern, P = .005). CONCLUSIONS The prognosis for Japanese patients with uveal malignant melanoma is poorer, and epithelioid cell-type melanoma is more common, than has been reported in white populations. Mitotic figures are well correlated with poor prognosis, as are two microvascular patterns, the network pattern and the closed loop pattern.