J.C. Jeanny

Placental growth factor-1 and epithelial haemato–retinal barrier breakdown: potential implication in the pathogenesis of diabetic retinopathy

Aims/hypothesisDisruption of the retinal pigment epithelial (RPE) barrier contributes to sub-retinal fluid and retinal oedema as observed in diabetic retinopathy. High placental growth factor (PLGF) vitreous levels have been found in diabetic patients. This work aimed to elucidate the influence of PLGF-1 on a human RPE cell line (ARPE-19) barrier in vitro and on normal rat eyes in vivo.MethodsARPE-19 permeability was measured using transepithelial resistance and inulin flux under stimulation of PLGF-1, vascular endothelial growth factor (VEGF)-E and VEGF 165. Using RT-PCR, we evaluated the effect of hypoxic conditions or insulin on transepithelial resistance and on PLGF-1 and VEGF receptors. The involvement of mitogen-activated protein kinase (MEK, also known as MAPK)/extracellular signal-regulated kinase (ERK, also known as EPHB2) signalling pathways under PLGF-1 stimulation was evaluated by western blot analysis and specific inhibitors. The effect of PLGF-1 on the external haemato–retinal barrier was evaluated after intravitreous injection of PLGF-1 in the rat eye; evaluation was by semi-thin analysis and zonula occludens-1 immunolocalisation on flat-mounted RPE.ResultsIn vitro, PLGF-1 induced a reversible decrease of transepithelial resistance and enhanced tritiated inulin flux. These effects were specifically abolished by an antisense oligonucleotide directed at VEGF receptor 1. Exposure of ARPE-19 cells to hypoxic conditions or to insulin induced an upregulation of PLGF-1 expression along with increased transcellular permeability. The PLGF-1-induced RPE cell permeability involved the MEK signalling pathway. Injection of PLGF-1 in the rat eye vitreous induced an opening of the RPE tight junctions with subsequent sub-retinal fluid accumulation, retinal oedema and cytoplasm translocation of junction proteins.Conclusions/interpretaionOur results indicate that PLGF-1 may be a potential regulation target for the control of diabetic retinal and macular oedema.

Protection against light-induced retinal degeneration by an inhibitor of NO synthase

The existence of nitric oxide synthase (NOS) in retinal rod outer segments and pigmented epithelial cells suggests that NO in excess could impair the interaction between these cells, resulting in photoreceptor degeneration. To test this hypothesis, NG-nitro-L-arginine methyl ester (L-NAME), an NOS inhibitor, was intraperitoneally injected daily into rats subjected to constant illumination for 7 days in order to destroy their photoreceptors. By measuring photoreceptor nuclear layer thicknesses, we found that L-NAME partially protects (by up to 35%) against the degeneration of photoreceptors and acts to maintain their organization. Thus NO may be involved in the process by which photoreceptor degeneration results from constant illumination of the retina.

Basic fibroblast growth factor experimentally induced choroidal angiogenesis in the minipig.

Basic fibroblast growth factor (bFGF), a soluble mitogen, has been isolated and purified from various organs, including the retina. In vivo angiogenic activity of bFGF has been demonstrated with several assays. An experimental model of choroidal neovascularization was developed in the mini pig by perfusion of recombinant human bFGF through an osmotic minipump. Endogenous bFGF and bFGF receptors were localized in the normal pig retina by immunohistochemistry and autoradiography after binding. The perfusion of exogenous bFGF induced well-organized new vessels along the last 3 mm of the catheter in the suprachoroidal space. This neovascularization did not penetrate the normal Bruchs membrane. Vascular cells (identified by von Willebrand factor antibody staining) increased in number and in surface from the proximal part to the end of the intraocular catheter in all bFGF perfused eyes. In eyes perfused with phosphate buffered saline (controls), but not in the bFGF perfused eyes, an inflammatory response occurred (identified by a macrophage specific antibody). These results demonstrate that choroidal angiogenesis can be achieved without an inflammatory response by perfusing an excess of bFGF in the suprachoroidal space.

Localization of acidic fibroblast growth factor (aFGF) mRNA in mouse and bovine retina by in situ hybridization.

Acidic fibroblast growth factor (aFGF) mRNA has been detected in adult mouse or bovine retina by in situ hybridization with bovine aFGF cDNA clones. It is localized on ganglion cell layer, inner nuclear layer, photoreceptors and slightly on pigmented epithelium. This synthesis of aFGF in highly specialized retinal cell types is discussed in the framework on current views about the role of FGF in retinal cell biology.

Immunohistochemical analysis of fibroblast growth factor receptor in bovine retina

The distribution of fibroblast growth factor (FGF) receptor in bovine retina was established using a polyclonal antibody against the extracellular domain of this receptor. Different conditions of tissue fixation and development of the secondary antibody were tested. The ability of the antiserum to map precisely the receptor was obtained on fresh-frozen sections which had been treated with paraformaldehyde prior to incubation with this antiserum. Positive staining was confined mainly to the synaptic and ganglion axon layers. These results suggest that the FGF receptor might act in the transmitter stability, and the plasticity of synapses and ganglion cell axons in adult retina.

Immunochemical analysis of extracellular matrix during embryonic lens development of the cat fraser mouse

We have characterized the extracellular matrix present during early mouse-lens morphogenesis in Swiss and Cat Fraser mutant mice which produces a thicker capsule. In the two mouse strains, laminin was first detected when the optic vesicle and the head ectoderm are closely associated. At day 10, staining for laminin and fibronectin is especially concentrated at the border of the lens pit. At this stage, type IV collagen and proteoheparan sulphate have a similar distribution to laminin and fibronectin. In the two mouse strains, no major differences were observed in the intensity and the distribution of fluorescent basement-membrane components. This suggests that the overall increase in capsule thickness of the Cat Fraser mutant is more related to an increased cellular synthesis of capsule than to an abnormal distribution of one or more basement-membrane macromolecules.

Glial cell localization of acidic fibroblast growth factor-like immunoreactivity in the optic nerve of young adult and aged mammals.

The number of axons in the optic nerve decreases with age and this degeneration is greater in patients suffering from Alzheimers disease. Alterations in the role of neurotrophic factors could lead to this degeneration. Acidic fibroblast growth factor (aFGF)-like immunoreactivity was examined by indirect immunofluorescence on cryostat sections incubated with a rabbit polyclonal antiserum specific for aFGF. Staining was observed by photonic microscopy on optic nerves of Wistar rats (1- to 25-month-old), bovine animals (0.5- to 7-year-old) and normal human adults (24-, 34-, 54- and 84-year-old). In the three species studied, the results show that (1) glial cells were stained in the nuclear region and (2) aFGF-like immuno-reactivity was present over a large age span in adult subjects. Endogenous aFGF may have trophic effects on retinal ganglion cells and their axons throughout the adult life span.

Distribution of transferrin and transferrin receptor of the eype 1 in the process of formation of the rat eye retina in early postnatal ontogenesis

By the method of indirect immunohistochemistry, distribution of transferrin and of transferrin receptor of the type 1 (TFR1) was studied in the formed rat eye retina at the period of early postnatal ontogenesis (from birth to opening of eyelids). It has been established that the character of distribution of these proteins and intensity of specific staining change dependent on the retina formation stage. Retina of the newborn rat is characterized by diffuse transferrin distribution in nuclear retina layer (in the neuroblast layer-NBL) and in the ganglionic cell layer (GCL) as well as in the eye pigment epithelium (PE); relative immunoreactivity to transferrin is not high. At the 5th postnatal day, immunoreactivity to transferrin is maximal and is revealed both in nuclear and in plexiform layers of retina and in the eye PE, the greatest signal being characteristic of NBL. At the 10th postnatal day the transferrin signal intensity in retina decreases, specific staining is revealed in GCL, PE, and in the area of formed outer segments of photoreceptors. At the 15th postnatal day, transferrin is revealed in GCL, in outer and inner photoreceptor segments and in the eye PE. TFR1 is present in all retina layers at all stages of the retina formation; the relative immunoreactivity to TFR1 sharply rises beginning from the 10th postnatal day; correlation between distribution of transferrin and TFR1 is detected in the entire retina of newborn rats as well as in the external retina area at subsequent stages of its development. A possible role of transferrin at various stages of formation of retina is discussed.

Histopathology of Peripapillary Choroidal Neovascularization

The different therapeutic responses observed among choroidal neovascularization (CNV) of different etiologies, ages, and locations might be related to the presence of varied mediators. Two surgically removed peripapillary CNVs from two different patients were analyzed. One of the patients had received one intravitreous injection of bevacizumab 3 months earlier. CNV was analyzed using conventional histology and immunohistochemistry. Histological analysis showed intense neovascularization and epithelial and glial components. Vascular endothelial growth factor (VEGF) receptors were found in the endothelial cells and the epithelial cells of the CNV. VEGF was expressed in the patient who had not been previously treated with anti-VEGF. The CNV was deeply infiltrated by glial cells and invaded by microglial cells in one case. VEGF and VEGF receptors may be expressed, suggesting that therapies aiming at VEGF may be efficient only for a subtype of CNV and at a certain time point of their evolution.

Distribution of Proteins Providing Homeostasis of Iron Ions in Bovine Retina

Distribution of proteins providing homeostasis of iron ions in bovine retina was studied by methods of indirect immunohistochemistry, which allowed detection of localization of transferrin, ferritin, and transferrin receptor. In bovine retina, transferrin is revealed in the region of outer and inner segments of photoreceptors and in the external plexiform layer. Distributions of ferritin and transferrin receptor are identical; they are revealed in all layers of retina, the maximal immunoreactivity against these proteins is found in pigment epithelium, in the region of inner segments of photoreceptors, in the external plexiform and internal nuclear layers. The obtained results are discussed from the point of view of mechanisms providing with iron the cells of the outer and inner retina.