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Purpose The lysozyme 2 (Lyz2 or LysM) cre mouse is extensively used to achieve genetic manipulation in myeloid cells and it has been widely employed in retinal research. However, LysM has been recently described to be expressed in brain neurons and there is a debate on whether it is also expressed by resident microglia in addition to infiltrating macrophages. Methods We examined LysM-cre recombination in retinal tissue using a LysM-cre/tdTomato reporter mouse together with immunolabeling for several retinal cell markers. We further compared LysM-cre tdTomato recombination with that of Cdh5-cre driver, which is expressed in both endothelial and hematopoietic cells. Results LysM-cre was strongly expressed in most microglia/resident macrophages in neonatal retinas (P8) and to a lesser extent in microglia of adult retinas. In addition, there was some neuronal recombination (8 %) of LysM-cre specifically in adult retinal ganglion cells and amacrine cells. After retinal ischemia-reperfusion injury, LysM-cre was strongly expressed in microglia/infiltrating macrophages. Cdh5-cre was expressed in endothelial and myeloid cells of P8 pups retinas. Unexpectedly, Cdh5 showed additional expression in adult mouse retinal ganglion cells and brain neurons. Conclusions LysM-cre is expressed in macrophages and a subset of microglia together with a small but significant recombination of LysM-cre in the retinal neurons of adult mice. Cdh5 also showed some neuronal expression in both retina and brain of adult mice. These findings should be taken into consideration when interpreting results from central nervous system research using LysM-cre and Cdh5-cre mice.

Purpose Neovascularization (NV) is a sight-threatening complication of retinal ischemia in diabetes, retinal vein occlusion, and retinopathy of prematurity. Current treatment modalities, including laser photocoagulation and repeated intraocular injection of VEGF antagonists, are invasive and not always effective, and may carry side effects. We studied the use of hyperoxia as an alternative therapeutic strategy for regressing established vitreous NV in a mouse model of oxygen-induced ischemic retinopathy. Methods Hyperoxia treatment (HT, 75% oxygen) was initiated on postnatal day (P)17 after the onset of vitreous NV. Immunohistochemistry and quantitative PCR were used to assess retinal vascular changes in relation to apoptosis, and expression of VEGFR2 and inflammatory molecules. Effects of intravitreal injections of VEGF-A, VEGF-E, PlGF-1, and VEGF trap were also studied. Results HT selectively reduced NV by 70% within 24 hours. It robustly increased the level of cleaved caspase-3 in the vitreous NV between 6 and 18 hours and promoted infiltration of macrophage/microglial cells. The HT-induced apoptosis was preceded by a significant reduction in VEGFR2 expression within the NV and an increase in VEGFR2 within the surrounding neural tissue. Intravitreal VEGF-A and VEGF-E (VEGFR2 agonist) but not PlGF-1 (VEGFR1 agonist) prevented HT-induced apoptosis and regression of NV. In contrast, VEGF trap and VEGFR2 blockers mimicked the effect of HT. However, intravitreal VEGF trap induced increases in inflammatory molecules while HT did not have such unwanted effect. Conclusions HT may be clinically useful to specifically treat proliferative NV in ischemic retinopathy.

Diabetic retinopathy is the leading cause of acquired blindness among working-age adults in developed countries worldwide. Vision loss in this disease correlates strongly with increased vascular permeability.1 The retinal vascular leakage results in retinal edema that can lead to decreased visual acuity when it involves the macula. The overexpression of vascular endothelial cell growth factor (VEGF) plays a key role in this process (for review, see Ref. 2). The increased formation of reactive oxygen species (ROS) is thought to be a key event in the pathogenesis of diabetic retinopathy, probably through increasing VEGF expression (Liu J, et al. IOVS 2007;48:ARVO E-Abstract 4984).3,4 We have shown that treatments that reduce ROS formation also prevent the early signs of diabetic retinopathy in a streptozotocin (STZ) diabetic rat model.4,5 A major source of ROS in vascular cells is activity of NADPH oxidase. Vascular endothelial cells express all the components of phagocytic NADPH oxidase, including the catalytic subunit NOX2 (formerly known as gp91phox), p22phox, p47phox, p67phox, and Rac1.6 NADPH oxidase has been shown to be critically involved in the VEGF-induced angiogenesis7 and in the neovascularization that occurs in response to hindlimb ischemia.8 We have recently demonstrated that ROS derived from NADPH oxidase are important for hypoxia-induced VEGF expression and angiogenesis in a model of ischemic retinopathy. 9 Moreover, we have data showing that inhibiting NADPH oxidase or deleting NOX2 blocks diabetes-induced increases in leukocyte adhesion to the vessel wall and prevents breakdown of the blood–retinal barrier (BRB).10 Signal transducer and activator of transcription 3 (STAT3) is critically involved in a wide variety of biological processes, including inflammation and angiogenesis. The role of STAT3 in angiogenesis has been shown to involve its action in increasing VEGF expression.11–15 Recent studies have identified STAT3 as a direct transcriptional activator of the VEGF gene.12,13 Furthermore, STAT3 is required for PI3K-Akt-mediated expression of HIF-1, a key regulator of VEGF expression.16 Our group has recorded data that show that diabetes or high glucose treatment causes sustained activation of STAT3 by a mechanism involving activation of NADPH oxidase (Liu J, et al. IOVS 2007;48:ARVO E-Abstract 4984). Moreover, inhibiting STAT3 in the retina by transduction with dominant negative STAT3 blocks the actions of diabetes that causes overexpression of VEGF and breakdown of the BRB. Treatment with statins, inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the rate-limiting enzyme of cholesterol synthesis in the liver, has been shown to reduce the cardiovascular complications of diabetes. Clinical studies of diabetic patients have shown promising efficacy of statins in reducing signs of diabetic retinopathy.17 The mechanisms of these protective actions are not understood. However, studies in patients and experimental animal models have shown that in addition to lowering plasma lipid levels, statins are also highly effective in reducing the formation of ROS, limiting vascular inflammation and improving vascular endothelial cell function.18 In the present study, we investigated the impact of statin treatment on diabetes-induced upregulation of VEGF and ICAM-1 and vascular permeability in relation to activation of STAT3 and NADPH oxidase. We demonstrated in this study that treatment with statin blocks the effects of diabetes or high glucose in causing increases in VEGF and ICAM-1 expression and preserves the BRB in the diabetic rat retina by a process that involves blockade of NADPH oxidase-mediated activation of STAT3.

To investigate the effects of prolonged hyperoxia on vascular recovery and glia survival after experimentally induced retinopathy of prematurity (ROP) in the mouse.The effects of hyperoxia on revascularization and vitreous neovascularization were compared between mice raised in 75% oxygen from postnatal day (P)7 to P12, followed by room air recovery and mice raised in 75% oxygen from P7 to P27. The status of astrocytes and Müller cells was evaluated by glial fibrillary acidic protein (GFAP) immunohistochemistry on retinal wholemounts and serial sections. A window of susceptibility to oxygen-induced vaso-obliteration was defined by comparing the extent of retinal vaso-obliteration resulting from 2 days of hyperoxia beginning on P7, P9, P11, P13, or P15.Oxygen-induced vaso-obliteration of retinal capillaries was limited to the period between birth and P15. Paradoxically, revascularization was markedly accelerated and neovascularization markedly reduced in mice maintained in prolonged hyperoxia (P7-P27) compared with mice recovering in room air. The extended use of 75% oxygen during the recovery period was associated with preservation of astrocytes and Müller cells in the avascular retina.The antiangiogenic effect of hyperoxia on retinal capillaries is strongly dependent on postnatal age. A protocol of continuous 75% supplemental oxygen accelerates recovery of inner retinal vasculature and prevents vitreous neovascularization, by a mechanism that may involve preservation of inner retinal glia.

To determine transforming growth factor (TGF) beta effects on matrix metalloproteinases (MMPs) as a potential cause of the blood-retinal barrier breakdown at the onset of angiogenesis. Previously, glial cells were shown to play a role in the angiogenesis process and to express the angiogenic regulating factor TGF-beta, which becomes active under hypoxia conditions. Here, the authors demonstrate that retinal endothelial cells express MMP-9 when treated with TGF-beta or cocultured with glial cells and that both TGF-beta and MMP-9 increase endothelial cell permeability.Primary cultures of bovine retinal endothelial (BRE) cells grown on porous membranes were treated with TGF-beta or purified MMP-9, and permeability changes were assayed. The amount and distribution of the tight junction protein occludin also was analyzed by immunocytochemistry and Western blotting. Cell extracts or conditioned media from TGF-beta-treated BRE cells and from glial cell-BRE cocultures were analyzed for MMP-9 content by substrate gel electrophoresis (zymography) or Western blotting.Both TGF-beta and MMP-9 increased the permeability of BRE monolayers and reduced the levels of the junction protein occludin. The effect of MMP-9 on permeability was rapid, but the TGF-beta-induced permeability required longer incubation and was blocked by anti-TGF-beta and anti-MMP-9 antibodies as well as by TGF-beta latency-associated peptide. Zymography showed that MMP-9 activity, which was very low or absent in untreated BRE cultures, was dramatically increased by TGF-beta as well as by coculturing with either astrocytes or Müller glial cells. Anti-TGF-beta antibody blocked the TGF-beta effect, but not the coculture effect on MMP-9 production.These data indicate a direct correlation between TGF-beta-induced MMP-9 activity and increased endothelial cell permeability. Moreover, endothelial cell production of MMP-9 is regulated by glial cells through expression of TGF-beta or by direct cell-to-cell contact. During retinal disease, glial cell production of active TGF-beta may contribute to breakdown of the blood-retinal barrier by stimulating endothelial cell MMP-9 production.

Exposure of premature human infants to hyperoxia results in the obliteration of developing retina capillaries, leading to a vision-threatening retinopathy termed retinopathy of prematurity (ROP). The authors hypothesized that this process may be mediated in part by endothelial nitric oxide (NO)-derived oxidants such as peroxynitrite and tested this hypothesis in a mouse model of ROP.Normal mice, mice treated with the nitric oxide synthase (NOS) inhibitor N:(G)-nitro-L-arginine (L-NNA), and knockout mice carrying a homozygous targeted disruption of the gene for endothelial NOS (eNOS) were studied in an experimental model of ROP. Retinas were compared for extent of capillary obliteration in hyperoxia, vascular endothelial growth factor (VEGF) expression, nitrotyrosine formation, and vitreous neovascularization.Oxygen-induced retinal vaso-obliteration was significantly reduced by L-NNA treatment (43% decrease from controls). The eNOS-deficient mice showed a similar reduction in vaso-obliteration (46% decrease from controls), and vitreous neovascularization was also substantially reduced (threefold decrease). Retinal nitrotyrosine formation, a measure of in situ peroxynitrite modification of proteins, was significantly elevated in normal mice during hyperoxia, in a spatial and temporal pattern consistent with a role in oxygen-induced vaso-obliteration. This was not seen in eNOS-deficient mice. VEGF expression was similar in both groups of mice, although suppression in hyperoxia was slightly blunted in eNOS-deficient mice.These data suggest a role for NO and peroxynitrite in the pathogenesis of ROP. Therapies aimed at modulation of eNOS activity may have therapeutic potential for preventing ROP.

To determine the cellular route by which vascular endothelial cell growth factor (VEGF) increases the permeability of cultured retinal endothelial cells and to test whether nitric oxide (NO) production by NO synthase (NOS) is involved in signaling VEGF's permeability enhancing effects.Cultured bovine retinal microvascular endothelial (BRE) cells were used for bioassay of permeability function and its ultrastructural correlates. The role of NOS activity in VEGF's permeability enhancing effects was tested with the use of an NOS inhibitor. Because activity of endothelial NOS (eNOS) is thought to be regulated by its interaction with the caveolar protein caveolin-1, structural relationships between eNOS, caveolin-1, and the VEGF receptor FIk-1/KDR were analyzed with double-label immunofluorescence and cell fractionation procedures.Bioassays of permeability function and structure demonstrated that VEGF increases permeability of cultured BRE cells by an NOS-dependent process of transcytotic transport in caveolae. Double-label analysis showed that Flk-1/KDR and eNOS colocalize with caveolin-1 in plasma membrane caveolae. Cell fractionation and immunoblot analysis confirmed this effect. Densitometry showed that Flk-1/KDR, eNOS, and caveolin-1 levels were highest in caveolar fractions. Similar results were obtained in studies with bovine aortic endothelial cells.These results demonstrate that VEGF increases endothelial cell permeability by an eNOS-dependent mechanism of transcytosis in caveolae. Localization of Flk-1/KDR and eNOS with caveolin-1 suggests that VEGF signaling occurs within the caveolar compartment.

These experiments were designed to characterize tight junction formation by retinal pigment epithelial (RPE) cells in vitro and to compare the effects on this process of hormonally defined medium (HDM) and serum-containing medium.Formation of RPE tight junctions was analyzed in freshly isolated rat RPE cells maintained either in HDM or serum-containing medium. Junctions were evaluated functionally by measuring transepithelial electrical resistance (TER) and permeability and structurally by immunolocalization of the junction-associated actin microfilaments. Calcium dependency of the junctions was determined by reducing media calcium concentration.RPE cells cultured in serum-free HDM developed calcium-dependent tight junctions, which exhibited TER levels150 omega cm2 and low paracellular permeability. Serum-containing media inhibited tight junction formation as indicated by significant reductions in TER and increases in permeability. Junction-associated actin microfilaments and cell density were unchanged.Tight junction formation by RPE cells is inhibited by serum. This activity may play an important role in responses of the RPE layer to injury, contributing to the pathologic progression of blood-retinal barrier dysfunction.

In the genetically defective Royal College of Surgeons (RCS) rat model for retinal degeneration, a breakdown occurs in the retinal pigment epithelial (RPE) cell tight junctions just as the photoreceptors begin to degenerate. These experiments sought to determine the impact of the RPE genetic defect on this alteration in the RPE cell tight junctions.Retinal pigment epithelial cell cultures prepared from RCS and control rats were treated with hormonally defined medium (HDM), base medium conditioned by RCS or control retinas, or unconditioned base medium. The tight junctions formed by these cultures were assayed functionally by measuring transepithelial electrical resistance and permeability. Junction structure was evaluated by immunolocalization of the tight junction protein zonula occludens 1 and of the junction-associated actin microfilaments.Retinal pigment epithelial cultures from dystrophic rats formed structurally and functionally normal tight junctions when maintained in hormonally defined medium. The junctions remained stable when the medium bathing the apical surface was switched to base medium preconditioned by normal retinas. In contrast, cultures treated with medium preconditioned by degenerating dystrophic retinas or with unconditioned medium exhibited a breakdown in their tight junctions.Retinal pigment epithelial cells isolated from dystrophic RCS rats can form tight junctions normally in vitro. Normal, but not dystrophic, retinas release factors that support RPE tight junctions. Therefore, the junctional abnormality seen in dystrophic rat RPE cells in vivo is probably caused by the loss of trophic factors normally provided by the healthy neural retina rather than by a direct effect of the genetic defect on the tight junctions.

Photoreceptor degeneration is accompanied by the invasion of phagocytic cells into the outer retina of Royal College of Surgeons (RCS) rats. Previous studies suggested that these mononuclear phagocytes were blood-borne macrophages and not retinal pigment epithelial cells nor Müller glia. Thus, immunospecific markers were used to identify these cells and to determine their distribution in the dystrophic retina.Retinas from RCS and control (RCS-rdy+) rats were processed for immunocytochemistry using antibodies against phosphotyrosine, which labels both microglial cells and peripheral macrophages, and ED2, which labels peripheral macrophages only. As a positive control to demonstrate ED2-labeling of peripheral macrophages that enter the retina during injury, experiments were performed using needle-punctured Long Evans rat retinas.In normal animals, process-bearing, phosphotyrosine-reactive cells were restricted to the inner retinal layers and the outer plexiform layer. In early dystrophic retinas, phosphotyrosine-reactive cells also were observed in the outer retinal layers. The number of phosphotyrosine-labeled cells in the outer retina increased substantially in later stages of dystrophy. ED2-reactive cells were observed in normal or dystrophic retinas only after needle puncture.These findings suggest that phagocytic cells during the early stages of dystrophy in RCS rat retinas are derived from resident microglial cells, not from peripheral macrophages. The migration of microglial cells into the outer retina when photoreceptor cells begin to degenerate further suggests that they may play a major role in photoreceptor cell death in the dystrophic retina.

High levels of epidermal growth factor (EGF)-receptors have been reported in membrane homogenates of bovine retinas, but the biologic function and tissue target of EGF in the retina have not been established fully. Because EGF participation has been suggested in the mechanisms of wound healing and Müller cells undergo changes after retinal injury, the authors studied EGF receptor expression and functional role of this substance in cultured Müller cells. These cells (isolated from normal rats) were tested for the glial cell markers: vimentin, S-100 protein, and carbonic anhydrase C. These markers were found to be positive through all passages used in the experiments. The 125I-EGF binding in Müller cells was highly specific, concentration dependent, and saturable. Compared with 3T3 fibroblasts, Müller cells bound threefold more EGF. Binding kinetics and Scatchard analyses showed the higher level of binding was related to the greater number of receptors on these cells (Müller cells, 2.4 x 10(5) receptors/cell; 3T3 fibroblasts, 7.1 x 10(4) receptors/cell) rather than a change in affinity of the receptors to bind the ligand. Nonlinear-regression analyses suggested the presence of two classes of affinity sites. The high level of EGF-receptor expression in Müller cells was confirmed by western blot analyses that showed increased reactivity of the approximately 170-kilodalton receptor band to a monoclonal anti-EGF receptor antibody. Moreover, EGF treatment of Müller cells resulted in two- to threefold increase in DNA synthesis, as evidenced by 3H-thymidine uptake studies. These findings support a functional role for EGF in Müller cell proliferation in retinal disease.

To see whether or not blood-retinal barrier breakdown during diabetes was associated with breakdown of the endothelial cell tight junctions or with other membrane alterations in the cells comprising the wall of the retinal microvasculature, streptozotocin-induced diabetic rat retinas were studied using lanthanum tracer and freeze-fracture electron microscopic morphometry. This study showed that endothelial cell tight junction permeability to lanthanum and luminal surface area were normal in these diabetic rats. However, freeze-fracture morphometry showed several alterations in the diabetic retinal microvessels. First, the endothelial cell membranes had abnormally large (80-120 nm) plasmalemmal vesicles not evident in the control retinas, suggesting that membrane turnover was abnormal. Second, endothelial cell P-face membranes at the blood front contained more larger particles than those in the control rats (P less than 0.05), implying an alteration in endothelial cell luminal membrane composition. Third, endothelial cell P-face membranes in areas of close apposition with pericyte membranes showed abnormal areas of particle clearing not seen in the control animals, suggesting a change in pericyte-endothelial cell interactions. Finally, pericyte membranes facing the neural retina contained increased numbers of plasmalemmal vesicles compared with control membranes (P less than 0.05). Moreover, the association of these vesicles with collagen fibrils in the extracellular space suggested an alteration in extracellular matrix turnover.

We have investigated the effects of diabetes on retinal oxidative metabolism. Since activity of the mitochondrial enzyme, cytochrome oxidase, has been demonstrated to be a reliable indicator of oxidative metabolism and physiological activity, we used cytochemical techniques to study the activity of this enzyme in spontaneously diabetic, streptozotocin-diabetic, and control rat retinas. Light microscope results showed an increase in staining for cytochrome oxidase activity in the diabetic RPE cell layer as compared with the control. Quantitative electron microscope analysis showed a significant increase in RPE cells with highly reactive mitochondria as compared with the controls. Mitochondrial staining within the diabetic photoreceptor and retinal vascular endothelial cells was normal. RPE cell volume and surface area, as well as number and volume of mitochondria, were unchanged. This increase in oxidative enzyme activity is further evidence of RPE cell alteration in diabetes.

The basal laminae of capillaries, glomeruli, and nerves are thickened in diabetes. Previous studies have shown that diabetic tissues produce increased levels of basal lamina collagen and reduced levels of proteoglycans. Since heparan sulfate and chondroitin sulfate proteoglycans are thought to act as anionic barriers regulating passage of proteins across Bruch's membrane of the eye, the cationic electron microscope tracers, polyethyleneimine (PEI) and ruthenium red, were used to study the distribution of anionic sites in Bruch's membrane of spontaneously diabetic Bio-Breeding/Worcester (BB-W) rats and streptozotocin-diabetic rats. The distribution of the two tracers is similar. In Bruch's membrane of control rats, electron dense particles are present at regular intervals along both sides of the basal laminae of the retinal pigment epithelium (RPE) and of the choriocapillary endothelium (CE), and along collagen fibers in the zone between the two basal laminae. Within 3-6 months after the onset of hyperglycemia in both diabetic rat models, quantitative analysis shows a significant reduction in binding sites along both RPE and CE basal laminae, while thickness of both basal laminae is significantly increased. Because reductions in anionic binding sites along basal laminae in the renal glomerulus have been found to accompany changes in glomerular filtration, these changes suggest that filtration through Bruch's membrane is altered in diabetes.

A progressive failure in phagocytosis by the retinal pigment epithelium (RPE) occurs in the Royal College of Surgeons rat with inherited retinal degeneration. Another change that can be attributed to a defect in the RPE is a breakdown in the blood-retinal barrier. RPE cell junctions, which form a part of this barrier, become permeable to extracellular tracer during the dystrophic process. We have used the freeze-fracture technique to study the structure of RPE cell junctions in normal and dystrophic retinas. In normal retinas, tight junctions between RPE cells consisted of 8 to 16 anastomosing strands on the cytoplasmic membrane leaflet (P-face) and a complementary pattern of grooves on the external membrane leaflet (E-face). Gap-junctional aggregates of hexagonally packed P-face particles and complementary E-face pits were enclosed within the tight junctional strands. In dystrophic retinas changes were first seen at postnatal day 21. Subtle breaks in P-face tight-junctional strands became more pronounced with time. Eventually the tight junctions appeared to unravel from the gap junctional aggregates, which became isolated and appeared to break off into patches of particle aggregates. The increased density of background particles in the membranes adjacent to disassembling junctions suggested that junctional elements were being removed by dispersal. Endocytosis of junctional elements was observed in both dystrophic and control retinas but may be accelerated in the dystrophic retina. In the late stages of the dystrophy some RPE cell junctions appeared to have proliferated and occupied extensive areas of the RPE membrane.