Molecular Mechanisms of Oxidative Stress Relief by CAPE in ARPE-19 Cells.

Caffeic acid phenylethyl ester (CAPE) is an antioxidative agent originally derived from propolis. Oxidative stress is a significant pathogenic factor in most retinal diseases. Our previous study revealed that CAPE suppresses mitochondrial ROS production in ARPE-19 cells by regulating UCP2. The present study explores the ability of CAPE to provide longer-term protection to RPE cells and the underlying signal pathways involved. ARPE-19 cells were given CAPE pretreatment followed by t-BHP stimulation. We used in situ live cell staining with CellROX and MitoSOX to measure ROS accumulation; Annexin V-FITC/PI assay to evaluate cell apoptosis; ZO-1 immunostaining to observe tight junction integrity in the cells; RNA-seq to analyze changes in gene expression; q-PCR to validate the RNA-seq data; and Western Blot to examine MAPK signal pathway activation. CAPE significantly reduced both cellular and mitochondria ROS overproduction, restored the loss of ZO-1 expression, and inhibited apoptosis induced by t-BHP stimulation. We also demonstrated that CAPE reverses the overexpression of immediate early genes (IEGs) and activation of the p38-MAPK/CREB signal pathway. Either genetic or chemical deletion of UCP2 largely abolished the protective effects of CAPE. CAPE restrained ROS generation and preserved the tight junction structure of ARPE-19 cells against oxidative stress-induced apoptosis. These effects were mediated via UCP2 regulation of p38/MAPK-CREB-IEGs pathway.

Inhibition of Epigenetic Modifiers LSD1 and HDAC1 Blocks Rod Photoreceptor Death in Mouse Models of Retinitis Pigmentosa.

Epigenetic modifiers are increasingly being investigated as potential therapeutics to modify and overcome disease phenotypes. Diseases of the nervous system present a particular problem as neurons are postmitotic and demonstrate relatively stable gene expression patterns and chromatin organization. We have explored the ability of epigenetic modifiers to prevent degeneration of rod photoreceptors in a mouse model of retinitis pigmentosa (RP), using rd10 mice of both sexes. The histone modification eraser enzymes lysine demethylase 1 (LSD1) and histone deacetylase 1 (HDAC1) are known to have dramatic effects on the development of rod photoreceptors. In the RP mouse model, inhibitors of these enzymes blocked rod degeneration, preserved vision, and affected the expression of multiple genes including maintenance of rod-specific transcripts and downregulation of those involved in inflammation, gliosis, and cell death. The neuroprotective activity of LSD1 inhibitors includes two pathways. First, through targeting histone modifications, they increase accessibility of chromatin and upregulate neuroprotective genes, such as from the Wnt pathway. We propose that this process is going in rod photoreceptors. Second, through nonhistone targets, they inhibit transcription of inflammatory genes and inflammation. This process is going in microglia, and lack of inflammation keeps rod photoreceptors alive.SIGNIFICANCE STATEMENT Retinal degenerations are a leading cause of vision loss. RP is genetically very heterogeneous, and the multiple pathways leading to cell death are one reason for the slow progress in identifying suitable treatments for patients. Here we demonstrate that inhibition of LSD1and HDAC1 in a mouse model of RP leads to preservation of rod photoreceptors and visual function, retaining of expression of rod-specific genes, and with decreased inflammation, cell death, and Müller cell gliosis. We propose that these epigenetic inhibitors cause more open and accessible chromatin, allowing expression of neuroprotective genes. A second mechanism that allows rod photoreceptor survival is suppression of inflammation by epigenetic inhibitors in microglia. Manipulation of epigenetic modifiers is a new strategy to fight neurodegeneration in RP.

Novel Regulators of Retina Neovascularization: A Proteomics Approach.

The purpose of this study was to identify proteins that regulate vascular remodeling in an ROP mouse model. Pups were subjected to fluctuating oxygen levels and retinas sampled during vessel regression (PN12) or neovascularization (PN17) for comparative SWATH-MS proteomics using liquid chromatography-tandem mass spectrometry (LC-MS/MS). We developed a human retinal endothelial cell (HREC) ROP correlate to validate the expression of retina neovascular-specific markers. A total of 5191 proteins were identified in OIR retinas with 498 significantly regulated in elevated oxygen and 345 after a return to normoxia. A total of 122 proteins were uniquely regulated during vessel regression and 69 during neovascularization (FC ≥ 1.5; p ≤ 0.05), with several validated by western blot analyses. Expressions of 56/69 neovascular-specific proteins were confirmed in hypoxic HRECs with 23 regulated in the same direction as OIR neovascular retinas. These proteins control angiogenesis-related processes including matrix remodeling, cell migration, adhesion, and proliferation. RNAi and transfection overexpression studies confirmed that VASP and ECH1, showing the highest levels in hypoxic HRECs, promoted human umbilical vein (HUVEC) and HREC cell proliferation, while SNX1 and CD109, showing the lowest levels, inhibited their proliferation. These proteins are potential biomarkers and exploitable intervention tools for vascular-related disorders. The proteomics data set generated has been deposited to the ProteomeXchange/iProX Consortium with the Identifier:PXD029208.

Deletion of the Pedf gene leads to inflammation, photoreceptor loss and vascular disturbances in the retina.

Retinal diseases are often accompanied by inflammation, vascular abnormalities, and neurodegeneration that decrease vision. Treatment with exogenous PEDF is widely shown to alleviate these conditions leading us to hypothesize that loss of function of the PEDF gene disrupts these pathways and leads to visual loss. Measurements were carried out by detailed phenotyping of PEDF null mice to assess expression of immunomodulators, glia activation, systemic inflammation, vascular disturbances, and visual sensitivity often associated with retinal pathologies. With a deletion of the Pedf gene, there was increased expression of several immune modulators in Pedf-/- retinas and serum with IL-2 and GM-CSF upregulated in both. Increases in retina glia activation and macrophage infiltration, levels of serum c-reactive protein (CRP), numbers of white and red blood cells and platelets and decreased blood glucose levels were all features associated with PEDF null mice. With PEDF gene deletion, there was also a notable increase in apoptosis in early developing retinas (PN3), reduced thickness of the photoreceptor layer, swelling of the inner plexiform layer, reduced retinal sensitivity and steady-state reduced activation of Erk and Akt, two signaling pathways used by PEDF. There is a substantial body of animal data emphasizing utility of PEDF treatment in homeostatic regulation of retinal diseases, including diabetic retinopathy and age-related macular degeneration but there is little agreement or evidence on the role of endogenous PEDF in retinal diseases. Our findings strongly support the concept that a deletion of the PEDF gene makes the retina vulnerable to diseases, and argue that endogenous PEDF plays a critical role in limiting pathological events in the retina.

Uncoupling Proteins as Therapeutic Targets for Neurodegenerative Diseases.

Most of the major retinal degenerative diseases are associated with significant levels of oxidative stress. One of the major sources contributing to the overall level of stress is the reactive oxygen species (ROS) generated by mitochondria. The driving force for ROS production is the proton gradient across the inner mitochondrial membrane. This gradient can be modulated by members of the uncoupling protein family, particularly the widely expressed UCP2. The overexpression and knockout studies of UCP2 in mice have established the ability of this protein to provide neuroprotection in a number of animal models of neurological disease, including retinal diseases. The expression and activity of UCP2 are controlled at the transcriptional, translational and post-translational levels, making it an ideal candidate for therapeutic intervention. In addition to regulation by a number of growth factors, including the neuroprotective factors LIF and PEDF, small molecule activators of UCP2 have been found to reduce mitochondrial ROS production and protect against cell death both in culture and animal models of retinal degeneration. Such studies point to the development of new therapeutics to combat a range of blinding retinal degenerative diseases and possibly other diseases in which oxidative stress plays a key role.

PEDF is an endogenous inhibitor of VEGF-R2 angiogenesis signaling in endothelial cells.

Pigment epithelium derived factor (PEDF), an endogenous inhibitor of angiogenesis, targets the growth of aberrant blood vessels in many tissues, including the eye. In this study we show that PEDF prevented early mitogenic signals of vascular endothelial growth factor (VEGF-A) in primate retinal endothelial cells, blocking proliferation, migration and tube formation. PEDF inhibited the phosphorylation and activation of five major downstream VEGF-A signaling partners, namely phosphoinositide-3-OH Kinase (PI3K), AKT, FAK, Src (Y416), and PLC-γ. It did so by binding to the extracellular domain of VEGF-R2, blocking VEGF-A-induced tyrosine phosphorylation (Tyr 951 and Tyr 1175), and inhibiting VEGF-R2 receptor kinase activity. PEDF had no effect on the transcription or translation of VEGF-R2 in cultured HUVECs. PEDF also bound to the extracellular domain of VEGF-R1. We conclude that PEDF blocks the growth of new blood vessels, in part, by reducing VEGF-A activation of its key mitogenic receptor, VEGF-R2, and by preventing its downstream signals in endothelial cells.

Mitochondrial Uncoupling Protein 2 Knock-out Promotes Mitophagy to Decrease Retinal Ganglion Cell Death in a Mouse Model of Glaucoma.

Glaucoma is a neurodegenerative disorder characterized by mitochondrial dysfunction and an increase in oxidative damage, leading to retinal ganglion cell (RGC) death. The oxidative status of RGCs is regulated intrinsically and also extrinsically by retinal glia. The mitochondrial uncoupling protein 2 (UCP2) relieves oxidative and neuronal damage in a variety of neurodegenerative disease models. We hypothesized that deletion of Ucp2 in either RGCs or retinal glia would increase retinal damage and RGC death in a mouse model of glaucoma. Paradoxically, we found the reverse, and deletion of mitochondrial Ucp2 decreased oxidative protein modification and reduced RGC death in male and female mice. This paradox was resolved after finding that Ucp2 deletion also increased levels of mitophagy in cell culture and retinal tissue. Our data suggest that Ucp2 deletion facilitates increased mitochondrial function by improving quality control. An increase in mitochondrial function explains the resistance of Ucp2-deleted retinas to glaucoma and may provide a therapeutic avenue for other chronic neurodegenerative conditions.SIGNIFICANCE STATEMENT Many unsolved neurodegenerative conditions result from defects in mitochondrial function. Molecular tools that can manipulate mitochondria will therefore be central to developing neuroprotective therapies. Among the most potent regulators of mitochondrial function are the uncoupling proteins, particularly UCP2. In this manuscript, we show that, while loss of Ucp2 does increase mitochondrial membrane potential and the production of reactive oxygen species, it also initiates an increase in mitophagy that is ultimately neuroprotective. This novel protective consequence of uncoupling protein inhibition may lead to new therapeutic approaches to combat neurodegenerative disease, particularly because pharmacological compounds do exist that can selectively inhibit UCP2.

Histone Deacetylase 1 Is Essential for Rod Photoreceptor Differentiation by Regulating Acetylation at Histone H3 Lysine 9 and Histone H4 Lysine 12 in the Mouse Retina.

Histone acetylation has a regulatory role in gene expression and is necessary for proper tissue development. To investigate the specific roles of histone deacetylases (HDACs) in rod differentiation in neonatal mouse retinas, we used a pharmacological approach that showed that inhibition of class I but not class IIa HDACs caused the same phenotypic changes seen with broad spectrum HDAC inhibitors, most notably a block in the differentiation of rod photoreceptors. Inhibition of HDAC1 resulted in increase of acetylation of lysine 9 of histone 3 (H3K9) and lysine 12 of histone 4 (H4K12) but not lysine 27 of histone 3 (H3K27) and led to maintained expression of progenitor-specific genes such as Vsx2 and Hes1 with concomitant block of expression of rod-specific genes. ChiP experiments confirmed these changes in the promoters of a group of progenitor genes. Based on our results, we suggest that HDAC1-specific inhibition prevents progenitor cells of the retina from exiting the cell cycle and differentiating. HDAC1 may be an essential epigenetic regulator of the transition from progenitor cells to terminally differentiated photoreceptors.

Developmentally regulated linker histone H1c promotes heterochromatin condensation and mediates structural integrity of rod photoreceptors in mouse retina.

Mature rod photoreceptor cells contain very small nuclei with tightly condensed heterochromatin. We observed that during mouse rod maturation, the nucleosomal repeat length increases from 190 bp at postnatal day 1 to 206 bp in the adult retina. At the same time, the total level of linker histone H1 increased reaching the ratio of 1.3 molecules of total H1 per nucleosome, mostly via a dramatic increase in H1c. Genetic elimination of the histone H1c gene is functionally compensated by other histone variants. However, retinas in H1c/H1e/H1(0) triple knock-outs have photoreceptors with bigger nuclei, decreased heterochromatin area, and notable morphological changes suggesting that the process of chromatin condensation and rod cell structural integrity are partly impaired. In triple knock-outs, nuclear chromatin exposed several epigenetic histone modification marks masked in the wild type chromatin. Dramatic changes in exposure of a repressive chromatin mark, H3K9me2, indicate that during development linker histone plays a role in establishing the facultative heterochromatin territory and architecture in the nucleus. During retina development, the H1c gene and its promoter acquired epigenetic patterns typical of rod-specific genes. Our data suggest that histone H1c gene expression is developmentally up-regulated to promote facultative heterochromatin in mature rod photoreceptors.

Stat3 mediates LIF-induced protection of astrocytes against toxic ROS by upregulating the UPC2 mRNA pool.

Reactive oxygen species (ROS) have been implicated in various types of CNS damage, including stroke. We used a cultured astrocyte model to explore mechanisms of survival of CNS cells following ROS damage. We found that pretreatment with leukemia inhibitory factor (LIF) preserves astrocytes exposed to toxic levels of t-BHP by inhibiting an increase in intracellular ROS following t-BHP treatment. Astrocytes lacking functional Stat3 did not benefit from the pro-survival or antioxidant effects of LIF. Inhibition of mitochondrial uncoupling protein 2 (UCP2) using a chemical inhibitor or siRNA abrogates the prosurvival effects of LIF, indicating a critical role for UCP2 in modulation of mitochondrial ROS production in survival following ROS exposure. LIF treatment of astrocytes results in increased UCP2 mRNA that is accompanied by an increase in Stat3 binding to the UCP2 promoter region. Although treatment with LIF alone did not increase UCP2 protein, a combination of LIF treatment and ROS stress led to increased UCP2 protein levels. We conclude that LIF protects astrocytes from ROS-induced death by increasing UCP2 mRNA, allowing cells to respond to ROS stress by rapidly producing UCP2 protein that ultimately decreases endogenous mitochondrial ROS production.

Cell type-specific epigenetic signatures accompany late stages of mouse retina development.

We have used ChIP-seq to map the distribution of two important histone H3 modifications, H3K4me2 and H3K27me3, over the whole genome at multiple time points during late mouse retina development. We merged these data with our previous retina developmental expression profiles and show that there are several epigenetic signatures specific for different functional groups of genes. The main conclusion from our study is that epigenetic signatures defined by H3K4me2 and H3K27me3 can distinguish cell-type specific genes from widespread transcripts and may be reflective of cell specificity during retina maturation. Rod photoreceptor-specific genes have a striking signature, a de novo accumulation of H3K4me2 and a complete absence of H3K27me3. We were able to use this signature in an unbiased search of the whole genome and identified essentially all the known rod photoreceptor genes as well as a group of novel genes that have a high probability of being rod photoreceptor specific. Comparison of our genome-wide chromatin signature maps with available data sets for Polymerase-II (Pol-II) and CRX binding sites and DNase1 Hypersensitive Sites (DHS) for retina shows great agreement. Because our approach is not dependent on high levels of gene expression, it provides a new way of identifying cell type-specific genes, particularly genes that may be involved in retinal diseases.

CD4 positive T helper cells contribute to retinal ganglion cell death in mouse model of ischemia reperfusion injury.

Neuron degeneration is a common pathological process associated with many disease conditions in the central nervous system including retina. Although immune responses have been proposed as one potential element in triggering neural damage, the mechanism of action of specific immune components underlying the pathogenesis is unclear. In this study we focus on adaptive immune activities to evaluate CD4 positive helper cells in the retinal ganglion cell (RGC) degeneration in response to transient retinal ischemic/reperfusion (I/R) injury. Transient retinal ischemia was induced in four mouse strains with different immune backgrounds, including wild type mice from C57BL/6 and BABL/c strains, severe combined immunodeficient (SCID) mice lacking T and B lymphocytes, SCID mice with transferred wild type CD4+ T cells, and the STAT6 deficient mice without T helper 2 (TH2) cells. In SCID mice RGCs showed a strong resistance to cell death in response to I/R injury (89% ± 3% of the survival cells in contralateral eye) compared with C57BL/6 (p = 0.018) and BALB/C (p = 0.038) wild types. By transferring the mature CD4+ T cells from matched wild type into SCID mice, the resistance of RGCs to injury was significantly compromised (p < 0.05). Furthermore a significant resistance of RGCs to cell death (p < 0.05) accompanied with an overexpression of STAT1 and STAT3 was confirmed in STAT6 deficient mice in response to I/R injury compared with the wild type controls, indicating that TH2 cells maturation might be involved in RGC damage. Adaptive immunity carried by CD4 T cells plays an essential role in RGC degeneration.

Specific protein kinase C isoforms are required for rod photoreceptor differentiation.

The protein kinase C (PKC) family of enzymes regulates cell physiology through phosphorylation of serine and threonine residues of many proteins in most cell types. Here we identify PKC-ß1 and PKC-γ as isoforms that are essential for rod photoreceptor differentiation in mouse retinas. Using ex vivo retinal explants, we found that phorbol ester 12-myristate 13-acetate and insulin-like growth factor 1 (IGF1) induced rod differentiation, as defined by opsin or Crx expression, in a PKC-dependent manner days ahead of rod development in untreated explants. PKC-ß1 and PKC-γ were colocalized with proliferating cell nuclear antigen (PCNA)- and STAT3-positive progenitors through the later differentiation period. Pharmacological or genetic inhibition of either isoform resulted in a partial reduction in the appearance of rods, whereas removing both isoforms resulted in their complete absence. Furthermore, a significant decline of STAT3 tyrosine phosphorylation was observed by activation of PKC, while inhibition of PKC resulted in an increase of phosphorylated STAT3 along with a delayed cell cycle exit of progenitors with prolonged PCNA expression. In adult retinas, IGF1 activates PI-3 kinase (PI3K), but in neonatal retinas its action is identical to the action of an PI3K inhibitor. These data unveil a novel signaling cascade that coordinates and regulates rod differentiation through specific PKC isoforms in mammals.

Pigment epithelium-derived factor (PEDF) peptide eye drops reduce inflammation, cell death and vascular leakage in diabetic retinopathy in Ins2(Akita) mice.

Inflammation, neurodegeneration and microvascular irregularities are included in the spectrum of defects associated with diabetic retinopathy. Here, we evaluated intraocular deliverability features of two pigment epithelium-derived factor (PEDF) derivatives given as eye drops and their efficacy in modulating diabetes-induced retinal complications. The antiangiogenic PEDF60-77 (P60) and neuroprotective PEDF78-121 (P78) derivatives were applied to Ins2(Akita) mouse eyes once a week for 15 wks at the onset of hyperglycemia. Peptides, labeled with Alexa Fluor 488, were observed penetrating the cornea by 1-4 h and gained access to the ciliary body, retinal pigment epithelium (RPE)-choroid complex, retina microvasculature and vitreous. Peak vitreous levels were 0.2 µg/mL for P60 and 0.9 µg/mL for P78 after 0.5 and 4 h, respectively. Both peptides reduced vascular leakage by ~60% and increased zona occludens 1 (ZO1) and occludin expression in the microvasculature to nondiabetic levels. P60 induced pERK1/2 and P78 promoted pAKT in Muller glia, two signals that were dampened in diabetic conditions. Pharmacologically inhibiting AKT signaling in the retina blocked effects of the peptides on ZO1 and occludin expression. P78 reduced levels of 9/20 cytokines in diabetic vitreous including interferon (IFN)-γ, interleukin (IL)-6, IL-3 and tumor necrosis factor (TNF)-α. P60 lowered levels of 6/20 cytokines but was less effective than P78. Neuroprotective P78 prevented diabetes-induced microglia activation by ~60%, retinal ganglion cell (RGC) death by ~22% and inner plexiform layer thinning by ~13%. In summary, we provide evidence that PEDF bioactive derivatives gained access to the retina by topical delivery and validated their efficacy in reducing diabetic retinopathy complications. Our findings argue for glia regulation of microvascular leakage and an early root cause for RGC degeneration embedded in microglia activation.

Epigenetics and Degenerative Retinal Diseases: Prospects for New Therapeutic Approaches.

ABSTRACT: There is growing evidence that retinal degenerative diseases are accompanied by epigenetic changes in both deoxyribonucleic acid methylation and histone modification. Even in the monogenic disease retinitis pigmentosa, there is a cascade of changes in gene expression that correlate with epigenetic changes, suggesting that many of the symptoms, and degenerative changes, may be a result of epigenetic changes downstream from the genetic mutation. This is supported by data from studies of diabetic retinopathy and macular degeneration, 2 diseases where it has been difficult to define a single causative change. Initial studies with modifiers of deoxyribonucleic acid methylation suggest that they can provide therapeutic benefit. A number of drugs are available to inhibit specific epigenetic histone modifier enzymes, and these offer the possibility of new therapeutic approaches to retinal disease. Systemic treatment with inhibitors of histone demethylases and histone deacetylases have arrested rod degeneration in rodent models of retinitis pigmentosa. Some evidence has suggested that similar treatments may provide benefits for patients with diabetic retinopathy. Because differentiation of retinal stem cells is regulated in part by epigenetic mechanisms, it may also be possible to direct stem cell differentiation pathways through the use of selective epigenetic modifiers. This is predicted to provide a valuable avenue to accelerate the introduction of regenerative approaches to retinal disease. Epigenetic modifiers are poised to become a powerful new approach to treat retinal degenerative diseases.

[Purified mouse retinal ganglion cell culture].

OBJECTIVE: To establish a culture system for purified mouse retinal ganglion cells (RGC) culture in order to lay a foundation for the in vitro study of RGC. METHODS: It was a experiment study. Eight to twelve C57BL/6 mice on postnatal day 4 to 6 were used. The retinas were dissected and dissociated enzymatically to make a suspension of single cells. The retinal suspension was incubated in rat anti-mouse-macrophage antiserum for 5 minutes and incubated on a 100 mm anti-rat IgG panning plate at room temperature for 30 min twice. The nonadherent cells were removed with the suspension and placed on the Thy-1.2 panning plate at room temperature. After 45 min, plates were washed 6 - 10 times with phosphate-buffered saline and swirled moderately to dislodge nonadherent cells. Trypsin (0.125% solution) dissociation was used to remove adherent cells from the plate. Then cells were spun and resuspended in neurobasal medium containing some neurotrophic factors. The cell suspension was implanted in 24 well culture plates and cultured under 37°C in an incubator with 5% CO(2). RESULTS: Most of the cells adhered to the plate after 24 hours in culture and showed dendrites at different lengths. The dendrites grew longer with time. Most of the RGC can survive more than two weeks. CONCLUSION: Monoclonal antibody to the Thy-1.2 antigen can be used to purify the mice RGC.

Uncoupling proteins in the mitochondrial defense against oxidative stress.

Oxidative stress is a major component of most major retinal diseases. Many extrinsic anti-oxidative strategies have been insufficient at counteracting one of the predominant intrinsic sources of reactive oxygen species (ROS), mitochondria. The proton gradient across the inner mitochondrial membrane is a key driving force for mitochondrial ROS production, and this gradient can be modulated by members of the mitochondrial uncoupling protein (UCP) family. Of the UCPs, UCP2 shows a widespread distribution and has been shown to uncouple oxidative phosphorylation, with concomitant decreases in ROS production. Genetic studies using transgenic and knockout mice have documented the ability of increased UCP2 activity to provide neuroprotection in models of a number of diseases, including retinal diseases, indicating that it is a strong candidate for a therapeutic target. Molecular studies have identified the structural mechanism of action of UCP2 and have detailed the ways in which its expression and activity can be controlled at the transcriptional, translational and posttranslational levels. These studies suggest a number of ways in control of UCP2 expression and activity can be used therapeutically for both acute and chronic conditions. The development of such therapeutic approaches will greatly increase the tools available to combat a broad range of serious retinal diseases.

PEDF Gene Deletion Disrupts Corneal Innervation and Ocular Surface Function.

Purpose: The cornea is richly innervated by the trigeminal ganglion (TG) and its function supported by secretions from the adjacent lacrimal (LG) and meibomian glands (MG). In this study we examined how pigment epithelium-derived factor (PEDF) gene deletion affects the cornea structure and function. Methods: We used PEDF hemizygous and homozygous knockout mice to study effects of PEDF deficiency on corneal innervation assessed by beta tubulin staining, mRNA expression of trophic factors, and PEDF receptors by adjacent supporting glands, corneal sensitivity measured using a Cochet-Bonnet esthesiometer, and tear production using phenol red cotton thread wetting. Results: Loss of PEDF was accompanied by reduced corneal innervation and sensitivity, increased corneal surface injury and tear production, thinning of the corneal stroma and loss of stromal cells. PEDF mRNA was expressed in the cornea and its supporting tissues, the TG, LG, and MG. Deletion of one or both PEDF alleles resulted in decreased expression of essential trophic support in the TG, LG, and MG including nerve growth factor, brain-derived neurotrophic growth factor, and GDNF with significantly increased levels of NT-3 in the LG and decreased EGF expression in the cornea. Decreased transcription of the putative PEDF receptors, adipose triglyceride lipase, lipoprotein receptor-related protein 6, laminin receptor, PLXDC1, and PLXDC2 was also evident in the TG, LG and MG with the first three showing increased levels in corneas of the Pedf+/- and Pedf-/- mice compared to wildtype controls. Constitutive inactivation of ERK1/2 and Akt was pronounced in the TG and cornea, although their protein levels were dramatically increased in Pedf-/- mice. Conclusions: This study highlights an essential role for PEDF in corneal structure and function and confirms the reported rescue of exogenous PEDF treatment in corneal pathologies. The pleiotropic effects of PEDF deletion on multiple trophic factors, receptors and signaling molecules are strong indications that PEDF is a key coordinator of molecular mechanisms that maintain corneal function and could be exploited in therapeutic options for several ocular surface diseases.

Neuroprotection of retinal cells by Caffeic Acid Phenylethyl Ester（CAPE) is mediated by mitochondrial uncoupling protein UCP2.

Oxidative stress due to mitochondrial produced reactive oxygen species is a major cause of damage seen in many retinal degenerative diseases. Caffeic acid phenylethyl ester （CAPE） is protective agent in multiple tissues and is reported to have anti-oxidant properties. Systemically applied CAPE protected retinal ganglion cells from ischemic injury induced by increased intraocular pressure. CAPE provided complete protection for ARPE19 retinal pigment epithelial cells against tert-butyl hydrogen peroxide and reduced both basal and LPS-stimulated ROS production. The major effect of CAPE was mediated by the mitochondrial uncoupling protein UCP2 since both pharmacological inhibition of UCP2 and siRNA-induced knockdown removed the ability of CAPE to block ROS production. Based on common structural features, CAPE may be acting as a mimetic of the natural UCP2 homeostatic regulator 4-hydroxy-2-nonenal. CAPE may provide a valuable tool to treat oxidative stress-related damage in retinal and other degenerative diseases.

Protein kinase C regulates rod photoreceptor differentiation through modulation of STAT3 signaling.

The molecular signals governing retinal development remain poorly understood, but some key molecules that play important roles have been identified. Activation of STAT3 by cytokines such as LIF and CNTF specifically blocks differentiation of rod photoreceptors. Here we test the hypothesis that PKC activation promotes development of rod photoreceptors by inhibiting STAT3. Explant cultures of mouse retina were used to study the effects of PKC activation on rod development. The expression of opsin, a rod specific marker, is induced at an early stage in retina explants cultured in the presence of PMA and this effect is prevented by the PKC inhibitor Go7874. Histological experiments show that there is expression of PKC beta1, but not PKC-alpha in the outer nuclear layer between E17.5 and PN5. In vitro data derived from cell lines shows that activation of PKC results in reduction of STAT3 phosphorylation. In addition, inhibition of PKC results in increase STAT3 phosphorylation. We suggest that cross talk of signals between STAT3 and PKC may determine the differentiation of rods from retinal progeitors.