Heparanase Deficiency Is Associated with Disruption, Detachment, and Folding of the Retinal Pigment Epithelium.

PURPOSE: Pentosan polysulfate sodium (PPS; Elmiron) is a FDA-approved heparanase inhibitor for the treatment of bladder pain and interstitial cystitis. The chronic use of PPS has been associated with a novel pigmentary maculopathy, associated with discrete vitelliform deposits that exhibit hyperfluorescence, macular hyper-pigmentary spots, and foci of nodular RPE enlargement. Therefore, this study aimed to investigate the retinal morphology of heparanase knockout mice. MATERIAL AND METHODS: The retinal morphology of heparanase knock-out and age-matched control wild type mice of 3-, 9- and 15-weeks old was characterized by means of histological evaluation. Immuno-histological stains for RPE65, F4/80 and Ki67 were performed for investigating the RPE, inflammatory and proliferating cells, respectively. RESULTS: Histological analysis showed no changes in age-matched wild-type controls, whereas the eyes of heparanase null mice were characterized by alterations in RPE and neural retina, as manifest by RPE folds and choroidal thickening, detached RPE cells, thickening of the photoreceptor layer and retinal disorganization. The presence of discrete hyperfluorescent foci, however, was absent. The prevalence of the RPE/choroidal changes or protrusions seemed to progress over time and were correlated with more RPE65 signal rather than influx of F4/80- or Ki67-positive cells. These results indicate that the subretinal alterations were mostly RPE driven, without influx of inflammatory or proliferating cells. CONCLUSIONS: Our results indicate that heparanase deficiency in the mice leads to RPE folds, choroidal thickening, and retinal disorganization. The presence of discrete hyperfluorescent foci, a key characteristic of the human disease, was not observed. However, it can be concluded that some of the observations in mice are similar to those seen after chronic use of PPS in humans. These findings indicate that the toxicity observed in the presence of heparanase inhibitors is target-related and will preclude the clinical use of heparanase inhibition as a therapeutic intervention.

Hexokinase 2 is dispensable for photoreceptor development but is required for survival during aging and outer retinal stress.

Photoreceptor death is the ultimate cause of vision loss in many retinal degenerative conditions. Identifying novel therapeutic avenues for prolonging photoreceptor health and function has the potential to improve vision and quality of life for patients suffering from degenerative retinal disorders. Photoreceptors are metabolically unique among other neurons in that they process the majority of their glucose via aerobic glycolysis. One of the main regulators of aerobic glycolysis is hexokinase 2 (HK2). Beyond its enzymatic function of phosphorylating glucose to glucose-6-phosphate, HK2 has additional non-enzymatic roles, including the regulation of apoptotic signaling via AKT signaling. Determining the role of HK2 in photoreceptor homeostasis may identify novel signaling pathways that can be targeted with neuroprotective agents to boost photoreceptor survival during metabolic stress. Here we show that following experimental retinal detachment, p-AKT is upregulated and HK2 translocates to mitochondria. Inhibition of AKT phosphorylation in 661W photoreceptor-like cells results in translocation of mitochondrial HK2 to the cytoplasm, increased caspase activity, and decreased cell viability. Rod-photoreceptors lacking HK2 upregulate HK1 and appear to develop normally. Interestingly, we found that HK2-deficient photoreceptors are more susceptible to acute nutrient deprivation in the experimental retinal detachment model. Additionally, HK2 appears to be important for preserving photoreceptors during aging. We show that retinal glucose metabolism is largely unchanged after HK2 deletion, suggesting that the non-enzymatic role of HK2 is important for maintaining photoreceptor health. These results suggest that HK2 expression is critical for preserving photoreceptors during acute nutrient stress and aging. More specifically, p-AKT mediated translocation of HK2 to the mitochondrial surface may be critical for protecting photoreceptors from acute and chronic stress.

LOXL3 Function Beyond Amino Oxidase and Role in Pathologies, Including Cancer.

Lysyl oxidase like 3 (LOXL3) is a copper-dependent amine oxidase responsible for the crosslinking of collagen and elastin in the extracellular matrix. LOXL3 belongs to a family including other members: LOX, LOXL1, LOXL2, and LOXL4. Autosomal recessive mutations are rare and described in patients with Stickler syndrome, early-onset myopia and non-syndromic cleft palate. Along with an essential function in embryonic development, multiple biological functions have been attributed to LOXL3 in various pathologies related to amino oxidase activity. Additionally, various novel roles have been described for LOXL3, such as the oxidation of fibronectin in myotendinous junction formation, and of deacetylation and deacetylimination activities of STAT3 to control of inflammatory response. In tumors, three distinct roles were described: (1) LOXL3 interacts with SNAIL and contributes to proliferation and metastasis by inducing epithelial-mesenchymal transition in pancreatic ductal adenocarcinoma cells; (2) LOXL3 is localized predominantly in the nucleus associated with invasion and poor gastric cancer prognosis; (3) LOXL3 interacts with proteins involved in DNA stability and mitosis completion, contributing to melanoma progression and sustained proliferation. Here we review the structure, function and activity of LOXL3 in normal and pathological conditions and discuss the potential of LOXL3 as a therapeutic target in various diseases.

Involvement of premacular mast cells in the pathogenesis of macular diseases.

We previously reported on the elevated intravitreal activities of tryptase and chymase in association with idiopathic epiretinal membrane (ERM) and idiopathic macular hole (MH). In this present study, we investigated the potential intraocular production of these serine proteases, and measured and compared tryptase and chymase activities in the vitreous body and serum in ERM, MH, proliferative diabetic retinopathy (PDR), and rhegmatogenous retinal detachment (RRD) patients. In addition, nuclear staining with hematoxylin and eosin (H&E) and mast-cell staining with toluidine blue were performed on samples of the vitreous core and bursa premacularis (BPM) of MH. We also performed immunostaining on the above two regions of vitreous samples for MH with anti-tryptase antibody, anti-chymase antibody, anti-podoplanin antibody, anti-lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1) antibody, and anti-fibroblast antibody. Moreover, we performed immunostaining with anti-tryptase antibody and anti-chymase antibody on ERMs collected intraoperatively. Tryptase activity in the vitreous body was significantly higher in ERM and MH than in PDR. However, no significant differences were observed in the tryptase activity in the serum among these four diseases. Chymase activity in the vitreous body was significantly higher in MH than in the other three diseases, yet chymase activity in the serum was below detection limit in any of the diseases. Nuclear staining with H&E revealed an abundance of nuclei in the BPM region, but few in the surrounding area. Mast-cell staining with toluidine blue revealed that the BPM showed metachromatic staining. In immunostaining with anti-fibroblasts antibody, anti-tryptase antibody, anti-chymase antibody, anti-podoplanin antibody, and anti-LYVE-1 antibody, the BPM stained more strongly than the vitreous core. Tryptase and chymase-positive cells were also observed in ERM. These findings revealed that the presence of mast cells in the BPM potentially represent the source of these serine proteases. Moreover, the BPM, as a lymphatic tissue, may play an important role in the pathogenesis of macular disease.

Oral Selumetinib Does Not Negatively Impact Photoreceptor Survival in Murine Experimental Retinal Detachment.

Purpose: Mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signaling is neuroprotective in some retinal damage models but its role in neuronal survival during retinal detachment (RD) is unclear. In addition, serous RDs are a prevalent side effect of MEK inhibitors (MEKi), blocking MAPK/ERK signaling for treatment of certain cancers. We tested the hypothesis that MEKi treatment in experimental RD would increase photoreceptor death. Methods: The MEKi selumetinib was delivered daily to C57BL/6 mice at a clinically relevant dose (10 mg/mL) starting 1 day prior to creating RD with subretinal hyaluronic acid injection. Photoreceptor TUNEL and outer nuclear layer (ONL) thickness were analyzed. Phospho-ERK1/2 (pERK) distribution, glial fibrillary acidic protein (GFAP) accumulation, and Iba-1 (microglia/macrophages) were evaluated with immunofluorescence. Results: pERK accumulated in the Müller glia in detached retinas, but this was effectively blocked by selumetinib. Selumetinib did not induce serous RDs at day 1 and did not increase TUNEL positive photoreceptors or further decrease ONL thickness compared to controls. Retinal gliosis was not altered, but selumetinib did block the increase in intraretinal microglia/macrophage Iba-1 fluorescence intensity and acquisition of amoeboid morphology. Conclusions: MAPK/ERK is neuroprotective in some retinal damage models; in RD, selumetinib blocked Müller pERK accumulation and changed the retinal microglia/macrophage phenotype but did not alter photoreceptor survival. This is consistent with the relatively good visual acuity seen in patients developing transient retinal detachments on MEK inhibitor therapy. Compensation by other neuroprotective pathways in the retina during retinal detachment may occur in the presence of MEK inhibition.

Retinal Pigment Epithelium Atrophy 1 (rpea1): A New Mouse Model With Retinal Detachment Caused by a Disruption of Protein Kinase C, θ.

PURPOSE: Retinal detachments (RDs), a separation of the light-sensitive tissue of the retina from its supporting layers in the posterior eye, isolate retinal cells from their normal supply of nourishment and can lead to their deterioration and death. We identified a new, spontaneous murine model of exudative retinal detachment, nm3342 (new mutant 3342, also referred to as rpea1: retinal pigment epithelium atrophy 1), which we characterize herein. METHODS: The chromosomal position for the recessive nm3342 mutation was determined by DNA pooling, and the causative mutation was discovered by comparison of whole exome sequences of mutant and wild-type controls. The effects of the mutation were examined in longitudinal studies by clinical evaluation, electroretinography (ERG), light microscopy, and marker and Western blot analyses. RESULTS: New mutant 3342, nm3342, also referred to as rpea1, causes an early-onset, complete RD on the ABJ/LeJ strain background, and central exudative RD and late-onset RPE atrophy on the C57BL/6J background. The ERG responses were normal at 2 months of age but deteriorate as mice age, concomitant with progressive pan-retinal photoreceptor loss. Genetic analysis localized rpea1 to mouse chromosome 2. By high-throughput sequencing of a whole exome capture library of an rpea1/rpea1 mutant and subsequent sequence analysis, a splice donor site mutation in the Prkcq (protein kinase C, θ) gene, was identified, leading to a skipping of exon 6, frame shift and premature termination. Homozygotes with a Prkcq-targeted null allele (Prkcqtm1Litt) have similar retinal phenotypes as homozygous rpea1 mice. We determined that the PKCθ protein is abundant in the lateral surfaces of RPE cells and colocalizes with both tight and adherens junction proteins. Phalloidin-stained RPE whole mounts showed abnormal RPE cell morphology with aberrant actin ring formation. CONCLUSIONS: The homozygous Prkcqrpea1 and the null Prkcqtm1Litt mutants are reliable novel mouse models of RD and can also be used to study the effects of the disruption of PRKCQ (PKCθ) signaling in RPE cells.

Reactivation of the PI3K/Akt Signaling Pathway by the Bisperoxovanadium Compound bpV(pic) Attenuates Photoreceptor Apoptosis in Experimental Retinal Detachment.

PURPOSE: Phosphatase and tensin homology deleted on chromosome 10 (PTEN) is crucial in neuronal apoptosis. This study evaluated the role of PTEN in photoreceptor cell apoptosis caused by retinal detachment (RD). METHODS: A rat model of RD was established, and PTEN expression changes were detected at different time points by Western blotting and immunofluorescence. Some of the rats were given subretinal injections of bisperoxovanadium compound (bpV[pic]) after RD. We documented the expression and distribution of phospho-Akt (p-Akt) and B-cell lymphoma 2 (Bcl-2) in the retina by Western blot analysis and immunofluorescence. Levels of phosph-phosphoinositide-dependent kinase 1 (p-PDK1), phospho-Bcl-2 death promotor (p-BAD), cytosolic cytochrome c (Cyt c), and cleaved Caspase-3 were detected by Western blotting. We measured phosphatidylinositol 3,4,5-triphosphate (PIP3) by ELISA. Apoptosis of photoreceptors was detected using the TUNEL assay. The thickness of the outer nuclear layer (ONL) also was recorded. RESULTS: The expression of PTEN gradually increased after RD, peaking at 3 days and then decreasing to normal by 7 days after RD. Subretinal injection of bpV(pic) effectively reduced the apoptosis of photoreceptors and preserved the retinal thickness of the ONL after RD. Compared to vehicle-treated RD groups, levels of p-Akt and p-PDK1 were significantly upregulated in bpV-treated RD groups. In addition, bpV treatment increased the levels of p-BAD and Bcl-2, and decreased the expression levels of cytosolic Cyt c and cleaved caspase-3 after RD. CONCLUSIONS: Phosphatase and tensin homology deleted on chromosome 10 (PTEN) participates in the apoptosis of photoreceptors after RD. Blocking PTEN may reactivate the PI3K/Akt pathway and attenuate photoreceptor apoptosis by suppressing the mitochondrial pathway.

Protein tyrosine phosphatase 1B regulates the activity of retinal pigment epithelial cells.

PURPOSE: To determine whether protein tyrosine phosphatase 1B (PTP1B) is expressed in rat retinal pigment epithelium (RPE) cells, to evaluate whether inhibition of PTP1B contributes to initiation of RPE cells into an active state, and to investigate the signaling pathways involved in this process. METHODS: Rat retinas were detached by trans-scleral injection of 1.4% sodium hyaluronate into the subretinal space. Immunocytochemistry evaluated the expression of PTP1B in RPE cells located at normal and detached retinas. From the cultured RPE cells treated with TCS-401, cell proliferation was assessed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetracolium bromide assay, and the protein expression levels of cyclin A and cyclin D1 were determined. The effect of TCS-401 on cell differentiation was confirmed by immunostaining for α-smooth muscle actin and by western blot. Cell migration activity and PTP1B signaling mechanism were determined. Migration Assay was used to evaluate cell migration activity. PTP1B signaling mechanism was determined by use of PD98059 and LY294002. RESULTS: PTP1B was expressed in the RPE layer of the normal retina. After retinal detachment, weak immunolabeling of PTP1B was seen in the RPE cells. TCS-401 promoted the proliferation and expression of cyclin A and cyclin D1 in RPE cells. TCS-401 induced RPE cells to differentiate toward better contractility and motility. A migration assay proved that inhibiting PTP1B improved the migratory activity of RPE cells. TCS-401 activated extracellular signal-regulated kinase (Erk) and protein kinase B (Akt) phosphorylation. Pretreatment with PD98059 and LY294002 abolished TCS-401-induced activation of Erk, Akt, cell proliferation, and cell migration. CONCLUSIONS: PTP1B may be involved in regulating the active state of RPE cells. The inhibition of PTP1B promoted the proliferation, myofibroblast differentiation, and migration of RPE cells, and MEK/Erk and PI3K/Akt signaling pathways played important roles in the proliferation and migration process.

Macrophage- and RIP3-dependent inflammasome activation exacerbates retinal detachment-induced photoreceptor cell death.

Detachment of photoreceptors from the retinal pigment epithelium is seen in various retinal disorders, resulting in photoreceptor death and subsequent vision loss. Cell death results in the release of endogenous molecules that activate molecular platforms containing caspase-1, termed inflammasomes. Inflammasome activation in retinal diseases has been reported in some cases to be protective and in others to be detrimental, causing neuronal cell death. Moreover, the cellular source of inflammasomes in retinal disorders is not clear. Here, we demonstrate that patients with photoreceptor injury by retinal detachment (RD) have increased levels of cleaved IL-1ß, an end product of inflammasome activation. In an animal model of RD, photoreceptor cell death led to activation of endogenous inflammasomes, and this activation was diminished by Rip3 deletion. The major source of Il1b expression was found to be infiltrating macrophages in the subretinal space, rather than dying photoreceptors. Inflammasome inhibition attenuated photoreceptor death after RD. Our data implicate the infiltrating macrophages as a source of damaging inflammasomes after photoreceptor detachment in a RIP3-dependent manner and suggest a novel therapeutic target for treatment of retinal diseases.

Mammalian STE20-like kinase 2, not kinase 1, mediates photoreceptor cell death during retinal detachment.

Photoreceptor cell death is the definitive cause of vision loss in retinal detachment (RD). Mammalian STE20-like kinase (MST) is a master regulator of both cell death and proliferation and a critical factor in development and tumorigenesis. However, to date the role of MST in neurodegeneration has not been fully explored. Utilizing MST1(-/-) and MST2(-/-) mice we identified MST2, but not MST1, as a regulator of photoreceptor cell death in a mouse model of RD. MST2(-/-) mice demonstrated significantly decreased photoreceptor cell death and outer nuclear layer (ONL) thinning after RD. Additionally, caspase-3 activation was attenuated in MST2(-/-) mice compared to control mice after RD. The transcription of p53 upregulated modulator of apoptosis (PUMA) and Fas was also reduced in MST2(-/-) mice post-RD. Retinas of MST2(-/-) mice displayed suppressed nuclear relocalization of phosphorylated YAP after RD. Consistent with the reduction of photoreceptor cell death, MST2(-/-) mice showed decreased levels of proinflammatory cytokines such as monocyte chemoattractant protein 1 and interleukin 6 as well as attenuated inflammatory CD11b cell infiltration during the early phase of RD. These results identify MST2, not MST1, as a critical regulator of caspase-mediated photoreceptor cell death in the detached retina and indicate its potential as a future neuroprotection target.

Retinal MMP-12, MMP-13, TIMP-1, and TIMP-2 expression in murine experimental retinal detachment.

PURPOSE: Matrix metalloproteinases (MMPs) and their inhibitors play a role in the pathobiology of retinal detachment (RD) and proliferative vitreoretinopathy (PVR). Proliferative vitreoretinopathy is facilitated by chronic retinal detachment and involves excessive deposition of extracellular matrix (ECM) proteins. Matrix metalloproteinase-2 and -13 are important modulators of the ECM which have not been evaluated in RD. The purpose of this study was to investigate the retinal expression of select MMPs, including MMP-12, MMP-13, and associated inhibitors in a murine model of retinal detachment. METHODS: Transient or chronic retinal detachments (RDs) were induced by subretinal injection of either saline (SA) or hyaluronic acid (HA) in C57BL/6 mice. To confirm that the HA-RD model has features consistent with PVR-like changes, glial activation and subretinal fibrosis were evaluated with immunofluorescence, dilated fundus examination, and spectral-domain optical coherence tomography (SD-OCT). Gene expression was quantified by qRT-PCR. Proteins were assayed by immunoblot and immunohistochemistry. RESULTS: Hyaluronic acid RD eyes developed gliosis and subretinal fibrosis on dilated exam, SD-OCT, and immunofluorescence analysis. Gene expression of Mmp-12 and Mmp-13, and Timp-1 was strongly upregulated at all time points in RD compared with controls. Timp-2, Mmp-2, and Mmp-9 expression was modest. Hyaluronic acid RDs exhibited more MMP and TIMP expression than SA-RDs. MMP-12, -13, and TIMP-1 proteins were elevated in RDs compared with controls. Immunohistochemistry revealed moderate to strong MMP-13 levels in subretinal space macrophages. CONCLUSIONS: Fibrosis can develop in the HA-RD model. There is an upregulation of select MMPs that may modulate the wound healing process following RD.

Intravitreal injection of soluble erythropoietin receptor exacerbates photoreceptor cell apoptosis in a rat model of retinal detachment.

PURPOSE: To evaluate the effects of intravitreal injection of soluble erythropoietin (EPO) receptor (sEPOR) on photoreceptor cell apoptosis in an animal model of retinal detachment (RD). METHODS: Various dosages of sEPOR (2, 20, or 200 ng) were injected into the vitreous cavities of normal rats. Three days after injection, retinal function was measured by flash electroretinography (ERG). On day 7, histopathology and retinal morphology were examined by light and transmission electron microscopy (TEM), respectively. Rat models of RD were successfully established by injection of 1.4% sodium hyaluronate into the subretinal space, followed by immediate injection of phosphate-buffered saline (PBS) or sEPOR into the vitreous cavity. On day 3, photoreceptor cell apoptosis was evaluated using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), and caspase-3 activity assayed by Western blotting and immunofluorescence. Light microscopic examination of retinal histopathology was used to determine the thickness of the outer nuclear layer (ONL) 14 days after establishment of RD. RESULTS: There were no significant differences in the latency and amplitude of maximal a, b and oscillatory potential (OP) wave responses by flash ERG before or 3 days after sEPOR injection (p > 0.05). Retinal tissues showed no obvious pathological changes by either light or transmission electron microscopy. Both Western blotting and immunofluorescence indicated consistent sEPOR enhanced caspase-3 activation aggravated apoptosis of photoreceptor cells in RD rat retinas. On day 14, RD ONLs were thinner, according to increasing dosages of sEPOR. CONCLUSION: Intravitreal injection of sEPOR exacerbates photoreceptor cell apoptosis in RD models via activation of caspase-3.

Interleukin-6 and matrix metalloproteinase expression in the subretinal fluid during proliferative vitreoretinopathy: correlation with extent, duration of RRD and PVR grade.

PURPOSE: To investigate interleukin (IL)-6 protein levels in the subretinal fluid (SRF) of patients with rhegmatogenous retinal detachment (RRD) complicated by proliferative vitreoretinopathy (PVR); to correlate the IL-6 levels with matrix metalloproteinases (MMP)-1, -2, -3, -8, -9 and tissue inhibitor of metalloproteinases (TIMP)-1 with respect to RRD extent, duration and PVR grade. METHODS: Thirty-one SRF samples from 31 eyes of 31 patients with RRD complicated with PVR and five SRF samples from five eyes of five patients suffering from RRD not complicated with PVR were collected during treatment by scleral buckling. Enzyme-Linked Immunosorbent Assay was employed for the measurement of IL-6, MMP-1, -3, -8 and TIMP-1 levels while the enzymatic activity of MMP-2 and MMP-9 was assessed by gelatin zymography. RESULTS: Protein levels of IL-6 (p=0.050), MMP-1 (p=0.001), MMP-3 (p=0.005), MMP-8 (p=0.003), TIMP-1 (p=0.001) as well as enzymatic activity of proMMP-2 (p=0.001), MMP-2 (p=0.023) and MMP-9 (p=0.015), were significantly higher in the SRF of PVR patients compared to controls. IL-6 levels correlated significantly with TIMP-1 (r=0.528, p=0.035). Regarding clinical parameters of the detachment, IL-6 levels correlated with RRD extent (r=0.592, p=0.016), but not with RRD duration (p=0.857) and PVR grade (p=0.594). Regression analysis revealed positive correlations between IL-6 and MMP-2. CONCLUSIONS: There was a significant correlation between IL-6 and TIMP-1 levels in the SRF of PVR patients. The findings of this study are in agreement with relevant studies concerning IL-6 involvement in the modulation of MMP expression and are indicative of IL-6 and MMP activity during PVR, mainly that of MMP-2 and TIMP-1.

Increased integration of transplanted CD73-positive photoreceptor precursors into adult mouse retina.

PURPOSE. Retinal degeneration initiated by loss of photoreceptors is the prevalent cause of visual impairment and blindness in industrialized countries. Transplantation of photoreceptor cells represents a possible replacement strategy. This study determined that identification of cell surface antigens can assist in enriching photoreceptor precursors for transplantation. METHODS. The expression profile of rod photoreceptors at postnatal day 4 was investigated by microarray analysis to identify photoreceptor-specific cell surface antigens. For enrichment of transplantable photoreceptors, neonatal retinas from rod photoreceptor-specific reporter mice were dissociated, and the rods were purified by magnetic associated cell sorting (MACS) with CD73 antibodies. MAC-sorted cell fractions were transplanted into the subretinal space of adult wild-type mice. The number of rod photoreceptors contained in unsorted, CD73-negative, and CD73-positive cell fractions were quantified in vitro and after grafting in vivo. RESULTS. Microarray analysis revealed that CD73 is a marker for rod photoreceptors. CD73-based MACS resulted in enrichment of rods to 87%. Furthermore, in comparison with unsorted cell fractions, CD73-positive MAC-sorted cells showed an approximately three-fold increase in the number of integrated, outer segment-forming photoreceptors after transplantation. CONCLUSIONS. CD73-based MACS is a reliable method for the enrichment of integrating photoreceptors. Purification via cell surface markers represents a new tool for the separation of transplantable photoreceptor precursors from a heterogeneous cell population, avoiding the need of reporter gene expression in target cells.

Heat shock protein 70 (HSP70) is critical for the photoreceptor stress response after retinal detachment via modulating anti-apoptotic Akt kinase.

Photoreceptor apoptosis is a major cause of vision loss in many ocular diseases. Significant progress has been made to elucidate the molecular pathways involved in this process, yet little is known about proteins counteracting these apoptotic pathways. It is established that heat shock proteins (HSPs) function as molecular helper proteins (chaperones) by preventing protein aggregation and facilitating refolding of dysfunctional proteins, critical to the survival of all organisms. Here, we investigated the role of HSP70 on photoreceptor survival after experimental retinal detachment (RD) in mice and rats. We found that HSP70 was up-regulated after RD and associated with phosphorylated Akt, thereby preventing its dephosphorylation and further activation of cell death pathways. Administration of quercetin, which inhibits HSP70 and suppresses Akt phosphorylation significantly increased photoreceptor apoptosis. Similarly, RD-induced photoreceptor apoptosis was augmented in mice carrying hypomorphic mutations of the genes encoding HSP70. On the other hand, administration of geranylgeranylacetone, which induces an increase in HSP70 significantly decreased photoreceptor apoptosis after RD through prolonged activation of Akt pathway. Thus, HSP70 may be a favorable potential target to increase photoreceptor cell survival after RD.

RhoA inactivation prevents photoreceptor axon retraction in an in vitro model of acute retinal detachment.

PURPOSE: An early injury response to retinal detachment is disruption of synaptic connectivity between photoreceptors and second-order neurons. Most dramatic is the retraction of rod cell axons and their terminals away from the outer synaptic layer and toward their cell bodies. This study tested whether axonal retraction in detached retina was due to the activation of the small GTPase RhoA and was preventable using RhoA antagonists. METHODS: Retinal detachments were created in in vitro preparations of porcine eyecups. RhoA activation was determined with a Rhotekin binding assay. To block axon retraction, drugs were applied to neural retinal explants either before or after detachment from the retinal pigment epithelium. Presynaptic movement was quantified by image analysis of double-labeled retinas examined with confocal microscopy. RESULTS: Active RhoA increases transiently after detachment followed by morphologic evidence of axonal retraction over the next 24 hours. Pretreating the retina with a RhoA antagonist, CT-04, or a Rho kinase inhibitor, Y27632, at multiple concentrations significantly inhibited axonal retraction. Reducing calcium influx through L-type calcium channels with nicardipine also blocked retraction. To create a more plausible therapeutic scenario, drug treatments were delayed and applied after retinal detachment. The Rho kinase inhibitor, but not nicardipine, significantly blocked rod axonal retraction when applied up to 6 hours after detachment. CONCLUSIONS: Thus, RhoA and downstream Rho kinase activity constitute part of the mechanism that produces rod axonal retraction in retinal explants. Treatments that manipulate RhoA signaling may promote synaptic stability after retinal detachment.

Correlation of matrix metalloproteinase levels with the grade of proliferative vitreoretinopathy in the subretinal fluid and vitreous during rhegmatogenous retinal detachment.

PURPOSE: We investigated the activity of matrix metalloproteinase (MMP)-2 and -9 and their latent pro-forms (proMMP-2, -9), and protein levels of MMP-1, -3, -8 and tissue inhibitor of MMPs (TIMP)-1 in the subretinal fluid (SRF) and vitreous of patients with rhegmatogenous retinal detachment (RRD). Potential correlations with proliferative vitreoretinopathy (PVR) grade were determined. METHODS: Thirty-seven SRF and 32 vitreous samples from RRD patients and nine vitreous samples from human organ donors (controls), were collected and assayed for MMP-1, -3, -8/TIMP-1 levels using enzyme-linked immunosorbent assay (ELISA), and for proMMP-2, -9, MMP-2, -9 activity employing gelatine zymography. RESULTS: ProMMP-2, -9, MMP-1, -3, -9, TIMP-1 were significantly higher in the SRF and vitreous of RRD patients compared to the vitreous of organ donors. MMP-8 levels were higher in RRD patients' SRF. Regarding PVR grade, MMPs and TIMP-1 were differentially present in SRF and vitreous. PVR grade correlated significantly with the levels of MMP-2 in SRF, while proMMP-2, MMP-1, -2, -3, -8, -9 and TIMP-1 levels correlated with PVR grade in the vitreous. CONCLUSION: MMP/TIMP-1 levels are elevated in SRF and vitreous during RRD. Significant correlations between PVR grade and MMP-2 in SRF and proMMP-2, MMP-1, -2, -3, -8, -9 and TIMP-1 levels in vitreous were revealed. Investigation of MMP activity in vitreous may provide more valid conclusions compared to SRF pertaining to the role of the MMPs during RRD. The observations of the present study suggest a possible role for MMPs and TIMP-1 in PVR pathophysiology.

Gene transcription profile of the detached retina (An AOS Thesis).

PURPOSE: Separation of the neurosensory retina from the retinal pigment epithelium (RPE) yields many morphologic and functional consequences, including death of the photoreceptor cells, Müller cell hypertrophy, and inner retinal rewiring. Many of these changes are due to the separation-induced activation of specific genes. In this work, we define the gene transcription profile within the retina as a function of time after detachment. We also define the early activation of kinases that might be responsible for the detachment-induced changes in gene transcription. METHODS: Separation of the retina from the RPE was induced in Brown-Norway rats by the injection of 1% hyaluronic acid into the subretinal space. Retinas were harvested at 1, 7, and 28 days after separation. Gene transcription profiles for each time point were determined using the Affymetrix Rat 230A gene microarray chip. Transcription levels in detached retinas were compared to those of nondetached retinas with the BRB-ArrayTools Version 3.6.0 using a random variance analysis of variance (ANOVA) model. Confirmation of the significant transcriptional changes for a subset of the genes was performed using microfluidic quantitative real-time polymerase chain reaction (qRT-PCR) assays. Kinase activation was explored using Western blot analysis to look for early phosphorylation of any of the 3 main families of mitogen-activated protein kinases (MAPK): the p38 family, the Janus kinase family, and the p42/p44 family. RESULTS: Retinas separated from the RPE showed extensive alterations in their gene transcription profile. Many of these changes were initiated as early as 1 day after separation, with significant increases by 7 days. ANOVA analysis defined 144 genes that had significantly altered transcription levels as a function of time after separation when setting a false discovery rate at < or =0.1. Confirmatory RT-PCR was performed on 51 of these 144 genes. Differential transcription detected on the microarray chip was confirmed by qRT-PCR for all 51 genes. Western blot analysis showed that the p42/p44 family of MAPK was phosphorylated within 2 hours of retinal-RPE separation. This phosphorylation was detachment-induced and could be inhibited by specific inhibitors of MAPK phosphorylation. CONCLUSIONS: Separation of the retina from the RPE induces significant alteration in the gene transcription profile within the retina. These profiles are not static, but change as a function of time after detachment. These gene transcription changes are preceded by the activation of the p42/p44 family of MAPK. This altered transcription may serve as the basis for many of the morphologic, biochemical, and functional changes seen within the detached retina.

XIAP effects on retinal detachment-induced photoreceptor apoptosis [corrected].

PURPOSE: To evaluate the ability of X-linked inhibitor of apoptosis (XIAP) gene therapy to provide neuroprotection to cells of the outer nuclear layer (ONL) of the retina after retinal detachment. METHODS: Subretinal injections of a recombinant adenoassociated virus (rAAV) encoding either XIAP or green fluorescent protein (GFP; injection control) were performed in the left eye of Brown Norway rats. Two weeks later, retinal detachments were created at the site of viral injection by delivering sodium hyaluronate into the subretinal space. Retinal tissue was harvested at 24 hours after retinal detachment and was analyzed for caspase 3 and 9 activity. Histologic analysis was conducted on samples taken at 3 days and 2 months after detachment to confirm the presence of XIAP or GFP expression and to assess levels of apoptosis and changes in retinal thickness. RESULTS: Caspase assays performed 24 hours after detachment confirmed an expected increase in caspase 3 and 9 activity in the detached regions of GFP-treated retinas, whereas XIAP-treated detached retinas behaved comparably to attached controls. TUNEL analysis of 3-day tissue samples showed fewer apoptotic cells in XIAP-treated detachments than in GFP-treated detachments. At 2 months after the detachment, histology and immunohistochemistry confirmed the preservation of the ONL at sites of XIAP overexpression, whereas the GFP-treated detached retinas had significantly deteriorated. CONCLUSIONS: The results suggest that XIAP confers structural neuroprotection of photoreceptors for at least 2 months after retinal detachment.