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.

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.

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.

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.

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.

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.

Viral delivery of heme oxygenase-1 attenuates photoreceptor apoptosis in an experimental model of retinal detachment.

This study was designed to evaluate the efficacy of subretinal injection of recombinant adeno-associated virus vector expressing heme oxygenase-1 (rAAV-HO-1) in attenuating photoreceptor apoptosis induced by experimental retinal detachment (RD) in Sprague-Dawley rats. Our results disclosed that subretinal rAAV-HO-1 delivery achieved localized high HO-1 gene expression in retinal outer nuclear layer (ONL) compared with rAAV-lacZ-injected eyes and eyes with RD left untreated both at 2 (p=0.003) and 28 (p=0.007) days of RD. The ONL thickness (p=0.018) and mean photoreceptor nuclei count (p=0.009) in eyes receiving rAAV-HO-1 injection was significantly higher than in rAAV-lacZ-injected or eyes with RD left untreated at 28 days of RD. There were fewer apoptotic photoreceptor nuclei at 2 (p=0.008) and 5 (p=0.018) days of RD and less activated caspase-3 expression (p=0.008) at 2 days of RD in rAAV-HO-1 treated eyes than in control eyes. These data supported that gene transfer approach might attenuate photoreceptor apoptosis caused by RD with a resultant better ONL preservation.

Lipocalin-like prostaglandin D synthase in subretinal fluid of detached retinas in humans.

BACKGROUND: The presence of lipocalin-like prostaglandin D synthase (L-PGDS), a dominantly brain derived protein in the cerebrospinal fluid (CSF) with a low concentration in serum, in the subretinal fluid (SRF) of detached retinas, has not been reported. L-PGDS has been demonstrated in the ciliary body and the retinal pigment epithelium in rats. METHODS: SRF was sampled during surgery (scleral buckling) of rhegmatogenous retinal detachments in 20 eyes. Biochemical and nephelometric analysis for L-PGDS concentration were performed. Albumin concentrations were determined in the same samples. RESULTS: L-PGDS concentrations in SRF (mean 18.9 mg/L +/- 14.8 mg/L) were markedly higher than the normal L-PGDS concentration in serum (<0.55 mg/L) and appears to decrease with the duration of the retinal detachment (P = 0.0064). CONCLUSIONS: As the subretinal space is not accessible in attached retinas there are no data on normal levels of L-PGDS in the subretinal space. The L-PGDS concentrations measured in the SRF of detached retinas are on average 34 times higher than normal L-PGDS serum concentration and about in the range of normal L-PGDS CSF concentration (15.3 mg/L). As the concentrations of L-PGDS exceed normal serum levels, a choriocapillary transudation is highly improbable as the source of L-PGDS in SRF of detached retinas.

Lysyl oxidase activity in the ocular tissues and the role of LOX in proliferative diabetic retinopathy and rhegmatogenous retinal detachment.

PURPOSE: Lysyl oxidase (LOX) cross-links the side chain of collagen and elastin and thereby contributes to extracellular matrix (ECM) integrity. ECM remodeling is seen in various ocular diseases. Until now, there have been no reports on the LOX enzyme's activity in ocular tissues. The purpose of this study was to estimate LOX activity and expression in human donor ocular tissues and to measure the specific activity of LOX in the vitreous of proliferative diabetic retinopathy (PDR) and rhegmatogenous retinal detachment (RRD). METHOD: Human donor eyeballs obtained from an eye bank were used to study tissue distribution of LOX. Human vitreous specimens were obtained during vitreoretinal surgery from PDR (n = 16) and RRD (n = 10). LOX activity was estimated by N-acetyl-3,7-dihydroxyphenoxazine assay, immunohistochemistry, and real-time polymerase chain reaction (RT-PCR). Matrix metalloprotease (MMP)-2 and -9 were quantified in the vitreous by sandwich enzyme-linked immunosorbent assay (ELISA). RESULTS: The specific activity of LOX in ocular tissues was on the order of vitreous, iris ciliary body, lens, choroid RPE, and retina, which were comparable by mRNA expression and immunolocalization. The vitreous level of LOX activity decreased significantly in PDR and RRD, with an increase in total MMP-2 and -9 levels compared with normal donor vitreous. CONCLUSIONS: LOX activity showed a statistically significant decrease in the vitreous of PDR and RRD relative to control specimens. This effect can contribute to the inadequate collagen cross-linking that causes the ECM changes that occur in these diseases.

Phosphorylation of extracellular signal-regulated kinase and p27(KIP1) after retinal detachment.

PURPOSE: The roles of the extracellular signal-regulated kinase (ERK) pathway in the expression of cyclin D1 and p27(KIP1), the phosphorylation of p27(KIP1), and proliferation activity were examined after retinal detachment. METHODS: Normal eyes and eyes at 15 min, 2 and 4 days after retinal detachment in C57Bl6 mice were examined by immunohistochemistry using anti-phosphorylated (p) ERK1/2, anti-cyclin D1, anti-p27(KIP1), anti-p27(KIP1) phosphorylated at serine 10 (S10-phospho-p27), and anti-proliferating cell nuclear antigen (PCNA) antibodies with or without treatment with a specific ERK inhibitor, PD98059. Mouse Müller cells were isolated and examined for alteration of p27(KIP1) and cyclin D1 after exposure of basic fibroblast growth factor (bFGF) with and without treatment of PD98059 using Western blotting. RESULTS: In the normal retina, nuclear immunoreactivity for p27(KIP1), but not S10-phospho-p27 or pERK1/2, was observed in the middle sublayer of the inner nuclear layer (INL), where Müller glial cells are situated. At 15 min after the retinal detachment, p27(KIP1), S10-phospho-p27 and pERK1/2-positive nuclei were noted in the INL, whereas immunoreactivity for pERK1/2 or S10-phospho-p27 was not observed after treatment with PD98095. Cyclin D1 was induced in the INL 2 days after the retinal detachment, and the induction was inhibited by PD98059. At 4 days after the detachment, p27(KIP1) immunoreactivity was not observed, and cyclin D1 and PCNA were expressed. The disappearance of p27(KIP1) was suppressed, whereas expression of cyclin D1 and PCNA was not observed in mice treated with PD98059. Exposure of bFGF relatively decreased the expression level of p27(KIP1) and increased the level of cyclin D1 in mouse Müller cells, compared with control level. Induction of cyclin D1 and decrease in p27(KIP1) were inhibited with treatment of PD98059. CONCLUSION: Phosphorylation of ERK and expression of p27(KIP1) and cyclin D1 are involved in the proliferation of Müller cells after retinal detachment.

Measurement of activities in two different angiotensin II generating systems, chymase and angiotensin-converting enzyme, in the vitreous fluid of vitreoretinal diseases: a possible involvement of chymase in the pathogenesis of macular hole patients.

PURPOSE: To investigate possible involvement of chymase and angiotensin-converting enzyme (ACE) in the pathogenesis of vitreoretinal diseases, both of which are related to the production of angiotensin II. METHODS: We measured chymase and ACE activities in the vitreous in the 54 affected eyes of 54 patients who had undergone vitreous surgery for idiopathic macular holes (MH, n = 14), proliferative diabetic retinopathy (PDR, n = 14), idiopathic epiretinal membranes (ERM, n = 13), and rhegmatogenous retinal detachment (RRD, n = 13). RESULTS: Chymase activities in the vitreous from patients with MH, PDR, ERM, and RRD were 1.87 +/- 0.53, 0.06 +/- 0.04, 0.40 +/- 0.12, and 0.08 +/- 0.03 (mean +/- SE) mU/mg protein, respectively, and ACE activities in the vitreous humor were 0.18 +/- 0.09, 0.30 +/- 0.07, 0.01 +/- 0.01, and 0.03 +/- 0.02 (mean +/- SE) mU/mg protein, respectively. Chymase activity was significantly elevated in MH among these diseases (p < 0.01, Scheffe), and ACE was significantly activated in PDR compared to ERM and RRD (p < 0.05, Scheffe). CONCLUSIONS: Our results suggest that two different angiotensin II generating systems are activated in human vitreous humor; an increased activity of chymase may play a possible role in the formation of macular holes.

FAS-mediated apoptosis and its relation to intrinsic pathway activation in an experimental model of retinal detachment.

PURPOSE: To determine whether the FAS-mediated apoptosis pathway becomes activated in the retina after retinal detachment and to investigate the temporal relationship between the activation of the FAS-pathway and the intrinsic apoptosis pathway involving caspase-9 and cytochrome c. METHODS: Experimental retinal detachments were created in Brown-Norway rats by injecting 10% hyaluronic acid into the subretinal space. Retinal tissue was harvested at 2, 4, 8, 24, 72, and 168 hours after creation of the detachment. Immunoprecipitation was performed to assess for FAS-receptor/FAS-ligand complex formation, and activation of caspase-8 and BID (a member of the Bcl-2 family of proteins) was assessed by Western blot analysis. A caspase-9 activity assay and immunoprecipitation of the caspase-9/cytochrome c complex were performed at these same time points. Specific pathway inhibition was performed with the caspase-9 inhibitor zLEHD.fmk or neutralizing antibodies against either the FAS-receptor or FAS-ligand. Transcription levels of FAS and intrinsic pathway intermediates were assessed as a function of time after retinal detachment by using quantitative real-time polymerase chain reaction. RESULTS: Retinal detachment resulted in the time-dependent formation of the FAS-receptor/FAS-ligand complex that preceded the peak of caspase-9 activity and caspase-9/cytochrome c complex formation. Cleavage of caspase-8 and truncation of BID were also observed. Injection of zLEHD.fmk into the subretinal space of a detached retina resulted in decreased caspase-9 activity, as did injection of anti-FAS-receptor antibody into either the subretinal space or the vitreous. Retinal detachment resulted in the transcriptional upregulation of the FAS-receptor, FAS-ligand, caspase-8 and BID, but not caspase-9 and cytochrome c. CONCLUSIONS: The FAS-mediated apoptosis pathway becomes activated and transcriptionally upregulated after retinal detachment. The peak of FAS activation precedes that of the intrinsic pathway, and inhibition of FAS activation can decrease caspase-9 activity.

Neuron specific enolase in retinal detachment.

PURPOSE: Neuron Specific Enolase (NSE) is released following central nervous system (CNS) distress. As retina is part of the CNS, NSE levels were measured in the subretinal fluid (SRF), aqueous, and serum of patients with primary rhegmatogenous retinal detachment (RD). METHODS: Radioimmunoassay was used to determine NSE levels in the SRF, aqueous, and serum of 13 patients (28-92 years old, mean = 71 years) with RD. As controls, NSE was measured in the aqueous of 6 patients undergoing cataract surgery and in serum of 18 patients without ophthalmological or neurological diseases. RESULTS: SRF levels of NSE ranged from 50-200 microg/l (mean +/- s.d. = 150 +/- 57). NSE levels in aqueous from patients with RD were 2-140 microg/l (mean +/- s.d. = 39 +/- 42), significantly higher than in controls (0-6 microg/l; mean +/- s.d. = 1.58 +/- 2.24; p = 0.04). Serum NSE levels in RD patients ranged from 6.5-80 microg/l (mean +/- s.d. = 26 +/- 21) and was significantly higher than in controls (5.3 +/- 1.66 microg/l; p = 0.005). CONCLUSIONS: Retinal neuron injury in retinal detachment (RD) releases sufficient Neuron Specific Enolase (NSE) to be detected in subretinal fluid, aqueous, and even in serum. Thus, NSE could index disease severity in RD and provide a means by which to assess the response to neuroprotection in RD.

[Activities of trypsin-like serine proteases and their inhibitors in blood and subretinal fluid of patients with different stages of proliferative vitreoretinopathy in rhegmatogenous retinal detachment]. / Izuchenie aktivnosti serinovykh proteinaz tipa tripsina i ikh ingibitorov v krovi i subretinal'noi zhidkosti u bol'nykh s razlichnymi stadiiami proliferativnoi vitreoretinopatii pri regmatogennoi otsloike setchatoi obolochki.

Concentrations of protein, trypsin-like and antitryptic activities, and the level of alpha 2-macroglobulin were measured in the subretinal fluid (SRF) and blood of patients with different stages of proliferative vitreoretinopathy (PVR). The content of protein in SRF of patients with retinal detachment with PVR was by an order of magnitude higher than in the vitreous body and increased from stage B to stage D1, which indicates increased permeability of the blood-eye barrier. Trypsin-like proteolytic enzymes and their main inhibitors are present in the SRF, and changes in their activity indicate augmentation of the inflammatory component of PVR from stage B to stage C3 and transition to a process similar to tissue cicatrization during stage D1. Changes in the proteinase-inhibitor balance in the peripheral blood in PVR are characteristic of a sluggish inflammatory process which activates from stage B to stage C and eventuates in stage D1. Increase in the blood level of alpha 2-macroglobulin during stages C3 and D1 seems to indicate the development of total autoimmune reaction. Antiinflammatory steroid therapy before surgery had a favorable impact on the course of the postoperative period.

Detached retina affects morphologic and biochemical changes in the retina adjacent to bullous retinal detachment in rabbits.

PURPOSE: Long-term results, more than 10 years after successful retinal detachment surgery, have shown gradually decreasing visual acuity in some cases. It is unclear if reduced functional recovery postoperatively is caused by anatomic changes or biochemical disorders. To determine the etiology of the reduced visual acuity, we cytochemically examined the changes in the cellular responses of the edges of retinal detachments. METHODS: We histochemically studied the glucose-6-phosphatase (G6P) and 5'-nucleotidase (5'-Nase) activity in the rabbit retina. Experimental rhegmatogenous retinal detachment was produced in a rabbit model after partial vitrectomy, followed by retinal tear formation. RESULTS: Although 5'-Nase activity gradually decreased during the period of detachment, activity was still detectable after 24 weeks. G6P activity increased in the region of the detached neural retina. Around the border of the detached retina, the decrease in 5'-Nase activity extended approximately 140 micrometers into the adjacent attached retina at 2 weeks after detachment and 270 micrometers at 24 weeks. CONCLUSIONS: These observations suggest that some anatomical and biochemical damages may occur in the retina adjacent to bullous retinal detachment and may explain the reduction in postoperative vision in some clinical cases.