Sphingomyelinases in retinas and optic nerve heads: Effects of ocular hypertension and ischemia.

Sphingomyelinases (SMase), enzymes that catalyze the hydrolysis of sphingomyelin to ceramide, are important sensors for inflammatory cytokines and apoptotic signaling. Studies have provided evidence that increased SMase activity can contribute to retinal injury. In most tissues, two major SMases are responsible for stress-induced increases in ceramide: acid sphingomyelinase (ASMase) and Mg2+-dependent neutral sphingomyelinase (NSMase). The purposes of the current study were to determine the localization of SMases and their substrates in the retina and optic nerve head and to investigate the effects of ocular hypertension and ischemia on ASMase and NSMase activities. Tissue and cellular localization of ASMase and NSMase were determined by immunofluorescence imaging. Tissue localization of sphingomyelin in retinas was further determined by Matrix-Assisted Laser Desorption/Ionization mass spectrometry imaging. Tissue levels of sphingomyelins and ceramide were determined by liquid chromatography with tandem mass spectrometry. Sphingomyelinase activities under basal conditions and following acute ischemic and ocular hypotensive stress were measured using the Amplex Red Sphingomyelinase Assay Kit. Our data show that ASMase is in the optic nerve head and the retinal ganglion cell layer. NSMase is in the optic nerve head, photoreceptor and retinal ganglion cell layers. Both ASMase and NSMase were identified in human induced pluripotent stem cell-derived retinal ganglion cells and optic nerve head astrocytes. The retina and optic nerve head each exhibited unique distribution of sphingomyelins with the abundance of very long chain species being higher in the optic nerve head than in the retina. Basal activities for ASMase in retinas and optic nerve heads were 54.98 ± 2.5 and 95.6 ± 19.5 mU/mg protein, respectively. Ocular ischemia significantly increased ASMase activity to 86.2 ± 15.3 mU/mg protein in retinas (P = 0.03) but not in optic nerve heads (81.1 ± 15.3 mU/mg protein). Ocular hypertension significantly increased ASMase activity to 121.6 ± 7.3 mU/mg protein in retinas (P < 0.001) and 267.0 ± 66.3 mU/mg protein in optic nerve heads (P = 0.03). Basal activities for NSMase in retinas and optic nerve heads were 12.3 ± 2.1 and 37.9 ± 8.7 mU/mg protein, respectively. No significant change in NSMase activity was measured following ocular ischemia or hypertension. Our results provide evidence that both ASMase and NSMase are expressed in retinas and optic nerve heads; however, basal ASMase activity is significantly higher than NSMase activity in retinas and optic nerve heads. In addition, only ASMase activity was significantly increased in ocular ischemia or hypertension. These data support a role for ASMase-mediated sphingolipid metabolism in the development of retinal ischemic and hypertensive injuries.

Evidence for ceramide induced cytotoxicity in retinal ganglion cells.

Ceramides are bioactive compounds that play important roles in regulating cellular responses to extracellular stimuli and stress. Previous studies have shown that ceramides contribute to retinal degeneration associated with ischemic and ocular hypertensive stress. Acid sphingomyelinase (ASMase) is one of the major enzymes responsible for the stress-induced generation of ceramides. The goals of this study are to investigate the effects of ceramides on retinal ganglion cells (RGCs) and of ASMase inhibition in ocular hypertensive mice. Induced pluripotent stem cell (iPSC)-derived RGCs and primary cultures of human optic nerve head astrocytes were used to characterize the response to C2-ceramide. Microbead-induced ocular hypertension in the ASMase heterozygote mouse model was used to confirm the physiological relevance of in vitro studies. In mice, RGC function and morphology were assessed with pattern ERG (pERG) and immunofluorescence. The addition of C2-ceramide to iPSC-derived RGCs produced a significant concentration- and time-dependent reduction in cell numbers when compared to control cultures. While the addition of C2-ceramide to astrocytes did not affect viability, it resulted in a 2.6-fold increase in TNF-α secretion. The addition of TNF-α or conditioned media from C2-ceramide-treated astrocytes to RGC cultures significantly reduced cell numbers by 56.1 ± 8.4% and 24.7 ± 4.8%, respectively. This cytotoxic response to astrocyte-conditioned media was blocked by TNF-α antibody. In ASMase heterozygote mice, functional and morphological analyses of ocular hypertensive eyes reveal significantly less RGC degeneration when compared with hypertensive eyes from wild-type mice. These results provide evidence that ceramides can induce RGC cell death by acting directly, as well as indirectly via the secretion of TNF-α from optic nerve head astrocytes. In vivo studies in mice provide evidence that ceramides derived through the activity of ASMase contribute to ocular hypertensive injury. Together these results support the importance of ceramides in the pathogenesis of ocular hypertensive injury to the retina.

Ischemic preconditioning, retinal neuroprotection and histone deacetylase activities.

Increased histone deacetylase (HDAC) activity and the resulting dysregulation of protein acetylation is an integral event in retinal degenerations associated with ischemia and ocular hypertension. This study investigates the role of preconditioning on the process of acetylation in ischemic retinal injury. Rat eyes were unilaterally subjected to retinal injury by 45 min of acute ischemia, and retinal neuroprotection induced by 5 min of an ischemic preconditioning (IPC) event. HDAC activity was evaluated by a fluorometric enzymatic assay with selective isoform inhibitors. Retinal localization of acetylated histone-H3 was determined by immunohistochemistry on retina cross sections. Cleaved caspase-3 level was evaluated by Western blots. Electroretinogram (ERG) analyses were used to assess differences in retinal function seven days following ischemic injury. In control eyes, analysis of HDAC isoforms demonstrated that HDAC1/2 accounted for 28.4 ± 1.6%, HDAC3 for 42.4 ± 1.5% and HDAC6 activity 27.3 ± 3.5% of total activity. Following ischemia, total Class-I HDAC activity increased by 21.2 ± 6.2%, and this increase resulted solely from a rise in HDAC1/2 activity. No change in HDAC3 activity was measured. Activity of Class-II HDACs and HDAC8 was negligible. IPC stimulus prior to ischemic injury also suppressed the rise in Class-I HDAC activity, cleaved caspase-3 levels, and increased acetylated histone-H3 in the retina. In control animals 7 days post ischemia, ERG a- and b-wave amplitudes were significantly reduced by 34.9 ± 3.1% and 42.4 ± 6.3%, respectively. In rats receiving an IPC stimulus, the ischemia-induced decline in ERG a- and b-wave amplitudes was blocked. Although multiple HDACs were detected in the retina, these studies provide evidence that hypoacetylation associated with ischemic injury results from the selective rise in HDAC1/2 activity and that neuroprotection induced by IPC is mediated in part by suppressing HDAC activity.

Essential roles of grp94 in gut homeostasis via chaperoning canonical Wnt pathway.

Increasing evidence points to a role for the protein quality control in the endoplasmic reticulum (ER) in maintaining intestinal homeostasis. However, the specific role for general ER chaperones in this process remains unknown. Herein, we report that a major ER heat shock protein grp94 interacts with MesD, a critical chaperone for the Wnt coreceptor low-density lipoprotein receptor-related protein 6 (LRP6). Without grp94, LRP6 fails to export from the ER to the cell surface, resulting in a profound loss of canonical Wnt signaling. The significance of this finding is demonstrated in vivo in that grp94 loss causes a rapid and profound compromise in intestinal homeostasis with gut-intrinsic defect in the proliferation of intestinal crypts, compromise of nuclear ß-catenin translocation, loss of crypt-villus structure, and impaired barrier function. Taken together, our work has uncovered the role of grp94 in chaperoning LRP6-MesD in coordinating intestinal homeostasis, placing canonical Wnt-signaling pathway under the direct regulation of the general protein quality control machinery in the ER.

Receptor mediated disruption of retinal pigment epithelium function in acute glycated-albumin exposure.

Diabetic macular edema (DME) is a major cause of visual impairment. Although DME is generally believed to be a microvascular disease, dysfunction of the retinal pigment epithelium (RPE) can also contribute to its development. Advanced glycation end-products (AGE) are thought to be one of the key factors involved in the pathogenesis of diabetes in the eye, and we have previously demonstrated a rapid breakdown of RPE function following glycated-albumin (Glyc-alb, a common AGE mimetic) administration in monolayer cultures of fetal human RPE cells. Here we present new evidence that this response is attributed to apically oriented AGE receptors (RAGE). Moreover, time-lapse optical coherence tomography in Dutch-belted rabbits 48 h post intravitreal Glyc-alb injections demonstrated a significant decrease in RPE-mediated fluid resorption in vivo. In both the animal and tissue culture models, the response to Glyc-alb was blocked by the relatively selective RAGE antagonist, FPS-ZM1 and was also inhibited by ZM323881, a relatively selective vascular endothelial growth factor receptor 2 (VEGF-R2) antagonist. Our data establish that the Glyc-alb-induced breakdown of RPE function is mediated via specific RAGE and VEGF-R2 signaling both in vitro and in vivo. These results are consistent with the notion that the RPE is a key player in the pathogenesis of DME.

Acetylation: a lysine modification with neuroprotective effects in ischemic retinal degeneration.

Neuroretinal ischemic injury contributes to several degenerative diseases in the eye and the resulting pathogenic processes involving a series of necrotic and apoptotic events. This study investigates the time and extent of changes in acetylation, and whether this influences function and survival of neuroretinal cells following injury. Studies evaluated the time course of changes in histone deacetylase (HDAC) activity, histone-H3 acetylation and caspase-3 activation levels as well as retinal morphology and function (electroretinography) following ischemia. In addition, the effect of two HDAC inhibitors, trichostatin-A and valproic acid were also investigated. In normal eyes, retinal ischemia produced a significant increase in HDAC activity within 2 h that was followed by a corresponding significant decrease in protein acetylation by 4 h. Activated caspase-3 levels were significantly elevated by 24 h. Treatment with HDAC inhibitors blocked the early decrease in protein acetylation and activation of caspase-3. Retinal immunohistochemistry demonstrated that systemic administration of trichostatin-A or valproic acid, resulted in hyperacetylation of all retinal layers after systemic treatment. In addition, HDAC inhibitors provided a significant functional and structural neuroprotection at seven days following injury relative to vehicle-treated eyes. These results provide evidence that increases in HDAC activity is an early event following retinal ischemia, and are accompanied by corresponding decreases in acetylation in advance of caspase-3 activation. In addition to preserving acetylation status, the administration of HDAC inhibitors suppressed caspase activation and provided structural and functional neuroprotection in model of ischemic retinal injury. Taken together these data provide evidence that decrease in retinal acetylation status is a central event in ischemic retinal injury, and the hyperacetylation induced by HDAC inhibition can provide acute neuroprotection.

C-type natriuretic peptide protects the retinal pigment epithelium against advanced glycation end product-induced barrier dysfunction.

In diabetic retinopathy, vision loss is usually secondary to macular edema, which is thought to depend on the functional integrity of the blood-retina barrier. The levels of advanced glycation end products in the vitreous correlate with the progression of diabetic retinopathy. Natriuretic peptides (NP) are expressed in the eye and their receptors are present in the retinal pigment epithelium (RPE). Here, we investigated the effect of glycated-albumin (Glyc-alb), an advanced glycation end product model, on RPE-barrier function and the ability of NP to suppress this response. Transepithelial electrical resistance (TEER) measurements were used to assess the barrier function of ARPE-19 and human fetal RPE (hfRPE) monolayers. The monolayers were treated with 0.1-100 µg/ml Glyc-alb in the absence or presence of 1 pM to 100 nM apical atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), or C-type natriuretic peptide (CNP). Glyc-alb induced a significant reduction in TEER within 2 hours. This response was concentration-dependent (EC(50)= 2.3 µg/ml) with a maximal reduction of 40 ± 2% for ARPE-19 and 27 ± 7% for hfRPE at 100 µg/ml 6 hours post-treatment. One hour pretreatment with ANP, BNP, or CNP blocked the reduction in TEER induced by Glyc-alb (100 µg/ml). The suppression of the Glyc-alb response by NP was dependent on the generation of cyclic guanosine monophosphate and exhibited a rank order of agonist potency consistent with the activation of natriuretic-peptide-receptor-2 (NPR2) subtype (CNP >> BNP ≥ ANP). Our data demonstrate that Glyc-alb is effective in reducing RPE-barrier function, and this response is suppressed by NP. Moreover, these studies support the idea that NPR2 agonists can be potential candidates for treating retinal edema in diabetic patients.

Bradykinin activation of extracellular signal-regulated kinases in human trabecular meshwork cells.

Bradykinin stimulation of B(2) kinin receptors has been shown to promote matrix metallo-proteinase (MMP) secretion from trabecular meshwork cells and to increase conventional outflow facility. Because acute secretion of MMPs can be dependent on the activity of extracellular signal-regulated MAP kinases (ERK1/2), experiments were performed to determine bradykinin effects on ERK1/2 in cultured human trabecular meshwork cells and the relationship of these effects to MMP-9 release. Treatment of cells with bradykinin produced a rapid 4-to 6-fold increase in ERK1/2 phosphorylation. Stimulation of ERK1/2 activity peaked within 2 min and then declined to control levels by 60 min. The response maximum occurred with 100nM bradykinin and the estimated EC50 was 0.7nM. Treatment of cells with the B2 kinin receptor agonist, Tyr8- bradykinin, also stimulated ERK1/2 phosphorylation while the B1 agonist, Lys- [Des-Arg9]- bradykinin had no significant effect. In addition, activation of ERK1/2 by bradykinin or Tyr8- bradykinin was blocked by the selective B2 receptor antagonist, Hoe-140. Inhibition of MAP kinase kinase (MEK) with U0126 also blocked bradykinin-induced ERK1/2 phosphorylation. Suppression of protein kinase C activity with the nonselective inhibitor, GF109203X, or by down-regulation with phorbol ester, diminished, but did not eliminate, bradykinin activation of ERK1/2. A similar decrease of ERK1/2 stimulation was observed when Src kinase was inhibited by 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2). Finally, blockade of bradykinin-induced ERK1/2 activation substantially reduced the peptide's action to stimulate MMP-9 release into the extracellular environment. The data demonstrate that bradykinin promotes ERK1/2 activation in human trabecular meshwork cells. The effect is mediated by B2 kinin receptors, involves two different signaling pathways, and results in increased secretion of MMP-9.

Expression of the kallikrein/kinin system in human anterior segment.

Tissue kallikrein acts on the substrate, low molecular weight kininogen, to liberate bradykinin in a variety of tissues. Bradykinin stimulation of B(2) kinin receptors has been shown to initiate signaling in trabecular meshwork cells and increase conventional outflow facility. The objective of the present study was to determine if the components for kinin generation and response are expressed in tissues of the human anterior segment. Expression of mRNA encoding tissue kallikrein (KK), low molecular weight kininogen, and B(1) and B(2) kinin receptors was examined in human ciliary smooth muscle (CM), trabecular meshwork (TM) and non-pigmented epithelial (NPE) cells using RT-PCR. Expression of component proteins was also investigated by immunohistochemical analyses performed on parasagittal sections of human anterior segment and TM cells, and by immunoblot. KK mRNA was detected in NPE cells and in cultured CM and TM cells from multiple donors. Each cell type also expressed mRNAs encoding both B(1) and B(2) kinin receptors. Immunohistochemical analysis of KK protein in sectioned anterior segment supported the RT-PCR results. Intense KK immunofluorescence was observed in the epithelial lining of the ciliary body and KK protein was also detected in the ciliary muscle. KK protein expression within the TM was demonstrated by analyses of TM tissue and cultured TM cells. The presence of KK along with B(1) and B(2) receptor proteins was confirmed by immunoblots of cell lysates prepared from CM, NPE or TM cells. Finally, both CM and TM cells were found to possess enzymes for bradykinin inactivation. These data demonstrate that key components for kinin generation and regulation are localized within the human anterior segment. Further, multiple cell types express both B(1) and B(2) kinin receptors and are targets for kinin action. The results support the possibility that kinins produced within the eye may contribute to the regulation of aqueous outflow.

Role of PKCepsilon in PGF2alpha-stimulated MMP-2 secretion from human ciliary muscle cells.

Studies were designed to examine the roles of individual protein kinase C (PKC) isoforms in the prostaglandin F(2alpha) (PGF(2alpha))-induced matrix metalloproteinase-2 (MMP-2) secretion from human ciliary muscle cells. Studies utilized primary cultures of human ciliary muscle cells. Individual PKC isoforms were detected by Western blotting, using PKC-isoform-specific antibodies. To evaluate MMP-2 secretion, cells were serum-starved overnight, treated with PGF(2alpha) (1 micromol/L) for 4 h and the media analyzed for MMP-2 by Western blotting. To assess ERK1/2 activation, cells were serum-starved overnight, treated with PGF(2alpha) (1 micromol/L) for 5 min and cell lysates analyzed for ERK1/2 phosphorylation by Western blot analysis. To evaluate the roles of individual PKC isoforms, cells were pretreated with PKC inhibitors or siRNAs prior to the addition of PGF(2alpha). In cultured human ciliary muscle cells, the PKC isoforms exhibiting the highest level of expression were PKCalpha, epsilon, iota and lambda. The delta and eta isoforms exhibited moderate levels of expression and beta, gamma, and phi were not detected. The administration of PGF(2alpha) (1 micromol/L) primarily induced the translocation of PKCepsilon from cytosol to the membrane fraction, as well as increased MMP-2 secretion and ERK1/2 phosphorylation. The secretion of MMP-2 was inhibited by pretreatment with the broad-range PKC inhibitor, chelerythrine chloride; however, this response was not blocked by Go-6976, an inhibitor of conventional PKC isoforms. The PGF(2alpha)-induced secretion of MMP-2 was also blocked by pretreatment with the PKCepsilon-selective peptide translocation inhibitor, EAVSLKPT, or the transfection of siRNA-targeting PKCepsilon. The activation of ERK1/2 was inhibited by chelerythrine and the PKCepsilon translocation inhibitor. Human ciliary muscle cells express the alpha, epsilon, iota and lambda PKC isoforms. Stimulation of FP receptors in these cells activates PKCepsilon, resulting in ERK1/2 activation and an eventual increase in MMP-2 secretion. These data support the idea that the activation of FP receptors in vivo modulate uveoscleral outflow through the PKCepsilon-dependent secretion of MMPs.

S-nitrosoglutathione prevents interphotoreceptor retinoid-binding protein (IRBP(161-180))-induced experimental autoimmune uveitis.

PURPOSE: Experimental autoimmune uveitis (EAU), an animal model of human uveitis, is an organ-specific autoimmune disease mediated by various inflammatory cytokines. In particular, tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta and interferon (IFN)-gamma are known to play a role in its pathogenesis. S-nitrosothiol S-nitrosoglutathione (GSNO), a slow nitric oxide (NO) donor, was reported to have beneficial effects in inflammatory disease in ischemia-reperfusion injury. The efficacy of GSNO treatment on interphotoreceptor retinoid-binding protein (IRBP)-induced EAU was investigated, using functional, histologic, and immunologic readouts. METHODS: Mice were immunized with a single injection of IRBP(161180) peptide to induce EAU, followed by a daily treatment with GSNO (1 mg/kg). Electroretinogram (ERG) analysis, histopathology, and immunologic responses to IRBP were analyzed. The effects of GSNO treatment on the antigen-specific T-cell recall responses and their cytokine production were determined. RESULTS: A single immunization of IRBP(161180) peptide led to significant structural damage of the retina and concomitant elimination of ERGs. Daily oral GSNO treatment from days 1-14 following immunization was found to be effective against IRBP-induced EAU. Histopathologic and ERG analysis both demonstrated significant retinal protection in GSNO-treated mice. The GSNO treatment of EAU animals significantly attenuated the levels of TNF-alpha, IL-1beta, IFN-gamma, and IL-10 in retinas, as measured by quantitative real-time polymerase chain reaction analysis. The splenocytes isolated from EAU- and GSNO-treated mice had lower antigen-specific T-cell proliferation in response to IRBP protein, and their cytokine production was inhibited. CONCLUSIONS: The oral administration of GSNO significantly suppressed the levels of inflammatory mediators in the retinas of EAU mice. This suppression was associated with the maintenance of normal retinal histology and function. These results clearly demonstrated the therapeutic potential of GSNO in EAU, and provide new insights for the treatment of human uveitis.

The rat Apg3p/Aut1p homolog is upregulated by ischemic preconditioning in the retina.

PURPOSE: Retinas can be protected from subsequent severe ischemic injury by ischemic preconditioning. Ischemic preconditioning is dependent on gene expression and protein synthesis; however, it is not clear which genes are important in this process. In this study, we have identified and characterized the rat homolog of yeast Apg3p/Aut1p, an important autophagy protein encoded by the autophagy 3-like (APG3L) gene. We have also further characterized the homologous human APG3L gene. METHODS: A fragment of the rat Apg3 cDNA was identified by mRNA differential display from hypoxia-treated E1A-NR3, an immortalized cell line derived from rat retinal cells that manifests phenotypes of retinal neurons. The full length of rat Apg3 (rApg3) cDNA sequence (about 1.4 kb) encoding 341 amino acids was cloned from a rat retinal cDNA library and characterized using Southern and northern blot analysis, and a global GenBank search. Protein expression was determined by western blotting, and immunohistochemistry. Ischemic preconditioning was achieved by ligation of the retinal arteries of the right eye for 5 min followed by 5 h reperfusion. The prolonged retinal ischemia was induced by ligation of the retinal arteries for 45 min followed by 5 h reperfusion. The full-length homologous human APG3L gene was cloned and sequenced from a human genomic DNA library. RESULTS: The combination of genomic Southern blot analysis and a global GenBank search indicated that rat APG3L is a single copy gene. Rat Apg3 mRNA is expressed in the retina at a high level but is also detected in other tissues. In the process of comparing the rat and human APG3L genes we showed that the organization of the human APG3L gene includes a unique transcriptional start site, a coding region with 12 translated exons and 11 introns and is located on human chromosome 3q13.1. Subcellular localization studies showed that recombinant rat autophagocytosis protein (Apg3p) is a cytosolic protein. Rat Apg3 mRNA level was upregulated by ischemic preconditioning but downregulated by prolonged ischemia. CONCLUSIONS: Our results suggest that the upregulation of rApg3 is a specific response to ischemic preconditioning rather than to retina ischemia, and autophagy may contribute to the neuroprotective effect of ischemic preconditioning in the retina.

Kinin modulation of conventional outflow facility in the bovine eye.

The aim of this study was to investigate the effects of bradykinin on conventional outflow facility in relation to kinin effects on matrix metalloproteinase (MMP) secretion. Conventional outflow facility was measured in isolated bovine segments perfused at a constant pressure of 10 mmHg. Experiments were also performed in primary cultures of bovine trabecular meshwork cells to assess bradykinin effects on the secretion of MMP-9 assessed by western blot. Administration of bradykinin (10(-7) M) to perfused anterior segments produced a 50% increase in outflow facility above basal levels. The effect was slow to develop, requiring 100 min for a significant increase in facility and 4 h for the peak response to be observed. Pretreatment of anterior segments with the B(2) kinin receptor antagonist, HOE-140 (10(-6) M), or with the nonselective MMP inhibitor, GM6001 (10(-5) M) blocked the response to bradykinin (10(-7) M). Treatment of cultured trabecular meshwork cells with bradykinin (10(-7) M) for 120 min stimulated secretion of MMP-9 into the extracellular media, and this response was inhibited by HOE-140 (10(-6) M). These results demonstrate that bradykinin activates B(2) kinin receptors to increase conventional outflow in the perfused bovine eye and provide evidence that secretion and activation of MMPs within the conventional pathway may mediate the effect.

Suppression of Acid Sphingomyelinase Protects the Retina from Ischemic Injury.

PURPOSE: Acid sphingomyelinase (ASMase) catalyzes the hydrolysis of sphingomyelin to ceramide and mediates multiple responses involved in inflammatory and apoptotic signaling. However, the role ASMase plays in ischemic retinal injury has not been investigated. The purpose of this study was to investigate how reduced ASMase expression impacts retinal ischemic injury. METHODS: Changes in ceramide levels and ASMase activity were determined by high performance liquid chromatography-tandem mass spectrometry analysis and ASMase activity. Retinal function and morphology were assessed by electroretinography (ERG) and morphometric analyses. Levels of TNF-α were determined by ELISA. Activation of p38 MAP kinase was assessed by Western blot analysis. RESULTS: In wild-type mice, ischemia produced a significant increase in retinal ASMase activity and ceramide levels. These increases were associated with functional deficits as measured by ERG analysis and significant structural degeneration in most retinal layers. In ASMase+/- mice, retinal ischemia did not significantly alter ASMase activity, and the rise in ceramide levels were significantly reduced compared to levels in retinas from wild-type mice. In ASMase+/- mice, functional and morphometric analyses of ischemic eyes revealed significantly less retinal degeneration than in injured retinas from wild-type mice. The ischemia-induced increase in retinal TNF-α levels was suppressed by the administration of the ASMase inhibitor desipramine, or by reducing ASMase expression. CONCLUSIONS: Our results demonstrate that reducing ASMase expression provides partial protection from ischemic injury. Hence, the production of ceramide and subsequent mediators plays a role in the development of ischemic retinal injury. Modulating ASMase may present new opportunities for adjunctive therapies when treating retinal ischemic disorders.

Histone Deacetylase Inhibition Restores Retinal Pigment Epithelium Function in Hyperglycemia.

In diabetic individuals, macular edema is a major cause of vision loss. This condition is refractory to insulin therapy and has been attributed to metabolic memory. The retinal pigment epithelium (RPE) is central to maintaining fluid balance in the retina, and this function is compromised by the activation of advanced glycation end-product receptors (RAGE). Here we provide evidence that acute administration of the RAGE agonist, glycated-albumin (gAlb) or vascular endothelial growth factor (VEGF), increased histone deacetylase (HDAC) activity in RPE cells. The administration of the class I/II HDAC inhibitor, trichostatin-A (TSA), suppressed gAlb-induced reductions in RPE transepithelial resistance (in vitro) and fluid transport (in vivo). Systemic TSA also restored normal RPE fluid transport in rats with subchronic hyperglycemia. Both gAlb and VEGF increased HDAC activity and reduced acetyl-α-tubulin levels. Tubastatin-A, a relatively specific antagonist of HDAC6, inhibited gAlb-induced changes in RPE cell resistance. These data are consistent with the idea that RPE dysfunction following exposure to gAlb, VEGF, or hyperglycemia is associated with increased HDAC6 activity and decreased acetyl-α-tubulin. Therefore, we propose inhibiting HDAC6 in the RPE as a potential therapy for preserving normal fluid homeostasis in the hyperglycemic retina.

Progressive Early Breakdown of Retinal Pigment Epithelium Function in Hyperglycemic Rats.

PURPOSE: Diabetic macular edema (DME), an accumulation of fluid in the subretinal space, is a significant cause of vision loss. The impact of diabetes on the breakdown of the inner blood-retina barrier (BRB) is an established event that leads to DME. However, the role of the outer BRB in ocular diabetes has received limited attention. We present evidence that the breakdown of normal RPE function in hyperglycemia facilitates conditions conducive to DME pathogenesis. METHODS: Brown Norway rats (130-150 g) were injected intraperitoneally with streptozotocin (STZ; 60 mg/kg) to induce hyperglycemia. After 4 weeks, Evans blue (EB) dye was injected intravenously to determine whether there was leakage of albumin into the retina. Subretinal saline blebs (0.5-1 µL) were placed 4 and 9 weeks after STZ injection, and time-lapse optical coherence tomography tracked the resorption rate. In a subset of rats, intravitreal bevacizumab, a humanized monoclonal antibody targeted to VEGF, was given at 5 weeks and resorption was measured at 9 weeks. RESULTS: The ability of the RPE to transport fluid was reduced significantly after 4 and 9 weeks of hyperglycemia with a reduction of over 67% at 9 weeks. No EB dye leakage from inner retinal vessels was measured in hyperglycemic animals compared to control. The intravitreal administration of bevacizumab at week 5 significantly increased the rate of fluid transport in rats subjected to hyperglycemia for 9 weeks. CONCLUSIONS: These results demonstrate that chronic hyperglycemia altered RPE fluid transport, in part dependent on the actions of VEGF. These results support the idea that RPE dysfunction is an early event associated with hyperglycemia that contributes to fluid accumulation in DME.

Difference in ischemic regulation of vascular endothelial growth factor and pigment epithelium--derived factor in brown norway and sprague dawley rats contributing to different susceptibilities to retinal neovascularization.

The present study compared susceptibilities of Sprague Dawley (SD) and Brown Norway (BN) rats with ischemia-induced retinal neovascularization. An exposure to constant hyperoxia followed by normoxia induced significant retinal neovascularization in BN rats but not in SD rats, as demonstrated by fluorescein retinal angiography, measurement of avascular area, and count of preretinal vascular cells. These results indicate a rat strain difference in susceptibility to retinal neovascularization. To understand the molecular basis responsible for the strain difference, we have measured the levels of pigment epithelium-derived factor (PEDF), an angiogenic inhibitor, and vascular endothelial growth factor (VEGF), a major angiogenic stimulator in the retina. The hyperoxia-treated BN rats showed a significant reduction in retinal PEDF, but they showed a substantial increase of VEGF at both the protein and RNA levels, resulting in an increased VEGF-to-PEDF ratio. Hyperoxia-treated SD rats showed changes in PEDF and VEGF levels that were less in magnitude and of shorter duration than in BN rats. In age-matched normal BN and SD rats, however, there was no detectable difference in the basal VEGF-to-PEDF ratio between the strains. These observations support the idea that different regulation of angiogenic inhibitors and stimulators under ischemia are responsible for the differences in susceptibility to ischemia-induced retinal neovascularization in SD and BN rats.

Acute effects of PGF2alpha on MMP-2 secretion from human ciliary muscle cells: a PKC- and ERK-dependent process.

PURPOSE: Studies were designed to evaluate the cellular mechanisms associated with prostaglandin (PG)F(2alpha)-induced matrix metalloproteinase (MMP)-2 secretion from human ciliary muscle (HCM) cells. METHODS: The secretion and activity of MMP-2 was determined by Western blot analysis and zymography, using conditioned medium and HCM cells. ERK1/2 activity was measured by in-gel kinase assay and Western blot analysis with anti-phospho-ERK1/2 antibodies. RESULTS: PGF(2alpha) increased the secretion of MMP-2 in a dose-dependent manner with an EC(50) of 2.7 x 10(-8) M. The addition of 1 muM PGF(2alpha) also increased MMP-2 secretion in a time-dependent manner with maximum secretion occurring at 4 hours after administration. At 4 hours, the maximum increase in MMP-2 secretion and activity were 112% +/- 32% and 88% +/- 18%, respectively. The secretory action of PGF(2alpha) was inhibited by pretreatment with a protein kinase C (PKC) inhibitor, chelerythrine chloride; the FP receptor antagonist, AL-8810; and the MEK inhibitor, PD-98059. The addition of PGF(2alpha) and latanoprost acid increased ERK1/2 activity by 117% +/- 12% and 75% +/- 9%, respectively. The PGF(2alpha)- and latanoprost-acid-induced ERK1/2 activation was blocked by the presence of PKC inhibitors and downregulation of PKC by prolonged incubation with a phorbol ester. CONCLUSIONS: These data provide evidence that FP receptor activation leads to an increase in the secretion and activation of MMP-2 through PKC- and ERK1/2-dependent pathways. FP-agonist-induced activation of ERK1/2 was blocked by PKC inhibitors, indicating that PKC activation is required for ERK1/2 activation and MMP-2 secretion from HCM cells. In the ciliary muscle, the functional responses to ERK1/2 activation include secretion of MMP-2, supporting the hypothesis that increases in uveoscleral outflow facility induced by PG administration involves the secretion and activation of MMP-2.

Hsp27 upregulation by HIF-1 signaling offers protection against retinal ischemia in rats.

PURPOSE: Previous work from the authors' laboratory has shown that Hsp27 is specifically upregulated after retinal ischemic preconditioning (IPC), and this upregulation acts as a key cytoprotective factor in preventing retinal ischemic damage. The regulatory mechanisms involved in the upregulation of Hsp27 after IPC are unknown. The purpose of this study was to explore the transcriptional events responsible for the upregulation of Hsp27 after IPC. METHODS: CoCl(2) was used to test for Hsp27 expression after hypoxic stimulus. The promoter and first intron regions of the human Hsp27 gene were cloned by PCR and characterized by deletion analysis by using a reporter assay. In vitro results were then applied to an in vivo model of retinal ischemia to determine whether CoCl(2) upregulates rHsp27 and protects the retina from ischemic injury. RESULTS: CoCl(2) upregulated Hsp27 in cultured retinal neurons. Promoter-intron reporter assays using various DNA deletion constructs indicated that several HIF-1 binding sites were necessary for CoCl(2)-induced expression of the Hsp27 gene. Furthermore, CoCl(2) upregulated Hsp27 in the rat retina and protected the rat retina from ischemic injury. CONCLUSIONS: These data provide evidence that Hsp27 is regulated by hypoxic signaling through HIF-1 activation and support the idea that an early event in IPC is the activation of HIF-1. These findings are significant, because this is the first time HIF-1 activation has been associated with the protective effects of IPC and with Hsp27 upregulation.

Modulation of conventional outflow facility by the adenosine A1 agonist N6-cyclohexyladenosine.

PURPOSE: Studies have shown that the activation of adenosine A(1) receptors lower intraocular pressure primarily by increasing total outflow facility. The purpose of this study was to investigate the actions of the adenosine A(1) agonist N(6)-cyclohexyladenosine (CHA) on conventional outflow facility. METHODS: Conventional outflow facility was evaluated in isolated bovine anterior segments, perfused at a constant pressure of 10 mm Hg. After overnight perfusion to establish a stable baseline, the concentration- and time-dependent changes in outflow facility induced by CHA were determined. To confirm the involvement of adenosine A(1) receptors and matrix metalloproteinases (MMP) in any change in facility, the responses to CHA were evaluated in preparations treated with the adenosine A(1) receptor antagonist, 8-cyclopentyl-1,3-dimethylxanthine (CPT), or the nonselective MMP inhibitor GM-6001. RESULTS: The administration of CHA (10 microM) to perfused anterior segments produced a 28% increase in outflow facility over basal levels. This response was relatively slow to develop with no significant change in outflow facility measured until after 60 minutes of CHA infusion. The peak response to CHA infusion occurred between 3 and 4 hours after CHA administration. Analysis of the CHA concentration-response curves demonstrated that this increase in outflow facility was concentration-dependent, with an EC(50) of 0.28 microM. Pretreatment with the adenosine A(1) receptor antagonist CPT (10 microM) or the nonselective MMP inhibitor GM-6001 (10 microM) blocked the response to CHA (1 microM). When compared with control eyes, no significant change in baseline facility was measured in eyes perfused with CPT or GM-6001. CONCLUSIONS: These studies demonstrate that the adenosine agonist CHA significantly increases conventional outflow facility in the perfused bovine eye. Analysis of the CHA concentration-response curve and inhibition of the CHA-induced increase in outflow facility by the adenosine A(1) antagonist confirms that this response is mediated by the activation of adenosine A(1) receptors. The inhibition of the CHA-induced increase in outflow facility by the MMP inhibitor GM-6001 provides evidence that the secretion and activation of MMPs within the conventional outflow pathway play a central role in the ocular hypotensive action of adenosine A(1) agonists.