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.

Islet Harvest in Carbon Monoxide-Saturated Medium for Chronic Pancreatitis Patients Undergoing Islet Autotransplantation.

Stresses encountered during human islet isolation lead to unavoidable ß-cell death after transplantation. This reduces the chance of insulin independence in chronic pancreatitis patients undergoing total pancreatectomy and islet autotransplantation. We tested whether harvesting islets in carbon monoxide-saturated solutions is safe and can enhance islet survival and insulin independence after total pancreatectomy and islet autotransplantation. Chronic pancreatitis patients who consented to the study were randomized into carbon monoxide (islets harvested in a carbon monoxide-saturated medium) or control (islets harvested in a normal medium) groups. Islet yield, viability, oxygen consumption rate, ß-cell death (measured by unmethylated insulin DNA), and serum cytokine levels were measured during the peri-transplantation period. Adverse events, metabolic phenotypes, and islet function were measured prior and at 6 months post-transplantation. No adverse events directly related to the infusion of carbon monoxide islets were observed. Carbon monoxide islets showed significantly higher viability before transplantation. Subjects receiving carbon monoxide islets had less ß-cell death, decreased CCL23, and increased CXCL12 levels at 1 or 3 days post transplantation compared with controls. Three in 10 (30%) of the carbon monoxide subjects and none of the control subjects were insulin independent. This pilot trial showed for the first time that harvesting human islets in carbon monoxide-saturated solutions is safe for total pancreatectomy and islet autotransplantation patients.

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.

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.

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.

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.

Lack of Acid Sphingomyelinase Induces Age-Related Retinal Degeneration.

BACKGROUND: Mutations of acid sphingomyelinase (ASMase) cause Niemann-Pick diseases type A and B, which are fatal inherited lipid lysosomal storage diseases, characterized with visceral organ abnormalities and neurodegeneration. However, the effects of suppressing retinal ASMase expression are not understood. The goal of this study was to determine if the disruption of ASMase expression impacts the retinal structure and function in the mouse, and begin to investigate the mechanisms underlying these abnormalities. METHODS: Acid sphingomyelinase knockout (ASMase KO) mice were utilized to study the roles of this sphingolipid metabolizing enzyme in the retina. Electroretinogram and morphometric analysis were used to assess the retinal function and structure at various ages. Sphingolipid profile was determined by liquid chromatography-mass spectrometry. Western blots evaluated the level of the autophagy marker LC3-II. RESULTS: When compared to control animals, ASMase KO mice exhibited significant age-dependent reduction in ERG a- and b-wave amplitudes. Associated with these functional deficits, morphometric analysis revealed progressive thinning of retinal layers; however, the most prominent degeneration was observed in the photoreceptor and outer nuclear layer. Additional analyses of ASMase KO mice revealed early reduction in ERG c-wave amplitudes and increased lipofuscin accumulation in the retinal pigment epithelium (RPE). Sphingolipid analyses showed abnormal accumulation of sphingomyelin and sphingosine in ASMase KO retinas. Western blot analyses showed a higher level of the autophagosome marker LC3-II. CONCLUSIONS: These studies demonstrate that ASMase is necessary for the maintenance of normal retinal structure and function. The early outer retinal dysfunction, outer segment degeneration, accumulation of lipofuscin and autophagosome markers provide evidence that disruption of lysosomal function contributes to the age-dependent retinal degeneration exhibited by ASMase KO mice.

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 preserves retinal ganglion cell structure and function in a chronic model of ocular hypertension.

PURPOSE: The current studies investigate if the histone deacetylase (HDAC) inhibitor, valproic acid (VPA), can limit retinal ganglion cell (RGC) degeneration in an ocular-hypertensive rat model. METHODS: Intraocular pressure (IOP) was elevated unilaterally in Brown Norway rats by hypertonic saline injection. Rats received either vehicle or VPA (100 mg/kg) treatment for 28 days. Retinal ganglion cell function and number were assessed by pattern electroretinogram (pERG) and retrograde FluoroGold labeling. Western blotting and a fluorescence assay were used for determination of histone H3 acetylation and HDAC activity, respectively, at 3-day, 1-week, and 2-week time points. RESULTS: Hypertonic saline injections increased IOPs by 7 to 14 mm Hg. In vehicle-treated animals, ocular hypertension resulted in a 29.1% and 39.4% decrease in pERG amplitudes at 2 and 4 weeks, respectively, and a 42.9% decrease in mean RGC density at 4 weeks. In comparison, VPA treatment yielded significant amplitude preservation at 2 and 4 weeks and showed significant RGC density preservation at 4 weeks. No significant difference in RGC densities or IOPs was measured between control eyes of vehicle- and VPA-treated rats. In ocular-hypertensive eyes, class I HDAC activity was significantly elevated within 1 week (13.3 ± 2.2%) and histone H3 acetylation was significantly reduced within 2 weeks following the induction of ocular hypertension. CONCLUSIONS: Increase in HDAC activity is a relatively early retinal event induced by elevated IOP, and suppressing HDAC activity can protect RGCs from ocular-hypertensive stress. Together these data provide a basis for developing HDAC inhibitors for the treatment of optic neuropathies.

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.

FR-190997, a nonpeptide bradykinin B2-receptor partial agonist, is a potent and efficacious intraocular pressure lowering agent in ocular hypertensive cynomolgus monkeys.

Preclinical Research FR-190997 (8-[2,6-dichloro-3-[N-[(E)-4-(N-methylcarbamoyl) cinnaminoacetyl]-N-methylamino]benzyloxy]-2-methyl-4- (2-pyridylmethoxy) quinoline), a nonpeptide bradykinin (BK) B2-receptor-selective agonist, represents a novel class of ocular hypotensive agents. FR-190997 exhibited a high affinity for the human cloned B2-receptor (Ki = 9.8 nM) and a relatively high potency (EC50 = 155 nM) for mobilizing intracellular Ca(2+) ([Ca(2+)]i) in human ocular cells from nonpigmented ciliary epithelium; trabecular meshwork [h-TM]; ciliary muscle [h-CM] that are involved in regulating intraocular pressure (IOP). Unlike BK, FR-190997 behaved as a partial agonist (Emax = 38-80%) in these cells and its [Ca(2+)]i-mobilizing effects were blocked by the B2-receptor-selective antagonists (HOE-140, Ki = 0.8-7 nM; WIN-64338, Ki = 157-425 nM). FR-190997 stimulated the production of prostaglandins (PGs) in h-CM and h-TM cells (EC50 = 15-19 nM; Emax = 27-33%); an effect that was reduced by the cyclooxygenase-2 inhibitor bromfenac, and by HOE-140. FR-190997 also induced pro-matrix metalloproteinase (MMP)-1 and MMP-3 release from h-CM cells. FR-190997 significantly lowered IOP (37% [P < 0.001] with 30 µg, 24 h post-topical ocular dosing) in ocular hypertensive eyes of conscious Cynomolgus monkeys. This effect was reduced by bromfenac and completely blocked by a B2-antagonist. FR-190997 primarily stimulated uveoslceral outflow (UVSO) of aqueous humor (2.6 to 3.9-fold above baseline). In conclusion, FR-190997 is a B2-receptor selective partial agonist that activates phospholipase C, mobilizes [Ca(2+)]; induces PG and pro-MMP production, and that profoundly lowers IOP by promoting UVSO in ocular hypertensive Cynomolgus monkey eyes.

Evidence of a novel mechanism for partial γ-secretase inhibition induced paradoxical increase in secreted amyloid ß protein.

BACE1 (ß-secretase) and α-secretase cleave the Alzheimer's amyloid ß protein (Aß) precursor (APP) to C-terminal fragments of 99 aa (CTFß) and 83 aa (CTFα), respectively, which are further cleaved by γ-secretase to eventually secrete Aß and Aα (a.k.a. P3) that terminate predominantly at residues 40 and 42. A number of γ-secretase inhibitors (GSIs), such as N-[N-(3,5-Difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester (DAPT), have been developed with the goal of reducing Aß to treat Alzheimer's disease (AD). Although most studies show that DAPT inhibits Aß in a dose-dependent manner several studies have also detected a biphasic effect with an unexpected increase at low doses of DAPT in cell cultures, animal models and clinical trials. In this article, we confirm the increase in Aß40 and Aß42 in SH-SY5Y human neuroblastoma cells treated with low doses of DAPT and identify one of the mechanisms for this paradox. We studied the pathway by first demonstrating that stimulation of Aß, a product of γ-secretase, was accompanied by a parallel increase of its substrate CTFß, thereby demonstrating that the inhibitor was not anomalously stimulating enzyme activity at low levels. Secondly, we have demonstrated that inhibition of an Aß degrading activity, endothelin converting enzyme (ECE), yielded more Aß, but abolished the DAPT-induced stimulation. Finally, we have demonstrated that Aα, which is generated in the secretory pathway before endocytosis, is not subject to the DAPT-mediated stimulation. We therefore conclude that impairment of γ-secretase can paradoxically increase Aß by transiently skirting Aß degradation in the endosome. This study adds to the growing body of literature suggesting that preserving γ-secretase activity, rather than inhibiting it, is important for prevention of neurodegeneration.

Vascular endothelial growth factor modulates the function of the retinal pigment epithelium in vivo.

PURPOSE: Retinal edema, the accumulation of extracellular fluid in the retina is usually attributed to inner blood retina barrier (BRB) leakage. Vascular endothelial growth factor plays an important role in this process. The effects of VEGF on the outer BRB, the RPE, however, have received limited attention. Here, we present a methodology to assess how VEGF modulates the integrity of the RPE barrier in vivo. METHODS: Control subretinal blebs (1-5 µL) and blebs containing VEGF (1-100 µg/mL), placental growth factor (PlGF; 100 µg/mL), or albumin (100-1000 µg/mL) were injected into New Zealand White or Dutch Belted rabbits with IOP maintained at 10, 15, or 20 mm Hg. One-hour intravitreal pretreatment with ZM323881 (10 µM/L) was used to inhibit the VEGF response. Fluid resorption was followed by optical coherence tomography for 1 hour. Retinal pigment epithelium leakage was assessed by fluorescein angiography. RESULTS: Increasing IOP resulted in an elevated rate of bleb resorption, while increasing albumin concentration in the bleb decreased the rate of resorption. Vascular endothelial growth factor, but not PlGF, caused a significant, concentration-dependent decrease in the rate of fluid resorption, which was reversed by ZM323881. Compared with albumin-filled blebs, VEGF-filled blebs showed accelerated early-phase leakage from the choroid. CONCLUSIONS: Consistent with a localized modulation of RPE function, VEGF induced a significant reduction in fluid resorption and an increase in hydraulic conductivity. Our results establish VEGF as a major cytokine regulating RPE barrier properties in vivo and indicate that the RPE is a principal factor in the pathogenesis of retinal edema.

Pigment epithelium-derived factor decreases outflow facility.

PURPOSE: Pigment epithelium-derived factor (PEDF) regulates blood-retinal barrier function. As a constituent of aqueous humor, the role of PEDF in conventional outflow function is unknown. The goals of the study were to examine the effects of PEDF on barrier function of cultured Schlemm's canal (SC) endothelia and outflow facility in mouse eyes in situ. METHODS: To model the inner wall of SC, transendothelial electrical resistance (TEER) of human SC and porcine angular aqueous plexus (AAP) cells was monitored. To examine an intact conventional outflow pathway, enucleated eyes from culled C57BL/6 mice were perfused with PEDF using a computer-controlled system. Purified PEDF (0.1 and 1 µg/mL) was perfused at four different pressure steps (4, 8, 15, 20 mm Hg), measuring flow to determine outflow facility (slope of flow/pressure relationship). RESULTS: Pigment epithelium-derived factor increased TEER of porcine AAP cells in a dose-dependent fashion (0.3-3 µg/mL), and 1 µg/mL recombinant PEDF or conditioned media from pigmented retinal pigment epithelial monolayers stabilized TEER of human SC monolayers over time (0-48 hours). In perfusion experiments, we observed a 43.7% decrease in outflow facility (0.016 vs. 0.029 µL/min/mm Hg, P = 4.5 × 10⁻5) in eyes treated with 1 µg/mL PEDF compared to vehicle-perfused controls, and a 19.9% decrease (0.021 vs. 0.027 µL/min/mm Hg, P = 0.003) at 100 ng/mL PEDF. CONCLUSIONS: Pigment epithelium-derived factor increased barrier function in both the in vitro and in situ models of the inner wall of SC. Modification of PEDF signaling in SC cells may be therapeutically exploited to increase outflow facility in people with ocular hypertension or decrease outflow facility in those with hypotony.

Inhibition of HDAC2 protects the retina from ischemic injury.

PURPOSE: Protein acetylation is an essential mechanism in regulating transcriptional and inflammatory events. Studies have shown that nonselective histone deacetylase (HDAC) inhibitors can protect the retina from ischemic injury in rats. However, the role of specific HDAC isoforms in retinal degenerative processes remains obscure. The purpose of this study was to investigate the role of HDAC2 isoform in a mouse model of ischemic retinal injury. METHODS: Localization of HDAC2 in mice retinas was evaluated by immunohistochemical analyses. To investigate whether selective reduction in HDAC2 activity can protect the retina from ischemic injury, Hdac2⁺/⁻ mice were utilized. Electroretinographic (ERG) and morphometric analyses were used to assess retinal function and morphology. RESULTS: Our results demonstrated that HDAC2 is primarily localized in nuclei in inner nuclear and retinal ganglion cell layers, and HDAC2 activity accounted for approximately 35% of the total activities of HDAC1, 2, 3, and 6 in the retina. In wild-type mice, ERG a- and b-waves from ischemic eyes were significantly reduced when compared to pre-ischemia baseline values. Morphometric examination of these eyes revealed significant degeneration of inner retinal layers. In Hdac2⁺/⁻ mice, ERG a- and b-waves from ischemic eyes were significantly greater than those measured in ischemic eyes from wild-type mice. Morphologic measurements demonstrated that Hdac2⁺/⁻ mice exhibit significantly less retinal degeneration than wild-type mice. CONCLUSIONS: This study demonstrated that suppressing HDAC2 expression can effectively reduce ischemic retinal injury. Our results support the idea that the development of selective HDAC2 inhibitors may provide an efficacious treatment for ischemic retinal injury.

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.

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.

Preservation of retina ganglion cell function by morphine in a chronic ocular-hypertensive rat model.

PURPOSE: The current study examined if opioid-receptor-activation by morphine can improve retinal function and retinal ganglion cell (RGC) integrity in a chronic glaucoma rat model. METHODS: IOP was raised in Brown Norway rats by injecting hypertonic saline into the limbal venous system. Rats were treated daily with 1 mg/kg morphine for 28 days at 24-hour intervals; animals were examined for changes in IOP by a TonoLab tonometer. Pattern-ERG (PERG) was obtained in response to contrast-reversal of patterned visual stimuli. RGCs were visualized by fluorogold retrograde-labeling. Changes in the expression pattern of TNF-α and caspases were measured by Western blotting. RESULTS: A significant IOP elevation was seen as early as 7 days, and maintained for up to 8 weeks, after surgery. PERG amplitudes were significantly reduced in ocular-hypertensive eyes (15.84±0.74 µvolts) when compared with normal eyes (19±0.86 µvolts). PERG deficits in hypertensive eyes were reversed by morphine treatment (18.23±0.78 µvolts; P<0.05). In untreated rats, a 24% reduction in labeled RGCs was measured in the hypertensive eye compared with the normal eye. This reduction in RGC labeling was significantly ameliorated in the presence of morphine. In retinal samples, TNF-α, caspase-8, and caspase-3 expressions were significantly upregulated in ocular hypertensive eyes, but completely inhibited in the morphine-treated animals. CONCLUSIONS: These data provide evidence that activation of opioid receptors can provide significant improvement in PERG and RGC integrity against glaucomatous injury. Mechanistic data provide clues that activation of one or more opioid receptors can reduce glaucomatous-injury via suppression of TNF-α and caspase activation.

Human retinal pigment epithelium cells as functional models for the RPE in vivo.

PURPOSE: The two most commonly used in vitro models of the retinal pigment epithelium (RPE) are fetal human RPE (fhRPE) and ARPE-19 cells; however, studies of their barrier properties have produced contradictory results. To compare their utility as RPE models, their morphologic and functional characteristics were analyzed. METHODS: Monolayers of both cell types were grown on permeable membrane filters. Barrier function and cellular morphology were assessed by transepithelial resistance (TER) measurements and immunohistochemistry. Protein expression was evaluated by immunoblotting and ELISA assays, and retinoid metabolism characterized by HPLC. RESULTS: Both cultures developed tight junctions. However, only the fhRPE cells were pigmented, uniform in size and shape, expressed high levels of RPE markers, metabolized all-trans retinal, and developed high TER (>400 Ωcm(2)). The net secretion of pigment-epithelium-derived factor (PEDF) was directed apically in both cultures, but fhRPE cells exhibited secretion rates a thousand-fold greater than in ARPE-19 cells. The net secretion of vascular endothelial growth factor (VEGF) was significantly higher in fhRPE cultures and the direction of this secretion was basolateral; while net secretion was apical in ARPE-19 cells. In fresh media, VEGF-E reduced TER in both cultures; however, in conditioned media fhRPE cells did not respond to VEGF-E administration, but retreatment of the conditioned media with anti-PEDF antibodies allowed fhRPE cells to fully respond to VEGF-E. CONCLUSIONS: Properties of fhRPE cells align with a functionally normal RPE in vivo, while ARPE-19 cells resemble a pathologic or aged RPE. These results suggest a utility for both cell types in understanding distinct, particular aspects of RPE function.