Dr. Crosson serves as a member of the Executive Board and provides consulting on many aspects of vision science with a specific focus on pharmacology and animal models of elevated intraocular pressure, retinal degenerations and macular edema. Dr. Crosson is a Professor in the Department of Ophthalmology at the Medical University of South Carolina (MUSC). He is also Director of the Ola B. Williams Glaucoma Therapeutic Development Center and Senior Associate Dean for Research at MUSC. His research interests include ischemia, neovascularization, and neuroprotection. Current research in his laboratory is targeting pharmacological treatment for elevated intraocular pressure, retinal degenerations and macular edema.
Sphingomyelinases in retinas and optic nerve heads: Effects of ocular hypertension and ischemia
Exp Eye Res. 2022 Nov;224:109250. doi: 10.1016/j.exer.2022.109250. Epub 2022 Sep 16.
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
Exp Eye Res. 2021 Oct;211:108762. doi: 10.1016/j.exer.2021.108762. Epub 2021 Sep 7.
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.
Histone Deacetylase Inhibition Restores Retinal Pigment Epithelium Function in Hyperglycemia
PLoS One. 2016 Sep 12;11(9):e0162596. doi: 10.1371/journal.pone.0162596. eCollection 2016.
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
Invest Ophthalmol Vis Sci. 2016 Aug 1;57(10):4476-84. doi: 10.1167/iovs.16-19717.
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
Invest Ophthalmol Vis Sci. 2016 May 1;57(6):2706-13. doi: 10.1167/iovs.15-18397.
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
Exp Eye Res. 2016 May;146:269-275. doi: 10.1016/j.exer.2016.03.026. Epub 2016 Apr 6.
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
PLoS One. 2015 Jul 13;10(7):e0133032. doi: 10.1371/journal.pone.0133032. eCollection 2015.
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
Exp Eye Res. 2015 Aug;137:50-6. doi: 10.1016/j.exer.2015.06.004. Epub 2015 Jun 10.
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
Invest Ophthalmol Vis Sci. 2014 Oct 30;55(11):7486-93. doi: 10.1167/iovs.14-14792.
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
Exp Eye Res. 2014 Oct;127:124-31. doi: 10.1016/j.exer.2014.07.012. Epub 2014 Jul 23.
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.
Vascular endothelial growth factor modulates the function of the retinal pigment epithelium in vivo
Invest Ophthalmol Vis Sci. 2014 Apr 9;55(4):2269-75. doi: 10.1167/iovs.13-13334.
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
Invest Ophthalmol Vis Sci. 2013 Oct 11;54(10):6655-61. doi: 10.1167/iovs.13-12766.
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
Invest Ophthalmol Vis Sci. 2013 Jun 12;54(6):4072-80. doi: 10.1167/iovs.12-11529.
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
Proc Natl Acad Sci U S A. 2013 Apr 23;110(17):6877-82. doi: 10.1073/pnas.1302933110. Epub 2013 Apr 9.
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 β-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
J Pharmacol Exp Ther. 2013 Jan;344(1):96-102. doi: 10.1124/jpet.112.199307. Epub 2012 Oct 19.
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
Invest Ophthalmol Vis Sci. 2012 Jun 28;53(7):4289-98. doi: 10.1167/iovs.12-9467.
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
Invest Ophthalmol Vis Sci. 2011 Nov 4;52(12):8614-20. doi: 10.1167/iovs.11-8021.
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.
Bradykinin activation of extracellular signal-regulated kinases in human trabecular meshwork cells
Exp Eye Res. 2011 Jun;92(6):495-501. doi: 10.1016/j.exer.2011.03.009. Epub 2011 Mar 21.
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.
Opioid receptor activation: suppression of ischemia/reperfusion-induced production of TNF-α in the retina
Invest Ophthalmol Vis Sci. 2011 Apr 20;52(5):2577-83. doi: 10.1167/iovs.10-5629. Print 2011 Apr.
PURPOSE: The detrimental role of TNF-α in ischemia-induced tissue damage is known. The authors study examined whether opioid receptor activation alters TNF-α levels in the postischemic retina.
METHODS: Retinal ischemia was induced by raising the intraocular pressure above systolic blood pressure (155-160 mm Hg) for 45 minutes. Rats were pretreated with the opioid receptor agonist morphine (1 mg/kg; intraperitoneally) before injury. Selected animals were pretreated with the opioid antagonist naloxone (3 mg/kg; intraperitoneally). Human optic nerve head (ONH) astrocytes and rat microglial cells were treated with morphine (0.1-1 μM) for 24 hours and then treated with 10 μg/mL or 30 ng/mL lipopolysaccharide (LPS), respectively. TNF-α was measured by ELISA. Opioid receptor subtypes in astrocytes and microglia were determined by Western blot analysis.
RESULTS: There was a time-dependent increase in TNF-α production; the maximum production occurred at 4 hours after ischemia and localized to the inner retinal regions. Ischemia-induced TNF-α production was significantly inhibited by morphine. In astrocytes and microglia, LPS triggered a robust increase in the release of TNF-α, which was significantly inhibited (P < 0.05) by morphine. Naloxone reversed the morphine-induced suppression of TNF-α production in vivo and in vitro. Both ONH astrocytes and microglial cells expressed δ-, κ-, and μ-opioid receptor subtypes.
CONCLUSIONS: These data provide evidence that the production of TNF-α after ischemia/reperfusion injury is an early event and that opioid receptor activation reduces the production of TNF-α. Immunohistochemistry data and in vitro studies provide evidence that ONH astrocytes and microglial cells are the primary sources for the TNF-α production under ischemic/inflammatory conditions. Activation of one or more opioid receptors can reduce ischemic/reperfusion injury by the suppression of TNF-α production.
Influence of race and age on aqueous humor levels of transforming growth factor-beta 2 in glaucomatous and nonglaucomatous eyes
J Ocul Pharmacol Ther. 2011 Oct;27(5):477-80. doi: 10.1089/jop.2010.0100. Epub 2010 Oct 29.
PURPOSE: To evaluate the influence of race and age on aqueous humor levels of transforming growth factor-beta 2 (TGF-β2).
METHODS: Patients >40 years of age and undergoing cataract or glaucoma surgery without associated significant intraocular pathology were selected for this study. In bilateral cases, only the first operated eye was included for evaluation. At the time of surgery, a small amount of aqueous was withdrawn. The concentration of total TGF-β2 was measured by enzyme-linked immunosorbent assay in duplicate by a masked observer.
RESULTS: Fifty-five aqueous humor samples were analyzed from subjects with an average age of 68.05 ± 10.94 years. Overall median TGF-β2 concentration was 247.03 pg/mL. The median concentration of TGF-β2 was higher in eyes with glaucoma than in eyes without glaucoma (269.39 vs. 165.56 pg/mL, respectively; P = 0.001). Subgroup analysis found no significant difference between African American and Caucasian American subjects in the nonglaucomatous or glaucomatous subgroups. Age showed positive correlation with TGF-β2 in nonglaucomatous eyes (r(2) = 0.44, P = 0.019). No correlation between age and TGF-β2 was noted in the glaucoma group (r(2) = 0.02, P = 0.343).
CONCLUSION: The aqueous humor concentration of TGF-β2 was significantly higher in eyes with glaucoma than in eyes without glaucoma. No significant difference between the aqueous humor levels of TGF-β2 from African American and Caucasian American subjects could be measured. However, a significant and positive correlation between age and aqueous humor concentration of TGF-β2 in the eyes of nonglaucomatous subjects was measured. These results are consistent with the idea that elevated levels of TGF-β2 within the anterior segment contribute to the development of glaucoma. In addition, the increased risk for developing glaucoma as one ages may in part be related to the rise of this cytokine.
Inhibition of histone deacetylase protects the retina from ischemic injury
Invest Ophthalmol Vis Sci. 2010 Jul;51(7):3639-45. doi: 10.1167/iovs.09-4538. Epub 2010 Feb 17.
PURPOSE. The pathogenesis of retinal ischemia results from a series of events involving changes in gene expression and inflammatory cytokines. Protein acetylation is an essential mechanism in regulating transcriptional and inflammatory events. The purpose of this study was to investigate the neuroprotective action of the histone deacetylase (HDAC) inhibitor trichostatin A (TSA) in a retinal ischemic model. METHODS. To investigate whether HDAC inhibition can reduce ischemic injury, rats were treated with TSA (2.5 mg/kg intraperitoneally) twice daily on days 0, 1, 2, and 3. Seven days after ischemic injury, morphometric and electroretinographic (ERG) analyses were used to assess retinal structure and function. Western blot and immunohistochemical analyses were used to evaluate TSA-induced changes in histone-H3 acetylation and MMP secretion. RESULTS. In vehicle-treated animals, ERG a- and b-waves from ischemic eyes were significantly reduced compared with contralateral responses. In addition, histologic examination of these eyes revealed significant degeneration of inner retinal layers. In rats treated with TSA, amplitudes of ERG a- and b-waves from ischemic eyes were significantly increased, and normal inner retina morphology was preserved. Ischemia also increased the levels of retinal TNF-alpha, which was blocked by TSA treatment. In astrocyte cultures, the addition of TNF-alpha (10 ng/mL) stimulated the secretion of MMP-1 and MMP-3, which were blocked by TSA (100 nM). CONCLUSIONS. These studies provide the first evidence that suppressing HDAC activity can protect the retina from ischemic injury. This neuroprotective response is associated with the suppression of retinal TNF-alpha expression and signaling. The use of HDAC inhibitors may provide a novel treatment for ischemic retinal injury.
INO-8875, an adenosine A1 agonist, lowers intraocular pressure through the conventional outflow pathway
Investigative Ophthalmology & Visual Science, 51(13), pp.3238-3238
Novel class of LIM-kinase 2 inhibitors for the treatment of ocular hypertension and associated glaucoma
J Med Chem. 2009 Nov 12;52(21):6515-8. doi: 10.1021/jm901226j.
The discovery of a pyrrolopyrimidine class of LIM-kinase 2 (LIMK2) inhibitors is reported. These LIMK2 inhibitors show good potency in enzymatic and cellular assays and good selectivity against ROCK. After topical dosing to the eye in a steroid induced mouse model of ocular hypertension, the compounds reduce intraocular pressure to baseline levels. The compounds also increase outflow facility in a pig eye perfusion assay. These results suggest LIMK2 may be an effective target for treating ocular hypertension and associated glaucoma.
Opioid receptor-activation: retina protected from ischemic injury
Invest Ophthalmol Vis Sci. 2009 Aug;50(8):3853-9. doi: 10.1167/iovs.08-2907. Epub 2009 Mar 25.
PURPOSE: In nonocular systems, activation of opioid receptors has been shown to ameliorate tissue damage induced by ischemic stress. The current study was an investigation of whether opioid receptors activated by endogenous or exogenous agonists can ameliorate ischemic retinal injury.
METHODS: In an investigation of whether endogenous opioid receptor-activation reduces ischemic injury, the effects of the opioid antagonist naloxone (3 mg/kg; IP) on retinal neuroprotection induced by ischemic preconditioning (IPC) were evaluated. Whether exogenous opioid administration can reduce ischemic retinal injury was determined by pretreating rats with morphine (0.01-10 mg/kg) before injury. Morphometric and electroretinogram (ERG) analyses were used to assess the differences in retinal structure and function. The expression of opioid receptor subtypes was evaluated by Western blot and immunohistochemical analyses.
RESULTS: In control animals, 7 days after ischemic retinal injury, ERG a- and b-wave amplitudes were significantly reduced (23% and 41%, respectively). In addition, degeneration of the inner retina resulted in a 34% reduction in overall retina thickness. In animals receiving IPC before ischemic injury, ERG wave forms and retinal morphology were preserved. Pretreatment with naloxone reversed both the functional and structural retinal protection induced by IPC. In animals treated with morphine 24-hours before ischemic injury, ERG waveforms were preserved in a dose-dependent fashion (ED(50) = 0.18 mg/kg), and this protective response was reversed by naloxone pretreatment. Immunohistochemical and Western blot data demonstrated that the delta-, kappa-, and mu-opioid receptor subtypes are expressed in the retina.
CONCLUSIONS: These data provide evidence that activation of one (or more) opioid receptor(s) facilitates the development of IPC within the retina and can reduce ischemic retina injury.
Expression of the kallikrein/kinin system in human anterior segment
Exp Eye Res. 2009 Aug;89(2):126-32. doi: 10.1016/j.exer.2009.02.016. Epub 2009 Mar 4.
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.
Latanoprost-induced changes in rat intraocular pressure: direct or indirect?
J Ocul Pharmacol Ther. 2008 Aug;24(4):367-72. doi: 10.1089/jop.2008.0042.
INTRODUCTION: The topical application of prostaglandin F(2 ) (FP)-receptor agonists has been shown to significantly lower intraocular pressure (IOP) in humans and is now considered the first-line treatment for ocular hypertension. Despite the prominent role FP-receptor agonists play in the treatment of glaucoma, our understanding of how these agents lower IOP remains incomplete. The present study was designed to evaluate the role of matrix metalloproteinase (MMP) activation and the cytokine, tumor necrosis factor alpha (TNF-alpha), in latanoprost-induced changes in IOP.
METHODS: Changes in IOP following an acute topical administration of latanoprost (60 ng) in normotensive Brown Norway rats were evaluated by means of a commercially available rebound tonometer. To examine the role of MMPs and TNF-alpha in this response, the rats were pretreated with a broad-spectrum MMP inhibitor, GM-6001 (100 microg), or the TNF-alpha inhibitor, thalidomide (25 microg).
RESULTS: The topical administration of latanoprost (60 ng) alone produced a biphasic change in ipsilateral IOP: an initial hypertension (4.21 +/- 0.52), followed by a prolonged hypotension (-4.79 +/- 0.65). In rats, pretreatment with GM-6001 blocked the latanoprost-induced reduction in IOP but did not prevent the initial rise in IOP. Pretreatment with thalidomide also blocked the ocular hypotension induced by latanoprost; however, thalidomide pretreatment enhanced the duration of the initial hypertension.
CONCLUSIONS: These results provide evidence that the secretion and activation of MMPs and the release of TNF-alpha play a central role in the ocular hypotension induced by FP-agonists. The administration of FP-agonists appears to lower IOP directly by inducing the activation of MMPs within the ciliary body, leading to improved uveoscleral outflow and indirectly through the release of TNF within the ciliary body. Secreted TNF-alpha may then activate TNF-receptors in the uvea and trabecular meshwork, increasing both uveoscleral and conventional outflow.
Role of PKCepsilon in PGF2alpha-stimulated MMP-2 secretion from human ciliary muscle cells
J Ocul Pharmacol Ther. 2008 Jun;24(3):268-77. doi: 10.1089/jop.2008.0014.
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.
VEGF modulation of retinal pigment epithelium resistance
Exp Eye Res. 2007 Dec;85(6):762-71. doi: 10.1016/j.exer.2007.08.010. Epub 2007 Aug 24.
Fluid accumulation into the subretinal space and the development of macular edema is a common condition in age-related macular degeneration, diabetic retinopathy, and following ocular surgery, or injury. Vascular endothelial growth factor (VEGF) and other cytokines have been implicated in the disruption of retinal pigment epithelium (RPE) barrier function and a reduction in the regulated removal of subretinal fluid; however, the cellular and molecular events linking these agents to the disruption of barrier function have not been established. In the current study, cultures of ARPE-19 and primary porcine retinal pigment epithelium (RPE) cells were utilized to investigate the effects of the VEGF-induced modifications to the barrier properties of the RPE. The barrier function was determined by transepithelial resistance (TER) measurements and morphology of the RPE monolayers. In both ARPE-19 and primary porcine RPE cells the administration of VEGF produced a significant drop in TER, and this response was only observed following apical administration. Maximum reduction in TER was reached 5h post VEGF administration. These responses were concentration-dependent with an EC(50) of 502pg/mL in ARPE-19 cells and 251pg/mL in primary porcine cells. In both ARPE-19 and primary RPE cells, the response to VEGF was blocked by pretreatment with the relatively selective VEGF-R2 antagonists, SU5416 or ZM323881, or the protein tyrosine kinase inhibitor, genistein. Administration of the relatively selective VEGF-R2 agonist, VEGF-E, also reduced TER in a concentration-dependent manner (EC(50) of 474pg/mL), while VEGF-R1 agonist, placental growth factor (PlGF), did not significantly alter the TER. Immunolocalization studies demonstrated that confluent monolayers exhibited continuous cell-to-cell ZO-1 protein contacts and apical localization of the VEGF-R2 receptors. These data provide evidence that the VEGF-induced breakdown of RPE barrier function is mediated by the activation of apically-oriented VEGF-R2 receptors. Thus, VEGF-mediated increases in RPE permeability are initiated by a rise in intraocular levels of VEGF.
S-nitrosoglutathione prevents interphotoreceptor retinoid-binding protein (IRBP(161-180))-induced experimental autoimmune uveitis
J Ocul Pharmacol Ther. 2007 Jun;23(3):221-31. doi: 10.1089/jop.2007.0023.
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
Mol Vis. 2006 Oct 26;12:1292-302.
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
J Ocul Pharmacol Ther. 2006 Oct;22(5):310-6. doi: 10.1089/jop.2006.22.310.
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.
Effects of latanoprost on rodent intraocular pressure
Exp Eye Res. 2006 Dec;83(6):1453-8. doi: 10.1016/j.exer.2006.08.004. Epub 2006 Oct 6.
The aim of the present study was to evaluate the effects of the prostaglandin F2 alpha analog, latanoprost, on the intraocular pressure (IOP) in rodent eyes. Rodents have been increasingly used in glaucoma research; however, conflicting results regarding the actions of prostaglandins on rodent IOP have been published. In Wistar rats, a single dose of latanoprost (60 ng) produced a biphasic change in IOP: an initial rise in pressure (2.1+/-0.7 mmHg) peaking at 2 h, followed by a prolonged hypotension with a peak reduction in IOP (5.2+/-0.7 mmHg) at 5 h. Both the hyper and hypotensive actions of latanoprost were dose-related with ED50 values of 108 and 5.2 ng, respectively. These responses were antagonized by pretreatment with 4% pilocarpine. In Brown Norway rats and C57BL/6 mice, a single dose of latanoprost also produced a biphasic response in IOP with an initial rise in pressure peaking between 1 and 2 h, followed by prolonged hypotension from 4 to 8 h. These results demonstrate that in rodents the IOP response to topical latanoprost is characterized by an initial hypertension followed by a prolonged hypotension. This prolonged hypotension is similar to that measured in monkeys and humans. Taken together, these results support the idea that rodents can serve as in vivo models to study the actions of ocular hypotensive agents, such as prostaglandins.
Modulation of conventional outflow facility by the adenosine A1 agonist N6-cyclohexyladenosine
Invest Ophthalmol Vis Sci. 2005 Oct;46(10):3795-9. doi: 10.1167/iovs.05-0421.
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.
Acute effects of PGF2alpha on MMP-2 secretion from human ciliary muscle cells: a PKC- and ERK-dependent process
Invest Ophthalmol Vis Sci. 2005 May;46(5):1706-13. doi: 10.1167/iovs.04-0993.
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
Invest Ophthalmol Vis Sci. 2005 Mar;46(3):1092-8. doi: 10.1167/iovs.04-0043.
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.
Heat shock protein 27 delays Ca2+-induced cell death in a caspase-dependent and -independent manner in rat retinal ganglion cells
Invest Ophthalmol Vis Sci. 2005 Mar;46(3):1085-91. doi: 10.1167/iovs.04-0042.
PURPOSE: Hsp27 is a well-characterized and studied antiapoptotic protein. A recent study reported that Hsp27 is upregulated in the retina after retinal ischemic preconditioning. The timing of this upregulation of Hsp27 correlates with the protective effects of the treatment. It was the goal of the current study to determine what role Hsp27 plays in this protection.
METHODS: The rat homologue of Hsp27 (rHsp27) was overexpressed in a transformed rat retinal ganglion cell line and subjected to ischemic stress and calcium overload.
RESULTS: The overexpression of rHsp27 increased cell survival and inhibited caspase-3 activation. However, the inhibition of caspase-3 alone had no effect on cell survival. Proteomic analysis after Ca(2+) overload identified four proteins that were repeatedly associated with rHsp27. These proteins include actin, Hsp70, eEF-1alpha, and SPIN-2. No association with cytochrome c or any caspase enzymes was detected.
CONCLUSIONS: The results indicate that Hsp27 protects the retinal cells by both caspase-dependent and -independent mechanisms.
Outflow resistance of the Baerveldt glaucoma drainage implant and modifications for early postoperative intraocular pressure control
J Glaucoma. 2004 Oct;13(5):396-9. doi: 10.1097/01.ijg.0000131759.48295.5c.
PURPOSE: To determine outflow resistance of the Baerveldt glaucoma implant using different tube configurations.
METHODS: Outflow resistance of 6 tube configurations (C1- C6) of Baerveldt implants was measured under conditions of constant pressure perfusion. Pressures ranged from 2 to 55 mm Hg. Venting slits were created using a 7-0 Vicryl, spatulated suture-needle. Seton tubes were occluded by threading a retrograde suture approximately 1.5 cm into the lumen.
RESULTS: At pressures between 2 and 55 mm Hg, mean outflow resistance of the normally configured seton (ie, open tube; C1) was 0.41 (+/- 0.6) mm Hg/microL/min. Resistance was unchanged (mean 0.41 (+/- 0.4) mm Hg/microL/min) by the addition of 4 venting slits (C2) to the seton tube. Occlusion of the open seton tube with a 3-0 Supramid suture (C3) significantly increased (P < 0.001) mean outflow resistance to 14.99 (+/- 0.6) mm Hg/microL/min. Occlusion of the tube with a 4-0 Supramid suture (C4) significantly increased (P < 0.001) mean outflow resistance to 1.09 (+/- 0.5) mm Hg/microL/min. In implants where tubes were occluded with a 3-0 Supramid suture, the addition of venting slits (C5) significantly decreased (P = 0.038) mean outflow resistance to 8.98 (+/- 0.4) mm Hg/microL/min. In tubes occluded with a 4-0 Supramid suture, the addition of venting slits (C6) decreased mean outflow resistance to 0.98 (+/- 0.6) mm Hg/microL/min.
CONCLUSIONS: Although these results cannot be directly correlated to the clinical setting, they do show that outflow resistance can be modified at the time of surgery by changing tube configuration of the Baerveldt glaucoma implant. Configuration C5 (3-0 Supramid with venting slits) closely approximates the outflow rate in the normal intraocular pressure range.
Evidence for multiple P2Y receptors in trabecular meshwork cells
J Pharmacol Exp Ther. 2004 May;309(2):484-9. doi: 10.1124/jpet.103.060319. Epub 2004 Jan 23.
The purpose of this study was to determine whether functional purinergic P2 receptors are present in trabecular meshwork cells. The human trabecular cell line HTM-3 and cultured bovine trabecular cells were used to assess the effects of P2 agonists on intracellular Ca(2+) levels, extracellular signal-regulated kinase (ERK1/2) activation, and P2Y receptor expression. ATP, UTP, ADP, and 2-methyl-thio-adenosine triphosphate (2-MeS-ATP) each produced a concentration-dependent increase in intracellular Ca(2+) in bovine trabecular cells and the HTM-3 cell line. The addition of UDP did not produce any detectable rise in intracellular Ca(2+). Pretreatment with the P2Y(1) receptor antagonist 2'-deoxy-N(6)-methyladenosine-3',5'-diphosphate (MRS-2179) blocked the ADP- and 2-MeS-ATP-induced rise in intracellular Ca(2+). However, the ATP- or UTP-induced rise in intracellular Ca(2+) was not inhibited by MRS-2179 pretreatment. The addition of ADP, 2-MeS-ATP, ATP, or UTP were also found to activate the ERK1/2 signaling pathway. This activation of ERK1/2 was blocked by pretreatment with the mitogen-activated protein kinase kinase inhibitor 1,4-diamino-2,3-dicyano-1,4-bis(o-aminophenylmercapto)butadiene (U-0126) or the protein kinase C inhibitor chelerythrine chloride, but not by MRS-2179. Analysis of mRNA from HTM-3 cells by reverse transcription-polymerase chain reaction revealed the expression of P2Y(1), P2Y(4), and P2Y(11) receptor subtypes. These data demonstrate that multiple P2Y receptors are present in trabecular cells. Our results are consistent with the idea that the mobilization of intracellular Ca(2+)results from the activation of P2Y(1) and P2Y(4) receptors, whereas the activation of the ERK1/2 pathway results from the activation of P2Y(4) receptors alone. However, a role for the P2Y(11) receptors in mobilization of Ca(2+), or activation of the ERK1/2 pathway, cannot be discounted.
Residual cleaner after normal cleaning of laser in situ keratomileusis instruments
J Cataract Refract Surg. 2003 Sep;29(9):1727-32. doi: 10.1016/s0886-3350(03)00513-3.
PURPOSE: To determine whether residual cleaner could be detected in the rinse solution of surgical instruments after a standard cleaning protocol.
SETTING: Magill Research Center for Vision Correction, Storm Eye Institute, Medical University of South Carolina, Charleston, South Carolina, USA.
METHODS: The wavelength for maximum absorbance of 5 cleaners (Endozime) [The Ruhof Corp.], Enzol/Cidezyme [Advanced Sterilization Products], and Klenzyme [Steris Co.] enzymatic detergents; Palmolive Ultra dishwashing liquid [Colgate-Palmolive Co.]; and Universal concentrated surgical instrument cleaner and lubricant [B. Graczyk, Inc.]) was determined. Identically designed stainless-steel and titanium instruments were cleaned using a standardized protocol. Water temperatures of 25 degrees C (room temperature) or 40 degrees C (warm temperature) were used to rinse the instruments. The amount of residual cleaner in each rinse solution and remaining on each instrument at the completion of the cleaning procedure as a percentage of the total cleaner was determined.
RESULTS: Residues of all cleaners were detected using a standard protocol involving rinse solutions at 25 degrees C. Increasing the temperature of the rinse solutions significantly reduced the cleaner residues (P<.05, Friedman repeated-measures analysis of variance on ranks test and Student-Newman-Keuls test) regardless of the instrument material. No significant difference was detected in the residual cleaners on stainless-steel and titanium instruments.
CONCLUSIONS: Lower levels of cleaner residue were found on surgical instruments after a standard cleaning protocol using warm rinse water. Because cleaner residue has been reported to cause inflammation (eg, diffuse lamellar keratitis) after laser in situ keratomileusis (LASIK), it is advisable to use cleaners and cleaning protocols that result in acceptable cleaning without detectable levels of cleaner residue to avoid potentially harmful effects to the cornea after LASIK.
Photodynamic therapy with verteporfin in a rabbit model of corneal neovascularization
Invest Ophthalmol Vis Sci. 2003 Jul;44(7):2954-8. doi: 10.1167/iovs.02-0572.
PURPOSE: To determine the efficacy of photodynamic therapy (PDT) with verteporfin (Visudyne; Novartis AG, Basel, Switzerland) for treatment of corneal neovascularization in a rabbit eye model.
METHODS: Corneal neovascularization was induced in Dutch belted rabbits by placing an intrastromal silk suture near the limbus. Verteporfin was administered by intravenous injection at a dose of 1.5 mg/kg, and the pharmacokinetics of verteporfin distribution in the anterior segment or PDT-induced (laser energy levels 17, 50, and 150 J/cm(2)) regression of corneal blood vessels were then determined. To assess PDT-induced toxicity of the anterior segment, corneal and iris/ciliary body histology, and IOP were evaluated after PDT.
RESULTS: Verteporfin accumulation in vascularized regions of the cornea and the iris/ciliary body tissue were time dependent and maximum levels achieved at 60 minutes after injection. In rabbits, PDT of corneal vessels using laser energy of 17 or 50 J/cm(2) resulted in 30% to 50% regression of corneal neovascularization; however, in these animals, a rapid regrowth of new blood vessels occurred between 3 and 5 days. In the rabbits receiving PDT using laser energies of 150 J/cm(2), the mean vessel regression was 56%. During the nine days of the laser therapy follow-up period, no vessel regrowth was observed in these rabbits. Histologic examination of the anterior segment after PDT (150 J/cm(2)) showed localized degeneration of the corneal blood vessels without observable change in other anterior segment structures.
CONCLUSIONS: These results provide evidence that PDT can produce significant regression of neovascular corneal vessels with no observable toxicity to the anterior segments. However, the optimal laser energy necessary to induce long-term regression (150 J/cm(2)) was three times that used to treat choroidal neovascularization.
Intraocular adenosine levels in normal and ocular-hypertensive patients
J Ocul Pharmacol Ther. 2003 Apr;19(2):113-9. doi: 10.1089/108076803321637645.
PURPOSE: Adenosine receptors modulate several ocular responses; however, our understanding of factors that influence ocular extracellular adenosine levels is limited. The objective of this study was to evaluate how changes in intraocular pressure (IOP) influence endogenous levels of the purines adenosine and inosine, in the aqueous humor of normal and ocular-hypertensive patients.
PATIENTS AND METHODS: Informed consent was obtained from 51 individuals undergoing cataract extraction or glaucoma surgical procedures. IOP was measured immediately prior to surgery. At the start of the surgical procedure, an aqueous sample of 75-100 microL was obtained. Purine levels were determined by reverse-phase HPLC.
RESULTS: In normotensive individuals, mean aqueous adenosine and inosine levels were 5.2 +/- 1.1 and 19.4 +/- 2.2 ng/100 microL, respectively. No significant correlation between IOP and purine concentration was measured in this group. In ocular hypertensive individuals, the mean aqueous adenosine and inosine concentration was significantly elevated when compared to normotensive individuals. In the ocular hypertensive individual, this elevation in adenosine level was significantly correlated with IOP (r(2) = 0.42).
CONCLUSIONS: These results demonstrate that in ocular hypertensive individuals, aqueous adenosine concentration is correlated with IOP. As the activation of adenosine receptors can modulate IOP and retinal blood flow, adenosine release during periods of ocular hypertension may play an important role in the physiological responses to elevated IOP.
Retinal preconditioning and the induction of heat-shock protein 27
Invest Ophthalmol Vis Sci. 2003 Mar;44(3):1299-304. doi: 10.1167/iovs.02-0235.
PURPOSE: Brief periods of ischemia have been shown to protect the retina from potentially damaging periods of ischemia. This phenomenon has been termed ischemic preconditioning or ischemic tolerance. In the present study the cellular changes in levels of heat shock protein (Hsp)27, -70, and -90 mRNA and expression of Hsp in the rat retina associated with ischemic preconditioning were evaluated.
METHODS: Unilateral retinal ischemia was created in Long-Evans and Sprague-Dawley rats for 5 minutes. Rats were then left for 1 hour to 7 days, to allow the retina to reperfuse. Retinas were dissected, the mRNA and protein isolated, and Northern and Western blot analyses conducted to detect changes in expression of Hsp27, -70, and -90. Immunohistochemical studies were used to identify retinal regions where Hsp changes occurred. Selected animals were subjected to a second ischemic event, 60 minutes in duration, to correlate the changes in expression of Hsp with functional protection of the retina from ischemic injury.
RESULTS: In control and sham-treated animals retinal Hsp27, -70, and -90 mRNAs were detectable. Five hours after retinal preconditioning, levels of Hsp27 mRNA were elevated above control levels, and 24 hours later, mRNA levels increased 200% over basal levels. Hsp27 expression remained elevated for up to 72 hours and then began to return to control levels. Hsp27 protein levels were increased by 200% over basal levels 24 hours after retinal preconditioning, remained at this level for 72 hours, and then returned to control levels. In contrast, no consistent change in Hsp70 or -90 mRNA or protein levels was observed during the course of the study. Immunohistochemical studies demonstrated that the increase in expression of Hsp27 was localized to neuronal and non-neuronal cells in the inner layers of the retina. Electroretinography studies demonstrated a strong correlation between the protection of retinal function from ischemic injury and the expression of Hsp27.
CONCLUSIONS: These results provide evidence that the induction of Hsp27 is a gene-specific event associated with ischemic preconditioning in the retina. This increase in expression of Hsp27 occurs in both neuronal and non-neuronal retinal cells, and appears to be one component of the neuroprotective events induced by ischemic preconditioning in the retina.
Bradykinin enhancement of PGE2 signalling in bovine trabecular meshwork cells
Exp Eye Res. 2003 Mar;76(3):283-9. doi: 10.1016/s0014-4835(02)00313-5.
Kinins and prostaglandins activate signalling pathways in cells of the trabecular meshwork and have opposing effects on outflow resistance to aqueous humor. Consequently, interactions between these pathways may be important in the regulation of intraocular pressure. In the present study, the influence of bradykinin on PGE(2) signalling was examined in primary cultures of bovine trabecular meshwork cells. Incubation of cells with bradykinin produced a concentration-dependent (EC(50)=3.6+/-0.7 nM) elevation of intracellular free Ca(2+). At a maximal concentration of 100 nM, the increase in Ca(2+) was rapid, peaking in 30 sec, and then slowly returned to baseline. This effect was completely inhibited in cells pretreated with the selective B(2) kinin receptor antagonist, Hoe-140. Treatment of trabecular meshwork cells with PGE(2), in comparison, had no effect on cellular Ca(2+) but produced a concentration-dependent increase in adenosine 3', 5'-cyclic monophosphate (cAMP) formation. Bradykinin had no effect on basal cAMP. However, incubation of cells with PGE(2) in combination with bradykinin resulted in a 3- to 5-fold enhancement of PGE(2)-stimulated cAMP production. Bradykinin enhancement of cAMP stimulation was concentration-dependent with an EC(50) of 3.6+/-1.8 nM. Treatment of cells with bradykinin increased the response maximum for PGE(2) signalling, while the EC(50) for PGE(2) was not changed. This action of bradykinin was again blocked in cells pretreated with Hoe-140. Bradykinin also produced a 2- to 3-fold increase in isoproterenol and cholera toxin-stimulated cAMP accumulation. However, when adenylyl cyclase was stimulated directly with forskolin, bradykinin failed to alter cAMP production. These results indicate that bradykinin activates B(2) kinin receptors in trabecular meshwork cells to amplify PGE(2)-stimulated cAMP formation by facilitating the interaction between activated G(s) and the catalytic unit of adenylyl cyclase.
Adenosine A1 receptor modulation of MMP-2 secretion by trabecular meshwork cells
Invest Ophthalmol Vis Sci. 2002 Sep;43(9):3016-20.
PURPOSE: Studies have shown that adenosine A(1) agonists can lower IOP in rabbits, mice, and monkeys, and this response is mediated in part by increases in outflow facility. The purpose of this project was to evaluate the response of trabecular meshwork cells to the addition of the adenosine A(1) receptor agonist N(6)-cyclohexyladenosine (CHA).
METHODS: The human trabecular meshwork (HTM-3) cell line and primary cultures of bovine trabecular meshwork (BTM) cells were used in these studies. Cells were treated with CHA, and the secretion of matrix metalloproteinase (MMP)-2 or the activation of extracellular signal-regulated kinase (ERK1/2) was determined.
RESULTS: Treatment of HTM-3 and BTM cells with CHA (0.1 micro M) resulted in a time-dependent secretion of MMP-2 that was measurable as early as 30 minutes after treatment and reached a maximum by 2 hours. This CHA-induced secretion of MMP-2 was inhibited by the adenosine A(1) receptor antagonist 8-cyclopentyl-1,3-dimethylxanthine (CPT) and by the ERK1/2 pathway inhibitor U0126. Treatment of HTM-3 cells with CHA produced a rapid dose-dependent activation of ERK1/2 with an EC(50) of 5.7 nM. The CHA-induced activation of ERK1/2 was inhibited by pretreatment with the adenosine A(1) antagonist CPT and by the ERK pathway inhibitor U0126.
CONCLUSIONS: The addition of the adenosine A(1) agonist CHA stimulates the secretion of MMP-2 from trabecular meshwork cells. This secretory response involves the activation of adenosine A(1)-linked stimulation of ERK1/2. These results provide evidence for the existence of functional adenosine A(1) receptors in the trabecular cells and that the activation of these receptors stimulates secretion of MMP-2.
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
Diabetes. 2002 Apr;51(4):1218-25. doi: 10.2337/diabetes.51.4.1218.
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.
Synthesis, pharmacokinetics, efficacy, and rat retinal toxicity of a novel mitomycin C-triamcinolone acetonide conjugate
J Med Chem. 2002 Feb 28;45(5):1122-7. doi: 10.1021/jm010511b.
A novel conjugate of mitomycin C (MMC) and triamcinolone acetonide (TA) was synthesized using glutaric acid as a linker molecule. To determine the rate of hydrolysis, the conjugate was dissolved in aqueous solution and the rate of appearance of free MMC and TA was determined by high-performance liquid chromatography analysis. Antiproliferative activity of the MMC-TA conjugate and parent compounds was assessed using an NIH 3T3 fibroblast cell line. Cell growth was quantified using the MTT assay. Kinetic analysis of the hydrolysis rate demonstrated that the conjugate had a half-life of 23.6 h in aqueous solutions. The antiproliferative activities of the MMC-TA conjugate and MMC were both concentration dependent, with similar IC(50) values of 2.4 and 1.7 microM, respectively. However, individual responses at concentrations above 3 microM showed that the conjugate was less active than MMC alone. TA alone showed only limited inhibition of cell growth. Studies evaluating intravitreal injection of the conjugate demonstrate that this agent produced no measurable toxicity. Our data provide evidence that the MMC-TA conjugate could be used as a slow-release drug delivery system. This could in turn be used to modulate a posttreatment wound healing process or to treat various proliferative diseases.