Gary W. McCollum

Role of Cytosolic Phospholipase A2 in Retinal Neovascularization

PURPOSE To identify and characterize the role of cytosolic phospholipase A(2) (cPLA(2)) in retinal angiogenesis using relevant cell-based assays and a rodent model of retinopathy of prematurity. METHODS The phosphorylation states of cPLA(2) and p38 MAP kinase and the expression of COX-2 were assessed by Western blot analysis in rat Müller cells. The activities of PLA(2) enzymes in rat retinal lysates were assessed using a commercially available assay. Prostaglandin E(2) (PGE(2)) and VEGF levels in Müller cell-conditioned medium and in retinal tissue samples were measured by ELISA. Rat retinal microvascular endothelial cell proliferation was measured using a BrdU assay. Efficacy of the cPLA(2) inhibitor CAY10502 was tested using the rat model of oxygen-induced retinopathy (OIR) in which neovascularization (NV) was assessed by computer-assisted image analysis. RESULTS In Müller cells, hypoxia increased the phosphorylation of cPLA(2) and p38 MAP kinase by 4-fold and 3-fold respectively. The cPLA(2) inhibitor CAY10502 decreased hypoxia-induced PGE(2) and VEGF levels in Müller cell-conditioned medium by 68.6% (P < 0.001) and 46.6% (P < 0.001), respectively. Retinal cPLA(2) activity peaked 1 day after oxygen exposure in OIR rats. CAY10502 (250 nM) decreased OIR-induced retinal PGE(2) and VEGF levels by 69% (P < 0.001) and 40.2% (P < 0.01), respectively. Intravitreal injection of 100 nM CAY10502 decreased retinal NV by 53.1% (P < 0.0001). CONCLUSIONS cPLA(2) liberates arachidonic acid, the substrate for prostaglandin (PG) production by the cyclooxygenase enzymes. PGs can exert a proangiogenic influence by inducing VEGF production and by stimulating angiogenic behaviors in vascular endothelial cells. Inhibition of cPLA(2) inhibits the production of proangiogenic PGs. Thus, cPLA(2) inhibition has a significant influence on pathologic retinal angiogenesis.

Genetic deletion of COX-2 diminishes VEGF production in mouse retinal Müller cells

Non-steroidal anti-inflammatory drugs (NSAIDs), which inhibit COX activity, reduce the production of retinal VEGF and neovascularization in relevant models of ocular disease. We hypothesized that COX-2 mediates VEGF production in retinal Müller cells, one of its primary sources in retinal neovascular disease. The purpose of this study was to determine the role of COX-2 and its products in VEGF expression and secretion. These studies have more clearly defined the role of COX-2 and COX-2-derived prostanoids in retinal angiogenesis. Müller cells derived from wild-type and COX-2 null mice were exposed to hypoxia for 0-24 h. COX-2 protein and activity were assessed by western blot analysis and GC-MS, respectively. VEGF production was assessed by ELISA. Wild-type mouse Müller cells were treated with vehicle (0.1% DMSO), 10 microM PGE(2), or PGE(2) + 5 microM H-89 (a PKA inhibitor), for 12 h. VEGF production was assessed by ELISA. Hypoxia significantly increased COX-2 protein (p < 0.05) and activity (p < 0.05), and VEGF production (p < 0.0003). COX-2 null Müller cells produced significantly less VEGF in response to hypoxia (p < 0.05). Of the prostanoids, PGE(2) was significantly increased by hypoxia (p < 0.02). Exogenous PGE(2) significantly increased VEGF production by Müller cells (p < 0.0039), and this effect was inhibited by H-89 (p < 0.055). These data demonstrate that hypoxia induces COX-2, prostanoid production, and VEGF synthesis in Müller cells, and that VEGF production is at least partially COX-2-dependent. Our study suggests that PGE(2), signaling through the EP(2) and/or EP(4) receptor and PKA, mediates the VEGF response of Müller cells.

The role of cytochrome P450 epoxygenases in retinal angiogenesis.

PURPOSE The purpose of this study was to investigate the role(s) of cytochrome P450 epoxygenases (CYPs) and their products, the epoxyeicosatrienoic acids (EETs), in hypoxia-induced VEGF production and pathologic retinal angiogenesis. METHODS Human retinal astrocytes, Müller cells, and retinal microvascular endothelial cells (HRMEC) were exposed to hypoxia, and relative CYP2C expression was measured by RT-PCR. Astrocyte and Müller cell VEGF production was measured by ELISA after exposure to hypoxia and treatment with the general CYP inhibitor, SKF-525a. Human retinal microvascular endothelial cells were treated with the CYP product, 11,12-epoxyeicosatrienoic acid [EET], or SKF-525a in the presence or absence of VEGF. Proliferation of HRMEC and tube formation were assayed. Oxygen-induced retinopathy (OIR) was induced in newborn rats. Retinal CYP2C11 and CYP2C23 expression were measured by RT-PCR. The OIR rats received SKF-525a by intravitreal injection and preretinal neovascularization (NV) was quantified. Retinal VEGF protein levels were measured by ELISA. RESULTS Human retinal astrocytes were the only cells to exhibit significant induction of CYP2C8 and CYP2C9 mRNA expression by hypoxia. Astrocytes, but not Müller cells, exhibited reduced hypoxia-induced VEGF production when treated with SKF-525a. 11,12-EET induced HRMEC proliferation and tube formation, and SKF-525a inhibited VEGF-induced proliferation. Oxygen-induced retinopathy induced expression of CYP2C23, but had no effect on CYP2C11. SKF-525a inhibited retinal NV and reduced retinal VEGF levels in OIR rats. CONCLUSIONS The CYP-derived 11,12-EET may exhibit a proangiogenic biological function in the retina following stimulation by hypoxia in astrocytes. Inhibition of CYP may provide a rational therapy against retinal NV, because it can reduce VEGF production and VEGF-induced angiogenic responses in endothelial cells.

In Vivo Imaging of Retinal Hypoxia in a Model of Oxygen-Induced Retinopathy.

Ischemia-induced hypoxia elicits retinal neovascularization and is a major component of several blinding retinopathies such as retinopathy of prematurity (ROP), diabetic retinopathy (DR) and retinal vein occlusion (RVO). Currently, noninvasive imaging techniques capable of detecting and monitoring retinal hypoxia in living systems do not exist. Such techniques would greatly clarify the role of hypoxia in experimental and human retinal neovascular pathogenesis. In this study, we developed and characterized HYPOX-4, a fluorescence-imaging probe capable of detecting retinal-hypoxia in living animals. HYPOX-4 dependent in vivo and ex vivo imaging of hypoxia was tested in a mouse model of oxygen-induced retinopathy (OIR). Predicted patterns of retinal hypoxia were imaged by HYPOX-4 dependent fluorescence activity in this animal model. In retinal cells and mouse retinal tissue, pimonidazole-adduct immunostaining confirmed the hypoxia selectivity of HYPOX-4. HYPOX-4 had no effect on retinal cell proliferation as indicated by BrdU assay and exhibited no acute toxicity in retinal tissue as indicated by TUNEL assay and electroretinography (ERG) analysis. Therefore, HYPOX-4 could potentially serve as the basis for in vivo fluorescence-based hypoxia-imaging techniques, providing a tool for investigators to understand the pathogenesis of ischemic retinopathies and for physicians to address unmet clinical needs.

Peroxisome Proliferator-Activated Receptor-β/δ Regulates Angiogenic Cell Behaviors and Oxygen-Induced Retinopathy

PURPOSE To develop new therapies against ocular neovascularization (NV), we tested the effect of peroxisome proliferator-activated receptor-β/δ (PPAR-β/δ) agonism and antagonism on angiogenic behaviors and in human retinal microvascular endothelial cells (HRMEC) and on preretinal NV in rat oxygen-induced retinopathy (OIR). METHODS HRMECs were treated with the PPAR-β/δ agonist GW0742 and the antagonist GSK0660. Messenger RNA levels of a PPAR-β/δ target gene, angiopoietin-like-4 (angptl4) were assayed by qRT-PCR. HRMEC proliferation and tube formation were assayed according to standard protocols. OIR was induced in newborn rats by exposing them to alternating 24-hour episodes of 50% and 10% oxygen for 14 days. OIR rats were treated with GW0742 or GSK0660. Angptl4 protein levels were assessed by ELISA and preretinal NV was quantified by adenosine diphosphatase staining. RESULTS GW0742 significantly increased angptl4 mRNA, and GSK0660 significantly decreased angptl4 mRNA. GW0742 had no effect on HRMEC proliferation, but caused a significant and dose-responsive increase in tube formation. GSK0660 significantly reduced serum-induced HRMEC proliferation and tube formation in a dose-dependent manner. Intravitreal injection of GW0742 significantly increased total retinal Angptl4 protein, but intravitreal injection of GSK0660 had no effect. Intravitreal injection of GW0742 significantly increased retinal NV, as did GW0742 administered by oral gavage. Conversely, both intravitreal injection and intraperitoneal injection of GSK0660 significantly reduced retinal NV. CONCLUSIONS PPAR-β/δ activation exacerbates, and its inhibition reduces, preretinal NV. PPAR-β/δ may regulate preretinal NV through a prodifferentiation/maturation mechanism that depends on Angptl4. Pharmacologic inhibition of PPAR-β/δ may provide a rational basis for therapeutic targeting of ocular NV.

Modulation of VEGF-Induced Retinal Vascular Permeability by Peroxisome Proliferator-Activated Receptor-β/δ

PURPOSE Vascular endothelial growth factor (VEGF)-induced retinal vascular permeability contributes to diabetic macular edema (DME), a serious vision-threatening condition. Peroxisome proliferator-activated receptor β/δ (PPARβ/δ) antagonist/reverse agonist, GSK0660, inhibits VEGF-induced human retinal microvascular endothelial cell (HRMEC) proliferation, tubulogenesis, and oxygen-induced retinal vasculopathy in newborn rats. These VEGF-induced HRMEC behaviors and VEGF-induced disruption of endothelial cell junctional complexes may well share molecular signaling events. Thus, we sought to examine the role of PPARβ/δ in VEGF-induced retinal hyperpermeability. METHODS Transendothelial electrical resistance (TEER) measurements were performed on HRMEC monolayers to assess permeability. Claudin-1/Claudin-5 localization in HRMEC monolayers was determined by immunocytochemistry. Extracellular signal-regulated protein kinases 1 and 2 (Erk 1/2) phosphorylation, VEGF receptor 1 (VEGFR1) and R2 were assayed by Western blot analysis. Expression of VEGFR1 and R2 was measured by quantitative RT-PCR. Last, retinal vascular permeability was assayed in vivo by Evans blue extravasation. RESULTS Human retinal microvascular endothelial cell monolayers treated with VEGF for 24 hours showed decreased TEER values that were completely reversed by the highest concentration of GSK0660 (10 μM) and PPARβ/δ-directed siRNA (20 μM). In HRMEC treated with VEGF, GSK0660 stabilized tight-junctions as evidenced by Claudin-1 staining, reduced phosphorylation of Erk1/2, and reduced VEGFR1/2 expression. Peroxisome proliferator-activated receptor β/δ siRNA had a similar effect on VEGFR expression and Claudin-1, supporting the specificity of GSK0660 in our experiments. Last, GSK0660 significantly inhibited VEGF-induced retinal vascular permeability and reduced retinal VEGFR1and R2 levels in C57BL/6 mice. CONCLUSIONS These data suggest a protective effect for PPARβ/δ antagonism against VEGF-induced vascular permeability, possibly through reduced VEGFR expression. Therefore, antagonism/reverse agonism of PPARβ/δ siRNA may represent a novel therapeutic methodology against retinal hyperpermeability and is worthy of future investigation.

Proteomic Analysis of the Retina: Removal of RPE Alters Outer Segment Assembly and Retinal Protein Expression

The mechanisms that regulate the complex physiological task of photoreceptor outer segment assembly remain an enigma. One limiting factor in revealing the mechanism(s) by which this process is modulated is that not all of the role players who participate in this process are known. The purpose of this study was to determine some of the retinal proteins that likely play a critical role in regulating photoreceptor outer segment assembly. To do so, we analyzed and compared the proteome map of tadpole Xenopus laevis retinal pigment epithelium (RPE)‐supported retinas containing organized outer segments with that of RPE‐deprived retinas containing disorganized outer segments. Solubilized proteins were labeled with CyDye fluors followed by multiplexed two‐dimensional separation. The intensity of protein spots and comparison of proteome maps was performed using DeCyder software. Identification of differentially regulated proteins was determined using nanoLC‐ESI‐MS/MS analysis. We found a total of 27 protein spots, 21 of which were unique proteins, which were differentially expressed in retinas with disorganized outer segments. We predict that in the absence of the RPE, oxidative stress initiates an unfolded protein response. Subsequently, downregulation of several candidate Müller glial cell proteins may explain the inability of photoreceptors to properly fold their outer segment membranes. In this study, we have used identification and bioinformatics assessment of proteins that are differentially expressed in retinas with disorganized outer segments as a first step in determining probable key molecules involved in regulating photoreceptor outer segment assembly.

High Glucose-induced Retinal Pericyte Apoptosis Depends on Association of GAPDH and Siah1.

Background: Pericyte cell death occurs early in diabetic retinopathy, but the cause remains unknown. Results: High glucose-induced retinal pericyte apoptosis is mediated, in part, by the association of GAPDH and Siah1. Conclusion: Inhibition of the GAPDH/Siah1 pro-apoptotic pathway inhibits high glucose-induced human pericyte death. Significance: Understanding the mechanism of diabetes-induced pericyte apoptosis could direct development of new therapeutic strategies for early diabetic retinopathy. Diabetic retinopathy (DR) is a leading cause of blindness worldwide, and its prevalence is growing. Current therapies for DR address only the later stages of the disease, are invasive, and have limited effectiveness. Retinal pericyte death is an early pathologic feature of DR. Although it has been observed in diabetic patients and in animal models of DR, the cause of pericyte death remains unknown. A novel pro-apoptotic pathway initiated by the interaction between glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the E3 ubiquitin ligase, seven in absentia homolog 1 (Siah1), was recently identified in ocular tissues. In this article we examined the involvement of the GAPDH/Siah1 interaction in human retinal pericyte (hRP) apoptosis. HRP were cultured in 5 mm normal glucose, 25 mm l- or d-glucose for 48 h (osmotic control and high glucose treatments, respectively). Siah1 siRNA was used to down-regulate Siah1 expression. TAT-FLAG GAPDH and/or Siah1-directed peptides were used to block GAPDH and Siah1 interaction. Co-immunoprecipitation assays were conducted to analyze the effect of high glucose on the association of GAPDH and Siah1. Apoptosis was measured by Annexin V staining and caspase-3 enzymatic activity assay. High glucose increased Siah1 total protein levels, induced the association between GAPDH and Siah1, and led to GAPDH nuclear translocation. Our findings demonstrate that dissociation of the GAPDH/Siah1 pro-apoptotic complex can block high glucose-induced pericyte apoptosis, widely considered a hallmark feature of DR. Thus, the work presented in this article can provide a foundation to identify novel targets for early treatment of DR.

Linoleic Acid is a Diabetes-relevant Stimulator of Retinal Inflammation in Human Retinal Muller Cells and Microvascular Endothelial Cells

OBJECTIVE To determine the effect of oleic acid and linoleic acid on the production and secretion of specific diabetic retinopathy- (DR-) related cytokines: vascular endothelial growth factor (VEGF), interleukin-6 (IL-6), and interleukin-8 (IL-8) by human retinal glial cells, retinal endothelial cells, and retinal pigment epithelial cells. These expression profiles will be compared to those obtained by treatment of the same cell types with elevated D-glucose, a diabetes-relevant stimulus often used in retinal cell culture experiments. METHODS Primary cultures of human retinal Müller cells, astrocytes, and microvascular endothelial cells (RMEC) and a human retinal pigment epithelial cell line (ARPE-19) were treated with oleic acid, linoleic acid, elevated D-glucose, or L-glucose as an osmotic control. VEGF, IL-6, and IL-8 concentrations in conditioned media were determined by colorimetric ELISA and normalized to total cellular protein. RESULTS In the conditioned medium of human Müller cells, linoleic and oleic acid increased VEGF production by 6.4-fold and 9.9-fold, respectively. Linoleic acid also significantly increased IL-6 by 2.9-fold and IL-8 by 5.7-fold. L-glucose and D-glucose both increased VEGF by 3.1-fold in Müller cell conditioned medium. Linoleic acid increased IL-8 concentrations by 56% in human RMEC conditioned medium. Human retinal astrocytes and ARPE-19 were unaffected by all stimuli. CONCLUSIONS Linoleic and oleic acid induce inflammatory mediators believed to be involved in the pathogenesis of diabetic retinopathy (DR). In culture, the free fatty acid insults, particularly linoleic acid, significantly increased cytokine production by Müller cells. In summary, these data identified Müller cells as the primary producer of these inflammatory mediators when treated with unsaturated fatty acids. This study also demonstrates that elevated glucose is an inadequate stimulus for assessing the production of inflammatory mediators. Therefore this study provides a novel in vitro model system of the dyslipidemia-induced inflammation occurring in DR.

Epoxygenated Fatty Acids Inhibit Retinal Vascular Inflammation

The objective of the present study was to assess the effect of elevating epoxygenated fatty acids on retinal vascular inflammation. To stimulate inflammation we utilized TNFα, a potent pro-inflammatory mediator that is elevated in the serum and vitreous of diabetic patients. In TNFα-stimulated primary human retinal microvascular endothelial cells, total levels of epoxyeicosatrienoic acids (EETs), but not epoxydocosapentaenoic acids (EDPs), were significantly decreased. Exogenous addition of 11,12-EET or 19,20-EDP when combined with 12-(3-adamantane-1-yl-ureido)-dodecanoic acid (AUDA), an inhibitor of epoxide hydrolysis, inhibited VCAM-1 and ICAM-1 expression and protein levels; conversely the diol product of 19,20-EDP hydrolysis, 19,20-DHDP, induced VCAM1 and ICAM1 expression. 11,12-EET and 19,20-EDP also inhibited leukocyte adherence to human retinal microvascular endothelial cell monolayers and leukostasis in an acute mouse model of retinal inflammation. Our results indicate that this inhibition may be mediated through an indirect effect on NFκB activation. This is the first study demonstrating a direct comparison of EET and EDP on vascular inflammatory endpoints, and we have confirmed a comparable efficacy from each isomer, suggesting a similar mechanism of action. Taken together, these data establish that epoxygenated fatty acid elevation will inhibit early pathology related to TNFα-induced inflammation in retinal vascular diseases.