Megan E. Capozzi

Circadian Perinatal Photoperiod Has Enduring Effects on Retinal Dopamine and Visual Function

Visual system development depends on neural activity, driven by intrinsic and light-sensitive mechanisms. Here, we examined the effects on retinal function due to exposure to summer- and winter-like circadian light cycles during development and adulthood. Retinal light responses, visual behaviors, dopamine content, retinal morphology, and gene expression were assessed in mice reared in seasonal photoperiods consisting of light/dark cycles of 8:16, 16:8, and 12:12 h, respectively. Mice exposed to short, winter-like, light cycles showed enduring deficits in photopic retinal light responses and visual contrast sensitivity, but only transient changes were observed for scotopic measures. Dopamine levels were significantly lower in short photoperiod mice, and dopaminergic agonist treatment rescued the photopic light response deficits. Tyrosine hydroxylase and Early Growth Response factor-1 mRNA expression were reduced in short photoperiod retinas. Therefore, seasonal light cycles experienced during retinal development and maturation have lasting influence on retinal and visual function, likely through developmental programming of retinal dopamine.

Molecular probes for imaging of hypoxia in the retina.

Hypoxia has been associated with retinal diseases which lead the causes of irreversible vision loss, including diabetic retinopathy, retinopathy of prematurity, and age-related macular degeneration. Therefore, technologies for imaging hypoxia in the retina are needed for early disease detection, monitoring of disease progression, and assessment of therapeutic responses in the patient. Toward this goal, we developed two hypoxia-sensitive imaging agents based on nitroimidazoles which are capable of accumulating in hypoxic cells in vivo. 2-nitroimidazole or Pimonidazole was conjugated to fluorescent dyes to yield the imaging agents HYPOX-1 and HYPOX-2. Imaging agents were characterized in cell culture and animal models of retinal vascular diseases which exhibit hypoxia. Both HYPOX-1 and -2 were capable of detecting hypoxia in cell culture models with >10:1 signal-to-noise ratios without acute toxicity. Furthermore, intraocular administration of contrast agents in mouse models of retinal hypoxia enabled ex vivo detection of hypoxic tissue. These imaging agents are a promising step toward translation of hypoxia-sensitive molecular imaging agents in preclinical animal models and patients.

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.

Molecular Imaging of Retinal Disease

Imaging of the eye plays an important role in ocular therapeutic discovery and evaluation in preclinical models and patients. Advances in ophthalmic imaging instrumentation have enabled visualization of the retina at an unprecedented resolution. These developments have contributed toward early detection of the disease, monitoring of disease progression, and assessment of the therapeutic response. These powerful technologies are being further harnessed for clinical applications by configuring instrumentation to detect disease biomarkers in the retina. These biomarkers can be detected either by measuring the intrinsic imaging contrast in tissue, or by the engineering of targeted injectable contrast agents for imaging of the retina at the cellular and molecular level. Such approaches have promise in providing a window on dynamic disease processes in the retina such as inflammation and apoptosis, enabling translation of biomarkers identified in preclinical and clinical studies into useful diagnostic targets. We discuss recently reported and emerging imaging strategies for visualizing diverse cell types and molecular mediators of the retina in vivo during health and disease, and the potential for clinical translation of these approaches.

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.

The role of the NFAT signaling pathway in retinal neovascularization.

PURPOSE The purpose of the present study was to investigate the role of nuclear factor of activated T cells (NFAT), a transcription factor downstream of VEGF, in angiogenic cell behaviors of human retinal microvascular endothelial cells (HRMEC), and to assess the efficacy of NFAT signaling inhibitors in a rat model of oxygen-induced retinopathy (OIR). METHODS Human retinal microvascular endothelial cells were treated with VEGF in the presence or absence of the NFAT inhibitor of NFAT-calcineurin association-6 (INCA-6), and NFAT translocation was evaluated using immunocytochemistry (ICC). Human retinal microvascular endothelial cells were treated with increasing doses of INCA-6, and cell proliferation and tube formation were assessed. Rats subjected to OIR were administered increasing doses of INCA-6 or the CN inhibitor FK-506, and the retinal neovascular area was measured. RESULTS Nuclear factor of activated T-cells c1 was translocated to the nucleus of HRMEC treated with VEGF, and INCA-6 treatment blocked translocation. Inhibitor of NFAT-calcineurin association-6inhibited HRMEC proliferation and tube formation in a dose-dependent manner. Both INCA-6 and FK-506 treatment significantly reduced pathologic neovascularization in OIR. CONCLUSIONS This investigation has demonstrated that in HRMEC, NFATc1 is activated downstream of VEGF signaling and NFAT signaling plays a key role in angiogenic cell behaviors. In addition, NFAT inhibition is shown to be highly efficacious in an OIR model. These findings indicate that the NFAT signaling pathway may serve as a suitable therapeutic target for the treatment of neovascular eye disease.

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.