Amany Tawfik

Suppression of retinal peroxisome proliferator-activated receptor γ in experimental diabetes and oxygen-induced retinopathy: role of NADPH oxidase.

PURPOSE Recently, the authors have shown that NADPH oxidase is positively correlated with increased leukocyte adhesion and vascular leakage in diabetes and neovascularization in oxygen-induced retinopathy (OIR). Peroxisome proliferator-activated receptor gamma (PPARgamma) agonists have been shown to prevent vascular inflammation and leakage in an experimental model of diabetes. The goal of this study was to investigate whether there is a link between NADPH oxidase and PPARgamma that leads to vascular dysfunction in diabetic retina or OIR. METHODS Diabetes was induced with streptozotocin in wild-type mice or NOX2 knockout mice. One group of wild-type mice was treated with apocynin. Bovine retinal endothelial cells (BRECs) were treated with normal glucose (5 mM) or high glucose (25 mM) in the presence or absence of superoxide dismutase (SOD) or NADPH oxidase inhibitors (apocynin or diphenyleneiodonium [DPI]). Western blotting and immunofluorescence were used to evaluate PPARgamma expression. Activation of nuclear factor (NF)kappaB was measured using the transcription factor assay kit and Western blot analysis of phospho-NFkappaB. PPARgamma expression was also tested in OIR and lipopolysaccharide-induced retinal inflammation. RESULTS Retinal expression of PPARgamma was suppressed in experimental models of diabetes, OIR, and retinal inflammation. This was associated with the activation of NFkappaB in the diabetic retina. These effects were prevented by apocynin or deletion of NOX2. PPARgamma expression was also suppressed in endothelial cells treated with high glucose, and this was prevented by apocynin, DPI, and SOD. CONCLUSIONS Suppression of PPARgamma is involved in the pathogenesis of diabetic retinopathy and OIR. NADPH oxidase could be an upstream mediator of these changes.

Increased Expression and Activity of 12-Lipoxygenase in Oxygen-Induced Ischemic Retinopathy and Proliferative Diabetic Retinopathy: Implications in Retinal Neovascularization

OBJECTIVE Arachidonic acid is metabolized by 12-lipoxygenase (12-LOX) to 12-hydroxyeicosatetraenoic acid (12-HETE) and has an important role in the regulation of angiogenesis and endothelial cell proliferation and migration. The goal of this study was to investigate whether 12-LOX plays a role in retinal neovascularization (NV). RESEARCH DESIGN AND METHODS Experiments were performed using retinas from a murine model of oxygen-induced ischemic retinopathy (OIR) that was treated with and without the LOX pathway inhibitor, baicalein, or lacking 12-LOX. We also analyzed vitreous samples from patients with and without proliferative diabetic retinopathy (PDR). Western blotting and RT-PCR were used to assess the expression of 12-LOX, vascular endothelial growth factor (VEGF), and pigment epithelium–derived factor (PEDF). Liquid chromatography–mass spectrometry was used to assess the amounts of HETEs in the murine retina and human vitreous samples. The effects of 12-HETE on VEGF and PEDF expression were evaluated in Müller cells (rMCs), primary mouse retinal pigment epithelial cells, and astrocytes. RESULTS Retinal NV during OIR was associated with increased 12-LOX expression and 12-, 15-, and 5-HETE production. The amounts of HETEs also were significantly higher in the vitreous of diabetic patients with PDR. Retinal NV was markedly abrogated in mice treated with baicalein or mice lacking 12-LOX. This was associated with decreased VEGF expression and restoration of PEDF levels. PEDF expression was reduced in 12-HETE–treated rMCs, astrocytes, and the retinal pigment epithelium. Only rMCs and astrocytes showed increased VEGF expression by 12-HETE. CONCLUSIONS 12-LOX and its product HETE are important regulators of retinal NV through modulation of VEGF and PEDF expression and could provide a new therapeutic target to prevent and treat ischemic retinopathy.

GADD34 induces cell death through inactivation of Akt following traumatic brain injury

Neuronal cell death contributes significantly to the pathology of traumatic brain injury (TBI) irrespective of the mode or severity of the injury. Activation of a pro-survival protein, Akt, is known to be regulated by an E3 ligase TRAF6 through a process of ubiquitination-coupled phosphorylation at its T308 residue. Here we show that upregulation of a pro-apototic protein, GADD34, attenuates TRAF6-mediated Akt activation in a controlled cortical impact model of TBI in mice. TBI induces the expression of GADD34 by stimulating binding of a stress inducible transcription factor, ATF4, to the GADD34 promoter. GADD34 then binds with TRAF6 and prevents its interaction with Akt. This event leads to retention of Akt in the cytosol and prevents phosphorylation at the T308 position. Finally, in vivo depletion of GADD34 using a lentiviral knockdown approach leads to a rescue of Akt activation and markedly attenuates TBI-induced cell death.

Late-onset inner retinal dysfunction in mice lacking sigma receptor 1 (σR1).

PURPOSE Sigma receptor 1 (σR1) is expressed abundantly in the eye, and several reports suggest that this putative molecular chaperone plays a role in lens cell survival, control of intraocular pressure (IOP), and retinal neuroprotection. The present study examined the consequence of the absence of σR1 on ocular development, structure, and function. METHODS Wild-type (σR1⁺/⁺), heterozygous (σR1⁺/⁻), and homozygous (σR1⁻/⁻, knockout) mice aged 5 to 59 weeks were subjected to comprehensive electrophysiological testing and IOP measurement. The eyes were examined by light and electron microscopy and subjected to morphometric examination and detection of apoptosis. RESULTS Cornea and lens of σR1⁻/⁻ mice were similar to wild-type mice in morphologic appearance at all ages examined, and IOP was within normal limits. Comprehensive ERG and morphometric analyses initially yielded normal findings in the σR1⁻/⁻ mice compared with those in the wild-type. By 12 months, however, significantly decreased ERG b-wave amplitudes and diminished negative scotopic threshold responses, consistent with inner retinal dysfunction, were detected in σR1⁻/⁻ mice. Concomitant with these late-onset changes were increased TUNEL- and active caspase 3-positive cells in the inner retina and significant loss of cells in the ganglion cell layer, particularly in the central retina. Before these functional and structural abnormalities, there was ultrastructural evidence of axonal disruption in the optic nerve head of σR1⁻/⁻ mice as early as 6 months of age, although there were no alterations observed in retinal vascularization in σR1⁻/⁻ mice. CONCLUSIONS These data suggest that lack of σR1 leads to development of late-onset retinal dysfunction with similarities to optic neuropathy.

Age-related changes in visual function in cystathionine-beta-synthase mutant mice, a model of hyperhomocysteinemia

Homocysteine is an amino acid required for the metabolism of methionine. Excess homocysteine is implicated in cardiovascular and neurological disease and new data suggest a role in various retinopathies. Mice lacking cystathionine-beta-synthase (cbs(-/-)) have an excess of retinal homocysteine and develop anatomical abnormalities in multiple retinal layers, including photoreceptors and ganglion cells; heterozygous (cbs(+/-)) mice demonstrate ganglion cell loss and mitochondrial abnormalities in the optic nerve. The purpose of the present study was to determine whether elevated homocysteine, due to absent or diminished cbs, alters visual function. We examined cbs(-/-) (3 weeks) and cbs(+/-) mice (5, 10, 15, 30 weeks) and results were compared to those obtained from wild type (WT) littermates. Conventional dark- and light-adapted ERGs were recorded, along with dc-ERG to assess retinal pigment epithelial (RPE) function. The visual evoked potential (VEP) was used to assess transmission to the visual cortex. The amplitudes of the major ERG components were reduced in cbs(-/-) mice at age 3 weeks and VEPs were delayed markedly. These findings are consistent with the early retinal disruption observed anatomically in these mice. In comparison, at 3 weeks of age, responses of cbs(+/-) mice did not differ significantly from those of WT mice. Functional abnormalities were not observed in cbs(+/-) mice until 15 weeks of age, at which time amplitude reductions were noted for the ERG a- and b-wave and the light peak component, but not for other components generated by the RPE. VEP implicit times were delayed in cbs(+/-) mice at 15 and 30 weeks, while VEP amplitudes were unaffected. The later onset of functional defects in cbs(+/-) mice is consistent with a slow loss of ganglion cells reported previously in the heterozygous mutant. Light peak abnormalities indicate that RPE function is also compromised in older cbs(+/-) mice. The data suggest that severe elevations of homocysteine are associated with marked alterations of retinal function while modest homocysteine elevation is reflected in milder and delayed alterations of retinal function. The work lays the foundation to explore the role of homocysteine in retinal diseases such as glaucoma and optic neuropathy.

Homocysteine-Mediated Modulation of Mitochondrial Dynamics in Retinal Ganglion Cells

PURPOSE To evaluate the effect of excess homocysteine on the regulation of retinal ganglion cell mitochondrial dynamics. METHODS Mice deficient in cystathionine-β-synthase (cbs) were used as a model of hyperhomocysteinemia. Gene and protein expression analyses of Opa1 and Fis1 were performed on cbs⁺/⁻ neural retinas. Mitochondria within retinal ganglion cell axons underwent systematic ultrastructural analysis to measure area, length, width, and the distance between the mitochondria and the axon wall. Primary mouse ganglion cells were cultured, treated with homocysteine, and assessed for levels of Opa1 and Fis1 protein, the number of mitochondria per length of neurite, and levels of cleaved caspase-3. RESULTS Opa1 and Fis1 protein levels in cbs⁺/⁻ neural retinas were elevated to 191.00% ± 26.40% and 226.20% ± 4.57%, respectively, compared with wild-type. Mitochondria of cbs⁺/⁻ retinas were smaller in all parameters studied, including area (0.32 ± 0.01 μm² vs. 0.42 ± 0.02 μm²), compared with wild-type. Primary ganglion cells treated with homocysteine had elevations in Opa1 and Fis1 proteins, a significantly higher number of mitochondria per length of neurite (0.1781 ± 0.017 vs. 0.1156 ± 0.012), and significantly higher levels of cleaved caspase-3 compared with control. CONCLUSIONS This study provides the first evidence that homocysteine-induced ganglion cell loss involves the dysregulation of mitochondrial dynamics, both in vivo and in vitro. The present data suggest increased mitochondrial fission as a novel mechanism of homocysteine toxicity to neurons. Of particular relevance are glaucoma and Alzheimers disease, neurodegenerative diseases that are associated with hyperhomocysteinemia and, more recently, have implicated increased mitochondrial fission in their pathogeneses.

A lipidomic screen of hyperglycemia-treated HRECs links 12/15-Lipoxygenase to microvascular dysfunction during diabetic retinopathy via NADPH oxidase.

Retinal hyperpermeability and subsequent macular edema is a cardinal feature of early diabetic retinopathy (DR). Here, we investigated the role of bioactive lipid metabolites, in particular 12/15-lipoxygenase (LOX)-derived metabolites, in this process. LC/MS lipidomic screen of human retinal endothelial cells (HRECs) demonstrated that 15-HETE was the only significantly increased metabolite (2.4 ± 0.4-fold, P = 0.0004) by high glucose (30 mM) treatment. In the presence of arachidonic acid, additional eicosanoids generated by 12/15-LOX, including 12- and 11-HETEs, were significantly increased. Fluorescein angiography and retinal albumin leakage showed a significant decrease in retinal hyperpermeability in streptozotocin-induced diabetic mice lacking 12/15-LOX compared with diabetic WT mice. Our previous studies demonstrated the potential role of NADPH oxidase in mediating the permeability effect of 12- and 15-HETEs, therefore we tested the impact of intraocular injection of 12-HETE in mice lacking the catalytic subunit of NADPH oxidase (NOX2). The permeability effect of 12-HETE was significantly reduced in NOX2−/− mice compared with the WT mice. In vitro experiments also showed that 15-HETE induced HREC migration and tube formation in a NOX-dependent manner. Taken together our data suggest that 12/15-LOX is implicated in DR via a NOX-dependent mechanism.

Alterations of Retinal Vasculature in Cystathionine-Beta-Synthase Mutant Mice, a Model of Hyperhomocysteinemia

PURPOSE Mice with moderate/severe hyperhomocysteinemia due to deficiency or absence of the cbs gene encoding cystathionine-beta-synthase (CBS) have marked retinal disruption, ganglion cell loss, optic nerve mitochondrial dysfunction, and ERG defects; those with mild hyperhomocysteinemia have delayed retinal morphological/functional phenotype. Excess homocysteine is a risk factor for cardiovascular diseases; however, it is not known whether excess homocysteine alters retinal vasculature. METHODS Cbs(+/+), cbs(+/-), and cbs(-/-) mice (age ∼3 weeks) were subjected to angiography; retinas were harvested for cryosections, flat-mount preparations, or trypsin digestion and subjected to immunofluorescence microscopy to visualize vessels using isolectin-B4, to detect angiogenesis using anti-VEGF and anti-endoglin (anti-CD105) and activated glial cells (anti-glial fibrillary acidic protein [anti-GFAP]) and to investigate the blood-retinal barrier using the tight junction markers zonula occludens-1 (ZO-1) and occludin. Expression of vegf was determined by quantitative RT-PCR (qRT-PCR) and immunoblotting. Human retinal endothelial cells (HRECs) were treated with excess homocysteine to analyze permeability. RESULTS Angiography revealed vascular leakage in cbs(-/-) mice; immunohistochemical analysis demonstrated vascular patterns consistent with ischemia; isolectin-B4 labeling revealed a capillary-free zone centrally and new vessels with capillary tufts midperipherally. This was associated with increased vegf mRNA and protein, CD105, and GFAP in cbs(-/-) retinas concomitant with a marked decrease in ZO-1 and occludin. Homocysteine-treated HRECs showed increased permeability. CONCLUSIONS Severe elevation of homocysteine in cbs(-/-) mutant mice is accompanied by alterations in retinal vasculature (ischemia, neovascularization, and incompetent blood-retinal barrier). The marked disruption of retinal structure and decreased visual function reported in cbs(-/-) mice may reflect vasculopathy as well as neuropathy.

Pigment epithelium-derived factor inhibits retinal microvascular dysfunction induced by 12/15-lipoxygenase-derived eicosanoids

We recently demonstrated that 12/15-lipoxygenase (LOX) derived metabolites, hydroxyeicosatetraenoic acids (HETEs), contribute to diabetic retinopathy (DR) via NADPH oxidase (NOX) and disruption of the balance in retinal levels of the vascular endothelial growth factor (VEGF) and pigment epithelium-derived factor (PEDF). Here, we test whether PEDF ameliorates retinal vascular injury induced by HETEs and the underlying mechanisms. Furthermore, we pursue the causal relationship between LOX-NOX system and regulation of PEDF expression during DR. For these purposes, we used an experimental eye model in which normal mice were injected intravitreally with 12-HETE with/without PEDF. Thereafter, fluorescein angiography (FA) was used to evaluate the vascular leakage, followed by optical coherence tomography (OCT) to assess the presence of angiogenesis. FA and OCT reported an increased vascular leakage and pre-retinal neovascularization, respectively, in response to 12-HETE that were not observed in the PEDF-treated group. Moreover, PEDF significantly attenuated the increased levels of vascular cell and intercellular adhesion molecules, VCAM-1 and ICAM-1, elicited by 12-HETE injection. Accordingly, the direct relationship between HETEs and PEDF has been explored through in-vitro studies using Müller cells (rMCs) and human retinal endothelial cells (HRECs). The results showed that 12- and 15-HETEs triggered the secretion of TNF-α and IL-6, as well as activation of NFκB in rMCs and significantly increased permeability and reduced zonula occludens protein-1 (ZO-1) immunoreactivity in HRECs. All these effects were prevented in PEDF-treated cells. Furthermore, interest in PEDF regulation during DR has been expanded to include NOX system. Retinal PEDF was significantly restored in diabetic mice treated with NOX inhibitor, apocynin, or lacking NOX2 up to 80% of the control level. Collectively, our findings suggest that interfering with LOX-NOX signaling opens up a new direction for treating DR by restoring endogenous PEDF that carries out multilevel vascular protective functions.

Sigma receptor 1 activation attenuates release of inflammatory cytokines MIP1γ, MIP2, MIP3α, and IL12 (p40/p70) by retinal Müller glial cells.

The high‐affinity sigma receptor 1 (σR1) ligand (+)‐pentazocine ((+)‐PTZ) affords profound retinal neuroprotection in vitro and in vivo by a yet‐unknown mechanism. A common feature of retinal disease is Müller cell reactive gliosis, which includes cytokine release. Here, we investigated whether lipopolysaccharide (LPS) stimulates cytokine release by primary mouse Müller cells and whether (+)‐PTZ alters release. Using a highly sensitive inflammatory antibody array we observed significant release of macrophage inflammatory proteins (MIP1γ, MIP2, MIP3α) and interleukin‐12 (IL12 (p40/p70)) in LPS‐treated cells compared to controls, and a significant decrease in secretion upon (+)‐PTZ treatment. Müller cells from σR1 knockout mice demonstrated increased MIP1γ, MIP2, MIP3α and IL12 (p40/p70) secretion when exposed to LPS compared to LPS‐stimulated WT cells. We investigated whether cytokine secretion was accompanied by cytosolic‐to‐nuclear NFκB translocation and whether endothelial cell adhesion/migration was altered by released cytokines. Cells exposed to LPS demonstrated increased NFκB nuclear location, which was reduced significantly in (+)‐PTZ‐treated cells. Media conditioned by LPS‐stimulated‐Müller cells induced leukocyte‐endothelial cell adhesion and endothelial cell migration, which was attenuated by (+)‐PTZ treatment. The findings suggest that release of certain inflammatory cytokines by Müller cells can be attenuated by σR1 ligands providing insights into the retinal neuroprotective role of this receptor.