Julian Nussbaum

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.

GPR109A as an anti-inflammatory receptor in retinal pigment epithelial cells and its relevance to diabetic retinopathy.

PURPOSE Retinal pigment epithelium (RPE) expresses GPR109A, a receptor for the vitamin niacin and the ketone body β-hydroxybutyrate (β-HB). Because diabetes results in elevated levels of β-HB, here we studied expression of the receptor in diabetic retina. We also investigated its functional relevance in RPE. METHODS Retinal expression of GPR109A in diabetic mice and postmortem human eyes was evaluated by quantitative PCR (qPCR). ARPE-19 cells and primary wild-type and Gpr109a(-/-) mouse RPE cells were exposed to TNF-α in the presence or absence of niacin or β-HB, followed by analysis of IL-6 and Ccl2 expression via real-time qPCR and ELISA. RESULTS GPR109A expression was increased in diabetic mouse and human retina. TNF-α increased the expression and secretion of IL-6 and Ccl2 in ARPE-19 cells. Niacin and β-HB suppressed these effects, implicating GPR109A as the target responsible for mediation of the observed effects. Primary RPE cells from wild-type mice behaved similarly. In contrast, GPR109A ligands failed to suppress TNF-α-induced expression and secretion of IL-6 and Ccl2 in primary RPE cells from Gpr109a(-/-) mice, confirming that the observed anti-inflammatory effects were mediated specifically by Gpr109a. CONCLUSIONS GPR109A plays an anti-inflammatory role in RPE and its expression is upregulated in diabetes. Inflammation is a key causative factor in the pathogenesis of diabetic retinopathy. We speculate that the increased expression of GPR109A and elevation of its ligand β-HB in diabetes are mechanisms by which the tissue attempts to fight inflammation in this disease. Pharmacological activation of GPR109A may therefore have therapeutic potential in clinical management of diabetic retinopathy.

Targeting neovascularization in ischemic retinopathy: Recent advances

Pathological retinal neovascularization (RNV) is a common microvascular complication in several retinal diseases including retinopathy of prematurity, diabetic retinopathy, age-related macular degeneration and central vein occlusion. The current therapeutic modalities of RNV are invasive and although they may slow or halt the progression of the disease, they are unlikely to restore normal acuity. Therefore, there is an urgent need to develop treatment modalities that are less invasive and thus associated with fewer procedural complications and systemic side effects. This review article summarizes our understanding of the pathophysiology and current treatment of RNV in ischemic retinopathies, lists potential therapeutic targets and provides a framework for the development of future treatment modalities.

Pronerve Growth Factor Induces Angiogenesis via Activation of TrkA: Possible Role in Proliferative Diabetic Retinopathy

Proliferative diabetic retinopathy (PDR) is the leading cause of blindness in working age Americans. We demonstrated that diabetes disturbs the homeostasis of nerve growth factor (NGF) resulting in accumulation of its precursor proNGF. Increases in proNGF were positively correlated with progression of diabetic retinopathy, having the highest level in ocular fluids from PDR patients compared to nondiabetic patients. Here, we attempted to evaluate the contribution and the possible mechanism of proNGF to PDR. The angiogenic response of aqueous humor samples from PDR patients was examined in human retinal endothelial cells in the presence or absence of anti-proNGF antibody. Additional cultures were treated with mutant-proNGF in the presence of specific pharmacological inhibitors of TrkA and p75NTR receptors. PDR-aqueous humor samples exerted significant angiogenic response including cell proliferation, migration, and alignment into tube-like structures. These effects were significantly reduced by anti-proNGF antibody but not by IgG. Treatment of retinal endothelial cells with mutant-proNGF activated phosphorylation of TrkA and p38MAPK; however, it did not alter p75NTR expression. Inhibition of TrkA but not p75NTR significantly reduced mutant-proNGF-induced cell proliferation, cell migration, and tube formation. Taken together, these results provide evidence that proNGF can contribute to PDR at least in part via activation of TrkA.

Protective effects of agonists of growth hormone-releasing hormone (GHRH) in early experimental diabetic retinopathy

Significance The studies described here are relevant to the cure of diabetic retinopathy, a leading cause of blindness with currently limited therapeutic options. Here we provided evidence showing that agonists of growth hormone-releasing hormone (GHRH) can significantly diminish retinal neurovascular injury characterizing the early stages of diabetic retinopathy through antioxidant and anti-inflammatory effects. The results of the presented studies provide information on the potential therapeutic effects of GHRH agonists and shed light on the role of hypothalamic hormones in retinal physiology and their effect on visual disorders. In addition, our findings suggest protective effects of GHRH analogs in other disease conditions affecting retinal neuronal cells and, possibly, other nonretinal neurons. The potential therapeutic effects of agonistic analogs of growth hormone-releasing hormone (GHRH) and their mechanism of action were investigated in diabetic retinopathy (DR). Streptozotocin-induced diabetic rats (STZ-rats) were treated with 15 μg/kg GHRH agonist, MR-409, or GHRH antagonist, MIA-602. At the end of treatment, morphological and biochemical analyses assessed the effects of these compounds on retinal neurovascular injury induced by hyperglycemia. The expression levels of GHRH and its receptor (GHRH-R) measured by qPCR and Western blotting were significantly down-regulated in retinas of STZ-rats and in human diabetic retinas (postmortem) compared with their respective controls. Treatment of STZ-rats with the GHRH agonist, MR-409, prevented retinal morphological alteration induced by hyperglycemia, particularly preserving survival of retinal ganglion cells. The reverse, using the GHRH antagonist, MIA-602, resulted in worsening of retinal morphology and a significant alteration of the outer retinal layer. Explaining these results, we have found that MR-409 exerted antioxidant and anti-inflammatory effects in retinas of the treated rats, as shown by up-regulation of NRF-2-dependent gene expression and down-regulation of proinflammatory cytokines and adhesion molecules. MR-409 also significantly down-regulated the expression of vascular endothelial growth factor while increasing that of pigment epithelium-derived factor in diabetic retinas. These effects correlated with decreased vascular permeability. In summary, our findings suggest a neurovascular protective effect of GHRH analogs during the early stage of diabetic retinopathy through their antioxidant and anti-inflammatory properties.

Increased expression and activity of 12-lipoxygenase in oxygen-induced ischemic retinopathy and proliferative diabetic retinopathy: implications in retinal neovascularization. Diabetes 2011;60:614–624

Al-Shabrawey M, Mussell R, Kahook K, Tawfik A, Eladl M, Sarthy V, Nussbaum J, El-Marakby A, Park SY, Gurel Z, Sheibani N, Maddipati KR. Increased expression and activity of 12-lipoxygenase in oxygen-induced ischemic retinopathy and proliferative diabetic retinopathy: implications in retinal neovascularization. Diabetes 2011;60:614–624 Several editorial …