Invasion of Human Retinal Pigment Epithelial Cells by Porphyromonas gingivalis leading to Vacuolar/Cytosolic localization and Autophagy dysfunction In-Vitro.

Recent epidemiological  studies link Periodontal disease(PD) to age-related macular degeneration (AMD). We documented earlier that Porphyromonas gingivalis(Pg), keystone oral-pathobiont, causative of PD, efficiently invades human gingival epithelial and blood-dendritic cells. Here, we investigated the ability of dysbiotic Pg-strains to invade human-retinal pigment epithelial cells(ARPE-19), their survival, intracellular localization, and the pathological effects, as dysfunction of RPEs leads to AMD. We show that live, but not heat-killed Pg-strains adhere to and invade ARPEs. This involves early adhesion to ARPE cell membrane, internalization and localization of Pg within single-membrane vacuoles or cytosol, with some nuclear localization apparent. No degradation of Pg or localization inside double-membrane autophagosomes was evident, with dividing Pg suggesting a metabolically active state during invasion. We found significant downregulation of autophagy-related genes particularly, autophagosome complex. Antibiotic protection-based recovery assay further confirmed distinct processes of adhesion, invasion and amplification of Pg within ARPE cells. This is the first study to demonstrate invasion of human-RPEs, begin to characterize intracellular localization and survival of Pg within these cells. Collectively, invasion of RPE by Pg and its prolonged survival by autophagy evasion within these cells suggest a strong rationale for studying the link between oral infection and AMD pathogenesis in individuals with periodontitis.

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

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.

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 p75(NTR) 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 p75(NTR) expression. Inhibition of TrkA but not p75(NTR) 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.

Targeting Neovascularization in Ischemic Retinopathy: Recent Advances.

Pathological retinal neovascularization (RNV) is a common micro-vascular 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, which are less invasive and therefore 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.

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 ß-hydroxybutyrate (ß-HB). Because diabetes results in elevated levels of ß-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 ß-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 ß-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 ß-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.

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.

Virtual reality in ophthalmology training.

Current training models are limited by an unstructured curriculum, financial costs, human costs, and time constraints. With the newly mandated resident surgical competency, training programs are struggling to find viable methods of assessing and documenting the surgical skills of trainees. Virtual-reality technologies have been used for decades in flight simulation to train and assess competency, and there has been a recent push in surgical specialties to incorporate virtual-reality simulation into residency programs. These efforts have culminated in an FDA-approved carotid stenting simulator. What role virtual reality will play in the evolution of ophthalmology surgical curriculum is uncertain. The current apprentice system has served the art of surgery for over 100 years, and we foresee virtual reality working synergistically with our current curriculum modalities to streamline and enhance the resident's learning experience.

Comparison of a digital retinal imaging system and seven-field stereo color fundus photography to detect diabetic retinopathy in the primary care environment.

BACKGROUND AND OBJECTIVE: Because patients with diabetes mellitus may visit their primary care physician regularly but not their ophthalmologist, a retinal risk assessment in the primary care setting could improve the screening rate for diabetic retinopathy. An imaging system for use in the primary care setting to identify diabetic retinopathy requiring referral to an ophthalmologist was evaluated. PATIENTS AND METHODS: In a masked prospective study, images were obtained from 11 patients with diabetes mellitus using both the digital retinal imaging system and seven-field stereo color fundus photography. The ability to obtain gradable images and to identify diabetic retinal lesions was compared. RESULTS: Of all images, 85% of digital retinal imaging system images and 88% of seven-field images were gradable. Agreement based on "no retinopathy" versus "any retinopathy" was excellent (Kappa = 0.96). Agreement based on "microaneurysms or less retinopathy" versus "retinal hemorrhages or worse retinopathy" was very good (Kappa = 0.83). CONCLUSIONS: The agreement between the digital retinal imaging system and seven-field photography indicates that the digital retinal imaging system may be useful to screen for diabetic retinopathy.