Topical administration of adrenergic receptor pharmaceutics and nerve growth factor

Topical application of nerve growth factor (NGF) and adrenergic receptor pharmaceutics are currently in use for corneal ulcers and glaucoma. A recent interest in the neuroprotective abilities of NGF has led to a renewed interest in NGF as a therapeutic for retinal and choroidal diseases. NGF can promote cell proliferation through actions of the TrkA receptor or promote apoptosis through receptor p75NTR. This understanding has led to novel interest in the role of NGF for diseases of the posterior eye. The role of β-adrenergic receptor agonists and antagonists for treatments of glaucoma, diabetic retinopathy, and their potential mechanisms of action, are still under investigation. This review discusses the current knowledge and applications of topical NGF and adrenergic receptor drugs for ocular disease

Sympathetic innervation regulates basement membrane thickening and pericyte number in rat retina

PURPOSE. To determine whether loss of sympathetic innervation alters basement membrane thickness and pericyte loss. METHODS. Sympathetic innervation to the eye was destroyed by surgical removal of the right superior cervical ganglion in rats. Basement membrane changes were assessed by real-time PCR and electron microscopy. The number of pericytes was measured by immunofluorescent staining for NG2 proteoglycan. Steady-state mRNA levels were also evaluated for platelet-derived growth factor-BB (PDGF-BB). RESULTS. Loss of sympathetic innervation caused a significant increase in steady state mRNA levels of fibronectin and a 15% increase in laminin-␤1 mRNA 3 weeks after surgical sympathectomy. Protein expression also increased at this point. In addition, capillary basement membrane thickness increased significantly. NG2 proteoglycan staining decreased significantly in pericytes in the sympathectomized rat retina. Steady state mRNA for PDGF-BB decreased significantly 6 weeks after surgery. CONCLUSIONS. Sympathetic nerves may be compromised in diabetes, and these findings suggest that they may regulate some complications of diabetic retinopathy. Gene expression levels of fibronectin and laminin-␤1 changed between 1 and 3 weeks. These data are supported by electron microscopy, which showed the increase in basement membrane thickness in vivo. Loss of sympathetic innervation to the eye also caused a decrease in the number of pericytes. Steady state mRNA expression of PDGF-BB was reduced, suggesting a mechanism for the loss of pericytes in the sympathectomized retina. Overall, these results suggest that sympathetic nerve alterations may function in some complications observed in diabetic retinopathy, and this may be a suitable model to investigate therapies for this disorder. 1,2 The principal characteristics of diabetic retinopathy are thickening of the basement membrane in the retina, loss of pericytes, increased proliferation of endothelial cells, and formation of microaneurysms, which can lead to neovascularization. 3 However, it is still unclear how these alterations are caused by chronic diabetes. Alterations to sympathetic innervation in the eye could contribute to diabetes-induced change. Sympathetic nerves are significantly altered in diabetes. 6 These changes are mediated by ␤-adrenergic receptors, since administration of propranolol causes changes similar to those noted in sympathectomy. 7 Even though the retina is autoregulated, we and others To test this hypothesis, female Sprague-Dawley rats underwent surgical removal of the superior cervical ganglion, followed by assessment of gene and protein expression of two key basement membrane components (laminin-␤1 and fibronectin), electron microscopy to determine basement membrane thickness, and evaluation of the number of pericytes. Steady state mRNA expression was also assessed for the growth factor PDGF-BB. MATERIALS AND METHODS Surgical Sympathectomy Twenty-six female Sprague-Dawley rats were anesthetized intraperitoneally at postnatal day 60 with a mixture of ketamine (60 mg/kg), atropine (0.54 mg/kg), and xylazine (8 mg/kg). The right superior cervical ganglion was removed aseptically by previously described methods. 11 Right eye ptosis was used to confirm denervation, and only rats displaying good ptosis were used in the experiments. Retinal samples were taken 1, 3, and 6 weeks after sympathectomy. The contralateral or left eye served as an intra-animal control. All surgical procedures were approved by the Institutional Animal Care and Use Committee at Southern Illinois University-Carbondale and conform to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and NIH guidelines. RNA Isolation and Reverse Transcription RNA was isolated from retinal samples of six rats at each time point (TriReagent; Molecular Research Center, Inc., Cincinnati, OH), by using chloroform and isopropanol. RNA purity was detected by agarose gel electrophoresis, and RNA concentration was measured spectrophotometrically. Reverse transcription of 1 g RNA for cDNA synthesis was performed (Improm II Kit; Promega, Madison, WI). The reaction mixture consisted of diethyl pyrocarbonate (DEPC) water, 5ϫ reaction buffer (Improm II; Promega), 25 mM MgCl 2, 10 mM dNTP, and 20 U RNAsin. Strands were extended for 60 minutes at 42°C, and the reverse transcriptase enzyme was heat inactivated at 70°C for 15 minutes. RNase A inhibitor (0.2 L; 10 mg/mL) was added, followed by incubation for 30 minutes at 37°C. Samples were stored at Ϫ20°C for real-time PCR. From the Departments of 1 Physiology an

Epac1 Blocks NLRP3 Inflammasome to Reduce IL-1 β

Inflammation is an important component of diabetic retinal damage. We previously reported that a novel β-adrenergic receptor agonist, Compound 49b, reduced Toll-like receptor 4 (TLR4) signaling in retinal endothelial cells (REC) grown in high glucose. Others reported that TLR4 activates high-mobility group box 1 (HMGB1), which has been associated with the NOD-like receptor 3 (NLRP3) inflammasome. Thus, we hypothesized that Epac1, a downstream mediator of β-adrenergic receptors, would block TLR4/HMGB1-mediated stimulation of the NLRP3 inflammasome, leading to reduced cleavage of caspase-1 and interleukin-1 beta (IL-1β). We generated vascular specific conditional knockout mice for Epac1 and used REC grown in normal and high glucose treated with an Epac1 agonist and/or NLRP3 siRNA. Protein analyses were done for Epac1, TLR4, HMGB1, NLRP3, cleaved caspase-1, and IL-1β. Loss of Epac1 in the mouse retinal vasculature significantly increased all of the inflammatory proteins. Epac1 effectively reduced high glucose-induced increases in TLR4, HMGB1, cleaved caspase-1, and IL-1β in REC. Taken together, the data suggest that Epac1 reduces formation of the NLRP3 inflammasome to reduce inflammatory responses in the retinal vasculature

miR-146a Attenuates Inflammatory Pathways Mediated by TLR4/NF-κB and TNFα to Protect Primary Human Retinal Microvascular Endothelial Cells Grown in High Glucose

Pathological mechanisms underlying diabetic retinopathy are still not completely understood. Increased understanding of potential cellular pathways responsive to hyperglycemia is essential to develop novel therapeutic strategies for diabetic retinopathy. A growing body of evidence shows that microRNA (miRNA) play important roles in pathological mechanisms involved in diabetic retinopathy, as well as possessing potential as novel therapeutic targets. The hypothesis of this study was that miR-146a plays a key role in attenuating hyperglycemia-induced inflammatory pathways through reduced TLR4/NF-κB and TNFα signaling in primary human retinal microvascular endothelial cells (REC). We cultured human REC in normal (5 mM) glucose or transferred to high glucose medium (25 mM) for 3 days. Transfection was performed on REC with miRNA mimic (hsa-miR-146a-5p). Our results demonstrate that miR-146a expression was decreased in human REC cultured in high glucose. Overexpression of miR-146a using mimics reduced the levels of TLR4/NF-κB and TNFα in REC cultured in high glucose. Both MyD88-dependent and -independent signaling were decreased by miR-146a overexpression in REC in high glucose conditions. The results suggest that miR-146a is a potential therapeutic target for reducing inflammation in REC through inhibition of TLR4/NF-κB and TNFα. Our study will contribute to understanding of diabetic retinal pathology, as well as providing important clues to develop therapeutics for clinical applications

TNFα inhibits IGFBP-3 through activation of p38α and casein kinase 2 in human retinal endothelial cells.

We recently reported a reciprocal relationship between tumor necrosis factor alpha (TNFα) and insulin-like receptor growth factor binding protein 3 (IGFBP-3) in whole retina of normal and IGFBP-3 knockout mice. A similar relationship was also observed in cultured retinal endothelial cells (REC). We found that TNFα significantly reduced IGFBP-3 levels and vice-versa, IGFBP-3 can lower TNFα and TNFα receptor expression. Since IGFBP-3 is protective to the diabetic retina and TNFα is causative in the development of diabetic retinopathy, we wanted to better understand the cellular mechanisms by which TNFα can reduce IGFBP-3 levels. For these studies, primary human retinal endothelial cells (REC) were used since these cells undergo TNFα-mediated apoptosis under conditions of high glucose conditions and contribute to diabetic retinopathy. We first cultured REC in normal or high glucose, treated with exogenous TNFα, then measured changes in potential signaling pathways, with a focus on P38 mitogen-activated protein kinase alpha (P38α) and casein kinase 2 (CK2) as these pathways have been linked to both TNFα and IGFBP-3. We found that TNFα significantly increased phosphorylation of P38α and CK2. Furthermore, specific inhibitors of P38α or CK2 blocked TNFα inhibition of IGFBP-3 expression, demonstrating that TNFα reduces IGFBP-3 through activation of P38α and CK2. Since TNFα and IGFBP-3 are key mediators of retinal damage and protection respectively in diabetic retinopathy, increased understanding of the relationship between these two proteins will offer new therapeutic options for treatment