Anticancer Activity and Mechanisms of Action of MAPK pathway inhibitors

Given these functions, microglial activation represents an important player in the pathogenesis of DR

Given these functions, microglial activation represents an important player in the pathogenesis of DR. activation. To test this, diabetic rats were treated intravitreally with A717, a specific AGA-neutralizing antibody, or murine IgG. Relative to nondiabetic rats, diabetic rats (IgG-treated) manifested 3.9- and 7.9-fold increases in Iba-1 and tumor necrosis factor (TNF)- mRNAs, respectively. Treatment of diabetic rats with A717 significantly attenuated overexpression of these mRNAs. Intravitreal injection of AGA per se in normal rats resulted in increases of Iba-1 expression and TNF- release. FLNA Guided by these results, a cultured retinal microglia model was developed to study microglial response after AGA treatment and the mechanistic basis behind this response. The results showed that formation of reactive oxygen species and subsequent activation of ERK and P38, but not Jun NH2-terminal kinase, are molecular events underpinning retinal microglial TNF- release during AGA treatment. CONCLUSIONS These results provide Pitolisant hydrochloride new insights in understanding the pathogenesis of early DR, showing that the accumulated AGA within the diabetic retina elicits the microglial activation and secretion of TNF-. Thus, intervention trials with agents that neutralize AGA effects may emerge as a new therapeutic approach to modulate early pathologic pathways long before the occurrence of vision loss among patients with diabetes. During the past decade, it has become clear that inflammation is a key feature in diabetes that leads to long-term complications in specific organs, in particular the eye and kidney (1). In the eye, the major complication is diabetic retinopathy (DR), which is the leading cause of blindness in the western world and affects approximately three fourths of diabetic patients within 15 years after onset of the disease (2). The recommended treatment for these patients has been laser photocoagulation, which is an invasive procedure with considerable limitations and adverse effects. Therefore, there is a great need for the development of new noninvasive therapies to treat those affected by DR. These therapies can be discovered by unraveling the pathophysiology of DR. As a consequence of diabetes, retinal microglia, a subtype of glial-immune sentinel cells prestationed in the tissue, become reactive, leading to the release of soluble cytotoxins that contribute to neuronal and vascular cell death and ultimately the progression of DR (3). However, the underlying mechanism of microglial activation during diabetes is still incompletely understood. In recent years, human and animal studies have elucidated that many effects of hyperglycemia are mediated by glycated proteins (4). Amadori-glycated albumin (AGA) is the prominent form of circulating glycated proteins in vivo, and its concentration is significantly increased after diabetes, reaching its maximum in 5C7 weeks (5). AGA arises from the nonenzymatic condensation reaction between a reducing sugar and susceptible amino groups. This modification confers properties to AGA that are not possessed by the native, nonglycated albumin, such as the promotion of the inflammatory response and Pitolisant hydrochloride the activation of different mitogen-activated protein kinase (MAPK) cascades in several cell types (6C9). These MAPKs, including extracellular signalCrelated kinase (ERK), Jun NH2-terminal kinases (JNKs), and P38, can be independently or simultaneously activated depending on the target cells (8C10). On the basis of these properties of AGA, a growing body of evidence now supports the causal role of AGA in the development of many complications associated with diabetes (11C13). In relation to DR, elevated AGA has been documented in the retinal capillaries of diabetic patients with retinopathy (14) and in the retina of diabetic rats (15). Treatment of diabetic mice with A717 antibody, which specifically recognizes AGA, ameliorated retinal basement membrane thickening (16). Furthermore, treatment of diabetic rats with 2-(3-chlorophenylamino)-phenylacetic acid, which inhibits the nonenzymatic glycation of albumin, mitigated vitreous changes in angiogenic cytokines associated with the development of DR (17). Therefore, AGA is believed to possess biologic characteristics that are linked to Pitolisant hydrochloride the DR pathogenesis and might be involved in the activation of retinal microglia. In the present work, we aimed to study the ability of AGA to induce retinal microglial activation and their secretion of inflammatory cytokines both in vivo and in vitro. RESEARCH DESIGN AND METHODS All procedures with animals were performed in accordance with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research and Medical College of Georgia guidelines. Diabetes was induced in male SD rats by intravenous injection of streptozocin (STZ) (60 mg/kg) and confirmed by urine-glucose levels 350 mg/dL. Eyes were used for immunofluorescence or Western examination at 8 weeks of diabetes. For intravitreal injections, the procedure was essentially the same as previously described (18). The A717 intravitreal injection scheme was chosen on the basis of.