ABSTRACT
Elucidation of the basic mechanisms underlying human disease pathogenesis depends on the findings afforded to us through in vivo and in vitro approaches. While there are inherent limitations in any model system, 2D in vitro culture systems tend to be particularly restricted due to their static nature. Here, we adapted a flow-based hollow-fiber cartridge system to better understand the cellular influences of human retinal microvascular endothelial cells and mouse-derived neutrophils under high glucose conditions similar to those observed in diabetes. Analyses by western blot and flow cytometry indicate that pro-inflammatory molecules known to be associated with the pathogenesis of diabetic retinopathy were significantly elevated following high glucose exposure, including VEGF, ICAM-1, and ROS. Changes in mitochondrial potential were also observed. Further, we demonstrate that this innovative system allows for cross-species co-culture as well as long-term culturing conditions. This in vitro modeling system not only mimics the retinal microvasculature, it also allows for the examination of cellular interactions and mechanisms that contribute to diabetic retinopathy, a visually debilitating complication of diabetes.
Subject(s)
Coculture Techniques/methods , Diabetic Retinopathy/pathology , Hyperglycemia/pathology , Neutrophils/pathology , Retinal Vessels/cytology , Animals , Coculture Techniques/instrumentation , Endothelial Cells , Equipment Design , Female , Humans , Hyperglycemia/complications , Intercellular Adhesion Molecule-1/metabolism , Mice, Inbred C57BL , Neutrophils/cytology , Oxidative Stress , Reactive Oxygen Species/metabolism , Retinal Vessels/pathology , Vascular Endothelial Growth Factor A/metabolismABSTRACT
Vasoactive intestinal peptide (VIP) has been shown to regulate corneal inflammation. Formyl peptide receptor 2 (FPR2) is a transmembrane protein belonging to the GPCR family. Ligands include pro-resolving lipids, lipoxin A4 (LXA4) and resolvin D1 (RvD1). The current study focuses on the effect of VIP regarding the FPR2 receptor axis in improving disease outcome in a mouse model of bacterial keratitis. Infection was induced in C57BL/6 (B6) mice using P. aeruginosa (PA) ATCC 19660. Mice received topical treatment (VIP or PBS) 3× daily after infection. Mean clinical scores, bacterial plate counts, Griess and myeloperoxidase (MPO) assays indicate that topical VIP effectively abrogates the disease response. Findings also reveal that VIP influences FPR2 pathway activation independent of archetypal VIP receptors. Exploring the immunoresolving role of FPR2, its ligand RvD1 and related enzymes (5-LOX, 12/15-LOX), our results suggest a mechanism by which VIP treatment influences the disease response in bacterial keratitis, which could offer a therapeutic point of intervention for enhancing this pro-resolving circuit.
Subject(s)
Homeodomain Proteins/metabolism , Keratitis/metabolism , Keratitis/microbiology , Pseudomonas aeruginosa/physiology , Receptors, Formyl Peptide/metabolism , Vasoactive Intestinal Peptide/pharmacology , Animals , Disease Models, Animal , Female , Inflammation Mediators/metabolism , Mice , Mice, Inbred C57BLABSTRACT
With increasing multidrug resistance and contraindication for corticosteroid use, the goal of this study was to develop thymosin beta-4 (Tß4) as an adjunctive therapy to antibiotics for the treatment of bacterial keratitis that effectively promotes enhanced wound healing, host defense, and inflammation resolution. Disease outcome was assessed by clinical score, slit lamp photography, and histopathology. Cytokine profile, bacterial load, PMN infiltration, and Griess and reactive oxygen species (ROS) levels were determined. Adjunct Tß4 treatment resulted in a significant improvement compared to PBS, Tß4, and most remarkably, ciprofloxacin, correlating with changes in mediators of inflammation and wound healing. Collectively, these data provide evidence that wound healing is an essential aspect in the development of new therapies to treat corneal infection. Use of adjunctive Tß4 provides a more efficacious approach for bacterial keratitis by addressing both the infectious pathogen and deleterious host response.
