Geldanamycin treatment reduces the inflammatory changes in the LPS–induced uveitis rat model

Abstract

Abstract: : Purpose: Heat–shock protein (hsp)90 is the central component of a molecular chaperone complex that interacts with various intracellular proteins in order to preserve their 3–dimensional conformation to a functionally competent state. Hsp90 inhibitors, such as geldanamycin (GA), constitute promising investigational agents, as they have been recently shown to exhibit antitumor and anti–inflammatory activities in a variety of models.The object of the present study was to investigate the anti–inflammatory effects of GA in endotoxin–induced uveitis (EIU) in rats. Materials and Methods: Female Lewis rats received a single intraperitoneal injection of 1mg/kg GA or vehicle. EIU was induced 24 hours later by a footpad injection of 200mg/kg lipopolysaccharide (LPS). Twenty–four hours after the administration of LPS, leukocyte adhesion was evaluated in vitro with quantitative endothelial cell–leukocyte adhesion assays and ex–vivo with concanavalin A lectin staining of retinal flatmounts. Leukocyte activation was quantified with a myeloperoxidase (MPO) activity assay and blood–retinal barrier breakdown was assessed by Evans Blue extravasation. Retinal levels of VEGF, TNF–a and leukocyte total levels of the LPS receptor CD14 were quantified with an ELISA method, whereas membranous CD14 levels were assessed with membrane precipitation and subsequent immunoblotting. Retinal activity of NF–kB and HIF–1a was quantified with a modified ELISA method.Results: Geldanamycin treatment significantly suppressed the LPS–induced increase in leukocyte adhesion both in vitro and ex–vivo, as well as MPO activity, vascular leakage and the LPS–induced increase of NF–kB and HIF–1a activity and VEGF and TNF–a levels in the retina. Although GA treatment did not reduce the LPS–induced increase in total CD14 levels in the leukocytes, it significantly decreased membrane CD14 levels. Conclusions: Geldanamycin treatment suppresses the inflammatory changes in the LPS–induced uveitis model by decreasing leukocyte adhesion and activation, blood–retinal barrier breakdown and the increase in crucial proinflammatory cytokines such as VEGF and TNF–a, most likely through the observed effect on NF–kB and HIF–1a activation. GA and its analogs has demonstrated a favorable profile in phase I clinical trials in cancer patients and represent promising therapeutic agents for the treatment of eye diseases characterized by inflammation.

Keywords: inflammation • retinitis • uveitis–clinical/animal model