Blockade of Endothelinergic Receptors Reduces Stress Pathway Signaling in Endotoxin-Induced Uveitis

Abstract

Purpose:: We have previously described that retinal endothelin receptors (ET-A and ET-B) are increased in endotoxin-induced uveitis. These increases, as well as uveitic manifestations, can be reduced by treatment with Tezosentan, a mixed antagonist of endothelin receptors. Our present work aims to understand the relationship of endothelinergic receptors with the activation of different stress pathways involved in neuroinflammation.

Methods:: BALB-c mice received two doses of Tezosentan (sc, 10mg/kg), 1 hour before and 24 hours after LPS injection (ip, 5mg/kg). Controls received saline at the same times. Animals were euthanized 1 hour after the last Tezosentan injection. Antibodies against ET-A, ET-B, Phospho-JNK, c-jun, NF-KB, and Protein Kinase C (PKC) were used for immunohistochemistry or western blotting of retinal samples.

Results:: Untreated animals showed Phospho-SAPK/JNK immunoreactivity in the inner plexiform layer (IPL), cells of inner nuclear layer (INL) and ganglion cell layer (GCL). LPS markedly increased expression levels in the inner retina and induced labeling of horizontal cells. These levels were reduced by Tezosentan. c-Jun signal was only detected after LPS, in GCL cell nuclei and endothelia. Tezosentan strikingly reduced c-Jun immunoreactivity. In control retinas, NF-KB appeared in the cytoplasm of cells in the GCL. Immunoreactivity increased and translocated to cell nuclei after LPS. NF-KB labeling was also reduced by Tezosentan. PKC, mainly present in rod bipolars, also increased after LPS. Unexpectedly, Tezosentan induced a much stronger increase of PKC immunoreactivity. These changes were corroborated in Western Blot assay.

Conclusions:: Phosphorylation of c-Jun, and the activation of its upstream kinases is required for many inflammatory responses. Thus, the increase of phospho-SAPK/JNK immunoreactivity in the inner retina together with the appearance of c-Jun immunostaining is probably associated with the increase of vascular permeability. Both LPS responses were similarly reduced by Tezosentan. This antagonist also reduced NF-KB nuclear translocation, indicating that endothelinergic mechanisms probably regulate most arms of the inflammatory response. Since ET-1 is a known activator of the PKC alpha isoform, its increase after Tezosentan appears as a paradoxical effect that could perhaps be associated with neuroprotection, as has been described in ischemia models.

Keywords: uveitis-clinical/animal model • retina • stress response