AMPK Activation by 5-Amino-4-Imidazole Carboxamide Riboside-1-β-D-Ribofuranoside Attenuates Alkali Injury-Induced Corneal Fibrosis.

Abstract

PurposeIncreased TGF-β1 synthesis after corneal alkali injury is implicated in corneal fibrosis, as it promotes transdifferentiation of keratocytes into myofibroblasts. The activation of 5′-adenosine monophosphate-activated protein kinase (AMPK) by 5-amino-4-imidazole carboxamide riboside-1-β-d-ribofuranoside (AICAR) inhibits TGF-β1-induced fibrosis in other cell types. We investigated the antifibrotic effect of AICAR in corneal fibroblasts after alkali injury.MethodsMouse models of corneal alkali burn, produced by placing 2-mm-diameter filter paper soaked in 0.1-N NaOH on the right cornea for 30 seconds, were treated with the test drugs 4× daily for 21 days. The central cornea was scanned by optical coherence tomography (OCT). Corneal tissues were obtained and processed for western blotting and immunohistochemistry. For in vitro analysis, primary human corneal fibroblasts were treated directly with TGF-β1 to induce fibrosis, with or without AICAR pretreatment. Myofibroblast activation and extracellular matrix (ECM) protein synthesis were detected by western blotting, real-time PCR, and collagen gel contraction assay. Signaling proteins were analyzed by western blotting.ResultsAlkali injury induced the upregulation of TGF-β1 expression, which led to increased α-smooth muscle actin (α-SMA) and fibronectin synthesis and myofibroblast differentiation. AMPK activation by AICAR significantly suppressed TGF-β1 and ECM protein expression. The antifibrotic effect of AICAR was AMPK dependent, as treatment with the AMPK inhibitor Compound C attenuated the antifibrotic response.ConclusionsAMPK activation by AICAR suppresses the myofibroblast differentiation and ECM synthesis that occur after alkali injury in corneal fibroblasts.