Acadesine Suppresses Tnf-Alpha Induced Complement Component 3 (C3), In Retinal Pigment Epithelial (Rpe) Cells

RationaleAge-related macular degeneration (AMD) is the most prevalent form of irreversible blindness in the developed world. Aging, inflammation and complement dysregulation affecting the retinal pigment epithelium (RPE), are considered significant contributors in its pathogenesis and several evidences have linked tumor necrosis factor alpha (TNF-alpha) and complement component 3 (C3) with AMD. Acadesine, an analog of AMP and an AMP-activated protein kinase (AMPK) activator, has been shown to have cytoprotective effects in human clinical trials as well as having anti-inflammatory and anti-vascular exudative effects in animals. The purpose of this study was to evaluate if acadesine is able to suppress TNF-alpha induced C3 in RPE cells.MethodsARPE-19 and human primary RPE cells were cultured and allowed to grow to confluence. TNF-alpha was used for C3 induction in the presence or absence of acadesine. Small molecule inhibitors and siRNA were used to determine if acadesine exerts its effect via the extracellular or intracellular pathway and to evaluate the importance of AMPK for these effects. The expression level of C3 was determined by immunoblot analysis.ResultsAcadesine suppresses TNF-alpha induced C3 in a dose dependent manner. When we utilized the adenosine receptor inhibitor dipyridamole (DPY) along with acadesine, acadesine's effects were abolished, indicating the necessity of acadesine to enter the cell in order to exert it's action. However, pretreatment with 5-iodotubericidin (5-Iodo), an adenosine kinase (AK) inhibitor, didn't prevent acadesine from decreasing TNF-alpha induced C3 expression suggesting that acadesine does not exert its effect through AMP conversion and subsequent activation of AMPK. Consistent with this, knockdown of AMPK alpha catalytic subunit did not affect the inhibitory effect of acadesine on TNF-alpha upregulation of C3.ConclusionsOur results suggest that acadesine suppresses TNF-alpha induced C3, likely through an AMPK-independent pathway, and could have potential use in complement over activation diseases.