Dataset Information

Acadesine suppresses TNF-? induced complement component 3 (C3), in retinal pigment epithelial (RPE) cells.

ABSTRACT:

Rationale

Age-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-?) 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-? induced C3 in RPE cells.

Methods

ARPE-19 and human primary RPE cells were cultured and allowed to grow to confluence. TNF-? 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.

Results

Acadesine suppresses TNF-? 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-? 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 ? catalytic subunit did not affect the inhibitory effect of acadesine on TNF-? upregulation of C3.

Conclusions

Our results suggest that acadesine suppresses TNF-? induced C3, likely through an AMPK-independent pathway, and could have potential use in complement over activation diseases.

SUBMITTER: Efstathiou NE

PROVIDER: S-EPMC7757886 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Publications

Acadesine suppresses TNF-α induced complement component 3 (C3), in retinal pigment epithelial (RPE) cells.

Efstathiou Nikolaos E NE Moustafa Giannis A GA Maidana Daniel E DE Konstantinou Eleni K EK Notomi Shoji S Barbisan Paulo R T PRT Georgakopoulos Constantine D CD Miller Joan W JW Vavvas Demetrios G DG

PloS one 20201223 12

<h4>Rationale</h4>Age-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-α) and complement component 3 (C3) with AMD. Acadesine, an analog of AMP and an AMP-activated protein kinase (AMPK) activator, has been shown ...[more]

PMID: 33362238

Similar Datasets

Project description:BackgroundAge-related changes in the retina are often accompanied by visual impairment but their mechanistic details remain poorly understood.MethodologyProteomic studies were pursued toward a better molecular understanding of retinal pigment epithelium (RPE) aging mechanisms. RPE cells were isolated from young adults (3-4 month-old) and old (24-25 month-old) F344BN rats, and separated into subcellular fractions containing apical microvilli (MV) and RPE cell bodies (CB) lacking their apical microvilli. Proteins were extracted in detergent, separated by SDS-PAGE, digested in situ with trypsin and analyzed by LC MS/MS. Select proteins detected in young and old rat RPE were further studied using immunofluorescence and Western blot analysis.Principal findingsA total of 356 proteins were identified in RPE MV from young and 378 in RPE MV from old rats, 48% of which were common to each age group. A total of 897 proteins were identified in RPE CB from young rats and 675 in old CB, 56% of which were common to each age group. Several of the identified proteins, including proteins involved in response to oxidative stress, displayed both quantitative and qualitative changes in overall abundance during RPE aging. Numerous proteins were identified for the first time in the RPE. One such protein, collectrin, was localized to the apical membrane of apical brush border of proximal tubules where it likely regulates several amino acid transporters. Elsewhere, collectrin is involved in pancreatic β cell proliferation and insulin secretion. In the RPE, collectrin expression was significantly modulated during RPE aging. Another age-regulated, newly described protein was DJ-1, a protein extensively studied in brain where oxidative stress-related functions have been described.Conclusions/significanceThe data presented here reveals specific changes in the RPE during aging, providing the first protein database of RPE aging, which will facilitate future studies of age-related retinal diseases.