Adenosine and ATP differentially control osteogenic gene expression patterns in a cardiac valve cell line.

INTRODUCTION

Calcific aortic valve disease (CAVD) involves the osteogenic transition of valve interstitial cells (VICs) and is influenced by purinergic signaling. ATP is considered pro-calcific, whereas adenosine (Ado) has been reported as anti- or pro-calcific. Extracellular conversion of ATP to Ado by ecto-enzymes (including ecto-5'-nucleotidase, CD73) may shape receptor signaling in VICs.

MATERIAL AND

METHODS

We quantified osteogenic and apoptotic markers in the murine valve-like cell line tsA58-AVM under osteogenic conditions after treatment with ATP or Ado, selective Adora1/Adora2A agonists, and/or CD73.

RESULTS

Runt-related transcription factor 2, isoform II (Runx2-II) expression decreased by 31% and 17% after 16 hours of exposure to 0.35 μM and 3.5 μM Ado, respectively. Adora1 activation (MeCCPA, 0.1 μM) reduced Runx2-II by 49-57% on days 1-5, and Adora2A activation (PSB0777, 1 μM) reduced Runx2-II by 17-63% at days 1-5. Repeated ATP addition (100 μM) increased osteopontin (SPP1) by 7.7- and 3.3-fold and osteocalcin (BGLAP) by 14.4- and 4.0-fold on days 5 and 12, respectively; cotreatment with CD73 (0.1 ng/μL) reduced or normalized these changes. ATP also doubled the Bcl-2- associated X protein (Bax) to B-cell lymphoma 2 protein (BCL2) ratio, an effect that was prevented by CD73.

CONCLUSIONS

Purinergic ligands and receptors directly regulate VIC osteogenic differentiation and apoptosis in vitro without exogenous inflammatory stimulation. Modulation of the ATP-Ado axis may represent a therapeutic target to slow CAVD progression.