Abstract Body: Background: Arterial calcification due to deficiency of CD73 (ACDC) is a rare genetic disease that leads to medial arterial calcification (MAC) in lower-extremity arteries. ACDC is caused by inactivating mutations in the gene encoding the CD73 enzyme, which converts extracellular adenosine monophosphate to adenosine. We observed reduced CD73 levels and elevated forkhead box O1 protein (FOXO1) and tissue non-specific alkaline phosphatase (TNAP) expression in both ACDC and non-ACDC patients with MAC, suggesting that the mechanisms underlying this rare genetic disorder may also contribute to common forms of MAC. Protein arginine methyltransferases (PRMTs) add methyl groups to arginine residues, and are regulated by metabolites produced in the methionine cycle, which adenosine feeds into. S-adenosylhomocysteine hydrolase (SAHH) is a bidirectional enzyme that converts adenosine and homocysteine to s-adenosylhomocysteine (SAH), an endogenous inhibitor of PRMTs. This study tested the hypothesis that reduced adenosine increases PRMT activity, which mediates FOXO1 nuclear localization and MAC in human coronary smooth muscle cells (SMCs).

Methods: PRMT group I and II activity was measured in control and ACDC fibroblasts at baseline and after 6 hours of adenosine treatment by ELISA. To investigate the functional role of PRMTs in vascular calcification, human coronary SMCs were treated daily with PRMT inhibitors (MS023 for group I PRMTs; GSK591 for group II PRMTs), and calcification was assessed by Alizarin Red staining on day 14. qRT-PCR was used to measure changes in transcript levels of SAHH, SMC dedifferentiation (KLF4), matrix remodeling (COL1A1, COL3A1, ELN), and mineralization (TNAP) markers on day 7. FOXO1 nuclear localization was quantified by immunofluorescence staining.

Results: SAHH expression was lower in ACDC fibroblasts, and exogenous adenosine significantly decreased group I PRMT activity compared to controls. In human coronary SMCs, pharmacological inhibition of group I PRMTs prevented vascular calcification and reduced KLF4 and TNAP expression. No change was observed in the expression of matrix remodeling markers. FOXO1 nuclear localization increased on day 3 of osteogenic treatment, suggesting early activation of FOXO1 signaling.

Conclusions: Our data suggest that group I PRMTs play a critical role in driving MAC and that adenosine, a product of CD73, suppresses their activity. It remains to be determined which group I PRMT regulates FOXO1.