Abstract 7: S-nitrosylation Affects Differentiation of Mesenchymal Stem Cells

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Introduction: Bone marrow-derived mesenchymal stem cells (BMMSCs) appear to play an important role in the formation of atherosclerotic lesions. Circulating BMMSCs are attracted to arterial wall where they undergo adipogenic or osteogenic differentiation to form ectopic aggregates of fat and bone. The factors that determine the direction of BMMSC differentiation are poorly understood. We assessed the role of nitric oxide (NO), an inhibitor of lipid formation and vascular calcification, in BMMSC differentiation. Hypothesis: S-nitrosylation mediated-NO signaling controls the balance between adipogenic and osteogenic differentiation of BMMSCs. Methods: We isolated BMMSCs from wild type mice (WT) and mice deficient in S-nitrosoglutathione reductase (GSNOR -/- ), an enzyme that governs levels of S-nitrosylation by promoting protein denitrosylation. Cells were cultured in either adipogenic or osteogenic differentiation media followed by functional and gene expression assays. Results: MSCs derived from GSNOR -/- mice had impaired fat droplet formation and lower expression of the adipogenic markers PPARγ (1329±415.3-fold increase in WT vs. 158±65.61-fold in GSNOR -/- , P<0.05) and FABP4 (11.06±3.29-fold in WT vs. 4.06±0.62-fold in GSNOR -/- , P<0.05). Conversely, GSNOR -/- MSCs exhibited enhanced osteogenic differentiation as indicated by greater calcium deposition and higher expression of the osteogenic marker Osteopontin (1.48±0.17-fold in WT vs. 16.18±5.26-fold in GSNOR -/- , P<0.05). Interestingly, GSNOR -/- cells had higher baseline expression of Osteopontin, Osteocalcin and Runx2. Treatment of GSNOR -/- MSCs with L-NAME, a nitric oxide synthase (NOS) inhibitor, resulted in decreased osteogenic differentiation without affecting adipogenic differentiation. Conclusion: Our results indicate that GSNOR enhances adipogenic differentiation and impairs osteogenic differentiation of BMMSCs in vitro and suggest that interaction of GSNOR- and NOS-mediated processes regulates the balance between adipogenic and osteogenic differentiation of BMMSCs.