Abstract 239: Nitric Oxide Suppresses the Angiogeneic Potential of Mesenchymal Stem Cells

Abstract

Introduction: Mesenchymal stem cells (MSCs) participate in blood vessel formation which is crucial for tissue regeneration. However, the mechanism involved in regulating MSC angiogenesis remains elusive. We examined the role of Nitric Oxide (NO) in this process by comparing angiogenesis by MSCs derived from wild type mice and mice lacking S-nitrosoglutathione reductase (GSNOR-/-), an enzyme that increases intracellular GSNO and enhances endogenous S-nitrosylation (a ubiquitous redox-related modification of cysteine thiol by NO) Hypothesis: Nitric oxide signaling pathways are mediated via small molecular thiols that regulate MSC angiogenesis. Method: Bone marrow-derived MSCs were isolated from wild type and GSNOR-/- mice and grown for 7 days in endothelial growth media followed by 24h in Matrigel (in vitro angiogenesis assay) in the presence of either vehicle, 15µM L-NAME (a nitric oxide synthase (NOS) inhibitor) or 500µM SNAP (a nitric oxide donor). Human (h)MSCs were similarly treated with vehicle, 15µM L-NAME and 100µM SNAP. MSC NO production and NOS expression were assessed. Results: Both types of MSCs exhibited similar levels of NO production and NOS expression, however, in the Matrigel Assay, MSCs from GSNOR-/- mice formed fewer (29.9±12.0 vs.. 50.0±15.4, p<0.001) and shorter (62.8 ± 34.6 µm vs. 105.9±57.0 µm, p<0.001) tubes than MSCs derived from wild type mice. L-NAME treatment normalized GSNOR-/- tube number (49.9 ± 14.8, p<0.001) and length (82.8±42.5 µm, p<0.001). Treatment of wild type MSCs with SNAP impaired tube formation (15.6±9.0, p<0.001) and length (57.1±24.9 µm, p<0.001). Similarly, hMSCs treated with SNAP exhibited impaired tube formation, both in number (25.0±4.9 vs. 49.2±8.1, p<0.001) and length (81±29.1 vs. 128.0±52.5 µm, p<0.001) compared to vehicle treated MSCs. L-NAME had no effect on hMSCs . Conclusion: These findings are consistent with a paradoxical negative influence of NO/GSNO on vascular formation by MSCs and reveal a novel mechanism whereby NO/GSNO inhibits MSC dependent angiogenesis. These findings have implications for disease states characterized by excessive or dysregulated angiogenesis; moreover NO deficient environments may trigger MSC mediated angiogenesis.