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Acta Pharmacologica Sinica 2008 May; 29 (5): 580-586

Nitroglycerin enhances proliferation and osteoblastic differentiation in human mesenchymal stem cells via nitric oxide pathway¹

Li HUANG², Ni QIU², Che ZHANG^2,4, Hong-yan WEI², Ya-lin LI², Hong-hao ZHOU², Zhou-sheng XIAO^2,3,5

²Institute of Clinical Pharmacology, Central South University, Changsha 410078, China; ³The Kidney Institute, University of Kansas Medical Center, Kansas City, Kansas 66160, USA; ⁴Department of Pharmacy, Taihe Hospital Affiliated to Yunyang Medical College, Shiyan 442000, China

1 This study was supported by grants from the Teaching and Research Award Program for Outstanding Young Teachers (TRAPOYT) in Higher Education Institutions of MOE, China (No 30040002); the National Natural Science Foundation of China (No 30171085); and the National Institutes of Health, USA (No RO1-AR049712). 5Correspondence to Prof Zhou-sheng XIAO. Ph n 1-913-588-0703. Fax 1-913-588-9251. E-mail zxiao@kumc.edu Received 2007-06-28 Accepted 2008-01-16 doi: 10.1111/j.1745-7254.2008.00778.x

Abstract Abstract Aim: To investigate the effect of nitroglycerin (NTG) on cell proliferation and osteoblastic differentiation of human bone marrow-derived mesenchymal stem cells (HBMSC) and its mechanisms. Methods: Primary HBMSC were cultured in osteogenic differentiation medium consisting of phenol red-free a-minimum essential media plus 10% fetal bovine serum (dextran-coated charcoal stripped) supplemented with 10 nmol/L dexamethasone, 50 mg/L ascorbic acid, and 10 mmol/L b-glycerophosphate for inducing osteoblastic differentiation. The cells were treated with NTG (0.1_10 µmol/L) alone or concurrent incubation with different nitric oxide synthase (NOS) inhibitors. Nitric oxide (NO) production was measured by using a commercial NO kit. Cell proliferation was measured by 5-bromodeoxyuridine (BrdU) incorporation. The osteoblastic differentiation of HBMSC culture was evaluated by measuring cellular alkaline phosphatase (ALP) activity and calcium deposition, as well as osteoblastic markers by real-time RT-PCR. Results: The treatment of HBMSC with NTG (0.1_10 µmol/L) led to a dose-dependent increase of NO production in the conditional medium. The release of NO by NTG resulted in increased cell proliferation and osteoblastic differentiation of HBMSC, as evidenced by the increment of the BrdU incorporation, the induction of ALP activity in the early stage, and the calcium deposition in the latter stage. The increment of NO production was also correlated with the upregulation of osteoblastic markers in HBMSC cultures. However, the stimulatory effect of NTG (10 µmol/L) could not be abolished by either NG-nitro-L-arginine methyl ester, an antagonist of endothelial NOS, or 1400W, a selective blocker of inducible NOS activity. Conclusion: NTG stimulates cell proliferation and osteoblastic differentiation of HBMSC through a direct release of NO, which is independent on intracellular NOS activity. Key words nitroglycerin; human bone marrow-derived mesenchymal stem cells; nitric oxide

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