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 α-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 β-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.
Similar content being viewed by others
Article PDF
References
Collaborative Group on Hormone Factors in Breast Cancer. Breast cancer and hormone replacement therapy: collaborative reanalysis of data from 51 epidemiological studies of 52 705 women with breast cancer and 108 411 women without breast cancer. Lancet 1997; 350: 1047–59.
Qu Q, Perälä-Heape M, Kapanen A, Dahllund J, Salo J, Väänänen HK, et al. Estrogen enhances differentiation of osteoblasts in mouse bone marrow culture. Bone 1998; 400: 201–9.
Aguirre J, Buttery L, O'Shaughnessy M, Afzal F, Fernandez de Marticorena I, Hukkanen M, et al. Endothelial nitric oxide synthase gene-deficient mice demonstrate marked retardation in postnatal bone formation, reduced bone volume, and deficient in osteoblasts maturation and activity. Am J Pathol 2001; 158: 247–57.
Hukkanen M, Platts LA, Lawes T, Girgis SI, Konttinen YT, Goodship AE, et al. Effect of nitric oxide donor nitroglycerin on bone mineral density in a rat model of estrogen deficiency-induced osteopenia. Bone 2003; 32: 1–8.
Wimalawansa SJ . Nitroglycerin therapy is as efficacious as standard estrogen replacement therapy (Premarin) in prevention of oophorectomy-induced bone loss: a human pilot clinical study. J Bone Miner Res 2000; 15: 2240–4.
Jiang X, Gittens SA, Chang Q, Zhang X, Chen C, Zhang Z . The use of tissue-engineered bone with human bone morphogenetic protein-4-modified bone-marrow stromal cells in repairing mandibular defects in rabbits. Int J Oral Maxillofac Surg 2006; 35: 1133–9.
Mankani MH, Kuznetsov SA, Wolfe RM, Marshall GW, Robey PG . In vivo bone formation by human bone marrow stromal cells: reconstruction of the mouse calvarium and mandible. Stem Cells 2006; 24: 2140–9.
Dai ZJ, Li YL, Quarles LD, Xiao ZS . Resveratrol enhances proliferation and osteoblastic differentiation in human mesenchymal stem cells via ER-dependent ERK1/2 activation. Phytomedicine 2007; 14: 806–14.
Xiao ZS, Simpson LG, Quarles LD . IRES-dependent translational control of Cbfa1/Runx2 expression. J Cell Biochem 2003; 88: 493–505.
Wang FH, Jin WF, Gao JJ . The evaluation of pharmacodynamic action to drugs for osteoporosis. Chin J Osteoporosis 1999; 5: 58–62.
Pan W, Quarles LD, Xiao ZS . Genistein stimulates the osteoblastic differentiation via NO/cGMP in bone marrow culture. J Cell Biochem 2005; 94: 307–16.
Xiao ZS, Quarles LD, Chen QQ, Yu YH, Qu XP, Jiang CH, et al. Effect of asymmetric dimethylarginine on osteoblastic differentiation. Kidney Int 2001; 60: 1699–704.
Song LH, Pan W, Yu YH, Quarles LD, Zhou HH, Xiao ZS . Resveratrol prevents CsA inhibition of proliferation and osteoblastic differentiation of mouse bone marrow-derived mesenchymal stem cells through an ER/NO/cGMP pathway. Toxicol In Vitro 2006; 20: 915–22.
Wimalawansa SJ, Chapa MT, Yallampalli C, Zhang R, Simmons DJ . Prevention of corticosteroid-induced bone loss with nitric oxide donor nitroglycerin in male rats. Bone 1997; 21: 275–80.
Wimalawansa SJ, De Marco G, Gangula P, Yallampalli C . Nitric oxide donor alleviates ovariectomy-induced bone loss. Bone 1996; 18: 301–4.
Steensberg A, Keller C, Hillig T, Frøsig C, Wojtaszewski JF, Pedersen BK, et al. Nitric oxide production is a proximal signaling event controlling exercise-induced mRNA expression in human skeletal muscle. FASEB 2007; 21: 2683–94.
Sanada M, Taguchi A, Higashi Y, Tsuda M, Kodama I, Yoshizumi M, et al. Forearm endothelial function and bone mineral loss in postmenopausal women. Atherosclerosis 2004; 176: 387–92.
He MH, Wang ZL, Wu J, Chen T, Pang W, Yang XM . Changes of serum nitric oxide and nitric oxide synthase in ovariectomy rats model of osteoporosis after nitroglycerin therapy. Chin J Endemiol 2004; 23: 438–40.
Jamal SA, Browner WS, Bauer DC, Cummings SR . Intermittent use of nitrates increases bone mineral density: the study of osteoporotic fractures. J Bone Min Res 1998; 13: 1755–9.
Lander HM, Ogiste JS, Pearce SF, Levi R . Nitric oxide-stimulated guanine nucleotide exchange on p21ras. J Biol Chem 1995; 270: 7017–20.
Feelisch M, Stamler JS . Methods in nitric oxide research. Chichester: Wiley & Sons 1996.
Kanamaru Y, Takada T, Saura R, Mizuno K . Effect of nitric oxide on mouse clonal osteogenic cell, MC3T3-E1, proliferation in vitro. Kobe J Med Sci 2001; 47: 1–11.
Inoue A, Hiruma Y, Hirose S, Yamaguchi A, Hagiwara H . Reciprocal regulation by cyclic nucleotides of the differentiation of rat osteoblast-like cells and mineralization of nodules. Biochem Biophys Res Commun 1995; 215: 1104–10.
Mancini L, Moradi-Bidhendi N, Becherini L, Martineti V, MacIntyre I . The biphasic effects of nitric oxide in primary rat osteoblasts are cGMP dependent. Biochem Biophys Res Commun 2000; 274: 477–81.
Pilz RB, Casteel DE . Regulation of gene expression by cyclic GMP. Circ Res 2003; 93: 1034–46.
Zaragoza C, López-Rivera E, García-Rama C, Saura M, Martínez-Ruíz A, Lizarbe TR, et al. Cbfa-1 mediates nitric oxide regulation of MMP-13 in osteoblasts. J Cell Sci 2006; 119: 1896–902.
Ducy P, Zhang R, Geoffroy V, Ridall AL, Karsenty G . Osf2/Cbfα1: a transcriptional activator of osteoblast differentiation. Cell 1997; 89: 747–54.
Afzal F, Polak J, Buttery L . Endothelial nitric oxide synthase in the control of osteoblastic mineralizing activity and bone integrity. J Pathol 2004; 202: 503–10.
Xiao ZS, Zhou HH . Progress in Cbfα1 gene and its regulation. Chin Pharmacol Bull 2001; 17: 365–8.
Author information
Authors and Affiliations
Corresponding author
Additional information
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).
Rights and permissions
About this article
Cite this article
Huang, L., Qiu, N., Zhang, C. et al. Nitroglycerin enhances proliferation and osteoblastic differentiation in human mesenchymal stem cells via nitric oxide pathway. Acta Pharmacol Sin 29, 580–586 (2008). https://doi.org/10.1111/j.1745-7254.2008.00778.x
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1111/j.1745-7254.2008.00778.x
Keywords
This article is cited by
-
Nitric oxide regulates multiple functions and fate of adult progenitor and stem cells
Journal of Physiology and Biochemistry (2015)