Nitroglycerin enhances proliferation and osteoblastic differentiation in human mesenchymal stem cells via nitric oxide pathway.
OBJECTIVE
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 alpha-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 beta-glycerophosphate for inducing osteoblastic differentiation. The cells were treated with NTG (0.1-10 micromol/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 micromol/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 micromol/L) could not be abolished by either N(G ) -nitro-L-arginine methyl ester, an antagonist of endothelial NOS, or 1400W, a selective blocker of inducible NOS activity.


CONCLUSIONS
NTG stimulates cell proliferation and osteoblastic differentiation of HBMSC through a direct release of NO, which is independent on intracellular NOS activity.
Huang
Li
L
Institute of Clinical Pharmacology, Central South University, Changsha 410078, China.
Qiu
Ni
N
Zhang
Che
C
Wei
Hong-Yan
HY
Li
Ya-Lin
YL
Zhou
Hong-Hao
HH
Xiao
Zhou-Sheng
ZS
D001854:Bone Marrow Cells / Q000166:cytology; D002454:Cell Differentiation / Q000187:drug effects* / Q000235:genetics / Q000502:physiology; D049109:Cell Proliferation / Q000187:drug effects*; D002478:Cells, Cultured; D017077:Culture Media, Conditioned; D004305:Dose-Response Relationship, Drug; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D009569:Nitric Oxide / Q000235:genetics / Q000378:metabolism*; D005996:Nitroglycerin / Q000378:metabolism / Q000494:pharmacology*; D010006:Osteoblasts / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism
D017077:Culture Media, Conditioned; D009569:Nitric Oxide; D005996:Nitroglycerin