Endothelial nitric oxide synthase is dynamically expressed during bone marrow stem cell differentiation into endothelial cells.
This study was designed to investigate the developmental expression of endothelial nitric oxide synthase (eNOS) during stem cell differentiation into endothelial cells and to examine the functional status of the newly differentiated endothelial cells. Mouse adult multipotent progenitor cells (MAPCs) were used as the source of stem cells and were induced to differentiate into endothelial cells with vascular endothelial growth factor (VEGF) in serum-free medium. Expression of eNOS in the cells during differentiation was evaluated with real-time PCR, nitric oxide synthase (NOS) activity, and Western blot analysis. It was found that eNOS, but no other NOS, was present in undifferentiated MAPCs. eNOS expression disappeared in the cells immediately after induction of differentiation. However, eNOS expression reoccurred at day 7 during differentiation. Increasing eNOS mRNA, protein content, and activity were observed in the cells at days 14 and 21 during differentiation. The differentiated endothelial cells formed dense capillary networks on growth factor-reduced Matrigel. VEGF-stimulated phosphorylation of extracellular signal-regulated kinase (ERK)-1 and ERK-2 occurred in these cells, which was inhibited by NOS inhibitor N(G)-nitro-L-arginine methyl ester. In conclusion, these data demonstrate that eNOS is present in MAPCs and is dynamically expressed during the differentiation of MAPCs into endothelial cells in vitro.
Liu
Zhenguo
Z
Davis Heart & Lung Research Institute, The Ohio State University Medical Center, Columbus, OH 43210, USA. zhenguo.liu@osumc.edu
Jiang
Yuehua
Y
Hao
Hong
H
Gupta
Kalpna
K
Xu
Jian
J
Chu
Ling
L
McFalls
Edward
E
Zweier
Jay
J
Verfaillie
Catherine
C
Bache
Robert J
RJ
D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology / Q000378:metabolism*; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology*; D002478:Cells, Cultured; D015536:Down-Regulation / Q000187:drug effects; D042783:Endothelial Cells / Q000502:physiology; D004730:Endothelium, Vascular / Q000166:cytology* / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D051379:Mice; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D039902:Multipotent Stem Cells / Q000166:cytology / Q000378:metabolism*; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D052250:Nitric Oxide Synthase Type III / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D042461:Vascular Endothelial Growth Factor A / Q000494:pharmacology
D004791:Enzyme Inhibitors; D042461:Vascular Endothelial Growth Factor A; D052250:Nitric Oxide Synthase Type III; D048049:Extracellular Signal-Regulated MAP Kinases; D019950:Mitogen-Activated Protein Kinase 1; D019331:NG-Nitroarginine Methyl Ester