Document Detail

Activation of the transcription factor HIF-1 and its target genes, VEGF, HO-1, iNOS, during fracture repair.
MedLine Citation:
PMID:  15050899     Owner:  NLM     Status:  MEDLINE    
One of the immediate sequelae of bone fracture is regional hypoxia resulting from vasculature disruption. Hypoxia stabilizes and activates the transcription factor hypoxia inducible factor-1alpha (HIF-1alpha), which ultimately leads to HIF-1-regulated gene expression. Because nothing is known about HIF-1 involvement in bone regeneration, we performed a series of experiments to elucidate the expression pattern of HIF-1alpha and selected HIF-1 target genes using a rat femoral fracture model. Callus samples were obtained on postfracture days (PFD) 3, 5, 7, 10, 14, and 21. Quantitative RT-PCR (qRT-PCR) was employed to quantify the temporal mRNA expression patterns of HIF-1alpha, vascular endothelial growth factor (VEGF), inducible nitric oxide synthase (iNOS), and heme oxygenase-1 (HO-1). Elevated HIF-1alpha and VEGF expression was seen at all time points, with peak increases of approximately 6- and 2-fold relative to the intact bone present on PFD 10 for HIF-1alpha and VEGF, respectively. Robust activation of iNOS was detected solely on PFD 10 (6.8-fold) with all other time points showing slight downregulation. HO-1 expression peaked on PFD 3 (4.5-fold) with no significant changes on any other PFD. Western blot analysis verified the temporal expression patterns with HIF-1alpha protein expression showing a steady rise to a PFD 10 peak of approximately 18-fold. Similarly, the expression patterns for VEGF and HO-1 showed increases of approximately 4-fold at their PFD 10 and PFD 3 peaks, respectively. Immunohistochemical analysis of PFD 10 callus sections revealed coexpression of HIF-1alpha and VEGF in proliferating chondrocytes and active osteoblasts. Immunostaining for HO-1 on PFD 3 callus sections demonstrated strong expression in hematoma macrophages and vascular endothelial cells. Taken together, these experiments demonstrate for the first time that HIF-1alpha is upregulated at both transcriptional and translational levels in the fracture callus and indicate that PFD 10 may be a key angiogenic time point in the developing rat fracture callus.
D E Komatsu; M Hadjiargyrou
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Publication Detail:
Type:  Journal Article; Research Support, U.S. Gov't, P.H.S.    
Journal Detail:
Title:  Bone     Volume:  34     ISSN:  8756-3282     ISO Abbreviation:  Bone     Publication Date:  2004 Apr 
Date Detail:
Created Date:  2004-03-30     Completed Date:  2004-12-22     Revised Date:  2008-11-21    
Medline Journal Info:
Nlm Unique ID:  8504048     Medline TA:  Bone     Country:  United States    
Other Details:
Languages:  eng     Pagination:  680-8     Citation Subset:  IM    
Department of Biomedical Engineering, Stony Brook University, NY 11794-2580, USA.
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MeSH Terms
Bony Callus / metabolism,  pathology
DNA-Binding Proteins / genetics,  metabolism*
Fracture Healing / genetics*
Gene Expression Profiling
Heme Oxygenase (Decyclizing) / genetics*,  metabolism
Heme Oxygenase-1
Hypoxia-Inducible Factor 1
Hypoxia-Inducible Factor 1, alpha Subunit
Nitric Oxide Synthase / genetics*
Nitric Oxide Synthase Type II
Nuclear Proteins / genetics,  metabolism*
RNA, Messenger / genetics,  metabolism
Rats, Sprague-Dawley
Transcription Factors / genetics,  metabolism*
Transcriptional Activation / genetics*
Vascular Endothelial Growth Factor A / genetics*,  metabolism
Grant Support
R03 AR047603-01/AR/NIAMS NIH HHS; R03 AR047603-02/AR/NIAMS NIH HHS; R03 AR047603-03/AR/NIAMS NIH HHS; R03AR476032/AR/NIAMS NIH HHS
Reg. No./Substance:
0/DNA-Binding Proteins; 0/Hif1a protein, rat; 0/Hypoxia-Inducible Factor 1; 0/Hypoxia-Inducible Factor 1, alpha Subunit; 0/Nuclear Proteins; 0/RNA, Messenger; 0/Transcription Factors; 0/Vascular Endothelial Growth Factor A; EC Oxide Synthase; EC Oxide Synthase Type II; EC protein, rat; EC Oxygenase (Decyclizing); EC Oxygenase-1

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