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3238206 
Journal Article 
Activation of the transcription factor HIF-1 and its target genes, VEGF, HO-1, iNOS, during fracture repair 
Komatsu, DE; Hadjiargyrou, M 
2004 
Yes 
Bone
ISSN: 8756-3282
EISSN: 1873-2763 
34 
4 
680-688 
WOS:000220922900012 
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 NOS was detected solely on PFD 10 (6.8-fold) with all other time points showing
slight downregulation. HO-I 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. (C) 2004 Elsevier Inc. All rights reserved. 
angiogenesis; bone; callus; fracture; HIF-1 alpha; hypoxia