Enhanced bone regeneration associated with decreased apoptosis in mice with partial HIF-1alpha deficiency.
HIF-1alpha activates genes under hypoxia and was hypothesized to regulate bone regeneration. Surprisingly, HIF-1alpha+/- fracture calluses are larger, stronger, and stiffer than HIF-1alpha+/+ calluses because of decreased apoptosis. These data identify apoptosis inhibition as a means to enhance bone regeneration.


BACKGROUND
Bone regeneration subsequent to fracture involves the synergistic activation of multiple signaling pathways. Localized hypoxia after fracture activates hypoxia-inducible factor 1alpha (HIF-1alpha), leading to increased expression of HIF-1 target genes. We therefore hypothesized that HIF-1alpha is a key regulator of bone regeneration.


METHODS
Fixed femoral fractures were generated in mice with partial HIF-1alpha deficiency (HIF-1alpha+/-) and wildtype littermates (HIF-1alpha+/+). Fracture calluses and intact contralateral femurs from postfracture days (PFDs) 21 and 28 (N=5-10) were subjected to microCT evaluation and four-point bending to assess morphometric and mechanical properties. Molecular analyses were carried out on PFD 7, 10, and 14 samples (N=3) to determine differential gene expression at both mRNA and protein levels. Finally, TUNEL staining was performed on PFD 14 samples (N=2) to elucidate differential apoptosis.


RESULTS
Surprisingly, fracture calluses from HIF-1alpha+/- mice exhibited greater mineralization and were larger, stronger, and stiffer. Microarray analyses focused on hypoxia-induced genes revealed differential expression (between genotypes) of several genes associated with the apoptotic pathway. Real-time PCR confirmed these results, showing higher expression of proapoptotic protein phosphatase 2a (PP2A) and lower expression of anti-apoptotic B-cell leukemia/lymphoma 2 (BCL2) in HIF-1alpha+/+ calluses. Subsequent TUNEL staining showed that HIF-1alpha+/+ calluses contained larger numbers of TUNEL+ chondrocytes and osteoblasts than HIF-1alpha+/- calluses.


CONCLUSIONS
We conclude that partial HIF-1alpha deficiency results in decreased chondrocytic and osteoblastic apoptosis, thereby allowing the development of larger, stiffer calluses and enhancing bone regeneration. Furthermore, apoptosis inhibition may be a promising target for developing new treatments to accelerate bone regeneration.
Komatsu
David E
DE
Department of Biomedical Engineering, Stony Brook University, Stony Brook, NY, USA.
Bosch-Marce
Marta
M
Semenza
Gregg L
GL
Hadjiargyrou
Michael
M
D000818:Animals; D030801:Animals, Genetically Modified; D017209:Apoptosis / Q000235:genetics; D001861:Bone Regeneration / Q000235:genetics; D002146:Bony Callus / Q000378:metabolism; D019902:Chondrocytes; D005260:Female; D005264:Femoral Fractures / Q000235:genetics / Q000378:metabolism; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation / Q000235:genetics; D000860:Hypoxia / Q000235:genetics / Q000378:metabolism; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000172:deficiency* / Q000378:metabolism; D051379:Mice; D020411:Oligonucleotide Array Sequence Analysis; D010006:Osteoblasts / Q000378:metabolism*
C497443:Hif1a protein, mouse; D051795:Hypoxia-Inducible Factor 1, alpha Subunit