J Bone Metab.  2014 Aug;21(3):169-188. 10.11005/jbm.2014.21.3.169.

Microarray Analysis of Gene Expression Reveals that Cyclo-oxygenase-2 Gene Therapy Up-regulates Hematopoiesis and Down-regulates Inflammation During Endochondral Bone Fracture Healing

Affiliations
  • 1Research Service (151), Jerry L. Pettis Memorial Veterans Administration Medical Center, Loma Linda, CA, USA. Charles.Rundle@va.gov
  • 2Department of Medicine, Loma Linda University School of Medicine, Loma Linda, CA, USA.

Abstract

BACKGROUND
Cyclo-oxygenase-2 (Cox-2) is an inflammatory mediator that is necessary for the tissue repair, including bone fracture healing. Although the application of Cox-2 gene therapy to a murine closed femoral fracture has accelerated bony union, but the beneficial effect was not observed until the endochondral stage of bone repair that is well after the inflammatory stage normally subsides.
METHODS
To identify the molecular pathways through which Cox-2 regulates fracture healing, we examined gene expression profile in fracture tissues in response to Cox-2 gene therapy during the endochondral bone repair phase. Cox-2 gene therapy was applied to the closed murine femur fracture model. Microarray analysis was performed at 10 days post-fracture to examine global gene expression profile in the fracture tissues during the endochondral bone repair phase. The entire repertoire of significantly expressed genes was examined by gene set enrichment analysis, and the most up-regulated individual genes were evaluated further.
RESULTS
The genes that normally promote inflammation were under-represented in the microarray analysis, and the expression of several inflammatory chemokines was significantly down-regulated. There was an up-regulation of two key transcription factor genes that regulate hematopoiesis and erythropoiesis. More surprisingly, there was no significant up-regulation in the genes that are normally involved in angiogenesis or bone formation. However, the expression of two tissue remodeling genes was up-regulated.
CONCLUSIONS
The down-regulation of the inflammatory genes in response to Cox-2 gene therapy was unexpected, given the pro-inflammatory role of prostaglandins. Cox-2 gene therapy could promote bony union through hematopoietic precursor proliferation during endochondral bone repair and thereby enhances subsequently fracture callus remodeling that leads to bony union of the fracture gap.

Keyword

Cyclooxygenase 2; Fracture healing; Gene expression; Genetic therapy; Microarray analysis

MeSH Terms

Bony Callus
Chemokines
Cyclooxygenase 2
Down-Regulation
Erythropoiesis
Femoral Fractures
Femur
Fracture Healing
Fractures, Bone*
Gene Expression*
Genetic Therapy*
Hematopoiesis*
Inflammation*
Microarray Analysis*
Osteogenesis
Prostaglandins
Transcription Factors
Transcriptome
Up-Regulation
Chemokines
Cyclooxygenase 2
Prostaglandins
Transcription Factors

Figure

  • Fig. 1 Real-time reverse transcription-polymerase chain reaction determination of prostaglandin E receptor (PTGER) gene expression in response to cyclo-oxygenase-2 (Cox-2) transgene at 10 days post-fracture. The three samples in each group size were from the same individuals that underwent microarray analysis. Statistics were performed by t-Test. PTGER, prostaglandin E2 receptors.

  • Fig. 2 Correlation of microarray and quantitative real-time reverse transcription-polymerase chain reaction measurements of gene expression for selected genes. The list of genes is shown in Table 2. The correlation coefficient was determined to be r=0.8. The genes below the 1-fold expression boundary displayed negatively regulated expression by each approach. RT-PCR, reverse transcription-polymerase chain reaction.

  • Fig. 3 A model proposed for the functions of the genes expressed during fracture callus development in response to cyclo-oxygenase-2 (Cox-2) gene therapy (box). The stages of fracture callus healing normally present at 10 days post-fracture are indicated. IM, intramembranous; Cox-2, cyclo-oxygenase-2; PGE2, prostaglandin E2.


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