In vitro liver tissue model established from transgenic mice: role of HIF-1alpha on hypoxic gene expression.
The instability of the hepatocyte phenotype in vitro has limited the ability to quantitatively investigate regulation of stress responses of the liver. Here, we adopt a tissue-engineering approach to form stable liver tissue in vitro by forming collagen "sandwich" cultures of transgenic murine hepatocytes harboring a regulatory gene of interest flanked by loxP sites. The floxed gene is excised in a subset of cultures by transfection with adenovirus carrying the gene for Cre-recombinase, thereby generating wild-type and null liver tissues from a single animal. In this study, we specifically investigated the role of hypoxia inducible factor 1 alpha (HIF-1alpha) in the hepatocellular response to hypoxia. Using high-density oligonucleotide arrays, we examined genome-wide gene expression after 8 h of hypoxia in wild-type and HIF- 1alpha null hepatocyte cultures. We identified more than 130 genes differentially expressed under hypoxia involved in metabolic adaptation, angiogenic signaling, immediate early response, and cell cycle regulation. Real-time polymerase chain reaction analysis verified that known hypoxia-responsive genes such as glucose transporter-1 and vascular endothelial growth factor were induced in a HIF-1alpha-dependent manner under hypoxia. Our results demonstrate the potential to integrate in vitro tissue models with transgenic and microarray technologies for the study of physiologic stress responses.
Allen
Jared W
JW
Department of Bioengineering, University of California at San Diego, La Jolla, California, USA.
Khetani
Salman R
SR
Johnson
Randall S
RS
Bhatia
Sangeeta N
SN
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D018929:Cell Culture Techniques; D015687:Cell Hypoxia / Q000502:physiology*; D002478:Cells, Cultured; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D016678:Genome; D051272:Glucose Transporter Type 1 / Q000235:genetics; D022781:Hepatocytes / Q000166:cytology*; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics / Q000502:physiology*; D008099:Liver / Q000166:cytology*; D051379:Mice; D008822:Mice, Transgenic; D008954:Models, Biological; D020411:Oligonucleotide Array Sequence Analysis; D016133:Polymerase Chain Reaction; D023822:Tissue Engineering / Q000379:methods; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics
D051272:Glucose Transporter Type 1; C497443:Hif1a protein, mouse; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D042461:Vascular Endothelial Growth Factor A