Upregulated hypoxia-inducible factor-1 DNA binding activity to the vascular endothelial growth factor-A promoter mediates increased vascular permeability in donor lung grafts.
BACKGROUND
Transplantation-induced hypoxia results in enhanced vascular permeability and tissue vascular endothelial growth factor (VEGF) and endothelin-1 (ET-1) overexpression in donor lung grafts. Promoter studies have uncovered a hypoxia-inducible factor (HIF)-1 binding site (HBS) in 5'-flanking region of VEGF gene that regulates the hypoxia-induced expression of VEGF; and ET-1 potently stimulates VEGF-A production. We hypothesized that HIF-1 regulates VEGF-mediated vascular permeability in lung grafts.


METHODS
We studied the mRNA and protein expression of HIF-1 and its protein-binding capacity to the HBS of the VEGF gene in biopsies of preserved donor and control lungs, using real-time reverse transcription-polymerase chain reaction, Western blotting, and electrophoretic mobility shift assay. Wet-to-dry lung weight ratio was measured in donor and control lungs.


RESULTS
While HIF-1 alpha mRNA expression was unchanged, HIF-1 beta was downregulated (p < 0.05) in donor versus control lungs. Protein expression of both, HIF-1 alpha and -beta was significantly upregulated in donor lung grafts. HIF-1 binding to the HBS of the VEGF promoter as well as tissue fluid content were increased in donor lung biopsies versus controls (p < 0.05).


CONCLUSIONS
These data indicate that upregulated HIF-1 DNA binding activity to the HBS of VEGF-A most likely contributes to elevated VEGF levels in preserved lung grafts. Unchanged HIF-1 alpha mRNA expression did not affect HIF-1 alpha protein levels. Endothelin-1 increases HIF-1 alpha accumulation and activates HIF-1 transcription complex in vitro. Therefore, ET-1-mediated increased HIF-1 alpha protein stability most likely leads to transcriptional activation of VEGF during lung graft preservation. Targeting HIF might be of benefit to counteract edema formation in preserved lung grafts.
Abraham
Dietmar
D
Laboratory for Cardiovascular Research, Department of Anatomy, University of Vienna, Vienna, Austria.
Krenn
Katharina
K
Seebacher
Gernot
G
Paulus
Patrick
P
Klepetko
Walter
W
Aharinejad
Seyedhossein
S
D051784:Aryl Hydrocarbon Receptor Nuclear Translocator; D001665:Binding Sites; D015153:Blotting, Western; D004268:DNA-Binding Proteins / Q000235:genetics / Q000502:physiology*; D024202:Electrophoretic Mobility Shift Assay; D019332:Endothelin-1 / Q000502:physiology; D006801:Humans; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D016040:Lung Transplantation / Q000502:physiology*; D009687:Nuclear Proteins / Q000235:genetics / Q000502:physiology*; D012333:RNA, Messenger / Q000032:analysis; D018336:Receptors, Aryl Hydrocarbon / Q000235:genetics / Q000502:physiology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014157:Transcription Factors / Q000235:genetics / Q000502:physiology; D015854:Up-Regulation; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics / Q000502:physiology*
C497396:ARNT protein, human; D004268:DNA-Binding Proteins; D019332:Endothelin-1; C497442:HIF1A protein, human; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D009687:Nuclear Proteins; D012333:RNA, Messenger; D018336:Receptors, Aryl Hydrocarbon; D014157:Transcription Factors; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D051784:Aryl Hydrocarbon Receptor Nuclear Translocator