Hypoxia inducible factor-1alpha is increased in ischemic retina: temporal and spatial correlation with VEGF expression.
OBJECTIVE
Hypoxia inducible factor-1 (HIF-1) is a transcription factor composed of HIF-1alpha and HIF-1beta subunits. HIF-1 transactivates multiple genes whose products play key roles in oxygen homeostasis, including vascular endothelial growth factor (VEGF). This study was designed to determine whether HIF-1 levels are increased in ischemic retina and whether there is a correlation with increased expression of VEGF.


METHODS
C57BL/6J mice were killed at time points that span retinal vascular development (PO to adult), or on postnatal day (P) 7 they were placed in a 75% oxygen environment for 5 days and then removed to room air and killed after 0, 2, or 6, or 24 hours and 5 or 14 days. Eyes were frozen, and retinas were isolated and used for immunoblot analysis, or eyes were sectioned for immunohisto chemical staining for HIF-1alpha or HIF-1beta, or for in situ hybridization for VEGF.


RESULTS
Immunoblots of retinal lysates showed low levels of HIF-1alpha at PO that were markedly increased at P4, remained high throughout the period of retinal vascular development and then decreased to an intermediate level in adults. HIF-1beta levels were relatively constant at all time points. In mice with oxygen-induced ischemic retinopathy, HIF-1alpha levels were increased in the retina. The peak of increase occurred at 2 hours, and levels returned to baseline by 24 hours. Immunohistochemistry showed increased staining for HIF-1alpha throughout the hypoxic inner retina, but not in the normoxic outer retina. There was no modulation of HIF-1beta levels. There was constitutive expression of VEGF mRNA in the inner nuclear layer that was increased 6 hours after the onset of hypoxia and remained elevated for several days.


CONCLUSIONS
There are increased levels of HIF-1alpha in ischemic retina that show temporal and spatial correlation with increased expression of VEGF. These findings are consistent with the hypothesis that HIF-1 plays a role in upregulation of VEGF in ischemic retina.
Ozaki
H
H
Department of Ophthalmology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21287-9277, USA.
Yu
A Y
AY
Della
N
N
Ozaki
K
K
Luna
J D
JD
Yamada
H
H
Hackett
S F
SF
Okamoto
N
N
Zack
D J
DJ
Semenza
G L
GL
Campochiaro
P A
PA
D000375:Aging / Q000378:metabolism; D000818:Animals; D017931:DNA Primers / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000378:metabolism*; D016228:Endothelial Growth Factors / Q000378:metabolism*; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D015151:Immunoblotting; D007124:Immunoenzyme Techniques; D017403:In Situ Hybridization; D007511:Ischemia / Q000378:metabolism*; D008222:Lymphokines / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D009687:Nuclear Proteins / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D012164:Retinal Diseases / Q000378:metabolism* / Q000473:pathology; D012171:Retinal Vessels / Q000254:growth & development / Q000378:metabolism* / Q000473:pathology; D014157:Transcription Factors / Q000378:metabolism*; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors
D017931:DNA Primers; D004268:DNA-Binding Proteins; D016228:Endothelial Growth Factors; C497443:Hif1a protein, mouse; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D008222:Lymphokines; D009687:Nuclear Proteins; D012333:RNA, Messenger; D014157:Transcription Factors; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors