Hypoxia regulates vascular endothelial growth factor receptor KDR/Flk gene expression through adenosine A2 receptors in retinal capillary endothelial cells.
OBJECTIVE
Vascular endothelial growth factor (VEGF) is an endothelial cell-specific angiogenic factor that serves an important role in numerous ischemic retinopathies. The authors studied the hypoxic gene regulation of two known VEGF receptors (KDR and Flt) and its mechanism in cultured bovine retinal endothelial cells (BREC).


METHODS
Confluent monolayers of BREC were exposed to various oxygen concentrations using a computer-controlled, infrared, water-jacked CO2 incubator with reduced oxygen control. Northern blot analysis and 125I-VEGF binding analysis were used to identify hypoxia-induced alterations of VEGF receptor at mRNA and protein levels.


RESULTS
KDR was detectable by Northern blot analysis in BREC, whereas Flt was not. Hypoxia decreased KDR gene expression in a dose-and time-dependent manner with maximal inhibition to 0.5 +/- 0.2% (P = 0.019) of normoxic control observed after 24 hours exposure to 0% oxygen and with significant inhibition at oxygen concentrations below 5%. Blockade of oxygen respiration decreased KDR mRNA expression to 58% +/- 7.1% of control (P = 0.001) after 3 hours. CPA, a stable adenosine A1 receptor (A1R) agonist, did not affect KDR mRNA expression at A1R stimulatory concentrations, but it decreased KDR mRNA levels to 30% +/- 4.9% (P = 0.002) of control at higher concentrations that react with A2R. DPMA, an adenosine A2 receptor (A2R) agonist, decreased KDR mRNA in a dose-dependent manner with an EC50 of 5 to 10 nM. A1R antagonists, 8-cyclolentyl-1,3-dipropylxanthine and 8-phenyltheophylline, did not inhibit the hypoxic response of KDR mRNA at A1R inhibitory concentrations but did inhibit the response at A2R effective doses (P = 0.001). The A2R antagonist, CSC, inhibited the KDR hypoxic response by 42% +/- 7.8% (P = 0.008) at 10 microM. Specific VEGF binding to BREC was decreased from 15.1% +/- 0.3% to 12.7% +/- 0.4% per milligram protein (P < 0.001) after exposure to 1% oxygen for 24 hours. In contrast, long-term exposure to 1% oxygen (72 hours) resulted in an increase of VEGF binding from 13.5% +/- 1.1% to 18.3% +/- 0.8% per milligram protein (P < 0.001). Scatchard analysis detected a decrease of receptor binding sites without change in binding affinity after 30 hours of exposure to hypoxia but demonstrated an increase in specific binding sites (4.2 +/- 0.6 x 10(4) sites/cell to 6.7 +/- 1.0 x 10(4) sites/cell, P = 0.049) with unaltered receptor affinity after 72 hours of hypoxic exposure.


CONCLUSIONS
These data suggest that hypoxia induces an initial decline in KDR mRNA levels and VEGF binding sites as mediated through adenosine binding to the A2R. Exposure to prolonged periods of hypoxia, however, results in an increase in VEGF binding sites by an as yet unidentified mechanism.
Takagi
H
H
Research Division, Beetham Eye Institute, Joslin Diabetes Center, Boston, Massachusetts 02215, USA.
King
G L
GL
Ferrara
N
N
Aiello
L P
LP
D000241:Adenosine / Q000378:metabolism; D000818:Animals; D015152:Blotting, Northern; D002196:Capillaries; D002417:Cattle; D015687:Cell Hypoxia; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D016228:Endothelial Growth Factors / Q000378:metabolism; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects; D008222:Lymphokines / Q000378:metabolism; D010100:Oxygen / Q000494:pharmacology*; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis / Q000235:genetics; D012333:RNA, Messenger / Q000378:metabolism; D020794:Receptor Protein-Tyrosine Kinases / Q000096:biosynthesis* / Q000235:genetics; D017978:Receptors, Growth Factor / Q000096:biosynthesis* / Q000235:genetics; D018047:Receptors, Purinergic P1 / Q000378:metabolism*; D040262:Receptors, Vascular Endothelial Growth Factor; D012171:Retinal Vessels / Q000187:drug effects / Q000378:metabolism*; D013997:Time Factors; D042461:Vascular Endothelial Growth Factor A; D040281:Vascular Endothelial Growth Factor Receptor-1; D042442:Vascular Endothelial Growth Factors
D016228:Endothelial Growth Factors; D008222:Lymphokines; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D017978:Receptors, Growth Factor; D018047:Receptors, Purinergic P1; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D020794:Receptor Protein-Tyrosine Kinases; D040262:Receptors, Vascular Endothelial Growth Factor; D040281:Vascular Endothelial Growth Factor Receptor-1; D000241:Adenosine; D010100:Oxygen