Hypoxia and vascular endothelial growth factor stimulate angiogenic integrin expression in bovine retinal microvascular endothelial cells.
OBJECTIVE
Integrins alphavbeta3 and alphavbeta5 are cell-to-matrix adhesion molecules that have been reported to mediate vascular cell proliferation and migration. The authors investigated the regulation of expression of these angiogenic integrins by hypoxia and vascular endothelial growth factor (VEGF) in retinal microvascular endothelial cells in culture.


METHODS
Cultured bovine retinal capillary endothelial cells were exposed to human recombinant VEGF under normoxic (95% air, 5% CO2) conditions to assess the effects of VEGF. Hypoxia studies were performed under lower oxygen concentration (0.5%-1.5% O2) induced by nitrogen replacement in constant 5% CO2 conditions. Integrin family mRNA and protein expression were assessed by northern blot analysis and immunoprecipitation.


RESULTS
VEGF (25 ng/ml) increased integrin alphav, beta3, and 35 mRNA after 24 hours 6.1+/-0.8-fold (P < 0.001), 5.9+/-1.1-fold (P < 0.001), and 1.9+/-0.2-fold (P < 0.01), respectively. Similarly, hypoxia stimulated gene expression of integrin alphav and beta3 after 24 hours by 5.1+/-1.7-fold (P < 0.01) and 3.0+/-0.5-fold (P < 0.01), respectively, and integrin beta5 after 9 hours 1.4+/-0.2-fold (P < 0.05). This hypoxia-induced, integrin alphav mRNA elevation was inhibited significantly by anti-VEGF neutralizing antibody. Also, a conditioned medium from confluent endothelial cells maintained under hypoxic conditions for 24 hours produced a 7.1+/-1.1-fold increase (P < 0.001) in integrin alphav mRNA expression after 24 hours, which was reversed by anti-VEGF neutralizing antibody. Induction of integrin alphav by VEGF and hypoxia was confirmed in the protein level.


CONCLUSIONS
These data suggest that hypoxia stimulates expression of vascular integrins alphavbeta3 and alphavbeta5 in retinal microvascular endothelial cells partially through autocrine-paracrine action of VEGF induced by the hypoxic state.
Suzuma
K
K
Department of Ophthalmology and Visual Sciences, Kyoto University Graduate School of Medicine, Japan.
Takagi
H
H
Otani
A
A
Honda
Y
Y
D000818:Animals; D015703:Antigens, CD / Q000378:metabolism; D015152:Blotting, Northern; D002417:Cattle; D015687:Cell Hypoxia / Q000502:physiology; D002478:Cells, Cultured; D017931:DNA Primers / Q000737:chemistry; D004587:Electrophoresis, Agar Gel; D016228:Endothelial Growth Factors / Q000276:immunology / Q000494:pharmacology*; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism*; D015870:Gene Expression; D039564:Integrin alphaV; D016023:Integrins / Q000378:metabolism*; D008222:Lymphokines / Q000276:immunology / Q000494:pharmacology*; D012333:RNA, Messenger / Q000378:metabolism; D019030:Receptors, Vitronectin / Q000378:metabolism*; D011994:Recombinant Proteins / Q000494:pharmacology; D012171:Retinal Vessels / Q000187:drug effects / Q000378:metabolism*; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors
D015703:Antigens, CD; D017931:DNA Primers; D016228:Endothelial Growth Factors; D039564:Integrin alphaV; D016023:Integrins; D008222:Lymphokines; D012333:RNA, Messenger; D019030:Receptors, Vitronectin; D011994:Recombinant Proteins; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; C064660:integrin alphaVbeta5