[Skip to Content]
[Skip to Content Landing]
December 1995

Hypoxic Regulation of Vascular Endothelial Growth Factor in Retinal Cells

Author Affiliations

From the Research Division (Drs Aiello and Tagaki and Ms Northrup) and Beetham Eye Institute (Dr Aiello), Joslin Diabetes Center, Boston, Mass; Department of Ophthalmology, Harvard Medical School, Boston (Drs Aiello and Iwamoto); Department of Cardiovascular Research, Genentech Inc, San Francisco, Calif (Dr Keyt); and Division of Ophthalmology, Brigham & Women's Hospital, Boston (Dr Iwamoto).

Arch Ophthalmol. 1995;113(12):1538-1544. doi:10.1001/archopht.1995.01100120068012

Background:  Vascular endothelial growth factor (VEGF) is an angiogenic protein and vasopermeability factor whose intraocular concentrations are closely correlated with active neovascularization in patients with diabetes mellitus, central retinal vein occlusion, retinopathy of prematurity, and rubeosis iridis.

Objective:  To determine whether hypoxia could induce expression of VEGF in retinal cells, which then promotes retinal endothelial cell proliferation.

Methods:  Retinal pigment epithelial cells, pericytes, and microvascular endothelial cells were exposed to hypoxic conditions in vitro, and RNA expression of VEGF was evaluated by Northern blot analysis. The VEGF-specific proliferative potential of the medium was measured by means of retinal endothelial cell growth assays and VEGF-neutralizing VEGF receptor IgG chimeric protein.

Results:  The VEGF RNA levels increased within 4 hours and reached elevations of threefold to 30-fold after 18 hours of hypoxia (0% to 5% oxygen, 5% carbon dioxide, 90% to 95% nitrogen) in all cell types (.01<P<.03). Stimulation was dependent on oxygen concentration. The VEGF RNA levels were normalized by reinstitution of normoxia for 24 hours (P<.004). Medium conditioned by hypoxic retinal pericytes and retinal pigment epithelial cells stimulated retinal endothelial cell growth by 20% (P=.04), and this stimulation was entirely inhibited by VEGF-neutralizing receptor chimeric protein (P=.02).

Conclusion:  Hypoxia increases VEGF expression in retinal cells, which promotes retinal endothelial cell proliferation, suggesting that VEGF plays a major role in mediating intraocular neovascularization resulting from ischemic retinal diseases.

Michaelson IC.  The mode of development of the vascular system of the retina, with some observations on its significance for certain retinal diseases . Trans Ophthalmol Soc U K . 1948;68:137-180.
Ashton N, Ward B, Serpell G.  Effect of oxygen on developing retinal vessels with particular reference to the problem of retrolental fibroplasia . Br J Ophthalmol . 1954;38:397-432.Article
Ferrara N, Houck KA, Jakeman LB, Leung DW.  Molecular and biological properties of the vascular endothelial growth factor family of proteins . Endocr Rev . 1992;13:18-32.Article
Leung DW, Cachianes G, Kuang WJ, Goeddel DV, Ferrara N.  Vascular endothelial growth factor is a secreted angiogenic mitogen . Science . 1989;246:1306-1309.Article
Berse B, Brown LF, Van de Water L, Dvorak HF, Senger DR.  Vascular permeability factor (vascular endothelial growth factor) gene is expressed differentially in normal tissues, macrophages, and tumors . Mol Biol Cell . 1992;3:211-220.Article
Connolly DT, Olander JV, Heuvelmand D, et al.  Human vascular permeability factor: isolation from U937 cells . Mol Biol Cell . 1992;2:211-220.
Dvorak HF, Sioussat TM, Brown LF, et al.  Distribution of vascular permeability factor (vascular endothelial growth factor) in tumors: concentration in tumor blood vessels . J Exp Med . 1991;174:1275-1278.Article
Senger DR, Perruzzi CA, Feder J, Dvorak HF.  A highly conserved vascular permeability factor secreted by a variety of human and rodent tumor cell lines . Cancer Res . 1986;46:5629-5632.
Plate KH, Breier G, Weich HA, Risau W.  Vascular endothelial growth factor is a potential tumour angiogenesis factor in human gliomas in vivo . Nature . 1992;359:845-848.Article
Shweiki D, Itin A, Softer D, Keshet E.  Vascular endothelial growth factor induced by hypoxia may mediate hypoxia-initiated angiogenesis . Nature . 1992;359:843-845.Article
Thieme H, Aiello LP, Takagi H, Ferrara N, King GL.  Comparative analysis of vascular endothelial growth factor (VEGF) receptors on retinal and aortic microvascular endothelial cells . Diabetes . 1995;44:98-103.Article
Jakeman LB, Winer J, Bennett GL, Altar CA, Ferrara N.  Binding sites for vascular endothelial growth factor are localized on endothelial cells in adult rat tissues . J Clin Invest . 1992;89:244-253.Article
De Vries C, Escobedo JA, Ueno H, Houck K, Ferrara N, Williams LT.  The fms-like tyrosine kinase, a receptor for vascular endothelial growth factor . Science . 1992;255:989-991.Article
Simorre-Pinatel V, Guerrin M, Chollet P, Penary M, Clamens S, Plouet J.  Vasculotropin-VEGF stimulates retinal capillary endothelial cells through an autocrine pathway . Invest Ophthalmol Vis Sci . 1994;35:3393-3400.
Aiello LP, Avery RL, Arrigg PG, et al.  Vascular endothelial growth factor in ocular fluid of patients with diabetic retinopathy and other retinal disorders . N Engl J Med . 1994;331:1480-1487.Article
Adamis AP, Shima DT, Yeo KT, et al.  Synthesis and secretion of vascular permeability factor/vascular endothelial growth factor by human retinal pigment epithelial cells . Biochem Biophys Res Commun . 1993;193:631-638.Article
Miller JW, Adamis AP, Shima DT, et al.  Vascular endothelial growth factor/vascular permeability factor is temporally and spatially correlated with ocular angiogenesis in a primate model . Am J Pathol . 1994;145:574-584.
Shima D, Adamis AP, Yeo KT, Yeo TK, Berse B, Brown L.  Hypoxic regulation of VEGF mRNA and protein secretion by human RPE cells . Invest Ophthalmol Vis Sci . 1995;34( (suppl) ):900. Abstract.
King GL, Goodman S, Buzney S, Moses A, Kahn CR.  Receptors and growth promoting effects of insulin and insulin-like growth factors on cells from bovine retinal capillaries and aorta . J Clin Invest . 1985;75:1028-1036.Article
Nayak RC, Berman AB, George KL, Eisenbarth GS, King GL.  A monoclonal antibody (3G5)-defined ganglioside antigen is expressed on the cell surface of microvascular pericytes . J Exp Med . 1988;167:1003-1015.Article
King GL, Berman AB, Bonner-Weir S, Carson MP.  Regulation of vascular permeability in cell culture . Diabetes . 1987;36:1460-1467.Article
Park JE, Chen HH, Winer J, Houck KA, Ferrara N.  Placenta growth factor: potentiation of vascular endothelial growth factor bioactivity, in vitro and in vivo, and high affinity binding to FLT-1 but not to FLK-1/KDR . J Biol Chem . 1994;269:25646-25654.
Aiello LP, Robinson GS, Lin Y, Nishio Y, King GL.  Identification of multiple genes in bovine retinal pericytes altered by exposure to elevated levels of glucose by using mRNA differential display . Proc Natl Acad Sci U S A . 1994;91:6231-6235.Article
Masiakowski P, Breathnach R, Bloch J, Gannon F, Krust A, Chambon P.  Cloning of cDNA sequences of hormone-regulated genes from the MFC-7 human breast cancer cell line . Nucleic Acids Res . 1982;10:7895-7903.Article
Senger DR, Connolly DT, Van de Water L, Feder J, Dvorak HF.  Purification and NH2-terminal amino acid sequence of guinea pig tumor-secreted vascular permeability factor . Cancer Res . 1990;50:1774-1778.
Tischer E, Mitchell R, Hartman T, et al.  The human gene for vascular endothelial growth factor: multiple protein forms are encoded through alternative exon splicing . J Biol Chem . 1991;266:11947-11954.
Houck KA, Leung DW, Rowland AM, Winer J, Ferrara N.  Dual regulation of vascular endothelial growth factor bioavailability by genetic and proteolytic mechanisms . J Biol Chem . 1992;267:26031-26037.
Pepper MS, Ferrara N, Orci L, Montesano R.  Potent synergism between vascular endothelial growth factor and basic fibroblast growth factor in the induction of angiogenesis in vitro . Biochem Biophys Res Commun . 1992;189:824-831.Article
Malecaze F, Clamens S, Simorre-Pinatel V, et al.  Detection of vascular endothelial growth factor messenger RNA and vascular endothelial growth factor-like activity in proliferative diabetic retinopathy . Arch Ophthalmol . 1994;112:1476-1482.Article
Adamis AP, Miller JW, Bernal MT, et al.  Increased vascular endothelial growth factor levels in the vitreous of eyes with proliferative diabetic retinopathy . Am J Ophthalmol . 1994;118:445-450.
Pierce EA, Avery RL, Foley ED, Aiello LP, Smith LEH.  Vascular endothelial growth factor/vascular permeability factor expression in a mouse model of retinal neovascularization . Proc Natl Acad Sci U S A . 1 995;92:905-909.Article
Keyt BA, Berleau L, Nguyen H, Henzel W, Ferrara N.  Partial hydrolysis of vascular endothelial growth factor: plasmin cleavage yields 110 amino acid bioactive form of VEGF . In: Program and abstracts of the VIII International Symposium on the Biology of Vascular Cells ; August 28 to September 4, 1994; Heidelberg, Germany. Page 48. Abstract.
Muthukrishnan L, Warder E, McNeil PL.  Basic fibroblast growth factor is efficiently released from cytosolic storage site through plasma membrane disruptions of endothelial cells . J Cell Physiol . 1991;148:1-16.Article
Vlodavsky I, Folkman J, Sullina R, et al.  Endothelial cell-derived basic fibroblast growth factor: synthesis and deposition into subendothelial extracellular matrix . Proc Natl Acad Sci U S A . 1987;84:2292-2296.Article
Hayreh SS, Rojas P, Podhajsky P.  Ocular neovascularization with retinal vascular occlusion, III: incidence of ocular neovascularization with retinal vein occlusion . Ophthalmology . 1983;89:780-784.
Cullinan-Bove K, Koos RD.  Vascular endothelial growth factor/vascular permeability factor expression in the rat uterus: rapid stimulation by estrogen correlates with estrogen-induced increases in uterine capillary permeability and growth . Endocrinology . 1993;133:829-837.
Klein BE, Moss SE, Klein R.  Effect of pregnancy on progression of diabetic retinopathy . Diabetes Care . 1990;13:34-40.Article
Davidorf FH, Chambers RB.  Diabetic retinopathy during pregnancy . Clin Perinatol . 1993;20:571-581.
Blom ML, Green WR, Schachat AP.  Diabetic retinopathy: a review . Del Med J . 1994;66:379-388.
Grunwald JE, Brucker AJ, Petrig BL, Riva CE.  Retinal blood flow regulation and the clinical response to panretinal photocoagulation in proliferative diabetic retinopathy . Ophthalmology . 1989;96:1518-1522.Article
Stefansson E.  Oxygen and diabetic eye disease . Graefes Arch Clin Exp Ophthalmol . 1990;228:120-123.Article
Glaser BM, Campochiaro PA, Davis JRJ, Jerdan JA.  Retinal pigment epithelial cells release inhibitors of neovascularization . Ophthalmology . 1987;94:780-784.Article
Kim KJ, Li B, Winer J, et al.  Inhibition of vascular endothelial growth factor-induced angiogenesis suppresses tumour growth in vivo . Nature . 1993;362:841-844.Article
The Diabetic Retinopathy Study Research Group.  Preliminary report on effects of photocoagulation therapy . Am J Ophthalmol . 1976;81:383.
Early Treatment Diabetic Retinopathy Study Group.  Results of the ETDRS . Ophthalmology . 1991;98:741-840.Article