Cloning and mRNA expression of vascular endothelial growth factor in ischemic retinas of Macaca fascicularis.
OBJECTIVE
To identify and isolate cDNAs for the alternatively spliced vascular endothelial growth factor (VEGF) mRNAs present in retina and to compare the relative levels of the splice variants and localization of VEGF mRNA in nonischemic and ischemic adult simian retinas.


METHODS
Retinas of cynomolgous monkeys were made ischemic by laser occlusion of the main branch retinal veins. Reverse transcription-polymerase chain reaction was used to amplify the VEGF coding region of RNA from ischemic and control retinas, and amplification products were analyzed by agarose gel electrophoresis, Southern blot, and nucleotide sequencing. Analysis of VEGF mRNA expression was accomplished by in situ hybridization.


RESULTS
Control and ischemic retinas produce mRNAs that correspond to the 121 and 165 amino acid diffusible isoforms of VEGF, and relatively low levels of VEGF189, the heparin-binding isoform. There was no significant difference in the levels of the alternatively spliced VEGF transcripts between control and ischemic retinas. Cloning and sequencing revealed that simVEGF cDNAs are 99% identical to human VEGFs and are predicted to encode proteins identical to their respective human homologues. In situ hybridization of nonischemic retinas revealed a low level of VEGF mRNA in retinal ganglion cells and in the inner nuclear layer. VEGF mRNA levels were elevated in ischemic retinas as early as 1 day after laser vein occlusion, when there was a small but reproducible increase in signal. The expression peaked at approximately 13 days, coincident with maximal iris neovascularization, and was significantly reduced by 28 days, when the iris vessels largely regressed.


CONCLUSIONS
The elevation of simVEGF121 and VEGF165 in ischemic retinas is consistent with a role for diffusible, retina-derived angiogenic factors in the development of ocular neovascularization.
Shima
D T
DT
Program in Biological and Biomedical Sciences, Harvard Medical School, Boston, Massachusetts, USA.
Gougos
A
A
Miller
J W
JW
Tolentino
M
M
Robinson
G
G
Adamis
A P
AP
D'Amore
P A
PA
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D003001:Cloning, Molecular; D017931:DNA Primers / Q000737:chemistry; D018076:DNA, Complementary / Q000032:analysis / Q000302:isolation & purification; D004587:Electrophoresis, Agar Gel; D016228:Endothelial Growth Factors / Q000096:biosynthesis* / Q000235:genetics; D015870:Gene Expression; D017403:In Situ Hybridization; D007498:Iris / Q000098:blood supply / Q000378:metabolism; D007511:Ischemia / Q000209:etiology / Q000378:metabolism*; D017075:Laser Coagulation; D008222:Lymphokines / Q000096:biosynthesis* / Q000235:genetics; D008252:Macaca fascicularis; D008969:Molecular Sequence Data; D009389:Neovascularization, Pathologic / Q000209:etiology / Q000378:metabolism; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000096:biosynthesis* / Q000235:genetics; D012160:Retina / Q000378:metabolism*; D012170:Retinal Vein Occlusion / Q000150:complications / Q000601:surgery; D012171:Retinal Vessels; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors
D017931:DNA Primers; D018076:DNA, Complementary; D016228:Endothelial Growth Factors; D008222:Lymphokines; D012333:RNA, Messenger; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors