Person:

D'Amore, Patricia

PURPOSE: 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.

Hypoxia-induced VEGF governs both physiological retinal vascular development and pathological retinal neovascularization. In the current paper, the mechanisms of physiological and pathological neovascularization are compared and contrasted. During pathological neovascularization, both the absolute and relative expression levels for VEGF164 increased to a greater degree than during physiological neovascularization. Furthermore, extensive leukocyte adhesion was observed at the leading edge of pathological, but not physiological, neovascularization. When a VEGF164-specific neutralizing aptamer was administered, it potently suppressed the leukocyte adhesion and pathological neovascularization, whereas it had little or no effect on physiological neovascularization. In parallel experiments, genetically altered VEGF164-deficient (VEGF120/188) mice exhibited no difference in physiological neovascularization when compared with wild-type (VEGF+/+) controls. In contrast, administration of a VEGFR-1/Fc fusion protein, which blocks all VEGF isoforms, led to significant suppression of both pathological and physiological neovascularization. In addition, the targeted inactivation of monocyte lineage cells with clodronate-liposomes led to the suppression of pathological neovascularization. Conversely, the blockade of T lymphocyte–mediated immune responses with an anti-CD2 antibody exacerbated pathological neovascularization. These data highlight important molecular and cellular differences between physiological and pathological retinal neovascularization. During pathological neovascularization, VEGF164 selectively induces inflammation and cellular immunity. These processes provide positive and negative angiogenic regulation, respectively. Together, new therapeutic approaches for selectively targeting pathological, but not physiological, retinal neovascularization are outlined.