DIFFERENTIAL EXPRESSION of VASCULAR ENDOTHELIAL GROWTH FACTOR (VEGF) mRNA SPLICE VARIANTS in NEONATAL HYPEROXIC INJURY • 1543

Neonatal hyperoxic lung injury results in persistent abnormalities of the alveolar microcirculation. VEGF is an angiogenic growth factor that is specifically mitogenic for vascular endothelial cells. In previous work we found that alveolar type II cells in neonatal rabbits expressed VEGF mRNA and that hyperoxic injury decreased lung VEGF expression. Alternative splicing of the primary VEGF mRNA produces at least four splice variants. The resulting protein isoforms have differing biochemical properties, including affinity for VEGF receptors and the extracellular matrix. Our current objectives were to determine the relative abundance of VEGF mRNA splice variants in neonatal lung and the effects of hyperoxia on splicing. Neonatal rabbits were exposed to 100% oxygen for 9 days and recovered in 60% oxygen for 3 days. VEGF splice variants were detected by semi-quantitative RT-PCR using primers that hybridize to sequences that flanked the alternatively spliced regions. In lung, we found 3 PCR products that correspond to 3 of the VEGF splice variants(VEGF₁₈₃, VEGF₁₆₅ and VEGF₁₂₁). Correcting for the size of the PCR fragments, we found that VEGF₁₈₃ was the most abundant splice variant in control lung (53% of total VEGF mRNA), followed by VEGF₁₆₅ (30%) and VEGF₁₂₁ (17%). Hyperoxic injury disproportionately decreased lung VEGF₁₈₃, which fell to 14% of total VEGF message. The amounts of VEGF₁₆₅ and VEGF₁₂₁ mRNA appeared unaffected, but their relative proportions increased to 54% and 32% of total VEGF message, respectively. By 3 days recovery, VEGF₁₈₃ was again the predominate splice variant (62%). These findings were confirmed by ribonuclease protection assay. These data show that neonatal lung VEGF mRNA splice variants are differentially regulated in oxygen injury, suggesting differential biologic functions that may contribute to hyperoxic microvascular abnormalities. We speculate that oxygen may regulate VEGF mRNA splicing either directly or through differential effects on lung cell survival.

Supported by RO1 HL-54632 (WMM) and HL-02630 (RMR)