Exposure to hyperoxia decreases the expression of vascular endothelial growth factor and its receptors in adult rat lungs

Abstract

Exposure to high levels of inspired oxygen leads to respiratory failure and death in many animal models. Endothelial cell death is an early finding, before the onset of respiratory failure. Vascular endothelial growth factor (VEGF) is highly expressed in the lungs of adult animals. In the present study, adult Sprague-Dawley rats were exposed to >95% FiO2 for 24 or 48 hours. Northern blot analysis revealed a marked reduction in VEGF mRNA abundance by 24 hours, which decreased to less than 50% of control by 48 hours. In situ hybridization revealed that VEGF was highly expressed in distal airway epithelial cells in controls but disappeared in the oxygen-exposed animals. Immunohistochemistry and Western blot analyses demonstrated that VEGF protein was decreased at 48 hours. TUNEL staining demonstrated the presence of apoptotic cells coincident with the decline in VEGF. Abundance of VEGF receptor mRNAs (Flt-1 and KDR/Flk) decreased in the late time points of the study (48 hours), possibly secondary to the loss of endothelial cells. We speculate that VEGF functions as a survival factor in the normal adult rat lung, and its loss during hyperoxia contributes to the pathophysiology of oxygen-induced lung damage.

Figure 1.

Northern blot analysis of VEGF mRNA from rat lung tissue. VEGF was expressed in control tissue and significantly decreased after exposure to hyperoxia (10 μg of total mRNA per lane). C, control; 24, 24 hours >95% FiO₂; 48, 48 hours >95% FiO₂.

Figure 2.

In situ hybridization for VEGF mRNA using digoxigenin-labeled probes. A: Control. Multiple positively stained cells (arrowheads) are seen in the distal airway epithelium. B: Exposure for 24 hours of >95% FiO₂. Only a few positive cells are seen compared with control tissue. C: Exposure for 48 hours of >95% FiO₂. VEGF mRNA expression can no longer be identified. Magnification, ×200.

Figure 3.

RNAse protection assay using VEGF₁₈₈ riboprobe showed a similar distribution of protected bands of 564, 341, and 151 bp in RNA from lung tissue of both control animals and animals exposed to 24 or 48 hours of >95% FiO₂. These bands are consistent with the presence of VEGF₁₈₈, VEGF₁₆₄, and possibly VEGF₁₂₀.

Figure 4.

Immunohistochemistry for VEGF protein. A and B: Control. VEGF immunoreactivity was seen in bronchial and distal epithelial cells. Magnification, ×200 (A) and ×1000 (B). C and D: Exposure for 48 hours to >95% FiO₂. No immunoreactivity in bronchial or distal epithelium. Magnification, ×200 (C) and ×1000 (D). E: TUNEL stain for apoptosis. Positive apoptotic staining is indicated by arrow. Magnification, ×1000. a, airway; v, vessel.

Figure 5.

Summary of Northern blot analyses showing the relative abundance of Flt-1 mRNA in lung tissue from animals exposed to >95% FiO₂ for 24 or 48 hours (24h and 48h, respectively) compared with mRNA from lung tissue of control animals (C). Results were corrected for loading and normalized to control. There was a greater than 50% reduction in Flt-1 mRNA after 48 hours of exposure to hyperoxia. n = 6; *P < 0.05.

Figure 6.

Summary of Northern blot analyses showing the relative abundance of KDR/Flk mRNA in lung tissue from animals exposed to >95% FiO₂ for 24 or 48 hours (24h and 48h, respectively) compared with mRNA from lung of control animals (C). Results were corrected for loading and normalized to control. After 48 hours of exposure to hyperoxia, KDR/Flk mRNA was reduced to 58% of control value. n = 6; *P < 0.05.