Activation of hypoxia-inducible factors in hyperoxia through prolyl 4-hydroxylase blockade in cells and explants of primate lung

Abstract

Preterm neonates with respiratory distress syndrome (RDS) often develop a chronic form of lung disease called bronchopulmonary dysplasia (BPD), characterized by decreased alveolar and vascular development. Ventilator treatment with supraphysiological O2 concentrations (hyperoxia) contribute to the development of BPD. Hyperoxia down-regulates and hypoxia up-regulates many angiogenic factors in the developing lung. We investigated whether angiogenic responses could be augmented through enhancement of hypoxia-inducible factors 1alpha and 2alpha (HIF-1alpha and -2alpha, respectively) via blockade of prolyl hydroxylase domain-containing proteins (HIF-PHDs) in human microvascular endothelial cells from developing and adult lung, in epithelial A549 cells, and in fetal baboon explants in relative or absolute hyperoxia. PHD inhibitor (FG-4095) and positive control dimethyloxaloylglycine (DMOG), selective and nonselective HIF-PHD inhibitors, respectively, enhanced HIF-1alpha and -2alpha, vascular endothelial growth factor (VEGF), and platelet-endothelial cell adhesion molecule 1 expression in vitro in 95% and 21% O2. Furthermore, VEGF receptor fms-like tyrosine kinase 1 (Flt-1) was elevated, whereas kinase insert domain-containing receptor/fetal liver kinase 1 (KDR) was diminished in endothelial, but not epithelial, cells. Intracellular Flt-1 and KDR locations were unchanged by PHD blockade. Like VEGF, FG-4095 and DMOG increased angiogenesis in vitro, both in 95% and 21% O2, an effect that could be blocked through either Flt-1 or KDR. Notably, FG-4095 was effective in stimulating HIFs and VEGF also in fetal baboon lung explants. FG-4095 or DMOG treatment appeared to stimulate the feedback loop promoting HIF degradation in that PHD-2 and/or -3, but not PHD-1, were enhanced. Through actions characterized above, FG-4095 could have desirable effects in enhancing lung growth in BPD.

Fig. 1.

Effect of FG-4095 (125 μM) or DMOG (1 mM) on HIF-1α (A) and -2α (B) proteins in 21% and 95% O₂ in HLMVE-A cells (24 h). Data are shown as means (SD, n = 3). *, P ≤ 0.01 vs. control in 21% and 95% O₂; #, P ≤ 0.01 FG-4095 vs. DMOG in 21% and 95% O₂.

Fig. 2.

Assessment of VEGF (A), Flt-1 (B), and KDR (C) in HLMVE-A cells treated with FG-4095 (125 μM) or DMOG (1 mM) in 21% and 95% O₂ (24 h). (A) VEGF mRNA was analyzed by real-time PCR, and the change in gene expression was determined by calculating ΔCT, where the threshold cycle (CT) value of the target gene (VEGF) was subtracted from that of the housekeeping gene malate dehydrogenase (MDH). Each unit of ΔCT represents a 2-fold change in VEGF mRNA. (B) Flt-1 and (C) KDR proteins (pg per mg total cell protein) measured by ELISA. Data are expressed as means (SD, n = 3–6). *, P ≤ 0.05 vs. control in 21% and 95% O₂; #, P ≤ 0.01 vs. DMOG in 21% and 95% O₂; ⁁, P ≤ 0.001 control in 21% vs. 95% O₂.

Fig. 3.

Localization of KDR protein in untreated (A, D, and G) and FG-4095-(125 μM) (B, E, and H) or DMOG- (1 mM) (C, F, and I) treated HLMVE-A cells in 21% O₂. KDR was localized to cytosol, extracellular membrane, and cis-Golgi. (A–C) KDR stained with FITC; (D–F) cis-Golgi stained with Texas red; and (G–I) merged images of KDR and Golgi. Nuclei were stained with gold-DAPI (blue). All pictures were taken with ×40 magnification, and digital images were saved by using identical color intensity settings. [Bar (A), 10 μm.]

Fig. 4.

Assessment of in vitro angiogenesis following PHD blockade. (A and B) Angiogenesis assays of HLMVE-F cells after treatment with FG-4095 (125 μM), DMOG (1 mM), or VEGF (20 ng/ml) in 21% and 95% O₂ (7 h). (A) a, control; b, FG-4095; c, DMOG; and d, VEGF treatment in 95% O₂. Cell branch points were counted in 21% (black bars) and 95% (white bars) O₂, and data are presented as percent change from control (means, SD; n = 21–28; magnification, ×6.3). * and #, P ≤ 0.001 vs. control in 21% O₂. (C) Role of Flt-1 and KDR in FG-4095- or DMOG-induced angiogenesis. HLMVE-A cells were treated with control IgG (black bars) or neutralizing antibodies against Flt-1 (gray bars) or KDR (white bars). Cell branch points were counted in 21% O₂, and data are presented as percent change compared with untreated control (means, SD, n = 22–40). *, P ≤ 0.05 vs. IgG-treated cells; #, P ≤ 0.001 in cells treated with neutralizing Flt-1 antibody vs. IgG treated cells; °, P ≤ 0.001 in cells treated with neutralizing KDR antibody vs. IgG treated cells; +, P ≤ 0.01 vs. control in cells treated with neutralizing Flt-1 antibody; •, P ≤ 0.001 vs. control in cells treated with neutralizing KDR antibody.

Fig. 5.

Evaluation of FG-4095 actions in lung explants of fetal (125 d) baboons ex vivo (24 h). (A) lung HIF-1α protein, (B) lung HIF-2α protein, (C) medium VEGF protein, and (D) lung PHD-2 protein after PHD blockade. Values are means (SD) from three or four separate experiments; *, P ≤ 0.05 vs. control in 21% O₂.