The pentose phosphate pathway mediates hyperoxia-induced lung vascular dysgenesis and alveolar simplification in neonates
Jiannan Gong, Zihang Feng, Abigail L. Peterson, Jennifer F. Carr, Xuexin Lu, Haifeng Zhao, Xiangming Ji, You-Yang Zhao, Monique E. De Paepe, Phyllis A. Dennery, Hongwei Yao
Jiannan Gong, Zihang Feng, Abigail L. Peterson, Jennifer F. Carr, Xuexin Lu, Haifeng Zhao, Xiangming Ji, You-Yang Zhao, Monique E. De Paepe, Phyllis A. Dennery, Hongwei Yao
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Research Article Pulmonology

The pentose phosphate pathway mediates hyperoxia-induced lung vascular dysgenesis and alveolar simplification in neonates

Abstract

Dysmorphic pulmonary vascular growth and abnormal endothelial cell (EC) proliferation are paradoxically observed in premature infants with bronchopulmonary dysplasia (BPD), despite vascular pruning. The pentose phosphate pathway (PPP), a metabolic pathway parallel to glycolysis, generates NADPH as a reducing equivalent and ribose 5-phosphate for nucleotide synthesis. It is unknown whether hyperoxia, a known mediator of BPD in rodent models, alters glycolysis and the PPP in lung ECs. We hypothesized that hyperoxia increases glycolysis and the PPP, resulting in abnormal EC proliferation and dysmorphic angiogenesis in neonatal mice. To test this hypothesis, lung ECs and newborn mice were exposed to hyperoxia and allowed to recover in air. Hyperoxia increased glycolysis and the PPP. Increased PPP, but not glycolysis, caused hyperoxia-induced abnormal EC proliferation. Blocking the PPP reduced hyperoxia-induced glucose–derived deoxynucleotide synthesis in cultured ECs. In neonatal mice, hyperoxia-induced abnormal EC proliferation, dysmorphic angiogenesis, and alveolar simplification were augmented by nanoparticle-mediated endothelial overexpression of phosphogluconate dehydrogenase, the second enzyme in the PPP. These effects were attenuated by inhibitors of the PPP. Neonatal hyperoxia augments the PPP, causing abnormal lung EC proliferation, dysmorphic vascular development, and alveolar simplification. These observations provide mechanisms and potential metabolic targets to prevent BPD-associated vascular dysgenesis.

Authors

Jiannan Gong, Zihang Feng, Abigail L. Peterson, Jennifer F. Carr, Xuexin Lu, Haifeng Zhao, Xiangming Ji, You-Yang Zhao, Monique E. De Paepe, Phyllis A. Dennery, Hongwei Yao

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Figure 6

The PPP enhances lung EC proliferation in mice exposed to hyperoxia as neonates.

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The PPP enhances lung EC proliferation in mice exposed to hyperoxia as n...
C57BL/6J neonatal mice (<12 hours old) were exposed to air or hyperoxia (95% O2) for 3 days and were then allowed for recover in room air until P14. (A) EdU was i.p. injected at 50 mg/kg daily for 3 days before sacrificing. Lung tissues were utilized for EdU staining, along with costaining with vWF. Scale bar: 20 μm. n = 5 per group. (B) Double immunofluorescence was conducted to determine the abundance of PCNA in vWF+ cells in mouse lungs. Scale bar: 20 μm. n = 5 per group. (C) 6-AN (5 and 10 mg/kg, i.p.) or DHEA (10 and 20 mg/kg, i.p.) were administered daily in mice from P9 to P13. (D) 6-AN (10 mg/kg, i.p.) or DHEA (20 mg/kg, i.p.) were administered daily in mice from P12 to P13. (C and D) Lung tissues were utilized for double immunofluorescence of EdU incorporation and vWF. Numbers of EdU+ and vWF+ cells were counted in 3 randomly selected high-power fields (HPF) for each sample. n = 5 per group. (E) EdU incorporation was measured by flow cytometry in LMVECs isolated from hyperoxia-exposed mice treated with 6-AN (10 mg/kg) or DHEA (20 mg/kg) between P9 and P13. n = 5 per group. (F) Nanoparticles mixed with plasmid DNA expressing pgd or empty vector under the control of human CDH5 promoter was administered into normoxia-exposed mice via a retro-orbital injection at P9. At P14, immunofluorescence was performed to detect colocalization of PGD and vWF in mouse lungs. Pgd OE, pgd overexpression. Scale bar: 20 μm. n = 5 per group. (G) Immunofluorescence of EdU incorporation and vWF was performed in hyperoxia-exposed mice injected with nanoparticles mixed with plasmid DNA expressing pgd or empty vector under the control of human CDH5 promoter. Numbers of EdU+/vWF+ cells were counted in 3 randomly selected high-power fields (HPF) for each sample. n = 5 per group. Data are expressed as mean ± SEM. **P < 0.01, ***P < 0.001 versus air (A–E), vector (F), or air/vector (G); †P < 0.05, ††P < 0.01 versus hyperoxia/vehicle (C–E) or hyperoxia/vector (G) using 1-tailed t test (A, B, and F) or ANOVA followed by Tukey-Kramer test (C–E, and G).