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

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.

Primary mouse LMVECs were exposed to hyperoxia for 24 h followed by air recovery for 24 h (refers to O2). Untargeted metabolomics analysis was performed by mass spectrometry. (A) Overall clustering for the correlation between air and hyperoxia groups. Scale is based on colors from green to red representing negative and positive correlations, respectively.
(B) The -log10 t-test P values were plotted against the log2 ratio as a volcano plot of 1383 metabolic features detected in cells exposed to hyperoxia after normalization into air group. Each metabolite is represented as a dot. The upper right and upper left areas of this plot represent 2-fold increase or decrease, respectively, in hyperoxia-exposed cells compared to the air group (P<0.05).
N=4 in air and N=6 in hyperoxia.
3 Supplemental Figure 2. Protein levels of key glycolytic enzymes in cultured lung ECs. Primary mouse LMVECs were exposed to hyperoxia for 24 h followed by air recovery for 24 h (refers to O2). Western blot was performed to determine the levels of GAPDH, PKM, and PFKFB3 proteins.
Data are expressed as mean ± SEM. N=4 per group. The t-test was used for detecting statistical differences (A and B). 4

Supplemental Figure 3. Hyperoxic exposure did not affect glucose uptake in cultured lung
ECs. MFLM-91U cells were exposed to hyperoxia for 24 h followed by air recovery for 24 h (refers to O2). (A and B) Levels of glut1 and glut4 proteins were detected using Western blot. N=4 per group. (C) 2-NBDG uptake was measured by flow cytometry. N=8 in air and N=10 in hyperoxia. C57BL/6J neonatal mice (<12 h old) were exposed to air or hyperoxia (95% O2) for 3 days, and then allowed for recover in room air until pnd14. (D) mRNA and (E) protein levels of glut1 and glut4 in mouse lungs were determined by qRT-PCR and Western blot, respectively. N=6 per group (D) and N=4 per group (E). Data are expressed as mean ± SEM. The t-test was used for detecting statistical differences (A-E). 5

Supplemental Figure 4. 2-DG decreases glycolysis in cultured lung ECs, and hyperoxic exposure influences glucose-derived nucleotides in cells and ATP levels in mouse lungs. (A)
ECAR was measured by the Seahorse Analyzer after 2-DG incubation (3 and 6 mM, 12 h) during air recovery phase in primary LMVECs. N=8 per group. (B) MFLM-91 cells were transfected with scramble or pgd siRNA for 48 h. Gene expression of pgd was measured by qRT-PCR. N=3 per group. (C) MFLM-91U cells were exposed to hyperoxia for 24 h followed by air recovery for 24 h (refers to O2). 13 C-labled NTPs were measured by mass spectrometry when cells were incubated with 20 mM U-13 C-glucose for 24 h during air recovery phase. N=5 per group. (D) C57BL/6J neonatal mice (<12 h old) were exposed to air or hyperoxia (95% O2) for 3 days, and then allowed for recover in room air until pnd7. Mean linear intercept (Lm) and radial alveolar count (RAC) were measured in mouse lungs. N=6 per group. Data are expressed as mean ± SEM. ** P<0.01, *** P<0.001 vs air using ANOVA followed by Tukey-Kramer test (A-C).