Oxygen-Dependent Changes in the N-Glycome of Murine Pulmonary Endothelial Cells

Abstract

Supplemental oxygen is frequently used together with mechanical ventilation to achieve sufficient blood oxygenation. Despite the undoubted benefits, it is vigorously debated whether too much oxygen can also have unpredicted side-effects. Uncertainty is also due to the fact that the molecular mechanisms are still insufficiently understood. The lung endothelium is covered with an exceptionally broad glycocalyx, carrying N- and O-glycans, proteoglycans, glycolipids and glycosaminoglycans. Glycan structures are not genetically determined but depend on the metabolic state and the expression level and activity of biosynthetic and glycan remodeling enzymes, which can be influenced by oxygen and the redox status of the cell. Altered glycan structures can affect cell interactions and signaling. In this study, we investigated the effect of different oxygen conditions on aspects of the glycobiology of the pulmonary endothelium with an emphasis on N-glycans and terminal sialylation using an in vitro cell culture system. We combined a proteomic approach with N-glycan structure analysis by LC-MS, qRT-PCR, sialic acid analysis and lectin binding to show that constant and intermittent hyperoxia induced time dependent changes in global and surface glycosylation. An siRNA approach identified St6gal1 as being primarily responsible for the early transient increase of α2-6 sialylated structures in response to hyperoxia.

Effects of Hyperoxia and Hyperoxic Oscillations on the Proteome of Murine Lung Microvascular Endothelium

“…Constant and intermittent hyperoxia were shown to have similar trends in mRNA expression in several cases, despite differences in absolute values, as seen for Eea1, Tomm34, Tjp2, Parva1, Nucb1, Aifm1, Nans, Uap1, Memo1, Fhl1, S100a11 and Pcna. Obvious differences were observed for Snx1 at 24 h, Kpna1 at 72 h, and Ppp1 at 72 h. Generally, it seems that a fast response (4 h) of lung endothelial cells to hyperoxia is an attempt to maintain homeostasis by counter-regulating a potential detrimental effect of high oxygen, as exemplified by a reduction in endocytosis (Eea1), a strengthened barrier by increasing tight junction proteins (Tjp2), an increased importance of calcium signaling and homeostasis (Nucb1, Ip3r3, S100a11), an increase in the biosynthesis of sialic acids (Nans, Uap1) (investigated in more detail by our group in a previous study [ 13 ]), and an attempt to improve the fidelity of DNA replication and DNA repair (Pcna). Many of these effects are reversed at later time points.…”

“…We have described our proteomic methodology in a previous paper from our lab [ 13 ]. Individual steps and settings are summarized here.…”

The Proteome of Extracellular Vesicles Released from Pulmonary Microvascular Endothelium Reveals Impact of Oxygen Conditions on Biotrauma

“…Prior to exposure, medium was replaced with a secretion medium (basal medium EGM2 containing only antibiotics; serum-free). Six-well cell culture plates with 2 mL secretion medium/well were exposed to different gas conditions in a custom-designed exposure unit as described in [55]. Gas exposure was performed for 48 h at 37 • C using the following gas conditions: 21% O 2 , 2% O 2 , 10% O 2 , 40% O 2 , 95% O 2 , 0-21% O 2 , 0-95% O 2 , all with 5% CO 2 , rest N 2 .…”

Oxidative Stress and Inflammation in Acute and Chronic Lung Injuries

“…Hyperoxia-induced circulating extracellular vesicles containing high amounts of gasdermin Dp30, an inflammasome-mediated cell death executor protein that may contribute to the pathological features of bronchopulmonary dysplasia, have been demonstrated in neonatal rats [ 354 ]. Using an in vitro murine cell culture system, a short period of hyperoxia was shown to promote a disturbance in N-glycolysis, especially in lung endothelial cells, suggesting a potential impairment of receptor signaling on the endothelial inner surface and interaction with immune cells [ 355 ].…”