Is Necrotizing Enterocolitis and Spontaneous Intestinal Perforation Part of Same Disease Spectrum - New Insights?

The diagnosis of NEC is based on the presence of pneumatosis, dilated bowel loops, portal venous gas, or pneumoperitoneum on the abdominal x-ray. Published studies suggest that the appearance of pneumatosis most likely depends on the gestational age, with a shift occurring between 27-28 weeks. For infants of gestational age under 27 weeks, pneumoperitoneum is the most likely presentation of bowel injury due to the thin bowel wall and the colonization of the gut with the non-gas-producing bacteria. Assessment of postoperative morbidity and white matter injury on the brain MRI at term equivalent age in a cohort of preterm infants failed to identify differences between SIP and NEC groups when confirmed by histology. These findings illustrate the difficulty in conclusively identifying cases as SIP or NEC, particularly when gestational age is considered and raise speculation that both conditions lie on the same spectrum of intestinal injury.

The Current State of Neonatal Neurodevelopmental Follow-up Programs in North America: A Children's Hospitals Neonatal Consortium Report.

OBJECTIVE: This study aimed to determine neonatal neurodevelopmental follow-up (NDFU) practices across academic centers. STUDY DESIGN: This study was a cross-sectional survey that addressed center-specific neonatal NDFU practices within the Children's Hospitals Neonatal Consortium (CHNC). RESULTS: Survey response rate was 76%, and 97% of respondents had a formal NDFU program. Programs were commonly staffed by neonatologists (80%), physical therapists (77%), and nurse practitioners (74%). Median gestational age at birth identified for follow-up was ≤32 weeks (range 26-36). Median duration was 3 years (range 2-18). Ninety-seven percent of sites used Bayley Scales of Infant and Toddler Development, but instruments used varied across ages. Scores were recorded in discrete electronic data fields at 43% of sites. Social determinants of health data were collected by 63%. Care coordination and telehealth services were not universally available. CONCLUSION: NDFU clinics are almost universal within CHNC centers. Commonalities and variances in practice highlight opportunities for data sharing and development of best practices. KEY POINTS: · Neonatal NDFU clinics help transition high-risk infants home.. · Interdisciplinary neonatal intensive care unit follow-up brings together previously separated outpatient service lines.. · This study reviews the current state of neonatal NDFU in North America..

Outcomes by disease onset, sex, and intervention in neonates with SIP and surgical NEC.

BACKGROUND: Outcomes of infants following surgical necrotizing enterocolitis (NEC) and spontaneous intestinal perforation (SIP) categorized by the age of onset, interventions, and sex are not well defined. METHODS: Retrospective comparison of infants categorized by age of onset (NEC at <10, 10-20, and >20 days) and SIP at <7 versus ≥7 days), sex, and intervention [Penrose Drain (PD) vs. laparotomy]. RESULTS: A total of 114 infants had NEC and 37 had SIP. On multinomial logistic regression, infants with NEC/SIP onset >20 days had significantly lower odds of small bowel involvement (aOR = 0.07, 95% CI: 0.01-0.33, p = 0.001), higher necrosis (aOR = 3.59, 95% CI: 1.34-9.65, p = 0.012) and higher CRP (p = 0.004) than onset <10 days. Initial laparotomy was associated with more bowel loss (24.1 cm [12.3; 40.6] vs.12.1 [8.00; 23.2]; p = 0.001), small and large intestine involvement (47.1% vs 17.2%; p = 0.01), and ileocecal valve resection (42% vs. 19.4%; p = 0.036) than initial PD therapy. Females underwent fewer small bowel resections (52.3% vs 73.6%; p = 0.025) but had higher surgical morbidity (53.7% vs. 24.7%.; p = 0.001) than males. CONCLUSION: Clinical, radiological, and histopathological presentation and outcomes in preterm infants with surgical NEC/SIP are associated with age of disease onset, sex, and initial intervention. IMPACT: Neonates with surgical NEC onset >20 days had more severe necrosis, inflammation, kidney injury, and bowel loss than those with <10 days. Initial laparotomy was associated with later age onset, more bowel loss, and ileocecal valve resection compared to initial PD treatment, but not with differences in mortality or length of stay. Female sex was associated with lower maturity, more placental malperfusion, less often small bowel involvement, lower pre-NEC hematocrit as well as higher surgical morbidity than males. Whether the management of surgical NEC and SIP should differ by the age of onset requires further investigation.

Association of Hospital Resource Utilization With Neurodevelopmental Outcomes in Neonates With Hypoxic-Ischemic Encephalopathy.

Importance: Intercenter variation exists in the management of hypoxic-ischemic encephalopathy (HIE). It is unclear whether increased resource utilization translates into improved neurodevelopmental outcomes. Objective: To determine if higher resource utilization during the first 4 days of age, quantified by hospital costs, is associated with survival without neurodevelopmental impairment (NDI) among infants with HIE. Design, Setting, and Participants: Retrospective cohort analysis of neonates with HIE who underwent therapeutic hypothermia (TH) at US children's hospitals participating in the Children's Hospitals Neonatal Database between 2010 and 2016. Data were analyzed from December 2021 to December 2022. Exposures: Infants who survived to 4 days of age and had neurodevelopmental outcomes assessed at greater than 11 months of age were divided into 2 groups: (1) death or NDI and (2) survived without NDI. Resource utilization was defined as costs of hospitalization including neonatal neurocritical care (NNCC). Data were linked with Pediatric Health Information Systems to quantify standardized costs by terciles. Main Outcomes and Measures: The main outcome was death or NDI. Characteristics, outcomes, hospitalization, and NNCC costs were compared. Results: Among the 381 patients who were included, median (IQR) gestational age was 39 (38-40) weeks; maternal race included 79 (20.7%) Black mothers, 237 (62.2%) White mothers, and 58 (15.2%) mothers with other race; 80 (21%) died, 64 (17%) survived with NDI (combined death or NDI group: 144 patients [38%]), and 237 (62%) survived without NDI. The combined death or NDI group had a higher rate of infants with Apgar score at 10 minutes less than or equal to 5 (65.3% [94 of 144] vs 39.7% [94 of 237]; P < .001) and a lower rate of infants with mild or moderate HIE (36.1% [52 of 144] vs 82.3% [195 of 237]; P < .001) compared with the survived without NDI group. Compared with low-cost centers, there was no association between high- or medium-hospitalization cost centers and death or NDI. High- and medium-EEG cost centers had lower odds of death or NDI compared with low-cost centers (high vs low: OR, 0.30 [95% CI, 0.16-0.57]; medium vs low: OR, 0.29 [95% CI, 0.13-0.62]). High- and medium-laboratory cost centers had higher odds of death or NDI compared with low-cost centers (high vs low: OR, 2.35 [95% CI, 1.19-4.66]; medium vs low: OR, 1.93 [95% CI, 1.07-3.47]). High-antiseizure medication cost centers had higher odds of death or NDI compared with low-cost centers (high vs. low: OR, 3.72 [95% CI, 1.51-9.18]; medium vs low: OR, 1.56 [95% CI, 0.71-3.42]). Conclusions and Relevance: Hospitalization costs during the first 4 days of age in neonates with HIE treated with TH were not associated with neurodevelopmental outcomes. Higher EEG costs were associated with lower odds of death or NDI yet higher laboratory and antiseizure medication costs were not. These findings serve as first steps toward identifying aspects of NNCC that are associated with outcomes.

CpG methylation patterns in placenta and neonatal blood are differentially associated with neonatal inflammation.

BACKGROUND: Infants born extremely premature are at increased risk for health complications later in life for which neonatal inflammation may be a contributing biological driver. Placental CpG methylation provides mechanistic information regarding the relationship between prenatal epigenetic programming, prematurity, neonatal inflammation, and later-in-life health. METHODS: We contrasted CpG methylation in the placenta and neonatal blood spots in relation to neonatal inflammation in the Extremely Low Gestational Age Newborn (ELGAN) cohort. Neonatal inflammation status was based on the expression of six inflammation-related proteins, assessed as (1) day-one inflammation (DOI) or (2) intermittent or sustained systemic inflammation (ISSI, inflammation on ≥2 days in the first 2 postnatal weeks). Epigenome-wide CpG methylation was assessed in 354 placental samples and 318 neonatal blood samples. RESULTS: Placental CpG methylation displayed the strongest association with ISSI (48 CpG sites) but was not associated with DOI. This was in contrast to CpG methylation in blood spots, which was associated with DOI (111 CpG sites) and not with ISSI (one CpG site). CONCLUSIONS: Placental CpG methylation was strongly associated with ISSI, a measure of inflammation previously linked to later-in-life cognitive impairment, while day-one neonatal blood methylation was associated with DOI. IMPACT: Neonatal inflammation increases the risk of adverse later-life outcomes, especially in infants born extremely preterm. CpG methylation in the placenta and neonatal blood spots were evaluated in relation to neonatal inflammation assessed via circulating proteins as either (i) day-one inflammation (DOI) or (ii) intermittent or sustained systemic inflammation (ISSI, inflammation on ≥2 days in the first 2 weeks). Tissue specificity was observed in epigenetic-inflammatory relationships: placental CpG methylation was associated with ISSI, neonatal blood CpG methylation was associated with DOI. Supporting the placental origins of disease framework, placental epigenetic patterns are associated with a propensity for ISSI in neonates.

New body composition reference charts for preterm infants.

BACKGROUND: The American Academy of Pediatrics (AAP) has recommended that nutritional management of the preterm infant should aim to achieve body composition that replicates the in utero fetus, but intrauterine body composition reference charts for preterm infants are lacking. OBJECTIVE: Our objective was to create body composition reference curves for preterm infants that approximate the body composition of the in utero fetus from 30 to 36 wk of gestation. DESIGN: A total of 223 ethnically diverse infants born at 30 + 0 to 36 + 6 wk of gestation were enrolled. Inclusion and exclusion criteria were specified so that the sample would represent healthy appropriately growing fetuses (e.g., singleton, birth weight appropriate for their gestational age, and medically stable). Cross-sectional reference values were generated for fat mass (FM), fat-free mass (FFM), and percentage body fat (PBF) by gestational age (GA), with the use of air-displacement plethysmography (ADP) and the lambda-mu-sigma method for percentile estimation. RESULTS: GA-specific percentile values and a percentile and z score calculator for FFM, FM, and PBF are presented. These values aligned closely with ADP centile values published for term infants from 36 to 38 wk of gestation. The medians were also similar to the mean values for the reference fetus derived from chemical analysis previously. CONCLUSIONS: To our knowledge, these are the first body composition reference charts for total FM and FFM at birth in preterm infants to assist in following AAP guidelines. Future work will test the clinical utility of body composition monitoring for improving nutritional management in this population. This trial was registered at clinicaltrials.gov as NCT02855814.

Regulation of fibroblast growth factor 2 expression in oxygen-induced retinopathy.

PURPOSE: Fibroblast growth factor (FGF) 2 is a potent endothelial cell mitogen and survival factor that is postulated to participate in the pathogenesis of retinopathy of prematurity (ROP). The purpose of the current study was to determine the transcriptional and translational regulation of FGF2 expression in oxygen-induced retinopathy (OIR), the animal model of ROP. METHODS: We examined FGF2 protein and mRNA expression and optokinetic visual responses in transgenic mice possessing a dual-luciferase bicistronic transgene containing a 5'-internal ribosome entry site (IRES) of FGF2. RESULTS: We found that retinal FGF2 protein isoform expression varies with age but not in response to OIR. Analysis of luciferase, protein, and mRNA data indicate that FGF2 protein expression is translationally repressed during the vaso-obliterative phase of OIR, possibly by inhibiting elongation. At the transition from vaso-obliteration to neovascularization, heightened FGF2 protein expression corresponds to maintenance of IRES activity and diminished cap-dependent translational activity. During neovascularization, FGF2 expression is primarily regulated by transcription. Mice recovering from OIR display alterations in visual optokinetic responses and increased FGF2 protein expression at 6 weeks of age. CONCLUSIONS: In total, these findings illustrate the complexity of translational and transcriptional regulation of FGF2 protein expression in OIR. The augmentation of FGF2 expression and reduced optokinetic responses during the resolution of surface vasculopathy may indicate a role for FGF2 in the maintenance of neuroretinal function in OIR/ROP.

Potential influence of total parenteral nutrition on body composition at discharge in preterm infants.

OBJECTIVE: This study was undertaken to assess the potential influence of total parenteral nutrition (TPN) on body composition (BC) in preterm infants. STUDY DESIGN: This prospective, observational study of infants born <35 weeks measured BC at discharge using air displacement plethysmography. The % body fat (BF) at discharge was correlated with variables gestational age (GA), severity of illness, days on oxygen, time to regain birth weight and duration of TPN. RESULT: The 61 patients enrolled had a %BF at discharge of 13.9%. GA and TPN days correlated with %BF for the entire group. Multiple regression analysis identified that the time to regain birth weight added to the effect of GA, but not TPN. Isolating the influence of TPN in a subgroup of similarly aged infants (30-35 weeks) did not reveal a difference in body composition at the time of discharge between infants who did or did not receive TPN. CONCLUSION: These findings fail to demonstrate a clear influence of TPN on the increased accrual of BF in premature infants and implicate gestational modification in nutrient/caloric utilization as a principle regulator of body composition in premature newborns.

Hyperoxia-induced alterations in the pulmonary proteome of juvenile rats.

High inspired concentrations of oxygen (hyperoxia) are often necessary to counteract tissue hypoxia during the treatment of ARDS. Reactive oxygen species generated by hyperoxic therapy may influence the expression of the pulmonary proteome and the application of discovery proteomics to the hyperoxic lung has the potential to divulge mechanisms regulating the expression of specific proteins integral to lung injury and repair. The present study examined the proteome derived from 30-day-old Sprague-Dawley rats exposed to room air (RA) and 95% O2 (Ox) for 24-72 hours using 2-dimensional difference in-gel electrophoresis (2D-DIGE) coupled with MALDI-ToF/ToF mass spectrometry. A total of 870 protein spots were visualized by 2D-DIGE across all gels. Mass spectral analysis identified 51 proteins representing 187 of the 214 significantly altered spots. Molecular and cellular function analysis grouped the identified proteins into free radical scavenging, cell death, cell-to-cell signaling, and cellular movement categories. The majority of the differences in the protein spots between RA and Ox occurred at 72 hours, with albumin, annexin A6 (AnxA6), and transferrin being increased, and mitochondrial Lon peptidase 1 being decreased by at least 20%. In Ox animals, AnxA6 protein expression increased three-fold without an increase in mRNA expression. Bioinformatic analysis of the AnxA6 transcript revealed the presence of a putative internal ribosome entry site within the 5'-untranslated region. These findings indicate that hyperoxia induces significant alterations in the pulmonary proteome which are temporally related. In addition, hyperoxia selectively enhances the expression of some proteins whose transcripts contain sequence motifs, which impart translational regulation.

Hyperglycemia mediates a shift from cap-dependent to cap-independent translation via a 4E-BP1-dependent mechanism.

Diabetes and its associated hyperglycemia induce multiple changes in liver function, yet we know little about the role played by translational control of gene expression in mediating the responses to these conditions. Here, we evaluate the hypothesis that hyperglycemia-induced O-GlcNAcylation of the translational regulatory protein 4E-BP1 alters hepatic gene expression through a process involving the selection of mRNA for translation. In both streptozotocin (STZ)-treated mice and cells in culture exposed to hyperglycemic conditions, expression of 4E-BP1 and its interaction with the mRNA cap-binding protein eIF4E were enhanced in conjunction with downregulation of cap-dependent and concomitant upregulation of cap-independent mRNA translation, as assessed by a bicistronic luciferase reporter assay. Phlorizin treatment of STZ-treated mice lowered blood glucose concentrations and reduced activity of the cap-independent reporter. Notably, the glucose-induced shift from cap-dependent to cap-independent mRNA translation did not occur in cells lacking 4E-BP1. The extensive nature of this shift in translational control of gene expression was revealed using pulsed stable isotope labeling by amino acids in cell culture to identify proteins that undergo altered rates of synthesis in response to hyperglycemia. Taken together, these data provide evidence for a novel mechanism whereby O-GlcNAcylation of 4E-BP1 mediates translational control of hepatic gene expression.

Hyperoxia-induced activation of the integrated stress response in the newborn rat lung.

Diverse environmental stresses stimulate eukaryotic translation initiation factor 2α (eIF2α) phosphorylation, leading to a stress-resistant state characterized by global attenuation of protein synthesis and induction of cytoprotective genes. The signal transduction network culminating in these effects is referred to as the integrated stress response (ISR) or, when initiated by misfolded proteins within the endoplasmic reticulum (ER), the unfolded protein response (UPR). Given that we previously reported that exposure of 4-day-old Sprague-Dawley rats to 95% O(2) (Ox) diminishes global pulmonary protein synthesis and increases eIF2α phosphorylation, we conducted the current study to determine whether Ox activates the ISR or UPR. We found that Ox-induced alterations in ER morphology of alveolar type II cells and interstitial fibroblasts were not associated with activation of the UPR sensors PERK or activating transcription factor (ATF) 6 or with X-box binding protein-1 mRNA splicing in whole lung extracts. Exposure to Ox enhanced ATF4 immunoreactivity and nuclear protein content, followed by a 2- and 5-fold increase in ATF3 protein and mRNA expression, respectively. The accumulation of nuclear ATF4 protein coincided with induction of glutamate-cysteine ligase catalytic subunit, an ISR-responsive gene. Immunohistochemistry revealed that changes in ATF3/4 expression were prominent in the alveolus, whereas primary cell culture implicated epithelial and endothelial cells as targets. Finally, induction of ISR intermediates in the intact lung occurred in the absence of the phosphorylation of PKR, JNK, ERK1/2, and p38 MAPK. These findings demonstrate that Ox activates the ISR within the newborn lung and highlight regional and cell-specific alterations in the expression ISR transcription factors that regulate redox balance.

Hyperoxia inhibits protein synthesis and increases eIF2α phosphorylation in the newborn rat lung.

Prolonged exposure to hyperoxia contributes to aberrant lung growth in premature infants. Of the deleterious effects induced by hyperoxia, alterations in protein synthesis are likely to be of great importance to the developing lung. Regulation of mRNA translation occurs predominantly at the level of initiation via control of mRNA/ribosome binding by proteins known as eukaryotic initiation factors (eIF). Although hyperoxia is known to suppress mRNA translation in adult lungs, little is known regarding the effects in newborns or the involved mechanism. This study was performed to determine the effect of exposure to 95% O(2) on pulmonary protein synthesis in 4-day-old Sprague-Dawley rat pups. We found that hyperoxia suppressed the incorporation of [(3)H]phenylalanine into lung protein over time, resulting in a 23% reduction after 72 h compared with pups reared in room air. This effect was preceded by a shift in total lung RNA to lower order polysomes. Hyperoxia increased eIF4G-eIF4E binding, a surrogate maker of eIF4F complex assembly, and initially activated, then suppressed, the phosphorylation of ribosomal S6 kinase 1 and ribosomal S6 protein, downstream targets of mammalian target of rapamycin. Exposure to 95% O(2) enhanced the phosphorylation of the translational repressor eIF2α in whole lung extracts and the immunoreactivity of phosphorylated eIF2α in epithelial cells. Cell culture studies further demonstrated that hyperoxia increases eIF2α phosphorylation in lung epithelial cells, but not in lung fibroblasts. These findings illustrate that hyperoxia-induced suppression of mRNA translation in the newborn lung is accompanied by increased phosphorylation of eIF2α in the epithelium.

Hydrogen peroxide impairs insulin-stimulated assembly of mTORC1.

Oxidants are well recognized for their capacity to reduce the phosphorylation of the mammalian target of rapamycin (mTOR) substrates, eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) and p70 S6 kinase 1 (S6K1), thereby hindering mRNA translation at the level of initiation. mTOR functions to regulate mRNA translation by forming the signaling complex mTORC1 (mTOR, raptor, GbetaL). Insulin signaling to mTORC1 is dependent upon phosphorylation of Akt/PKB and the inhibition of the tuberous sclerosis complex (TSC1/2), thereby enhancing the phosphorylation of 4E-BP1 and S6K1. In this study we report the effect of H(2)O(2) on insulin-stimulated mTORC1 activity and assembly using A549 and bovine aortic smooth muscle cells. We show that insulin stimulated the phosphorylation of TSC2 leading to a reduction in raptor-mTOR binding and in the quantity of proline-rich Akt substrate 40 (PRAS40) precipitating with mTOR. Insulin also increased 4E-BP1 coprecipitating with mTOR and the phosphorylation of the mTORC1 substrates 4E-BP1 and S6K1. H(2)O(2), on the other hand, opposed the effects of insulin by increasing raptor-mTOR binding and the ratio of PRAS40/raptor derived from the mTOR immunoprecipitates in both cell types. These effects occurred in conjunction with a reduction in 4E-BP1 phosphorylation and the 4E-BP1/raptor ratio. siRNA-mediated knockdown of PRAS40 in A549 cells partially reversed the effect of H(2)O(2) on 4E-BP1 phosphorylation but not on S6K1. These findings are consistent with PRAS40 functioning as a negative regulator of insulin-stimulated mTORC1 activity during oxidant stress.

Hyperoxia enhances VEGF release from A549 cells via post-transcriptional processes.

Exposure of animals to hyperoxia decreases lung VEGF mRNA expression concomitant with an acute increase in VEGF protein within the epithelial lining fluid (ELF). The VEGF concentration in ELF is in excess of that found in the plasma, leading to the hypothesis that hyperoxia stimulates the release of VEGF protein from stores within the extracellular matrix. To test this hypothesis in a cell culture system, we exposed A549 cells to 95% O(2) (Ox) for 48 h followed by recovery in room air (RA) for 24 h. We found that Ox increased VEGF protein two- to threefold within the medium at 48 h of exposure and during recovery. Heparin clearing revealed the medium to contain a 50/50 mixture of the heparin-binding (VEGF(165)) and heparin-nonbinding (VEGF(121)) proteins and that Ox increased both proteins equally. Transcriptional activation of VEGF seems unlikely to explain the increase in VEGF protein, as expression of full-length and splice variant VEGF mRNA was unchanged by hyperoxia. Analysis of cell-associated VEGF proteins found that Ox increased the expression of VEGF(121) and VEGF(165) proteins. Blocking binding sites with exogenous heparin enhanced VEGF protein in the medium from RA-grown cells, whereas heparinase digestion of bound VEGF revealed a greater reserve of VEGF protein in RA cells. Collectively these findings indicate that hyperoxia enhances the expression of VEGF(121/165) proteins and facilitates the release of VEGF(165) from cell-associated stores. Increases in VEGF in ELF may represent an adaptive response fostering cell survival and type II cell proliferation in O(2)-induced lung injury.

Roles of mitogen-activated protein kinase signal-integrating kinases 1 and 2 in oxidant-mediated eIF4E phosphorylation.

Oxidative stress alters cellular metabolic processes including protein synthesis. The eukaryotic initiation factor, eIF4E, acts in the rate-limiting steps of initiation and promotes nuclear export. Phosphorylation of eIF4E by mitogen activated protein kinase signal-integrating kinases 1 and 2 (Mnk) influences the affinity of eIF4E for the 5'-mRNA cap and fosters nuclear export activity. Although phosphorylation of eIF4E on Ser209 is observed following oxidant exposure, the contribution of Mnk isoforms and the significance of phosphorylation remain elusive. Using a Mnk inhibitor and fibroblasts derived from Mnk knockout mice, we demonstrate that that H2O2 enhances eIF4E phosphorylation in cells containing Mnk1. In contrast, cells containing only Mnk2 show little change or a decrease in eIF4E phosphorylation in response to H2O2. H2O2 also shifted eIF4GI protein from the nucleus to the cytoplasm suggesting that the increases in eIF4E phosphorylation may reflect enhanced substrate availability to cytoplasmic Mnk1. In Mnk1(+/+) cells, H2O2 also enhanced eIF4E phosphorylation in the nucleus to a greater degree than in the cytoplasm, an effect not observed in cells containing Mnk2. In response to H2O2, all MEFs showed increased eIF4E:4E-BP1 and 4E-BP2:eIF4E binding and reduced eIF4E:eIF4GI binding. We also observed a dramatic increase in the amount of Mnk1 associated with eIF4E following affinity chromatography. These changes coincided with a smaller reduction in global protein synthesis in response to H2O2 in the DKO cells. These findings suggest that changes in eIF4GI distribution may enhance eIF4E phosphorylation and that the presence of either Mnk1 or 2 or any degree of eIF4E phosphorylation negatively regulates global protein synthesis in response to oxidant stress.

Cyclic stretch attenuates effects of hyperoxia on cell proliferation and viability in human alveolar epithelial cells.

The treatment of severe lung disease often requires the use of high concentrations of oxygen coupled with the need for assisted ventilation, potentially exposing the pulmonary epithelium to both reactive oxygen species and nonphysiological cyclic stretch. Whereas prolonged hyperoxia is known to cause increased cell injury, cyclic stretch may result in either cell proliferation or injury depending on the pattern and degree of exposure to mechanical deformation. How hyperoxia and cyclic stretch interact to affect the pulmonary epithelium in vitro has not been previously investigated. This study was performed using human alveolar epithelial A549 cells to explore the combined effects of cyclic stretch and hyperoxia on cell proliferation and viability. Under room air conditions, cyclic stretch did not alter cell viability at any time point and increased cell number after 48 h compared with unstretched controls. After exposure to prolonged hyperoxia, cell number and [(3)H]thymidine incorporation markedly decreased, whereas evidence of oxidative stress and nonapoptotic cell death increased. The combination of cyclic stretch with hyperoxia significantly mitigated the negative effects of prolonged hyperoxia alone on measures of cell proliferation and viability. In addition, cyclic stretch resulted in decreased levels of oxidative stress over time in hyperoxia-exposed cells. Our results suggest that cyclic stretch, as applied in this study, can minimize the detrimental effects of hyperoxia on alveolar epithelial A549 cells.

Hyperoxia alters the expression and phosphorylation of multiple factors regulating translation initiation.

Hyperoxia is cytotoxic and depresses many cellular metabolic functions including protein synthesis. Translational control is exerted primarily during initiation by two mechanisms: 1) through inhibition of translation initiation complex formation via sequestration of the cap-binding protein, eukaryotic initiation factor (eIF) 4E, with inhibitory 4E-binding proteins (4E-BP); and 2) by prevention of eIF2-GTP-tRNA(i)(Met) formation and eIF2B activity by phosphorylated eIF2alpha. In this report, exposure of human lung fibroblasts to 95% O2 decreased the incorporation of thymidine into DNA at 6 h and the incorporation of leucine into protein beginning at 12 h. The reductions in DNA and protein synthesis were accompanied by increased phosphorylation of eIF4E protein and reduced phosphorylation of 4E-BP1. At 24 h, hyperoxia shifted 4E-BP1 phosphorylation to lesser-phosphorylated isoforms, increased eIF4E expression, and increased the association of eIF4E with 4E-BP1. Although hyperoxia did not change eIF2alpha expression, it increased its phosphorylation at Ser51, but not until 48 h. In addition, the activation of eIF2alpha was not accompanied by the formation of stress granules. These findings suggest that hyperoxia diminishes protein synthesis by increasing eIF4E phosphorylation and enhancing the affinity of 4E-BP1 for eIF4E.

Oxidant-induced hypertrophy of A549 cells is accompanied by alterations in eukaryotic translation initiation factor 4E and 4E-binding protein-1.

Control of protein synthesis resides at the level of eukaryotic translation initiation (eIF) complex formation. Complex formation is regulated by the mRNA cap-binding protein, eIF4E, whose activity is influenced by phosphorylation and binding to 4E-binding protein 1 (4E-BP1). To provide a link between alterations in protein synthesis and the pathogenesis of oxidant-mediated lung disease, we investigated the effect of hydrogen peroxide (H2O2) on actively growing A549 cells. Cells were exposed to 200 or 400 microM H2O2 for 4 h and then assessed for changes in proliferation, protein synthesis, and eIF4E and 4E-BP1 status over 72 h. We found that both concentrations of H2O2 inhibited [3H]thymidine incorporation and cell division while inducing a G2/M-predominant growth arrest within 24 h. In addition, H2O2 increased cell size, [3H]leucine incorporation/cell, and total cell protein. Although time had little effect on eIF4E and 4E-BP1 expression and phosphorylation state of control cells, H2O2 induced a 2- to 3-fold increase in eIF4E and 4E-BP1 expression, a 5-fold increase in eIF4E phosphorylation, and a shift in the distribution of 4E-BP1 phosphorylation favoring lesser phosphorylated forms. These findings suggest that oxidant-mediated alterations in protein synthesis and cell morphology occur in concert with changes in factors known to regulate translation kinetics.