Extremely low gestational age neonates (ELGANs) are born in a relatively hyperoxic environment with weak antioxidant defenses, placing them at high risk for mitochondrial dysfunction affecting multiple organ systems including the nervous, respiratory, ocular, and gastrointestinal systems. The brain and lungs are highly affected by mitochondrial dysfunction and dysregulation in the neonate, causing white matter injury (WMI) and bronchopulmonary dysplasia (BPD), respectively. Adequate mitochondrial function is important in providing sufficient energy for organ development as it relates to alveolarization and axonal myelination and decreasing oxidative stress via reactive oxygen species (ROS) and reactive nitrogen species (RNS) detoxification. Peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) is a master regulator of mitochondrial biogenesis and function. Since mitochondrial dysfunction is at the root of WMI and BPD pathobiology, exploring therapies that can regulate PGC-1α activity may be beneficial. This review article describes several promising therapeutic agents that can mitigate mitochondrial dysfunction through direct and indirect activation and upregulation of the PGC-1α pathway. Metformin, resveratrol, omega 3 fatty acids, montelukast, L-citrulline, and adiponectin are promising candidates that require further pre-clinical and clinical studies to understand their efficacy in decreasing the burden of disease from WMI and BPD in preterm infants.
Introduction Bronchopulmonary dysplasia (BPD) is a chronic lung disease initiated by inflammation and oxidative stress during the early stages of lung development, disrupting alveolar growth. BPD damages alveolar Type 1 (AT1) epithelial cells, causing decreased alveolarization and impaired gas exchange. Recently, it was shown that AT1 cells undergo cell reprogramming into surfactant producing alveolar Type 2 cells (AT2) in hyperoxic and hypoxic lung injury models, causing significant structural damage in the neonatal lung. While current treatments have improved neonatal care, they cause significant side effects. L‐citrulline, a naturally occurring amino acid, protects the newborn rat lung from injury, preserving alveolar growth in various neonatal BPD models. Due to the significance of inflammation in BPD, we sought to investigate the effect of inflammatory stimuli, lipopolysaccharide (LPS) and tumour necrosis factor α (TNF‐α), and L‐citrulline treatment on AT1 cell programming in vitro. We hypothesize that L‐citrulline treatment will prevent inflammation‐induced AT1 cell reprogramming in vitro. Methods AT1 cells were isolated from newborn rats on postnatal day 4 using fluorescence activated cell sorting. AT1 cells were pre‐treated with L‐citrulline (4mM/mL or 10mM/mL) for 1hr prior to LPS (10µg/mL), TNF‐α (5ng/mL), or a combination of LPS and TNF‐α for 24hrs in vitro. Whole cell lysates were prepared for immunoblotting. AT1 cells were also assayed for the level of injury‐induced cell viability using the MTT assay. Results AT1 cell lysates from the LPS+TNF‐α group had significantly decreased RTI‐40 protein expression compared to the control group (p<0.04 LPS+TNF‐α vs. control, n=6). In the presence of L‐citrulline, RTI‐40 protein expression of cells exposed to LPS+ TNF‐α was maintained (p<0.02 L‐citrulline+LPS+TNF‐α vs. LPS+TNF‐α, n=4) (Figure 1). AT1 cell viability in LPS, TNF‐α, and the combined exposure group was comparable to the control group (n=8) (Figure 2). Conclusion Our preliminary data strongly suggests that L‐citrulline treatment mitigates inflammation‐induced cell reprogramming in neonatal AT1 cells. Our findings show that the injury‐induced decrease in RTI‐40 expression is not due to reduced AT1 cell viability, rather due to phenotypic changes. Given the importance of AT1 cells in alveolarization, gas exchange, and BPD pathogenesis, characterizing the cell specific effects of L‐citrulline on AT1 cell reprograming is considerably critical and highly relevant to identifying a safe and effective treatment for premature infants that can mitigate the effects of inflammation on the developing lung.
Background: Activated macrophages can produce macrophage extracellular traps (METs) - DNA strands coated with cytotoxic components that are released to immobilize pathogens and control infection. However, METs can also contribute to the inflammatory process and lead to severe tissue damage. Bronchopulmonary dysplasia (BPD) is a chronic lung disease in premature infants initiated by inflammation and oxidative stress. Gram-negative bacteria and candida increase the risk of developing BPD and are strong inducers of extracellular traps. Macrophages are the most abundant immune cells in the neonatal lung tissue, responsible for controlling the inflammatory process. We aimed to determine whether inflammation triggers METosis in a neonatal model of inflammatory lung injury. Methods: Alveolar macrophages (AMs) from bronchoalveolar lavage fluid (BALF) were isolated from Sprague Dawley rat pups on postnatal day (PND) 8. In vitro - AMs were incubated with METosis agonists (lipopolysaccharide [LPS] and phorbol myristate acetate [PMA]) or control (media) for 4h. Cells were fixed and stained for markers of METosis (myeloperoxidase [MPO] and citrullinated histone H3 [CitH3]) and DNA (DAPI) for immunofluorescence imaging. In vivo - PND6-9 pups were treated with LPS or saline intrapharyngeally and DNase enzyme or saline intraperitoneally. BALF was collected on PND10. BALF was analysed using immunofluoresence imaging and picogreen assay. Results: Immunofluorescence imaging of neonate AMs showed increased expression of METosis markers MPO, CitH3, and DNA strands with in vitro exposure to METosis agonists PMA or LPS compared to control (n =3). In vivo LPS exposure also showed evidence of increased extracellular trap formation in the BALF collected from neonate rat lung compared to control (n=3; p<0.0001); further, rats exposed to both LPS and DNAse enzyme showed decreased extracellular trap formation (p = 0.002) in comparison to the rats exposed to LPS and saline. Conclusions: To the best of our knowledge, this study is first to establish (i) rat AMs undergo METosis, (ii) a plausible role for METosis in lung injury (iii), and the potential relevance of METosis in lung injury during early development. Novel therapies targeting METosis formation may reduce lung inflammation contributing to BPD in premature infants. This work was supported by the Women's Auxiliary Fund, The Hospital for Sick Children, Toronto, Ontario, Canada This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.
Introduction: Bronchopulmonary dysplasia (BPD) is the most common lung disease in premature infants. Inflammation and oxidative stress during the early stages of lung development damage alveolar Type 1 (AT1) and Type 2 (AT2) epithelial cells that line the alveoli, leading to decreased alveolarization and surfactant production. Unlike AT2 cells, the role of AT1 cells in inflammation is not understood. Since inflammation initiates BPD pathogenesis, we sought to characterize the profile of inflammatory markers produced by neonatal AT1 cells in response to lipopolysaccharide (LPS) and tumour necrosis factor α (TNF-α). Methods: AT1 cells were isolated from neonatal rats on postnatal day 4 using fluorescence activated cell sorting. Cells were serum starved for 24hrs and then exposed to LPS (5, 10, 25μg/mL), TNF-α (5, 10, 25ng/mL), or a combination of LPS and TNF-α (10μg/mL and 5ng/mL) for 18h in vitro. Conditioned media was collected for cytokine and chemokine analysis by multiplex ELISA. Results: AT1 cells exposed to both LPS and TNF-α produced a larger inflammatory response than those exposed to LPS or TNF-α alone. After 18h, AT1 cells from the combined LPS and TNF-α group increased the production of IL-6 by 243-fold, CCL2 by 34-fold, and CCL3 by 9-fold in comparison to the control (all p<0.05, n=4). IL-1β and IFN-γ, and the anti-inflammatory cytokines, IL-4 and IL-10, were not detected in any experimental groups at this time point. Conclusion: This is the first study that characterizes the response of neonatal AT1 cells to inflammatory stimuli. Our preliminary findings show that LPS and TNF-α stimulate the production of the pro-inflammatory cytokines and chemokines IL-6, CCL2, and CCL3 in primary AT1 cells. These inflammatory markers are strongly associated with the development of BPD and are predictors of adverse pulmonary outcomes in neonates. Developing therapies that regulate the inflammatory response produced by AT1 cells may be beneficial in mitigating BPD pathogenesis. This work was funded by the Women's Auxiliary of the Hospital for Sick Children. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.
Introduction Bronchopulmonary dysplasia (BPD), a chronic lung disease in premature infants resulting from inflammation and oxidative stress, leads to arrested pulmonary development and lifelong respiratory morbidities. Macrophages are the most abundant immune cells in the neonatal lung, orchestrating inflammation, maintaining homeostasis, and supporting tissue remodeling and repair. Macrophages can polarize into specific phenotypes in response to the micro‐environment. M1 macrophages are pro‐inflammatory and pathogen killing whereas, M2 macrophages are anti‐inflammatory and support normal lung development. Existing therapies for BPD have inherent side effects. L‐Citrulline (L‐CIT), a non‐essential amino acid, that is safe and well‐tolerated by infants, reduces oxidative stress and lung inflammation. Thus, we hypothesize that L‐CIT modulates macrophage polarization to an M2 phenotype in the presence of lipopolysaccharide (LPS) induced lung injury during early development. Methods Sprague Dawley rat pups received saline (SAL) or L‐CIT (2.5 g/kg) intraperitoneally during postnatal day (PND) 4‐9. In addition, pups were treated with LPS (5mg/kg) or SAL intrapharyngeally during PND 5‐8. Thus, we had 4 treatment groups, ie., SAL+SAL (controls), LPS+SAL, LPS+L‐CIT and L‐CIT+SAL. Pups were euthanized on PND 9. Bronchoalveolar Lavage Fluid (BALF): BALF was cytospinned, and cells were counted following hematoxylin and eosin staining. BALF supernatant was collected, and an arginase activity assay was performed for M2 marker expression. Lung homogenate: Lungs were dissected, homogenized and processed for western blotting of M2 (CD206) and M1 (Arg2) markers normalized to rat macrophage marker (F4/80). Results BALF analysis: The number of alveolar macrophages infiltrated in the BALF of animals treated with L‐CIT+LPS (n=4) and those treated with SAL+LPS (n=4) were similar; both were significantly higher than control (p<.001; p<.0001, respectively). Arginase activity in the BALF was significantly higher in animals treated with L‐CIT+LPS (p<.05; n= 4) compared to LPS alone (p<.05; n=4). Lung homogenate analysis: LPS+SAL treated animals had lower levels of protein expression for CD206 (M2 marker) (p<.05; n=4) compared to control animals. Whereas L‐CIT+LPS treated animals had increased expression of CD206 (M2 marker) (p<.05; n=4) and lower expression of Arg2 (M1 marker) (p<.001; n=4) compared to control animals. Conclusion In BPD pathogenesis, there is a major shift towards M1 polarization due to the presence of pro‐inflammatory stimuli in the microenvironment, leading to arrested lung development. Our preliminary findings support a role for L‐CIT in regulating macrophage polarization, favoring an anti‐inflammatory M2 phenotype in the presence of inflammatory injury. This further suggests that L‐CIT, a safe nutritional supplement, could reduce inflammatory lung injury during early development.
Introduction Premature infants are at risk for bronchopulmonary dysplasia (BPD), a disorder characterized by abnormal pulmonary development. Inflammation is one major component in causing BPD, which can further lead to abnormal vascular remodelling, causing pulmonary hypertension (PH). During this process, pulmonary artery smooth muscle cell (PASMC) proliferation and hypertrophy contribute to the thickening of the pulmonary artery wall, leading to heightened blood pressures. 5‐hydroxytryptamine (5‐HT) or serotonin has been implicated in PH and is involved in the pathogenesis of PH in adults. Meanwhile, L‐citrulline, a nitric oxide donor, has been effective in reducing PH in acute models of BPD. The role of 5‐HT signalling on PASMC proliferation during early pulmonary vascular development is currently unknown. Thus, the objective of this study is to establish the effect of 5‐HT on PASMC proliferation in a newborn model of early lung development, and to elucidate the interaction of 5‐HT on proliferation of PASMCs in the presence of LPS and L‐citrulline. Methods Sprague Dawley rat pups were grown and euthanized on postnatal day 4, after which the pulmonary arteries (PA) were isolated. Primary cultures of PASMCs were grown from PA explants and treated with varying concentrations of 5‐HT. Proliferation and cell viability assays were performed to measure growth and cell viability after exposure to 5‐HT and L‐citrulline (4mM). Lysate samples were also prepared with the cells to measure protein expression via Western blotting for Cyclin D1, an important protein involved in regulation of the cell cycle, and phosphorylated AKT (p‐AKT), which is involved in cell survival. To investigate the effects of inflammation on 5‐HT signalling, PASMCs were exposed to lipopolysaccharide (LPS, 3 µg/mL) and lysate samples were prepared to measure changes in the expression of the 5‐HT2A receptor. Results Proliferation assays illustrated a dose‐dependent increase in growth of PASMCs exposed to 5‐HT, reaching a peak 150% growth rate (p < 0.05). Western blots revealed a three‐fold increase in Cyclin D1 expression (p < 0.01) and two‐fold increase in p‐AKT expression (p < 0.05) in PASMCs exposed to 5‐HT compared to protein expression of control PASMCs. Both changes were abrogated in PASMCs exposed to both L‐citrulline and 5‐HT, bringing expression of both proteins to control levels. When exposed to LPS, PASMCs have heightened expression of 5‐HT2A, reaching nearly two times higher than in control PASMCs. This effect is also reversed in PASMCs exposed to LPS and L‐citrulline (p < 0.05). Conclusion Our preliminary data support a role for 5‐HT in pathogenesis on BPD‐PH in the presence and absence of inflammation in a newborn model of lung development, and L‐citrulline may be an attractive safe therapeutic option to reverse or mitigate inflammation induced PH in a newborn model, which could later be translated to human infants.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
334 Leonard St
Brooklyn, NY 11211
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.