Congenital diaphragmatic hernia (CDH) is a severe birth defect that is characterized by pulmonary hypoplasia and pulmonary hypertension (PHTN). PHTN secondary to CDH is a result of vascular ...remodeling, a structural alteration in the pulmonary vessel wall that occurs in the fetus. Factors involved in vascular remodeling have been reported in several studies, but their interactions remain unclear. To help understand PHTN pathophysiology and design novel preventative and treatment strategies, we have conducted a systematic review of the literature and comprehensively analyzed all factors and pathways involved in the pathogenesis of pulmonary vascular remodeling secondary to CDH in the nitrofen model. Moreover, we have linked the dysregulated factors with pathways involved in human CDH. Of the 358 full-text articles screened, 75 studies reported factors that play a critical role in vascular remodeling secondary to CDH. Overall, the impairment of epithelial homeostasis present in pulmonary hypoplasia results in altered signaling to endothelial cells, leading to endothelial dysfunction. This causes an impairment of the crosstalk between endothelial cells and pulmonary artery smooth muscle cells, resulting in increased smooth muscle cell proliferation, resistance to apoptosis, and vasoconstriction, which clinically translate into PHTN.
To determine the global prevalence for congenital diaphragmatic hernia (CDH) and identify CDH-related risk factors.
Using a defined strategy, a systematic review of the literature was conducted ...according to PRISMA guidelines, searching for population-based epidemiological studies to evaluate the prevalence of CDH globally and per country. Studies containing overlapping populations or timeframes were excluded. CDH-related risk factors were calculated by meta-analysis using RevMan5.3 and expressed as risk ratio and 95% confidence interval.
Prevalence: Of 8230 abstracts screened, 30 full-text articles published between 1980 and 2019 were included. The overall prevalence of CDH was 2.3 in 10,000 births (16,710 CDH babies in 73,663,758 livebirths). Risk factors: From 9 studies we found that male sex RR 1.38 (1.05–1.80), p=0.02 and maternal age >35 years RR 1.69 (1.26–2.25), p=0.0004 were associated with CDH. Conversely, maternal black ethnicity resulted as a protective factor RR 0.82 (0.77–0.89, p<0.00001.
This study reveals that there is a worldwide paucity of population-based studies, and those studies that report on prevalence and risk factors come from a small number of countries. The prevalence of CDH varies within and across geographical world regions. The main risk factors for CDH identified are male sex and older maternal age. More epidemiological studies, involving more world regions, are needed to identify possible strategies to help strengthen our understanding of the risk factors, provide clinicians with the tools necessary for prenatal and postnatal counseling, and inform policy makers on how to strategize CDH care in different parts of the world.
Systematic review and meta-analysis.
Level III.
Necrotizing enterocolitis (NEC) is characterized by intestinal injury and impaired mucin synthesis. We recently showed that breast milk exosomes from rodents promote intestinal cell viability, ...epithelial proliferation, and stem cell activity, but whether they also affect mucus production is unknown. Therefore, the aim of this study was to investigate the effects of bovine milk-derived exosomes on goblet cell expression in experimental NEC and delineate potential underlying mechanisms of action. Exosomes were isolated from bovine milk by ultracentrifugation and confirmed by Nanoparticle Tracking Analysis and through the detection of exosome membrane markers. To study the effect on mucin production, human colonic LS174T cells were cultured and exposed to exosomes. Compared to control, exosomes promoted goblet cell expression, as demonstrated by increased mucin production and relative expression levels of goblet cell expression markers trefoil factor 3 (TFF3) and mucin 2 (MUC2). In addition, exosome treatment enhanced the expression of glucose-regulated protein 94 (GRP94), the most abundant intraluminal endoplasmic reticulum (ER) chaperone protein that aids in protein synthesis. Furthermore, experimental NEC was induced in mouse pups by hyperosmolar formula feeding, lipopolysaccharide administration and hypoxia exposure on postnatal days 5-9. Milk exosomes were given with each gavage feed. NEC was associated with ileal morphological injury and reduction in MUC2+ goblet cells and GRP94+ cells per villus. Exosome administration to NEC pups prevented these changes. This research highlights the potential novel application of milk-derived exosomes in preventing the development of NEC in high-risk infants when breast milk is not available.
The transcriptional pathways activated downstream of vascular endothelial growth factor (VEGF) signaling during angiogenesis remain incompletely characterized. By assessing the signals responsible ...for induction of the Notch ligand delta-like 4 (DLL4) in endothelial cells, we find that activation of the MAPK/ERK pathway mirrors the rapid and dynamic induction of
transcription and that this pathway is required for
expression. Furthermore, VEGF/ERK signaling induces phosphorylation and activation of the ETS transcription factor ERG, a prerequisite for
induction. Transcription of
coincides with dynamic ERG-dependent recruitment of the transcriptional co-activator p300. Genome-wide gene expression profiling identified a network of VEGF-responsive and ERG-dependent genes, and ERG chromatin immunoprecipitation (ChIP)-seq revealed the presence of conserved ERG-bound putative enhancer elements near these target genes. Functional experiments performed
and
confirm that this network of genes requires ERK, ERG and p300 activity. Finally, genome-editing and transgenic approaches demonstrate that a highly conserved ERG-bound enhancer located upstream of
(which encodes a transcription factor implicated in sprouting angiogenesis) is required for its VEGF-mediated induction. Collectively, these findings elucidate a novel transcriptional pathway contributing to VEGF-dependent angiogenesis.
The regulatory elements controlling gene expression during acute inflammation are not fully elucidated. Here we report the identification of a set of NF-κB-bound elements and common chromatin ...landscapes underlying the acute inflammatory response across cell-types and mammalian species. Using primary vascular endothelial cells (human/mouse/bovine) treated with the pro-inflammatory cytokine, Tumor Necrosis Factor-α, we identify extensive (~30%) conserved orthologous binding of NF-κB to accessible, as well as nucleosome-occluded chromatin. Regions with the highest NF-κB occupancy pre-stimulation show dramatic increases in NF-κB binding and chromatin accessibility post-stimulation. These 'pre-bound' regions are typically conserved (~56%), contain multiple NF-κB motifs, are utilized by diverse cell types, and overlap rare non-coding mutations and common genetic variation associated with both inflammatory and cardiovascular phenotypes. Genetic ablation of conserved, 'pre-bound' NF-κB regions within the super-enhancer associated with the chemokine-encoding CCL2 gene and elsewhere supports the functional relevance of these elements.
Extracellular vesicles (EVs) derived from amniotic fluid stem cells (AFSCs) mediate anti-apoptotic, pro-angiogenic, and immune-modulatory effects in multiple disease models, such as skeletal muscle ...atrophy and Alport syndrome. A source of potential variability in EV biological functions is how EV are isolated from parent cells. Currently, a comparative study of different EV isolation strategies using conditioned medium from AFSCs is lacking. Herein, we examined different isolation strategies for AFSC-EVs, using common techniques based on differential sedimentation (ultracentrifugation), solubility (ExoQuick, Total Exosome Isolation Reagent, Exo-PREP), or size-exclusion chromatography (qEV). All techniques isolated AFSC-EVs with typical EV morphology and protein markers. In contrast, AFSC-EV size, protein content, and yield varied depending on the method of isolation. When equal volumes of the different AFSC-EV preparations were used as treatment in a model of lung epithelial injury, we observed a significant variation in how AFSC-EVs were able to protect against cell death. AFSC-EV enhancement of cell survival appeared to be dose dependent, and largely uninfluenced by variation in EV-size distributions, relative EV-purity, or their total protein content. The variation in EV-mediated cell survival obtained with different isolation strategies emphasizes the importance of testing alternative isolation techniques in order to maximize EV regenerative capacity.
Severe COVID-19 infection results in bilateral interstitial pneumonia, often leading to acute respiratory distress syndrome (ARDS) and pulmonary fibrosis in survivors. Most patients with severe ...COVID-19 infections who died had developed ARDS. Currently, ARDS is treated with supportive measures, but regenerative medicine approaches including extracellular vesicle (EV)-based therapies have shown promise. Herein, we aimed to analyse whether EV-based therapies could be effective in treating severe pulmonary conditions that affect COVID-19 patients and to understand their relevance for an eventual therapeutic application to human patients. Using a defined search strategy, we conducted a systematic review of the literature and found 39 articles (2014-2020) that reported effects of EVs, mainly derived from stem cells, in lung injury models (one large animal study, none in human). EV treatment resulted in: (1) attenuation of inflammation (reduction of pro-inflammatory cytokines and neutrophil infiltration, M2 macrophage polarization); (2) regeneration of alveolar epithelium (decreased apoptosis and stimulation of surfactant production); (3) repair of microvascular permeability (increased endothelial cell junction proteins); (4) prevention of fibrosis (reduced fibrin production). These effects were mediated by the release of EV cargo and identified factors including miRs-126, −30b-3p, −145, −27a-3p, syndecan-1, hepatocyte growth factor and angiopoietin-1. This review indicates that EV-based therapies hold great potential for COVID-19 related lung injuries as they target multiple pathways and enhance tissue regeneration. However, before translating EV therapies into human clinical trials, efforts should be directed at developing good manufacturing practice solutions for EVs and testing optimal dosage and administration route in large animal models.
Abstract Congenital diaphragmatic hernia (CDH) is a birth defect characterized by incomplete closure of the diaphragm, herniation of abdominal organs into the chest, and compression of the lungs and ...the heart. Besides complications related to pulmonary hypoplasia, 1 in 4 survivors develop neurodevelopmental impairment, whose etiology remains unclear. Using a fetal rat model of CDH, we demonstrated that the compression exerted by herniated organs on the mediastinal structures results in decreased brain perfusion on ultrafast ultrasound, cerebral hypoxia with compensatory angiogenesis, mature neuron and oligodendrocyte loss, and activated microglia. In CDH fetuses, apoptosis was prominent in the subventricular and subgranular zones, areas that are key for neurogenesis. We validated these findings in the autopsy samples of four human fetuses with CDH compared to age- and sex-matched controls. This study reveals the molecular mechanisms and cellular changes that occur in the brain of fetuses with CDH and creates opportunities for therapeutic targets.
Despite advances in intensive care, several neonatal conditions typically due to prematurity affect vital organs and are associated with high mortality and long-term morbidities. Current treatment ...strategies for these babies are only partially successful or are effective only in selected patients. Regenerative medicine has been shown to be a promising option for these conditions at an experimental level, but still warrants further exploration for the development of optimal treatment. Although stem cell-based therapy has emerged as a treatment option, studies have shown that it is associated with potential risks and hazards, especially in the fragile population of babies. Recently, extracellular vesicles (EVs) have emerged as an attractive therapeutic alternative that holds great regenerative potential and is cell-free. EVs are nanosized particles endogenously produced by cells that mediate intercellular communication through the transfer of their cargo. Currently, EVs are garnering considerable attention as they are the key effectors of stem cell paracrine signaling and can epigenetically regulate target cell genes through the release of RNA species, such as microRNA. Herein, we review the emerging literature on the therapeutic potential of EVs derived from different sources for the treatment of neonatal conditions that affect the brain, retinas, spine, lungs, and intestines and discuss the challenges for the translation of EVs into clinical practice.
Necrotizing enterocolitis (NEC) is an inflammatory gastrointestinal disease primarily affecting preterm neonates. Neonates with NEC suffer from a degree of neurodevelopmental delay that is not ...explained by prematurity alone. There is a need to understand the pathogenesis of neurodevelopmental delay in NEC. In this study, we assessed the macroscopic and microscopic changes that occur to brain cell populations in specific brain regions in a neonatal mouse model of NEC. Moreover, we investigated the role of intestinal inflammation as part of the mechanism responsible for the changes observed in the brain of pups with NEC.
Brains of mice were assessed for gross morphology and cerebral cortex thickness (using histology). Markers for mature neurons, oligodendrocytes, neural progenitor cells, microglia, and astrocytes were used to quantify their cell populations in different regions of the brain. Levels of cell apoptosis in the brain were measured by Western blotting and immunohistochemistry. Endoplasmic reticulum (ER) stress markers and levels of pro-inflammatory cytokines (in the ileum and brain) were measured by RT-qPCR and Western blotting. A Pearson test was used to correlate the levels of cytokines (ELISA) in the brain and ileum and to correlate activated microglia and astrocyte populations to the severity of NEC.
NEC pups had smaller brain weights, higher brain-to-body weight ratios, and thinner cortices compared to control pups. NEC pups had increased levels of apoptosis and ER stress. In addition, NEC was associated with a reduction in the number of neurons, oligodendrocytes, and neural progenitors in specific regions of the brain. Levels of pro-inflammatory cytokines and the density of activated microglia and astrocytes were increased in the brain and positively correlated with the increase in the levels pro-inflammatory cytokines in the gut and the severity of NEC damage respectively.
NEC is associated with severe changes in brain morphology, a pro-inflammatory response in the brain that alters cell homeostasis and density of brain cell populations in specific cerebral regions. We show that the severity of neuroinflammation is associated with the severity of NEC. Our findings suggest that early intervention during NEC may reduce the chance of acute neuroinflammation and cerebral damage.
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