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Port Wine Birthmarks (PWBs) are a congenital vascular malformation on the skin, occurring in 1–3 per 1000 live births. We have recently generated PWB-derived induced pluripotent stem cells (iPSCs) as clinically relevant disease models. The metabolites associated with the pathological phenotypes of PWB-derived iPSCs are unknown, and so we aim to explore them in this study. Metabolites were separated by ultra-performance liquid chromatography and screened with electrospray ionization mass spectrometry. Orthogonal partial least-squares discriminant, multivariate, and univariate analyses were used to identify differential metabolites (DMs). KEGG analysis was used to determine the enrichment of metabolic pathways. A total of 339 metabolites was identified. There were 22 DMs, among which nine were downregulated—including sphingosine—and 13 were upregulated, including glutathione in PWB iPSCs, as compared to controls. Pathway enrichment analysis confirmed the upregulation of glutathione and the downregulation of sphingolipid metabolism in PWB-derived iPSCs as compared to normal ones. We next examined the expression patterns of the key molecules associated with glutathione metabolism in PWB lesions. We found that hypoxia-inducible factor 1α (HIF1α), glutathione S-transferase Pi 1 (GSTP1), γ-glutamyl transferase 7 (GGT7), and glutamate cysteine ligase modulatory subunit (GCLM) were upregulated in PWB vasculatures as compared to blood vessels in normal skin. Other significantly affected metabolic pathways in PWB iPSCs included pentose and glucuronate interconversions; amino sugar and nucleotide sugars; alanine, aspartate, and glutamate; arginine, purine, D-glutamine, and D-glutamate; arachidonic acid, glyoxylate, and dicarboxylate; nitrogen, aminoacyl-tRNA biosynthesis, pyrimidine, galactose, ascorbate, and aldarate; and starch and sucrose. Our data demonstrated that there were perturbations in sphingolipid and cellular redox homeostasis in PWB vasculatures, which could facilitate cell survival and pathological progression. Our data implied that the upregulation of glutathione could contribute to laser-resistant phenotypes in some PWB vasculatures.

Lesional iPSCs-derived ECs can resemble pathological vascular phenotypes of PWB. Our data demonstrate multiple pathways, such as Hippo and Wnt, NF-kappa B, TNF, MAPK, and cholesterol metabolism, are dysregulated. This data suggests new therapeutics to be developed for targeting such dysregulated pathways for treatment of PWB.

BACKGROUND: Port wine birthmark (PWB) is a congenital vascular malformation resulting from developmentally defective endothelial cells (ECs). Developing clinically relevant disease models for PWB studies is currently an unmet need. OBJECTIVE: Our study aims to generate PWB-derived induced pluripotent stem cells (iPSCs) and iPSC-derived ECs that preserve disease-related phenotypes. METHODS: PWB iPSCs were generated by reprogramming lesional dermal fibroblasts and differentiated into ECs. RNA-seq was performed to identify differentially expressed genes (DEGs) and enriched pathways. The functional phenotypes of iPSC-derived ECs were characterized by capillary-like structure (CLS) formation in vitro and Geltrex plug-in assay in vivo . RESULTS: Human PWB and control iPSC lines were generated through reprogramming of dermal fibroblasts by introducing the "Yamanaka factors" (Oct3/4, Sox2, Klf4, c-Myc) into them; the iPSCs were successfully differentiated into ECs. These iPSCs and their derived ECs were validated by expression of a series of stem cell and EC biomarkers, respectively. PWB iPSC-derived ECs showed impaired CLS in vitro with larger perimeters and thicker branches as compared to control iPSC-derived ECs. In the plug-in assay, perfused human vasculature formed by PWB iPSC- derived ECs showed bigger perimeters and greater densities than those formed by control iPSC- derived ECs in severe combined immune deficient (SCID) mice. The transcriptome analysis showed that dysregulated pathways of stem cell differentiation, Hippo, Wnt, and focal adhesion persisted through differentiation of PWB iPSCs to ECs. Functional enrichment analysis showed that Hippo and Wnt pathway-related PWB DEGs are enriched for vasculature development, tube morphology, endothelium development, and EC differentiation. Further, members of the zinc finger (ZNF) gene family were overrepresented among the DEGs in PWB iPSCs. ZNF DEGs confer significant functions in transcriptional regulation, chromatin remodeling, protein ubiquitination, and retinoic acid receptor signaling. Furthermore, NF-kappa B, TNF, MAPK, and cholesterol metabolism pathways were dysregulated in PWB ECs as readouts of impaired differentiation. CONCLUSIONS: PWB iPSC-derived ECs render a novel and clinically-relevant disease model by retaining pathological phenotypes. Our data demonstrate multiple pathways, such as Hippo and Wnt, NF-kappa B, TNF, MAPK, and cholesterol metabolism, are dysregulated, which may contribute to the development of differentiation-defective ECs in PWB. BULLETED STATEMENTS: What is already known about this topic?: Port Wine Birthmark (PWB) is a congenital vascular malformation with an incidence rate of 0.1 - 0.3 % per live births.PWB results from developmental defects in the dermal vasculature; PWB endothelial cells (ECs) have differentiational impairments.Pulse dye laser (PDL) is currently the preferred treatment for PWB; unfortunately, the efficacy of PDL treatment of PWB has not improved over the past three decades.What does this study add?: Induced pluripotent stem cells (iPSCs) were generated from PWB skin fibroblasts and differentiated into ECs.PWB ECs recapitulated their pathological phenotypes such as forming enlarged blood vessels in vitro and in vivo.Hippo and Wnt pathways were dysregulated in PWB iPSCs and ECs.Zinc-finger family genes were overrepresented among the differentially expressed genes in PWB iPSCs.Dysregulated NF-kappa B, TNF, MAPK, and cholesterol metabolism pathways were enriched in PWB ECs.What is the translational message?: Targeting Hippo and Wnt pathways and Zinc-finger family genes could restore the physiological differentiation of ECs.Targeting NF-kappa B, TNF, MAPK, and cholesterol metabolism pathways could mitigate the pathological progression of PWB.These mechanisms may lead to the development of paradigm-shifting therapeutic interventions for PWB.

BackgroundTransmural healing (TH) is associated with better long-term outcomes in Crohn disease (CD), whereas pretreatment ileal gene signatures encoding myeloid inflammatory responses and extracellular matrix production are associated with stricturing. We aimed to develop a predictive model for ileal TH and to identify ileal genes and microbes associated with baseline luminal narrowing (LN), a precursor to strictures.Materials and methodsBaseline small bowel imaging obtained in the RISK pediatric CD cohort study was graded for LN. Ileal gene expression was determined by RNASeq, and the ileal microbial community composition was characterized using 16S rRNA amplicon sequencing. Clinical, demographic, radiologic, and genomic variables were tested for association with baseline LN and future TH.ResultsAfter controlling for ileal location, baseline ileal LN (odds ratio [OR], 0.3; 95% confidence interval [CI], 0.1-0.8), increasing serum albumin (OR, 4; 95% CI, 1.3-12.3), and anti-Saccharomyces cerevisiae antibodies IgG serology (OR, 0.97; 95% CI, 0.95-1) were associated with subsequent TH. A multivariable regression model including these factors had excellent discriminant power for TH (area under the curve, 0.86; positive predictive value, 80%; negative predictive value, 87%). Patients with baseline LN exhibited increased Enterobacteriaceae and inflammatory and extracellular matrix gene signatures, coupled with reduced levels of butyrate-producing commensals and a respiratory electron transport gene signature. Taxa including Lachnospiraceae and the genus Roseburia were associated with increased respiratory and decreased inflammatory gene signatures, and Aggregatibacter and Blautia bacteria were associated with reduced extracellular matrix gene expression.ConclusionsPediatric patients with CD with LN at diagnosis are less likely to achieve TH. The association between specific microbiota, wound healing gene programs, and LN may suggest future therapeutic targets.

Ileal strictures are the major indication for resective surgery in Crohn's disease (CD). We aimed to define ileal gene programs present at diagnosis linked with future stricturing behavior during five year follow-up, and to identify potential small molecules to reverse these gene signatures. Antimicrobial serologies and pre-treatment ileal gene expression were assessed in a representative subset of 249 CD patients within the RISK multicenter pediatric CD inception cohort study, including 113 that are unique to this report. These data were used to define genes associated with stricturing behavior and for model testing to predict stricturing behavior. A bioinformatics approach to define small molecules which may reverse the stricturing gene signature was applied. 19 of the 249 patients developed isolated B2 stricturing behavior during follow-up, while 218 remained B1 inflammatory. Using deeper RNA sequencing than in our prior report, we have now defined an inflammatory gene signature including an oncostatin M co-expression signature, tightly associated with extra-cellular matrix (ECM) gene expression in those who developed stricturing complications. We further computationally prioritize small molecules targeting macrophage and fibroblast activation and angiogenesis which may reverse the stricturing gene signature. A model containing ASCA and CBir1 serologies and a refined eight ECM gene set was significantly associated with stricturing development by year five after diagnosis (AUC (95th CI) = 0.82 (0.7-0.94)). An ileal gene program for macrophage and fibroblast activation is linked to stricturing complications in treatment naïve pediatric CD, and may inform novel small molecule therapeutic approaches.

Background and Purpose: Fibrotic lung remodelling after a respiratory viral infection represents a debilitating clinical sequela. Studying or managing viral–fibrotic sequela remains challenging, due to limited therapeutic options and lack of understanding of mechanisms. This study determined whether protein disulfide isomerase A3 (PDIA3) and secreted phosphoprotein 1 (SPP1), which are associated with pulmonary fibrosis, can promote influenza‐induced lung fibrotic remodelling and whether inhibition of PDIA3 or SPP1 can resolve viral‐mediated fibrotic remodelling. Experimental Approach: A retrospective analysis of TriNetX data sets was conducted. Serum from healthy controls and influenza A virus (IAV)‐infected patients was analysed. An inhibitor of PDIA3, punicalagin, and a neutralizing antibody for SPP1 were administered in mice. Macrophage cells treated with macrophage colony‐stimulating factor (M‐CSF) were used as a cell culture model. Key Results: The TriNetX data set showed an increase in lung fibrosis and decline in lung function in flu‐infected acute respiratory distress syndrome (ARDS) patients compared with non‐ARDS patients. Serum samples revealed a significant increase in SPP1 and PDIA3 in influenza‐infected patients. Lung PDIA3 and SPP1 expression increased following viral infection in mouse models. Punicalagin administration 2 weeks after IAV infection in mice caused a significant decrease in lung fibrosis and improved oxygen saturation. Administration of neutralizing SPP1 antibody decreased lung fibrosis. Inhibition of PDIA3 decreased SPP1secretion from macrophages, in association with diminished disulfide bonds in SPP1. Conclusion and Implications: The PDIA3–SPP1 axis promotes post‐influenza lung fibrosis in mice and that pharmacological inhibition of PDIA3 or SPP1 can treat virus‐induced lung fibrotic sequela. [ABSTRACT FROM AUTHOR]

Idiopathic pulmonary fibrosis (IPF) is marked by the activation of fibroblasts, leading to excessive production and deposition of extracellular matrix (ECM) within the lung parenchyma. Despite the pivotal role of ECM overexpression in IPF, potential negative regulators of ECM production in fibroblasts have yet to be identified. Semaphorin class 3B (SEMA3B), a secreted protein highly expressed in lung tissues, has established roles in axonal guidance and tumor suppression. However, the role of SEMA3B in ECM production by fibroblasts in the pathogenesis of IPF remains unexplored. Here, we show the downregulation of SEMA3B and its cognate binding receptor, neuropilin 1 (NRP1), in IPF lungs compared with healthy controls. Notably, the reduced expression of SEMA3B and NRP1 is associated with a decline in lung function in IPF. The downregulation of SEMA3B and NRP1 transcripts was validated in the lung tissues of patients with IPF, and two alternative mouse models of pulmonary fibrosis. In addition, we show that transforming growth factor-β (TGFβ) functions as a negative regulator of SEMA3B and NRP1 expression in lung fibroblasts. Furthermore, we demonstrate the antifibrotic effects of SEMA3B against TGFβ-induced ECM production in IPF lung fibroblasts. Overall, our findings uncovered a novel role of SEMA3B in the pathogenesis of pulmonary fibrosis and provided novel insights into modulating the SEMA3B-NRP1 axis to attenuate pulmonary fibrosis. NEW & NOTEWORTHY: The excessive production and secretion of collagens and other extracellular matrix proteins by fibroblasts lead to the scarring of the lung in severe fibrotic lung diseases. This study unveils an antifibrotic role for semaphorin class 3B (SEMA3B) in the pathogenesis of idiopathic pulmonary fibrosis. SEMA3B functions as an inhibitor of transforming growth factor-β-driven fibroblast activation and reduced levels of SEMA3B and its receptor, neuropilin 1, are associated with decreased lung function in idiopathic pulmonary fibrosis. [ABSTRACT FROM AUTHOR]

Introduction: There is significant unexplained variability in behavioral and executive functioning after pediatric traumatic brain injury (TBI). Prior research indicates that there are likely genetic contributions; however, current research is limited. The purpose of this study is to use a systems biology informed approach to characterize the genomic signature related to behavioral and executive functioning ~12 months after moderate through severe TBI in children. Methods: Participants were from two prospective cohorts of children with severe TBI (Cohort #1) and moderate-severe TBI and an orthopedic injury (OI) group (Cohort #2). Participants included 196 children (n = 72 and n = 124 total from each respective cohort), ranging in age between 0--17 years at the time of injury. In total, 86 children had severe TBI, 49 had moderate TBI, and 61 had an OI. Global behavioral functioning assessed via the Child Behavior Checklist and executive function assessed via the Behavioral Rating Inventory of Executive Function at ~ 12 months post injury served as outcomes. To test for a genomic signature, we compared the number of nominally significant (p < 0.05) polymorphisms associated with the outcomes in our systems biology identified genes to a set 10,000 permutations using control genes (e.g., not implicated by systems biology). We used the ToppFun application from Toppgene Suite to identify enriched biologic processes likely to be associated with behavioral and executive function outcomes. Results: At 12 months post injury, injury type (TBI vs OI) by polymorphism interaction was significantly enriched in systems biology selected genes for behavioral and executive function outcomes, suggesting these genes form a genomic signature. Effect sizes of the associations from our genes of interest ranged from .2--.5 for the top 5% of variants. Systems biology analysis of the variants associated with the top 5% effect sizes indicated enrichment in several specific biologic processes and systems. Discussion: Findings indicate that a genomic signature may explain heterogeneity of behavioral and executive outcomes after moderate and severe TBI. This work provides the foundation for constructing genomic signatures and integrating systems biology and genetic information into future recovery, prognostic, and treatment algorithms. [ABSTRACT FROM AUTHOR]