Gene Expression In Epithelial Cells Research Articles

Effects of pectin's degree of methyl esterification on TLR2-mediated IL-8 secretion and tight junction gene expression in intestinal epithelial cells: influence of soluble TLR2.

This study investigates the anti-inflammatory effects of pectins with different degrees of methyl esterification (DM) on intestinal epithelial cells (IECs) expressing low and high levels of TLR2. It also studies the influence of soluble TLR2 (sTLR2) which may be enhanced in patients with inflammatory bowel syndrome on the inflammation-attenuating effects of pectins. Also, it examines the impact of pectins on tight junction gene expression in IECs. Lemon pectins with DM18 and DM88 were characterized, and their effects on TLR2-1-induced IL8 gene expression and secretion were investigated in low-TLR2 expressing Caco-2 and high-TLR2 expressing DLD-1 cells. The results demonstrate that both DM18 and DM88 pectins can counteract TLR2-1-induced IL-8 expression and secretion, with more pronounced effects observed in DLD-1 cells expressing high levels of TLR2. Furthermore, the presence of sTLR2 does not interfere with the attenuating effects of low DM18 pectin and may even support its anti-inflammatory effects in Caco-2 cells. The impact of pectins and sTLR2 on tight junction gene expression also demonstrates cell-type-dependent effects. Overall, these findings suggest that low DM pectins possess potent anti-inflammatory properties and may influence tight junction gene expression in IECs, thereby contributing to the maintenance of gut homeostasis.

Gene expression in calcium triggered healthy primary limabal epithelial cells, in vitro

Aims/Purpose: Calcium supplementation can induce differentiation of several cell types. The tear film has an important role in maintenance of the ocular surface homeostasis with an ionic calcium concentration of 0.4 mM‐1.0 mM. The effect of calcium concentration on healthy primary limbal epithelial cells (LECs) has not been analysed, yet. Our purpose was to investigate the effect of calcium supplementation on gene expression of primary limbal epithelial cells (LECs) of healthy donor corneas, in vitro.Methods: Primary LECs were isolated from corneoscleral rims of healthy donors (n = 6) and were cultured in serum free low calcium (0.06 mM Ca+2) Keratocyte growth medium 3 (KGM3). The primary LECs were then supplemented by 0.3 mM‐2.0 mM Ca+2 concentrations for 72 h. Control LEC cultures were supplemented by low calcium (0.06 mM Ca+2) KGM3 medium. After cell harvesting, RNA and protein were extracted from treated and control LECs to perform qPCR and western blot analysis.Results: The calcium supplementation had no significant effect on PAX6 mRNA expression after 72 h. The differentiation marker and adhesion protein DSG1 and the cornea specific keratin K3 expression did not change significantly through Ca+2 supplementation (p ≥ 0.5). The retinoic acid signalling component fatty acid binding protein FABP5 expression (p ≥ 0.5) the wound healing and vascularization marker CAV1 gene expression (p ≥ 0.5) did not change significantly through Ca+2 supplementation, after 72 h.Conclusions: Calcium supplementation for 72 h did not change PAX6 expression and did not trigger differentiation of healthy primary limbal epithelial cells. We plan to analyse the effect of longer incubation time in the future in order to trigger a full differentiated status of limbal epithelial cells, using calcium supplementation.

Corneal epithelial cell markers in congenital aniridia

Congenital aniridia is a rare panocular disease in which almost all structures of the eye are affected. The observed corneal abnormalities are called aniridia‐associated keratopathy (AAK). AAK is characterized by progressive limbal stem cell deficiency (LSCD), epithelial thinning and erosions, corneal ulceration, vascularization, inflammation and pannus formation. Although in some aspects AAK resembles other types of LSCD, through its specific genetic origin, alterations in corneal epithelial cell gene expression differ from other types of corneal diseases. Primary limbal epithelial cells of aniridia subjects lack expression of KRT3 and KRT12 mRNA and protein, and DSG1, ADH7, FABP5, CRABP2, SPINK7 and ALDH1A1 mRNA expression is decreased in these cells. However, miRNA 138‐5p expression is upregulated. This epithelial behaviour can also be mimicked using an siRNA based aniridia primary limbal epithelial cell model.

Specific corneal epithelial cell markers in congenital aniridia

Congenital aniridia is a rare panocular disease in which almost all structures of the eye are affected. The observed corneal abnormalities are called aniridia‐associated keratopathy (AAK). AAK is characterized by progressive limbal stem cell deficiency (LSCD), epithelial thinning and erosions, corneal ulceration, vascularization, inflammation and pannus formation. Although in some aspects AAK resembles other types of LSCD, through its specific genetic origin, alterations in corneal epithelial cell gene expression differ from other types of corneal diseases. Primary limbal epithelial cells of aniridia subjects lack expression of KRT3 and KRT12 mRNA and protein, and DSG1, ADH7, FABP5, CRABP2, SPINK7 and ALDH1A1 mRNA expression is decreased in these cells. However, miRNA 138‐5p expression is upregulated. This epithelial behaviour can also be mimicked using an siRNA based aniridia primary limbal epithelial cell model.

Farnesol Inhibits PI3 Kinase Signaling and Inflammatory Gene Expression in Primary Human Renal Epithelial Cells.

Chronic inflammation and elevated cytokine levels are closely associated with the progression of chronic kidney disease (CKD), which is responsible for the manifestation of numerous complications and mortality. In addition to conventional CKD therapies, the possibility of using natural compounds with anti-inflammatory potential has attracted widespread attention in scientific research. This study aimed to study the potential anti-inflammatory effects of a natural oil compound, farnesol, in primary human renal proximal tubule epithelial cell (RPTEC) culture. Farnesol was encapsulated in lipid-based small unilamellar vesicles (SUVs) to overcome its insolubility in cell culture medium. The cell attachment of empty vesicles (SUVs) and farnesol-loaded vesicles (farnesol-SUVs) was examined using BODIPY, a fluorescent dye with hydrophobic properties. Next, we used multiple protein, RNA, and protein phosphorylation arrays to investigate the impact of farnesol on inflammatory signaling in RPTECs. The results indicated that farnesol inhibits TNF-α/IL-1β-induced phosphorylation of the PI3 kinase p85 subunit and subsequent transcriptional activation of the inflammatory genes TNFRSF9, CD27, TNFRSF8, DR6, FAS, IL-7, and CCL2. Therefore, farnesol may be a promising natural compound for treating CKD.

Transcriptomic profiling of Polycystic Kidney Disease identifies paracrine factors in the early cyst microenvironment

Initial cysts that are formed upon Pkd1 loss in mice impose persistent stress on surrounding tissue and trigger a cystic snowball effect, in which local aberrant PKD-related signaling increases the likelihood of new cyst formation, ultimately leading to accelerated disease progression. Although many pathways have been associated with PKD progression, the knowledge of early changes near initial cysts is limited. To perform an unbiased analysis of transcriptomic alterations in the cyst microenvironment, microdomains were collected from kidney sections of iKsp-Pkd1del mice with scattered Pkd1-deletion using Laser Capture Microdissection. These microdomains were defined as F4/80-low cystic, representing early alterations in the cyst microenvironment, F4/80-high cystic, with more advanced alterations, or non-cystic. RNA sequencing and differential gene expression analysis revealed 953 and 8088 dysregulated genes in the F4/80-low and F4/80-high cyst microenvironment, respectively, when compared to non-cystic microdomains. In the early cyst microenvironment, several injury-repair, growth, and tissue remodeling-related pathways were activated, accompanied by mild metabolic changes. In the more advanced F4/80-high microdomains, these pathways were potentiated and the metabolism was highly dysregulated. Upstream regulator analysis revealed a series of paracrine factors with increased activity in the early cyst microenvironment, including TNFSF12 and OSM. In line with the upstream regulator analysis, TWEAK and Oncostatin-M promoted cell proliferation and inflammatory gene expression in renal epithelial cells and fibroblasts in vitro. Collectively, our data provide an overview of molecular alterations that specifically occur in the cyst microenvironment and identify paracrine factors that may mediate early and advanced alterations in the cyst microenvironment.

Dermatophagoides farinae microRNAs released to external environments via exosomes regulate inflammation-related gene expression in human bronchial epithelial cells.

Dermatophagoides farinae (DFA) is an important species of house dust mites (HDMs) that causes allergic diseases. Previous studies have focused on allergens with protein components to explain the allergic effect of HDMs; however, there is little knowledge on the role of microRNAs (miRNAs) in the allergic effect of HDMs. This study aimed to unravel the new mechanism of dust mite sensitization from the perspective of cross-species transport of extracellular vesicles-encapsulated miRNAs from HDMs. Small RNA (sRNA) sequencing was performed to detect miRNAs expression profiles from DFA, DFA-derived exosomes and DFA culture supernatants. A quantitative fluorescent real-time PCR (qPCR) assay was used to detect miRNAs expression in dust specimens. BEAS-2B cells endocytosed exosomes were modeled in vitro to detect miRNAs from DFA and the expression of related inflammatory factors. Representative dfa-miR-276-3p and dfa-novel-miR2 were transfected into BEAS-2B cells, and then differentially expressed genes (DEGs) were analyzed by RNA sequencing. Protein-protein interaction (PPI) network analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and Gene Ontology (GO) terms enrichment analyses were performed on the first 300 nodes of DEGs. sRNA sequencing identified 42 conserved miRNAs and 66 novel miRNAs in DFA, DFA-derived exosomes, and DFA culture supernatants. A homology analysis was performed on the top 18 conserved miRNAs with high expression levels. The presence of dust mites and miRNAs from HDMs in living environment were also validated. Following uptake of DFA-derived exosomes by BEAS-2B cells, exosomes transported miRNAs from DFA to target cells and produced pro-inflammatory effects in corresponding cells. RNA sequencing identified DEGs in dfa-miR-276-3p and dfa-novel-miR2 transfected BEAS-2B cells. GO and KEGG enrichment analyses revealed the role of exosomes with cross-species transporting of DFA miRNAs in inflammatory signaling pathways, such as JAK-STAT signaling pathway, PI3K/AKT signaling pathway and IL-6-mediated signaling pathway. Our findings demonstrate the miRNAs expression profiles in DFA for the first time. The DFA miRNAs are delivered into living environments via exosomes, and engulfed by human bronchial epithelial cells, and cross-species regulation may contribute to inflammation-related processes.

Antioxidant effect of lactic acid bacteria in human bronchial epithelial cells exposed to cigarette smoke.

Chronic lung diseases are a major and increasing global health problem, commonly caused by cigarette smoke. We aimed to explore the antioxidant effects of lactic acid bacteria (LAB) against cigarette smoke in bronchial epithelial cells. The antioxidant effects of 21 heat-killed (HK) LAB strains were tested in cigarette smoke stimulated BEAS-2B cells and 3-D bronchospheres organoids. We showed that HK Lactiplantibacillus plantarum BGPKM22 possesses antioxidant activity against cigarette smoke, resistance to hydrogen peroxide, and free radical neutralizing activity. We demonstrated that HK BGPKM22 inhibited cigarette smoke induced expression of the Aryl hydrocarbon receptor (AhR) and Nuclear factor erythroid 2 related factor 2 (Nrf2) genes. The cell-free supernatant (SN) of BGPKM22 fully confirmed the effects of HK BGPKM22. For the first time, we revealed that HK and SN of L. plantarum BGPKM22 possess antioxidant activity and modulate AhR and Nrf2 gene expression in bronchial epithelial cells exposed to cigarette smoke.

Spatial Transcriptomic Analysis Reveals Altered Gene Expression in Glomerular Parietal Epithelial Cells Following Tubular Injury

Spatial Transcriptomic Analysis Reveals Altered Gene Expression in Glomerular Parietal Epithelial Cells Following Tubular Injury

Estradiol mediates colonic epithelial protection in aged mice after stroke and is associated with shifts in the gut microbiome

ABSTRACT The gut is a major source of bacteria and antigens that contribute to neuroinflammation after brain injury. Colonic epithelial cells (ECs) are responsible for secreting major cellular components of the innate defense system, including antimicrobial proteins (AMP) and mucins. These cells serve as a critical regulator of gut barrier function and maintain host-microbe homeostasis. In this study, we determined post-stroke host defense responses at the colonic epithelial surface in mice. We then tested if the enhancement of these epithelial protective mechanisms is beneficial in young and aged mice after stroke. AMPs were significantly increased in the colonic ECs of young males, but not in young females after experimental stroke. In contrast, mucin-related genes were enhanced in young females and contributed to mucus formation that maintains the distance between the host and gut bacteria. Bacterial community profiling was done using universal amplification of 16S rRNA gene sequences. The sex-specific colonic epithelial defense responses after stroke in young females were reversed with ovariectomy and led to a shift from a predominately mucin response to the enhanced AMP expression seen in males after stroke. Estradiol (E2) replacement prior to stroke in aged females increased mucin gene expression in the colonic ECs. Interestingly, we found that E2 treatment reduced stroke-associated neuronal hyperactivity in the insular cortex, a brain region that interacts with visceral organs such as the gut, in parallel to an increase in the composition of Lactobacillus and Bifidobacterium in the gut microbiota. This is the first study demonstrating sex differences in host defense mechanisms in the gut after brain injury.

Comparative Effects of Cyclosporine and Voclosporin on Primary Human Proximal Tubular Epithelial Cell (PTEC) Gene Expression

Comparative Effects of Cyclosporine and Voclosporin on Primary Human Proximal Tubular Epithelial Cell (PTEC) Gene Expression

Chlamydial YAP activation in host endocervical epithelial cells mediates pro-fibrotic paracrine stimulation of fibroblasts.

Chronic or repeated infection of the female upper genital tract by C. trachomatis can lead to severe fibrotic sequelae, including tubal factor infertility and ectopic pregnancy. However, the molecular mechanisms underlying this effect are unclear. In this report, we define a transcriptional program specific to C. trachomatis infection of the upper genital tract, identifying tissue-specific induction of host YAP-a pro-fibrotic transcriptional cofactor-as a potential driver of infection-mediated fibrotic gene expression. Furthermore, we show that infected endocervical epithelial cells stimulate collagen production by fibroblasts and implicate chlamydial induction of YAP in this effect. Our results define a mechanism by which infection mediates tissue-level fibrotic pathology via paracrine signaling and identify YAP as a potential therapeutic target for the prevention of Chlamydia-associated scarring of the female genital tract.

Acellular ex vivo lung perfusate silences pro-inflammatory signaling in human lung endothelial and epithelial cells

BackgroundIschemia–reperfusion injury is a key complication following lung transplantation. The clinical application of ex vivo lung perfusion (EVLP) to assess donor lung function has significantly increased the utilization of “marginal” donor lungs with good clinical outcomes. The potential of EVLP on improving organ quality and ameliorating ischemia–reperfusion injury has been suggested.MethodsTo determine the effects of ischemia–reperfusion and EVLP on gene expression in human pulmonary microvascular endothelial cells and epithelial cells, cell culture models were used to simulate cold ischemia (4 °C for 18 h) followed by either warm reperfusion (DMEM + 10% FBS) or EVLP (acellular Steen solution) at 37 °C for 4 h. RNA samples were extracted for bulk RNA sequencing, and data were analyzed for significant differentially expressed genes and pathways.ResultsEndothelial and epithelial cells showed significant changes in gene expressions after ischemia–reperfusion or EVLP. Ischemia–reperfusion models of both cell types showed upregulated pro-inflammatory and downregulated cell metabolism pathways. EVLP models, on the other hand, exhibited downregulation of cell metabolism, without any inflammatory signals.ConclusionThe commonly used acellular EVLP perfusate, Steen solution, silenced the activation of pro-inflammatory signaling in both human lung endothelial and epithelial cells, potentially through the lack of serum components. This finding could establish the basic groundwork of studying the benefits of EVLP perfusate as seen from current clinical practice.

Fish Collagen Peptides Enhance Thymopoietic Gene Expression, Cell Proliferation, Thymocyte Adherence, and Cytoprotection in Thymic Epithelial Cells via Activation of the Nuclear Factor-κB Pathway, Leading to Thymus Regeneration after Cyclophosphamide-Induced Injury.

Prolonged thymic involution results in decreased thymopoiesis and thymic output, leading to peripheral T-cell deficiency. Since the thymic-dependent pathway is the only means of generating fully mature T cells, the identification of strategies to enhance thymic regeneration is crucial in developing therapeutic interventions to revert immune suppression in immunocompromised patients. The present study clearly shows that fish collagen peptides (FCPs) stimulate activities of thymic epithelial cells (TECs), including cell proliferation, thymocyte adhesion, and the gene expression of thymopoietic factors such as FGF-7, IGF-1, BMP-4, VEGF-A, IL-7, IL-21, RANKL, LTβ, IL-22R, RANK, LTβR, SDF-1, CCL21, CCL25, CXCL5, Dll1, Dll4, Wnt4, CD40, CD80, CD86, ICAM-1, VCAM-1, FoxN1, leptin, cathepsin L, CK5, and CK8 through the NF-κB signal transduction pathway. Furthermore, our study also revealed the cytoprotective effects of FCPs on TECs against cyclophosphamide-induced cellular injury through the NF-κB signaling pathway. Importantly, FCPs exhibited a significant capability to facilitate thymic regeneration in mice after cyclophosphamide-induced damage via the NF-κB pathway. Taken together, this study sheds light on the role of FCPs in TEC function, thymopoiesis, and thymic regeneration, providing greater insight into the development of novel therapeutic strategies for effective thymus repopulation for numerous clinical conditions in which immune reconstitution is required.

PDZ Peptide of the ZO-1 Protein Significantly Increases UTP-Induced MUC8 Anti-Inflammatory Mucin Overproduction in Human Airway Epithelial Cells.

Mucus hyperproduction and hypersecretion are observed often in respiratory diseases. MUC8 is a glycoprotein synthesized by epithelial cells and generally expressed in the respiratory track. However, the physiological mechanism by which extracellular nucleotides induce MUC8 gene expression in human airway epithelial cells is unclear. Here, we show that UTP could induce MUC8 gene expression through P2Y2-PLCβ3-Ca2+ activation. Because the full-length cDNA sequence of MUC8 has not been identified, a specific siRNA-MUC8 was designed based on the partial cDNA sequence of MUC8. siRNA-MUC8 significantly increased TNF-α production and decreased IL-1Ra production, suggesting that MUC8 may downregulate UTP/P2Y2-induced airway inflammation. Interestingly, the PDZ peptide of ZO-1 protein strongly abolished UTP-induced TNF-α production and increased IL-1Ra production and MUC8 gene expression. In addition, the PDZ peptide dramatically increased the levels of UTP-induced ZO proteins and TEER (trans-epithelial electrical resistance). These results show that the anti-inflammatory mucin MUC8 may contribute to homeostasis, and the PDZ peptide can be a novel therapeutic candidate for UTP-induced airway inflammation.

Differential dependence on microbiota of IL-23/IL-22-dependent gene expression between the small- and large-intestinal epithelia.

In the intestine, interleukin (IL)-23 and IL-22 from immune cells in the lamina propria contribute to maintenance of the gut epithelial barrier through the induction of antimicrobial production and the promotion of epithelial cell proliferation. Several previous studies suggested that some of the functions of the IL-23/IL-22 axis on intestinal epithelial cells are shared between the small and large intestines. However, the similarities and differences of the IL-23/IL-22 axis on epithelial cells between these two anatomical sites remain unclear. Here, we comprehensively analyzed the gene expression of intestinal epithelial cells in the ileum and colon of germ-free, Il23-/- , and Il22-/- mice by RNA-sequencing. We found that while the IL-23/IL-22 axis is largely dependent on gut microbiota in the small intestine, it is much less dependent on it in the large intestine. In addition, the negative regulation of lipid metabolism in the epithelial cells by IL-23 and IL-22 in the small intestine was revealed, whereas the positive regulation of epithelial cell proliferation by IL-23 and IL-22 in the large intestine was highlighted. These findings shed light on the intestinal site-specific role of the IL-23/IL-22 axis in maintaining the physiological functions of intestinal epithelial cells.

Alveolar epithelial cell growth hormone releasing hormone receptor in alveolar epithelial inflammation

Purpose: Growth hormone-releasing hormone (GHRH) is a 44-amino acid peptide that regulates growth hormone (GH) secretion. We hypothesized that GHRH receptor (GHRH-R) in alveolar type 2 (AT2) cells could modulate pro-inflammatory and possibly subsequent pro-fibrotic effects of lipopolysaccharide (LPS) or cytokines, such that AT2 cells could participate in lung inflammation and fibrosis. Methods: We used human alveolar type 2 (iAT2) epithelial cells derived from induced pluripotent stem cells (iPSC) to investigate how GHRH-R modulates gene and protein expression. We tested iAT2 cells’ gene expression in response to LPS or cytokines, seeking whether these mechanisms caused endogenous production of pro-inflammatory molecules or mesenchymal markers. Quantitative real-time PCR (RT-PCR) and Western blotting were used to investigate differential expression of epithelial and mesenchymal markers. Result: Incubation of iAT2 cells with LPS increased expression of IL1-β and TNF-α in addition to mesenchymal genes, including ACTA2, FN1 and COL1A1. Alveolar epithelial cell gene expression due to LPS was significantly inhibited by GHRH-R peptide antagonist MIA-602. Incubation of iAT2 cells with cytokines like those in fibrotic lungs similarly increased expression of genes for IL1-β, TNF-α, TGFβ-1, Wnt5a, smooth muscle actin, fibronectin and collagen. Expression of mesenchymal proteins, such as N-cadherin and vimentin, were also elevated after prolonged exposure to cytokines, confirming epithelial production of pro-inflammatory molecules as an important mechanism that might lead to subsequent fibrosis. Conclusion: iAT2 cells clearly expressed the GHRH-R. Exposure to LPS or cytokines increased iAT2 cell production of pro-inflammatory factors. GHRH-R antagonist MIA-602 inhibited pro-inflammatory gene expression, implicating iAT2 cell GHRH-R signaling in lung inflammation and potentially in fibrosis.

Several Alphaherpesviruses Interact Similarly with the NF-κB Pathway and Suppress NF-κB-Dependent Gene Expression.

Alphaherpesvirus infection is associated with attenuation of different aspects of the host innate immune response that is elicited to confine primary infections at the mucosal epithelia. Here, we report that infection of epithelial cells with several alphaherpesviruses of different species, including herpes simplex virus 1 and 2 (HSV-1 and HSV-2), feline alphaherpesvirus 1 (FHV-1), and bovine alphaherpesvirus 1 (BoHV-1) results in the inactivation of the responses driven by the nuclear factor kappa B (NF-κB) pathway, considered a pillar of the innate immune response. The mode to interact with and circumvent NF-κB-driven responses in infected epithelial cells is seemingly conserved in human, feline, and porcine alphaherpesviruses, consisting of a persistent activation of the NF-κB cascade but a potent repression of NF-κB-dependent transcription activity, which relies on replication of viral genomes. However, BoHV-1 apparently deviates from the other investigated members of the taxon in this respect, as BoHV-1-infected epithelial cells do not display the persistent NF-κB activation observed for the other alphaherpesviruses. In conclusion, this study suggests that inhibition of NF-κB transcription activity is a strategy used by several alphaherpesviruses to prevent NF-κB-driven responses in infected epithelial cells. IMPORTANCE The current study provides a side-by-side comparison of the interaction of different alphaherpesviruses with NF-κB, a key and central player in the (proinflammatory) innate host response, in infected nontransformed epithelial cell lines. We report that all studied viruses prevent expression of the hallmark NF-κB-dependent gene IκB, often but not always via similar strategies, pointing to suppression of NF-κB-dependent host gene expression in infected epithelial cells as a common and therefore likely important aspect of alphaherpesviruses.

Single Cell Multiomics Identifies Cells and Genetic Networks Underlying Alveolar Capillary Dysplasia.

Rationale: Alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV) is a lethal developmental disorder of lung morphogenesis caused by insufficiency of FOXF1 (forkhead box F1) transcription factor function. The cellular and transcriptional mechanisms by which FOXF1 deficiency disrupts human lung formation are unknown. Objectives: To identify cell types, gene networks, and cell-cell interactions underlying the pathogenesis of ACDMPV. Methods: We used single-nucleus RNA and assay for transposase-accessible chromatin sequencing, immunofluorescence confocal microscopy, and RNA in situ hybridization to identify cell types and molecular networks influenced by FOXF1 in ACDMPV lungs. Measurements and Main Results: Pathogenic single-nucleotide variants and copy-number variant deletions involving the FOXF1 gene locus in all subjects with ACDMPV (n = 6) were accompanied by marked changes in lung structure, including deficient alveolar development and a paucity of pulmonary microvasculature. Single-nucleus RNA and assay for transposase-accessible chromatin sequencing identified alterations in cell number and gene expression in endothelial cells (ECs), pericytes, fibroblasts, and epithelial cells in ACDMPV lungs. Distinct cell-autonomous roles for FOXF1 in capillary ECs and pericytes were identified. Pathogenic variants involving the FOXF1 gene locus disrupt gene expression in EC progenitors, inhibiting the differentiation or survival of capillary 2 ECs and cell-cell interactions necessary for both pulmonary vasculogenesis and alveolar type 1 cell differentiation. Loss of the pulmonary microvasculature was associated with increased VEGFA (vascular endothelial growth factor A) signaling and marked expansion of systemic bronchial ECs expressing COL15A1 (collagen type XV α 1 chain). Conclusions: Distinct FOXF1 gene regulatory networks were identified in subsets of pulmonary endothelial and fibroblast progenitors, providing both cellular and molecular targets for the development of therapies for ACDMPV and other diffuse lung diseases of infancy.

P-325 Evidence for dysregulation of oncogene pathways at the gene expression level in the epithelium of deep infiltrating endometriosis compared to adenomyosis

Abstract Study question How does the gene expression profile of epithelium cells differ between deep infiltrating endometriosis (DIE) and adenomyosis (FA)? Summary answer The gene expression of epithelial cells from DIE reveals a significant upregulation of the PI3K-pathway and downregulation of the RAS-pathway compared to epithelium from FA. What is known already Both DIE and FA, are histologically considered benign but have features of malignant diseases, such as a tendency for local tissue invasion. Recent next generation sequencing studies have detected driver mutations in cancer-associated genes such as PIK3CA, ARID1A, PPP2R1A and KRAS in the epithelium of DIE, as well as in KRAS in epithelium of FA. Furthermore, identical mutations in the KRAS gene were detected in coexisting adenomyotic and endometriotic lesions, supporting the theory of a common molecular mechanism. The gene expression differences between these two entities were therefore investigated in the present study. Study design, size, duration This is an experimental analysis of 7 adenomyosis and 19 DIE samples (histologically confirmed), that were formalin-fixed and paraffin-embedded (FFPE) collected between 2003 and 2018. These were provided by the tissue bank of the National Centre for Tumour Diseases (Heidelberg, Germany), in accordance with the Ethics Committee of the University of Heidelberg (approval number S-362/2017). In addition, the medical and epidemiological data of the corresponding patients were retrospectively analyzed based on the clinical data collected. Participants/materials, setting, methods For this study, the epithelia of FFPE samples were microdissected in a laser-guided fashion. After RNA extraction, the expression of 770 genes was analyzed using the nCounter Technology with the Human PanCancer Pathways Panel (Nanostring). All genes with an adjusted false discovery rate of p < 0.05 and a fold change of < 0.66 or > 1.5 were considered differentially expressed and subjected to functional annotation and clustering using DAVID bioinformatics resources. Main results and the role of chance Our analysis revealed a total of 162 differentially expressed genes, that were either significantly increased (n = 116) or decreased (n = 46) in epithelium of DIE compared to that of FA (with log2 fold changes of < 0.66 or > 1.5 and an adjusted p-value of < 0.05). Gene ontology and KEGG pathway analysis for genes with increased expression in DIE compared with FA revealed significant upregulation of genes belonging to the PI3K-pathway and the focal adhesion pathway, as well as pathways related to viral infection, endocrine resistance, and malignancy. The upregulated pathways in adenomyosis mainly included the RAS-pathway. Limitations, reasons for caution The average age of DIE patients was lower, but many of them were hormonally inactive due to GnRH analogues, which mitigates any age-related differences. Furthermore, only relative expression was studied between the groups without comparison to normal endometrium. Overall, the sample size is relatively small and further studies are needed. Wider implications of the findings The expression of different signaling pathways in the two entities could, for example, explain the different sensitivity of the two diseases to certain therapeutic approaches. While DIE responds well to therapy with the progestogen dienogest, progesterone resistance is often described in adenomyosis patients. Trial registration number not applicable