Biology subjects

Keijzer, R.

Publications and source records attributed to Keijzer, R..

5 recordsLinked to original sources

bioRxiv2025

Dysregulated circRNA expression profile and associated miRNA sponging in abnormal lung development in congenital diaphragmatic hernia

RationaleCircular RNAs (circRNAs) can function as disease biomarkers. Their profile in abnormal lung development in congenital diaphragmatic hernia (CDH) is unknown. ObjectiveTo evaluate circRNA expression profile in CDH-associated abnormal lung development. MethodsWe profiled circRNAs in rat CDH and control lungs at embryonic day (E)15 and E21 by microarray. We validated identified circRNAs using back-splice junction amplicon sequencing, RT-qPCR, and in situ hybridization. We modified a CircRNA Function prediction Tool to predict CircRNA::micro(mi)RNA::messenger(m)RNA interactions and compared these with Oxford Nanopore RNA sequencing and existing human CDH datasets. Measurements and Main ResultsMicroarrays revealed a unique circRNA biosignature during CDH lung development. CircAnp32e was expressed in a sex-specific and spatiotemporal expression pattern in the epithelium at E15. The predicted mature sequence of circAnp32e overlapped >90% with its human orthologue. CircRNA::miRNA::mRNA interaction networks in E15 and E21 revealed enrichment in inflammation/infection, smooth muscle cell function, cell proliferation/cell cycle regulation, and response to hypoxia pathways. Parental genes of differential expressed circRNAs at E15 enriched pathways linked to cell proliferation/cell cycle/cancer, while at end-gestation, inflammation and cardiovascular processes were also overrepresented. Rat and human CDH lungs showed overlapping pathways with additional enrichment for RNA processing and protein binding/modification in humans. ConclusionA unique circRNA signature during abnormal lung development in CDH may mediate inflammatory responses, smooth-muscle-cell function, and cell proliferation regulation via miRNA sponging. Overlap of downstream pathways in rat and human CDH suggest conserved functions across species. CircRNAs may serve as biomarkers to guide prenatal management and mitigate aberrant lung development in CDH.

developmental biology↗

bioRxiv2025

FindBacksplice: a Tool for Locating Circular RNA Backsplice Coordinates

Circular RNAs (circRNAs) are generated through back-splicing, a process where a backsplice junction (BSJ) is formed, based on the circRNAs unique sequence. BSJs are highly conserved and can be mapped to chromosomal coordinates. Current platforms determine these coordinates from bulk RNA sequencing data. We aimed to develop a tool capable of determining backsplice coordinates based on BSJ sequences for any species and genome version. MotivationcircRNAs are emerging as an important regulator of cellular differentiation and other biologically important processes. Common tools for circRNA analyses require a circular RNAs backsplice coordinates. These can be accessed from public databases. However, the coordinates are specific to a version of a species genome, and are unavailable for many model organisms. ResultsWe have developed a Python-based, command line tool, FindBacksplice, which produces backsplice coordinates for any available genome, based on a circRNAs BSJ sequence. Implemented in Python, this script is integrated with BLAST for use in existing pipelines. We were able to find valid locations of backsplices for known human BSJs in the rat genome and produce backsplice coordinates for use in existing pipelines. Availability and implementationFindBacksplice is available at github.com/m-kraljevic/findbacksplice

bioinformatics↗

bioRxiv2023

Overactivated epithelial NF-κB disrupts lung development in human and nitrofen CDH

Background & ObjectiveAbnormal lung development is the main cause of morbidity and mortality in neonates with congenital diaphragmatic hernia (CDH), a common birth defect (1:2500) of largely unknown pathobiology. Recent studies discovered that inflammatory processes, and specifically NF-{kappa}B associated pathways are enriched in human and experimental CDH. However, the molecular signaling of NF-{kappa}B in abnormal CDH lung development and its potential as a therapeutic target requires further investigation. Methods & ResultsUsing sections and hypoplastic lung explant cultures from the nitrofen rat model of CDH and human fetal CDH lungs, we demonstrate that NF-{kappa}B and its downstream transcriptional targets are hyperactive during abnormal lung formation in CDH. NF-{kappa}B activity was especially elevated in the airway epithelium of nitrofen and human CDH lungs at different developmental stages. Fetal rat lung explants had impaired pseudoglandular airway branching after exposure to nitrofen, together with increased phosphorylation and transcriptional activity of NF-{kappa}B. Dexamethasone, the broad and clinically applicable anti-inflammatory NF-{kappa}B antagonist, rescued lung branching and normalized NF-{kappa}B signaling in hypoplastic lung explants. Moreover, specific NF-{kappa}B inhibition with curcumenol similarly rescued ex vivo lung hypoplasia and restored NF-{kappa}B signaling. Lastly, we showed that prenatal intraperitoneal dexamethasone administration to pregnant rat dams carrying fetuses with hypoplastic lungs, significantly improves lung branching and normalizes NF-{kappa}B in vivo. Conclusions: Our results indicate that NF-{kappa}B is aberrantly activated in human and nitrofen CDH lungs. Anti-inflammatory treatment with dexamethasone and/ or specific NF-{kappa}B inhibition should be investigated further as a therapeutic avenue to target lung hypoplasia in CDH.

developmental biology↗

bioRxiv2022

A tracheal aspirate-derived airway basal cell model reveals a pro-inflammatory epithelial defect in congenital diaphragmatic hernia

RationaleCongenital diaphragmatic hernia (CDH) is characterized by incomplete closure of the diaphragm and lung hypoplasia. The pathophysiology of lung defects in CDH is poorly understood. ObjectivesTo establish a translational model of human airway epithelium in CDH for pathogenic investigation and therapeutic testing. MethodsWe developed a robust methodology of epithelial progenitor derivation from tracheal aspirates of newborns. Basal stem cells (BSCs) from CDH patients and preterm and term, non-CDH controls were derived and analyzed by bulk RNA-sequencing, ATAC-sequencing, and air-liquidinterface differentiation. Lung sections from fetal human CDH samples and the nitrofen rat model of CDH were subjected to histological assessment of epithelial defects. Therapeutics to restore epithelial differentiation were evaluated in human epithelial cell culture and the nitrofen rat model of CDH. Measurements and Main ResultsTranscriptomic and epigenetic profiling of CDH and non-CDH basal stem cells reveals a disease-specific, proinflammatory signature independent of severity or hernia size. In addition, CDH basal stem cells exhibit defective epithelial differentiation in vitro that recapitulates epithelial phenotypes found in fetal human CDH lung samples and fetal tracheas of the nitrofen rat model of CDH. Furthermore, steroid treatment normalizes epithelial differentiation phenotypes of human CDH basal stem cells in vitro and in nitrofen rat tracheas in vivo. ConclusionsOur findings have identified an underlying proinflammatory signature and BSC differentiation defects as a potential therapeutic target for airway epithelial defects in CDH.

developmental biology↗

bioRxiv2022

Proteomic Profiling of Hypoplastic Lungs Suggests an Underlying Inflammatory Response in the Pathogenesis of Abnormal Lung Development in Congenital Diaphragmatic Hernia

The pathogenesis of lung hypoplasia in congenital diaphragmatic hernia (CDH), a common birth defect, is poorly understood. The diaphragmatic defect can be repaired surgically, but the abnormal lung development contributes to a high mortality in these patients. To better understand the underlying pathobiology, we used the nitrofen rat model of CDH and characterized the proteome of hypoplastic CDH lungs at the alveolar stage (E21). Amongst the 218 significantly altered proteins between CDH and control lungs were Tenascin C, CREBBP, LYN and STAT3. We showed that Tenascin C was decreased around the distal airway branches in nitrofen and human fetal CDH lungs. In contrast, STAT3 was significantly increased in the airway epithelium of nitrofen lungs at E21. STAT3 inhibition after direct nitrofen exposure to fetal rat lung explants (E14.5) partially reversed the hypoplastic lung phenotype ex vivo by increasing peripheral lung budding. Moreover, we demonstrated that several STAT3 associated cytokines (IL-15, IL-9, IL-2) are increased in fetal tracheal aspirates of CDH survivors compared to non-survivors after fetoscopic tracheal occlusion. Using pathway analysis for significantly altered proteins in our proteomic analysis, we observed an enrichment in inflammatory response associated with Epstein Barr Virus and cytokine signaling in nitrofen CDH lungs. However, we were unable to detect EBV mRNA via in-situ Hybridization in human CDH lungs. With our unbiased proteomics approach, we show for the first time that inflammatory processes are likely underlying the pathogenesis of abnormal lung development in CDH.

developmental biology↗