Biology subjects

Mokry, M.

Publications and source records attributed to Mokry, M..

At least 19 recordsLinked to original sources

bioRxiv2025

A CROP-seq screen of histone modifying enzymes reveals histone demethylase Kdm5c regulates inflammatory macrophage activation.

BackgroundMacrophages adopt activation states along a spectrum from pro- to anti-inflammatory, enabling appropriate responses to pathogens and environmental cues. Dysregulated inflammatory macrophage activation contributes to diseases including sepsis, rheumatoid arthritis, cancer, and atherosclerosis. Epigenetic processes such as DNA methylation and histone modification prime macrophages for activation, and several histone modifying enzymes (HMEs) have been implicated in this regulation. ObjectiveTo systematically identify histone modifying enzymes that regulate inflammatory macrophage activation. MethodsWe performed a CRISPR knockout screen with single-cell RNA-seq readout (CROP-seq) targeting 92 macrophage-expressed HMEs in immortalized LPS-activated mouse bone marrow-derived macrophages (BMDMs). The resulting single-cell transcriptomes were analyzed to identify significant perturbations. Kdm5c was selected for experimental validation in mouse BMDMs, and its expression pattern was compared with macrophage subsets from human atherosclerotic plaques using scRNA-seq data. ResultsThe CROP-seq screen identified Prmt6, Carm1, Kat2b, and Kdm5c as top regulators of inflammatory macrophage activation. Validation in a KO cell line revealed loss of Kdm5c suppressed inflammatory tone at baseline but led to an exaggerated transcriptional response to LPS stimulation, indicating a role for Kdm5c in balancing tonic and inducible activation. A weighted gene module derived from Kdm5c-deficient macrophages was enriched in inflammatory macrophages in human atherosclerotic plaques. ConclusionOur findings demonstrate the value of CROP-seq screening to dissect the epigenetic control of macrophage activation. We also identify Kdm5c-mediated histone demethylation as a key mechanism modulating inflammatory macrophage activation.

molecular biology↗

bioRxiv2025

A collection of patient-derived intestinal organoid lines reveals epithelial phenotypes associated with genetic drivers of pediatric inflammatory bowel disease

Pediatric Inflammatory Bowel Disease (IBD) is a chronic condition characterized by per-sistent intestinal inflammation in children and adolescents. Despite a rising global incidence, the underlying causes and optimal management strategies for pediatric IBD are still not fully understood. Compared to adult IBD, pediatric IBD frequently presents with distinct disease phenotypes, and is more commonly linked to rare monogenic variants that cause intestinal epithelial barrier dysfunction or affect the function of mucosal immune cells. While more than 100 genes have been associated with early-onset IBD, the roles of many of these genes in the intestinal epithelium and the mechanisms by which genetic variants contribute to disease remain poorly defined. Here we aimed to improve our understanding of intestinal epithelium dysfunction in early-onset IBD by conducting extensive molecular and cellular characterization to gain insights into patient-specific epithelial phenotypes and identify therapeutic targets. We generated intestinal epithelial organoids (IEOs) from 94 pediatric IBD patients, representing diverse clinical characteristics and including those with monogenic variants (BTK n=4, TTC7A n=3, IL10RA n=1, LRBA n=1, STXBP2 n=1, TTC37 n=1, TRNT1 n=1, PLCG2 n=1, DKC1 n=1, POLA1 n=1), and 46 non-IBD controls. This effort resulted in the largest RNA-seq dataset of pediatric IBD intestinal epithelial organoids to date, encompassing both baseline conditions and post-immunological stimulation, serving as a valuable resource for future research. We observed that IEOs effectively initiate inflammation upon stimulation with bacterial lysate, regardless of disease status, origin, or mutation status. Inflammatory stimulation triggered single-gene upregulation of IBD-linked SERPINA1 and LIFR across the IBD population compared to controls, suggesting their role in intestinal epithelial innate immune responses. However, co-expression network analysis showed no consistent transcriptional signatures across the entire IBD group at the systems level. Instead, differences emerged between controls and specific genotypes (TTC7A, STXBP2, LRBA), with STXBP2 and LRBA sharing an upregulated transcriptional response of IL-1 and SLC30-mediated zinc trafficking pathways. These findings underscore the potential of IEOs as a valuable model for studying IBD and offer key insights that could guide the development of targeted therapies for both monogenic and non-monogenic forms of IBD. O_FIG O_LINKSMALLFIG WIDTH=158 HEIGHT=200 SRC="FIGDIR/small/659052v2_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@10a4d9org.highwire.dtl.DTLVardef@b6cdadorg.highwire.dtl.DTLVardef@179216dorg.highwire.dtl.DTLVardef@181a4e4_HPS_FORMAT_FIGEXP M_FIG C_FIG

genetics↗

bioRxiv2025

Distinct heterozygous TTC7A missense variants lead to different intestinal epithelial phenotypes in pediatric IBD

Pathogenic mutations in Tetratricopeptide repeat domain 7A (TTC7A) result in gastrointestinal and immunological disorders of which the pathobiology is not fully understood. Previous case reports indicate that TTC7A plays an important role in preserving intestinal epithelial integrity, but thus far only few variants have been investigated and it is unclear if different variants exert the same effects. Here, we aim to study the effects of different variants on the intestinal epithelium. We present three instances of pediatric inflammatory bowel disease (IBD), displaying varying clinical symptoms and severity levels, and associated with different heterozygous missense mutations in TTC7A. Intestinal organoids derived from patients show dissimilar epithelial phenotypes and exhibit differences in growth, morphology, apicobasal polarity, responses to specific drugs, TTC7A expression, and transcriptional profiles. The findings of our study suggest differences in pathobiology between individuals with different TTC7A mutations. This investigation enhances our comprehension of TTC7A-related conditions and can have implications for developing targeted therapies for TTC7A-associated disorders.

genetics↗

bioRxiv2025

Kynurenine and NAD+ Pathways are Associated with Macrophage Content and Polarization in Carotid Plaques

Background and AimsMetabolism dictates macrophage function and plays a central role in atherosclerotic plaque progression. The kynurenine pathway, which metabolizes the majority of the essential amino acid tryptophan, plays a pivotal role in regulating immune responses and supporting NAD+ synthesis, essential for cellular energy metabolism. Higher circulating kynurenine levels are associated with cardiovascular disease, yet their role in atherosclerotic plaques is unclear. This study aims to investigate the underlying mechanisms driving increased kynurenine concentrations in plaques and to determine whether kynurenine serves as a mere biomarker of low-grade inflammation or reflects specific macrophage-driven metabolic alterations that could position it as a potential therapeutic target. MethodsWe used histological and transcriptomic data from two biobanks: the Athero Express Biobank (AE; n=91) and Maastricht human plaque study (MaasHPS, n= 26). Macrophages were identified through CD68 staining in AE, and M1/M2-like macrophage subtypes were distinguished by iNOS/CD68 and arginase/CD68 expression in MAASHPS. Primary human monocyte-derived cultured macrophages were polarized into M1- and M2-like phenotypes for using IFN-{gamma} and IL-4, respectively. Tryptophan, kynurenine and/or NAD+ concentrations in plaques were quantified usingliquid chromatography and metabolomics analyses. ResultsKynurenine concentrations were significantly higher in plaques with greater macrophage density (p = 0.023). Transcriptomic analysis in AE revealed upregulation of IDO2, AFMID, and KYNU in plaques with increased macrophage infiltration (p < 0.05), but not IDO1 (p = 0.16). In the MAASHPS biobank, higher IDO1, KYNU, and KMO expression correlated negatively with M2 marker positive macrophages (p < 0.001), while HAAO correlated positively (p < 0.01). In vitro, M1-like macrophages showed increased IDO1 and reduced QPRT expression compared to M2-like macrophages. We found that this disruption in kynurenine pathway gene expression led to decreased NAD+ concentrations in M1-like macrophages compared to M2-like macrophages in vitro. ConclusionHigher kynurenine levels in atherosclerotic plaques are increased by the increased presence of M1 macrophages, likely driven by both an increased IDO1 activity and reduced QPRT gene expression. This leads to decreased concentrations of NAD+, potentially determining the phenotype of the macrophages. Future studies should address whether modulation of the kynurenine pathway restores NAD+ metabolism and leads to a decrease in inflammation and an increased stable plaque phenotype.

immunology↗

bioRxiv2025

Female-biased vascular smooth muscle cell gene regulatory networks predict MYH9 as a key regulator of fibrous plaque phenotype

Atherosclerosis, a chronic inflammatory condition driving coronary artery disease (CAD), manifests in two primary plaque types: unstable atheromatous plaques and stable fibrous plaques. While significant research has focused on atheromatous plaques, recent studies emphasize the growing importance of fibrous plaques, particularly in females under 50 years of age, where erosion on fibrous plaques significantly contributes to coronary thrombosis. The molecular mechanisms underlying sex differences in atherosclerotic plaque characteristics, including vascular smooth muscle cell (VSMC) contributions, remain understudied. Therefore, we utilized sex-specific gene regulatory networks (GRNs) derived from VSMC gene expression data from 119 male and 32 female heart transplant donors to identify molecular drivers of fibrous plaques. GRN analysis revealed two female-biased networks in VSMC, GRNfloralwhite and GRNyellowgreen, enriched for inflammatory signaling and actin remodeling pathways, respectively. Single-cell RNA sequencing of carotid plaques from female and male patients confirmed the sex specificity of these networks in VSMCs. Further sub cellular phenotyping of the single-cell RNA sequencing revealed a sex-specific gene expression signature within GRNyellowgreen for VSMCs enriched for contractile and vasculature development pathways. Bayesian network modeling of the GRNyellowgreen identified MYH9 as a key driver gene. Indeed, elevated MYH9 protein expression in atherosclerotic plaques was associated with higher smooth muscle cell content and lower lipid content in female plaques, suggesting its involvement in fibrous plaque formation. Further proteomic analysis confirmed MYH9s upregulation in female fibrous plaques only and its correlation with stable plaque features. These findings provide novel insights into sex-specific molecular mechanisms regulating fibrous plaque formation.

genomics↗

bioRxiv2025

Human colon stem cells are the principal epithelial responders to bacterial antigens

Intestinal epithelial cells (IECs) are capable of mounting an adequate antimicrobial inflammatory response to pathogens while tolerating commensals. The underlying regulatory mechanisms of immune sensitivity remain incompletely understood, particularly in the context of human IECs. To enhance our understanding of the immune response of IECs to bacterial epithelial barrier breach, we investigated whether epithelial responsiveness is contingent on cell identity and cell polarization. We exposed human intestinal organoids to bacterial antigens to study their immune responses. Notable discrepancies were observed in the specific reactions exhibited by intestinal stem cells (ISCs) and enterocytes. It was determined that basolateral exposure of IECs to bacterial antigens resulted in a robust response, whereas apical exposure elicited a significantly more modest response. We identified ISCs as the responders, while the reaction of enterocytes was found to be attenuated. The regulation of bacterial responsiveness in enterocytes occurs at multiple levels, including the modulation of NF{kappa}B activation and post-transcriptional control of mRNA stability. Our findings demonstrate that differentiated non-responsive enterocytes can be sensitized to bacterial antigens through the activation of the WNT pathway. These findings extend the crucial role of WNT signaling for intestinal epithelial homeostasis and regulation of stem cell maintenance, proliferation, differentiation, and tissue architecture in the gut. Additionally, they reveal a new function of WNT signaling in regulating microbial responses within the intestinal environment.

immunology↗

bioRxiv2024

IFNγ induces epithelial reprogramming driving CXCL11-mediated T cell migration

The cytokine interferon-gamma (IFN{gamma}) plays a multifaceted role in intestinal immune responses ranging from anti-to pro-inflammatory depending on the setting. Here, using a 3D co-culture system based on human intestinal epithelial organoids, we explore the capacity of IFN{gamma}-exposure to reprogram intestinal epithelia and thereby directly modulate lymphocyte responses. IFN{gamma} treatment of organoids led to transcriptional reprogramming, marked by a switch to a pro-inflammatory gene expression profile, including transcriptional upregulation of the chemokines CXCL9, CXCL10, and CXCL11. Proteomic analysis of organoid-conditioned medium post-treatment confirmed chemokine secretion. Furthermore, IFN{gamma}-treatment of organoids led to enhanced T cell migration in a CXCL11-dependent manner without affecting T cell activation status. Taken together, our results suggest a specific role for CXCL11 in T cell recruitment that can be targeted to prevent T cell trafficking to the inflamed intestine.

immunology↗

bioRxiv2023

Colchicine promotes atherosclerotic plaque stability independently of inflammation

Atherosclerosis is a chronic inflammatory disease which is driven in part by the aberrant trans-differentiation of vascular smooth muscle cells (SMCs). No therapeutic drug has been shown to reverse detrimental SMC-derived cell phenotypes into protective phenotypes, a hypothesized enabler of plaque regression and improved patient outcome. Herein, we describe a novel function of colchicine in the beneficial modulation of SMC-derived cell phenotype, independent of its conventional anti-inflammatory effects. Using SMC fate mapping in an advanced atherosclerotic lesion model, colchicine induced plaque regression by converting pathogenic SMC-derived macrophage-like and osteoblast-like cells into protective myofibroblast-like cells which thickened, and thereby stabilized, the fibrous cap. This was dependent on Notch3 signaling in SMC-derived plaque cells. These findings may help explain the success of colchicine in clinical trials relative to other anti-inflammatory drugs. Thus, we demonstrate the potential of regulating SMC phenotype in advanced plaque regression through Notch3 signaling, in addition to the canonical anti-inflammatory actions of drugs to treat atherosclerosis.

cell biology↗

bioRxiv2023

Identification of endothelial-to-mesenchymal transition gene signatures in single-cell transcriptomics of human atherosclerotic tissue

RationaleEndothelial cells can differentiate into mesenchymal-like cells via endothelial to mesenchymal transition (EndoMT). In murine models, cell transitions of EndoMT have been assessed with lineage tracing techniques. Knowledge on molecular mechanisms of EndoMT in human vascular lesions is scarce as studies in human atherosclerosis are limited by observational study designs such as histo-pathological studies. ObjectiveWe aim to identify a human EndoMT gene expression signature by combining experimentally induced in vitro EndoMT with lineage-traced pathways from atherosclerotic mice and extrapolate this to human plaque scRNA-seq data. Methods and resultsFirst, we stimulated human coronary artery endothelial cells (HCAEC) with TNF and TFG{beta} to trigger EndoMT. We executed transcriptomic analyses and defined multiple temporal patterns of gene expression changes during EndoMT. We used Cdh5-CreERT2 Rosa-eYFP apoE-/- lineage traced mouse scRNA-seq data to demonstrate that the temporal in vitro gene expression changes are reflected in EndoMT trajectories in mice plaque tissue. Finally, we constructed three candidate EndoMT lineages across multiple subpopulations of ECs and SMCs in human carotid scRNA-seq data (n=46). We examined gene expression over the course of these lineages and identified 73 markers for the presence of EndoMT such as NRG1 and DEPP1. ConclusionThis study reveals the gene expression profile of EndoMT trajectories in human atherosclerotic plaques by combining RNA-seq data from in vitro models with single-cell transcriptomic datasets. Our gene expression atlas of EndoMT in atherosclerosis could serve as a reference for future studies, providing novel inroads to study atherosclerotic mechanisms for the development of novel therapies.

molecular biology↗

bioRxiv2023

Chemotherapy-induced intestinal injury promotes Galectin-9-driven modulation of T cell function

The intestine is vulnerable to chemotherapy-induced toxicity due to its high epithelial proliferative rate, making gut toxicity an off-target effect in several cancer treatments, including conditioning regimens for allogeneic hematopoietic cell transplantation (allo-HCT). In allo-HCT, intestinal damage is an important factor in the development of Graft-versus-Host Disease (GVHD), an immune complication in which donor immune cells attack the recipients tissues. Here, we developed a novel human intestinal organoid-based 3D model system to study the direct effect of chemotherapy-induced intestinal epithelial damage on T cell behavior. Chemotherapy treatment using busulfan, fludarabine, and clofarabine led to damage responses in organoids resulting in increased T cell migration, activation, and proliferation in ex-vivo co-culture assays. We identified galectin-9 (Gal-9), a beta-galactoside-binding lectin released by damaged organoids, as a key molecule mediating T cell responses to damage. Increased levels of Gal-9 were also found in the plasma of allo-HCT patients who later developed acute GVHD, supporting the predictive value of the model system in the clinical setting. This study highlights the potential contribution of chemotherapy-induced epithelial damage to the pathogenesis of intestinal GVHD through direct effects on T cell activation and trafficking promoted by galectin-9.

immunology↗

bioRxiv2023

Human primary plaque cell cultures to study mechanisms of atherosclerosis

Plaque smooth muscle cells are critical players in the initiation and advancement of atherosclerotic disease. They produce extracellular matrix (ECM) components, which play a role in lesion progression and stabilization. Despite clear phenotypic differences between plaque smooth muscle cells and vascular smooth muscle cells (VSMCs), VSMCs are still widely used as a model system in atherosclerotic research. Here we present a conditioned outgrowth method to isolate plaque smooth muscle cells. We obtained plaque cells from 27 donors (24 carotid and 3 femoral endarterectomies). We show that these cells keep their proliferative capacity for eight passages, are transcriptionally stable, retain donor-specific gene expression programs, and express extracellular matrix proteins (FN1, COL1A1, DCN) and smooth muscle cell markers (ACTA2, MYH11, CNN1). Single-cell transcriptomics of plaque tissue and cultured cells reveals that cultured plaque cells closely resemble the myofibroblast fraction of plaque smooth muscle cells. Chromatin immunoprecipitation sequencing (ChIP-seq) shows the presence of histone H3 lysine 4 dimethylation (H3K4me2) at the MYH11 promoter, pointing to their smooth muscle cell origin. Finally, we demonstrated that plaque cells can be efficiently transduced (>97%) and are capable to take up oxidized LDL (oxLDL) and undergo calcification. In conclusion, we present a method to isolate and culture primary human plaque cells that retain plaque myofibroblast-like cells phenotypical and functional capabilities - making them a suitable in vitro model for studying selected mechanisms of atherosclerosis.

cell biology↗

bioRxiv2023

Female gene networks are expressed in myofibroblast-like smooth muscle cells in vulnerable atherosclerotic plaques.

Women presenting with coronary artery disease (CAD) more often present with fibrous atherosclerotic plaques, which are currently understudied. Phenotypically modulated smooth muscle cells (SMCs) contribute to atherosclerosis in women. How these phenotypically modulated SMCs shape female versus male plaques is unknown. Here, we show sex-stratified gene regulatory networks (GRNs) from human carotid atherosclerotic tissue. Prioritization of these networks identified two main SMC GRNs in late-stage atherosclerosis. Single-cell RNA-sequencing mapped these GRNs to two SMC phenotypes: a phenotypically modulated myofibroblast-like SMC network and a contractile SMC network. The myofibroblast-like GRN was mostly expressed in plaques that were vulnerable in females. Finally, mice orthologs of the female myofibroblast-like genes showed retained expression in advanced plaques from female mice but were downregulated in male mice during atherosclerosis progression. Female atherosclerosis is driven by GRNs that promote a fibrous vulnerable plaque rich in myofibroblast-like SMCs.

bioinformatics↗

bioRxiv2022

Functional Specialization of Specific DC Subsets in the Synovial Fluid of JIA Patients

Dendritic cells (DC) are crucial for initiating and shaping immune responses. So far, little is known about heterogeneity and functional specialization of human DC subsets in (local) inflammatory conditions. We profiled conventional (c)DC1, cDC2 and monocytes based on phenotype, transcriptome and function from a local inflammatory site, namely synovial fluid (SF) from patients suffering from a chronic inflammatory condition, Juvenile Idiopathic Arthritis (JIA). cDC1, a relatively small dendritic cell subset in blood, are strongly enriched in SF. SF cDC1 showed a quiescent immune signature without a clear inflammatory profile low expression of PRR, chemokine, and cytokine receptors, and poor induction of T cell proliferation and cytokine production, but selective production of IFN{lambda} upon polyinosinic:polycytidylic acid exposure. In stark contrast, cDC2 and monocytes from the same environment, showed a pro-inflammatory transcriptional profile, high levels of (spontaneous) pro-inflammatory cytokine production, and strong induction of T cell proliferation and cytokine production, including IL-17. Although the cDC2 and monocytes showed an overlapping transcriptional core profile, there were clear differences in the transcriptional landscape and functional features, indicating that these cell types retain their lineage identity in chronic inflammatory conditions. In conclusion, our findings suggest that at the site of inflammation, there is specific functional programming of human DC subsets, especially cDC2. In contrast, the enriched subset of cDC1 remains relatively quiescent and seemingly unchanged under inflammatory conditions, pointing to a more regulatory role.

immunology↗

bioRxiv2021

Epigenetic changes in autoimmune monocytes contribute to disease and can be targeted by JAKinhibition

How the local inflammatory environment regulates epigenetic changes in the context of autoimmune diseases remains unclear. Here we assessed the transcriptional and active enhancer profile of monocytes derived from the inflamed joints of Juvenile Idiopathic Arthritis (JIA) patients, a model well-suited for studying autoimmune diseases. RNA-sequencing analysis demonstrated that synovial-derived monocytes display an activated phenotype, characterized by increased expression of surface activation markers and multiple chemokines and cytokines. This increased expression is regulated on the epigenetic level, as ChIP-sequencing indicated increased H3K27 acetylation of genes upregulated in synovial monocytes. IFN signaling-associated genes are increased and epigenetically altered in synovial monocytes, suggesting a role for IFN in establishing the inflammatory phenotype. Treatment of human synovial monocytes with the Janus-associated kinase (JAK) inhibitor Ruxolitinib, which inhibits IFN signaling, transformed the activated enhancer landscape and reduced disease-associated gene expression, thereby inhibiting the inflammatory phenotype. Taken together, these data provide novel insight into epigenetic regulation of autoimmune disease-patient derived monocytes and suggest that altering the epigenetic profile can be a therapeutic approach for the treatment of autoimmune diseases.

immunology↗

bioRxiv2021

Locational memory of macrovessel vascular cells is transcriptionally imprinted

The locational predisposition of vascular pathologies illustrates the need for a better insight into vascular heterogeneity. To investigate the transcriptomic basis of angiodiversity, we isolated and analyzed transcriptomes from endothelial cells and vascular smooth muscle cells from nine different adult canine macrovessels: the aorta, coronary artery, vena cava, portal vein, femoral artery, femoral vein, saphenous vein, pulmonary vein, and pulmonary artery. We identified both reported and novel expression patterns defining specialized adult blood vessels. Our findings also show that adult vascular cells in culture express a remarkably high number of transcription factors crucial to organ development in the embryo. The persistent expression of these genes in culture indicates that these genes are not regulated by the flow or surrounding cell types but are rather fixed in the molecular memory. Therefore, our findings prompt the re-thinking of the extrapolation of results from single-origin endothelial cell systems.

cell biology↗

bioRxiv2021

Human placental bed transcriptomic profiling reveals inflammatory activation of endothelial cells in preeclampsia

RationaleFunctional characteristics of endothelial cells (ECs) within the human placental bed are unknown and may provide insight into the adaptive biology of ECs in disorders of vascular remodelling like preeclampsia. ObjectiveTo determine transcriptional profiles of human placental bed ECs and systemic biomarker profiles in women with normal pregnancy, and women with preeclampsia, a condition characterized by extensive EC dysfunction, poor development of spiral arteries underlying the placenta and long-term susceptibility to atherosclerosis and hypertension. Methods & resultsWe obtained biopsy samples from the uterine placental bed, of five women with preeclampsia with fetal growth restriction (FGR) due to impaired spiral artery development and four controls undergoing Caesarean section. CD31+CD146+ ECs were isolated and sorted by flow cytometry for RNA-sequencing using CEL-Seq2 protocol. Data were analyzed by unsupervised clustering, gene set enrichment (GSEA) and pathway analysis. 67 circulating biomarkers of EC function and inflammation were measured in 20 women with preeclampsia with FGR and 20 controls by multiplex immunoassay. Transcriptional profiling showed various differentially expressed genes (FDR<0.05) in placental bed ECs of preeclampsia patients, with enhanced activity of pathways associated with vasoconstriction, platelet activation and innate immunity. GSEA was suggestive of a VEGF- and PlGF deprived state of preeclampsia-derived ECs. Moreover, the transcriptomic profile was similar to that of human umbilical vein endothelial cells (HUVECs) treated with plasma from preeclampsia patients, pointing towards a central role for circulating factors in EC dysfunction. Unsupervised clustering of subjects by EC-related circulating factors identified distinct profiles for healthy pregnancy and preeclampsia, in particular for those women with low platelets and elevated liver enzymes, which was predominantly driven by sFLT-1, endoglin, PlGF, leptin, SAA-1 and sICAM-1. ConclusionsWe revealed inflammatory activation of EC and a key role for systemic factors in EC dysfunction in women with preeclampsia associated with impaired spiral artery development.

immunology↗

bioRxiv2021

Sex-dependent gene regulation of human atherosclerotic plaques by DNA methylation and transcriptome integration points to smooth muscle cell involvement in women.

Sex differences are evident in the clinical presentation and underlying histology of atherosclerotic disease with women developing more stable atherosclerotic lesions than men. It is unknown whether this is explained by sex differences in gene regulation in cellular compartments of atherosclerotic plaques. To study sex differences in gene regulation we performed genome-wide DNA methylation and transcriptomics analysis on plaques of 485 carotid endarterectomy patients (31% female). Sex-differential DNA methylation at 4,848 sites in the autosome was enriched for cell-fate commitment and developmental processes, and its deconvolution predicted more smooth muscle cells in females, as compared to more immune cells in males. RNA-sequencing of the same plaques corroborated the sex differences in DNA methylation predicted cell-types, in which genes that were higher expressed in females were enriched for TGF-beta signaling and extracellular matrix biology. In addition, female-biased genes were enriched for targeting by regulatory loci based on sex differential methylation. Lastly, by using single-cell RNA sequencing we showed that these female-biased genes are mostly expressed in smooth muscle cells, and higher expressed in smooth muscle cells from female (predominantly stable) plaques as compared to male (relatively unstable) plaques. Our approach identified female-biased genes in smooth muscle cells in fibrous atherosclerotic plaques. This points towards new mechanisms in smooth muscle cell biology of stable atherosclerotic plaques and offers new directions for research to develop new sex-specific therapeutics for atherosclerotic disease.

genomics↗

bioRxiv2020

Transcriptional regulation profiling reveals disrupted lipid metabolism in failing hearts with a pathogenic phospholamban mutation

BackgroundThe R14del mutation in the phospholamban (PLN) gene is associated with various types of cardiomyopathies and increases the risk of developing life-threatening ventricular arrhythmias. In this study, we focused on a homogeneous Dutch founder cohort of genetic cardiomyopathy due to PLN R14del mutation and aimed to study the influence of epigenetic changes from a multi-dimensional perspective. ResultsUsing cardiac tissue of PLN R14del patients and donors, we identified differentially acetylated promoters and enhancers (H3K27ac ChIPseq), annotated enriched transcription factor (TF) binding motifs located in those regions, and identified differentially expressed genes (RNA-seq). In line with the fibrofatty replacement in PLN R14del hearts at the histological level, our integrative analysis detected the downregulation of key TF regulators in fatty acid oxidation (FAO) metabolisms and their downstream target in PLN R14del hearts as compared to controls. We further examined heart tissue using immunofluorescence staining (IF) and to confirm the mitochondrial lipid abnormalities in the PLN R14del hearts. Furthermore, we observed the accumulation and deformation of lipid droplets and a disrupted morphology of mitochondria, the key organelle where FAO takes place, in PLN R14del heart using transmission electron microscopy (TEM). ConclusionUsing multi-omics approaches, we successfully obtained a unique list of chromatin regions and genes, including TF-coding genes, which played important roles in the metabolism-related signalling in PLN R14del hearts.

genomics↗