single donor human umbilical vein endothelial cell huvec lines  (PromoCell)

Journal: medRxiv

Article Title: Machine Learning and Micro Capture-C resolve GWAS associations revealing endothelial stress pathways in Coronary Artery Disease

doi: 10.64898/2025.12.18.25342557

Figure Lengend Snippet: Machine Learning predicts the effect of genetic variants in coronary artery cell types. A. REnformer was trained on publicly available scATAC data from primary coronary artery tissue samples. REnformer was then used to predict the genome accessibility for 9 coronary artery cell types. An example locus around the MMP2 gene is shown. The tracks show the ground truth scATAC data (labelled “ATAC” followed by the cell type), and directly below the REnformer prediction for that cell type. B. The effect on genome accessibility of 21,348 CAD associated lead SNPs and high linkage disequilibrium candidate causal SNPs is predicted by REnformer across 9 coronary artery cell types as well as HUVECs. Predicted coverage (Y-axis) shows the predicted chromatin accessibility at a given SNP position. Alt-Ref prediction (X-axis) shows the predicted effect size of the candidate CAD SNP. Empirical significance cut-offs of 0.01 were calculated for each cell type; variants below the cut-off (loss of function) are coloured blue, variants above the cut-off (gain of function) are coloured red. C. Example REnformer predictions at the chr1:q25.1 locus for SNP rs1879454 (reference allele = C, alternate allele =A). The position of rs1879454 is shown by a blue triangle. D. The prediction difference for rs1879454 where the reference allele has been subtracted from the alternate allele. E. Feature attribution for the REnformer predictions in panel C. The relative contribution of each base pair in the reference and alternate genome to the prediction is shown by height (positive or negative) of the relevant letter. The variant position is shown by a blue triangle.

Article Snippet: Single donor human umbilical vein endothelial cell (HUVEC) lines were purchased from Promocell (C-12200) or were isolated from umbilical cord tissue samples provided by the Anthony Nolan Trust, who received ethical approval from the East Midlands-Derby, UK Research Ethics Committee and written, informed consent from the donor’s parents.

Techniques: Variant Assay

Journal: medRxiv

Article Title: Machine Learning and Micro Capture-C resolve GWAS associations revealing endothelial stress pathways in Coronary Artery Disease

doi: 10.64898/2025.12.18.25342557

Figure Lengend Snippet: A. Genome regulatory elements including enhancers (Enh), CTCF sites, and promoters (Pr) were determined for HUVECs using REgulamentary. B. Chart showing the number of intersections between REgulamentary defined regulatory elements and candidate causal variants.

Article Snippet: Single donor human umbilical vein endothelial cell (HUVEC) lines were purchased from Promocell (C-12200) or were isolated from umbilical cord tissue samples provided by the Anthony Nolan Trust, who received ethical approval from the East Midlands-Derby, UK Research Ethics Committee and written, informed consent from the donor’s parents.

Techniques:

Journal: medRxiv

Article Title: Machine Learning and Micro Capture-C resolve GWAS associations revealing endothelial stress pathways in Coronary Artery Disease

doi: 10.64898/2025.12.18.25342557

Figure Lengend Snippet: REnformer prioritises candidate causal variants in HUVECs. REnformer predictions are shown for 4 example candidate causal variants: rs604723 (A), rs2508619 (B), rs582384 (C), rs17293632 (D). In each panel, the prioritised SNP is labelled above the gene annotation, and other non-prioritised SNPs are shown as vertical black lines. The REnformer prediction (alt-ref) is shown below the gene annotation and directly above ATACseq data for HUVECs. Publicly available ChIPseq tracks (H3K4me3, H3K4me1, H3K27ac, CTCF) are also shown to aid identification of the genome regulatory elements. REnformer feature attribution for the 40bp around the prioritised SNP (yellow highlight) is shown below the genome tracks in each panel; the reference allele feature attribution is shown above the alternate allele. Statistically significant transcription factor binding motifs are shown below the feature attribution in B, C, and D. The identity of the transcription factor binding the affected motif in A is unknown.

Article Snippet: Single donor human umbilical vein endothelial cell (HUVEC) lines were purchased from Promocell (C-12200) or were isolated from umbilical cord tissue samples provided by the Anthony Nolan Trust, who received ethical approval from the East Midlands-Derby, UK Research Ethics Committee and written, informed consent from the donor’s parents.

Techniques: Binding Assay

Journal: medRxiv

Article Title: Machine Learning and Micro Capture-C resolve GWAS associations revealing endothelial stress pathways in Coronary Artery Disease

doi: 10.64898/2025.12.18.25342557

Figure Lengend Snippet: Analysis of the endothelial enhancer-promoter map for CAD genetics. A. String diagram showing genes linked by MCC to a HUVEC enhancer containing a candidate causal CAD SNP. Interactions between a captured MCC enhancer and gene promoters were called using the bespoke MCC peakcaller MCC_LoTron. Gene promoters were defined using REgulamentary. Nodes which are enriched for specified GO terms are highlighted by colour. The thickness of the connecting line corresponds to the strength of the interaction evidence between two nodes. B. Gene Ontology analysis (biological processes enrichment) of the MCC derived gene network.

Article Snippet: Single donor human umbilical vein endothelial cell (HUVEC) lines were purchased from Promocell (C-12200) or were isolated from umbilical cord tissue samples provided by the Anthony Nolan Trust, who received ethical approval from the East Midlands-Derby, UK Research Ethics Committee and written, informed consent from the donor’s parents.

Techniques: Derivative Assay

Journal: medRxiv

Article Title: Machine Learning and Micro Capture-C resolve GWAS associations revealing endothelial stress pathways in Coronary Artery Disease

doi: 10.64898/2025.12.18.25342557

Figure Lengend Snippet: POINTseq detects the changes in MCC linked output genes. A. Schematic overview of experimental approach. MCC links candidate causal enhancer SNPs to the promoters of output genes. Long read sequencing is used to phase SNPs and reconstruct the alleles from the donor cells. ATACseq and POINTseq data is re-mapped to the reconstructed alleles and reads are assigned to the allele they are derived from based on corresponding SNPs. The amount of signal coming from each allele can then be measured and compared, demonstrating the effect of a casual SNP in an enhancer on gene transcriptional output. B. Example POINTseq data tracks compared to traditional RNAseq. POINTseq, polyA-, and polyA+ RNAseq were performed on 3 HUVEC donors. Reads are split between forward and reverse DNA strand, mapped to the hg38 reference genome, and displayed as bigwig files. POINTseq offers far greater transcriptional gene coverage as introns are retained. D. Plot of POINTseq measured allelic skew in genes linked by MCC to candidate causal enhancer SNPs. Direction of change is displayed with respect to the reference allele versus the alternate allele (x-axis). Significance (log10 p-value) is displayed on the y-axis. Detected significant genes are labelled.

Article Snippet: Single donor human umbilical vein endothelial cell (HUVEC) lines were purchased from Promocell (C-12200) or were isolated from umbilical cord tissue samples provided by the Anthony Nolan Trust, who received ethical approval from the East Midlands-Derby, UK Research Ethics Committee and written, informed consent from the donor’s parents.

Techniques: Sequencing, Derivative Assay

Journal: Nature Communications

Article Title: Environmental enrichment and physical exercise prevent stress-induced social avoidance and blood-brain barrier alterations via Fgf2

doi: 10.1038/s41467-025-68058-9

Figure Lengend Snippet: A , Experimental timeline for inflammatory insult with TNF-α and FGF2 co-treatment. Once confluent (4 days), HBEC-5i or bEnd.3 were pretreated with FGF2 or vehicle for one hour and then stimulated with TNF-α or vehicle for up to 7 days. FGF2 alters mouse ( B ) and human ( C ) endothelial transcription in response to acute TNF-α stimulation. FGF2 promotes faster restoration of TNF-α-induced Cldn5 loss in mouse ( D ) and human ( E ) endothelial cells. Chronic stimulation with TNF-α leads to a reduction in endothelial monolayer integrity measured by trans-endothelial electrical resistance (TEER) in mouse ( F ) and human ( G ) endothelial cells. FGF2 co-treatment preserves normal TEER despite TNF-α. H 7 days of TNF-α treatment promotes spikes and discontinuities in Cldn5 tight junction strands in bEnd.3, which is reversed by FGF2 treatment (scalebar = 20 μm). Data represent mean ± s.e.m., and each experiment was replicated at least twice on independent samples. Group comparisons were evaluated with two-way ANOVA followed by Bonferroni’s post hoc tests; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: The human brain microvascular endothelial cell line HBEC-5i (ATCC CRL-3245, male donor according to https://www.cellosaurus.org/CVCL_4D10 ) and the mouse brain endothelial cell line bEnd.3 (ATCC CRL-2299) were subcultured and stored in banks at −150 ° C. Cells were thawed as needed and cultured in DMEM/F12 supplemented with 10% fetal bovine serum, 25 ug/mL gentamicin (Gibco), and 1X endothelial cell growth supplement (ScienCell).

Techniques:

Journal: Nature Communications

Article Title: Environmental enrichment and physical exercise prevent stress-induced social avoidance and blood-brain barrier alterations via Fgf2

doi: 10.1038/s41467-025-68058-9

Figure Lengend Snippet: A 1 h pretreatment with Fgf2 increases serine-9 phosphorylation of GSK3β in HBEC-5i when compared to no treatment (CTRL 0 h). TNF-α treatment induces rapid, transient dephosphorylation of GSK3β, but this effect is not reversed by Fgf2 coadministration. Each dot represents a replicate ( n = 3). B 1 h Fgf2 pretreatment diminishes basal β-catenin phosphorylation when compared to no treatment (CTRL 0 h). Further, while TNF-α induces a rapid reduction in phosphorylated β-catenin, Fgf2 reverses this dynamic upon inflammatory activation ( n = 3). C In health control endothelial cells (top), β-catenin interacts with VE-Cadherin at the cell membrane, and this complex inhibits Cldn5 transcriptional suppression by FOXO1. Excess cytosolic β-catenin is phosphorylated by GSK3β, targeting it for degradation. When stimulated with TNFα, unbound β-catenin complexes with FOXO1, leading to suppression of Cldn5 expression (bottom, red arrow), while a small amount is targeted for degradation. Meanwhile, when FGF2 is co-administered with TNF-α (bottom, blue arrow), our results suggest that unbound β-catenin is strongly redirected toward GSK3β-mediated phosphorylation. D 30 min of TNF-α is sufficient to induce β-catenin distribution at tight junctions ( n = 4 replicates) with representative images on the right ( E ) (scalebar = 20 μm). F Fgf2 attenuates TNF-α-induced reductions in the wound healing capacity of HBEC-5i ( n = 4 replicates) (**** p < 0.0001). Data represent mean ± s.e.m., and each experiment was replicated at least twice on independent samples. Group comparisons were evaluated with one or two-way ANOVA followed by Bonferroni’s post hoc tests or two-tailed t-tests with Welch’s correction when appropriate; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: The human brain microvascular endothelial cell line HBEC-5i (ATCC CRL-3245, male donor according to https://www.cellosaurus.org/CVCL_4D10 ) and the mouse brain endothelial cell line bEnd.3 (ATCC CRL-2299) were subcultured and stored in banks at −150 ° C. Cells were thawed as needed and cultured in DMEM/F12 supplemented with 10% fetal bovine serum, 25 ug/mL gentamicin (Gibco), and 1X endothelial cell growth supplement (ScienCell).

Techniques: Phospho-proteomics, De-Phosphorylation Assay, Activation Assay, Control, Membrane, Expressing, Two Tailed Test