Journal: PLOS Neglected Tropical Diseases

Article Title: In vitro and in vivo endothelial interactions of Leptospira species are markers of virulence

doi: 10.1371/journal.pntd.0013939

Figure Lengend Snippet: (top) HMEC-1 cells were grown to post-confluence, bacteria were added at an MOI of 20 and incubated for one hour. Non-associated bacteria were removed by washing and qPCR was performed to quantify associated bacteria. Strains associate with endothelial cells to varying extents, with 3/6 P1 + strains and 2/5 P1- species binding significantly more than Lb P. (bottom) HMEC-1 cells were grown to confluence on glass coverslips and infected at an MOI of 20 for 24 h. After washing to remove unbound bacteria, cells were fixed, stained for VE-cadherin, and mounted with Prolong Diamond Antifade Mountant with DAPI. Binary area was quantified as previously described and subtracted from uninfected controls to define “VE-cadherin disruption”. P1 + Leptospira (4/6) disrupt VE-cadherin more than other clades (3/10). Spearman correlation analysis determines cell association and VE-cadherin disruption correlate with r s = 0.644 and p = 0.0085. Strains are ordered based upon presence of virulence-associated genes in their genomes . Mean ± SEM is plotted. Each column is compared to LbP . * p < 0.05, # p < 0.01, & p < 0.001, $ p < 0.0001.

Article Snippet: Human dermal microvascular endothelial cells (HMEC-1) [ ] were obtained from ATCC (CRL-3243) and cultured as described previously [ , ] in MCDB medium at 37°C with 5% CO 2 .

Techniques: Bacteria, Incubation, Binding Assay, Infection, Staining, Disruption

Journal: bioRxiv

Article Title: Urban PM 2.5 at Realistic Environmental Concentrations Impairs Blood–Brain Barrier Integrity and Enhances LOX-1 Expression in Human Brain Endothelial Cells

doi: 10.64898/2026.01.29.702473

Figure Lengend Snippet: A . Physiological rationale: Ambient PM2.5 exposure is epidemiologically linked to increased ischemic stroke risk. This in vitro model simulates the real-life scenario of pre-existing PM2.5 exposure followed by ischemic stroke and subsequent reperfusion. B . Primary adult male HBMEC were exposed to 5, 15, 75, or 300 μg/m 3 PM 2.5 for 48h in total. To compare with the effects of physiological ischemic-like injury, some plates were exposed to hypoxia (1% O 2 ) and glucose deprived media (HGD) for 3h after the initial 24h incubation. Following HGD or normoxia, cells were reperfused with nutrient-enriched media and incubated with PM 2.5 at normoxic (21% O 2 ) conditions as a reference for resolution of ischemia. Barrier integrity, cell viability, reactive oxygen species (ROS), inflammation and LOX-1 expression was assessed. Figure created in BioRender.

Article Snippet: Primary adult male HBMEC were purchased from Innoprot (Spain, Catalog number: P10361, Lot number: 111224CS).

Techniques: In Vitro, Incubation, Expressing

Journal: bioRxiv

Article Title: Urban PM 2.5 at Realistic Environmental Concentrations Impairs Blood–Brain Barrier Integrity and Enhances LOX-1 Expression in Human Brain Endothelial Cells

doi: 10.64898/2026.01.29.702473

Figure Lengend Snippet: Adult male HBMEC were exposed to vehicle or PM 2.5 (5, 15, 75, or 300 μg/m 3 ) for 24h and incubated for 3h in normoxia- or hypoxia and glucose deprivation (HGD) followed by 24h reperfusion. A . Live cell count (CyQUANT nuclear stain) decreased when exposed to ≥75 μg/m 3 PM 2.5 compared to vehicle. HGD treatment reduced live cell count compared to normoxia but did not differ between particle treated groups. B . Reactive oxygen species (ROS) signal (DCHF-DA) normalized to live cell count. Relative ROS levels increased dose-dependently with PM 2.5 concentration, with significant increase observed at PM 2.5 ≥75 μg/m 3 , in comparison to normoxia vehicle. ROS levels were uniformly elevated following HGD across all doses in comparison to normoxia vehicle and significantly higher than untreated HBMEC. (n=12 technical replicates for vehicle and 5, n=8 technical replicates for 15, 75 and 300) C . Analysis of crystal violet-stained HBMEC shows a longer maximum cellular length when treated with ≥15 μg/m 3 PM 2.5 . (n=21-37 individual cells) D . Representative images of crystal violet-stained HBMEC visualizing a differentiated morphology in cells treated with higher PM 2.5 concentration, where cells appear more elongated and expanding towards neighbouring cells. Data presented as mean ± SD. Statistical significance assessed through Kruskal-Wallis test within treatment groups (Normoxia/HGD) and Mann-Whitney test between groups with different treatment (300 normoxia/vehicle HGD). *p<0.05. ***p<0.001. ****p<0.0001.

Article Snippet: Primary adult male HBMEC were purchased from Innoprot (Spain, Catalog number: P10361, Lot number: 111224CS).

Techniques: Incubation, Cell Characterization, CyQUANT Assay, Staining, Concentration Assay, Comparison, MANN-WHITNEY

Journal: bioRxiv

Article Title: Urban PM 2.5 at Realistic Environmental Concentrations Impairs Blood–Brain Barrier Integrity and Enhances LOX-1 Expression in Human Brain Endothelial Cells

doi: 10.64898/2026.01.29.702473

Figure Lengend Snippet: Western Blot assessment of adult male HBMEC exposed to vehicle, 5, 15, 75, or 300 μg/m 3 PM 2.5 during normoxia or ischemic-like injury with hypoxia, glucose deprivation and reperfusion (HGD). A . Representative Western Blot image of IL-6 and β-actin band migration. B . Signal quantification of 25kDa IL-6 shows no difference between PM 2.5 exposure or HGD treated group. C . Signal quantification of 17kDa IL-6 shows dose-dependency with higher IL-6 expression from higher PM 2.5 exposure, with significant increase ≥75 μg/m 3 and from HGD treatment compared to vehicle. D . Representative Western Blot image of LOX-1 and β-actin. E . Signal quantification of LOX-1 displays a dose-dependent increase in LOX-1 with exposure to ≥15 μg/m 3 PM 2.5 or HGD. (n=4-7 technical replicates). Data presented as mean +-SD. Statistical significance assessed by Kruskal-Wallis test. *p<0.05, **p<0.01.

Article Snippet: Primary adult male HBMEC were purchased from Innoprot (Spain, Catalog number: P10361, Lot number: 111224CS).

Techniques: Western Blot, Migration, Expressing

Journal: JACC: Basic to Translational Science

Article Title: Human Left Ventricle circRNA-miRNA-mRNA Network Analyses Reveal a Novel Proangiogenic Role for circNPHP1 Under Ischemic Conditions

doi: 10.1016/j.jacbts.2025.101468

Figure Lengend Snippet: CircNPHP1 Expression in the Human LV and Cultured ECs Cardiac CircNPHP1 levels are higher in patients with IHD with/without associated T2DM and in cultured endothelial cells (human cardiac microvascular endothelial cells and human umbilical vein endothelial cells) exposed to disease-mimicking conditions, such as hypoxia and hypoxia, combined with increased D-glucose level (High glu). (A) Preliminary screening of circRNAs (identified from predicted networks) in endothelial cells: human cardiac microvascular endothelial cells were cultured in normal conditions, hypoxia (1% Oxygen), and hypoxia-high glucose (High Glu) (25 mmol/L D-glucose) conditions, respectively. After 48 hours, cells were harvested for quantitative real-time polymerase chain reaction (qRT-PCR) for the analysis and validation of indicated circRNAs. RNA expression is relative to normoxia. 18S is used as housekeeping gene; n = 3. Data are expressed as mean ± SEM and were assessed by 1-way analysis of variance with Dunnett's post hoc test. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, and ns = not significant. CircSTX17 was also tested but the levels were undetectable; hence, it is not included in the plot. CircNPHP1 emerges as an interesting candidate for further exploration and validation. (B) Comparison of expression levels of circNPHP1 and its linear counterpart in different patient groups—RNAseq (LV biopsies, cohort 1). Log-transformed normalized (FPM) counts of circNPHP1 and linear NPHP1 are plotted as a boxplot showing the median and IQR. Comparison between patient groups and calculation of P values was done using the R package Limma. CircNPHP1 is significantly up-regulated in IHD+T2DM compared with IHD and non-IHD ( P < 0.05). Linear NPHP1 is expressed at very low levels and does not change between the different patient groups (IHD [n = 12], IHD+T2DM [n = 11], non-IHD [n = 12]). (C) qRT-PCR of circNPHP1 (left) and linear NPHP1 (right) of LV biopsies (cohort 1) from non-IHD (n = 12), IHD (n = 12), and IHD+T2DM (n = 11), and (D) of LV biopsies (cohort 2) from non-IHD (n = 10), IHD (n = 10), IHD+T2DM (n = 10) of patients. RNA expression is relative to non-IHD; 18S is used as housekeeping gene. Data are expressed as mean ± SEM and were assessed by Kruskal-Wallis test followed by Dunn's post hoc test. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, and ns = not significant. Abbreviations as in .

Article Snippet: Human cardiac microvascular endothelial cells (HCMECs) (Promocell: C-12285 [mono-doner], Lot numbers: 440Z021.5 [male], 440Z021.4 [male], 446Z001.1 [female], 492Z009.4 [female]) and human umbilical vein endothelial cells (HUVECs) (Promocell: C-12208 [poly-donor], Lot numbers: 447Z004, 450Z015, 466Z022, 503Z026) were cultured on 0.2% gelatin (Sigma-Aldrich) in endothelial cell growth medium MV and endothelial cell growth medium 2 (EGM2) (PromoCell).

Techniques: Expressing, Cell Culture, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Biomarker Discovery, RNA Expression, Comparison, Transformation Assay

Journal: JACC: Basic to Translational Science

Article Title: Human Left Ventricle circRNA-miRNA-mRNA Network Analyses Reveal a Novel Proangiogenic Role for circNPHP1 Under Ischemic Conditions

doi: 10.1016/j.jacbts.2025.101468

Figure Lengend Snippet: CircNPHP1 and Linear NPHP1 Expression in Different Cell Types (A) Single-cell and single-nuclei RNAseq data from healthy donor hearts (human heart atlas ) of NPHP1 gene as log2 transformed counts per million (CPM). Each dot indicates the data point as 1 donor (source: human heart atlas ). (B) RNAseq: A preliminary screen was carried out in different cells (sample size = 2 each) to determine absolute levels of circNPHP1 and linear NPHP1 in human umbilical vein endothelial cells (HUVECs or HU), human cardiac microvascular endothelial cells (HCMECs or HC), human AC16 cardiomyocytes (ACs), and cardiac fibroblasts (CFs). (C) HCMEC (left) and HUVEC cells (right) were cultured in either normal conditions, hypoxia (1% oxygen), or hypoxia combined with high glucose (25 Mm D-glucose). After 48 hours, cells were harvested for quantitative real-time polymerase chain reaction for the analysis of circNPHP1 (n = 3). Fold change in RNA expression is relative to normoxia; 18S is used as housekeeping gene. Data are expressed as mean ± SEM and were assessed by 1-way analysis of variance with Dunnett's post hoc test. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, and ns = not significant. (D) 2 μg of total RNA from HUVEC cells were digested with RNase R enzyme followed by quantitative real-time polymerase chain reaction analysis of circNPHP1 and linear NPHP1 (n = 3). Fold change in RNA expression is relative to undigested RNA; 18S is used as housekeeping gene. Data are expressed as mean ± SEM and were assessed by unpaired Student's t -test. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, and ns = not significant. Abbreviations as in .

Article Snippet: Human cardiac microvascular endothelial cells (HCMECs) (Promocell: C-12285 [mono-doner], Lot numbers: 440Z021.5 [male], 440Z021.4 [male], 446Z001.1 [female], 492Z009.4 [female]) and human umbilical vein endothelial cells (HUVECs) (Promocell: C-12208 [poly-donor], Lot numbers: 447Z004, 450Z015, 466Z022, 503Z026) were cultured on 0.2% gelatin (Sigma-Aldrich) in endothelial cell growth medium MV and endothelial cell growth medium 2 (EGM2) (PromoCell).

Techniques: Expressing, Transformation Assay, Cell Culture, Real-time Polymerase Chain Reaction, RNA Expression

Journal: bioRxiv

Article Title: Endothelial RRM2B-dependent mitochondrial DNA signalling drives doxorubicin-cardiotoxicity

doi: 10.64898/2026.01.19.700350

Figure Lengend Snippet: Consequences of Doxorubicin exposure in cardiac endothelial cells in vitro : senescence, mtDNA instability and inflammation. A. Experimental model of endothelial cells transiently exposed to Doxorubicin . B. Representative immunoblot image in HCMECs, not treated (NT), during Dox exposure (1d) and after drug withdrawal (8d). C. SA-β-galactosidase assay of HCMECs, not treated (NT), during Dox exposure (1d) and after drug withdrawal (8d). **** p<0.0001 in a two-sample t-test with Welch’s post hoc analysis for mean comparison. D. Quantification of mtDNA copy per cell determined by qPCR in HCMECs, not treated (NT), during Dox exposure (1d) and after drug withdrawal (8d). Hydroxyurea (HU) was added one day after Dox withdrawal. N=3 independent experiments. * p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001 in a one-way ANOVA test with Tukey’s post hoc analysis for mean comparison. E. Expression level of dGUOK and TK2 by qRT-PCR in HCMECs, during Dox exposure (1d) and after drug withdrawal (8d). TK2, thymidine kinase 2. dGUOK, deoxyguanosine kinase. N=3 independent experiments. * p<0.05 in a one-way ANOVA test with Tukey’s post hoc analysis for mean comparison. F. Representative immunoblot image and G. quantification of the canonical RRM2 and alternative p53 inducible RRM2B small RNR subunit in HCMECs, not treated (NT), during Dox exposure (1d) and after drug withdrawal (8d). N=3 independent experiments. **** p<0.0001 in in a one-way ANOVA test with Tukey’s post hoc analysis for mean comparison. H-J. Immunostaining of HCMECs not treated (NT) and after Dox withdrawal (8d). H. Representative super-resolution Airyscan confocal images of dsDNA (green) and HSP60 (red) Scale bar 10 µm. At bottom, the magnified images show mtDNA (green) and mitochondria (red), showing that most DNA foci are located within mitochondria with some foci in the cytoplasm of Dox treated HCMECs. Arrows indicate cytosolic dsDNA signals. I. Quantification of the colocalization coefficient between mtDNA and mitochondria. N=3 independent experiments. ** p<0.01 in a two-sample t-test with Welch’s post hoc analysis for mean comparison. J. Quantification of mitochondrial length. NT (n=317), 8d (n=379). N=3 independent experiments. **** p<0.0001 in a Mann-Whitney test. K. Expression levels of indicated genes measured by qRT-PCR in HCMECs, not treated (NT) and after drug withdrawal (8d). Hydroxyurea (HU) or Sting inhibitor H151 (H151) was added one day after Dox withdrawal. N=3 independent experiments. * p<0.05; ** p<0.01; *** p<0.001 in a one-way ANOVA test with Tukey’s post hoc analysis for mean comparison.

Article Snippet: Human endothelial cell lines HUVECs (pooled donors, C2519A, Lonza) and HCMECs (Caucasian male, C-12285 PromoCell) were used between passage 3 and 6.

Techniques: In Vitro, Western Blot, Comparison, Expressing, Quantitative RT-PCR, Immunostaining, MANN-WHITNEY

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