Journal: bioRxiv

Article Title: IFITM1 inhibits Henipavirus membrane fusion by trapping ephrinB2 receptors in fusion-unfavorable membrane nanodomains

doi: 10.64898/2026.05.06.723334

Figure Lengend Snippet: The role of endogenous IFITMs in NiV and HeV pseudovirus entry into human endothelial and epithelial cells. The endogenous IFITMs mRNA levels in HuMEC ( a-c ) and HEK293T cells ( g-i ). Cells were transfected with siRNAs targeting IFITM proteins and scrambled siRNA (NC). IFN-α2b was used to stimulate the expression of IFITM proteins. The mRNA expression levels were detected using qPCR and normalized to that of NC at the untreated condition (-IFN-α). d and j , endogenous IFITM1,2,3 proteins expression in HuMEC ( d ) and HEK293T cells ( j ) upon siRNA knockdown analyzed by Western Blot. IFITMs were detected by anti-IFITM antibodies, and GAPDH was a loading control. The entry of NiV/VSV pp and HeV/VSV pp to HuMEC ( e and f ) and HEK293T cells ( k and l ). NiV and HeV glycoproteins were pseudotyped to VSV particles in which the VSV-G gene was replaced with the Renilla luciferase gene. Virus entry was measured by luminescence intensity and normalized to that of scrambled siRNA at the untreated condition (NC, −IFN-α). Virus entry levels in siRNA-transfected cells were compared with those transfected with scrambled siRNA (NC) under respective −IFN-α and +IFN-α conditions. Virus entry in IFN-α–treated cells (+IFN-α) was further compared with that in resting cells (−IFN-α) and labeled with a bracket. Bars represent means ± SEM. Results from at least 3 independent experiments are shown. p values were obtained using one-way analysis of variance (ANOVA) with post hoc correction (nonsignificant [ns], p > 0.05; *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001).

Article Snippet: Human microvascular endothelial cells (HuMEC) (ATCC CRL-4060) were cultured in Vascular cell basal medium (ATCC, PCS-100-030) with Microvascular endothelial cell growth kit – BBE (ATCC, PCS-110-040) and 0.5 ug/ml puromycin (10 mg/ml stock, Gibco A1138-03).

Techniques: Transfection, Expressing, Knockdown, Western Blot, Control, Luciferase, Virus, Labeling

Journal: bioRxiv

Article Title: Glycan-coated nanoparticles mimicking the ischemic glycocalyx scavenge the complement system conferring protection after experimental ischemic stroke

doi: 10.64898/2026.03.30.715069

Figure Lengend Snippet: A ) The experimental plan for lectins binding to ihBMECs at the end of hypoxia or after re-oxygenation in absence/presence/. B ) Sensorgrams obtained with Quartz Crystal Microbalance showing the binding of ConA (upper panels) and WGA (lower) injected at four different concentration (0.7-2-6-18 µg/mL) over chip-adherent ihBMECs. The data show decreased binding at the end of the 16h of hypoxia and increased binding after the 4h of re-oxygenation either in presence or absence of MBL compared to normoxic ihBMECs. C ) Microphotographs of MBL (red) deposited on normoxic (left) or hypoxic (right) ihBMEC after re-oxygenation. Nuclei in blue (DAPI), scale bars 10 µm.

Article Snippet: Immortalized human brain microvascular endothelial cells (ihBMECs) (5000 cells/cm 2 ) (Innoprot) were seeded on black 96-well μ-plates (ibidi, Germany) with optically clear flat bottom, suitable for fluorescence microscopy.

Techniques: Binding Assay, Injection, Concentration Assay

Journal: bioRxiv

Article Title: Glycan-coated nanoparticles mimicking the ischemic glycocalyx scavenge the complement system conferring protection after experimental ischemic stroke

doi: 10.64898/2026.03.30.715069

Figure Lengend Snippet: A ) MBL detection on the soft and hard corona samples obtained after preincubation of GNPs with human serum. The MBL signal decreased in the soft corona concomitantly with the three washes (Soft C1-3) and no signal was captured in the fourth wash (Soft C.4). The presence of MBL in the Hard Corona (Hard C., i.e. the proteins remaining after the washings because of their high affinity for the GNPs,) was strong for Man-GNPs (black arrow), and much less for Glc-GNPs (white arrow). B ) The experimental plan for testing sugar-GNPs localization on ihBMEC. C ) 3D microphotographs of Man-GNPs (red, reflectance microscopy) and F-actin (phalloidin, green) in normoxic (CTRL) or hypoxic (HYP) ihBMECs undergone re-oxygenation in the presence of 40 µg/mL Man-GNPs in 30% human serum. Man-GNPs were internalized in the cytoplasm of ihBMECs. Nuclei in blue (DAPI), scale bar 10 µm. D ) Normoxic (CTRL) or hypoxic (HYPOXIA) ihBMECs undergone re-oxygenation in the presence of 5, 20 or 40 µg/mL Man-GNPs in 30% HS were analyzed by reflectance confocal microscopy for Man-GNPs and MBL co-localization. Microphotographs show that Man-GNPs (white, reflectance microscopy) and hMBL (red) did not co-localize (as seen in magnification of white frame, scale bar 1 µm). Phalloidin in green, nuclei in blue (DAPI), scale bar 10 µm.

Article Snippet: Immortalized human brain microvascular endothelial cells (ihBMECs) (5000 cells/cm 2 ) (Innoprot) were seeded on black 96-well μ-plates (ibidi, Germany) with optically clear flat bottom, suitable for fluorescence microscopy.

Techniques: Microscopy, Confocal Microscopy

Journal: bioRxiv

Article Title: Glycan-coated nanoparticles mimicking the ischemic glycocalyx scavenge the complement system conferring protection after experimental ischemic stroke

doi: 10.64898/2026.03.30.715069

Figure Lengend Snippet: A) Microphotographs of MBL (red) deposited on normoxic (CTRL) or hypoxic (HYPOXIA) ihBMECs undergone re-oxygenation in the presence of 5, 20 or 40 µg/mL Man-GNPs in 30% HS (w/Man-GNPs). Nuclei in blue (DAPI), scale bar 200 µm. B ) MBL deposition, measured as fluorescence intensity, was greater on hypoxic than normoxic cells exposed to 30% HS. This increase was significantly reduced when ihBMECs were exposed to 5 and 20 µg/mL of Man-GNPs. Data as mean with individual values ± SD (n= 4). Two-way ANOVA followed by Tukey’s multiple comparisons, **p<0.001, *p<0.05. C ) Overexpression of ICAM-1 in hypoxic ihBMECs was significantly reduced when the cells were exposed to 20 µg/mL of Man-GNPs, to a similar extent than exposure to MBL depleted HS 30% (Δ MBL). D ) Overexpression of MMP-2 in hypoxic ihBMECs was partially counteracted by 20 µg/mL of Man-GNPs. E ) Expression of IL-1α was not significantly changed in presence of Man-GNPs with or without hypoxia. Data from 3 independent experiments, presented as mean with individual values ± SD (n= 4-12). Two-way ANOVA followed by Tukey’s multiple comparisons, ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05.

Article Snippet: Immortalized human brain microvascular endothelial cells (ihBMECs) (5000 cells/cm 2 ) (Innoprot) were seeded on black 96-well μ-plates (ibidi, Germany) with optically clear flat bottom, suitable for fluorescence microscopy.

Techniques: Fluorescence, Over Expression, Expressing

Journal: bioRxiv

Article Title: Glycan-coated nanoparticles mimicking the ischemic glycocalyx scavenge the complement system conferring protection after experimental ischemic stroke

doi: 10.64898/2026.03.30.715069

Figure Lengend Snippet: A) The experimental plan to generate ihBMECs’ normoxic or hypoxic conditioned medium (NORM CM, HYP CM, respectively) and co-cultures of hIPSC-derived neurons, astrocytes and microglia. B ) Microphotographs of GFAP (astrocytes, green), MAP-2 (neurons, red) exposed for 24h to NORM CM or HYP CM +/− Man-GNPs. White arrows point to damaged neurons, i.e. circular cells without dendrites. Nuclei in blue (DAPI), scale bar 10 µm. C ) The quantification of stained volumes (in µm 3 ) showed a decrease of MAP-2 volumes in co-cultures exposed to HYP CM, which was counteracted by Man-GNPs. Data as mean ± SD. Each value is a random field of view (FOV) selected automatically from the overview image. Two-way ANOVA for repeated measures followed by Sidak’s multiple comparisons, ****p<0.0001 (n= 16 FOVs from two experimental replicates, empty rectangles indicate the mean of each replicate). D ) Microphotographs of GFAP (green) and nuclei (DAPI, blue) with a yellow line along which we calculated the FWHM reported in the graph. Width of the first ramification emerging from astrocytic soma was calculated at gray level’s half maximum (HM) and was larger in HYP CM compared to NORM CM or HYP CM + Man-GNPs. Data as mean gray levels of 8 cells per group ± SEM. Two-way ANOVA followed by Tukey’s multiple comparisons, ***p<0.001. Scale bars 10 µm. E ) Microphotographs of GFAP (green), β3-tubulin (neurons, red) and Iba1 (microglia, purple) exposed for 24h to NORM CM or HYP CM +/− Man-GNPs. Dashed squares indicate the magnified views of microglia on the right panels. Nuclei in blue (DAPI), scale bar 100 µm in full images, 20 µm in magnifications. White traces in the magnifications correspond to the Iba1 skeletonized signal. F ) The quantification of microglia morphological parameters showed increased number of branches and junctions after HYP CM exposure, which was counteracted by Man-GNPs. Data as violin plot. Each dot is individual microglia. Kruskal-Wallis test, **p<0.01, ***p<0.001 (n= 25-40 cells from 3 FOVs placed in one well). G ) Histograms of frequency distributions of the morphological parameters in E, shown with automatically chosen bin size.

Article Snippet: Immortalized human brain microvascular endothelial cells (ihBMECs) (5000 cells/cm 2 ) (Innoprot) were seeded on black 96-well μ-plates (ibidi, Germany) with optically clear flat bottom, suitable for fluorescence microscopy.

Techniques: Derivative Assay, Staining

Journal: medRxiv

Article Title: Feasibility of Endothelial Cell Isolation from Routine Coronary Function Testing in ANOCA Patients

doi: 10.64898/2026.04.09.26350551

Figure Lengend Snippet: A) Method to isolate and culture ECs from catheterization material used during coronary function testing. B) Representative morphology (I, passage 0) and immunofluorescence images of cultured ECs (II and III, passage 5) showing positivity for VE-cadherin (II), von Willebrand Factor (vWF) (II) and CD31 (III). C) Flow-cytometric characterization of cultured ECs (passage 1) in comparison with multiple reference cell populations, including human dermal microvascular ECs (HDMVEC), human cardiac microvascular ECs (HCMEC), human coronary artery ECs (HCAEC), human plaque myofibroblasts and mesenchymal stem cells (MSC). The plotted histograms depict the ‘relative counts’ on the y-axis and the ‘relative intensity’ on the x-axis

Article Snippet: Reference populations included human dermal microvascular ECs (HDMVEC; iXCells Biotechnologies, REF#10HU-019), human cardiac microvascular ECs (HCMEC; Sigma-Aldrich, REF#C-12285), human coronary artery ECs (HCAEC; Lonza, REF#CC-2585), human plaque myofibroblasts and mesenchymal stem cells (MSC; Cell Therapy Facility, University Medical Center Utrecht; code: MSC053P3_AL-MSC071P3_R).

Techniques: Immunofluorescence, Cell Culture, Comparison

Journal: bioRxiv

Article Title: Ubiquitin ligase CHFR impairs Tie2 signaling via K 48 -linked ubiquitylation and degradation of Akt1 in endothelial cells

doi: 10.64898/2026.03.31.715582

Figure Lengend Snippet: a HMEC were transfected with WT or K/R-mutant Akt1 constructs. At 36 h after transfection, cells were treated with LPS (5 μg/ml) for 0, 12, and 24h and then cells were used for IB to determine expression of Akt1 and VE-cadherin. Shown are mean values ± SEM (n = 3 independent experiments; two-way ANOVA followed by Tukey’s post hoc test). b TIME endothelial cells (telomerase-immortalized human dermal microvascular endothelial cell line) were transfected with WT or K/R mutant Akt1 constructs and stimulated with LPS (5 μg/ml) for 0 and 6 h. Confocal imaging showed that expression of K/R mutant Akt1 prevents degradation of VE-cadherin. c WT mice were injected (i.v.) with liposome-encapsulated pmCherry-tagged WT or K/R mutant Akt1 constructs. Lungs harvested 96 h after injection were subjected to cryosection and stained with EC marker antibody vWF (green). Confocal imaging confirms expression of pmCherry-Akt1 (red) plasmid in lung endothelial cells. d-f Liposome-mediated delivery of Akt1 (WT) or K/R-mutated Akt1 in WT mice prevents degradation of VE-cadherin, mitigates LPS-induced lung vascular leak (EBA uptake), and reduces PMN transmigration (MPO assay). Shown are mean values ± SEM ( n = 3 or n = 5 mice/group; two-way ANOVA followed by Tukey’s post hoc test). g Model for E3 ligase CHFR regulation of endothelial junctional barrier integrity. Under baseline condition, constitutive Ang1-Tie2 signaling in EC maintains endothelial junctional barrier through Akt1 activation-mediated inhibition of the transcription factor FoxO1 activation and Ang-2 expression. During vascular inflammatory conditions such as sepsis, TLR4 signaling induces the expression of E3 ligase CHFR in a FoxO1-dependent manner. Then the upregulated CHFR mediates K 48 -linked polyubiquitylation and degradation of Akt1 and VE-cadherin (Tiruppathi et al., 2023) to disassemble EC junctional barrier. CHFR-mediated loss of FoxO1 negative regulator Akt1 expression in EC leads to increased FoxO1 expression which in turn promotes sustained expression of Ang-2 in EC to induce life-threatening pulmonary edema.

Article Snippet: The telomerase-immortalized human dermal microvascular endothelial cell line (Lonza, catalog #CRL-4025) was maintained in Vascular Cell Basal Medium (ATCC ® PCS-100-030), supplemented with Microvascular Endothelial Cell Growth Kit-VEGF (ATCC ® PCS-110-041).

Techniques: Transfection, Mutagenesis, Construct, Expressing, Imaging, Injection, Staining, Marker, Plasmid Preparation, Transmigration Assay, MPO Assay, Activation Assay, Inhibition