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: Frontiers in Cell and Developmental Biology

Article Title: Trauma promotes heparan sulfate modifications and cleavage that disrupt homeostatic gene expression in microvascular endothelial cells

doi: 10.3389/fcell.2024.1390794

Figure Lengend Snippet: Top differentially expressed genes in flow conditioned primary human lung microvascular endothelial cells (HLMVEC) following exposure to heparinase III (HepIII) relative to vehicle control. Messenger RNA was collected from confluent monolayers of HLMVEC that were conditioned with 15 dyn/cm 2 for 48 h followed by exposure to heparinase III 500 mU/mL or vehicle for 6 h while remaining under shear stress ( n = 4 biological replicates per condition; two replicates were pooled to generate two samples per condition for RNAseq). (A) Heatmap representing top 40 differentially expressed genes in HLMVEC between heparinase III and vehicle. Each treatment group contains n = 2 RNA samples that were combined from HLMVEC within two ibidi channel slides, thus representing a total of n = 4 per condition. Colors represent gene expression z-score with red corresponding to upregulated and blue to downregulated. (B) Volcano plot depicting differential gene expression between HLMVEC exposed to heparinase III (positive log2 fold change) and vehicle (negative log2 fold change). Red genes meet figure thresholds of p ≤ 1 × 10 −3 and log2 fold change ≥|1| for the purposes of visualization. (C) Expression of the flow-responsive genes Krüppel-like factor 2 and 4 ( KLF2 , 4 ), endothelial nitric oxide synthase ( NOS3 ) and solute carrier family nine isoform A3 regulatory factor 2 ( SLC9A3R2 ) is reduced following heparinase III treatment. (D) Expression of angiopoietin-2 ( ANGPT2 ), endothelial cell-specific molecule-1 ( ESM1 , also known as endocan), and thrombospondin ( THBS1 ), markers of endothelial cell activation, is increased following heparinase III treatment.

Article Snippet: Primary human lung microvascular ECs (HLMVEC) (S540-05a, Cell Applications, San Diego, CA, United States) were conditioned under shear stress (15 dyn/cm 2 ) for 48 h as described previously ( ).

Techniques: Control, Shear, Gene Expression, Expressing, Activation Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: Trauma promotes heparan sulfate modifications and cleavage that disrupt homeostatic gene expression in microvascular endothelial cells

doi: 10.3389/fcell.2024.1390794

Figure Lengend Snippet: Targeted representation of gene set enrichment analysis (GSEA) in flow conditioned (15 dyn/cm 2 for 48 h) primary human lung microvascular endothelial cells (HLMVEC) after 6-h exposure to vehicle or heparinase III (HepIII, 500 mU/mL) while remaining under shear stress ( n = 4 biological replicates per condition; two replicates were pooled to generate two samples per condition for RNAseq). GSEA was performed using (A) GO: Biological Process and (B) KEGG datasets. Figure displays up to twenty pathways from GSEA that are most relevant to endothelial cell organization and function with lowest False discovery rate (FDR)-adjusted p values (FDR q value). Pathways were organized according to their contribution to cellular maintenance and bioenergetics; cell organization and adhesion; angiogenesis and wound healing; or response to biophysical cues. Pathways on presented on the left were enriched in HLMVEC after exposure to vehicle whereas pathways on the right were enriched in HLMVEC after exposure to heparinase III. Circle size corresponds with number of genes present in experimental samples that overlap with respective dataset pathways, and circle shading represents the −log10 (FDR q value) with darker shades representing lower q values.

Article Snippet: Primary human lung microvascular ECs (HLMVEC) (S540-05a, Cell Applications, San Diego, CA, United States) were conditioned under shear stress (15 dyn/cm 2 ) for 48 h as described previously ( ).

Techniques: Shear

Journal: Frontiers in Cell and Developmental Biology

Article Title: Trauma promotes heparan sulfate modifications and cleavage that disrupt homeostatic gene expression in microvascular endothelial cells

doi: 10.3389/fcell.2024.1390794

Figure Lengend Snippet: Differentially expressed genes that govern synthesis of heparan sulfate proteoglycans and glycosaminoglycans in flow conditioned (15 dyn/cm 2 for 48 h) primary human lung microvascular endothelial cells treated for 6 h with vehicle or heparinase III (HepIII, 500 mU/mL) while remaining under shear stress ( n = 4 biological replicates per condition; two replicates were pooled to generate two samples per condition for RNAseq). (A) Of the heparan sulfate proteoglycans found in the vascular endothelial apical glycocalyx, expression of syndecan 3 ( SDC3 ) and SDC4 were downregulated by heparinase III treatment. (B) Of the enzymes regulating hyaluronan expression in the endothelial glycocalyx, hyaluronan synthase isoform 2 ( HAS2 ) was upregulated while hyaluronidases 1 and 2 ( HYAL1 , 2 ) were downregulated by heparinase III treatment. (C) Of the enzymes that synthesize chondroitin sulfate expressed in the endothelial glycocalyx (commonly observed in SDC1 and SDC3) and that modify its sulfation, chondroitin sulfate synthase isoform 3 ( CHSY3 ) and chondroitin sulfate N -acetylgalactosaminylsulfotransferase isoform 1 ( CSGALNACT1 ) were upregulated while carbohydrate sulfotransferase isoform 15 ( CHST15 , catalyzing 6- O -sulfation of 4- O -sulfated N -acetylgalactosamine in chondroitin sulfate disaccharides) was downregulated following heparinase III treatment. (D) Of the enzymes that synthesize and modify heparan sulfate expressed in the endothelial glycocalyx, expression of N -deacetylase/ N -sulfotransferase isoform 1 ( NDST1 ) and glucuronic acid C5-epimerase ( GLCE ) (which also contributes to glucuronic acid epimerization to iduronic acid in chondroitin sulfate) were downregulated while heparan sulfate 3- O -sulfotransferase isoform 1 ( HS3ST1 ) and heparan sulfate 6- O -sulfotransferase isoform 3 ( HS6ST3 ) were upregulated following heparinase III treatment. We also found that heparinase III treatment suppressed heparanase ( HPSE ) expression. False discovery rate (FDR)-adjusted p values (FDR q values) are presented.

Article Snippet: Primary human lung microvascular ECs (HLMVEC) (S540-05a, Cell Applications, San Diego, CA, United States) were conditioned under shear stress (15 dyn/cm 2 ) for 48 h as described previously ( ).

Techniques: Shear, Expressing, Histone Deacetylase Assay