Journal: bioRxiv

Article Title: Using atorvastatin-induced vascular weakness to model brain haemorrhage in vascularised cerebral organoids

doi: 10.64898/2026.04.20.719465

Figure Lengend Snippet: ( A ) Phase contrast images of HBMECs after 24 hours of treatment with 100 μM atorvastatin (ATV). Scale bars 50 μm. ( B ) Cell survival after ATV treatment for 24 hours in both stationary and rotating culture conditions ( n =4 independent repeats). Scale bars 100 μm. ( C ) qPCR analysis of marker expression after ATV treatment in rotating cells shows an increase in VE-Cadherin and NG2 gene expression (non-significant, One-Way ANOVA). ( D ) Fluorescent images of CD31 (green) and ZO-1 (red) cellular expression after 24 hours with ATV and DMSO control. ( E ) Total expression of ZO-1 after treatment ( n =4 independent repeats, two-way ANOVA *** P =0.0009, **** P <0.0001) ( F ) Analysis of ZO-1 co-localised with CD31 on the cell surface when treated with ATV ( n =4 independent repeats, two-way ANOVA, *** P =0.0002). ( G ) Fluorescent images of CD31 (green) and VE-Cadherin (red) cellular expression after 24 hours of ATV treatment in both stationary (top panels) and rotating culture (bottom panels). Zoom inset shows the internalisation of both CD31 and VE-Cadherin from the cell surface when treated with ATV. Scale bars 20 μm. ( H ) Analysis of VE-Cadherin co-localised with CD31 on the cell surface when treated with ATV ( n =3 independent repeats, two-way ANOVA, * P <0.04). ( I ) Total area expression of CD31 and VE-Cadherin as a percentage area of DAPI in both stationary and rotating cells, ( n =3 independent repeats, no significance from analysis with a two-way ANOVA).

Article Snippet: Primary human brain microvascular endothelial cells (HBMECs) (Innoprot P10361) were maintained on 1% gelatin coated flasks in endothelial cell growth medium MV (PromoCell C-22020).

Techniques: Marker, Expressing, Gene Expression, Control

Journal: bioRxiv

Article Title: Using atorvastatin-induced vascular weakness to model brain haemorrhage in vascularised cerebral organoids

doi: 10.64898/2026.04.20.719465

Figure Lengend Snippet: ( A ) Representative images of filipin-stained HBMECs, after 24hours of drug treatment. Scale bars 50 μm. ( B ) Filipin expression as a percentage of area from 6 ROIs in 2 wells across 2 independent repeats after 24 hours with ATV, ** P =0.0081, t -test. ( C ) qPCR analysis of rotated HBMECs after 24 hours of drug treatment shows a significant increase in HMGCR expression ( n =5 independent repeats, * P =0.0323 One-Way ANOVA).

Article Snippet: Primary human brain microvascular endothelial cells (HBMECs) (Innoprot P10361) were maintained on 1% gelatin coated flasks in endothelial cell growth medium MV (PromoCell C-22020).

Techniques: Staining, Expressing

Journal: bioRxiv

Article Title: Using atorvastatin-induced vascular weakness to model brain haemorrhage in vascularised cerebral organoids

doi: 10.64898/2026.04.20.719465

Figure Lengend Snippet: ( A ) Phase images of tube formation in untreated and ATV-treated HBMECs. Scale bars 20 μm. ( B ) Analysis of the network parameter average vessel length shows a significant reduction with ATV treatment, both pre-tube formation and post (One-way ANOVA with Tukey’s post hoc multiple comparisons test ** P =0.003, *** P =0.0007, **** P <0.0001). ( C ) Cytotoxicity analysis of ATV treated tubes showed no difference from controls ( n =3, t -test).

Article Snippet: Primary human brain microvascular endothelial cells (HBMECs) (Innoprot P10361) were maintained on 1% gelatin coated flasks in endothelial cell growth medium MV (PromoCell C-22020).

Techniques:

Journal: bioRxiv

Article Title: Using atorvastatin-induced vascular weakness to model brain haemorrhage in vascularised cerebral organoids

doi: 10.64898/2026.04.20.719465

Figure Lengend Snippet: ( A ) Representative confocal images of whole organoids treated with ATV for 24 hours and the loss of VE-Cadherin expression from the surface. Scale bars 500 μm. ( B ) VE-Cadherin, not CD31 ( P =0.091), expressed as a percentage of DAPI was significantly reduced with ATV treatment compared to DMSO controls (two-tailed t -test, * P =0.011). ( C ) Size progression for 4 batches of organoids that were treated with ATV at day 40. ( D ) Vascular metrics were unchanged for CD31 ( n =15 organoids from 4 batches, non-significant t- test) with slightly more endpoints, signifying single cells. ( E ) Angiotool analysis of VE-Cadherin staining revealed shorter overall vessel length ( n =15 organoids from 4 batches, t- test, * P =0.0389)). ( F ) qPCR analysis of ATV-treated organoids showed a reduction in VE-Cadherin RNA expression; however, other markers of endothelial function are unchanged (One-Way ANOVA, n =5 organoids from 2 batches). ( G ) ATV treatment increased the expression of some cholesterol biosynthesis markers compared to DMSO controls, opposite to what was observed in HBMECs in 2D (One-Way ANOVA, n =5 organoids from 2 batches).

Article Snippet: Primary human brain microvascular endothelial cells (HBMECs) (Innoprot P10361) were maintained on 1% gelatin coated flasks in endothelial cell growth medium MV (PromoCell C-22020).

Techniques: Expressing, Two Tailed Test, Staining, RNA Expression

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