Journal: Cell Regeneration

Article Title: Dissecting endothelial cell heterogeneity with new tools

doi: 10.1186/s13619-025-00223-3

Figure Lengend Snippet: Organoid methods and applications

Article Snippet: 3D microvessel model , Breast cancer, HUVEC , No , Tumor-vascular dynamics , Silvestri et al. .

Techniques: Sequencing, Construct, In Vitro, Membrane, Biomarker Discovery, Organ Culture, Transplantation Assay, Cell Culture, Suspension, In Vivo Imaging, Immunohistochemical staining, Diagnostic Assay, Imaging, RNA Sequencing, Drug discovery

Journal: Nature

Article Title: Glycocalyx dysregulation impairs blood-brain barrier in ageing and disease.

doi: 10.1038/s41586-025-08589-9

Figure Lengend Snippet: Fig. 3 | Reduced brain endothelial mucin-type O-glycosylation increases BBB leakiness and brain bleeding. a, Overview of AAV-mediated C1galt1 knockdown paradigm. ITR, inverted terminal repeat. b, C1GALT1 expression and mucin-domain glycoprotein labelling in acutely isolated microvessels. Scale bars, 10 µm. c, Quantification of C1GALT1 expression in b (n = 5 mice per group, two-sided t-test; mean ± s.e.m.). d, Quantification of mucin-domain glycoprotein labelling in b (n = 5, two-sided t-test; mean ± s.e.m.). e, Sulfo-NHS-biotin leakage in the cortices of young mice transduced with AAV-EGFP and AAV-miR-C1galt1. Scale bars, 500 µm. f, Sulfo-NHS-biotin leakage (indicated by white arrowheads) from EGFP+ cortical vessels of AAV-miR-C1galt1-transduced mice. Scale bars, 50 µm. g, Quantification of vessel permeability in f (n = 5 mice per group; two-sided t-test; mean ± s.e.m.). h, Overview of luminal mucin-domain glycoprotein degradation paradigm using 48 h StcE treatment. H&E, haematoxylin and eosin. i, Whole-brain images of sulfo-NHS-biotin leakage in mice treated with StcE for 48 h.

Article Snippet: Microvessels were blocked in 3% normal donkey serum ( Jackson ImmunoResearch) with 0.3% Triton X-100 (Sigma) in TBS-T (1× TBS with 0.05% Tween-20) for 1 h, followed by 1 h incubation at room temperature with primary antibodies.

Techniques: Glycoproteomics, Knockdown, Expressing, Isolation, Transduction, Permeability

Journal: Nature

Article Title: Glycocalyx dysregulation impairs blood-brain barrier in ageing and disease.

doi: 10.1038/s41586-025-08589-9

Figure Lengend Snippet: Fig. 4 | Restoration of mucin-type O-glycosylation improves BBB function in aged mice. a, Overview of C1GALT1 and B3GNT3 overexpression paradigm and relevant AAV constructs. b, C1GALT1 expression in acutely isolated microvessels labelled with LEL. Scale bars, 10 µm. c, Quantification of b (n = 5 mice per group; two-sided t-test; mean ± s.e.m.). d, B3GNT3 expression in acutely isolated microvessels labelled with LEL. Scale bar = 10 µm. e, Quantification of d (n = 5 mice per group; one-way ANOVA with Dunnett’s post hoc test; mean ± s.e.m.). f, Quantification of mucin-domain glycoprotein labelling in acutely isolated microvessels (n = 5 mice per group; two-sided t-test; mean ± s.e.m.).

Article Snippet: Microvessels were blocked in 3% normal donkey serum ( Jackson ImmunoResearch) with 0.3% Triton X-100 (Sigma) in TBS-T (1× TBS with 0.05% Tween-20) for 1 h, followed by 1 h incubation at room temperature with primary antibodies.

Techniques: Glycoproteomics, Over Expression, Construct, Expressing, Isolation

Journal: bioRxiv

Article Title: The Batch-Resourcing Angiogenesis Tool (BRAT) to enable high-content microscopy screening of microvascular networks

doi: 10.1101/2025.01.24.634836

Figure Lengend Snippet: GFP-hAECs co-cultured with hAD-MSCs for 7 days to form microvascular networks, with images captured every 10 minutes, as . BRAT and AngioTool were applied to measure microvascular network (A) end points, (B) branch points, and (C) vessel area as a percentage of the field of view area. (D) BRAT can also measure average or total vessel diameter and length (distances between end or branch points). Hashed vertical lines represent media change times. Individual timepoint analysis (dots) are overlaid with a regression and 95% confidence intervals. These results are compared with REAVER in .

Article Snippet: Finally, co-cultures were counterstained with DAPI (4′,6-diamidino-2-phenylindole, dihydrochloride, A-21137, ThermoFisher) for 10 minutes, washed thrice with PBS, and then imaged for CD31-positive microvascular networks on a Nikon ECLIPSE Ti2 Inverted Microscope (using a 555 nm excitation laser, 590-650 nm detector).

Techniques: Cell Culture

Journal: bioRxiv

Article Title: The Batch-Resourcing Angiogenesis Tool (BRAT) to enable high-content microscopy screening of microvascular networks

doi: 10.1101/2025.01.24.634836

Figure Lengend Snippet: (A) 3D microvascular networks formed by GFP-hAECs and hAD-MSCs co-cultured in a hydrogel microchip. (B) A 3D microvascular network of CD31-stained umbilical cord blood-outgrowth ECs (BOECs) and hAD-MSCs co-cultured in a fibrin hydrogel. (C) BRAT shows hAECs form thicker, longer, but fewer microvessels in 3D hAD-MSC hydrogel co-culture as opposed to BOECs. (A, B) 100 µm scale bars. (C)’s dimensions are reported in µm using 0.81 µm/pixel (Etaluma 720S) or 0.73 µm/pixel (Nikon Ti2) conversion factors. (C) datapoints represent independent cultures. Barplots represent mean ± standard deviation. *p<0.05, **p<0.01, ***p<0.001. Additional BRAT-generated metrics can be found in Fig. S9.

Article Snippet: Finally, co-cultures were counterstained with DAPI (4′,6-diamidino-2-phenylindole, dihydrochloride, A-21137, ThermoFisher) for 10 minutes, washed thrice with PBS, and then imaged for CD31-positive microvascular networks on a Nikon ECLIPSE Ti2 Inverted Microscope (using a 555 nm excitation laser, 590-650 nm detector).

Techniques: Cell Culture, MicroChIP Assay, Staining, Co-Culture Assay, Standard Deviation, Generated

Journal: bioRxiv

Article Title: The Batch-Resourcing Angiogenesis Tool (BRAT) to enable high-content microscopy screening of microvascular networks

doi: 10.1101/2025.01.24.634836

Figure Lengend Snippet: (A) A comparison of BRAT versus the leading four open-source software’s accuracy, sensitivity (true-positive), and specificity (true-negative) of microvascular network (MVN) pixel segmentation versus gold-standard manual segmentation. (B) Accuracy and precision across automatically detected vessel length, area, diameter, and branch points with reference to manual segmentations. (C) The most challenging MVN image to segment from the REAVER dataset; an image of CD31 + MVN in cardiac tissue (left), beside a manual annotation (ground truth), BRAT’s automated annotation, and BRAT’s false-positive (FP; green) and false-negative (FN; red) incorrectly-labelled MVN pixels. (A, B) Boxplots represent mean, quartile ranges, while whiskers extend to show the distribution, less outlier points. The annotations above or below each plot indicate significant pairwise comparisons between groups with Bonferroni adjusted p-values (letters).

Article Snippet: Finally, co-cultures were counterstained with DAPI (4′,6-diamidino-2-phenylindole, dihydrochloride, A-21137, ThermoFisher) for 10 minutes, washed thrice with PBS, and then imaged for CD31-positive microvascular networks on a Nikon ECLIPSE Ti2 Inverted Microscope (using a 555 nm excitation laser, 590-650 nm detector).

Techniques: Comparison

Journal: bioRxiv

Article Title: The Batch-Resourcing Angiogenesis Tool (BRAT) to enable high-content microscopy screening of microvascular networks

doi: 10.1101/2025.01.24.634836

Figure Lengend Snippet: Co-cultured GFP-hAECs and hAD-MSCs form microvascular networks over 7 days, as imaged every 10 minutes. BRAT analysed all 886 timesteps, while AngioTool only analysed 89 timesteps. In the bioRxiv version, the video is a download in the ‘Supplemental Material’ tab on the right of the abstract.

Article Snippet: Finally, co-cultures were counterstained with DAPI (4′,6-diamidino-2-phenylindole, dihydrochloride, A-21137, ThermoFisher) for 10 minutes, washed thrice with PBS, and then imaged for CD31-positive microvascular networks on a Nikon ECLIPSE Ti2 Inverted Microscope (using a 555 nm excitation laser, 590-650 nm detector).

Techniques: Cell Culture