human umbilical cord endothelial cells huvec  (ATCC)

Journal: bioRxiv

Article Title: miR-23b neutralization in brain endothelium promotes blood-brain barrier repair through Wnt/β-catenin dependent and independent mechanisms

doi: 10.1101/2025.08.20.671398

Figure Lengend Snippet: (A) Schematic diagram of the anti-miR library screen to identify miRs/anti-miRs that regulate cell-cell contact and barrier properties in ECs. (B) Mouse lung endothelial cells were transduced using a lentiviral-based, miR-neutralizing shRNA library, selected for puromycin resistance, and clonally expanded. Each well represents the neutralization of a single endogenously expressed miR. Cells were then analyzed by trans-endothelial electrical resistance (TEER) by employing an Electric Cell-substrate Impedance Sensing (ECIS) plate reader to examine paracellular barrier properties. (C) Single high-titer miR-control, miR-23b, and anti-miR-23b lentiviruses were generated and stable miR-modulated human lung endothelial cells established. TEER was measured to assess the specific effect of miR-23b (n=12) (D) miR expression profiling was performed by miR specific qPCR analysis of mouse brain endothelial cells (BECs) from postnatal pups and adult brains as well as primary human brain microvascular endothelial cells (HBMEC) and shown as a heatmap (n=3). (E) Human HUVECs were transfected with miR control or anti-miR-23b mimics (oligonucleotides), and (F) stable HBMECs (parental, miR control, and anti-miR-23b) were generated. HUVEC or HBMEC cells were subjected to TEER by ECIS or EVOM3 analysis (n=4). Results shown as percent change SEM ± n = 4 or 8, independent biological replicates, *p < 0.05, **p < 0.01, and ***p < 0.001 by two-tailed Student’s t-test.

Article Snippet: Primary Human Umbilical Cord Vein Endothelial Cells (HUVEC, PCS-100-010, ATCC, Manassas, VA, USA) were cultured on 1% Gelatin (G9136, Sigma-Aldrich, St. Louis, MO, USA) coated T75 flasks in Endothelial Cell Growth Medium MV2 (C-22022, PromoCell, Heidelberg, Germany).

Techniques: shRNA, Neutralization, Electric Cell-substrate Impedance Sensing, Control, Generated, Expressing, Transfection, Two Tailed Test

Journal: bioRxiv

Article Title: miR-23b neutralization in brain endothelium promotes blood-brain barrier repair through Wnt/β-catenin dependent and independent mechanisms

doi: 10.1101/2025.08.20.671398

Figure Lengend Snippet: (A) miR-modulated HBEMC or hCMEC/D3 lines were generated (over-expressing anti-miR-23b, miR-23b, or control miR), whereas HUVECs were transiently transfected with anti-miR-23b, miR-23b, or control miR oligonucleotides. Stable and transiently transduced anti-miR-23b, miR-23b, and control miR cell lines were analyzed for changes in protein levels of key tight junction proteins (ZO-1, Claudin-5, and Occludin) by Western blot (WB) analysis. Quantification of WB results (n = 3/sample) normalized to GAPDH is shown as relative levels (left), and representative examples are shown (right). (B) Cartoon of key brain endothelial cell junctional proteins. (C) Representative immunofluorescence (IF) images of transduced primary HBMECs and transiently transfected HUVECs stained for ZO-1 (green), Claudin-5 (red), and Occludin (red) are shown on the left, and the quantification of relative fluorescence levels is shown on the right. (D) Stable anti-miR-23b and control miR cell lines were analyzed for the adherens junction protein VE-Cadherin by IF (left panel), WB (right, bottom panel), and quantified (top, right panel). Graphs are shown as mean ± SEM (n = 3, independent biological replicates), *p <0.05, **p < 0.01, ***p < 0.001, by two-tailed Student’s t-test.

Article Snippet: Primary Human Umbilical Cord Vein Endothelial Cells (HUVEC, PCS-100-010, ATCC, Manassas, VA, USA) were cultured on 1% Gelatin (G9136, Sigma-Aldrich, St. Louis, MO, USA) coated T75 flasks in Endothelial Cell Growth Medium MV2 (C-22022, PromoCell, Heidelberg, Germany).

Techniques: Generated, Expressing, Control, Transfection, Western Blot, Immunofluorescence, Staining, Fluorescence, Two Tailed Test

Journal: bioRxiv

Article Title: miR-23b neutralization in brain endothelium promotes blood-brain barrier repair through Wnt/β-catenin dependent and independent mechanisms

doi: 10.1101/2025.08.20.671398

Figure Lengend Snippet: (A) miR-modulated HBMEC or hCMEC/D3 lines were analyzed for changes in expression levels of Caveolin-1 and PLVAP by Western blotting. Representative examples are shown on the left, and quantification of results (n=3) normalized to GAPDH as relative levels are shown on the right. (B) Cartoon of brain endothelial cell junctional proteins and transcytosis, dependent on PVLAP expression. (C, D) Transcytosis assay employing labelled Albumin-594 (red), which is transported by transcytosis in HBMECs toward the brain using HBMECs overexpressing anti-miR-23b or controls. ( C ) Representative images of a single 3D tubule (top panel) and (D, left) complete 3-channel chip cultures (n=5), and quantification of albumin transcytosis (middle panel). (D, right) Confocal images of PLVAP/DAPI staining of anti-miR-23b or controls (right panel) and quantifications (middle panel). Graphs are shown as SEM ± of independent biological replicates, *p <0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, by two-tailed Student’s t-test.

Article Snippet: Primary Human Umbilical Cord Vein Endothelial Cells (HUVEC, PCS-100-010, ATCC, Manassas, VA, USA) were cultured on 1% Gelatin (G9136, Sigma-Aldrich, St. Louis, MO, USA) coated T75 flasks in Endothelial Cell Growth Medium MV2 (C-22022, PromoCell, Heidelberg, Germany).

Techniques: Expressing, Western Blot, Staining, Two Tailed Test

Journal: bioRxiv

Article Title: miR-23b neutralization in brain endothelium promotes blood-brain barrier repair through Wnt/β-catenin dependent and independent mechanisms

doi: 10.1101/2025.08.20.671398

Figure Lengend Snippet: (A) Mice were transduced with AAVBR1 anti-miR-23b or control viruses before stroke to specifically and efficiently target endothelial cells long-term. A representative image of AAVBR1-control-GFP (green) virus showing expression in brain capillaries co-stained with podocalyxin (red, vessel marker), three weeks post-infection. (B-C) 20-week-old male mice were injected with anti-miR-23b-AAVBR1 or control AAVBR1 7 days before the tMCAo surgery (75-minute occlusion). Some mice were sacrificed 4 days after tMCAo, and BBB damage was examined by analysis of serum IgG extravasation (green). (D) The leakage volume (left panel), average leakage area (middle panel), and average leakage intensity (right panel) for serum IgG extravasation (n=5 mice /group) are shown. (E) Some mice were exposed to tail-vein injections of a small tracer [biocytin-TMR (red); 890 Da] for 45 minutes before tissue harvest to visualize BBB permeability. Biocytin-TMR leakage volume (left panel), average leakage area (middle panel), and average leakage intensity (right panel) (n=3 mice/group). Finally, neurological scoring was monitored daily. A poor neurological score of> 5 is shown (F), and survival monitoring results are depicted in (G) (n=11 mice/group).

Article Snippet: Primary Human Umbilical Cord Vein Endothelial Cells (HUVEC, PCS-100-010, ATCC, Manassas, VA, USA) were cultured on 1% Gelatin (G9136, Sigma-Aldrich, St. Louis, MO, USA) coated T75 flasks in Endothelial Cell Growth Medium MV2 (C-22022, PromoCell, Heidelberg, Germany).

Techniques: Transduction, Control, Virus, Expressing, Staining, Marker, Infection, Injection, Permeability