human umbilical cord vein endothelial cells huvec  (PromoCell)

Journal: bioRxiv

Article Title: Endothelial expression of ZBTB16 protects against cardiac aging

doi: 10.1101/2025.10.08.681100

Figure Lengend Snippet: ( A ) siRNA mediated knockdown of ZBTB16 in human umbilical vein endothelial cells (HUVEC). Gene silencing was confirmed 72h after siRNA transfection (n=6). ( B ) Network formation assay of HUVEC transfected with siCtrl or siZBTB16 (n=4). ( C ) Quantification of Boyden Chamber transmigration assay using HUVEC after siCtrl Vs. siZBTB16 transfection. Data are normalized to siCtrl-treated cells without VEGFA. ( D ) Spheroid sprouting assay of HUVEC transfected with siCtrl or siZBTB16 followed by treatment with or without VEGFA (n=4 and n=5). Representative images are shown in the left panel. ( E ) Cell numbers were counted by visual assessment after 72h of knockdown (n=5). ( F ) Quantification of Annexin V and 7-AAD FACS. (n=3). ( G ) Representative images of senescence associated-β-galactosidase staining (senescence) at 72 h of siRNA treatment. Quantification is shown in the right panel (n=5). Data are shown as mean and error bar indicate the standard error of the mean. P-value was calculated by two-tailed Student’s t-test.

Article Snippet: Human umbilical cord vein endothelial cells (HUVEC) were purchased from PromoCell (C-122203) and cultured in endothelial basal medium (EBM, CC-3121, Lonza) supplemented with 10% fetal bovine serum (10100147, Gibco).

Techniques: Knockdown, Transfection, Tube Formation Assay, Transmigration Assay, Staining, Two Tailed Test

Journal: bioRxiv

Article Title: Endothelial expression of ZBTB16 protects against cardiac aging

doi: 10.1101/2025.10.08.681100

Figure Lengend Snippet: ( A ) Bulk RNA sequencing of ZBTB16 knockdown cells and siRNA control cells (n=5). Graph shows log2 fold-change of genes associated with senescence (based on SenMayo (PMID: 35974106)). Genes were significant (Bonferroni adjusted p-value < 0.05). Positive log2 fold-change indicates higher expression in ZBTB16 knockdown cells. Genes were annotated according to their biological function. ( B-D ). Fibroblasts treated 72h with supernatants of HUVEC, which were transfected with siCtrl or siZBTB16 for 72h (n=10). Arrows indicate COL1A1 expression. Quantification is shown in c for COL1A1 (72h) and d for aSMA (48h). ( E-F ) Collagen gel contraction assay at baseline (upper panel) and after 96h (bottom panel) of fibroblast gels treated with supernatants from siCtrl (left) or siZBTB16 (right). Red line indicates gel boundaries. Quantification of relative decrease in gel area is shown in F. ( G-I ) Cardiac tissue mimetics (CTMs) containing primary rat cardiomyocytes, fibroblasts and endothelial cells were treated with supernatants of siCtrl or siZBTB16-transfected HUVEC for 14d. Representative immunohistochemical stainings are shown in G. Quantification of alpha smooth muscle actinin (aSMA) (H) and collagen COL1A1 (I). ( J-L ) siCtrl or siZBTB16-transfected HUVEC were directly co-cultured with neonatal rat cardiomyocytes (n=8) for 96h and stained for DAPI (blue), Phalloidin (green), sarcomeric-actinin (red) and VE-cadherin (magenta). Arrows indicate cardiomyocytes depicted by high sarcomeric actinin content. Cardiomyocyte hypertrophy quantified in panel k, and cardiomyocyte beating frequency is shown in L. ( M-O ) Neonatal rat cardiomyocytes were cultured in the supernatant of HUVEC after siCtrl or siZBTB16 transfection. Contraction (peak time) and relaxation (return velocity time, return to baseline 90%) were determined using IonOptix. Single cardiomyocytes were analyzed in the presence and absence of phenylephrine (-PE: n=19 vs. n=22; +PE: n=22 vs. n=23). ( P ) Primary mouse cortical neurons were treated with supernatants of siCtrl or siZBTB16-transfected HUVEC. Quantification is shown in the right panel. ( Q ) Innervation as assessed histologically by TUJ1 (green) normalized to IB4 (red) in hearts of Zbtb16 +/+ and Zbtb16 +/- mice (n=6 vs. n=7). Quantification is shown in the right panel. Data are shown as mean and error bar indicate the standard error of the mean. P-value was calculated by two-tailed Student’s t-test.

Article Snippet: Human umbilical cord vein endothelial cells (HUVEC) were purchased from PromoCell (C-122203) and cultured in endothelial basal medium (EBM, CC-3121, Lonza) supplemented with 10% fetal bovine serum (10100147, Gibco).

Techniques: RNA Sequencing, Knockdown, Control, Expressing, Transfection, Collagen Gel Contraction Assay, Immunohistochemical staining, Cell Culture, Staining, Two Tailed Test

Journal: bioRxiv

Article Title: Endothelial expression of ZBTB16 protects against cardiac aging

doi: 10.1101/2025.10.08.681100

Figure Lengend Snippet: ( A-C ) ZBTB16 was overexpressed by lentiviral vectors in long-term passaged HUVEC (>P9) for >8 days. (A) Staining of HUVEC for acidic-β-galactosidase (n=5). (B) ZBTB16 was overexpressed in long-term passaged senescent HUVEC (>P12) prior to performing a network formation assay (n=4). (C) ZBTB16 was overexpressed in long-term passaged HUVEC (>P12). Supernatants were collected and transferred to human cardiac fibroblasts (n=5). After 72h, fibroblasts were stained for COL1A1 (grey), DAPI (blue), aSMA (red) and Phalloidin (green). Scale bar indicates 50 µm. ( D-I ) Overexpression of Zbtb16 in endothelial cells by targeted AAV9 vectors improves cardiac function in 18-month-old mice. (D) Zbtb16 expression in liver endothelial cells 4 weeks after AAV9 treatment. (E) Diastolic heart function and F ejection fraction 8 weeks after AAV9 treatment. Acidic β-galactosidase (G) of cardiac sections 8 weeks after AAV9 treatment. (H, I) Vascular fibrosis as assesses by Sirius red staining on heart section from 3-month-old (n=3), 18-month-old (n=3), 20-month-old (n=5) and 20-month-old mice after two months of ZBTB16-AAV9 or control treatment (n=6). Data are shown as mean and error bar indicate the standard error of the mean. P-value was calculated by two-tailed Student’s t-test or using One-way ANOVA with a post-hoc Tukey’s test (H).

Article Snippet: Human umbilical cord vein endothelial cells (HUVEC) were purchased from PromoCell (C-122203) and cultured in endothelial basal medium (EBM, CC-3121, Lonza) supplemented with 10% fetal bovine serum (10100147, Gibco).

Techniques: Staining, Tube Formation Assay, Over Expression, Expressing, Control, 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) 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