Journal: bioRxiv

Article Title: Uncertainty-Aware Traction Force Microscopy

doi: 10.1101/2024.07.05.602172

Figure Lengend Snippet: (A) Membrane labeling of HUVEC monolayers. (B) Fluorescent bead image (beads of size 0.2 µ m). Arrows indicate bead-related image artifacts (C) PIV-UQ displacement field u h (D) σ u,PIV map. White regions indicate ’bad’ PIV windows that were deleted and replaced as described in the main text. Uncertainty arrows denote the pointwise angular uncertainty corresponding to 1 circular std. dev. of bootstrapped PIV-UQ samples. (E) Inferred mean marginal posterior traction stress, Marginal posterior traction stress uncertainty field ( σ t ). Uncertainty arrows denote the pointwise angular uncertainty corresponding to 1 circular std. dev. of marginal posterior p ( t | u h ). Scale bar : 25 µ m

Article Snippet: Human vascular umbilical endothelial vein cells (HUVECs) (Cell Applications) were cultured in M199 supplemented with 10% (v/v) endothelial growth medium (Cell Applications), 10% (v/v) fetal bovine serum (Gibco), and 1% penicillin-streptomycin (Gibco).

Techniques: Membrane, Labeling

Journal: bioRxiv

Article Title: TAILS identifies candidate substrates and biomarkers of ADAMTS7, a therapeutic protease target in coronary artery disease

doi: 10.1101/2021.11.19.469331

Figure Lengend Snippet: Media collected from human coronary artery smooth muscle cells (SMC) or human umbilical vein endothelial cells (HUVEC) expressing control Luciferase (Luc), active mouse ADAMTS7 (WT) or catalytic mutant mouse ADAMTS7 E373Q (EQ) from three separate experiments. 20ml of media collected from each 15cm tissue culture dish. A , SMC1 media was pooled for each condition and split into technical replicates after concentration to generate technical replicates. B , expression of full-length (FL) ADAMTS7-3xFLAG constructs was verified in the conditioned media (CM) and whole cell lysate (WCL) by direct anti-Flag HRP western blot detection. * indicates mucin domain cleaved degradation product detected by c-terminal Flag tags. C , Replicates from SMC2 were collected from 3 dishes and processed separately. D , expression in the media from SMC2 replicates was verified by western blot. E , Replicates from HUVEC were collected from 2 dishes and processed separately. F , expression in the media from HUVEC replicates was verified by western blot. G , media preparation workflow for each replicate to generate input for total secretome and TAILS proteomics experiments. H , sample processing for TMT10 TAILS proteomics to identify neo-N-termini from the active ADAMTS7 enzyme condition. SMC1 TAILS experiment was digested with Trypsin only. SMC2 and HUVEC were digested with AspN or Trypsin before negative selection.

Article Snippet: The third experiment used input from cultured Human umbilical vein endothelial cells (HUVEC) from Lifeline Cell Technology grown in VascuLife Media and transduced in Lifeline Basal EC (+bFGF2 10ng/ml).

Techniques: Expressing, Control, Luciferase, Mutagenesis, Concentration Assay, Construct, Western Blot, Selection

Journal: bioRxiv

Article Title: TAILS identifies candidate substrates and biomarkers of ADAMTS7, a therapeutic protease target in coronary artery disease

doi: 10.1101/2021.11.19.469331

Figure Lengend Snippet: A-C , comparison of WT/EQ and WT/Luc regulated peptides from three independent TAILS experiments after removal of mouse ADAMTS7 peptides. A , TAILS SMC1 (p<0.01) B , TAILS SMC2 (p<0.05) C , TAILS HUVEC (p<0.05), dotted line reflects significance cut off on the -log10P scale. Regulated peptides enriched for ADAMTS7 WT activity and meeting all criterial for the high confidence candidate cleavage sites (signifincant for both WT/EQ and WT/Luc comparisons) are shown in green for each TAILS experiment.

Article Snippet: The third experiment used input from cultured Human umbilical vein endothelial cells (HUVEC) from Lifeline Cell Technology grown in VascuLife Media and transduced in Lifeline Basal EC (+bFGF2 10ng/ml).

Techniques: Comparison, Activity Assay

Journal: bioRxiv

Article Title: TAILS identifies candidate substrates and biomarkers of ADAMTS7, a therapeutic protease target in coronary artery disease

doi: 10.1101/2021.11.19.469331

Figure Lengend Snippet: Histograms showing the overlap between significantly regulated candidate cleavage sites from the SMC1 ( A ), SMC2 ( C ) and HUVEC ( E ) TAILS experiments. Candidate cleavage sites present in both the WT/EQ and WT/Luc comparisons were consistently associated with ADAMTS7 activity and are defined as high confidence cleavage sites (shown as green dots in ). The remaining regulated peptides were significant for one or more condition(s) in the histogram categories. Analysis of the cleavage sites using iceLogo shows the similarities between independent TAILS experiments for SMC1 ( B ), SMC2 ( D ) and HUVEC ( F ).

Article Snippet: The third experiment used input from cultured Human umbilical vein endothelial cells (HUVEC) from Lifeline Cell Technology grown in VascuLife Media and transduced in Lifeline Basal EC (+bFGF2 10ng/ml).

Techniques: Activity Assay

Journal: bioRxiv

Article Title: TAILS identifies candidate substrates and biomarkers of ADAMTS7, a therapeutic protease target in coronary artery disease

doi: 10.1101/2021.11.19.469331

Figure Lengend Snippet: A-D, Stringent cleavage site logo plots generated by WebLogo and amino acid counts for the TAILS high confidence candidate substrate cleavage sites and ADAMTS7 auto-cleavage sites. A, SMC1 candidates (p<0.01, +FC>1, n=179), B, SMC2 candidates (p<0.05, +FC>1, n=200), C, HUVEC candidates (p<0.05, +FC>1, n=118) and D, all unique ADAMTS7 auto-cleavage sites (n=75).

Article Snippet: The third experiment used input from cultured Human umbilical vein endothelial cells (HUVEC) from Lifeline Cell Technology grown in VascuLife Media and transduced in Lifeline Basal EC (+bFGF2 10ng/ml).

Techniques: Generated

Journal: bioRxiv

Article Title: TAILS identifies candidate substrates and biomarkers of ADAMTS7, a therapeutic protease target in coronary artery disease

doi: 10.1101/2021.11.19.469331

Figure Lengend Snippet: A-D , TAILS candidate substrate cleavage sites and ADAMTS7 auto-cleavage sites analyzed using iceLogo to generate heatmaps adjusted for the abundance of each amino acid in humans. A , SMC1 candidate heatmap (p<0.01, +FC>1, n=179), B , SMC2 candidate heatmap (p<0.05, +FC>1, n=200), C , HUVEC candidate heatmap (p<0.05, +FC>1, n=118). D , the heat map including all unique ADAMTS7 auto-cleavage sites (n=75) resembles the TAILS high confidence substrates. G-H , Amino acid frequency plots generated by WebLogo showing the similar distribution between experiments, however no amino acid was present more than 30% at any given position at the cleavage site.

Article Snippet: The third experiment used input from cultured Human umbilical vein endothelial cells (HUVEC) from Lifeline Cell Technology grown in VascuLife Media and transduced in Lifeline Basal EC (+bFGF2 10ng/ml).

Techniques: Generated

Journal: bioRxiv

Article Title: TAILS identifies candidate substrates and biomarkers of ADAMTS7, a therapeutic protease target in coronary artery disease

doi: 10.1101/2021.11.19.469331

Figure Lengend Snippet: A , Venn diagram showing the overlap of unique candidate cleavage sites from SMC1 and SMC2 high confidence sites. B , Venn diagram showing the overlap from all SMC and HUVEC TAILS datasets. C , Gene assignment of the 91 unique candidate cleavage sites identified from multiple TAILS experiments, including 24 unique sites from 16 different genes identified in all three TAILS datasets.

Article Snippet: The third experiment used input from cultured Human umbilical vein endothelial cells (HUVEC) from Lifeline Cell Technology grown in VascuLife Media and transduced in Lifeline Basal EC (+bFGF2 10ng/ml).

Techniques:

Journal: Korean Journal of Radiology

Article Title: In Vitro and In Vivo Imaging of Prostate Cancer Angiogenesis Using Anti-Vascular Endothelial Growth Factor Receptor 2 Antibody-Conjugated Quantum Dot

doi: 10.3348/kjr.2013.14.1.30

Figure Lengend Snippet: Confocal microscope images of HUVEC interacted to QDs-VEGFR2 and QDs. Green fluorescence indicates QDs interaction to cells. QDs-VEGFR2 bound to HUVEC, while there was no QD binds to HUVEC without VEGFR2 conjugation. A. HUVEC with QDs-VEGFR2. B. HUVEC with QDs only (left: overlaid image of DIC and fluorescent images, middle: green fluorescent [QDs] image, right: overlaid image of blue [DAPI] and green fluorescent images). HUVEC = human umbilical vein cord endothelial cells, DIC = differential interference contrast, DAPI = 4',6-diamidino-2-phenylindole, QDs = quantum dots, VEGFR2 = vascular endothelial growth factor receptor 2

Article Snippet: Human umbilical vein cord endothelial cells (HUVECs) were purchased from company (Innopharma Screen, Asan, Korea).

Techniques: Microscopy, Fluorescence, Conjugation Assay

Journal: PPAR Research

Article Title: Stimulation of Alpha 1 -Adrenergic Receptor Ameliorates Cellular Functions of Multiorgans beyond Vasomotion through PPAR δ

doi: 10.1155/2020/3785137

Figure Lengend Snippet: The effects of α 1 -AR stimulation on the expression of mitochondrial energetic molecules, oxidative phosphorylation, and biological functions in skeletal and cardiac muscle cells and liver cells. (a, b) The expression of p-AMPK and PPAR δ in C2C12, HL1, and HepG2 cells was stimulated with 1–30 μ M midodrine for the indicated times. (c) Cytosolic calcium mobilization after midodrine treatment in C2C12 and HL1 cells. Each cell type was pretreated with the calcium reactive dye Fluo-3 AM for 45 min and then stimulated with 30 μ M midodrine for the indicated times. Green fluorescence emitted by Fluo-3 AM was detected using confocal microscopy. (d) The phosphorylation of AMPK α at Thr172 and expression of PPAR δ in C2C12 and HL1 cells after pretreatment with the calcium/calmodulin-dependent protein kinase kinase antagonist STO-609 for 25 min and treatment with midodrine. (e) Fluorescence after using the CytoPainter mitochondrial staining kit in midodrine-treated and control C2C12 cells. Original magnification was 200x. (f) The measured activity of succinate dehydrogenase (SDH) in C2C12 cells. (G) Oxygen consumption rate (OCR) in C2C12 cells treated with midodrine (30 μ M), as measured by a Seahorse XFp analyzer. (h) ATP content in C2C12 cells treated with midodrine (30 μ M) cultured with low-glucose (5.56 mM) medium. (i) Glucose transporter (GLUT) 4 protein expression in C2C12 cells treated with high glucose (HG) and midodrine (HG+Mido), HG and insulin (HG+Insulin), and the control treatment (Ctrl). (j) The uptake of 2-deoxyglucose in C2C12 skeletal muscle cells treated with midodrine. (k) OCR (measured by the Seahorse XFp analyzer) in H9C2 cells treated with midodrine (30 μ M) and cultured with low-glucose (5.56 mM) medium. (l) ATP content in H9C2 cells treated with midodrine (30 μ M). Data are expressed as the mean ± standard deviation of triplicate experiments. AMPK: adenosine monophosphate-activated protein kinase; p-AMPK: phosphorylated AMPK; PPAR δ : peroxisome proliferator-activated receptor delta; PGC-1 α : peroxisome proliferator-activated receptor gamma coactivator 1-alpha; mGLUT4: GLUT4 expression of the cell membrane; tGLUT4: total cellular expression of GLUT4; Ctrl: an untreated control group; Mido: midodrine-treated group.

Article Snippet: L6 rat skeletal muscle, C2C12 mouse skeletal muscle, HL1 and H9C2 cardiac muscle, HUVEC human umbilical vein endothelial cell line, RAW 264.7 macrophages, and 3T3-L1 mouse preadipocyte cells were purchased from a Korean cell line bank (Seoul, Korea).

Techniques: Expressing, Phospho-proteomics, Fluorescence, Confocal Microscopy, Staining, Control, Activity Assay, Cell Culture, Standard Deviation, Membrane

Journal: PPAR Research

Article Title: Stimulation of Alpha 1 -Adrenergic Receptor Ameliorates Cellular Functions of Multiorgans beyond Vasomotion through PPAR δ

doi: 10.1155/2020/3785137

Figure Lengend Snippet: The effect of midodrine on the endothelial expression of p-AMPK and p-eNOS in HUVECs; OCR analyses in H9C2 cells; intracellular fat and the expression of PPAR δ , p-AMPK, and PGC-1 α in differentiated 3T3-L1 cells; and the effects of midodrine on mRNA levels of PPAR δ , AMPK α 1 , and mannose receptor and protein levels of mannose receptor and hexokinase II in RAW 264.7 macrophage cells treated with different concentrations of midodrine. (a) The expression of phosphorylated AMPK (p-AMPK) and phosphorylated endothelial nitric oxide synthase (p-eNOS) proteins in human umbilical vein endothelial cells (HUVECs) treated with cholesterol and palmitate, and the effects from the addition of GSK0660, a PPAR δ antagonist. Ctrl: the control group; CP: the cholesterol- and palmitate-treated group; CPM: the cholesterol-, palmitate-, and midodrine-treated group. (b) The maximal oxygen consumption rate (OCR) analysis as estimated using a Seahorse XFp analyzer and ATP content measured by ELISA in H9C2 cells. (c) The effect of compound C (1 μ M) on p-AMPK expression and PPAR δ expression. (d) The effect of midodrine on intracellular lipid deposits (Oil Red O staining result) and the protein levels of PPAR δ , AMPK, and PGC-1 α in differentiated 3T3-L1 cells treated with midodrine and GSK0660. (e) The effects of midodrine on mRNA levels of PPAR δ , AMPK α 1 , and mannose receptor and protein levels of mannose receptor and hexokinase II in RAW 264.7 macrophage cells treated with different concentrations of midodrine. Ctrl: untreated control group; Mido: midodrine-treated group; Mido+GSK0660: midodrine- and GSK0660-treated group.

Article Snippet: L6 rat skeletal muscle, C2C12 mouse skeletal muscle, HL1 and H9C2 cardiac muscle, HUVEC human umbilical vein endothelial cell line, RAW 264.7 macrophages, and 3T3-L1 mouse preadipocyte cells were purchased from a Korean cell line bank (Seoul, Korea).

Techniques: Expressing, Control, Enzyme-linked Immunosorbent Assay, Staining

Journal: eLife

Article Title: GIPC proteins negatively modulate Plexind1 signaling during vascular development

doi: 10.7554/eLife.30454

Figure Lengend Snippet:

Article Snippet: Cell line ( Homo sapiens ) , HUVEC (Normal Primary Human Umbilical Vein Endothelial Cells) , Lifeline Cell Technology , Cat. #FC-0003 , https://www.lifelinecelltech.com/shop/cells/human-endothelial-cells/umbilical-vein-endothelial-cells/huvec-fc-0003/.

Techniques: Transgenic Assay, Plasmid Preparation, Mutagenesis, Derivative Assay, Selection, Stable Transfection, Knock-Out, Recombinant, Control, shRNA, Sequencing, Construct, Synthesized, Positive Control, Sterility, Concentration Assay

Journal: Frontiers in Cardiovascular Medicine

Article Title: Therapeutic Effect of Pericytes for Diabetic Wound Healing

doi: 10.3389/fcvm.2022.868600

Figure Lengend Snippet: The expression of pericyte markers by flow cytometry was reduced in the diabetic condition. (A) Analysis of pericyte surface markers in the non-diabetic and diabetic conditions using flow cytometry [CD146-fluorescein isothiocyanate (FITC), Nestin-phycoerythrin (PE), and NG2-PE]. The results provided here are representative of those obtained in three independent studies. In the diabetic condition, Nestin and NG2-PE were seen to decrease by more than half. (B) Overlay of diabetic condition (green line) and non-diabetic (red line) condition flow cytometry. The diabetic condition showed the three pericyte markers to be decreased.

Article Snippet: Each well contained 300 μl of the human umbilical vein endothelial cells (HUVECs) and pericytes (10:1 ratio) cell suspension in the VascuNet Basal Assay Medium (ESI BIO, USA) ( ).

Techniques: Expressing, Flow Cytometry

Journal: Frontiers in Cardiovascular Medicine

Article Title: Therapeutic Effect of Pericytes for Diabetic Wound Healing

doi: 10.3389/fcvm.2022.868600

Figure Lengend Snippet: The group with pericytes showed improved tube formation and cell migration ability in the high glucose condition. (A) The in-vitro vascular, tube formation assay showed effects of exposure on FITC-tagged human umbilical vein endothelial cell (HUVEC) tube formation with and without PE-tagged human pericyte cells. The optimal observation time for tube formation was 8 h after seeding. In high glucose conditions, HUVECs with pericytes (10:1 ratio) showed active tube formation even compared to the without pericyte group. (B) The number of pieces in the assay was measured. (** P < 0.01 and *** P < 0.001 according to the Student's t -test). HUVEC tube formation capacity showed no significant difference in low glucose conditions. However, in high glucose conditions, the group with pericytes had a statistically significant increase in tube formation. (C) Photomicrographs were taken at 0, 12, and 24 h following initiation of the wound. HUVEC migration, often termed as wound closure ability, was more remarkable in the group with pericytes in both the high and low glucose conditions. (D) The quantitative approach applied to the wound closure assay, showing a histogram calculated based on the group breath ratio of the control group and the group with pericytes.

Article Snippet: Each well contained 300 μl of the human umbilical vein endothelial cells (HUVECs) and pericytes (10:1 ratio) cell suspension in the VascuNet Basal Assay Medium (ESI BIO, USA) ( ).

Techniques: Migration, In Vitro, Tube Formation Assay, Wound Closure Assay, Control

Journal: Frontiers in Cardiovascular Medicine

Article Title: Therapeutic Effect of Pericytes for Diabetic Wound Healing

doi: 10.3389/fcvm.2022.868600

Figure Lengend Snippet: The diabetes mellitus (DM)-with-pericyte group exhibited substantial wound healing in a mouse wound model. (A) Illustration of injection of collagen type 1 scaffold + 5 × 10 5 pericytes in the mouse wound. (B) Streptozotocin (STZ)-induced diabetic mice on blood glucose confirmed successful DM induction (**** P < 0.0001). DM-induced mice ( n = 10), control ( n = 10). (C) Incisions with a diameter of 4 mm on either side of the midline were produced. To avoid wound contraction, the wounds were circumscribed with donut-shaped silicone splints. As observed on day 7, wounds of the DM-with-pericyte group healed, whereas those of the DM-without-pericyte and control groups did not heal. (D) The Y-axis of the graph represents the wound size, when measured on days 0, 2, 7, and 14. It was verified that all the groups, except the DM-without-pericyte group, had wound closure by the time of sacrifice on day 14 and approximately 3.6 mm of the wound of the DM-without-pericyte group remained. The pericyte and DM-with-pericyte groups showed the same wound size on days 0, 2, 7, and 14. (E) Quantification of wound size at sacrifice time points (** P < 0.01). In comparison to the DM-without-pericyte group, the DM-with-pericyte group exhibited substantial wound healing.

Article Snippet: Each well contained 300 μl of the human umbilical vein endothelial cells (HUVECs) and pericytes (10:1 ratio) cell suspension in the VascuNet Basal Assay Medium (ESI BIO, USA) ( ).

Techniques: Injection, Control, Produced, Comparison

Journal: Frontiers in Cardiovascular Medicine

Article Title: Therapeutic Effect of Pericytes for Diabetic Wound Healing

doi: 10.3389/fcvm.2022.868600

Figure Lengend Snippet: The DM-with-pericyte group showed successful repair of the collagen layer and higher blood vessel formation capacity compared to the DM-without-pericyte group. (A) H&E staining. (B) Masson's trichrome staining for wound analysis at 14 days. The black square indicates the regenerated dermal collagen layer. Scale bars = 200 μm. (C) CD31 and Ki67 immunofluorescence of wounds at 14 days. In diabetes wounds, new vessels were significantly less visible than in normal mice. However, when diabetic mice were injected with pericytes, new vessels were observed more often. (D) α-smooth muscle actin (α-SMA), collagen 1 (Col 1), and Nestin immunohistochemistry of wounds at 14 day. Scale bar = 50 μm.

Article Snippet: Each well contained 300 μl of the human umbilical vein endothelial cells (HUVECs) and pericytes (10:1 ratio) cell suspension in the VascuNet Basal Assay Medium (ESI BIO, USA) ( ).

Techniques: Staining, Immunofluorescence, Injection, Immunohistochemistry