human dermal microvascular endothelial cells (mvecs, lonza)  (Lonza)

Journal: Cancers

Article Title: CAFs and TGF-β Signaling Activation by Mast Cells Contribute to Resistance to Gemcitabine/Nabpaclitaxel in Pancreatic Cancer

doi: 10.3390/cancers11030330

Figure Lengend Snippet: CM-HMC-1 and CM-CAF did not alter GEM/NAB-dependent inhibition of tumor angiogenesis but reduced the efficacy on tumor invasion. ( a ) Capillary morphogenesis. Both CM-HMC-1 and CM-CAF did not alter angiogenesis as demonstrated by microvascular formation and did not reduce the antiangiogenic potential of GEM/NAB. Pictures represent three different experiments. ( b ) Invasion assay. Both CM-HMC-1 and CM-CAF altered tumor invasion, by inducing the decrese and the increase of MIA PaCa-2 invasion, respectively. Both conditioned media reduced the efficacy of GEM/NAB-dependent inhibion of tumor invasion as reported in the histogram plot showing the recovery of tumor invasion on CM-HMC-1/CM-CAF + GEM/NAB treated cells. The pictures represent three different experiments.

Article Snippet: HMC-1 human mast cell line-1 cells were kindly provided by Prof. L. Macchia, University of Bari, CAF cells were purchased from Vitro Biopharma, and MVEC human dermal microvascular endothelial cells were purchased from Lonza, Switzerland.

Techniques: Inhibition, Invasion Assay

Journal: Annals of the Rheumatic Diseases

Article Title: Scleroderma fibroblasts suppress angiogenesis via TGF-β/caveolin-1 dependent secretion of pigment epithelium-derived factor

doi: 10.1136/annrheumdis-2017-212120

Figure Lengend Snippet: Suppression of angiogenesis in an organotypic co-culture assay by systemic sclerosis (SSc) fibroblasts is reversed by pigment epithelium-derived factor (PEDF) knockdown. (A, C) Images show representative microscopic fields from co-culture assays of human dermal microvascular endothelial cells (MVECs) (A) or human umbilical vein endothelial cells (HUVECs) (C) seeded onto confluent fibroblasts (FBs), healthy control (HC-FBs) or SSc (SSc-FBs), stained for the endothelial marker CD31 (fibroblasts are seen unstained in the background). Note that HUVECs reproduce the behaviour of MVECs in the organotypic assays. (B, D) Histograms show the number of tubules and total tubule length quantified using Angiosys software, represented as mean±SEM (n=12 microscopic fields at ×4 magnification from triplicate wells). (E) Representative western blot showing intracellular PEDF levels in SSc fibroblasts treated with GolgiPlug, non-silencing control (NS) or with PEDF depletion (shPEDF) by means of lentiviral short-hairpin RNA (sh). (F) Images show representative microscopic fields from co-culture assays of HUVECs seeded onto confluent SSc fibroblasts (SSc-FBs), non-silencing control (NS) or with PEDF depletion (shPEDF). (G) Quantification of the number of tubules and total tubule length represented as mean±SEM (n=12 microscopic fields at ×4 magnification from triplicate wells). **P<0.01, ***P<0.001 by unpaired t-test. Scale bars, 100 µm.

Article Snippet: Human dermal microvascular endothelial cells (MVECs) were purchased from PromoCell, UK, cultured in Endothelial Cell Growth Medium BulletKit (Lonza, Slough, UK) and used at passages 2–4.

Techniques: Co-culture Assay, Derivative Assay, Co-Culture Assay, Staining, Marker, Software, Western Blot, shRNA

Journal: PLoS ONE

Article Title: GDF5 Regulates TGFß-Dependent Angiogenesis in Breast Carcinoma MCF-7 Cells: In Vitro and In Vivo Control by Anti-TGFß Peptides

doi: 10.1371/journal.pone.0050342

Figure Lengend Snippet: Panel A . MVECs were exposed for 6 hours to CM from MCFTß or MCFM. Numbers on the upper right side indicate the percent field occupancy of capillary plexus (±SD), taking untreated MVECs incubated in their own culture medium as 100%. Since the overall morphology did not substantially differ between 6 and 18 h after MVEC seeding (apart of a better definition of tubule formation by capillary projections), quantification was performed at 6 h after seeding. Pictures show a typical out of 4 different experiments that gave similar results. Dx: Doxycycline. The bottom pictures of panel A show capillary morphogenesis of MVECs exposed to CM of Dx−/MCFTß in the presence of p17 (+P17), p144 (+P144) or of a scrambled oligopeptide (+scr), respectively. * p<0.05 (Student t-test), significantly different from mock. Panel B . Upper part: Matrigel sponges, containing 2.5×10 6 MCFTß or MCFM, were recovered five days post implantation in 4 mice, each one grafted with 2 different sponges. The anti-angiogenesis activity of p17 peptide was evaluated in the presence of MCFTß. The figure shows a typical experiment out of 4 different experiments that gave similar results, upon systemic administration (i.p.) of the peptide. Co-injecting p17 in the plug with MCFTß gave similar results (not shown). Dx: Doxycycline. Lower part: CD31 immunohistochemistry of sponges in the same experimental conditions shown in the upper part. Panel C . On the left: haemoglobin contents ± SD versus MCFM +Dx taken as 100% in explanted specimes from 4 different experiments. On the right: % vascularization ± SD in the same conditions. * p<0.05 (Student t-test), significantly different versus the relevant control.

Article Snippet: Human dermal microvascular endothelial cells (MVECs) were purchased from Lonza Ltd.

Techniques: Incubation, Activity Assay, Immunohistochemistry, Control

Journal: PLoS ONE

Article Title: GDF5 Regulates TGFß-Dependent Angiogenesis in Breast Carcinoma MCF-7 Cells: In Vitro and In Vivo Control by Anti-TGFß Peptides

doi: 10.1371/journal.pone.0050342

Figure Lengend Snippet: Panel A . RT-PCR showing ALK expression on 3 of the MVEC cell lines used. GAPDH: loading control. Bp is shown on the right. The picture shows the results of a typical experiment out of 3 replicas. Panel B. Quantification of ALK RNA as related to GAPDH. The histogram shows the results obtained in 3 different experiments performed in duplicate on each MVEC cell line, ± SD. * p<0.05 (Student t-test), significantly different among various ALK RNAs. Panel C . Western blots of total protein extracts from MVECs exposed to CM of MCFM and MCFTß for 30 min and 120 min, respectively, in the presence or absence of P17. Western blots were probed for phospho-SMAD1/5, SMAD5, phospho-SMAD2, SMAD2/3 and Tubulin (loading control) as indicated. Molecular weights are reported on the right. Also in this case the picture shows the results of a typical experiment out of 3 replicas.

Article Snippet: Human dermal microvascular endothelial cells (MVECs) were purchased from Lonza Ltd.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Control, Western Blot

Journal: PLoS ONE

Article Title: GDF5 Regulates TGFß-Dependent Angiogenesis in Breast Carcinoma MCF-7 Cells: In Vitro and In Vivo Control by Anti-TGFß Peptides

doi: 10.1371/journal.pone.0050342

Figure Lengend Snippet: Panel A . Activation of pro-angiogenic genes following 6 h MVEC stimulation with exogenously added recombinant TGFß (1 ng/ml). BMP7, bone morphogenetic protein-7; GDF5, growth and differentiation factor-5. Data shown were obtained in 3 different experiments performed in double, and are expressed as average value ±SD. * p<0.05 (Student t-test). Panel B . Activation of pro-angiogenic genes following 6 h MVEC stimulation with CM of MCFM and MCFTß cells in the absence and in the presence of peptide p17. White columns: fold change of gene expression under stimulation with Dx−/MCFTß-CM with respect to MCFM-CM, assumed as value 1. Grey columns: the same of white columns, in the presence of p17. Data shown were obtained in 3 different experiments performed in double, and are expressed as average value ±SD. * p<0.05 (Student t-test) with respect to values obtained with MCFM CM; ** p<0.05 in the presence of the inhibiting peptide with respect to stimulation by Dx−/MCFTß-CM. Panel C . Quantitative PCR of GDF5 and uPAR in control MVECs and in MVECs stimulated with 1 ng/ml TGFß. non targ: not-targeting siRNA; siGDF5, siRNA anti-GDF5. * p<0.05 (Student t-test) with respect to values obtained with not-targeting siRNA. Panel D. Western blotting of MVEC lysates (100 µg/lane), under control conditions (control) and following stimulation with 1 ng/ml TGFß alone (TGFß), TGFß+lipofectamine (lipo), TGFß+not targeting siRNA (siNT) and TGFß+GDF5-targeting siRNA (siDGF5). The primary antibodies were anti-GDF5 IgG and M2 polyclonal anti-uPAR antibody. Tubulin, loading control. Molecular weights are reported on the right.

Article Snippet: Human dermal microvascular endothelial cells (MVECs) were purchased from Lonza Ltd.

Techniques: Activation Assay, Recombinant, Gene Expression, Real-time Polymerase Chain Reaction, Control, Western Blot

Journal: PLoS ONE

Article Title: GDF5 Regulates TGFß-Dependent Angiogenesis in Breast Carcinoma MCF-7 Cells: In Vitro and In Vivo Control by Anti-TGFß Peptides

doi: 10.1371/journal.pone.0050342

Figure Lengend Snippet: Panel A . Capillary morphogenesis experiments in control MVECs (untreated), upon MCFTß CM stimulation alone, and in the presence of irrelevant IgG (+ Irrel IgG), 5 µg/ml anti-GDF5 antibody (anti-GDF5), not-targeting siRNA (siNT), GDF5-targeting siRNA (siGDF5), 5 µg/ml anti-BM7 antibody (anti BMP7), BMP7-targeting siRNA (siBMP7). Panel B . Representative experiment of siGDF5-dependent and anti-GDF5 antibody-dependent inhibition of angiogenesis in the Matrigel sponge model in mice. Panel C . On the left: angiogenesis in the sponges was quantified by evaluating the haemoglobin content of each sponge. On the right: % vascularization ± SD in the same conditions. Images were acquired with the ZEISS SR Stemi stereomicroscope. TR: transfection reagents alone; siNT: not-targeting siRNA. are the mean of three experiments (three animal for each condition, two Matrigel sponges in each animal). Graphs are shown as mean ± SD; * p<0.05 (Student t-test) with respect to both transfection reagents alone (TR) and not-targeting siRNA (siNT).

Article Snippet: Human dermal microvascular endothelial cells (MVECs) were purchased from Lonza Ltd.

Techniques: Control, Inhibition, Transfection