Journal: Nature Communications

Article Title: Targeting QKI-7 in vivo restores endothelial cell function in diabetes

doi: 10.1038/s41467-020-17468-y

Figure Lengend Snippet: Morphology of hiPSCs and their EC differentiated counterparts are shown by bright field microscopy. Scale bar: 50 μm ( a ). Flow cytometry showed the pure population of hiPS-derived ECs after MACS selection using CD144 magnetic beads ( b ). Immunofluorescence confocal image showing that the differentiated ECs expressed the EC-specific markers CD31, CD144, and ZO-1 localizing to cell–cell junction. QKI-7 displayed perinuclear cytoplasm localization. Scale bar: 25 μm ( c ). The expression of EC marker proteins CD31, CD144, KDR, and eNOS was shown by western blot ( d ). hiPS-ECs formed tube structure indicating their angiogenic capacity. Scale bar: 200 μm ( e ). Data are from n = 3 representative images. Source data are provided as a Source data file.

Article Snippet: Primary antibodies include QKI-7 (UC Davis/NIH NeuroMab Facility 73-200, WB 1:1000, ICC 1:100), CD144 (St John’s Laboratory STJ96234, WB 1:1000, ICC 1:200), CD31 (Abcam AB28364, WB 1:1000, ICC 1:20), KDR (R&D Systems MAB3571, WB 1:1000), FLK1 (Thermo Fisher Scientific MA5-15157, WB 1:1000), eNOS (Abcam AB76198, WB 1:1000), NLGN1 (Abcam ab153821, WB 1:1000), β-actin (R&D Systems MAB8928, WB 1:1000), and ZO-1 (Thermo Fisher Scientific 40-2200, ICC 1:200).

Techniques: Microscopy, Flow Cytometry, Derivative Assay, Selection, Magnetic Beads, Immunofluorescence, Expressing, Marker, Western Blot

Journal: Chinese Journal of Cancer Research

Article Title: Tim-3 promotes cell aggressiveness and paclitaxel resistance through NF-κB/STAT3 signalling pathway in breast cancer cells

doi: 10.21147/j.issn.1000-9604.2020.05.02

Figure Lengend Snippet: Effect of Tim-3 OE on tube formation of endothelial cells. (A) Tube formation ability of HUVECs cultured in conditioned medium from MDA-MB-231 Tim-3-overexpressing cells (P=0.014 vs . Scr); (B) Tube formation ability of HUVECs cultured in conditioned medium from MCF7 Tim-3-overexpressing cells (P=0.016 vs . Scr); (C) Protein levels of VEGFA, VEGFB and VEGFD following Tim-3 OE (left) and quantitative densitometric analysis (right); (D) mRNA expression of VEGFA and VEGFD genes in breast cancer cells. OE, overexpression; Tim-3, T-cell immunoglobulin and mucin-domain containing molecule-3; HUVEC, human umbilical vein endothelial cell; VEGF, vascular endothelial growth factor. * , P<0.05; ** , P<0.01; *** , P<0.001.

Article Snippet: ELISA was performed using the human VEGFC (cat. no. E-EL-H1600; Elabscience) and vascular endothelial growth factor receptor 2 (VEGFR2) ELISA kit (cat. no. E-EL-H1603; Elabscience), according to the manufacturer’s protocol.

Techniques: Cell Culture, Expressing, Over Expression

Journal: Chinese Journal of Cancer Research

Article Title: Tim-3 promotes cell aggressiveness and paclitaxel resistance through NF-κB/STAT3 signalling pathway in breast cancer cells

doi: 10.21147/j.issn.1000-9604.2020.05.02

Figure Lengend Snippet: VEGFC and VEGFR2 protein levels in conditioned media of stable cell lines indicated by ELISA. (A) VEGFC; (B) VEGFR2. VEGF, vascular endothelial growth factor; ELISA, enzyme-linked immunosorbent assay.

Article Snippet: ELISA was performed using the human VEGFC (cat. no. E-EL-H1600; Elabscience) and vascular endothelial growth factor receptor 2 (VEGFR2) ELISA kit (cat. no. E-EL-H1603; Elabscience), according to the manufacturer’s protocol.

Techniques: Stable Transfection, Enzyme-linked Immunosorbent Assay

Journal: Chinese Journal of Cancer Research

Article Title: Tim-3 promotes cell aggressiveness and paclitaxel resistance through NF-κB/STAT3 signalling pathway in breast cancer cells

doi: 10.21147/j.issn.1000-9604.2020.05.02

Figure Lengend Snippet: Schematic illustration of role of Tim-3 in breast cancer. Upregulation of Tim-3 not only promotes cell proliferation, migration and invasion, but also disrupts cell-cell tight junction, increases angiogenesis of endothelial cells and paclitaxel-resistance. Tim-3 functions in breast cancer cells by activating NF-κB/STAT3 pathway and downstream target genes. Tim-3, T-cell immunoglobulin and mucin-domain containing molecule-3; IL-6, interleukin 6; ZO, zona occludens; VEGF, vascular endothelial growth factor; CCND1, cyclin D1; MMP-1, matrix metalloproteinase-1.

Article Snippet: ELISA was performed using the human VEGFC (cat. no. E-EL-H1600; Elabscience) and vascular endothelial growth factor receptor 2 (VEGFR2) ELISA kit (cat. no. E-EL-H1603; Elabscience), according to the manufacturer’s protocol.

Techniques: Migration

Journal: Cells

Article Title: Fibrotic Changes and Endothelial-to-Mesenchymal Transition Promoted by VEGFR2 Antagonism Alter the Therapeutic Effects of VEGFA Pathway Blockage in a Mouse Model of Choroidal Neovascularization

doi: 10.3390/cells9092057

Figure Lengend Snippet: Primary human retinal endothelial cells (hREC) can be used to model endothelial-to-mesenchymal transition (EndoMT) in vitro. ( a ) Expression levels for the genes encoding the EndoMT-associated proteins SNAI1, SNAI2, α-SMA, FSP-1, vimentin, fibronectin, COL1A2, and COL3A1; and the endothelial differentiation markers VE-cadherin, CD31 and vascular endothelial growth factor receptor-2 (VEGFR2), are shown relative to the housekeeping gene HPRT1 and normalized to the control hREC as assessed by qPCR on day 6 of EndoMT induction. Data = mean ± SEM, ** p < 0.01, *** p < 0.001 compared to the control hREC by unpaired 2-tail t-test, n = 4 (VE-cadherin), 6 (collagen type III alpha 1 chain (COL3A1)), 8 (SNAI1, α-smooth muscle actin (α-SMA), fibroblast-specific protein 1 (FSP-1), CD31), 9 (vimentin), 10 (fibronectin, VEGFR2), and 11 (SNAI2, COL3A1) independent wells per group. ( b ) Histological analysis of EndoMT cells on day 6 of EndoMT induction. Phase contract microscopy and phalloidin staining (green) in the top four panels on the left illustrate the distinctions in cellular morphology between hREC and EndoMT cells. Alterations in expression and localization of endothelial differentiation markers CD31 and VE-cadherin and the mesenchymal markers vimentin, α-SMA, fibronectin and SNAI1 between hREC and EndoMT cells are also shown. Note the nuclear localization of SNAI1 (green, arrows) in EndoMT cells whereas the control hREC do not display SNAI1 expression. Isotype-matched IgG staining controls are shown on the bottom rows. Scale bars = 100 µm.

Article Snippet: Diluted DMSO vehicle control, 5 nmoles of the p38MAPK inhibitor SB203580 (Abcam), 2.5 or 5.0 μg of anti-VEGFR2 neutralizing antibody (MAB4431; R&D Systems, Minneapolis, MN, USA), or a combination of SB203580 plus anti-VEGFR2 neutralizing antibody was injected into both eyes.

Techniques: In Vitro, Expressing, Control, Microscopy, Staining

Journal: Cells

Article Title: Fibrotic Changes and Endothelial-to-Mesenchymal Transition Promoted by VEGFR2 Antagonism Alter the Therapeutic Effects of VEGFA Pathway Blockage in a Mouse Model of Choroidal Neovascularization

doi: 10.3390/cells9092057

Figure Lengend Snippet: Treatment with VEGFA at a high concentration significantly reduces expression of genes associated with EndoMT and restores gene expression associated with endothelial cell differentiation. After EndoMT induction using IL-1β (0.1 ng/mL), TNF-α (5.0 ng/mL), and TGF-β2 (5.0 ng/mL), EndoMT cells were cultured for seven days in medium containing 5% FBS and 1% penicillin/streptomycin in the presence or absence of VEGFA165 (50 ng/mL). Expression levels for the genes encoding the EndoMT-associated proteins SNAI2, FSP-1, vimentin, and COL3A1, as well as the endothelial differentiation markers CD31 and VEGFR2, are normalized to the housekeeping gene HPRT1 and shown relative to the hREC control as assessed by qPCR. ( n = 3 independent wells per group). Data = mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, 2-tail unpaired t-test.

Article Snippet: Diluted DMSO vehicle control, 5 nmoles of the p38MAPK inhibitor SB203580 (Abcam), 2.5 or 5.0 μg of anti-VEGFR2 neutralizing antibody (MAB4431; R&D Systems, Minneapolis, MN, USA), or a combination of SB203580 plus anti-VEGFR2 neutralizing antibody was injected into both eyes.

Techniques: Concentration Assay, Expressing, Gene Expression, Cell Differentiation, Cell Culture, Control

Journal: Cells

Article Title: Fibrotic Changes and Endothelial-to-Mesenchymal Transition Promoted by VEGFR2 Antagonism Alter the Therapeutic Effects of VEGFA Pathway Blockage in a Mouse Model of Choroidal Neovascularization

doi: 10.3390/cells9092057

Figure Lengend Snippet: Phospho-p38 MAPK inhibitors prevent and reverse EndoMT-associated gene expression in vitro. ( a ) To study possible prevention of EndoMT, hREC were treated with the p38 MAPK inhibitor SB203580 or SB202190 (10 μM) during EndoMT induction. Expression levels for the genes encoding the EndoMT-associated proteins SNAI1, SNAI2, α-SMA, FSP-1, vimentin, fibronectin, COL1A2 and COL3A1; and the endothelial differentiation markers VE-cadherin, CD31 and VEGFR2 were assessed by qPCR on day 6 of EndoMT induction. Data = mean ± SEM, * p < 0.05, ** p < 0.01, *** p < 0.001 by 2-tail unpaired t-test relative to the untreated EndoMT controls, n = 7 (COL3A1, VE-cadherin), 9 (α-SMA, FSP-1, vimentin), 10 (SNAI1, SNAI2, fibronectin, COL1A2, CD31), 12 (VEGFR2) independent wells. ( b ) To study reversal of EndoMT, EndoMT cells were treated with SB203580 (10 μM) for 9 days after EndoMT induction. Expression levels for the genes encoding EndoMT-associated proteins, and endothelial differentiation markers were assessed by qPCR after 9 days of treatment. Data = mean ± SEM, * p < 0.05, ** p < 0.01, *** p < 0.001 by 2-tail unpaired t-test, relative to the untreated EndoMT controls; n = 3 (COL1A2) and n = 6 (the rest of the genes) independent wells.

Article Snippet: Diluted DMSO vehicle control, 5 nmoles of the p38MAPK inhibitor SB203580 (Abcam), 2.5 or 5.0 μg of anti-VEGFR2 neutralizing antibody (MAB4431; R&D Systems, Minneapolis, MN, USA), or a combination of SB203580 plus anti-VEGFR2 neutralizing antibody was injected into both eyes.

Techniques: Gene Expression, In Vitro, Expressing

Journal: Cells

Article Title: Fibrotic Changes and Endothelial-to-Mesenchymal Transition Promoted by VEGFR2 Antagonism Alter the Therapeutic Effects of VEGFA Pathway Blockage in a Mouse Model of Choroidal Neovascularization

doi: 10.3390/cells9092057

Figure Lengend Snippet: Inhibition of VEGFR2 as a solo treatment promotes EndoMT, whereas anti-VEGFR2 therapy in combination with inhibition of EndoMT/fibrosis reduces CNV area and CNV number in a mouse model of wet AMD. ( a ) Eyes of P24 JR5558 mice were treated with a single intravitreal injection of vehicle, SB203580 (10 nmoles), anti-VEGFR2 neutralizing antibody (5 μg), or a combination of SB203580 and anti-VEGFR2 antibody. RPE–choroid eyecups with retina removed were harvested seven days post-injection, prepared as flat-mounts, and immunostained for the EndoMT marker vimentin (white) in addition to the vascular marker IB4 (green) in the CNV (left panels). Luminosity of the vimentin staining was quantified and normalized to CNV lesion area (right panel). ( b ) Eyes of P24 JR5558 mice were treated with a single intravitreal injection of vehicle, SB203580 (10 nmoles), anti-VEGFR2 neutralizing antibody (2.5 μg), or a combination of SB203580 and anti-VEGFR2 antibody. RPE–choroid eyecups were harvested seven days post-injection, prepared as flat-mounts, and immunostained for isolectin B4 (IB4, green) to visualize the vasculature in the CNV (top panels). Micrographs of the IB4-stained eyecups were used to quantify total CNV area per eye and the number of CNV lesions per eye (bottom panels). For micrographs in A, scale bars = 100 μm. For graphs, data = mean ± SEM. # p < 0.05, ### p < 0.001 by one-way ANOVA with Bunnett’s multiple comparison test compared to the vehicle control. * p < 0.05, ** p < 0.01, *** p < 0.001 by 2-tail unpaired t -test. n = 240 (Vehicle), 95 (SB), 83 (Anti-VEGFR2), and 186 (SB+Anti-VEGFR2) individual CNV per group in (a), and 28 (Vehicle), 16 (SB), 11 (Anti-VEGFR2), and 10 (SB+Anti-VEGFR2) individual eyes per group in ( b ). All dosing in animals was performed in a masked fashion and all image analysis was performed by a trained investigator who was masked to the identity of the treatment groups.

Article Snippet: Diluted DMSO vehicle control, 5 nmoles of the p38MAPK inhibitor SB203580 (Abcam), 2.5 or 5.0 μg of anti-VEGFR2 neutralizing antibody (MAB4431; R&D Systems, Minneapolis, MN, USA), or a combination of SB203580 plus anti-VEGFR2 neutralizing antibody was injected into both eyes.

Techniques: Inhibition, Injection, Marker, Staining, Comparison, Control