Journal: EMBO Molecular Medicine

Article Title: Angpt2/Tie2 autostimulatory loop controls tumorigenesis

doi: 10.15252/emmm.202114364

Figure Lengend Snippet: ANGPT2 plasma concentration in 69 NF‐NF‐PitNET patients and in 69 age‐ and gender‐matched healthy controls. Each dot represents one individual. Healthy controls: mean ± SEM = 0.8698 ± 0.05757; PitNET patients; mean ± SEM = 1.786 ± 0.1383; Difference between means = 0.9158 ± 0.1498; 95% confidence interval = 0.6181 to 1.213. *** P ‐value < 0.0001 by unpaired t ‐test with Welch’s correction. Correlation between circulating ANGP T2 levels and Ki67 LI (i.e., % of Ki67‐positive cells). All box plots show 25 th to 75 th percentiles (box) and 5 th to 95 th percentiles (whiskers). The line in the box represents the median. Results are expressed as mean ± SEM. ** P = 0.014 by one‐way ANOVA ( P = 0.007 by Kruskal–Wallis test). Expression of ANGPT1 and ANGPT2 in human unaffected anterior pituitary (control, n = 3) and in human NF‐PitNETs. Thirty‐seven human NF‐PitNETs were analyzed for ANGPT2 expression and 14 for ANGPT1. NF‐PitNET panels show representative cases. Original magnification: 400×; scale bar: 20 µm. Summary of the IHC results for human NF‐PitNETs. Expression of Tie2 receptor in human normal pituitary ( n = 3) and human NF‐PitNET tissues ( n = 10). Original magnification: 400×; scale bar: 20 µm.

Article Snippet: After incubation the primary antibodies, anti‐Ang‐2 (AF623, R&D) and anti‐Tie‐2 (PA5‐28582, Thermo Scientific) or the combination of anti‐Ang‐2 and anti‐P‐Tie‐2 (PC449, Millipore) was added to each sample and incubated overnight at 4°C.

Techniques: Concentration Assay, Expressing

Journal: EMBO Molecular Medicine

Article Title: Angpt2/Tie2 autostimulatory loop controls tumorigenesis

doi: 10.15252/emmm.202114364

Figure Lengend Snippet: qRT–PCR for Angpt1 and Angpt2 was performed on pituitary samples from tumor‐bearing MENX mutant rats ( n = 10, 12, respectively) and wild‐type control rats ( n = 5, 8). Data are expressed as mean ± SEM. * P = 0.049; ** P = 0.008 by t ‐test. IHC was performed on pituitary tissues from wild‐type ( n = 5) and MENX mutant rats ( n = 10) using antibodies against Angpt1, Angpt2. Representative stainings are shown. Original magnification: 400×; scale bar: 20 µm. N, normal area; T, tumor area. Expression of Angpt2, Tie2, and CD‐31 in rat NF‐PitNETs and associated ECs (used as positive control). Consecutive tissue sections of rat NF‐PitNETs ( n = 4) were stained with the indicated antibodies. One representative tumor is shown. The dashed shape indicates the vessel present in the consecutive slides. Original magnification: 200×; scale bar: 20 µm.

Article Snippet: After incubation the primary antibodies, anti‐Ang‐2 (AF623, R&D) and anti‐Tie‐2 (PA5‐28582, Thermo Scientific) or the combination of anti‐Ang‐2 and anti‐P‐Tie‐2 (PC449, Millipore) was added to each sample and incubated overnight at 4°C.

Techniques: Quantitative RT-PCR, Mutagenesis, Expressing, Positive Control, Staining

Journal: EMBO Molecular Medicine

Article Title: Angpt2/Tie2 autostimulatory loop controls tumorigenesis

doi: 10.15252/emmm.202114364

Figure Lengend Snippet: Expression of Angpt2 and Tie2 was assessed in Att20, GH3, LβT2 and αT3 cells by western blotting (WB) using specific antibodies. α‐Tubulin was included as loading control. Immunofluorescence (IF) of GH3 cells for Angpt2 (red) and Tie2 (green). Nuclei were counterstained with DAPI (blue). Original magnification: 400x; scale bar: 20 µm. Panels shown are representative of 3 independent experiments. Cell proliferation of GH3 cells transfected with si Angpt2 or scrRNA POOLs and incubated with rhANGPT2 (+rhANGPT2) or left untreated (−rhANGPT2) normalized against scrRNA‐transfected cells. *** P < 0.0001 (one‐way ANOVA). In samples parallel to (C), activated caspase‐3/7 was measured to assess for apoptosis. *** P < 0.0001; * P = 0.0411 (one‐way ANOVA). Cell proliferation of GH3 cells transfected with the individual siAngpt2 or scrRNA and incubated with rhANGPT2 (+rhANGPT2) or left untreated (−rhANGPT2) normalized against scrRNA‐transfected, untreated (scr‐rhANGPT2) cells. n.s., not significant; *** P < 0.0001; ** P < 0.002 (one‐way ANOVA). In samples parallel to (E), activated caspase‐3/7 was measured to assess for apoptosis. n.s., not significant; *** P < 0.0001 (one‐way ANOVA). Cell proliferation of GH3‐sh Angpt2 (#2) cells or cells transduced with shCtrl was measured 24h later and normalized against shCtrl. *** P < 0.0001 ( t ‐test). In samples parallel to (G), activated caspase‐3/7 was measured to assess for apoptosis. *** P < 0.0001 ( t ‐test). GH3 cells transfected with si Angpt2 or scrRNA POOLs were incubated with conditioned medium (CM) from rat primary PitNET cells for 24 h. CM was pre‐incubated with AMG386 (red bars), or left untreated (blue bars). Cell proliferation was measured and normalized against that of scrRNA‐transfected cells. n.s., not significant; *** P < 0.0001; ** P = 0.00116; * P = 0.0192 by two‐way ANOVA. Cell viability of isolated rat primary EC cells incubated with CM from isolated rat primary PitNET cells. The experiments was performed independently 2 times, each with 3 technical replicates. Results are expressed as mean ± SEM. * P < 0.0368 ( t ‐test). (C–I) Data are expressed as the mean ± SEM of three biological replicates, each with 3–6 technical replicates. Source data are available online for this figure.

Article Snippet: After incubation the primary antibodies, anti‐Ang‐2 (AF623, R&D) and anti‐Tie‐2 (PA5‐28582, Thermo Scientific) or the combination of anti‐Ang‐2 and anti‐P‐Tie‐2 (PC449, Millipore) was added to each sample and incubated overnight at 4°C.

Techniques: Expressing, Western Blot, Immunofluorescence, Transfection, Incubation, Transduction, Isolation

Journal: EMBO Molecular Medicine

Article Title: Angpt2/Tie2 autostimulatory loop controls tumorigenesis

doi: 10.15252/emmm.202114364

Figure Lengend Snippet: Zebrafish larvae were implanted with red fluorescence‐stained GH3 cells infected with unspecific shRNA (shCtrl) or with sh Angpt2 (#2). In the bottom panels, the red channel was omitted to highlight the tumor‐induced microvascular networks. Digital magnifications of graft regions (white boxes) are shown in the bottom left panels. Scale bar: 100 µm. Quantification of tumor‐induced vessel sprouting in zebrafish embryos engrafted with GH3 ‐shCtrl versus shC2 cells ( n = 20 each). Data were normalized against the mean of the control (shCtrl) group arbitrarily set to 1. A.U. arbitrary Units; * P = 0.0319 ( t ‐test). Example of flow cytometry data of primary rat pituitary cells from a tumor‐bearing MENX rat gated for cell surface Tie2 and CD31 expression (cells were pre‐gated for Tie2). Percentage of Tie2 + CD31 − (PitNET cells) and Tie2 + CD31 + (ECs) in the pituitary glands of 7 age‐matched (8 months) MENX rats. Data are expressed as the mean ± SEM. **** P < 0.0001 ( t ‐test). A representative graph displaying the Tie2 fluorescence intensity and counts of Tie2 + CD31 − and Tie2 + CD31 − cell populations the pituitary of one rat. Fluorescence intensities of the Tie2 + CD31 − and Tie2 + CD31 − cell populations across all 7 pituitary samples. Data are expressed as the mean ± SEM. n.s, not significant (Mann–Whitney test). Source data are available online for this figure.

Article Snippet: After incubation the primary antibodies, anti‐Ang‐2 (AF623, R&D) and anti‐Tie‐2 (PA5‐28582, Thermo Scientific) or the combination of anti‐Ang‐2 and anti‐P‐Tie‐2 (PC449, Millipore) was added to each sample and incubated overnight at 4°C.

Techniques: Fluorescence, Staining, Infection, shRNA, Flow Cytometry, Expressing, MANN-WHITNEY

Journal: EMBO Molecular Medicine

Article Title: Angpt2/Tie2 autostimulatory loop controls tumorigenesis

doi: 10.15252/emmm.202114364

Figure Lengend Snippet: Proximity ligation assay (PLA) was performed on FFPE sections of rat PitNETs ( n = 5) using antibodies against both Angpt2 and Tie2. Nuclei were counterstained with DAPI (blue). Original magnification: 400×; scale bar: 20 µm. Quantification of the interactions between Angpt2 and Tie2 in rat PitNET tissues versus negative controls obtained using only 1 antibody (Appendix Fig C). Box plots show 25 th to 75 th percentiles (box) and 5 th to 95 th percentiles (whiskers). The line in the box represents the median, whereas the “x” represents the mean. PLA was performed as in (A) on isolated rat primary NF‐PitNET stimulated with rhANGPT2. Quantification of Angpt2/Tie2 interactions in primary NF‐PitNET incubated with/without rhANGPT2. The total number of cells counted is reported in parenthesis (i.e., 212 and 345) as well as the number of positive cells (= showing at least one interaction). The graphs show the percentage of cells having the number of interactions reported on the x axis. Box plots show 25 th to 75 th percentiles (box) and 5 th to 95 th percentiles (whiskers). The line in the box represents the median, the “x” represents the mean, and the circle outlier points. Data information: (C, D) Pictures were taken with the same exposure time. Results shown are representative of the stainings results across all samples ( n = 4, n = 3, respectively). Source data are available online for this figure.

Article Snippet: After incubation the primary antibodies, anti‐Ang‐2 (AF623, R&D) and anti‐Tie‐2 (PA5‐28582, Thermo Scientific) or the combination of anti‐Ang‐2 and anti‐P‐Tie‐2 (PC449, Millipore) was added to each sample and incubated overnight at 4°C.

Techniques: Proximity Ligation Assay, Isolation, Incubation

Journal: EMBO Molecular Medicine

Article Title: Angpt2/Tie2 autostimulatory loop controls tumorigenesis

doi: 10.15252/emmm.202114364

Figure Lengend Snippet: Co‐IF for both P‐Tie2 (Tyr 1102/1108; red) and for Na + K + ATPase (green), used as plasma membrane marker, of GH3 cells transduced with unspecific shRNA (shCtrl) and sh Angpt2 (#2) and incubated with rhANGPT2 or left untreated. Original magnification: 400×; scale bar: 20 μm. Quantification of P‐Tie2 immunostaining intensity in cells shown in (A) shCtrl‐transduced GH3 cells (266.71 ± 12.12); in sh Angpt2 cells (176.04 ± 8.46); in control cells treated with rhANGPT2: shCtrl + rhANGPT2: 246.33 ± 9.79 (versus shCtrl, not significant by t ‐test); in treated knockdown cells: sh Angpt2 + rhANGPT2: 218.68 ± 18.16 (versus shC2; * P = 0.0421 by t ‐test). The experiment was performed twice each with three technical replicates. Intensities are expressed as arbitrary units ± SEM. *** P < 0.0001 by t ‐test. Tie2 and P‐Tie2 expression in serum‐starved GH3 cells transfected with siAngpt2 POOLs and stimulated with rhANGPT2 for the indicated times. Tie2 and P‐Tie2 expression in cells as in (C) stimulated with the indicated doses of rhANGPT2 for 30 min. Co‐IF for Angpt2 (red) and P‐Tie2 (green) of a representative rat primary NF‐PitNET. Nuclei were counterstained with DAPI. White arrows point to Angpt2‐positive (P‐Tie2 negative) cells in adjacent non‐tumor area. Scale bars: 50 µm. T, tumor area. Data information: (C, D) Blots shown in all panels are representative of three independent experiments. The numbers represent the ratio phospho/total proteins. Source data are available online for this figure.

Article Snippet: After incubation the primary antibodies, anti‐Ang‐2 (AF623, R&D) and anti‐Tie‐2 (PA5‐28582, Thermo Scientific) or the combination of anti‐Ang‐2 and anti‐P‐Tie‐2 (PC449, Millipore) was added to each sample and incubated overnight at 4°C.

Techniques: Marker, Transduction, shRNA, Incubation, Immunostaining, Expressing, Transfection

Journal: EMBO Molecular Medicine

Article Title: Angpt2/Tie2 autostimulatory loop controls tumorigenesis

doi: 10.15252/emmm.202114364

Figure Lengend Snippet: Pituitary tumor cells secrete mostly Angpt2 in the TME. Tumor‐associated ECs secrete mostly Angpt2 but also Angpt1. Thanks to the expression of Tie2 on their plasma membrane, tumor cells can respond to angiopoietins (secreted by both tumor and endothelial cells) and activate pro‐proliferative/pro‐survival pathways.

Article Snippet: After incubation the primary antibodies, anti‐Ang‐2 (AF623, R&D) and anti‐Tie‐2 (PA5‐28582, Thermo Scientific) or the combination of anti‐Ang‐2 and anti‐P‐Tie‐2 (PC449, Millipore) was added to each sample and incubated overnight at 4°C.

Techniques: Expressing

Journal: PLoS ONE

Article Title: Toll-Like Receptor 2 Induced Angiogenesis and Invasion Is Mediated through the Tie2 Signalling Pathway in Rheumatoid Arthritis

doi: 10.1371/journal.pone.0023540

Figure Lengend Snippet: Human dermal microvascular endothelial cell tubule formation on matrigel matrix following stimulation with Pam3CSK4 (1 ug/ml). (A) Representative image of baseline tube formation (left panel) and tube formation following stimulation with Pam3CSK4 (right panel). (B) Quantitative analysis of the number of connecting branches at baseline and in response to Pam3CSK4. The tube analysis was determined from 5 sequential fields (Magnification×40) focussing on the surface of the matrigel (n = 4). (C–D) The effect of Pam3CSK4 on angiopoietin-2 expression in HMVEC (n = 4) and RA synovial explants (n = 6). Data represented as the mean+/−sem. *p<0.05 significantly different from baseline. (E) Ang2, Tie2 and TLR2 expression in RA synovial tissue sections.

Article Snippet: The sections were incubated with primary antibodies against mouse-monoclonal Ang2 (R&D systems, UK), mouse-monoclonal Tie2 (R&D systems, UK) and mouse monoclonal anti-TLR2 antibody (kind gift from OPSONA therapeutics, Ireland) at room temperature for 1 hour.

Techniques: Expressing

Journal: Neuromolecular Medicine

Article Title: A Short Bout of Exercise Prior to Stroke Improves Functional Outcomes by Enhancing Angiogenesis

doi: 10.1007/s12017-019-08533-x

Figure Lengend Snippet: Immunohistochemical analyses of VEGFR2, Ang-2, vWF and CD34 in the peri-infarct area of the ischemic brain. a Representative merged images counterstained with DAPI (nuclear staining) (blue). Ang-2, VEGFR2, vWF and CD34 antibodies are represented by the green color (arrows). b Relative fluorescence intensity showed that Ang-2, VEGFR2, and CD34 were increased in the exercise groups. After exercise, vWF was decreased. No significant differences were observed between groups ( p > 0.05). Sham: ( n = 4); MCAO: ( n = 4); MCAO-30m: ( n = 4); MCAO-60m: ( n = 4). Bar = 50 µm. Error bars represent S.E.M

Article Snippet: The primary antibodies used for brain tissue were rabbit polyclonal angiopoietin 2 (Ang2; 1:500; Abcam; ab153934), rabbit polyclonal VEGF Receptor 2 (VEGFR2; 1:500; Novus; NB100-530), rabbit polyclonal Von Willebrand Factor (vWF; 1:500; Abcam; ab6994), and rabbit monoclonal CD34 (CD34; 1:500; Abcam; ab81289).

Techniques: Immunohistochemical staining, Staining, Fluorescence

Journal: Neuromolecular Medicine

Article Title: A Short Bout of Exercise Prior to Stroke Improves Functional Outcomes by Enhancing Angiogenesis

doi: 10.1007/s12017-019-08533-x

Figure Lengend Snippet: Western blot analysis of Ang2, VEGFR2, and VEGF in the ipsilateral ischemic striatum. Quantitative analysis showed a significant increase in Ang-2 and VEGFR2 expression in the ipsilateral ischemic striatum of the 30m and 60m exercise groups relative to the non-exercise group. The VEGF expression showed an increasing trend in the exercise group rats. Sham: ( n = 4); MCAO: ( n = 4); MCAO-30m: ( n = 4); MCAO-60m: ( n = 4). Data expressed as mean ± S.E.M. (* p < 0.05) were normalized to GAPDH levels. Error bars represent S.E.M

Article Snippet: The primary antibodies used for brain tissue were rabbit polyclonal angiopoietin 2 (Ang2; 1:500; Abcam; ab153934), rabbit polyclonal VEGF Receptor 2 (VEGFR2; 1:500; Novus; NB100-530), rabbit polyclonal Von Willebrand Factor (vWF; 1:500; Abcam; ab6994), and rabbit monoclonal CD34 (CD34; 1:500; Abcam; ab81289).

Techniques: Western Blot, Expressing

Journal: Biomedicines

Article Title: Differential Impact of Tumor Endothelial Angiopoietin-2 and Podoplanin in Lymphatic Endothelial Cells on HCC Outcomes with Tyrosine Kinase Inhibitor Treatment According to Sex

doi: 10.3390/biomedicines12071424

Figure Lengend Snippet: Comparison of Ang2, podoplanin, and CLEC-2 staining in female and male patients treated with TKIs (50 µm scale bar). ( A ) 63× image of Ang2 staining in tumor and endothelial tissue (red arrows for endothelia) of a female patient. ( B ) 63× image of Ang2 staining in tumor and endothelial tissue (red arrows for endothelia) of a male patient. ( C ) 63× image of podoplanin staining at the level of the lymphatic endothelium in a female patient. ( D ) 63× image of podoplanin staining at the level of the lymphatic endothelium in a male patient. ( E ) 63× image of CLEC-2 staining at the level of tumor tissue in a female patient. ( F ) 63× image of CLEC-2 staining at the level of tumor tissue in a male patient.

Article Snippet: Sections were then incubated with goat anti-Ang2 primary antibody (AF623, R&D Systems, Inc., Minneapolis, MN, USA) at working dilution of 1:50 or mouse anti-podoplanin primary antibody (05463645001, Roche Diagnostics, Monza, MB, Italy), or rabbit anti-CLEC-2 primary antibody (LSB12627, LifeSpan BioSciences, Inc., Shirley, MA, USA) at working dilution of 1:50.

Techniques: Comparison, Staining

Journal: Biomedicines

Article Title: Differential Impact of Tumor Endothelial Angiopoietin-2 and Podoplanin in Lymphatic Endothelial Cells on HCC Outcomes with Tyrosine Kinase Inhibitor Treatment According to Sex

doi: 10.3390/biomedicines12071424

Figure Lengend Snippet: Ang2, podoplanin, and CLEC-2 staining intensity (expressed as optical density (OD)) in HCC patients treated with TKIs stratified according to sex.

Article Snippet: Sections were then incubated with goat anti-Ang2 primary antibody (AF623, R&D Systems, Inc., Minneapolis, MN, USA) at working dilution of 1:50 or mouse anti-podoplanin primary antibody (05463645001, Roche Diagnostics, Monza, MB, Italy), or rabbit anti-CLEC-2 primary antibody (LSB12627, LifeSpan BioSciences, Inc., Shirley, MA, USA) at working dilution of 1:50.

Techniques: Staining

Journal: Biomedicines

Article Title: Differential Impact of Tumor Endothelial Angiopoietin-2 and Podoplanin in Lymphatic Endothelial Cells on HCC Outcomes with Tyrosine Kinase Inhibitor Treatment According to Sex

doi: 10.3390/biomedicines12071424

Figure Lengend Snippet: Receiver operating characteristic (ROC) curve analysis to evaluate the diagnostic power of the LEC podoplanin ( A ) and of endothelial Ang2 ( B ) to predict extra-hepatic spread. The area under the ROC curves (AUCs) was analyzed using the Hanley and McNeil method . The AUC values were 0.839 (95% CI 0.679 to 0.999; p < 0.0001) and 0.821 (95% CI 0.652 to 0.989; p < 0.0001).

Article Snippet: Sections were then incubated with goat anti-Ang2 primary antibody (AF623, R&D Systems, Inc., Minneapolis, MN, USA) at working dilution of 1:50 or mouse anti-podoplanin primary antibody (05463645001, Roche Diagnostics, Monza, MB, Italy), or rabbit anti-CLEC-2 primary antibody (LSB12627, LifeSpan BioSciences, Inc., Shirley, MA, USA) at working dilution of 1:50.

Techniques: Diagnostic Assay

Journal: Biomedicines

Article Title: Differential Impact of Tumor Endothelial Angiopoietin-2 and Podoplanin in Lymphatic Endothelial Cells on HCC Outcomes with Tyrosine Kinase Inhibitor Treatment According to Sex

doi: 10.3390/biomedicines12071424

Figure Lengend Snippet: Univariate and multivariate Cox regression analysis for survival.

Article Snippet: Sections were then incubated with goat anti-Ang2 primary antibody (AF623, R&D Systems, Inc., Minneapolis, MN, USA) at working dilution of 1:50 or mouse anti-podoplanin primary antibody (05463645001, Roche Diagnostics, Monza, MB, Italy), or rabbit anti-CLEC-2 primary antibody (LSB12627, LifeSpan BioSciences, Inc., Shirley, MA, USA) at working dilution of 1:50.

Techniques: