Journal: Nature Communications

Article Title: Pericyte-expressed Tie2 controls angiogenesis and vessel maturation

doi: 10.1038/ncomms16106

Figure Lengend Snippet: ( a ) Representative images of control BP (shCtr) and silenced for Tie2 (shTie2 I and shTie2 II) at the beginning ( t =0 h) and end ( t =6.5 h) of time-lapse microscopy during lateral-scratch wound assay. ( b ) Quantification of covered area by BP shTie2 I and shTie2 II in a lateral-scratch wound assay after 6.5 h normalized to BP shCtr. ( c ) Quantification of covered area by BP treated with supernatants of control (Ctr) or Ang2-overexpressing (pAng2) HUVEC in a lateral scratch wound assay after 6.5 h normalized to control. ( d ) Representative images of calpain 1 (CAPN1) staining of BP stimulated with recombinant human (rh) Ang2 for 1 h or left unstimulated (us). Nuclei were counterstained with Hoechst. ( e ) Quantification of calpain 1 activity measured by detection of fluorescent cleaved substrate in BP stimulated for 1 h with rhAng2 normalized to unstimulated (us) control ( n =3). ( f ) Western blot analysis of phosphorylated FOXO3A (pFOXO3A) and total FOXO3A in BP stimulated with rhAng1 or rhAng2 for 30 min. Tubulin served as loading control. ( g ) Representative images showing FOXO3A localization in BP stimulated with rhAng1 or rhAng2 for 30 min. Cells were further stained with actin (cytoskeleton staining) and Hoechst (nucleus staining). ( h ) Human phospho-receptor tyrosine kinase array for EphA2, EphB2 and EphB4 ephrin receptors from control BP (shCtr) and BP silenced for Tie2 (siTie2 I and siTie2 II). Intensity is shown as fold of siCtr. Representative western blot and array images are cropped versions and original images can be found in . Scale bars: 250 μm ( a ), 50 μm ( d , g ). Data are shown as mean±s.d. Statistics were performed using one-way ANOVA ( b ), Mann–Whitney U test ( c ) and Student’s t -test ( e ). * P <0.05, *** P <0.001.

Article Snippet: The membrane was blocked with 3% BSA/PBS for 1 h at RT followed by incubation with the corresponding primary antibody in 1% BSA/PBS-T overnight at 4 °C: pTyr (Millipore, #05-321, 1:500), Tie2 (R&D, #AF313, 1:1,000), Tie2 (R&D, #AF762), pAKT (Cell Signaling, #4060S, 1:1,000), AKT (Cell Signaling, #9272S, 1:1,000), FOXO1 (Cell Signaling, #9454, 1:1,000), pFOXO3A (Cell Signaling, #9466, 1:1,000), FOXO3A (Cell Signaling, #12829, 1:1,000), VEGFR2 (Cell Signaling, #2479, 1:1,000), tubulin (Sigma, #T8203, 1:5,000).

Techniques: Control, Time-lapse Microscopy, Scratch Wound Assay Assay, Staining, Recombinant, Activity Assay, Western Blot, MANN-WHITNEY

Journal: Nature Communications

Article Title: Pericyte-expressed Tie2 controls angiogenesis and vessel maturation

doi: 10.1038/ncomms16106

Figure Lengend Snippet: ( a ) Microarray-based expression screening of brain pericytes (BP), placenta pericytes (PP), pancreas pericytes (PA), lung pericytes (LP), muscle pericytes (MP) and human umbilical vein endothelial cells (HUVEC [HU]). ( b ) Semi-quantitative PCR of TEK ( Tie2 ), endothelial marker genes ( PECAM1 , CDH5 ) and pericyte marker genes ( CSPG4 , CD248 ) in HUVEC and BP; house keeping gene: GAPDH . ( c ) Representative images showing expression of Tie2 in HUVEC and BP. ( d ) Histogram of membrane-bound Tie2 expression in HUVEC and BP compared to isotype control measured by flow cytometry. ( e , g ) Western blot (WB) analysis of tyrosine phosphorylation (pTyr) and total Tie2 after Tie2 immunoprecipitation (IP) in BP ( e ) and HUVEC ( g ) upon stimulation with recombinant human (rh) Ang1 compared to unstimulated (us) cells. ( f , h ) Quantification of the ratio of phosphorylated Tie2 (pTie2) relative to total Tie2 protein in BP ( f ) and HUVEC ( h ) normalized to us control ( n =3). ( i , k ) Western blot analysis of AKT phosphorylation (Ser473) in control (shCtr) and Tie2-silenced (shTie2 I/shTie2 II) BP ( i ) and HUVEC ( k ) upon stimulation with rhAng1 or rhAng2. ( j , l ) Quantification of the ratio of pAKT to total AKT protein expression in BP ( j ) and HUVEC ( l ) normalized to unstimulated control ( n =3). Representative western blot images are cropped versions and original images can be found in . Scale bars: 20 μm ( c ). Data are shown as mean±s.d. Statistics were performed using Mann–Whitney U test (f,h) and one-way ANOVA ( j , l ). * P <0.05, ** P <0.01.

Article Snippet: The membrane was blocked with 3% BSA/PBS for 1 h at RT followed by incubation with the corresponding primary antibody in 1% BSA/PBS-T overnight at 4 °C: pTyr (Millipore, #05-321, 1:500), Tie2 (R&D, #AF313, 1:1,000), Tie2 (R&D, #AF762), pAKT (Cell Signaling, #4060S, 1:1,000), AKT (Cell Signaling, #9272S, 1:1,000), FOXO1 (Cell Signaling, #9454, 1:1,000), pFOXO3A (Cell Signaling, #9466, 1:1,000), FOXO3A (Cell Signaling, #12829, 1:1,000), VEGFR2 (Cell Signaling, #2479, 1:1,000), tubulin (Sigma, #T8203, 1:5,000).

Techniques: Microarray, Expressing, Real-time Polymerase Chain Reaction, Marker, Membrane, Control, Flow Cytometry, Western Blot, Phospho-proteomics, Immunoprecipitation, Recombinant, MANN-WHITNEY

Journal: Nature Communications

Article Title: Pericyte-expressed Tie2 controls angiogenesis and vessel maturation

doi: 10.1038/ncomms16106

Figure Lengend Snippet: ( a ) Representative images of control BP (shCtr) and silenced for Tie2 (shTie2 I and shTie2 II) at the beginning ( t =0 h) and end ( t =6.5 h) of time-lapse microscopy during lateral-scratch wound assay. ( b ) Quantification of covered area by BP shTie2 I and shTie2 II in a lateral-scratch wound assay after 6.5 h normalized to BP shCtr. ( c ) Quantification of covered area by BP treated with supernatants of control (Ctr) or Ang2-overexpressing (pAng2) HUVEC in a lateral scratch wound assay after 6.5 h normalized to control. ( d ) Representative images of calpain 1 (CAPN1) staining of BP stimulated with recombinant human (rh) Ang2 for 1 h or left unstimulated (us). Nuclei were counterstained with Hoechst. ( e ) Quantification of calpain 1 activity measured by detection of fluorescent cleaved substrate in BP stimulated for 1 h with rhAng2 normalized to unstimulated (us) control ( n =3). ( f ) Western blot analysis of phosphorylated FOXO3A (pFOXO3A) and total FOXO3A in BP stimulated with rhAng1 or rhAng2 for 30 min. Tubulin served as loading control. ( g ) Representative images showing FOXO3A localization in BP stimulated with rhAng1 or rhAng2 for 30 min. Cells were further stained with actin (cytoskeleton staining) and Hoechst (nucleus staining). ( h ) Human phospho-receptor tyrosine kinase array for EphA2, EphB2 and EphB4 ephrin receptors from control BP (shCtr) and BP silenced for Tie2 (siTie2 I and siTie2 II). Intensity is shown as fold of siCtr. Representative western blot and array images are cropped versions and original images can be found in . Scale bars: 250 μm ( a ), 50 μm ( d , g ). Data are shown as mean±s.d. Statistics were performed using one-way ANOVA ( b ), Mann–Whitney U test ( c ) and Student’s t -test ( e ). * P <0.05, *** P <0.001.

Article Snippet: The membrane was blocked with 3% BSA/PBS for 1 h at RT followed by incubation with the corresponding primary antibody in 1% BSA/PBS-T overnight at 4 °C: pTyr (Millipore, #05-321, 1:500), Tie2 (R&D, #AF313, 1:1,000), Tie2 (R&D, #AF762), pAKT (Cell Signaling, #4060S, 1:1,000), AKT (Cell Signaling, #9272S, 1:1,000), FOXO1 (Cell Signaling, #9454, 1:1,000), pFOXO3A (Cell Signaling, #9466, 1:1,000), FOXO3A (Cell Signaling, #12829, 1:1,000), VEGFR2 (Cell Signaling, #2479, 1:1,000), tubulin (Sigma, #T8203, 1:5,000).

Techniques: Control, Time-lapse Microscopy, Scratch Wound Assay Assay, Staining, Recombinant, Activity Assay, Western Blot, MANN-WHITNEY

Journal: Nature Communications

Article Title: Pericyte-expressed Tie2 controls angiogenesis and vessel maturation

doi: 10.1038/ncomms16106

Figure Lengend Snippet: ( a ) Microarray-based expression screening of brain pericytes (BP), placenta pericytes (PP), pancreas pericytes (PA), lung pericytes (LP), muscle pericytes (MP) and human umbilical vein endothelial cells (HUVEC [HU]). ( b ) Semi-quantitative PCR of TEK ( Tie2 ), endothelial marker genes ( PECAM1 , CDH5 ) and pericyte marker genes ( CSPG4 , CD248 ) in HUVEC and BP; house keeping gene: GAPDH . ( c ) Representative images showing expression of Tie2 in HUVEC and BP. ( d ) Histogram of membrane-bound Tie2 expression in HUVEC and BP compared to isotype control measured by flow cytometry. ( e , g ) Western blot (WB) analysis of tyrosine phosphorylation (pTyr) and total Tie2 after Tie2 immunoprecipitation (IP) in BP ( e ) and HUVEC ( g ) upon stimulation with recombinant human (rh) Ang1 compared to unstimulated (us) cells. ( f , h ) Quantification of the ratio of phosphorylated Tie2 (pTie2) relative to total Tie2 protein in BP ( f ) and HUVEC ( h ) normalized to us control ( n =3). ( i , k ) Western blot analysis of AKT phosphorylation (Ser473) in control (shCtr) and Tie2-silenced (shTie2 I/shTie2 II) BP ( i ) and HUVEC ( k ) upon stimulation with rhAng1 or rhAng2. ( j , l ) Quantification of the ratio of pAKT to total AKT protein expression in BP ( j ) and HUVEC ( l ) normalized to unstimulated control ( n =3). Representative western blot images are cropped versions and original images can be found in . Scale bars: 20 μm ( c ). Data are shown as mean±s.d. Statistics were performed using Mann–Whitney U test (f,h) and one-way ANOVA ( j , l ). * P <0.05, ** P <0.01.

Article Snippet: The membrane was blocked with 3% BSA/PBS for 1 h at RT followed by incubation with the corresponding primary antibody in 1% BSA/PBS-T overnight at 4 °C: pTyr (Millipore, #05-321, 1:500), Tie2 (R&D, #AF313, 1:1,000), Tie2 (R&D, #AF762), pAKT (Cell Signaling, #4060S, 1:1,000), AKT (Cell Signaling, #9272S, 1:1,000), FOXO1 (Cell Signaling, #9454, 1:1,000), pFOXO3A (Cell Signaling, #9466, 1:1,000), FOXO3A (Cell Signaling, #12829, 1:1,000), VEGFR2 (Cell Signaling, #2479, 1:1,000), tubulin (Sigma, #T8203, 1:5,000).

Techniques: Microarray, Expressing, Real-time Polymerase Chain Reaction, Marker, Membrane, Control, Flow Cytometry, Western Blot, Phospho-proteomics, Immunoprecipitation, Recombinant, MANN-WHITNEY

Journal: bioRxiv

Article Title: Endothelial TIE1 restricts angiogenic sprouting to coordinate vein assembly in synergy with its homologue TIE2

doi: 10.1101/2022.08.05.502976

Figure Lengend Snippet: A-C . RNAseq analysis of the skin tissues of Tie1 null ( Tie1 tm1a/tm1a ) and control mice. The Volcano plot of the differentially regulated genes was shown in A. GSEA analysis revealed that there was a significant decrease in the expression of venous genes but an increase in angiogenesis and hypoxia genes after Tie1 deletion. In contrast, there was no significant difference in the expression of artery genes (B). The subset of the upregulated angiogenesis genes and down-regulated venous genes (P < 0.05) was shown as heatmaps in C. D . Quantitative expression analysis of venous genes including Aplnr and Tek and arterial genes including Efnb2, Notch1 and Dll4 in skin and lung tissues of Tie1 mutant ( Tie1 Δ ICD/ Δ ICD ) and control mice (E15.5; ). E, F . Western blotting analysis and quantification of COUP-TFII and TIE2 protein normalized against beta-actin in lungs (COUP-TFII/beta-actin: Tie1 Δ ICD/ Δ ICD : 0.56 ± 0.13, n = 6; Control: 1.00 ± 0.11, n = 6. TIE2/beta-actin: Tie1 Δ ICD/ Δ ICD : 0.62 ± 0.17, n = 6; Control: 1.00 ± 0.17, n = 6), and skin of Tie1 mutant and control mice (COUP-TFII/beta-actin: Tie1 Δ ICD/ Δ ICD : 0.40 ± 0.13, n = 6; Control: 1.00 ± 0.13, n = 6. TIE2/beta-actin: Tie1 Δ ICD/ Δ ICD : 0.50 ± 0.24, n = 6; Control: 1.00 ± 0.18, n = 6). Quantification of AKT (Ser473) phosphorylation in lung is as follows (pAKT / tAKT, Tie1 Δ ICD/ Δ ICD : 0.70 ± 0.21, n=6; Control: 1.00 ± 0.02, n=6).

Article Snippet: The following primary antibodies were used in this study, including rabbit polyclonal anti-TIE2 (Santa Cruz sc-324), goat polyclonal anti-TIE2 (R&D Systems AF762), goat polyclonal anti-TIE1 (R&D Systems AF619), rabbit polyclonal anti-AKT (Cell Signaling Technology #9272), rabbit monoclonal anti-Phospho-AKT (Ser473, Cell Signaling Technology #4060), mouse monoclonal anti-COUP-TFII (R&D Systems #PP-H7147-00), mouse monoclonal to beta-Actin (Santa Cruz sc-47778).

Techniques: Control, Expressing, Mutagenesis, Western Blot, Phospho-proteomics

Journal: bioRxiv

Article Title: Endothelial TIE1 restricts angiogenic sprouting to coordinate vein assembly in synergy with its homologue TIE2

doi: 10.1101/2022.08.05.502976

Figure Lengend Snippet: A-C . Western blot analysis of COUP-TFII protein in HUVECs after siRNA mediated Tie1 knockdown. Two small interfering (siRNAs) targeting Tie1 were used in this study, including S14141 and S14142. Quantification of TIE1, TIE2 and COUP-TFII protein level was as follows. For S14141: COUP-TFII was decreased to 61.44 ± 27.32 % and TIE2 to 71.63 ± 25.52 % of the control respectively, when TIE1 expression was reduced to 15.11 ± 6.85 % of control (normalized by beta-actin; values from five independent experiments, n=15). For S14142: COUP-TFII was decreased to 53.92 ± 20.29 % and TIE2 to 72.39 ± 24.06 % of the control respectively, when TIE1 expression was reduced to 20.30 ± 6.12 % of control (normalized by beta-actin; values from three independent experiments, n=9). D . Tamoxifen intragastric (i.g.) administration and the analysis scheme. E, F . Analysis of blood vessels in the retinas of Tie1ICD ECKO/- , Tie1ICD ECKO/- ;Tek +/- and control mice between P7 and P21. Arrows point to haemangioma-like vascular tufts (E). Quantification of the vascularization index (F; ratio of vascularized area to total retina area normalized against the littermate controls at P7; Control: 100.00 ± 5.06, n=10; Tie1ICD ECKO/- : 80.00 ± 7.03, n=6; Tie1ICD ECKO/- ; Tek +/- : 55.92 ± 5.42, n=6; Control versus Tie1ICD ECKO/- : P =0.0001; Control versus Tie1ICD ECKO/- ;Tek +/- : P =0.0001; Tie1ICD ECKO/- versus Tie1ICD ECKO/- ;Tek +/- : P =0.0022). G-H . Visualization of retinal blood vessels of Tie1ICD ECKO/- ;Tek +/- mice by immunostaining for PECAM-1 at P9, P11, P15 and P21. Arrows point to veins and arrowheads to arteries (G). Analysis of the three layers of retinal blood vessels in Tie1ICD ECKO/- , Tie1ICD ECKO/- ;Tek +/- and control mice at P21 (H). Note that the vein-associated vascular defects were more severe in Tie1ICD ECKO/- ;Tek +/- mice compared with the Tie1 deletion alone. I . Schematic model shows that TIE1 and TIE2 act in a synergistic manner to regulate vein development via COUP-TFII. Scale bar: 200 μm in G; 50 μm in H.

Article Snippet: The following primary antibodies were used in this study, including rabbit polyclonal anti-TIE2 (Santa Cruz sc-324), goat polyclonal anti-TIE2 (R&D Systems AF762), goat polyclonal anti-TIE1 (R&D Systems AF619), rabbit polyclonal anti-AKT (Cell Signaling Technology #9272), rabbit monoclonal anti-Phospho-AKT (Ser473, Cell Signaling Technology #4060), mouse monoclonal anti-COUP-TFII (R&D Systems #PP-H7147-00), mouse monoclonal to beta-Actin (Santa Cruz sc-47778).

Techniques: Western Blot, Knockdown, Control, Expressing, Immunostaining

Journal: Investigative Ophthalmology & Visual Science

Article Title: A Small Molecule Inhibitor of VE-PTP Activates Tie2 in Schlemm's Canal Increasing Outflow Facility and Reducing Intraocular Pressure

doi: 10.1167/iovs.61.14.12

Figure Lengend Snippet: Expression of VE-PTP and activation of Tie2 by AKB-9778 in SC endothelial cells. ( A ) Adult murine eyes of either wildtype (VE-PTP +/+ ) or heterozygous knock-in mice expressing β-Galactosidase under the VE-PTP promotor (VE-PTP +/mut ) were cross-sectioned after X-Gal staining to visualize VE-PTP expression. ( B ) Sagittal sections of adult murine eyes were stained with antibodies specific for VE-PTP ( green ) or VE-cadherin ( red ). ( C ) Top panel shows labelling of conventional outflow tissues from human donor eyes restricted to the endothelial cells of Schlemm's canal (SC) using antibodies specific for VE-PTP ( violet ). Bottom panel shows selective staining of SC with antibodies against the vascular endothelial cell marker, CD31 ( red ). Sections underwent counterstaining with DAPI for cell nuclei localization. TM = trabecular meshwork, magnification bars = 50 µm. ( D ) Whole mount immunofluorescence analysis of adult murine eyes of VE-PTP +/+ or VE-PTP +/mut mice. Enucleated eyes were stained as whole mounts with X-gal or with antibodies to detect PECAM-1, Prox-1, and Tie2. The entire thickness of the limbus was visualized by confocal imaging and subsets of optical sections containing SC are shown. ( E ) Whole mount immunofluorescence staining of adult murine eyes topically treated with either AKB-9778 or vehicle for 1 hour and stained for PECAM-1, pY992-Tie2, and total Tie2. The entire thickness of the limbus was visualized by confocal imaging and subsets of optical sections depicting SC are shown. Scale bar = 20 µm; SC = Schlemm's canal; AV = aqueous vein; CB = ciliary body; BV = blood vessel; TM = trabecular meshwork.

Article Snippet: The following primary antibodies were used for immunofluorescence staining at the indicated concentrations: VE-PTP (rat-anti-mouse-VE-PTP clone 109.1, 10 µg/mL), (rabbit-anti-human VE-PTP, hVE-PTP 1-8 antibodies were generated in rabbits against a recombinant form of the extracellular fibronectin type III-like domains 1-8 of human VE-PTP and affinity purified using the antigen), VE-cadherin (rabbit-a-mouse-VE-cadherin, pAB42) Prox1 (goat-a-human-Prox1, R&D, AF-2727, 2 µg/mL); PECAM-1 (rat-a-mouse PECAM-1 clone 1 G5.1 (3 µg/mL) + clone 5D2.6 (1 µg/mL), (rabbit polyclonal anti-PECAM-1, Abcam, ab28364); Tie2 (goat-a-mouse-Tie2, R&D, AF762, 5 µg/mL); and phospho-Tie2 (rabbit-a-pY992-Tie2, R&D, AF2720, 10 µg/mL).

Techniques: Expressing, Activation Assay, Knock-In, Staining, Marker, Immunofluorescence, Imaging