Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Matrix mechanotransduction mediated by thrombospondin-1/integrin/YAP in the vascular remodeling

doi: 10.1073/pnas.1919702117

Figure Lengend Snippet: Comprehensive secretome analysis of smooth muscle cells under cyclic stretch. (A) Volcano plots of protein secretion between the two conditions: 1) stretch vs. static, 2) stretch vs. stretch+BFA, and 3) stretch+BFA vs. static. BFA (1 μM) was used to inhibit secretion. The P values in the unpaired Student’s t test were calculated and plotted against the fold change for all identified proteins. Each dot represents mean values (n = 3). (B) Heat map of secreted proteins induced by cyclic stretch. A list of all of the proteins is provided in SI Appendix, Fig. S2. (C) Functional enrichment analysis of 87 proteins, the negative log10 of the P value. The top enriched GO terms associated with molecular function (orange), biological process (green), and reactome pathway analysis (blue) are shown. (D) Biological network using IPA. Diagrams show the direct (solid lines) and indirect (dashed lines) interactions among proteins reported in blood vessel development (Top) and ECM and cell adhesion (Bottom). (E) Western blot for Thbs1 from CM of rat vascular SMCs in static and stretch (1 Hz, 20% strain, 20 h) with or without BFA (n = 3). The entire image of silver staining is provided in SI Appendix, Fig. S1A.

Article Snippet: Rat vascular SMCs (Lonza, R-ASM-580, isolated from the aorta of 150 to 200 g adult male Sprague-Dawley rats) were grown in Dulbecco's modified Eagle medium (DMEM) with 20% (vol/vol) fetal bovine serum (FBS) and 1× Antibiotic-Antimycotic (ThermoFisher Scientific).

Techniques: Functional Assay, Western Blot, Silver Staining

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Matrix mechanotransduction mediated by thrombospondin-1/integrin/YAP in the vascular remodeling

doi: 10.1073/pnas.1919702117

Figure Lengend Snippet: Thbs1 localizes to FAs and binds integrin αvβ1 under cyclic stretch. (A) Cyclic stretch alters localization of Thbs1 (n = 5). Representative immunostaining with phalloidin (red), Thbs1 (green), and DAPI (blue). (B) Representative image of immunostaining showing Thbs1 colocalization with p-paxillin under cyclic stretch (n = 5). Immunostained with p-paxillin (red), Thbs1 (green), and DAPI (blue). (C) Immunostaining showing Thbs1 localization at FAs in confluent conditions (n = 3). Immunostained with p-paxillin (red), Thbs1 (green), phalloidin (gray), and DAPI (blue). (D) Cartoon shows various binding domains of Thbs1 and its receptors. (E) Representative immunostaining of Thbs1 showing colocalization with integrins αv and β1 (white arrows) but not with integrin β3, CD47, or CD36 under cyclic stretch (n = 5). Immunostained for indicated antibodies (red), Thbs1 (green), and DAPI (blue). (F) Quantification of colocalization of Thbs1 with molecules shown in E using Imaris colocalization software. Bars are mean ± SEM. ***P < 0.001, two-way ANOVA. NS: not significant. (G) Immunoprecipitation (IP) with anti-Thbs1 or control immunoglobulin G (IgG) followed by Western blotting for integrins αv and β1 (n = 2). Coomassie Brilliant Blue (CBB) shows the heavy chain of IgG in IP lysates. IB: immunoblot. (H) PLA shows the clusters of Thbs1/integrin αv or Thbs1/integrin β1 (red dots; white arrowheads). Bottom shows highly magnified images of the white dashed box in Top. DAPI (blue) and phalloidin (green) are shown. In all experiments, rat vascular SMCs were subjected to cyclic stretch (1.0 Hz, 20% strain for 20 h in A, C, and E or 8 h in G and H). In A, C, E, and H, 80 to 100 cells were evaluated in each immunostaining. Two-way arrows indicate stretch directions. (Scale bars, 50 μm.)

Article Snippet: Rat vascular SMCs (Lonza, R-ASM-580, isolated from the aorta of 150 to 200 g adult male Sprague-Dawley rats) were grown in Dulbecco's modified Eagle medium (DMEM) with 20% (vol/vol) fetal bovine serum (FBS) and 1× Antibiotic-Antimycotic (ThermoFisher Scientific).

Techniques: Immunostaining, Binding Assay, Software, Immunoprecipitation, Western Blot

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Matrix mechanotransduction mediated by thrombospondin-1/integrin/YAP in the vascular remodeling

doi: 10.1073/pnas.1919702117

Figure Lengend Snippet: Deletion of Thbs1 affects maturation of focal adhesion and cell stiffness. (A) Wild-type cells with or without 1 μM of BFA or Thbs1KO cells were subjected to cyclic stretch (1.0 Hz, 20% strain, 8 h). Two-way arrows indicate the stretch direction. (Scale bars, 50 μm.) Phalloidin (red) is also shown. (B and C) The orientation of each cell was analyzed by measuring the orientation angle (θ) of the long axis (yellow bars in A) of the ellipse relative to the stretch axis (n = 3). (D) Thbs1KO cells show reduced FA formation. Immunostaining with p-paxillin (red), integrin αv (red), integrin β1 (red), Thbs1 (green), and DAPI (blue) (n = 3). Phalloidin (red) is also shown. White arrows indicate colocalization with Thbs1. Two-way arrows indicate stretch direction. (Scale bars, 50 μm.) (E) Representative immunostaining of CTRL or Thbs1KO cells with vinculin (red) in static or stretch condition. GFP-GRP1-PH (green) and DAPI (blue) are also shown. White arrows show vinculin deposition onto the tip of actin fibers (n = 3). Two-way arrows indicate stretch direction. (Scale bars, 50 μm.) (F) Immunostaining of CTRL or Thbs1KO cells with vinculin (green) and phalloidin (red). Rat vascular SMCs were subjected to cyclic stretch (1.0Hz, 20% strain for 8 h). Two-way arrows indicate stretch direction. (Scale bars, 25 μm.) Arrow heads (purple) show vinculin deposition onto the tip of actin fibers. (G) Young’s modulus of actin fibers in CTRL (n = 129) or Thbs1KO (n = 86) cells measured using atomic force microscopy. Representative topographic images and stiffness maps are shown. (H) Cell height and Young’s modulus are shown. Bars are means ± SEM. ***P < 0.001, unpaired t test. NS: not significant.

Article Snippet: Rat vascular SMCs (Lonza, R-ASM-580, isolated from the aorta of 150 to 200 g adult male Sprague-Dawley rats) were grown in Dulbecco's modified Eagle medium (DMEM) with 20% (vol/vol) fetal bovine serum (FBS) and 1× Antibiotic-Antimycotic (ThermoFisher Scientific).

Techniques: Immunostaining, Microscopy

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Matrix mechanotransduction mediated by thrombospondin-1/integrin/YAP in the vascular remodeling

doi: 10.1073/pnas.1919702117

Figure Lengend Snippet: Thbs1 mediates nuclear shuttling of YAP via integrin αvβ1 in a Rap2-dependent manner. (A) Representative immunostaining of CTRL or Thbs1KO cells in the static or stretch condition with YAP (green). Phalloidin (red) and DAPI (blue) are also shown. Two-way arrows indicate stretch direction. (Scale bars, 50 μm.) Quantification of YAP localization is shown on the Right. In each experiment 150 to 200 cells were evaluated (n = 3). YAP localization in the nucleus (green) or cytoplasm (red) is shown. (B) Thbs1KO cells treated with (+) or without (−) recombinant human THBS1 (rhTHBS1; 1 μg/mL) following 24 h of serum starvation, then subjected to uniaxial cyclic stretch (20% strain; 1 Hz) for 8 h. Representative immunostaining with YAP (green) and DAPI (blue). (Scale bars, 50 μm.) Quantification of YAP localization is shown on the Right. In each experiment 80 to 120 cells were evaluated (n = 3). (C) Western blot shows Thbs1, p-YAP, and p-LATS levels in CTRL and Thbs1KO cells in the static or stretch condition (n = 3). Quantification graphs are shown on the Right. Bars are means ± SEM. **P < 0.01, ***P < 0.001, one-way ANOVA. (D) Pull down of GTP-bound Rap2 from CTRL and Thbs1KO cells in static and stretch conditions using Ral-GDS-RBD agarose beads (n = 2). (E) Overexpression of Myc-Rap2A(WT), constitutively active Myc-Rap2A(G12V), or dominant-negative Myc-Rap2A(S17N) in CTRL and Thbs1KO cells in the static or stretch condition (n = 3). Representative immunostaining with Myc (red), YAP (green), and DAPI (blue). Two-way arrows indicate stretch direction. (Scale bars, 50 μm.) Quantification of YAP localization is shown on the Right. In each experiment 50 to 100 cells were evaluated (n = 3). YAP localization in the nucleus (green) or cytoplasm (red) is shown. (F) Scramble or targeted siRNA-treated rat vascular SMCs were subjected to cyclic stretch (n = 3). Representative immunostaining with YAP (green), phalloidin (red), and DAPI (blue). Two-way arrows indicate stretch direction. (Scale bars, 50 μm.) Quantification of YAP localization is shown on the Right. In each experiment 150 to 200 cells were evaluated (n = 3). qPCR data for confirming the knockdown of Itgαv, Itgβ1, and Itgβ3 are provided in SI Appendix, Fig. S10.

Article Snippet: Rat vascular SMCs (Lonza, R-ASM-580, isolated from the aorta of 150 to 200 g adult male Sprague-Dawley rats) were grown in Dulbecco's modified Eagle medium (DMEM) with 20% (vol/vol) fetal bovine serum (FBS) and 1× Antibiotic-Antimycotic (ThermoFisher Scientific).

Techniques: Immunostaining, Recombinant, Western Blot, Over Expression, Dominant Negative Mutation