Journal: bioRxiv

Article Title: Cell-cultured PDMS vascular model to allow placement of implant devices

doi: 10.1101/2025.01.20.634010

Figure Lengend Snippet: ( a ) Reflection and scattering of fluorescence on the stent surface with no cells in the vascular model. ( b , c ) Endothelial cell dynamics in the stented vascular models exposed to SS. Representative images of fluorescent-stained ECs in the model after stent placement and exposure to SS of 2.3 Pa for ( b ) 24 h and ( c ) 48 h. These images show the results of staining F-actin with a long-wavelength fluorescent label (594 nm) and VE-cadherin with a short-wavelength one (488 nm). Scale bars, 500 µm (wide view) and 100 µm (magnified view).

Article Snippet: In this study, we used the mouse monoclonal anti-VE-cadherin antibody (sc-9989, Santa Cruz Biotechnology, Dallas, TX, USA) as the primary antibody and Alexa Fluor 594 conjugated goat anti-mouse IgG antibody (A-11005, Thermo Fisher Scientific) as the secondary antibody.

Techniques: Fluorescence, Staining

Journal: Journal of Cell Science

Article Title: Shear stress-stimulated AMPK couples endothelial cell mechanics, metabolism and vasodilation

doi: 10.1242/jcs.262232

Figure Lengend Snippet: VE-cadherin is required for shear-induced AMPK activation. (A,B) Shear stimulated AMPK activation in the presence of VE-cadherin function-blocking antibodies. HUVECs were left resting (−) or exposed to shear stress (+) for the indicated times in the presence or absence of BV9, a VE-cadherin function-blocking antibody, or DMSO as a control. AMPK activation was examined by immunoblotting total cell lysates with an antibody that recognizes AMPK phosphorylated in its activation loop (pAMPK) or actin as a loading control. The blots were stripped and probed with antibodies that report on total AMPK (AMPK). The graph in B depicts the ratios of phosphorylated AMPK to total AMPK; the data are mean±s.e.m., n =3 biologically independent samples. (C,D) VE-cadherin inhibition. VE-cadherin expression was inhibited by treating HUVECs with lentiviruses encoding shRNAs targeting human VE-cadherin (shVE-Cadherin) or a scrambled sequence (shScramble). Wild-type parental cells were employed as a control. Total cell lysates were immunoblotted with antibodies against VE-cadherin to show the level of inhibition or actin as a loading control. Representative western blot images are shown C. The graphs in D depict the level of VE-cadherin normalized to total protein levels. The data are mean±s.e.m of an n =6 biologically independent samples. (E,F) Shear stress-induced AMPK activation in cells with depleted VE-cadherin levels. AMPK activation was examined as described above in A and B in wild-type, shScramble, or shVE-cadherin cells after 5 min of applied shear stress. The data are mean±s.e.m of an n =3 biologically independent samples. * P <0.05; ** P <0.01; *** P <0.001; ns, not significant [two-way ANOVA with Tukey's comparison (B,D); one-way ANOVA with Dunnett's test (F)].

Article Snippet: Primary antibodies used for immunoblotting were: monoclonal Thr-172 phospho-AMPK at 1:1000 (Cell Signaling; 40H9), monoclonal AMPK-α at 1:1000 (Cell Signaling; 2532), monoclonal Ser-1177 phospho-eNOS at 1:500 (Cell Signaling; 9571S), monoclonal eNOS at 1:500 (Cell Signaling; D9A5L), monoclonal VE-cadherin at 1:1000 (Cell Signaling; D87F2), monoclonal β-actin at 1:1000 (Cell Signaling; 8H10D10), monoclonal β-catenin at 1:1000 (BD Transduction; 610153) and monoclonal LKB1 at 1:1000 (Cell Signaling; 27D10; or Abcam EPR19379 for coimmunoprecipitation studies).

Techniques: Shear, Activation Assay, Blocking Assay, Control, Western Blot, Inhibition, Expressing, Sequencing, Comparison

Journal: Journal of Cell Science

Article Title: Shear stress-stimulated AMPK couples endothelial cell mechanics, metabolism and vasodilation

doi: 10.1242/jcs.262232

Figure Lengend Snippet: LKB1 is the upstream kinase responsible for shear-induced AMPK activation. (A,B) LKB1 inhibition using RNA interference. LKB1 expression was inhibited by treating HUVECs with lentiviruses encoding shRNAs targeting human LKB1 (shLKB1), or a scramble sequence (shScramble). Wild-type parental cells were employed as a control. Total cell lysates were immunoblotted with antibodies against LKB1 to show the level of inhibition or actin as a loading control. Representative western blots are shown in A. (B) The graph depicts the LKB1 levels normalized to the total protein levels; the data are mean±s.e.m., n =4 biologically independent samples. (C,D) Shear stress stimulated AMPK activation in cells depleted of LKB1. AMPK activity was monitored as described in the legend of Fig. 1A . In C, representative immunoblots are shown for cells under resting conditions (−) or exposed to 5 min of shear (+). The graph in D depicts the ratio of phosphorylated AMPK to total protein. The data are mean±s.e.m., n =3 biologically independent samples. (E,F) Shear-induced LKB1 coimmunoprecipitation with VE-cadherin. Cells were preincubated with DMSO or BV9 for 1 h and then were left resting (−) or exposed to shear stress (+) for 5 min. VE-cadherin was immunoprecipitated (IP), and the co-precipitation of LKB1 was examined by immunoblotting. The ratio of LKB1 recovered to VE-Cadherin immunoprecipitated is plotted in the graph (F). Data are mean±s.e.m., n =4 biologically independent samples. TCL, total cell lysates (5%). * P <0.05; ** P <0.01; **** P <0.0001; ns, not significant [one-way ANOVA with Dunnett's test (B,F); two-way ANOVA with Tukey's multiple comparison test (D)].

Article Snippet: Primary antibodies used for immunoblotting were: monoclonal Thr-172 phospho-AMPK at 1:1000 (Cell Signaling; 40H9), monoclonal AMPK-α at 1:1000 (Cell Signaling; 2532), monoclonal Ser-1177 phospho-eNOS at 1:500 (Cell Signaling; 9571S), monoclonal eNOS at 1:500 (Cell Signaling; D9A5L), monoclonal VE-cadherin at 1:1000 (Cell Signaling; D87F2), monoclonal β-actin at 1:1000 (Cell Signaling; 8H10D10), monoclonal β-catenin at 1:1000 (BD Transduction; 610153) and monoclonal LKB1 at 1:1000 (Cell Signaling; 27D10; or Abcam EPR19379 for coimmunoprecipitation studies).

Techniques: Shear, Activation Assay, Inhibition, Expressing, Sequencing, Control, Western Blot, Activity Assay, Immunoprecipitation, Comparison

Journal: Journal of Cell Science

Article Title: Shear stress-stimulated AMPK couples endothelial cell mechanics, metabolism and vasodilation

doi: 10.1242/jcs.262232

Figure Lengend Snippet: VE-cadherin stimulated AMPK is required for eNOS membrane localization and activation. (A–D) eNOS activation in the presence of (A,B) a VE-cadherin inhibitor or (C,D) shRNAs targeting signaling molecules downstream of VE-cadherin [i.e LKB1 (shLKB1), AMPKα1 (shAMPKα1) or a scramble sequence (shScramble)]. HUVECs were left untreated (DMSO) or treated with BV9 and left resting (−) or exposed to 12 dyne/cm 2 orbital shear (+) for the indicated times in minutes. The cells were lysed and probed with an eNOS Ser1177 phospho-specific antibody (peNOS) or actin as a loading control. The blots were stripped and reprobed with antibodies that recognize total eNOS. Representative immunoblots are shown in A and C. The graphs in B and D depict the ratio of phosphorylated protein to total protein; the data are mean±s.e.m., n =3 biologically independent samples. (E–G) eNOS localization when VE-cadherin stimulated AMPK is disrupted. Wild-type HUVECs or those expressing shRNAs against VE-cadherin (shVE-Cadherin), LKB1 (shLKB1) or AMPKα1 (shAMPKα1) were left resting (no shear) or exposed to shear for 30 min. The cells were fixed and stained with antibodies against eNOS (green) or VE-cadherin (magenta) and examined by confocal microscopy. Representative images are shown in E. Scale bar: 20 μm. The graphs beneath the images represent the average corrected fluorescence intensity of VE-cadherin (F) or eNOS (G). The data are mean±s.d., n =3 biologically independent samples, of which two or three fields of view (FOV) were analyzed with >100 junctions per FOV. * P <0.05; *** P <0.001; **** P <0.0001; ns, not significant (two-way ANOVA with Tukey's multiple comparison test).

Article Snippet: Primary antibodies used for immunoblotting were: monoclonal Thr-172 phospho-AMPK at 1:1000 (Cell Signaling; 40H9), monoclonal AMPK-α at 1:1000 (Cell Signaling; 2532), monoclonal Ser-1177 phospho-eNOS at 1:500 (Cell Signaling; 9571S), monoclonal eNOS at 1:500 (Cell Signaling; D9A5L), monoclonal VE-cadherin at 1:1000 (Cell Signaling; D87F2), monoclonal β-actin at 1:1000 (Cell Signaling; 8H10D10), monoclonal β-catenin at 1:1000 (BD Transduction; 610153) and monoclonal LKB1 at 1:1000 (Cell Signaling; 27D10; or Abcam EPR19379 for coimmunoprecipitation studies).

Techniques: Membrane, Activation Assay, Sequencing, Shear, Control, Western Blot, Expressing, Staining, Confocal Microscopy, Fluorescence, Comparison

Journal: Journal of Cell Science

Article Title: Shear stress-stimulated AMPK couples endothelial cell mechanics, metabolism and vasodilation

doi: 10.1242/jcs.262232

Figure Lengend Snippet: LKB1 and AMPKα1 are required for shear-induced junctional reinforcement. (A–C) Effect of AMPK inhibition of actin cytoskeletal reinforcement. Wild-type HUVECs or those expressing shRNAs against LKB1 (shLKB1), AMPKα1 (shAMPKα1) or a scrambled sequence (shScramble) were left resting (no shear) or exposed to shear for 30 min. The cells were fixed and stained with an antibody against VE-cadherin or phalloidin conjugated to Alexa Fluor 594 to visualize F-actin. Cells were examined by confocal microscopy. Representative images are shown in A. Scale bar: 20 μm. The graphs beneath the images represent the mean corrected fluorescence intensity of actin (B) or VE-cadherin (C) at the cellular junctions. The data are mean±s.d., n =3 biologically independent samples, of which two or three fields of view (FOV) were analyzed with >100 junctions per FOV. * P <0.05; ** P <0.01; *** P <0.001; **** P <0.0001; ns, not significant (two-way ANOVA with Tukey's multiple comparison test).

Article Snippet: Primary antibodies used for immunoblotting were: monoclonal Thr-172 phospho-AMPK at 1:1000 (Cell Signaling; 40H9), monoclonal AMPK-α at 1:1000 (Cell Signaling; 2532), monoclonal Ser-1177 phospho-eNOS at 1:500 (Cell Signaling; 9571S), monoclonal eNOS at 1:500 (Cell Signaling; D9A5L), monoclonal VE-cadherin at 1:1000 (Cell Signaling; D87F2), monoclonal β-actin at 1:1000 (Cell Signaling; 8H10D10), monoclonal β-catenin at 1:1000 (BD Transduction; 610153) and monoclonal LKB1 at 1:1000 (Cell Signaling; 27D10; or Abcam EPR19379 for coimmunoprecipitation studies).

Techniques: Shear, Inhibition, Expressing, Sequencing, Staining, Confocal Microscopy, Fluorescence, Comparison

Journal: Journal of Cell Science

Article Title: Shear stress-stimulated AMPK couples endothelial cell mechanics, metabolism and vasodilation

doi: 10.1242/jcs.262232

Figure Lengend Snippet: Increased metabolism fuels endothelial reinforcement and eNOS localization to cell–cell junctions. (A) Illustration of glycolytic inhibitors and their targets. (B) Mechanical or metabolic disruption and its effects on shear stress-stimulated glucose uptake. HUVECs were incubated in glucose free medium and were pretreated with DMSO or inhibitors against VE-cadherin (BV9), myosin II (blebbistatin), GLUT1 (WZB117) or phosphofructokinase fructose-bisphosphate 3 (3PO). The non-hydrolyzable glucose analog 2-NBDG was added to cells, and the cells were left resting or shear stress was applied for 1 h. Fluorometric analyses of the cell lysates were performed, and the amount of 2-NBDG uptake was quantified as the mean±s.e.m., n =3 biologically independent samples. (C) The effect of metabolic inhibitors on reinforcement of the actin cytoskeleton and enrichment of VE-cadherin and eNOS. HUVECs pretreated with DMSO or inhibitors against myosin II (blebbistatin), GLUT1 (WZB117) or phosphofructokinase fructose-bisphosphate 3 (3PO). Cells were then left under static (no shear) or exposed to shear stress (shear) conditions for 30 min. Cells were fixed and stained with Alexa Fluor 594–phalloidin (red) and antibodies against VE-cadherin (yellow) and eNOS (green). Representative images are shown in C. Scale bar: 20 μm. The graphs represent the average corrected fluorescence intensity of VE-cadherin (D), actin (E) and eNOS (F) in 100 junctions per condition. The data are mean±s.d., n =3 biologically independent samples, of which two or three fields of view (FOV) were analyzed with >100 junctions per FOV. ** P <0.01; *** P <0.001; **** P <0.0001; ns, not significant [two-way ANOVA with Tukey's multiple comparison test (B); two-way ANOVA with Dunnett's multiple comparison test (D–F)].

Article Snippet: Primary antibodies used for immunoblotting were: monoclonal Thr-172 phospho-AMPK at 1:1000 (Cell Signaling; 40H9), monoclonal AMPK-α at 1:1000 (Cell Signaling; 2532), monoclonal Ser-1177 phospho-eNOS at 1:500 (Cell Signaling; 9571S), monoclonal eNOS at 1:500 (Cell Signaling; D9A5L), monoclonal VE-cadherin at 1:1000 (Cell Signaling; D87F2), monoclonal β-actin at 1:1000 (Cell Signaling; 8H10D10), monoclonal β-catenin at 1:1000 (BD Transduction; 610153) and monoclonal LKB1 at 1:1000 (Cell Signaling; 27D10; or Abcam EPR19379 for coimmunoprecipitation studies).

Techniques: Disruption, Shear, Incubation, Staining, Fluorescence, Comparison

Journal: Journal of Cell Science

Article Title: Shear stress-stimulated AMPK couples endothelial cell mechanics, metabolism and vasodilation

doi: 10.1242/jcs.262232

Figure Lengend Snippet: Increased VE-cadherin mediated signaling to AMPK and metabolism support endothelial alignment. The effects of metabolic inhibitors (A–C) or shRNAs against components of the signal transduction cascade initiated by VE-cadherin (D–F) on endothelial alignment. HUVECs pretreated with DMSO or inhibitors against VE-cadherin (BV9), myosin II (blebbistatin), GLUT1 (WZB117) or phosphofructokinase fructose-bisphosphate 3 (3PO) (A–C) or HUVECs expressing shRNAs against VE-cadherin (shVE-cadherin), LKB1 (shLKB1) or a scramble sequence (shScramble) were then left resting (no shear) or exposed to shear stress (shear) for 48 h. For cells treated with inhibitors, new medium containing a fresh supply of inhibitors was added after 24 h. Cells were fixed and stained with Alexa Fluor 594–phalloidin and DAPI to visualize actin and the nucleus, respectively. Representative images are shown in A and D. Scale bars: 40 μm. Alignment was assessed based on whether the major axis of the cell and its Feret angle was within ±45° of the direction of flow and is quantified in B and E. Elongation was assessed by comparing the ratio of the length of the major axis (L major ) with the width of the minor axis (L minor ) and is quantified in C and F. >250 cells across three fields of view (FOV) per condition were assessed for alignment and 100 cells across three FOV per condition were assessed for elongation. The average percentage alignment and elongation is plotted as the mean ±s.e.m., n =3–5 biologically independent experiments. * P <0.05; ** P <0.01; *** P <0.001; **** P <0.0001 (one-way ANOVA with Dunnett's multiple comparison test).

Article Snippet: Primary antibodies used for immunoblotting were: monoclonal Thr-172 phospho-AMPK at 1:1000 (Cell Signaling; 40H9), monoclonal AMPK-α at 1:1000 (Cell Signaling; 2532), monoclonal Ser-1177 phospho-eNOS at 1:500 (Cell Signaling; 9571S), monoclonal eNOS at 1:500 (Cell Signaling; D9A5L), monoclonal VE-cadherin at 1:1000 (Cell Signaling; D87F2), monoclonal β-actin at 1:1000 (Cell Signaling; 8H10D10), monoclonal β-catenin at 1:1000 (BD Transduction; 610153) and monoclonal LKB1 at 1:1000 (Cell Signaling; 27D10; or Abcam EPR19379 for coimmunoprecipitation studies).

Techniques: Transduction, Expressing, Sequencing, Shear, Staining, Comparison