American Society of Hematology

Figure 3

$Figure 3. TIMP-2 regulation of VE-cadherin distribution and endothelial cell permeability is mediated by an integrin α3β1-Shp-1-cAMP–dependent pathway. (A) Quiescent hMVECs were treated with Ala + TIMP-2 (100nM) or forskolin (10μM) for the indicated time points. Translocation of VE-cadherin was assessed by Triton X-100 solubility. The Triton X-100 insoluble or soluble fraction was resolved by SDS-PAGE and Western blotted with anti–VE-cadherin antibodies. The integrated band intensities for the VE-cadherin (Triton-insoluble fraction) were normalized to untreated control cells at 30 minutes. Results are representative of 3 independent experiments. (B) Cells were treated with cycloheximide (CHX, 10μM) for 1 hour, followed by Ala + TIMP-2 for 30 minutes. Cell lysates were Western blotted with anti–VE-cadherin and antiactin antibodies. (C) Cells were treated with TIMP-2 or Ala + TIMP-2 for 30 minutes, and colocalization of VE-cadherin and actin was determined as described in “Immunofluorescence microscopy.” Scale bar represents 10 μm. (D) Cells were pretreated with orthovanadate (2μM) or SQ22536 (100μM) for 20 minutes and followed by Ala + TIMP-2 for 30 minutes. Results are representative of 3 independent experiments. (E) Cells were pretreated with orthovanadate or NSC87877 (2μM) for 20 minutes and followed by Ala + TIMP-2 (100nM) before VEGF-A (100 ng/mL) stimulation for 30 minutes, and assessed by confocal microscopy (left panel) and Western blot analysis (right panel). The Triton-insoluble fraction was Western blotted with anti–VE-cadherin antibodies. Anti-VE-cadherin immunoprecipitates (IP) were Western blotted with anti-phosphotyrosine or anti–VE-cadherin antibodies. Results are representative of 3 independent experiments. (F-G) Cells transfected with control, Shp-1, or integrin α3 siRNA were pretreated with Ala + TIMP-2 and followed by VEGF-A for 30 minutes. The Triton-insoluble or soluble fraction was Western blotted with anti-Shp-1, anti-integrin α3, or anti–VE-cadherin antibodies. Integrated density values were normalized to untreated controls. Results are representative of 3 independent experiments. (H) In vitro endothelial cell permeability was performed as described in “Permeability assay.” The results from 3 independent experiments (mean ± SD) are presented as the percentage of maximally induced permeability by VEGF-A in the absence of Ala + TIMP-2. **P < .01, compared with VEGF-A treatment alone.$

TIMP-2 regulation of VE-cadherin distribution and endothelial cell permeability is mediated by an integrin α3β1-Shp-1-cAMP–dependent pathway. (A) Quiescent hMVECs were treated with Ala + TIMP-2 (100nM) or forskolin (10μM) for the indicated time points. Translocation of VE-cadherin was assessed by Triton X-100 solubility. The Triton X-100 insoluble or soluble fraction was resolved by SDS-PAGE and Western blotted with anti–VE-cadherin antibodies. The integrated band intensities for the VE-cadherin (Triton-insoluble fraction) were normalized to untreated control cells at 30 minutes. Results are representative of 3 independent experiments. (B) Cells were treated with cycloheximide (CHX, 10μM) for 1 hour, followed by Ala + TIMP-2 for 30 minutes. Cell lysates were Western blotted with anti–VE-cadherin and antiactin antibodies. (C) Cells were treated with TIMP-2 or Ala + TIMP-2 for 30 minutes, and colocalization of VE-cadherin and actin was determined as described in “Immunofluorescence microscopy.” Scale bar represents 10 μm. (D) Cells were pretreated with orthovanadate (2μM) or SQ22536 (100μM) for 20 minutes and followed by Ala + TIMP-2 for 30 minutes. Results are representative of 3 independent experiments. (E) Cells were pretreated with orthovanadate or NSC87877 (2μM) for 20 minutes and followed by Ala + TIMP-2 (100nM) before VEGF-A (100 ng/mL) stimulation for 30 minutes, and assessed by confocal microscopy (left panel) and Western blot analysis (right panel). The Triton-insoluble fraction was Western blotted with anti–VE-cadherin antibodies. Anti-VE-cadherin immunoprecipitates (IP) were Western blotted with anti-phosphotyrosine or anti–VE-cadherin antibodies. Results are representative of 3 independent experiments. (F-G) Cells transfected with control, Shp-1, or integrin α3 siRNA were pretreated with Ala + TIMP-2 and followed by VEGF-A for 30 minutes. The Triton-insoluble or soluble fraction was Western blotted with anti-Shp-1, anti-integrin α3, or anti–VE-cadherin antibodies. Integrated density values were normalized to untreated controls. Results are representative of 3 independent experiments. (H) In vitro endothelial cell permeability was performed as described in “Permeability assay.” The results from 3 independent experiments (mean ± SD) are presented as the percentage of maximally induced permeability by VEGF-A in the absence of Ala + TIMP-2. **P < .01, compared with VEGF-A treatment alone.

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