VEGF-induced vascular permeability is mediated by FAK

Abstract

Endothelial cells (ECs) form cell-cell adhesive junctional structures maintaining vascular integrity. This barrier is dynamically regulated by vascular endothelial growth factor (VEGF) receptor signaling. We created an inducible knockin mouse model to study the contribution of the integrin-associated focal adhesion tyrosine kinase (FAK) signaling on vascular function. Here we show that genetic or pharmacological FAK inhibition in ECs prevents VEGF-stimulated permeability downstream of VEGF receptor or Src tyrosine kinase activation in vivo. VEGF promotes tension-independent FAK activation, rapid FAK localization to cell-cell junctions, binding of the FAK FERM domain to the vascular endothelial cadherin (VE-cadherin) cytoplasmic tail, and direct FAK phosphorylation of β-catenin at tyrosine-142 (Y142) facilitating VE-cadherin-β-catenin dissociation and EC junctional breakdown. Kinase inhibited FAK is in a closed conformation that prevents VE-cadherin association and limits VEGF-stimulated β-catenin Y142 phosphorylation. Our studies establish a role for FAK as an essential signaling switch within ECs regulating adherens junction dynamics.

Figure 1. FAK activity within ECs is required for VEGF-initiated vascular permeability (VP) in mice

(A) Progeny from FAK^fl/fl SCL-Cre-ER(T) and heterozygous FAK^KD/WT crosses are treated with tamoxifen to induce Cre expression to create mice with hemizygous FAK-WT or FAK-KD expression in ECs. (B) Tamoxifen treatment of Cre-ER(T)-positive FAK^fl/WT and FAK^fl/KD mice results in reduced FAK pY397 and FAK pY576 phosphorylation but no changes in Pyk2 expression or Pyk2 pY402 phosphorylation in FAK^fl/KD compared to FAK^fl/WT mice by immunoblotting of heart lysates. (C) VEGF-stimulated dermal VP was measured in Cre-ER(T)-positive FAK^fl/WT and FAK^fl/KD mice with or without tamoxifen pre-treatment. Leakage of circulating Evans blue dye was measured after 30 min. Shown are mean values +/− SEM (***, p<0.001). (D) Representative images of dermal dye leakage. VEGF or PBS injection sites are circled. Scale is 0.5 cm. (E) Quantification of VP. Data is plotted as a VEGF/PBS ratio of individual mice from two independent experiments. Box-whisker plots show the distribution of the data: black square, mean; bottom line, 25th percentile; middle line, median; top line, 75th percentile; and whiskers, 5th and 95th percentiles (**, p<0.01). See also Figure S1.

Figure 2. FAK-KD prevents VEGF-stimulated FAK activation, FAK association with VE-cadherin, and β-catenin Y142 phosphorylation in vivo

(A–F) Hearts of tamoxifen-treated FAK^fl/WT (FAK-WT) or FAK^fl/KD (FAK-KD) mice were analyzed 2 min after VEGF (0.2 mg/kg) or PBS tail vein injections. (A) VEGF-R2 immunoprecipitation (IP) shows equal phosphotyrosine (pY) content after VEGF administration. Reduced FAK Y397 and Y576 phosphorylation after VEGF stimulation of FAK-KD compared to FAK-WT mice. (B) Heart sections were analyzed by combined staining for ECs (CD31, green) and activated FAK (pY576, red). Shown are merged images with FAK activation occurring within ECs (yellow) upon VEGF stimulation of FAK-WT but not FAK-KD mice. Scale is 20 μm. (C) Mean correlation of pixel intensities of CD31 to pY576 FAK staining from ten full frame images of experimental groups in panel B (+/− SEM, ***, p<0.001). (D) Increased VE-cadherin/FAK association after VEGF stimulation in FAK-WT but not FAK-KD heart lysates. (E) A VE-cadherin/β-catenin complex is maintained in heart lysates upon VEGF stimulation of FAK-KD but not FAK-WT mice by IP analyses. (F) FAK-KD blocks basal and VEGF-stimulated β-catenin Y142 but not basal Y654 phosphorylation in heart lysates by anti-pY IP analyses. See also Figure S2.

Figure 3. Pharmacological FAK inhibition prevents VEGF-initiated VP and FAK modulation of VE-cadherin-β-catenin complex formation and phosphorylation in vivo

(A) Quantification of dermal VP after VEGF (400 ng) or PBS injection (2 sites each per mouse) with or without pre-treatment with FAK inhibitor (PF271, 30 mg/kg). Evan’s Blue dye leakage is plotted as a VEGF/PBS ratio. Box-whisker plots show the distribution of the data: black square, mean; bottom line, 25th percentile; middle line, median; top line, 75th percentile; and whiskers, 5th and 95th percentiles (*, p<0.05). (B) Representative images of dye leakage. VEGF or PBS injection sites are circled. Scale is 0.5 cm. (C–E) Hearts of vehicle or FAK inhibitor (FAK-I) pre-treated mice were analyzed 2 min after VEGF (0.2 mg/kg) or PBS tail vein injections by IP and immunoblotting. (C) FAK-I blocks FAK Y397 phosphorylation but not FAK expression. (D) VEGF-stimulated FAK/VE-cadherin association and VE-cadherin/β-catenin dissociation is prevented by FAK-I administration as determined by co-IP analyses. (E) FAK-I treated mice exhibit loss of β-catenin Y142 phosphorylation by anti-pY IP analyses. Increased basal but prevention of VEGF-stimulated β-catenin Y654 phosphorylation by FAK-I. See also Figure S3.

Figure 4. VEGF-stimulated paracellular permeability and adherens junction regulation are prevented by pharmacological FAK-I addition to human ECs

(A) Time course (5 to 60 min) of VEGF-increased human pulmonary artery endothelial cells (HPAEC) permeability to HRP-conjugated IgG. Inhibition of basal (*, p<0.05) and VEGF-stimulated (***, p<0.001) permeability by 1 μM PF271 (FAK-I) addition. Data is the mean +/− SD of twelve experimental points from two independent experiments. (B) Significant inhibition of VEGF-stimulated change in HPAEC cell index (cell monolayer electrical resistance) at 90 min by 1 μM FAK-I addition (p<0.0001) as measured by the Roche xCELLigence system. Values are normalized to DMSO controls and are means +/− SD from four independent Xcelligence chambers. (C) FAK-I blocks FAK (pY397) but not Src (pY416) or VEGF-R2 tyrosine phosphorylation after VEGF addition (50 ng/ml, 5 min) of human umbilical vein endothelial cells (HUVEC) lysates. (D) VEGF (50 ng/ml, 60 min) addition promotes cellular gaps (arrows) in a HUVEC monolayer that is prevented by FAK-I (1 μM) addition. Shown is anti-VE-cadherin (green) and actin (phalloidin, red) staining. Scale is 10 μm. (E) FAK/VE-cadherin association, β-catenin Y142 phosphorylation, but not β-catenin Y654 phosphorylation is blocked by FAK-I addition to HUVECs. (F) FAK-I prevents VEGF-stimulated VE-cadherin/β-catenin complex dissociation and increased paxillin phosphorylation by IP and immunoblotting analyses. (G) VEGF-stimulated FAK/VE-cadherin binding and increased FAK Y397 but not paxillin Y31 tyrosine phosphorylation occurs in the presence of the non-muscle myosin IIA inhibitor blebbistatin (20 μM). See also Figure S4.

Figure 5. Genetic inhibition of FAK prevents VEGF-stimulated permeability independent of Src

(A) FAK-KD ECs establish a barrier to FITC-dextran but lack a permeability response to VEGF. Values are means +/− SD from one of two independent experiments (*** p<0.001). (B) No differences in VEGF-R2, Pyk2 Y402, and Src Y416 tyrosine phosphorylation upon VEGF (50 ng/ml, 15 min) addition to FAK-WT and FAK-KD ECs by IP and immunoblotting analyses. FAK-WT but not FAK-KD phosphorylation at Y397 after VEGF addition. (C) FAK-KD mutation prevents FAK/VE-cadherin association, VE-cadherin/β-catenin dissociation, and β-catenin Y142 phosphorylation after VEGF stimulation. (D) FAK-I prevents VEGF-stimulated FAK/VE-cadherin association by co-IP analyses. (E) Mutation of β-catenin Y142 prevents VE-cadherin dissociation after VEGF stimulation. β-catenin constructs were transfected into HUVECs and VE-cadherin association determined by co-IP analyses. (F) Percent of VE-cadherin association with His-tagged β-catenin as determined by densitometry. Values are means +/−SD from two samples (p<0.05). See also Figure S5.

Figure 6. VEGF-stimulated recruitment of FAK to EC adherens junctions

(A) Staining for endogenous FAK (green) reveals nuclear, cytoplasmic, and focal adhesion distribution within starved HUVECs without overlap with VE-cadherin (red) at cell-cell junctions. Scale is 10 μm. (B) VEGF (50 ng/ml, 5 min) promotes FAK (green) and VE-cadherin (red) co-localization in HUVECs at cell-cell junctions (yellow, merge). Scale is 10 μm. Fluorescence intensity profiles of the indicated boxed region were obtained using ImageJ (v1.43). (C) Live-cell confocal microscopy (medial focal plane) reveals rapid VEGF-stimulated GFP-FAK accumulation (arrows) at HUVEC cell-cell junctions. Image montage (−30 to 120 seconds) before and after VEGF (50 ng/ml) addition. Scale is 10 μm.

Figure 7. FAK FERM mutations release conformational restraint in FAK and allow for direct FAK FERM binding to VE-cadherin cytoplasmic domain

(A) FAK FERM F2 lobe mutations (Y180A, M183A) promote VEGF-independent association of both FAK-WT and FAK-KD with VE-cadherin. The indicated GFP-FAK constructs were transfected in HUVECs and associations determined by co-IP analyses in starved or VEGF-stimulated cells. FAK FERM Y180/183A mutations do not promote phosphorylation of FAK-KD at Y397. (B) FAK FERM binds directly to VE-cadherin. In vitro translated GFP, FAK-WT, FAK-KD, GFP-FAK FERM, and FAK C-terminal domain were used in a direct binding assay with glutathione-S-transferase (GST) or GST-fusions of the VE-cadherin cytoplasmic domain (621-784). Streptavidin-HRP analyses show the amount of FAK bound (left) or 10% of input (right). GFP and GFP-FAK FERM contain a tandem affinity probe tag. (C) GFP-FAK FERM (1-402) associates with endogenous VE-cadherin. Co-IP analyses were performed using antibodies to GFP or VE-cadherin in adenovirus (Ad) infected HUVECs. (D) FAK FERM localizes to adherens junctions and to the nucleus in HUVECs. Cells were analyzed for VE-cadherin (red) and GFP-FAK-FERM (green) and the merged image is shown. Inset, enlarged area (boxed) shows co-localization at cell-cell junctions (yellow) in the merged image. TO-PRO-3 iodide (642/661) was used as a nuclear marker. Scale is 10 μm.