Conditional deletion of FAK in mice endothelium disrupts lung vascular barrier function due to destabilization of RhoA and Rac1 activities

Abstract

Loss of lung-fluid homeostasis is the hallmark of acute lung injury (ALI). Association of catenins and actin cytoskeleton with vascular endothelial (VE)-cadherin is generally considered the main mechanism for stabilizing adherens junctions (AJs), thereby preventing disruption of lung vascular barrier function. The present study identifies endothelial focal adhesion kinase (FAK), a nonreceptor tyrosine kinase that canonically regulates focal adhesion turnover, as a novel AJ-stabilizing mechanism. In wild-type mice, induction of ALI by intraperitoneal administration of lipopolysaccharide or cecal ligation and puncture markedly decreased FAK expression in lungs. Using a mouse model in which FAK was conditionally deleted only in endothelial cells (ECs), we show that loss of EC-FAK mimicked key features of ALI (diffuse lung hemorrhage, increased transvascular albumin influx, edema, and neutrophil accumulation in the lung). EC-FAK deletion disrupted AJs due to impairment of the fine balance between the activities of RhoA and Rac1 GTPases. Deletion of EC-FAK facilitated RhoA's interaction with p115-RhoA guanine exchange factor, leading to activation of RhoA. Activated RhoA antagonized Rac1 activity, destabilizing AJs. Inhibition of Rho kinase, a downstream effector of RhoA, reinstated normal endothelial barrier function in FAK-/- ECs and lung vascular integrity in EC-FAK-/- mice. Our findings demonstrate that EC-FAK plays an essential role in maintaining AJs and thereby lung vascular barrier function by establishing the normal balance between RhoA and Rac1 activities.

Keywords: acute lung injury; adherens junctions; endothelial barrier; focal adhesion kinase.

Fig. 1.

Focal adhesion kinase (FAK) protein expression is decreased during acute lung injury in mice. Lung lysates from wild-type mice exposed to vehicle or lipopolysaccharide (LPS, 30 mg/kg, 6 h) (A) or subjected to cecal ligation (control) or cecal ligation and puncture (CLP, 22 h) (B) were immunoblotted with anti-FAK or anti-β-actin (loading control) antibodies (Abs). Bar graphs show the densitometric analysis of FAK expression from 4 lungs/group (A and B); *statistically significant difference from vehicle or control lungs (P < 0.05).

Fig. 2.

Inducible deletion of endothelial FAK. A: schematic of tamoxifen-induced deletion of FAK in CRE-expressing FAK^fl/fl mice. Four-week-old FAK^fl/fl (control)- and FAK^fl/fl-expressing Cre mice were injected with 2 mg tamoxifen ip for 5 consecutive days, followed by a rest period of 5 days. Mice were used for experiments on the 11th day. B: genotype showing tamoxifen deletion of FAK. Tails from FAK^fl/fl or endothelial cell (EC)-FAK^−/− mice were digested, and FAK and Cre expression was determined using specific primers. Cre induces FAK deletion in Cre-expressing EC-FAK^−/− mice that is absent in FAK^fl/fl mice. C–E: assessment of endothelial FAK expression. Lungs harvested from FAK^fl/fl or EC-FAK^−/− mice were homogenized, and FAK mRNA and protein expression was quantified using FAK primers and FAK antibody (C). The bar graph shows mean ± SD of FAK expression normalized against the housekeeping gene GAPDH (for RNA) or β-actin (for protein). *Statistically significant difference from FAK^fl/fl lungs (P < 0.05, n = 4–5). D: FAK^fl/fl and EC-FAK^−/− lung sections were immunostained with anti-FAK (red) and anti-CD31 (green) Abs, followed by appropriate Alexa-fluor-conjugated secondary Abs to assess FAK expression in CD31⁺ endothelial cells. Scale bar, 20 μM. Bar graph shows %CD31⁺ endothelial cells that express FAK in EC-FAK null and FAK^fl/fl lungs from 6–7 individual vessels from multiple lung sections (E). F: lung sections from FAK^fl/fl and EC-FAK^−/− mice were either coimmunostained with anti-FAK (green) and anti-E-cadherin (red) or anti-FAK and anti-α-smooth muscle actin (α-SMA; red) Abs to confirm FAK expression in epithelial and smooth muscle cells, respectively. Arrows show similar FAK colocalization with E-cadherin or α-SMA in FAK^fl/fl and EC-FAK^−/− mice lungs. Scale bars, 10 μM. Images represent 3–4 mice/group. G: macrophages were isolated from bronchoalveloar lavage (BAL) of FAK^fl/fl and EC-FAK^−/− mice after which RNA was extracted. FAK expression was quantified using suitable primers as described in materials and methods. GAPDH was used as an internal control. Plot represents the mean ± SD from 4 mice. H: lung endothelial cells (LECs) isolated from FAK^fl/fl mice infected with β-gal (control, FAK^+/+) or Cre adenovirus (FAK^−/−) were lysed to assess FAK expression using actin as a loading control. Blot is representative of five individual isolations.

Fig. 3.

Endothelial FAK deletion disrupts lung vascular barrier function. A: loss of EC-FAK leads to diffuse lung hemorrhage. Top, representative images of buffer-perfused lungs harvested from EC-FAK^−/− and FAK^fl/fl mice. Bottom, mean ± SD of heme concentration from four FAK^fl/fl and EC-FAK^−/− mouse lungs. *Values significantly different from FAK^fl/fl lungs (P < 0.05). B and C: EC-FAK deletion increases lung microvascular permeability. Transendothelial albumin influx into lung parenchyma (B) and lung wet-to-dry weight ratio (C) were determined as described in materials and methods to quantify lung vascular protein permeability and lung edema formation, respectively. *Significant difference from FAK^fl/fl lungs (P < 0.05; n = 7–8 mice/group). D and E: tamoxifen does not affect lung vascular permeability or lung edema formation in wild-type (WT), CRE, or FAK^fl/fl mice. Tamoxifen was injected into WT, CRE, or FAK^fl/fl mice following the protocol described in Fig. 1A. Mice were killed at day 11 to determine lung wet-to-dry weight ratios (D) or transendothelial albumin influx into lung parenchyma (E). Data represent means ± SD from three groups of four lungs. F and G: EC-FAK deletion induces lung leukocyte infiltration and activation. Representative images of hematoxylin and eosin (H & E)-stained lung sections from multiple FAK^fl/fl and EC-FAK^−/− mice (F). Scale bars, 20 μm. G: top, plot shows mean ± SD leukocyte infiltration in the lungs. *Significant difference from FAK^fl/fl lungs (P < 0.05, n = 4 mice/group). Bottom, lungs from FAK^fl/fl or EC-FAK^−/− were harvested, and myeloperoxidase (MPO) activity was quantified as indicated in materials and methods. *Significant difference from FAK^fl/fl lungs (P < 0.05, n = 4 mice/group). H: endothelial FAK deletion does not alter mRNA expression of related kinases. RNA was extracted from FAK^fl/fl and EC-FAK^−/− mouse lungs. The expression of cSrc, Fyn, Pyk2, and FAK was analyzed by RT-PCR using specific primers. GAPDH was used as a loading control. Results represent data from three individual lungs. I–K: FAK deletion disrupts adherens junctions. FAK^+/+ and FAK^−/− LECs were immunostained with anti-vascular endothelial (VE)-cadherin (green) antibody or rhodamine phallodin (red) as described in materials and methods (I). Immunoblot from FAK^+/+ and FAK^−/− lysates was probed with indicated Abs (J). *Significant difference between FAK^+/+ vs. FAK^−/− LECs (n = 3; P < 0.05). K: plot shows mean ± SD of interendothelial gap area in FAK^+/+ and FAK^−/− monolayers. *Significant difference from FAK^+/+ monolayers (P < 0.05, n = 5).

Fig. 3.

Endothelial FAK deletion disrupts lung vascular barrier function. A: loss of EC-FAK leads to diffuse lung hemorrhage. Top, representative images of buffer-perfused lungs harvested from EC-FAK^−/− and FAK^fl/fl mice. Bottom, mean ± SD of heme concentration from four FAK^fl/fl and EC-FAK^−/− mouse lungs. *Values significantly different from FAK^fl/fl lungs (P < 0.05). B and C: EC-FAK deletion increases lung microvascular permeability. Transendothelial albumin influx into lung parenchyma (B) and lung wet-to-dry weight ratio (C) were determined as described in materials and methods to quantify lung vascular protein permeability and lung edema formation, respectively. *Significant difference from FAK^fl/fl lungs (P < 0.05; n = 7–8 mice/group). D and E: tamoxifen does not affect lung vascular permeability or lung edema formation in wild-type (WT), CRE, or FAK^fl/fl mice. Tamoxifen was injected into WT, CRE, or FAK^fl/fl mice following the protocol described in Fig. 1A. Mice were killed at day 11 to determine lung wet-to-dry weight ratios (D) or transendothelial albumin influx into lung parenchyma (E). Data represent means ± SD from three groups of four lungs. F and G: EC-FAK deletion induces lung leukocyte infiltration and activation. Representative images of hematoxylin and eosin (H & E)-stained lung sections from multiple FAK^fl/fl and EC-FAK^−/− mice (F). Scale bars, 20 μm. G: top, plot shows mean ± SD leukocyte infiltration in the lungs. *Significant difference from FAK^fl/fl lungs (P < 0.05, n = 4 mice/group). Bottom, lungs from FAK^fl/fl or EC-FAK^−/− were harvested, and myeloperoxidase (MPO) activity was quantified as indicated in materials and methods. *Significant difference from FAK^fl/fl lungs (P < 0.05, n = 4 mice/group). H: endothelial FAK deletion does not alter mRNA expression of related kinases. RNA was extracted from FAK^fl/fl and EC-FAK^−/− mouse lungs. The expression of cSrc, Fyn, Pyk2, and FAK was analyzed by RT-PCR using specific primers. GAPDH was used as a loading control. Results represent data from three individual lungs. I–K: FAK deletion disrupts adherens junctions. FAK^+/+ and FAK^−/− LECs were immunostained with anti-vascular endothelial (VE)-cadherin (green) antibody or rhodamine phallodin (red) as described in materials and methods (I). Immunoblot from FAK^+/+ and FAK^−/− lysates was probed with indicated Abs (J). *Significant difference between FAK^+/+ vs. FAK^−/− LECs (n = 3; P < 0.05). K: plot shows mean ± SD of interendothelial gap area in FAK^+/+ and FAK^−/− monolayers. *Significant difference from FAK^+/+ monolayers (P < 0.05, n = 5).

Fig. 4.

FAK deletion impairs normal balance between RhoA and Rac1 activities. A and B: effect of FAK deletion on RhoA and Rac1 activities. FAK^−/− and FAK^+/+ LECs were lysed, and the activity of RhoA or Rac1 was determined using rhotekin or PAK pull down beads (A). Data represent means ± SD of GTPase activities compared with those in FAK^+/+ LECs. *Significant difference from FAK^+/+ LECs (P < 0.05; n = 3). Human pulmonary artery endothelial cells (HPAECs) transfected with scrambled (siSc) or FAK (siFAK) small-interfering RNA (siRNA) were lysed to determine RhoA and Rac1 activities (B). In parallel, lysates were immunoblotted with anti-FAK and anti-actin Abs to assess FAK depletion in ECs using actin as a loading control (inset). Blot is representative of findings from three individual experiments. *Significant difference from siSc-transfected HPAECs (P < 0.05; n = 3). C: RhoA inhibits Rac1 activity. Scrambled and FAK siRNA-expressing HPAECs were incubated with 10 μM Y-27632 to inhibit Rho kinase (ROCK), a downstream effector of RhoA. After 30 min, cells were lysed, and Rac1 activity was determined to assess whether inhibition of ROCK restores normal Rac1 activity. *Significant difference from siSc-transfected HPAECs (P < 0.05; n = 3). In bar graph, Rac1 activity in Y-27632-pretreated siSc- or siFAK-transfected cells is plotted relative to their respective vehicle-treated values to better discern the level of restoration seen in siFAK cells after inhibiting ROCK. *Significant difference from siSc-transfected HPAECs or vehicle-treated siFAK cells (P < 0.05; n = 3). D: FAK depletion promotes RhoA interaction with p115RhoGEF. Lysates from scrambled and FAK siRNA-expressing HPAECs were immunoprecipitated with either control IgG or anti-RhoA antibody followed by immunoblotting with anti-p115RhoGEF and anti-RhoA Abs. The blot shown is representative of data from multiple experiments. *Significant difference from siSc-transfected HPAECs (P < 0.05; n = 4).

Fig. 5.

Blockade of RhoA reinstates lung vascular permeability. A: restoration of FAK expression in EC-FAK^−/− mice rescues lung vascular permeability. FAK^fl/fl or EC-FAK^−/− mice expressing indicated constructs were assessed for lung edema formation by determining lung wet-to-dry weight ratios. Graph represents means ± SD. *Significance from GFP or GFP-FAK transducing FAK^fl/fl lungs or GFP-FAK transducing EC-FAK^−/− lungs (P < 0.05, n = 4). Inset shows representative immunoblots of free GFP or GFP-FAK fusion protein found in lung lysates from EC-FAK^−/− mice with the aid of an anti-GFP antibody. A representative immunoblot from 4 mouse lungs/group is shown. All experiments were performed 48 h after injection of liposome-encapsulated mutants. B and C: inhibition of RhoA signaling rescues endothelial barrier function in FAK-depleted ECs and lungs. B: FAK^+/+ or FAK^−/− LEC monolayers were pretreated with vehicle or 10 μM ROCK inhibitor Y-27632 to inhibit ROCK. After 30 min, transendothelial albumin influx was determined using Evans blue-conjugated albumin. Experiments were performed in duplicate at least three times. *Significant difference from FAK^+/+ LECs (P < 0.05). C: FAK^fl/fl or EC-FAK^−/− mice were retro-orbitally injected with either vehicle or ROCK inhibitor Y-27632 (10 mg/kg). Mice were killed after 30 min, and lung wet-to-dry weight ratio was calculated. *Significance from PBS- or Y-27632-exposed FAK^fl/fl lungs or EC-FAK^−/− lungs receiving Y-27632 (P < 0.05; n = 3–4 mice/group). D: model of endothelial FAK regulation of lung vascular permeability described in text.