Inhibition of AMP-Activated Protein Kinase Accentuates Lipopolysaccharide-Induced Lung Endothelial Barrier Dysfunction and Lung Injury in Vivo

The aim of this study was to determine the role of AMP-activated protein kinase (AMPK) in lipopolysaccharide (LPS)-induced lung endothelial barrier dysfunction and lung injury in vivo. Both cultured human pulmonary artery endothelial cells (HPAECs) and experimental animals AMPK subunit α–deficient mice and wild-type (WT) control mice (C57BL/6J) were used. In cultured HPAECs, LPS increased endothelial permeability in parallel with a decrease in AMPK activity. Consistent with this observation, AMPK activation with the potent AMPK activator 5-aminoimidazole-4-carboxamide-1-d-ribofuranoside (AICAR) attenuated LPS-induced endothelial hyperpermeability in vitro. Intratracheal administration of LPS (1 mg/kg) in WT mice reduced AMPK phosphorylation at Thr172 in lung tissue extracts, increased protein content and cell count in bronchial alveolar lavage fluid, and increased Evans Blue dye infiltration into the lung. These same attributes were similarly enhanced in AMPKα-knockout mice, compared with WT mice. Pretreatment with AICAR reduced these lung injury indicators in LPS-treated WT mice. AMPK activation with AICAR attenuated LPS-induced endothelial hyperpermeability by activating the Rac/Cdc42/PAK pathway, with concomitant inhibition of the Rho pathway, and decreased VE-cadherin phosphorylation at Tyr658. We conclude that AMPK activity supports normal endothelial barrier function and that LPS exposure inhibits AMPK, thereby contributing to endothelial barrier dysfunction and lung injury. The aim of this study was to determine the role of AMP-activated protein kinase (AMPK) in lipopolysaccharide (LPS)-induced lung endothelial barrier dysfunction and lung injury in vivo. Both cultured human pulmonary artery endothelial cells (HPAECs) and experimental animals AMPK subunit α–deficient mice and wild-type (WT) control mice (C57BL/6J) were used. In cultured HPAECs, LPS increased endothelial permeability in parallel with a decrease in AMPK activity. Consistent with this observation, AMPK activation with the potent AMPK activator 5-aminoimidazole-4-carboxamide-1-d-ribofuranoside (AICAR) attenuated LPS-induced endothelial hyperpermeability in vitro. Intratracheal administration of LPS (1 mg/kg) in WT mice reduced AMPK phosphorylation at Thr172 in lung tissue extracts, increased protein content and cell count in bronchial alveolar lavage fluid, and increased Evans Blue dye infiltration into the lung. These same attributes were similarly enhanced in AMPKα-knockout mice, compared with WT mice. Pretreatment with AICAR reduced these lung injury indicators in LPS-treated WT mice. AMPK activation with AICAR attenuated LPS-induced endothelial hyperpermeability by activating the Rac/Cdc42/PAK pathway, with concomitant inhibition of the Rho pathway, and decreased VE-cadherin phosphorylation at Tyr658. We conclude that AMPK activity supports normal endothelial barrier function and that LPS exposure inhibits AMPK, thereby contributing to endothelial barrier dysfunction and lung injury. Vascular endothelial permeability plays a pivotal role in regulating many physiological and pathological processes, including angiogenesis, immunity, and inflammation.1Cowan C.E. Kohler E.E. Dugan T.A. Mirza M.K. Malik A.B. Wary K.K. Kruppel-like factor-4 transcriptionally regulates VE-cadherin expression and endothelial barrier function.Circ Res. 2010; 107: 959-966Crossref PubMed Scopus (85) Google Scholar In lungs, endothelial cells form a semipermeable barrier between the vessel lumen and underlying alveoli, thereby mediating the transmigration of blood cells and maintaining fluid homeostasis. The integrity of the endothelial cell (EC) monolayer therefore directly determines lung vascular permeability. For example, EC barrier dysfunction can lead to an increase in permeation of fluid and macromolecules into the interstitium and alveolar space, resulting in pulmonary edema, a major characteristic of acute lung injury. RhoA, Rac1, and Cdc42 are key members of the Rho GTPase family and are activated on binding GTP at the membrane.2Vogel S.W.M. Farhat K. Zieseniss A. Schnelle M. Le-Huu S. von Ahlen M. Malz C. Camenisch G. Katschinski D.M. Prolyl hydroxylase domain (PHD) 2 affects cell migration and F-actin formation via RhoA/rho-associated kinase-dependent cofilin phosphorylation.J Biol Chem. 2010; 285: 33756-33763Crossref PubMed Scopus (32) Google Scholar These proteins are intimately involved in regulating cell adhesion and cytoskeletal dynamics, both of which play an important role in endothelial barrier function.3Spindler V. Schlegel N. Waschke J. Role of GTPases in control of microvascular permeability.Cardiovasc Res. 2010; 87: 243-253Crossref PubMed Scopus (273) Google Scholar, 4Popoff M.R. Geny B. Multifaceted role of Rho, Rac, Cdc42 and Ras in intercellular junctions, lessons from toxins.Biochim Biophys Acta. 2009; 1788: 797-812Crossref PubMed Scopus (103) Google Scholar, 5Watanabe T. Sato K. Kaibuchi K. Cadherin-mediated intercellular adhesion and signaling cascades involving small GTPases.Cold Spring Harb Perspect Biol. 2009; 1: a003020Crossref Scopus (62) Google Scholar For example, Rac1 and Cdc42 are important in maintaining, stabilizing, and restoring the endothelial barrier.3Spindler V. Schlegel N. Waschke J. Role of GTPases in control of microvascular permeability.Cardiovasc Res. 2010; 87: 243-253Crossref PubMed Scopus (273) Google Scholar More specifically, Baumer et al6Baumer Y. Spindler V. Werthmann R.C. Bünemann M. Waschke J. Role of Rac 1 and cAMP in endothelial barrier stabilization and thrombin-induced barrier breakdown.J Cell Physiol. 2009; 220: 716-726Crossref PubMed Scopus (87) Google Scholar demonstrated that Rac1 is involved in mitigating endothelial hyperpermeability by a subset of agonists, including thrombin and lipopolysaccharide (LPS). In addition, activation of the Rho GTPases Cdc42 and Rac1 restores endothelium integrity after lung injury.7Zhao Y.D. Ohkawara H. Rehman J. Wary K.K. Vogel S.M. Minshall R.D. Zhao Y.Y. Malik A.B. Bone marrow progenitor cells induce endothelial adherens junction integrity by sphingosine-1-phosphate-mediated Rac1 and Cdc42 signaling.Circ Res. 2009; 105 (8 p following 704): 696-704Crossref PubMed Scopus (45) Google Scholar Although the contributions of Rho GTPases in maintaining endothelial barrier function are well established, how Rho GTPase is regulated in endothelial cells is largely unknown. AMP-activated protein kinase (AMPK) is a heterotrimeric serine/threonine kinase; a catalytic α subunit and regulatory β and γ subunits are important in maintaining the stability of the complex. AMPK belongs to a family of energy-sensing enzymes that function as fuel gauges, monitoring changes in the energy status of the cell.8Wu Y. Song P. Xu J. Zhang M. Zou M.H. Activation of protein phosphatase 2A by palmitate inhibits AMP-activated protein kinase.J Biol Chem. 2007; 282: 9777-9788Crossref PubMed Scopus (228) Google Scholar AMPK is activated in response to a variety of stressors that increase the intracellular ratio of AMP to ATP. On activation, AMPK phosphorylates a number of downstream targets, thereby affecting glucose metabolism, fatty acid oxidation, hepatic lipogenesis, and cholesterol synthesis.9Xie Z. Dong Y. Zhang M. Cui M.Z. Cohen R.A. Riek U. Neumann D. Schlattner U. Zou M.H. Activation of protein kinase C zeta by peroxynitrite regulates LKB1-dependent AMP-activated protein kinase in cultured endothelial cells.J Biol Chem. 2006; 281: 6366-6375Crossref PubMed Scopus (163) Google Scholar In addition to its regulatory role in metabolism, recent studies have demonstrated a role for AMPK in maintaining normal endothelial function.10Choi H.C. Song P. Xie Z. Wu Y. Xu J. Zhang M. Dong Y. Wang S. Lau K. Zou M.H. Reactive nitrogen species is required for the activation of the AMP-activated protein kinase by statin in vivo.J Biol Chem. 2008; 283: 20186-20197Crossref PubMed Scopus (84) Google Scholar For example, AMPK subunit α2 exerts protective effects against atherosclerosis by inhibiting the endoplasmic reticulum stress response in ECs.11Dong Y. Zhang M. Liang B. Xie Z. Zhao Z. Asfa S. Choi H.C. Zou M.H. Reduction of AMP-activated protein kinase alpha2 increases endoplasmic reticulum stress and atherosclerosis in vivo.Circulation. 2010; 121: 792-803Crossref PubMed Scopus (225) Google Scholar Thus, agents that enhance EC barrier function are of potential therapeutic value in a variety of pathological settings, including inflammatory disease, atherosclerosis, and tumor angiogenesis. The effects of AMPK on endothelial barrier function and vascular permeability have not been investigated previously. Thus, the aim of the present study was to investigate whether AMPK protects lung endothelial barrier function and mitigates acute lung injury in response to LPS. 5-Aminoimidazole-4-carboxamide-1-d-ribofuranoside (AICAR; also known as acadesine) was purchased from Toronto Chemicals (Toronto, ON, Canada). The Rac1 inhibitor NSC 23766 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies against Thr172-phosphorylated AMPKα, AMPKα, Ser307-phosphorylated LKB1, Ser79-phosphorylated acetyl coenzyme A carboxylase (ACC), vascular endothelial cadherin (VE-cadherin), and diphosphorylated myosin light chain (pp-MLC Thr18/Ser19) were purchased from Cell Signaling Technology (Danvers, MA). Antibody against LKB1 was purchased from Santa Cruz Biotechnology. Tyr658-phosphorylated VE-cadherin was purchased from Calbiochem (San Diego, CA). Texas Red phalloidin–conjugated and Alexa Fluor 488–conjugated secondary antibodies were purchased from Invitrogen (Carlsbad, CA). Bacterial LPS (Escherichia coli O55:B5) was purchased from Sigma-Aldrich (St. Louis, MO). Unless otherwise specified, all biochemical reagents were purchased from Sigma-Aldrich. Knockout (KO) mice lacking AMPK subunit α1 (α1-KO)12Jørgensen S.B. Viollet B. Andreelli F. Frøsig C. Birk J.B. Schjerling P. Vaulont S. Richter E.A. Wojtaszewski J.F. Knockout of the alpha2 but not alpha1 5’-AMP-activated protein kinase isoform abolishes 5-aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside but not contraction-induced glucose uptake in skeletal muscle.J Biol Chem. 2004; 279: 1070-1079Crossref PubMed Scopus (468) Google Scholar or AMPK subunit α2 (α2-KO)13Viollet B. Andreelli F. Jørgensen S.B. Perrin C. Geloen A. Flamez D. Mu J. Lenzner C. Baud O. Bennoun M. Gomas E. Nicolas G. Wojtaszewski J.F. Kahn A. Carling D. Schuit F.C. Birnbaum M.J. Richter E.A. Burcelin R. Vaulont S. The AMP-activated protein kinase alpha2 catalytic subunit controls whole-body insulin sensitivity.J Clin Invest. 2003; 111: 91-98Crossref PubMed Scopus (443) Google Scholar and their wild-type (WT) genetic controls were used. The two subunits are encoded by Prkaa1 and Prkaa2 (synonyms: Ampka1 and Ampka2). The mice were housed in temperature-controlled cages under a 12-hour light–dark cycle and were given free access to water and normal chow. Mice aged 8 to 10 weeks were used for subsequent experiments. A solution of LPS (1 mg/kg) in PBS was delivered intratracheally; PBS alone was used as a control. After 16 hours, bronchoalveolar lavage was performed by intratracheal injection of 1 mL of PBS solution, followed by gentle aspiration. The recovered fluids were processed for determination of total protein concentration and white blood cell counts. Lungs from challenged mice were collected for histological evaluation by H 299: L652-L663Crossref PubMed Scopus (56) Google Scholar In brief, Evans Blue dye (30 mL/kg) was injected into the external jugular vein 2 hours before sacrifice. At the end of the experiment, a thoracotomy was performed and the lungs were perfused with PBS containing 5 mmol/L EDTA, to remove blood. Both left and right lobes of the lung were excised. Evans Blue accumulation in the lung tissue was measured by spectrofluorometric analysis of lung tissue lysates according to a protocol described previously.15Birukov G.K. Cokic I. Fu P. Junjie X. Birukova A.A. Protective effects of iloprost in cellular and animal models of ventilator-induced lung injury (abstract).Virchows Archiv. 2009; 455: S84Google Scholar Human pulmonary artery endothelial cells (HPAECs) and n bovine pulmonary artery endothelial cells (BPAECs) were purchased from Cell Applications (San Diego, CA). For subsequent experiments, cell lines were used at passages 5 to 9. Cell lysates were immunoprecipitated with PAK-1 PBD (p21/Cdc42/Rac1-activated kinase p21-binding domain). Rac1/Cdc42 activation was evaluated using a Rac1/Cdc42 activation assay kit (Millipore, Billerica, MA) according to the manufacturer’s instructions. BPAEC monolayers were in solution for 10 followed by with After with cell monolayers were with antibodies in solution bovine in for 1 followed by staining with Alexa Fluor 488–conjugated secondary were with Texas for 1 of these were performed at After the were using (Santa Cruz Santa Cruz, and using a AMPK was immunoprecipitated from of protein using an against AMPK activity in was by the of into the as described Z. Dong Y. Zhang M. Cui M.Z. Cohen R.A. Riek U. Neumann D. Schlattner U. Zou M.H. Activation of protein kinase C zeta by peroxynitrite regulates LKB1-dependent AMP-activated protein kinase in cultured endothelial cells.J Biol Chem. 2006; 281: 6366-6375Crossref PubMed Scopus (163) Google Scholar, M.H. S. F. M.H. Cohen R.A. Activation of 5’-AMP-activated kinase is and activity of bovine endothelial Role of Biol Chem. 2003; PubMed Scopus Google Scholar was performed as described M. Dong Y. Xu J. Xie Z. Wu Y. Song P. M. Wu J. Zou M.H. the AMP-activated protein kinase in vascular cells via Res. 2008; PubMed Scopus Google Scholar of was performed according to the Santa Cruz Biotechnology the were in to a 10 in were with in containing For in was with of in After of at the were to the cells in mL The cells were with this for hours at After the was with normal and cells were cultured for AMPK was as described Xie Z. Viollet B. Zou M.H. Activation of the AMP-activated kinase by in by the of protein and endothelial 2006; PubMed Scopus Google Scholar of was by the into cells were with as described Xie Z. Viollet B. Zou M.H. Activation of the AMP-activated kinase by in by the of protein and endothelial 2006; PubMed Scopus Google Scholar was used as control. these of expression was are as were performed using or analysis of followed by the for or using Diego, CA). A value of was AMPK is important in maintaining normal endothelial function and LPS in endothelial barrier investigated whether LPS increases endothelial permeability WT mice with LPS reduced of Thr172-phosphorylated AMPK in lung compared with mice a role for LPS as an inhibitor of AMPK activity in vivo. We investigated the of LPS on endothelial barrier function in WT mice. Lung EC barrier dysfunction is by increased permeation of fluid and macromolecules into the interstitium and alveolar with that of control mice, LPS a increase in the white blood cell count the bronchoalveolar lavage fluid which is of an acute inflammatory response in the lungs LPS a increase in the total protein concentration in barrier and lung injury. of lung tissue from LPS-treated mice enhanced alveolar and infiltration of white blood cells into the lung interstitium and alveolar LPS injection infiltration of Evans from the vessel into the lung the of LPS on EC barrier dysfunction LPS AMPK activity in whether AMPK LPS-induced hyperpermeability and lung injury. Lung injury was in both WT and mice challenged with LPS. The subunit in lungs was α1 After LPS protein and cell were in mice in WT mice and Consistent with this infiltration of inflammatory cells into lung lung and were also in mice We investigated the effects of AICAR in mice, to potential effects of this AICAR alone not the of Evans and inflammatory cell infiltration in control WT mice and AICAR attenuated LPS-induced microvascular lung and decreased protein concentration AICAR not the decrease of protein concentration and cell count in LPS-treated or mice and EC cytoskeletal formation of between cells and increases endothelial permeability. We therefore whether LPS cytoskeletal in cultured In F-actin was into and were After with F-actin into stress in the of were These changes were with the of EC barrier by LPS. We the effects of AICAR on LPS-induced of endothelial barrier function in cultured AICAR not cytoskeletal in AICAR attenuated LPS-induced stress and formation A and attenuated LPS-induced cytoskeletal compared with control increased stress and compared with control Consistent with this AICAR decreased the phosphorylation of VE-cadherin at in a and A and of or increased the phosphorylation of VE-cadherin at The between Rho and Rac controls endothelial permeability. determine whether AICAR affects the Rho signaling pathway, a downstream of Rho AICAR LPS-induced phosphorylation of Thr172 is in the activation of the AMPK α1 and α2 and its phosphorylation to AMPK Z. Dong Y. R. Neumann D. Zou M.H. of LKB1 at by protein kinase is required for activation of the AMP-activated protein kinase in endothelial 2008; PubMed Scopus Google Scholar in a role for LPS in AMPK phosphorylation at this EC to LPS for and AMPK phosphorylation at Thr172 was by LPS phosphorylation of both AMPK at Thr172 and its downstream acetyl coenzyme A carboxylase at Consistent with this LPS decreased AMPK activity as as after LPS exposure We whether LPS inhibits AMPK by phosphorylation of its kinase LKB1 at LPS increased LKB1 phosphorylation at and therefore the effects of LPS on AMPK are of protein phosphatase AMPK in response to inflammatory as the effects of LPS on expression in LPS not expression in a or a and were with AICAR in a and after which AMPK and phosphorylation were via The of Thr172-phosphorylated AMPK increased in response to AICAR to 2 in a mmol/L as the concentration of the for AMPK and phosphorylation of AMPK at Thr172 and of at was after and at the AICAR the LPS-induced in phosphorylation of AMPK at Thr172 and of at We the or by which AMPK activation by AICAR LPS-induced cytoskeletal Rho family members Cdc42 and Rac1 endothelial barrier V. Schlegel N. Waschke J. Role of GTPases in control of microvascular permeability.Cardiovasc Res. 2010; 87: 243-253Crossref PubMed Scopus (273) Google Scholar, 4Popoff M.R. Geny B. Multifaceted role of Rho, Rac, Cdc42 and Ras in intercellular junctions, lessons from toxins.Biochim Biophys Acta. 2009; 1788: 797-812Crossref PubMed Scopus (103) Google Scholar, 5Watanabe T. Sato K. Kaibuchi K. Cadherin-mediated intercellular adhesion and signaling cascades involving small GTPases.Cold Spring Harb Perspect Biol. 2009; 1: a003020Crossref Scopus (62) Google Scholar by EC junction integrity after injury.7Zhao Y.D. Ohkawara H. Rehman J. Wary K.K. Vogel S.M. Minshall R.D. Zhao Y.Y. Malik A.B. Bone marrow progenitor cells induce endothelial adherens junction integrity by sphingosine-1-phosphate-mediated Rac1 and Cdc42 signaling.Circ Res. 2009; 105 (8 p following 704): 696-704Crossref PubMed Scopus (45) Google Scholar We therefore investigated the effects of AICAR on Rac1/Cdc42 in AICAR activated both Rac1 and Cdc42 in a A and These activation of Rac1 and the role of AICAR in the signaling pathway, the effects of AMPK on a downstream of Rac1 that plays a role in cell cytoskeletal cell and barrier A.A. Xing J. Fu P. Yakubov B. Dubrovskyi O. Fortune J.A. Klibanov A.M. Birukov K.G. Atrial natriuretic peptide attenuates LPS-induced lung vascular leak: role of PAK1.Am J Physiol Lung Cell Mol Physiol. 2010; 299: L652-L663Crossref PubMed Scopus (56) Google Scholar AICAR increased phosphorylation in in a and A and Consistent with this inhibition of or with to a in were with the Rac inhibitor NSC 23766 or with Rac and phosphorylation which as a downstream for Rac1 in The major of the present study is that AMPK inhibition to LPS-induced endothelial barrier dysfunction and lung injury. LPS AMPK activity both in and in vivo. lung tissue inflammatory cell lung and were in mice in WT mice, in mice, that AMPK lung endothelial barrier dysfunction and lung injury in vivo. which AMPK in the lungs, decreased pulmonary vascular permeability and the of LPS-induced acute lung injury. LPS-induced lung injury was in mice, that is important in maintaining endothelial Thus, AMPK activation by AICAR attenuates LPS-induced pulmonary vascular hyperpermeability and lung injury. The of AMPK on maintaining endothelial integrity to by For example, the of AMPK activation to enhance pulmonary vascular barrier function to to its with cytoskeletal including of and that activation to activation of the Rac downstream which plays a role in cell cytoskeletal cell and barrier A.A. Xing J. Fu P. Yakubov B. Dubrovskyi O. Fortune J.A. Klibanov A.M. Birukov K.G. Atrial natriuretic peptide attenuates LPS-induced lung vascular leak: role of PAK1.Am J Physiol Lung Cell Mol Physiol. 2010; 299: L652-L663Crossref PubMed Scopus (56) Google Scholar activation of the Rac inhibits Rho A.A. S. O. Birukov K.G. Signaling involved in pulmonary endothelial barrier Res. 2007; PubMed Scopus Google Scholar AICAR activated Rac and but LPS-induced phosphorylation of a downstream of Rho, inhibiting LPS-induced of the cell and cell Wu and R. M. N. M. Zhang Z. 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Carling D. skeletal insulin of 2006; PubMed Scopus Google Scholar In the present LPS not the of LPS-induced AMPK inhibition not to to LKB1, an kinase of In LPS increased the phosphorylation of LKB1 at a phosphorylation known to important for AMPK Z. Dong Y. Zhang J. R. Neumann D. Zou M.H. of the of LKB1 as a phosphorylation for its and endothelial cell Cell Biol. 2009; PubMed Scopus Google Scholar LKB1 is to lung cell by mediating Cdc42 and S. K. The tumor LKB1 regulates lung cell by mediating Cdc42 and Res. 2008; PubMed Scopus Google Scholar In addition, phosphorylation is in cell lines the human lung cell in which LKB1 function is S. K. The tumor LKB1 regulates lung cell by mediating Cdc42 and Res. 2008; PubMed Scopus Google Scholar, P. O. A. M. and of lung cells of the Res. 2003; Google Scholar this of LKB1 is by AMPK In the present study activation of AMPK protects against lung barrier dysfunction and accumulation by intratracheal LPS protective effects of AMPK against LPS-induced lung dysfunction are by lung vascular endothelium and by Rac GTPase and phosphorylation of and LPS inhibits AMPK expression in the lung. In addition to the endothelial effects described AMPK or AMPK activation as a therapeutic for by permeability