RESUMO
AIMS: Proliferation of vascular smooth muscle cells (VSMCs) is a hallmark of pulmonary hypertension (PH). Proliferative cells utilize purine bases from the de novo purine synthesis (DNPS) pathways for nucleotide synthesis; however, it is unclear whether DNPS plays a critical role in VSMC proliferation during development of PH. The last two steps of DNPS are catalysed by the enzyme 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/inosine monophosphate cyclohydrolase (ATIC). This study investigated whether ATIC-driven DNPS affects the proliferation of pulmonary artery smooth muscle cells (PASMCs) and the development of PH. METHODS AND RESULTS: Metabolites of DNPS in proliferative PASMCs were measured by liquid chromatography-tandem mass spectrometry. ATIC expression was assessed in platelet-derived growth factor-treated PASMCs and in the lungs of PH rodents and patients with pulmonary arterial hypertension. Mice with global and VSMC-specific knockout of Atic were utilized to investigate the role of ATIC in both hypoxia- and lung interleukin-6/hypoxia-induced murine PH. ATIC-mediated DNPS at the mRNA, protein, and enzymatic activity levels were increased in platelet-derived growth factor-treated PASMCs or PASMCs from PH rodents and patients with pulmonary arterial hypertension. In cultured PASMCs, ATIC knockdown decreased DNPS and nucleic acid DNA/RNA synthesis, and reduced cell proliferation. Global or VSMC-specific knockout of Atic attenuated vascular remodelling and inhibited the development and progression of both hypoxia- and lung IL-6/hypoxia-induced PH in mice. CONCLUSION: Targeting ATIC-mediated DNPS compromises the availability of purine nucleotides for incorporation into DNA/RNA, reducing PASMC proliferation and pulmonary vascular remodelling and ameliorating the development and progression of PH.
Assuntos
Hipertensão Pulmonar , Hipertensão Arterial Pulmonar , Camundongos , Animais , Roedores/metabolismo , Remodelação Vascular/fisiologia , Artéria Pulmonar , Purinas/metabolismo , Células Cultivadas , Hipóxia/metabolismo , RNA Mensageiro/metabolismo , Fator de Crescimento Derivado de Plaquetas/metabolismo , Proliferação de Células , Miócitos de Músculo Liso/metabolismoRESUMO
Acute lung injury (ALI) is characterized by lung vascular endothelial cell (EC) barrier compromise resulting in increased endothelial permeability and pulmonary edema. The infection of gram-negative bacteria that produce toxins like LPS is one of the major causes of ALI. LPS activates Toll-like receptor 4, leading to cytoskeleton reorganization, resulting in lung endothelial barrier disruption and pulmonary edema in ALI. However, the signaling pathways that lead to the cytoskeleton reorganization and lung microvascular EC barrier disruption remain largely unexplored. Here we show that LPS induces calpain activation and talin cleavage into head and rod domains and that inhibition of calpain attenuates talin cleavage, RhoA activation, and pulmonary EC barrier disruption in LPS-treated human lung microvascular ECs in vitro and lung EC barrier disruption and pulmonary edema induced by LPS in ALI in vivo. Moreover, overexpression of calpain causes talin cleavage and RhoA activation, myosin light chain (MLC) phosphorylation, and increases in actin stress fiber formation. Furthermore, knockdown of talin attenuates LPS-induced RhoA activation and MLC phosphorylation and increased stress fiber formation and mitigates LPS-induced lung microvascular endothelial barrier disruption. Additionally, overexpression of talin head and rod domains increases RhoA activation, MLC phosphorylation, and stress fiber formation and enhances lung endothelial barrier disruption. Finally, overexpression of cleavage-resistant talin mutant reduces LPS-induced increases in MLC phosphorylation in human lung microvascular ECs and attenuates LPS-induced lung microvascular endothelial barrier disruption. These results provide the first evidence that calpain mediates LPS-induced lung microvascular endothelial barrier disruption in ALI via cleavage of talin.
Assuntos
Lesão Pulmonar Aguda , Edema Pulmonar , Humanos , Lipopolissacarídeos/farmacologia , Calpaína/metabolismo , Talina/metabolismo , Pulmão/metabolismo , Lesão Pulmonar Aguda/induzido quimicamente , Lesão Pulmonar Aguda/metabolismo , Cadeias Leves de Miosina/metabolismo , Permeabilidade CapilarRESUMO
Chronic obstructive pulmonary disease (COPD) is a multisystemic respiratory disease that is associated with progressive airway and pulmonary vascular remodeling due to the increased proliferation of bronchial smooth muscles cells (BSMCs) and pulmonary arterial smooth muscle cells (PASMCs) and the overproduction of extracellular matrix (e.g., collagen). Cigarette smoke (CS) and several mediators, such as PDGF (platelet-derived growth factor) and IL-6, play critical roles in COPD pathogenesis. HDAC6 has been shown to be implicated in vascular remodeling. However, the role of airway HDAC6 signaling in pulmonary vascular remodeling in COPD and the underlying mechanisms remain undetermined. Here, we show that HDAC6 expression is upregulated in the lungs of patients with COPD and a COPD animal model. We also found that CS extract (CSE), PDGF, and IL-6 increase the protein levels and activation of HDAC6 in BSMCs and PASMCs. Furthermore, CSE and these stimulants induced deacetylation and phosphorylation of ERK1/2 and increased collagen synthesis and BSMC and PASMC proliferation, which were outcomes that were prevented by HDAC6 inhibition. Inhibition of ERK1/2 also diminished the CSE-, PDGF-, and IL-6-caused elevation in collagen levels and cell proliferation. Pharmacologic HDAC6 inhibition with tubastatin A prevented the CS-stimulated increases in the thickness of the bronchial and pulmonary arterial wall, airway resistance, emphysema, and right ventricular systolic pressure and right ventricular hypertrophy in a rat model of COPD. These data demonstrate that the upregulated HDAC6 governs the collagen synthesis and BSMC and PASMC proliferation that lead to airway and vascular remodeling in COPD.
Assuntos
Remodelação das Vias Aéreas , Desacetilase 6 de Histona/metabolismo , Sistema de Sinalização das MAP Quinases , Doença Pulmonar Obstrutiva Crônica/enzimologia , Remodelação Vascular , Animais , Citocinas/metabolismo , Modelos Animais de Doenças , Desacetilase 6 de Histona/antagonistas & inibidores , Humanos , Ácidos Hidroxâmicos/farmacologia , Indóis/farmacologia , Músculo Liso Vascular/enzimologia , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/enzimologia , Miócitos de Músculo Liso/patologia , Artéria Pulmonar/enzimologia , Artéria Pulmonar/patologia , Doença Pulmonar Obstrutiva Crônica/patologia , Ratos , Ratos Sprague-DawleyRESUMO
Acute lung injury (ALI) is an acute inflammatory process arises from a wide range of lung insults. A major cause of ALI is dysfunction of the pulmonary vascular endothelial barrier but the mechanisms involved are incompletely understood. The therapeutic potential of histone deacetylase (HDAC) inhibitors for the treatment of cardiovascular and inflammatory diseases is increasingly apparent, but the mechanisms by which HDACs regulate pulmonary vascular barrier function remain to be resolved. We found that specific Class IIa HDACs inhibitor, TMP269, significantly attenuated the lipopolysaccharide (LPS)-induced human lung microvascular endothelial cells (HLMVEC) barrier compromise in vitro and improved vascular barrier integrity and lung function in murine model of ALI in vivo. TMP269 decreased LPS-induced myosin light chain phosphorylation suggesting the role for Class IIa HDACs in LPS-induced cytoskeleton reorganization. TMP269 did not affect microtubule structure and tubulin acetylation in contrast to the HDAC6-specific inhibitor, Tubastatin A suggesting that Class IIa HDACs and HDAC6 (Class IIb) regulate endothelial cytoskeleton and permeability via different mechanisms. Furthermore, LPS increased the expression of ArgBP2 which has recently been attributed to HDAC-mediated activation of Rho. Depletion of ArgBP2 abolished the ability of LPS to disrupt barrier function in HLMVEC and both TMP269 and Tubastatin A decreased the level of ArgBP2 expression after LPS stimulation suggesting that both Class IIa and IIb HDACs regulate endothelial permeability via ArgBP2-dependent mechanism. Collectively, our data strongly suggest that Class IIa HDACs are involved in LPS-induced ALI in vitro and in vivo via specific mechanism which involved contractile responses, but not microtubule reorganization.
Assuntos
Lesão Pulmonar Aguda/induzido quimicamente , Lesão Pulmonar Aguda/enzimologia , Histona Desacetilases/metabolismo , Lesão Pulmonar Aguda/fisiopatologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Permeabilidade da Membrana Celular/efeitos dos fármacos , Modelos Animais de Doenças , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Endotoxinas , Frequência Cardíaca/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Lipopolissacarídeos , Pulmão/efeitos dos fármacos , Pulmão/patologia , Pulmão/fisiopatologia , Camundongos Endogâmicos C57BL , Microvasos/patologia , Modelos Biológicos , Oxigênio/metabolismo , Pneumonia/complicações , Pneumonia/patologia , Transdução de Sinais/efeitos dos fármacos , Proteínas rho de Ligação ao GTP/metabolismoRESUMO
Pulmonary hypertension (PH) is a progressive cardiovascular disorder in which local vascular inflammation leads to increased pulmonary vascular remodeling and ultimately to right heart failure. The HDAC inhibitor butyrate, a product of microbial fermentation, is protective in inflammatory intestinal diseases, but little is known regarding its effect on extraintestinal diseases, such as PH. In this study, we tested the hypothesis that butyrate is protective in a Sprague-Dawley (SD) rat model of hypoxic PH. Treatment with butyrate (220 mg/kg intake) prevented hypoxia-induced right ventricular hypertrophy (RVH), hypoxia-induced increases in right ventricular systolic pressure (RVSP), pulmonary vascular remodeling, and permeability. A reversal effect of butyrate (2200 mg/kg intake) was observed on elevated RVH. Butyrate treatment also increased the acetylation of histone H3, 25-34 kDa, and 34-50 kDa proteins in the total lung lysates of butyrate-treated animals. In addition, butyrate decreased hypoxia-induced accumulation of alveolar (mostly CD68+) and interstitial (CD68+ and CD163+) lung macrophages. Analysis of cytokine profiles in lung tissue lysates showed a hypoxia-induced upregulation of TIMP-1, CINC-1, and Fractalkine and downregulation of soluble ICAM (sICAM). The expression of Fractalkine and VEGFα, but not CINC-1, TIMP-1, and sICAM was downregulated by butyrate. In rat microvascular endothelial cells (RMVEC), butyrate (1 mM, 2 and 24 h) exhibited a protective effect against TNFα- and LPS-induced barrier disruption. Butyrate (1 mM, 24 h) also upregulated tight junctional proteins (occludin, cingulin, claudin-1) and increased the acetylation of histone H3 but not α-tubulin. These findings provide evidence of the protective effect of butyrate on hypoxic PH and suggest its potential use as a complementary treatment for PH and other cardiovascular diseases.
Assuntos
Butiratos/farmacologia , Hipertensão Pulmonar/etiologia , Hipertensão Pulmonar/fisiopatologia , Hipóxia/complicações , Pulmão/fisiopatologia , Pneumonia/fisiopatologia , Remodelação Vascular/efeitos dos fármacos , Acetilação/efeitos dos fármacos , Animais , Pressão Sanguínea/efeitos dos fármacos , Citocinas/metabolismo , Células Endoteliais/efeitos dos fármacos , Ventrículos do Coração/efeitos dos fármacos , Ventrículos do Coração/fisiopatologia , Hipertrofia Ventricular Direita/complicações , Hipertrofia Ventricular Direita/fisiopatologia , Pulmão/irrigação sanguínea , Pulmão/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/patologia , Microvasos/patologia , Pneumonia/complicações , Ratos Sprague-Dawley , Sístole/efeitos dos fármacos , Junções Íntimas/efeitos dos fármacos , Junções Íntimas/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
The vasa vasorum (VV), the microvascular network around large vessels, has been recognized as an important contributor to the pathological vascular remodeling in cardiovascular diseases. In bovine and rat models of hypoxic pulmonary hypertension (PH), we have previously shown that chronic hypoxia profoundly increased pulmonary artery (PA) VV permeability, associated with infiltration of inflammatory and progenitor cells in the arterial wall, perivascular inflammation, and structural vascular remodeling. Extracellular adenosine was shown to exhibit a barrier-protective effect on VV endothelial cells (VVEC) via cAMP-independent mechanisms, which involved adenosine A1 receptor-mediated activation of Gi-phosphoinositide 3-kinase-Akt pathway and actin cytoskeleton remodeling. Using VVEC isolated from the adventitia of calf PA, in this study we investigated in more detail the mechanisms linking Gi activation to downstream barrier protection pathways. Using a small-interference RNA (siRNA) technique and transendothelial electrical resistance assay, we found that the adaptor protein, engulfment and cell motility 1 (ELMO1), the tyrosine phosphatase Src homology region 2 domain-containing phosphatase-2, and atypical Gi- and Rac1-mediated protein kinase A activation are implicated in VVEC barrier enhancement. In contrast, the actin-interacting GTP-binding protein, girdin, and the p21-activated kinase 1 downstream target, LIM kinase, are not involved in this response. In addition, adenosine-dependent cytoskeletal rearrangement involves activation of cofilin and inactivation of ezrin-radixin-moesin regulatory cytoskeletal proteins, consistent with a barrier-protective mechanism. Collectively, our data indicate that targeting adenosine receptors and downstream barrier-protective pathways in VVEC may have a potential translational significance in developing pharmacological approach for the VV barrier protection in PH.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adenosina/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Células Endoteliais/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Vasa Vasorum/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Adenosina/farmacologia , Animais , Bovinos , Células Endoteliais/efeitos dos fármacos , Líquido Extracelular/efeitos dos fármacos , Líquido Extracelular/metabolismo , Masculino , Artéria Pulmonar/efeitos dos fármacos , Artéria Pulmonar/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Vasa Vasorum/efeitos dos fármacosRESUMO
Rationale: Glycolytic shift is implicated in the pathogenesis of pulmonary arterial hypertension (PAH). It remains unknown how glycolysis is increased and how increased glycolysis contributes to pulmonary vascular remodeling in PAH.Objectives: To determine whether increased glycolysis is caused by 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3) and how PFKFB3-driven glycolysis induces vascular remodeling in PAH.Methods: PFKFB3 levels were measured in pulmonary arteries of patients and animals with PAH. Lactate levels were assessed in lungs of animals with PAH and in pulmonary artery smooth muscle cells (PASMCs). Genetic and pharmacologic approaches were used to investigate the role of PFKFB3 in PAH.Measurements and Main Results: Lactate production was elevated in lungs of PAH rodents and in platelet-derived growth factor-treated PASMCs. PFKFB3 protein was higher in pulmonary arteries of patients and rodents with PAH, in PASMCs of patients with PAH, and in platelet-derived growth factor-treated PASMCs. PFKFB3 inhibition by genetic disruption and chemical inhibitor attenuated phosphorylation/activation of extracellular signal-regulated kinase (ERK1/2) and calpain-2, and vascular remodeling in PAH rodent models, and reduced platelet-derived growth factor-induced phosphorylation/activation of ERK1/2 and calpain-2, collagen synthesis and proliferation of PASMCs. ERK1/2 inhibition attenuated phosphorylation/activation of calpain-2, and vascular remodeling in Sugen/hypoxia PAH rats, and reduced lactate-induced phosphorylation/activation of calpain-2, collagen synthesis, and proliferation of PASMCs. Calpain-2 inhibition reduced lactate-induced collagen synthesis and proliferation of PASMCs.Conclusions: Upregulated PFKFB3 mediates collagen synthesis and proliferation of PASMCs, contributing to vascular remodeling in PAH. The mechanism is through the elevation of glycolysis and lactate that results in the activation of calpain by ERK1/2-dependent phosphorylation of calpain-2.
Assuntos
Proliferação de Células/efeitos dos fármacos , Músculo Liso Vascular/crescimento & desenvolvimento , Fosfofrutoquinase-2/sangue , Fosfofrutoquinase-2/metabolismo , Hipertensão Arterial Pulmonar/sangue , Hipertensão Arterial Pulmonar/fisiopatologia , Remodelação Vascular/fisiologia , Animais , Modelos Animais de Doenças , Humanos , Masculino , Camundongos , RatosRESUMO
Purinergic G-protein-coupled receptors are ancient and the most abundant group of G-protein-coupled receptors (GPCRs). The wide distribution of purinergic receptors in the cardiovascular system, together with the expression of multiple receptor subtypes in endothelial cells (ECs) and other vascular cells demonstrates the physiological importance of the purinergic signaling system in the regulation of the cardiovascular system. This review discusses the contribution of purinergic P2Y receptors to endothelial dysfunction (ED) in numerous cardiovascular diseases (CVDs). Endothelial dysfunction can be defined as a shift from a "calm" or non-activated state, characterized by low permeability, anti-thrombotic, and anti-inflammatory properties, to a "activated" state, characterized by vasoconstriction and increased permeability, pro-thrombotic, and pro-inflammatory properties. This state of ED is observed in many diseases, including atherosclerosis, diabetes, hypertension, metabolic syndrome, sepsis, and pulmonary hypertension. Herein, we review the recent advances in P2Y receptor physiology and emphasize some of their unique signaling features in pulmonary endothelial cells.
Assuntos
Doenças Cardiovasculares/metabolismo , Células Endoteliais/metabolismo , Endotélio/metabolismo , Neovascularização Patológica/metabolismo , Receptores Purinérgicos P2Y/metabolismo , Transdução de Sinais/fisiologia , Animais , Endotélio/patologia , Humanos , Inflamação/metabolismo , Inflamação/fisiopatologia , Estresse Oxidativo/fisiologia , Receptores Purinérgicos P2Y/fisiologiaRESUMO
Maintenance of the endothelial cell (EC) barrier is critical to vascular homeostasis and a loss of barrier integrity results in increased vascular permeability. While the mechanisms that govern increased EC permeability have been under intense investigation over the past several decades, the processes regulating the preservation/restoration of the EC barrier remain poorly understood. Herein we show that the extracellular purines, adenosine (Ado) and adenosine 5'-[γ-thio]-triphosphate (ATPγS) can strengthen the barrier function of human lung microvascular EC (HLMVEC). This ability involves protein kinase A (PKA) activation and decreases in myosin light chain 20 (MLC20) phosphorylation secondary to the involvement of MLC phosphatase (MLCP). In contrast to Ado, ATPγS-induced PKA activation is accompanied by a modest, but significant decrease in cyclic adenosine monophosphate (cAMP) levels supporting the existence of an unconventional cAMP-independent pathway of PKA activation. Furthermore, ATPγS-induced EC barrier strengthening does not involve the Rap guanine nucleotide exchange factor 3 (EPAC1) which is directly activated by cAMP but is instead dependent upon PKA-anchor protein 2 (AKAP2) expression. We also found that AKAP2 can directly interact with the myosin phosphatase-targeting protein MYPT1 and that depletion of AKAP2 abolished ATPγS-induced increases in transendothelial electrical resistance. Ado-induced strengthening of the HLMVEC barrier required the coordinated activation of PKA and EPAC1 in a cAMP-dependent manner. In summary, ATPγS-induced enhancement of the EC barrier is EPAC1-independent and is instead mediated by activation of PKA which is then guided by AKAP2, in a cAMP-independent mechanism, to activate MLCP which dephosphorylates MLC20 resulting in reduced EC contraction and preservation.
Assuntos
Trifosfato de Adenosina/análogos & derivados , Permeabilidade Capilar/efeitos dos fármacos , Microvasos/efeitos dos fármacos , Agonistas do Receptor Purinérgico P1/farmacologia , Receptores Purinérgicos P1/efeitos dos fármacos , Proteínas de Ancoragem à Quinase A/genética , Proteínas de Ancoragem à Quinase A/metabolismo , Trifosfato de Adenosina/farmacologia , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Impedância Elétrica , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Células HEK293 , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Microvasos/metabolismo , Cadeias Leves de Miosina/metabolismo , Fosfatase de Miosina-de-Cadeia-Leve/genética , Fosfatase de Miosina-de-Cadeia-Leve/metabolismo , Fosforilação , Receptores Purinérgicos P1/genética , Receptores Purinérgicos P1/metabolismo , Transdução de SinaisRESUMO
One of the early events in the progression of LPS-mediated acute lung injury in mice is the disruption of the pulmonary endothelial barrier resulting in lung edema. However, the molecular mechanisms by which the endothelial barrier becomes compromised remain unresolved. The SRY (sex-determining region on the Y chromosome)-related high-mobility group box (Sox) group F family member, SOX18, is a barrier-protective protein through its ability to increase the expression of the tight junction protein CLDN5. Thus, the purpose of this study was to determine if downregulation of the SOX18-CLDN5 axis plays a role in the pulmonary endothelial barrier disruption associated with LPS exposure. Our data indicate that both SOX18 and CLDN5 expression is decreased in two models of in vivo LPS exposure (intraperitoneal, intratracheal). A similar downregulation was observed in cultured human lung microvascular endothelial cells (HLMVECs) exposed to LPS. SOX18 overexpression in HLMVECs or in the mouse lung attenuated the LPS-mediated vascular barrier disruption. Conversely, reduced CLDN5 expression (siRNA) reduced the HLMVEC barrier-protective effects of SOX18 overexpression. The mechanism by which LPS decreases SOX18 expression was identified as transcriptional repression through binding of NF-κB (p65) to a SOX18 promoter sequence located between -1,082 and -1,073 bp with peroxynitrite contributing to LPS-mediated NF-κB activation. We conclude that NF-κB-dependent decreases in the SOX18-CLDN5 axis are essentially involved in the disruption of human endothelial cell barrier integrity associated with LPS-mediated acute lung injury.
Assuntos
Lesão Pulmonar Aguda/metabolismo , Permeabilidade Capilar , Células Endoteliais/metabolismo , Lipopolissacarídeos , Pulmão/irrigação sanguínea , NF-kappa B/metabolismo , Edema Pulmonar/metabolismo , Fatores de Transcrição SOXF/metabolismo , Lesão Pulmonar Aguda/induzido quimicamente , Lesão Pulmonar Aguda/genética , Lesão Pulmonar Aguda/patologia , Animais , Sítios de Ligação , Células Cultivadas , Claudina-5/genética , Claudina-5/metabolismo , Modelos Animais de Doenças , Regulação para Baixo , Células Endoteliais/patologia , Humanos , Masculino , Camundongos Endogâmicos C57BL , NF-kappa B/genética , Ácido Peroxinitroso/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Edema Pulmonar/induzido quimicamente , Edema Pulmonar/genética , Edema Pulmonar/patologia , Fatores de Transcrição SOXF/genética , Transdução de Sinais , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/metabolismoRESUMO
We have previously shown that Gs-coupled adenosine receptors (A2a) are primarily involved in adenosine-induced human pulmonary artery endothelial cell (HPAEC) barrier enhancement. However, the downstream events that mediate the strengthening of the endothelial cell (EC) barrier via adenosine signaling are largely unknown. In the current study, we tested the overall hypothesis that adenosine-induced Rac1 activation and EC barrier enhancement is mediated by Gs-dependent stimulation of cAMP-dependent Epac1-mediated signaling cascades. Adenoviral transduction of HPAEC with constitutively-active (C/A) Rac1 (V12Rac1) significantly increases transendothelial electrical resistance (TER) reflecting an enhancement of the EC barrier. Conversely, expression of an inactive Rac1 mutant (N17Rac1) decreases TER reflecting a compromised EC barrier. The adenosine-induced increase in TER was accompanied by activation of Rac1, decrease in contractility (MLC dephosphorylation), but not Rho inhibition. Conversely, inhibition of Rac1 activity attenuates adenosine-induced increase in TER. We next examined the role of cAMP-activated Epac1 and its putative downstream targets Rac1, Vav2, Rap1, and Tiam1. Depletion of Epac1 attenuated the adenosine-induced Rac1 activation and the increase in TER. Furthermore, silencing of Rac1 specific guanine nucleotide exchange factors (GEFs), Vav2 and Rap1a expression significantly attenuated adenosine-induced increases in TER and activation of Rac1. Depletion of Rap1b only modestly impacted adenosine-induced increases in TER and Tiam1 depletion had no effect on adenosine-induced Rac1 activation and TER. Together these data strongly suggest that Rac1 activity is required for adenosine-induced EC barrier enhancement and that the activation of Rac1 and ability to strengthen the EC barrier depends, at least in part, on cAMP-dependent Epac1/Vav2/Rap1-mediated signaling.
Assuntos
Adenosina/metabolismo , Endotélio Vascular/metabolismo , Pulmão/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Linhagem Celular , AMP Cíclico/metabolismo , Impedância Elétrica , Células Endoteliais/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Proteínas Proto-Oncogênicas c-vav/metabolismo , Artéria Pulmonar/metabolismo , Transdução de Sinais/fisiologia , Proteína 1 Indutora de Invasão e Metástase de Linfoma de Células T/metabolismoRESUMO
The pulmonary endothelium is the target of continuous physiological and pathological stimuli that affect its crucial barrier function. The regulation, defense, and repair of endothelial barrier function require complex biochemical processes. This review examines the role of endothelial phosphorylating enzymes, kinases, a class with profound, interdigitating influences on endothelial permeability and lung function.
Assuntos
Endotélio Vascular/enzimologia , Endotélio Vascular/fisiologia , Pulmão/enzimologia , Proteínas Quinases/metabolismo , Animais , Humanos , Transdução de SinaisRESUMO
Acute lung injury (ALI) is characterized by increased endothelial hyperpermeability. Protein nitration is involved in the endothelial barrier dysfunction in LPS-exposed mice. However, the nitrated proteins involved in this process have not been identified. The activation of the small GTPase RhoA is a critical event in the barrier disruption associated with LPS. Thus, in this study we evaluated the possible role of RhoA nitration in this process. Mass spectroscopy identified a single nitration site, located at Tyr(34) in RhoA. Tyr(34) is located within the switch I region adjacent to the nucleotide-binding site. Utilizing this structure, we developed a peptide designated NipR1 (nitration inhibitory peptide for RhoA 1) to shield Tyr(34) against nitration. TAT-fused NipR1 attenuated RhoA nitration and barrier disruption in LPS-challenged human lung microvascular endothelial cells. Further, treatment of mice with NipR1 attenuated vessel leakage and inflammatory cell infiltration and preserved lung function in a mouse model of ALI. Molecular dynamics simulations suggested that the mechanism by which Tyr(34) nitration stimulates RhoA activity was through a decrease in GDP binding to the protein caused by a conformational change within a region of Switch I, mimicking the conformational shift observed when RhoA is bound to a guanine nucleotide exchange factor. Stopped flow kinetic analysis was used to confirm this prediction. Thus, we have identified a new mechanism of nitration-mediated RhoA activation involved in LPS-mediated endothelial barrier dysfunction and show the potential utility of "shielding" peptides to prevent RhoA nitration in the management of ALI.
Assuntos
Lesão Pulmonar/enzimologia , Lesão Pulmonar/patologia , Proteína rhoA de Ligação ao GTP/metabolismo , Lesão Pulmonar Aguda/enzimologia , Lesão Pulmonar Aguda/patologia , Lesão Pulmonar Aguda/fisiopatologia , Sequência de Aminoácidos , Animais , Líquido da Lavagem Broncoalveolar , Citocinas/metabolismo , Modelos Animais de Doenças , Células Endoteliais/enzimologia , Células Endoteliais/patologia , Ativação Enzimática , Humanos , Lipopolissacarídeos , Lesão Pulmonar/induzido quimicamente , Lesão Pulmonar/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microvasos/patologia , Dados de Sequência Molecular , Óxido Nítrico Sintase Tipo III/metabolismo , Nitrosação , Peptídeos/metabolismo , Substâncias Protetoras/metabolismo , Tirosina/metabolismo , Proteína rhoA de Ligação ao GTP/químicaRESUMO
RATIONALE: Alveolar liquid clearance is regulated by Na(+) uptake through the apically expressed epithelial sodium channel (ENaC) and basolaterally localized Na(+)-K(+)-ATPase in type II alveolar epithelial cells. Dysfunction of these Na(+) transporters during pulmonary inflammation can contribute to pulmonary edema. OBJECTIVES: In this study, we sought to determine the precise mechanism by which the TIP peptide, mimicking the lectin-like domain of tumor necrosis factor (TNF), stimulates Na(+) uptake in a homologous cell system in the presence or absence of the bacterial toxin pneumolysin (PLY). METHODS: We used a combined biochemical, electrophysiological, and molecular biological in vitro approach and assessed the physiological relevance of the lectin-like domain of TNF in alveolar liquid clearance in vivo by generating triple-mutant TNF knock-in mice that express a mutant TNF with deficient Na(+) uptake stimulatory activity. MEASUREMENTS AND MAIN RESULTS: TIP peptide directly activates ENaC, but not the Na(+)-K(+)-ATPase, upon binding to the carboxy-terminal domain of the α subunit of the channel. In the presence of PLY, a mediator of pneumococcal-induced pulmonary edema, this binding stabilizes the ENaC-PIP2-MARCKS complex, which is necessary for the open probability conformation of the channel and preserves ENaC-α protein expression, by means of blunting the protein kinase C-α pathway. Triple-mutant TNF knock-in mice are more prone than wild-type mice to develop edema with low-dose intratracheal PLY, correlating with reduced pulmonary ENaC-α subunit expression. CONCLUSIONS: These results demonstrate a novel TNF-mediated mechanism of direct ENaC activation and indicate a physiological role for the lectin-like domain of TNF in the resolution of alveolar edema during inflammation.
Assuntos
Agonistas do Canal de Sódio Epitelial/metabolismo , Canais Epiteliais de Sódio/metabolismo , Peptídeos Cíclicos/metabolismo , Alvéolos Pulmonares/metabolismo , Edema Pulmonar/metabolismo , Estreptolisinas , Fator de Necrose Tumoral alfa/metabolismo , Animais , Proteínas de Bactérias , Agonistas do Canal de Sódio Epitelial/química , Canais Epiteliais de Sódio/química , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Peptídeos Cíclicos/química , Alvéolos Pulmonares/microbiologia , Edema Pulmonar/microbiologia , Fator de Necrose Tumoral alfa/químicaRESUMO
Aggressive treatment with antibiotics in patients infected with Streptococcus pneumoniae induces release of the bacterial virulence factor pneumolysin (PLY). Days after lungs are sterile, this pore-forming toxin can still induce pulmonary permeability edema in patients, characterized by alveolar/capillary barrier dysfunction and impaired alveolar liquid clearance (ALC). ALC is mainly regulated through Na(+) transport by the apically expressed epithelial sodium channel (ENaC) and the basolaterally expressed Na(+)/K(+)-ATPase in type II alveolar epithelial cells. Because no standard treatment is currently available to treat permeability edema, the search for novel therapeutic candidates is of high priority. We detected mRNA expression for the active receptor splice variant SV1 of the hypothalamic polypeptide growth hormone-releasing hormone (GHRH), as well as for GHRH itself, in human lung microvascular endothelial cells (HL-MVEC). Therefore, we have evaluated the effect of the GHRH agonist JI-34 on PLY-induced barrier and ALC dysfunction. JI-34 blunts PLY-mediated endothelial hyperpermeability in monolayers of HL-MVEC, in a cAMP-dependent manner, by means of reducing the phosphorylation of myosin light chain and vascular endothelial (VE)-cadherin. In human airway epithelial H441 cells, PLY significantly impairs Na(+) uptake, but JI-34 restores it to basal levels by means of increasing cAMP levels. Intratracheal instillation of PLY into C57BL6 mice causes pulmonary alveolar epithelial and endothelial hyperpermeability as well as edema formation, all of which are blunted by JI-34. These findings point toward a protective role of the GHRH signaling pathway in PLY-induced permeability edema.
Assuntos
Hormônio Liberador de Hormônio do Crescimento/agonistas , Edema Pulmonar/patologia , Estreptolisinas/toxicidade , Animais , Antígenos CD/metabolismo , Proteínas de Bactérias/toxicidade , Caderinas/metabolismo , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Regulação da Expressão Gênica , Hormônio Liberador de Hormônio do Crescimento/genética , Hormônio Liberador de Hormônio do Crescimento/metabolismo , Humanos , Ativação do Canal Iônico , Pulmão/metabolismo , Pulmão/patologia , Camundongos , Camundongos Endogâmicos C57BL , Microvasos/patologia , Cadeias Leves de Miosina/metabolismo , Permeabilidade , Fosforilação , Alvéolos Pulmonares/efeitos dos fármacos , Alvéolos Pulmonares/patologia , Edema Pulmonar/genética , Edema Pulmonar/fisiopatologia , Splicing de RNA/genética , Receptores de Neuropeptídeos/genética , Receptores de Neuropeptídeos/metabolismo , Receptores de Hormônios Reguladores de Hormônio Hipofisário/genética , Receptores de Hormônios Reguladores de Hormônio Hipofisário/metabolismo , Canais de Sódio/metabolismoRESUMO
Acute lung injury (ALI) is a severe hypoxemic respiratory insufficiency associated with lung leak, diffuse alveolar damage, inflammation, and loss of lung function. Decreased dimethylaminohydrolase (DDAH) activity and increases in asymmetric dimethylarginine (ADMA), together with exaggerated oxidative/nitrative stress, contributes to the development of ALI in mice exposed to LPS. Whether restoring DDAH function and suppressing ADMA levels can effectively ameliorate vascular hyperpermeability and lung injury in ALI is unknown, and was the focus of this study. In human lung microvascular endothelial cells, DDAH II overexpression prevented the LPS-dependent increase in ADMA, superoxide, peroxynitrite, and protein nitration. DDAH II also attenuated the endothelial barrier disruption associated with LPS exposure. Similarly, in vivo, we demonstrated that the targeted overexpression of DDAH II in the pulmonary vasculature significantly inhibited the accumulation of ADMA and the subsequent increase in oxidative/nitrative stress in the lungs of mice exposed to LPS. In addition, augmenting pulmonary DDAH II activity before LPS exposure reduced lung vascular leak and lung injury and restored lung function when DDAH activity was increased after injury. Together, these data suggest that enhancing DDAH II activity may prove a useful adjuvant therapy to treat patients with ALI.
Assuntos
Lesão Pulmonar Aguda/prevenção & controle , Amidoidrolases/metabolismo , Células Endoteliais/enzimologia , Terapia Genética , Lipopolissacarídeos , Pulmão/irrigação sanguínea , Microvasos/enzimologia , Edema Pulmonar/prevenção & controle , Lesão Pulmonar Aguda/induzido quimicamente , Lesão Pulmonar Aguda/enzimologia , Lesão Pulmonar Aguda/genética , Amidoidrolases/genética , Animais , Arginina/análogos & derivados , Arginina/metabolismo , Líquido da Lavagem Broncoalveolar/química , Permeabilidade Capilar , Células Cultivadas , Modelos Animais de Doenças , Células Endoteliais/patologia , Humanos , Pulmão/enzimologia , Pulmão/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microvasos/patologia , Estresse Oxidativo , Ácido Peroxinitroso/metabolismo , Edema Pulmonar/induzido quimicamente , Edema Pulmonar/enzimologia , Edema Pulmonar/genética , Superóxidos/metabolismo , Fatores de Tempo , Transfecção , Regulação para CimaRESUMO
Permeability of the endothelial monolayer is increased when exposed to the bacterial endotoxin LPS. Our previous studies have shown that heat shock protein (Hsp) 90 inhibitors protect and restore LPS-mediated hyperpermeability in bovine pulmonary arterial endothelial cells. In this study, we assessed the effect of Hsp90 inhibition against LPS-mediated hyperpermeability in cultured human lung microvascular endothelial cells (HLMVECs) and delineated the underlying molecular mechanisms. We demonstrate that Hsp90 inhibition is critical in the early phase, to prevent LPS-mediated hyperpermeability, and also in the later phase, to restore LPS-mediated hyperpermeability in HLMVECs. Because RhoA is a well known mediator of endothelial hyperpermeability, we investigated the effect of Hsp90 inhibition on LPS-mediated RhoA signaling. RhoA nitration and activity were increased by LPS in HLMVECs and suppressed when pretreated with the Hsp90 inhibitor, 17-allylamino-17 demethoxy-geldanamycin (17-AAG). In addition, inhibition of Rho kinase, a downstream effector of RhoA, protected HLMVECs from LPS-mediated hyperpermeability and abolished LPS-induced myosin light chain (MLC) phosphorylation, a target of Rho kinase. In agreement with these findings, 17-AAG or dominant-negative RhoA attenuated LPS-induced MLC phosphorylation. MLC phosphorylation induced by constitutively active RhoA was also suppressed by 17-AAG, suggesting a role for Hsp90 downstream of RhoA. Inhibition of Src family kinases also suppressed RhoA activity and MLC phosphorylation. Together, these data indicate that Hsp90 inhibition prevents and repairs LPS-induced lung endothelial barrier dysfunction by suppressing Src-mediated RhoA activity and signaling.
Assuntos
Células Endoteliais/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Proteínas de Choque Térmico HSP90/metabolismo , Lipopolissacarídeos/efeitos adversos , Quinases Associadas a rho/metabolismo , Animais , Benzoquinonas/farmacologia , Permeabilidade Capilar/efeitos dos fármacos , Células Cultivadas , Células Endoteliais/metabolismo , Humanos , Lactamas Macrocíclicas/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Cadeias Leves de Miosina/metabolismo , Fosforilação/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Proteína rhoA de Ligação ao GTP/metabolismo , Quinases da Família src/metabolismoRESUMO
Shear stress secondary to increased pulmonary blood flow (PBF) is elevated in some children born with congenital cardiac abnormalities. However, the majority of these patients do not develop pulmonary edema, despite high levels of permeability inducing factors. Previous studies have suggested that laminar fluid shear stress can enhance pulmonary vascular barrier integrity. However, little is known about the mechanisms by which this occurs. Using microarray analysis, we have previously shown that Sox18, a transcription factor involved in blood vessel development and endothelial barrier integrity, is up-regulated in an ovine model of congenital heart disease with increased PBF (shunt). By subjecting ovine pulmonary arterial endothelial cells (PAEC) to laminar flow (20 dyn/cm(2) ), we identified an increase in trans-endothelial resistance (TER) across the PAEC monolayer that correlated with an increase in Sox18 expression. Further, the TER was also enhanced when Sox18 was over-expressed and attenuated when Sox18 expression was reduced, suggesting that Sox18 maintains the endothelial barrier integrity in response to shear stress. Further, we found that shear stress up-regulates the cellular tight junction protein, Claudin-5, in a Sox18 dependent manner, and Claudin-5 depletion abolished the Sox18 mediated increase in TER in response to shear stress. Finally, utilizing peripheral lung tissue of 4 week old shunt lambs with increased PBF, we found that both Sox18 and Claudin-5 mRNA and protein levels were elevated. In conclusion, these novel findings suggest that increased laminar flow protects endothelial barrier function via Sox18 dependent up-regulation of Claudin-5 expression.
Assuntos
Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Pulmão/fisiopatologia , Fatores de Transcrição SOXF/metabolismo , Resistência ao Cisalhamento , Estresse Mecânico , Animais , Proliferação de Células , Células Endoteliais/patologia , Endotélio Vascular/patologia , Endotélio Vascular/fisiopatologia , Feminino , Humanos , Pulmão/irrigação sanguínea , Pulmão/metabolismo , Pulmão/patologia , Artéria Pulmonar/patologia , Ovinos , Proteínas de Junções Íntimas/metabolismo , Regulação para CimaRESUMO
Acute lung injury and acute respiratory distress syndrome (ALI/ARDS) affect 200,000 people a year in the USA. Pulmonary vascular and specifically endothelial cell (EC) barrier compromise is a hallmark of these diseases. We have recently shown that extracellular adenosine enhances human pulmonary (EC) barrier via activation of adenosine receptors (ARs) in cell cultures. On the basis of these data, we hypothesized that activation of ARs might exert barrier-protective effects in a model of ALI/ARDS in mice. To test this hypothesis, we examined the effects of pre- and posttreatment of adenosine and 5'-N-ethylcarboxamidoadenosine (NECA), a nonselective stable AR agonist, on LPS-induced lung injury. Mice were given vehicle or LPS intratracheally followed by adenosine, NECA, or vehicle instilled via the internal jugular vein. Postexperiment cell counts, Evans Blue Dye albumin (EBDA) extravasation, levels of proteins, and inflammatory cytokines were analyzed. Harvested lungs were used for histology and myeloperoxidase studies. Mice challenged with LPS alone demonstrated an inflammatory response typical of ALI. Cell counts, EBDA extravasation, as well as levels of proteins and inflammatory cytokines were decreased in adenosine-treated mice. Histology displayed reduced infiltration of neutrophils. NECA had a similar effect on LPS-induced vascular barrier compromise. Importantly, posttreatment with adenosine or NECA recovers lung vascular barrier and reduces inflammation induced by LPS challenge. Furthermore, adenosine significantly attenuated protein degradation of A2A and A3 receptors induced by LPS. Collectively, our results demonstrate that activation of ARs protects and restores vascular barrier functions and reduces inflammation in LPS-induced ALI.
Assuntos
Lesão Pulmonar Aguda/metabolismo , Adenosina/metabolismo , Endotélio/metabolismo , Receptores Purinérgicos P1/metabolismo , Lesão Pulmonar Aguda/induzido quimicamente , Adenosina-5'-(N-etilcarboxamida)/metabolismo , Animais , Líquido da Lavagem Broncoalveolar/citologia , Permeabilidade Capilar/efeitos dos fármacos , Contagem de Células , Citocinas/metabolismo , Células Endoteliais/metabolismo , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Interleucina-6/metabolismo , Lipopolissacarídeos , Pulmão/metabolismo , Pulmão/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Agonistas do Receptor Purinérgico P1/metabolismo , Síndrome do Desconforto Respiratório/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Endothelial cells (ECs) form a semi-permeable barrier between the interior space of blood vessels and the underlying tissues. Pulmonary endothelial barrier integrity is maintained through coordinated cellular processes involving receptors, signaling molecules, junctional complexes, and protein-regulated cytoskeletal reorganization. In acute lung injury (ALI) or its more severe form acute respiratory distress syndrome (ARDS), the loss of endothelial barrier integrity secondary to endothelial dysfunction caused by severe pulmonary inflammation and/or infection leads to pulmonary edema and hypoxemia. Pro-inflammatory agonists such as histamine, thrombin, bradykinin, interleukin 1ß, tumor necrosis factor α, vascular endothelial growth factor, angiopoietin-2, and platelet-activating factor, as well as bacterial toxins and reactive oxygen species, cause dynamic changes in cytoskeletal structure, adherens junction disorganization, and detachment of vascular endothelial cadherin (VE-cadherin) from the actin cytoskeleton, leading to an increase in endothelial permeability. Endothelial interactions with leukocytes, platelets, and coagulation enhance the inflammatory response. Moreover, inflammatory infiltration and the associated generation of pro-inflammatory cytokines during infection cause EC death, resulting in further compromise of the structural integrity of lung endothelial barrier. Despite the use of potent antibiotics and aggressive intensive care support, the mortality of ALI is still high, because the mechanisms of pulmonary EC barrier disruption are not fully understood. In this review, we summarized recent advances in the studies of endothelial cytoskeletal reorganization, inter-endothelial junctions, endothelial inflammation, EC death, and endothelial repair in ALI and ARDS, intending to shed some light on the potential diagnostic and therapeutic targets in the clinical management of the disease.