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1.
Dig Dis Sci ; 66(1): 238-246, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-32128647

RESUMO

BACKGROUND AND AIMS: Data on comparative efficacy of various available endoscopic ultrasound-guided liver biopsy (EUS-LB) needles are limited. We sought to compare the performance of a novel Franseen-tip 22G fine-needle biopsy (FNB) device to that of 19G needle platforms for liver parenchyma. METHODS: Consecutive patients referred for EUS and suspected to have hepatic parenchymal disease underwent EUS-LB using different EUS needles and were included in this retrospective study. Two blinded expert liver pathologists independently reviewed and reported on: total number of tissue fragments, length of longest fragment, number of complete and incomplete portal tracts (CPT and IPT), and specimen adequacy. RESULTS: A 22G Franseen-tip needle (A) was used in 30 patients; 19G Tru-Cut needle (B) in 50 patients; 19G reverse beveled non-Tru-Cut needle (C) in 27 patients; and a 19G flexible non-Tru-Cut needle (D) in 28 patients. In the order of needles, A, B, C and D, > 10 tissue fragments were obtained in 100%, 6%, 82%, and 96% samples, the mean number of CPTs was 6.9; 3.0; 7.3; and 16.9, length of longest fragment was 3.8, 4. 7, 3.9, and 8.4 mm, and specimen adequacy was 66.7%, 46%, 82.1%, and 81.5%, respectively. A positive correlation was obtained between number of CPTs and length of longest fragment in samples accrued by 19G needles. CONCLUSION: EUS-LB specimens using 22G Franseen-tip needle appear highly fragmented, leading to inferior specimen adequacy compared to 19G non-Tru-Cut needles. We also report on using length of longest fragment as an additional criterion for specimen adequacy as it positively correlates with number of CPTs standard.


Assuntos
Aspiração por Agulha Fina Guiada por Ultrassom Endoscópico/normas , Hepatopatias/diagnóstico por imagem , Agulhas/normas , Adulto , Aspiração por Agulha Fina Guiada por Ultrassom Endoscópico/métodos , Feminino , Humanos , Biópsia Guiada por Imagem/métodos , Biópsia Guiada por Imagem/normas , Hepatopatias/patologia , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Estudos Retrospectivos
2.
Mod Pathol ; 29(5): 489-99, 2016 05.
Artigo em Inglês | MEDLINE | ID: mdl-26916069

RESUMO

Increase in hepatic arterial flow in response to reduced portal flow (hepatic arterial buffer response) has been demonstrated experimentally and surgically. We provide pathologic evidence for hepatic arterial buffer response in non-cirrhotic patients with extrahepatic portal vein thrombosis and elucidate the histopathologic spectrum of non-cirrhotic portal vein thrombosis. Liver biopsies and resections from non-cirrhotic patients with extra-hepatic portal vein thrombosis were retrieved. Morphologic features, extent of CD34 staining, outer diameters, luminal diameters and wall thickness of hepatic arteries cut in cross-section and outer diameters of cross-sectioned paired bile ducts were compared with age- and gender-matched controls. There were 12 male and 9 female patients. Measurements of 280 and 193 arteries from patients and controls, respectively, demonstrated statistically significant (P<0.05) arterial dilatation (increase in percentage of arterial lumen to outer diameter) and arterial wall thinning in resection specimens of non-cirrhotic patients with extra-hepatic portal vein thrombosis. Subtle and/or focal dilatation of central veins, portal veins and sinusoids; focal trabecular thinning/thickening and mild ductular reaction were common findings in both the patient and control groups. Diffuse and obvious changes, and portal vein absence or attenuation were seen only in the patient group. Capillarization of sinusoids was not seen on CD34 stain. Two patients showed significant ductular reaction, one of who developed biliary strictures on follow-up. Hepatic arterial dilatation and wall thinning in non-cirrhotic patients with portal vein thrombosis provide pathologic evidence of hepatic arterial buffer response in the human liver. Obvious and diffuse sinusoidal dilatation and absence or attenuation of portal veins are highly suggestive of extrahepatic portal vein thrombosis in non-cirrhotic patients with portal hypertension. Periportal shunt vessels, hypervascular portal tracts, muscularized portal veins, large thick-walled or dilated arteries aid diagnosis but are rare findings. Normal or near-normal biopsies do not rule out portal vein thrombosis.


Assuntos
Artéria Hepática/patologia , Fígado/irrigação sanguínea , Fígado/patologia , Veia Porta/patologia , Trombose/patologia , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fluxo Sanguíneo Regional
3.
Am J Physiol Lung Cell Mol Physiol ; 309(4): L425-34, 2015 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-26092999

RESUMO

In addition to exerting a potent anti-elastase function, α-1 antitrypsin (A1AT) maintains the structural integrity of the lung by inhibiting endothelial inflammation and apoptosis. A main serpin secreted in circulation by hepatocytes, A1AT requires uptake by the endothelium to achieve vasculoprotective effects. This active uptake mechanism, which is inhibited by cigarette smoking (CS), involves primarily clathrin- but also caveola-mediated endocytosis and may require active binding to a receptor. Because circulating A1AT binds to high-density lipoprotein (HDL), we hypothesized that scavenging receptors are candidates for endothelial uptake of the serpin. Although the low-density lipoprotein (LDL) receptor-related protein 1 (LRP1) internalizes only elastase-bound A1AT, the scavenger receptor B type I (SR-BI), which binds and internalizes HDL and is modulated by CS, may be involved in A1AT uptake. Transmission electron microscopy imaging of colloidal gold-labeled A1AT confirmed A1AT endocytosis in both clathrin-coated vesicles and caveolae in endothelial cells. SR-BI immunoprecipitation identified binding to A1AT at the plasma membrane. Pretreatment of human lung microvascular endothelial cells with SR-B ligands (HDL or LDL), knockdown of SCARB1 expression, or neutralizing SR-BI antibodies significantly reduced A1AT uptake by 30-50%. Scarb1 null mice exhibited decreased A1AT lung content following systemic A1AT administration and reduced lung anti-inflammatory effects of A1AT supplementation during short-term CS exposure. In turn, A1AT supplementation increased lung SR-BI expression and modulated circulating lipoprotein levels in wild-type animals. These studies indicate that SR-BI is an important mediator of A1AT endocytosis in pulmonary endothelium and suggest a cross talk between A1AT and lipoprotein regulation of vascular functions.


Assuntos
Células Endoteliais/metabolismo , Receptores Depuradores Classe B/fisiologia , Fumar/metabolismo , alfa 1-Antitripsina/metabolismo , Animais , Ligação Competitiva , Células Cultivadas , Endocitose , Endotélio Vascular/patologia , Feminino , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout
4.
Am J Respir Cell Mol Biol ; 48(1): 87-93, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23024063

RESUMO

Key host responses to the stress induced by environmental exposure to cigarette smoke (CS) are responsible for initiating pathogenic effects that may culminate in emphysema development. CS increases lung ceramides, sphingolipids involved in oxidative stress, structural alveolar cell apoptosis, and inhibition of apoptotic cell clearance by alveolar macrophages, leading to the development of emphysema-like pathology. RTP801, a hypoxia and oxidative stress sensor, is also increased by CS, and has been recently implicated in both apoptosis and inflammation. We investigated whether inductions of ceramide and RTP801 are mechanistically linked, and evaluated their relative importance in lung cell apoptosis and airspace enlargement in vivo. As reported, direct lung instillation of either RTP801 expression plasmid or ceramides in mice triggered alveolar cell apoptosis and oxidative stress. RTP801 overexpression up-regulated lung ceramide levels 2.6-fold. In turn, instillation of lung ceramides doubled the lung content of RTP801. Cell sorting after lung tissue dissociation into single-cell suspension showed that ceramide triggers both endothelial and epithelial cell apoptosis in vivo. Interestingly, mice lacking rtp801 were protected against ceramide-induced apoptosis of epithelial type II cells, but not type I or endothelial cells. Furthermore, rtp801-null mice were protected from ceramide-induced alveolar enlargement, and exhibited improved static lung compliance compared with wild-type mice. In conclusion, ceramide and RTP801 participate in alveolar cell apoptosis through a process of mutual up-regulation, which may result in self-amplification loops, leading to alveolar damage.


Assuntos
Apoptose/fisiologia , Ceramidas/fisiologia , Proteínas de Ligação a DNA/fisiologia , Pulmão/patologia , Pulmão/fisiopatologia , Fatores de Transcrição/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Enfisema/etiologia , Enfisema/patologia , Enfisema/fisiopatologia , Enfisema/prevenção & controle , Células Endoteliais/patologia , Células Endoteliais/fisiologia , Células Epiteliais/patologia , Células Epiteliais/fisiologia , Feminino , Complacência Pulmonar/fisiologia , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Estresse Oxidativo , Receptores de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fumar/efeitos adversos , Fumar/patologia , Fumar/fisiopatologia , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética
5.
Am J Respir Crit Care Med ; 183(2): 215-25, 2011 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-20709815

RESUMO

RATIONALE: Adipose-derived stem cells express multiple growth factors that inhibit endothelial cell apoptosis, and demonstrate substantial pulmonary trapping after intravascular delivery. OBJECTIVES: We hypothesized that adipose stem cells would ameliorate chronic lung injury associated with endothelial cell apoptosis, such as that occurring in emphysema. METHODS: Therapeutic effects of systemically delivered human or mouse adult adipose stem cells were evaluated in murine models of emphysema induced by chronic exposure to cigarette smoke or by inhibition of vascular endothelial growth factor receptors. MEASUREMENTS AND MAIN RESULTS: Adipose stem cells were detectable in the parenchyma and large airways of lungs up to 21 days after injection. Adipose stem cell treatment was associated with reduced inflammatory infiltration in response to cigarette smoke exposure, and markedly decreased lung cell death and airspace enlargement in both models of emphysema. Remarkably, therapeutic results of adipose stem cells extended beyond lung protection by rescuing the suppressive effects of cigarette smoke on bone marrow hematopoietic progenitor cell function, and by restoring weight loss sustained by mice during cigarette smoke exposure. Pulmonary vascular protective effects of adipose stem cells were recapitulated by application of cell-free conditioned medium, which improved lung endothelial cell repair and recovery in a wound injury repair model and antagonized effects of cigarette smoke in vitro. CONCLUSIONS: These results suggest a useful therapeutic effect of adipose stem cells on both lung and systemic injury induced by cigarette smoke, and implicate a lung vascular protective function of adipose stem cell derived paracrine factors.


Assuntos
Tecido Adiposo/citologia , Células-Tronco Adultas/transplante , Lesão Pulmonar/terapia , Enfisema Pulmonar/terapia , Fumar/efeitos adversos , Transplante de Células-Tronco/métodos , Tecido Adiposo/transplante , Animais , Apoptose , Western Blotting , Técnicas de Cultura de Células , Modelos Animais de Doenças , Feminino , Citometria de Fluxo , Humanos , Inflamação/fisiopatologia , Inflamação/prevenção & controle , Lesão Pulmonar/etiologia , Lesão Pulmonar/fisiopatologia , Camundongos , Camundongos Endogâmicos C57BL , Alvéolos Pulmonares/fisiopatologia , Enfisema Pulmonar/etiologia , Enfisema Pulmonar/fisiopatologia , Transplante Heterólogo/métodos , Transplante Homólogo/métodos , Redução de Peso
6.
J Biol Chem ; 285(51): 40322-32, 2010 Dec 17.
Artigo em Inglês | MEDLINE | ID: mdl-20956540

RESUMO

A decreased clearance of apoptotic cells (efferocytosis) by alveolar macrophages (AM) may contribute to inflammation in emphysema. The up-regulation of ceramides in response to cigarette smoking (CS) has been linked to AM accumulation and increased detection of apoptotic alveolar epithelial and endothelial cells in lung parenchyma. We hypothesized that ceramides inhibit the AM phagocytosis of apoptotic cells. Release of endogenous ceramides via sphingomyelinase or exogenous ceramide treatments dose-dependently impaired apoptotic Jurkat cell phagocytosis by primary rat or human AM, irrespective of the molecular species of ceramide. Similarly, in vivo augmentation of lung ceramides via intratracheal instillation in rats significantly decreased the engulfment of instilled target apoptotic thymocytes by resident AM. The mechanism of ceramide-induced efferocytosis impairment was dependent on generation of sphingosine via ceramidase. Sphingosine treatment recapitulated the effects of ceramide, dose-dependently inhibiting apoptotic cell clearance. The effect of ceramide on efferocytosis was associated with decreased membrane ruffle formation and attenuated Rac1 plasma membrane recruitment. Constitutively active Rac1 overexpression rescued AM efferocytosis against the effects of ceramide. CS exposure significantly increased AM ceramides and recapitulated the effect of ceramides on Rac1 membrane recruitment in a sphingosine-dependent manner. Importantly, CS profoundly inhibited AM efferocytosis via ceramide-dependent sphingosine production. These results suggest that excessive lung ceramides may amplify lung injury in emphysema by causing both apoptosis of structural cells and inhibition of their clearance by AM.


Assuntos
Apoptose/efeitos dos fármacos , Ceramidas/farmacologia , Macrófagos Alveolares/metabolismo , Fumar/efeitos adversos , Animais , Membrana Celular/metabolismo , Membrana Celular/patologia , Ceramidases/metabolismo , Ceramidas/metabolismo , Relação Dose-Resposta a Droga , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Humanos , Células Jurkat , Macrófagos Alveolares/patologia , Masculino , Enfisema Pulmonar/metabolismo , Enfisema Pulmonar/patologia , Ratos , Ratos Sprague-Dawley , Proteínas rac1 de Ligação ao GTP/metabolismo
7.
Am J Respir Crit Care Med ; 181(4): 344-52, 2010 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-19965812

RESUMO

RATIONALE: Vascular endothelial growth factor receptor (VEGFR) inhibition increases ceramides in lung structural cells of the alveolus, initiating apoptosis and alveolar destruction morphologically resembling emphysema. The effects of increased endogenous ceramides could be offset by sphingosine 1-phosphate (S1P), a prosurvival by-product of ceramide metabolism. OBJECTIVES: The aims of our work were to investigate the sphingosine-S1P-S1P receptor axis in the VEGFR inhibition model of emphysema and to determine whether stimulation of S1P signaling is sufficient to functionally antagonize alveolar space enlargement. METHODS: Concurrent to VEGFR blockade in mice, S1P signaling augmentation was achieved via treatment with the S1P precursor sphingosine, S1P agonist FTY720, or S1P receptor-1 (S1PR1) agonist SEW2871. Outcomes included sphingosine kinase-1 RNA expression and activity, sphingolipid measurements by combined liquid chromatography-tandem mass spectrometry, immunoblotting for prosurvival signaling pathways, caspase-3 activity and terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling assays, and airspace morphometry. MEASUREMENTS AND MAIN RESULTS: Consistent with previously reported de novo activation of ceramide synthesis, VEGFR inhibition triggered increases in lung ceramides, dihydroceramides, and dihydrosphingosine, but did not alter sphingosine kinase activity or S1P levels. Administration of sphingosine decreased the ceramide-to-S1P ratio in the lung and inhibited alveolar space enlargement, along with activation of prosurvival signaling pathways and decreased lung parenchyma cell apoptosis. Sphingosine significantly opposed ceramide-induced apoptosis in cultured lung endothelial cells, but not epithelial cells. FTY720 or SEW2871 recapitulated the protective effects of sphingosine on airspace enlargement concomitant with attenuation of VEGFR inhibitor-induced lung apoptosis. CONCLUSIONS: Strategies aimed at augmenting the S1P-S1PR1 signaling may be effective in ameliorating the apoptotic mechanisms of emphysema development.


Assuntos
Alvéolos Pulmonares/efeitos dos fármacos , Enfisema Pulmonar/tratamento farmacológico , Receptores de Lisoesfingolipídeo/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Western Blotting , Células Cultivadas , Ceramidas/biossíntese , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Cloridrato de Fingolimode , Indóis/farmacologia , Lisofosfolipídeos/biossíntese , Lisofosfolipídeos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Reação em Cadeia da Polimerase , Propilenoglicóis/farmacologia , Alvéolos Pulmonares/fisiopatologia , Enfisema Pulmonar/fisiopatologia , Pirróis/farmacologia , Receptores de Lisoesfingolipídeo/fisiologia , Receptores de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptores de Fatores de Crescimento do Endotélio Vascular/efeitos dos fármacos , Receptores de Fatores de Crescimento do Endotélio Vascular/fisiologia , Transdução de Sinais/efeitos dos fármacos , Esfingosina/análogos & derivados , Esfingosina/biossíntese , Esfingosina/farmacologia
8.
Transplant Direct ; 7(7): e714, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34131586

RESUMO

The predictors of recurrent autoimmune hepatitis (R-AIH) after liver transplantation (LT) are heterogeneous with limited data to guide immunosuppression, with little data on impact of race. AIMS: To describe the incidence, predictors, and outcomes of R-AIH. METHODS: We studied patients undergoing LT for AIH during 2000-2017 at our center. Liver biopsies were performed for clinical indications. R-AIH was defined using clinical and histologic criteria. RESULTS: Among 75 patients undergoing LT for AIH (mean age 45 ± 16, 65% female individuals, 19% Black), 71 (95%) received antithymocyte globulin induction with tacrolimus-based immunosuppression. R-AIH developed in 20 (27%) patients at a median interval of 313 d (interquartile range, 155-1205). R-AIH was associated with level 2 HLA-DR mismatch (hazard ratio, 3.6; (95% confidence interval, 1.3-9.9; P = 0.01) and Black race (hazard ratio, 4.5; 95% confidence interval, 1.8-11.8; P = 0.002)] in the multivariable analysis. R-AIH developed in 62% of patients with level 2 HLA-DR mismatch on single-agent immunosuppression but in <20% of patients with no or 1 HLA-DR mismatch regardless of maintenance immunosuppression. R-AIH developed in 8 (57%) of 14 Black patients (71% on single-agent and 43% on dual-agent maintenance immunosuppression). Patient and graft survival were not impacted by R-AIH over a median follow-up of 8.3 y (interquartile range, 3-12). CONCLUSIONS: High-level HLA-DR mismatch and Black recipient race are associated with an increased risk of R-AIH. Immunosuppression did not predict R-AIH, but higher rates of disease recurrence with single-agent maintenance immunosuppression with these risk factors were observed and may guide maintenance immunosuppression in LT for AIH.

9.
FASEB J ; 23(9): 3149-58, 2009 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19423638

RESUMO

The integrity of lung alveoli is maintained by proper circulating levels of alpha-1 antitrypsin (A1AT). Next to cigarette smoking, A1AT deficiency is a major risk factor for lung emphysema development. We recently reported that in addition to neutralizing neutrophil elastases in the extracellular compartment, A1AT is internalized by lung endothelial cells and inhibits apoptosis. We hypothesized that the intracellular uptake of A1AT by endothelial cells may be required for its protective function; therefore, we studied the mechanisms of A1AT internalization by primary rat lung microvascular endothelial cells and the effect of cigarette smoke on this process both in vitro and in vivo (in mice). Purified A1AT was taken up intracellularly by endothelial cells in a time-dependent, dose-dependent, and conformer-specific manner and was detected in the cytoplasm of endothelial cells of nondiseased human lung sections. Despite a critical role for caveoli in endothelial cell endocytosis in general, specific inhibition of clathrin-mediated, but not caveoli-mediated, endocytosis profoundly decreased A1AT internalization and reversed the A1AT's antiapoptotic action. Further more, A1AT associated with clathrin heavy chains, but not with caveolin-1 in the plasma membrane fraction of endothelial cells. Interestingly, cigarette smoke exposure significantly inhibited A1AT uptake both in endothelial cells and in the mouse lung and altered the intracellular distribution of clathrin heavy chains. Our results suggest that clathrin-mediated endocytosis regulates A1AT intracellular function in the lung endothelium and may be an important determinant of the serpin's protection against developing cigarette smoke-induced emphysema.


Assuntos
Endocitose , Endotélio/fisiologia , Pulmão/citologia , Fumaça/efeitos adversos , alfa 1-Antitripsina/metabolismo , Animais , Clatrina/metabolismo , Enfisema/etiologia , Endotélio/metabolismo , Humanos , Camundongos , Ratos , Ratos Sprague-Dawley , Nicotiana , alfa 1-Antitripsina/sangue
10.
JCI Insight ; 5(14)2020 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-32573489

RESUMO

Cigarette smoking (CS) and genetic susceptibility determine the risk for development, progression, and severity of chronic obstructive pulmonary diseases (COPD). We posited that an incidental balanced reciprocal chromosomal translocation was linked to a patient's risk of severe COPD. We determined that 46,XX,t(1;4)(p13.1;q34.3) caused a breakpoint in the immunoglobulin superfamily member 3 (IGSF3) gene, with markedly decreased expression. Examination of COPDGene cohort identified 14 IGSF3 SNPs, of which rs1414272 and rs12066192 were directly and rs6703791 inversely associated with COPD severity, including COPD exacerbations. We confirmed that IGSF3 is a tetraspanin-interacting protein that colocalized with CD9 and integrin B1 in tetraspanin-enriched domains. IGSF3-deficient patient-derived lymphoblastoids exhibited multiple alterations in gene expression, especially in the unfolded protein response and ceramide pathways. IGSF3-deficient lymphoblastoids had high ceramide and sphingosine-1 phosphate but low glycosphingolipids and ganglioside levels, and they were less apoptotic and more adherent, with marked changes in multiple TNFRSF molecules. Similarly, IGSF3 knockdown increased ceramide in lung structural cells, rendering them more adherent, with impaired wound repair and weakened barrier function. These findings suggest that, by maintaining sphingolipid and membrane receptor homeostasis, IGSF3 is required for cell mobility-mediated lung injury repair. IGSF3 deficiency may increase susceptibility to CS-induced lung injury in COPD.


Assuntos
Fumar Cigarros/genética , Predisposição Genética para Doença , Imunoglobulinas/genética , Proteínas de Membrana/genética , Doença Pulmonar Obstrutiva Crônica/genética , Translocação Genética/genética , Apoptose/genética , Adesão Celular/genética , Movimento Celular/genética , Cromossomos Humanos Par 1/genética , Cromossomos Humanos Par 4/genética , Fumar Cigarros/efeitos adversos , Feminino , Regulação da Expressão Gênica/genética , Humanos , Integrina beta1/genética , Masculino , Pessoa de Meia-Idade , Mutação/genética , Polimorfismo de Nucleotídeo Único/genética , Doença Pulmonar Obstrutiva Crônica/induzido quimicamente , Doença Pulmonar Obstrutiva Crônica/patologia , Índice de Gravidade de Doença , Tetraspanina 29/genética
11.
Am J Respir Cell Mol Biol ; 41(3): 314-23, 2009 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19168702

RESUMO

The functional significance of the expression of cystic fibrosis transmembrane regulator (CFTR) on endothelial cells has not yet been elucidated. Since CFTR has been implicated in the regulation of intracellular sphingolipid levels, which are important regulators of endothelial cell apoptosis in response to various insults, we investigated the role of CFTR in the apoptotic responses of lung endothelial cells. CFTR was detected as a functional chloride channel in primary lung endothelial cells isolated from both pulmonary arteries (human or mouse) and bronchial arteries (sheep). Both specific CFTR inhibition with 2-(phenylamino) benzoic acid diphenylamine-2-carboxylic acid, 5-[(4-carboxyphenyl)methylene]-2-thioxo-3-[(3-trifluoromethyl)phenyl-4-thiazolidinone (CFTR(inh)-172), or 5-nitro-2-(3-phenylpropylamino)benzoic acid and CFTR knockdown significantly attenuated endothelial cell apoptosis induced by staurosporine or H(2)O(2). CFTR(inh)-172 treatment prevented the increases in the ceramide:sphingosine-1 phosphate ratio induced by H(2)O(2) in lung endothelial cells. Replenishing endogenous ceramides via sphingomyelinase supplementation restored the susceptibility of CFTR-inhibited lung endothelial cells to H(2)O(2)-induced apoptosis. Similarly, the anti-apoptotic phenotype of CFTR-inhibited cells was reversed by lowering the intracellular pH, and was reproduced by alkalinization before H(2)O(2) challenge. TUNEL staining and active caspase-3 immunohistochemistry indicated that cellular apoptosis was decreased in lung explants from patients with cystic fibrosis compared with those with smoking-induced chronic obstructive lung disease, especially in the alveolar tissue and vascular endothelium. In conclusion, CFTR function is required for stress-induced apoptosis in lung endothelial cells by maintaining adequate intracellular acidification and ceramide activation. These results may have implications in the pathogenesis of cystic fibrosis, where aberrant endothelial cell death may dysregulate lung vascular homeostasis, contributing to abnormal angiogenesis and chronic inflammation.


Assuntos
Apoptose/fisiologia , Ceramidas/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Fibrose Cística , Células Endoteliais/metabolismo , Concentração de Íons de Hidrogênio , Pulmão , Animais , Fibrose Cística/metabolismo , Fibrose Cística/patologia , Regulador de Condutância Transmembrana em Fibrose Cística/antagonistas & inibidores , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Células Endoteliais/citologia , Humanos , Peróxido de Hidrogênio/metabolismo , Pulmão/citologia , Pulmão/metabolismo , Camundongos , Oxidantes/metabolismo , Técnicas de Patch-Clamp , Esfingolipídeos/metabolismo
13.
Transplantation ; 100(8): 1699-704, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27136262

RESUMO

BACKGROUND: Donation after circulatory death (DCD) donor pool remains underutilized for liver transplantation (LT). We describe optimizing "modifiable risk factors," such as cold ischemia time (CIT) recipient warm ischemia time (WIT) and the use of thrombolytic flush at the time of procurement to minimize ischemic cholangiopathy (IC). METHODS: From July 2011 (era II), to improve outcomes after DCD LT, measures were taken to minimize CIT, operative time and recipient WIT along with the use of tissue plasminogen activator (tPA) flush during DCD procurements. Thirty consecutive DCD LTs were performed prospectively in era II. Outcomes were compared with 61 historic controls (era I). Reperfusion biopsies were evaluated for the presence of necrosis and biliary epithelial damage. RESULTS: Median CIT (4.9 [3.5-5.9] vs 6.4 [4.3-12]; P < 0.001), hepatectomy time (70 [42-120] vs 81 [58-207]; P = 0.02), and recipient WIT (16 [13-31] vs 24[15-40]; P < 0.001) were significantly shorter in era II. All patients in era II received tPA flushed liver grafts. None of the patients in era II developed IC (0% vs 18%; P = 0.013). There were fewer biliary complications in era II, and there was no increased risk of bleeding associated with the use of tPA. One-year graft survival was slightly better in era II (n = 24 patients with 1 year follow-up) (88% vs 80%; P = 0.14). CONCLUSIONS: Optimizing peritransplant conditions, such as shortening ischemic times with the use of thrombolytic donor flush, may prevent IC after DCD LT. With this approach, the DCD donor pool may be expanded.


Assuntos
Doenças Biliares/prevenção & controle , Isquemia Fria , Fibrinolíticos/administração & dosagem , Isquemia/prevenção & controle , Transplante de Fígado/métodos , Doadores de Tecidos/provisão & distribuição , Ativador de Plasminogênio Tecidual/administração & dosagem , Isquemia Quente , Adulto , Idoso , Doenças Biliares/diagnóstico , Doenças Biliares/etiologia , Causas de Morte , Isquemia Fria/efeitos adversos , Seleção do Doador , Feminino , Fibrinolíticos/efeitos adversos , Sobrevivência de Enxerto/efeitos dos fármacos , Humanos , Isquemia/diagnóstico , Isquemia/etiologia , Estimativa de Kaplan-Meier , Transplante de Fígado/efeitos adversos , Masculino , Pessoa de Meia-Idade , Duração da Cirurgia , Estudos Retrospectivos , Fatores de Risco , Fatores de Tempo , Ativador de Plasminogênio Tecidual/efeitos adversos , Resultado do Tratamento , Isquemia Quente/efeitos adversos , Adulto Jovem
14.
Pulm Circ ; 4(2): 260-8, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25006445

RESUMO

Abnormal lung microvascular endothelial vascular barrier function may contribute to pulmonary inflammation, such as that occurring during inhalation of cigarette smoke (CS). Cystic fibrosis transmembrane conductance regulator (CFTR), an anion channel expressed in both epithelial and endothelial cells, regulates the organization of tight junctions between epithelial cells and has also been implicated in the transport of sphingosine-1 phosphate (S1P), a vascular barrier-enhancing sphingolipid. Because CS has been shown to affect CFTR function, we hypothesized that CFTR function contributes to lung endothelial cell barrier and that CFTR dysfunction worsens CS-induced injury. CFTR inhibitors GlyH-101 or CFTRinh172 caused a dose-dependent increase in pulmonary or bronchial endothelial monolayer permeability, which peaked after 4 hours. CFTR inhibition was associated with both intercellular gaps and actin stress fiber formation compared with vehicle-treated cells. Increasing endothelial S1P, either by exogenous treatment or by inhibition of its degradation, significantly improved the barrier function in CFTR-inhibited monolayers. Both cultured lung endothelia and the lung microcirculation visualized in vivo with intravital two-photon imaging of transgenic mice deficient in CFTR showed that CFTR dysfunction increased susceptibility to CS-induced permeability. These results suggested that CFTR function might be required for lung endothelial barrier, including adherence junction stability. Loss of CFTR function, especially concomitant to CS exposure, might promote lung inflammation by increasing endothelial cell permeability, which could be ameliorated by S1P.

15.
PLoS One ; 9(4): e93979, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24743137

RESUMO

The homeostatic lung protective effects of alpha-1 antitrypsin (A1AT) may require the transport of circulating proteinase inhibitor across an intact lung endothelial barrier. We hypothesized that uninjured pulmonary endothelial cells transport A1AT to lung epithelial cells. Purified human A1AT was rapidly taken up by confluent primary rat pulmonary endothelial cell monolayers, was secreted extracellularly, both apically and basolaterally, and was taken up by adjacent rat lung epithelial cells co-cultured on polarized transwells. Similarly, polarized primary human lung epithelial cells took up basolaterally-, but not apically-supplied A1AT, followed by apical secretion. Evidence of A1AT transcytosis across lung microcirculation was confirmed in vivo by two-photon intravital microscopy in mice. Time-lapse confocal microscopy indicated that A1AT co-localized with Golgi in the endothelium whilst inhibition of the classical secretory pathway with tunicamycin significantly increased intracellular retention of A1AT. However, inhibition of Golgi secretion promoted non-classical A1AT secretion, associated with microparticle release. Polymerized A1AT or A1AT supplied to endothelial cells exposed to soluble cigarette smoke extract had decreased transcytosis. These results suggest previously unappreciated pathways of A1AT bidirectional uptake and secretion from lung endothelial cells towards the alveolar epithelium and airspaces. A1AT trafficking may determine its functional bioavailablity in the lung, which could be impaired in individuals exposed to smoking or in those with A1AT deficiency.


Assuntos
Células Endoteliais/citologia , Pulmão/citologia , Transcitose , alfa 1-Antitripsina/metabolismo , Animais , Células Endoteliais/efeitos dos fármacos , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Humanos , Camundongos , Ratos , Fumaça/efeitos adversos , Produtos do Tabaco/análise , Transcitose/efeitos dos fármacos
16.
J Clin Invest ; 121(6): 2470-9, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21576822

RESUMO

Pulmonary emphysema is a disease characterized by alveolar cellular loss and inflammation. Recently, excessive apoptosis of structural alveolar cells has emerged as a major mechanism in the development of emphysema. Here, we investigated the proapoptotic and monocyte chemoattractant cytokine endothelial monocyte-activating protein 2 (EMAPII). Lung-specific overexpression of EMAPII in mice caused simplification of alveolar structures, apoptosis, and macrophage accumulation, compared with that in control transgenic mice. Additionally, in a mouse model of cigarette smoke-induced (CS-induced) emphysema, EMAPII levels were significantly increased in murine lungs. This upregulation was necessary for emphysema development, as neutralizing antibodies to EMAPII resulted in reduced alveolar cell apoptosis, inflammation, and emphysema-associated structural changes in alveoli and small airways and improved lung function. The mechanism of EMAPII upregulation involved an apoptosis-dependent feed-forward loop, since caspase-3 instillation in the lung markedly increased EMAPII expression, while caspase inhibition decreased its production, even in transgenic EMAPII mice. These findings may have clinical significance, as both current smokers and ex-smoker chronic obstructive pulmonary disease (COPD) patients had increased levels of secreted EMAPII in the bronchoalveolar lavage fluid compared with that of nonsmokers. In conclusion, we suggest that EMAPII perpetuates the mechanism of CS-induced lung emphysema in mice and, given its secretory nature, is a suitable target for neutralization antibody therapy.


Assuntos
Citocinas/fisiologia , Proteínas de Neoplasias/fisiologia , Enfisema Pulmonar/genética , Proteínas de Ligação a RNA/fisiologia , Poluição por Fumaça de Tabaco/efeitos adversos , Adulto , Animais , Anticorpos Neutralizantes/uso terapêutico , Apoptose , Câmaras de Exposição Atmosférica , Bronquíolos/efeitos dos fármacos , Bronquíolos/patologia , Líquido da Lavagem Broncoalveolar/química , Caspase 3/toxicidade , Inibidores de Caspase , Citocinas/uso terapêutico , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Humanos , Imunização Passiva , Macrófagos/patologia , Masculino , Camundongos , Camundongos Endogâmicos , Camundongos Transgênicos , Pessoa de Meia-Idade , Proteínas de Neoplasias/uso terapêutico , Alvéolos Pulmonares/metabolismo , Alvéolos Pulmonares/patologia , Enfisema Pulmonar/etiologia , Enfisema Pulmonar/metabolismo , Proteínas de Ligação a RNA/uso terapêutico , Ratos , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/fisiologia , Proteínas Recombinantes de Fusão/uso terapêutico , Fumar/efeitos adversos , Fumar/metabolismo , Adulto Jovem
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