RESUMO
The European guidelines, which focus on clinical aspects of pulmonary hypertension (PH), provide only minimal information about the pathophysiological concepts of PH. Here, we review this topic in greater detail, focusing on specific aspects in the pathobiology, pathology and genetics, which include mechanisms of vascular inflammation, the role of transcription factors, ion channels/ion channel diseases, hypoxic pulmonary vasoconstriction, genetics/epigenetics, metabolic dysfunction, and the potential future role of histopathology of PH in the modern era of PH therapy. In addition to new insights in the pathobiology of this disease, this working group of the Cologne Consensus Conference also highlights novel concepts and potential new therapeutic targets to further improve the treatment options in PAH.
Assuntos
Conferências de Consenso como Assunto , Hipertensão Pulmonar/genética , Hipertensão Pulmonar/patologia , Ensaios Clínicos como Assunto/métodos , Alemanha/epidemiologia , Humanos , Hipertensão Pulmonar/epidemiologia , Hipertensão Pulmonar/metabolismo , Mediadores da Inflamação/metabolismo , Vasoconstrição/fisiologiaRESUMO
Cancer cells are reprogrammed to consume large amounts of glucose to support anabolic biosynthetic pathways. However, blood perfusion and consequently the supply with glucose are frequently inadequate in solid cancers. PEPCK-M (PCK2), the mitochondrial isoform of phosphoenolpyruvate carboxykinase (PEPCK), has been shown by us and others to be functionally expressed and to mediate gluconeogenesis, the reverse pathway of glycolysis, in different cancer cells. Serine and ribose synthesis have been identified as downstream pathways fed by PEPCK in cancer cells. Here, we report that PEPCK-M-dependent glycerol phosphate formation from noncarbohydrate precursors (glyceroneogenesis) occurs in starved lung cancer cells and supports de novo glycerophospholipid synthesis. Using stable isotope-labeled glutamine and lactate, we show that PEPCK-M generates phosphoenolpyruvate and 3-phosphoglycerate, which are at least partially converted to glycerol phosphate and incorporated into glycerophospholipids (GPL) under glucose and serum starvation. This pathway is required to maintain levels of GPL, especially phosphatidylethanolamine (PE), as shown by stable shRNA-mediated silencing of PEPCK-M in H23 lung cancer cells. PEPCK-M shRNA led to reduced colony formation after starvation, and the effect was partially reversed by the addition of dioleyl-PE. Furthermore, PEPCK-M silencing abrogated cancer growth in a lung cancer cell xenograft model. In conclusion, glycerol phosphate formation for de novo GPL synthesis via glyceroneogenesis is a newly characterized anabolic pathway in cancer cells mediated by PEPCK-M under conditions of severe nutrient deprivation.
Assuntos
Glicerol/metabolismo , Neoplasias/metabolismo , Fosfoenolpiruvato Carboxiquinase (ATP)/metabolismo , Fosfolipídeos/metabolismo , Células A549 , Animais , Glucose/metabolismo , Glutamina/metabolismo , Xenoenxertos , Humanos , Ácido Láctico/metabolismo , Masculino , Camundongos , Camundongos Nus , Fosfoenolpiruvato Carboxiquinase (ATP)/genética , Fosfolipídeos/químicaRESUMO
The cancer stem cell (CSC) and epithelial-to-mesenchymal transition (EMT) models have been closely associated and used to describe both the formation of metastasis and therapy resistance. We established a primary lung cell culture from a patient in a clinically rare and unique situation of primary resistant disease. This culture consisted of two biologically profoundly distinct adenocarcinoma cell subpopulations, which differed phenotypically and genotypically. One subpopulation initiated and sustained in spheroid cell culture (LT22s) whereas the other subpopulation was only capable of growth and proliferation under adherent conditions (LT22a). In contrast to our expectations, LT22s were strongly associated with the epithelial phenotype, and expressed additionally CSC markers ALDH1 and CD133, whereas the LT22a was characterized as mesenchymal with lack of CSC markers. The LT22s cells also demonstrated an invasive behavior and mimicked gland formation. Finally, LT22s were more resistant to Cisplatin than LT22a cells. We demonstrate a primary lung adenocarcinoma cell culture derived from a patient with resistant disease, with epithelial aggressive subpopulation of cells associated with stem cell features and therapy resistance. Our findings challenge the current model associating CSC and disease resistance mainly to mesenchymal cells and may have important clinical implications.
Assuntos
Adenocarcinoma/patologia , Transição Epitelial-Mesenquimal , Neoplasias Pulmonares/patologia , Células-Tronco Neoplásicas/patologia , Antígeno AC133/genética , Antígeno AC133/metabolismo , Adulto , Família Aldeído Desidrogenase 1 , Animais , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Galinhas , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos , Feminino , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Retinal Desidrogenase/genética , Retinal Desidrogenase/metabolismo , Células Tumorais CultivadasRESUMO
Lung cancer is the leading cause of cancer deaths worldwide; survival times are poor despite therapy. The role of the two-pore domain K+ (K2P) channel TASK-1 (KCNK3) in lung cancer is at present unknown. We found that TASK-1 is expressed in non-small cell lung cancer (NSCLC) cell lines at variable levels. In a highly TASK-1 expressing NSCLC cell line, A549, a characteristic pH- and hypoxia-sensitive non-inactivating K+ current was measured, indicating the presence of functional TASK-1 channels. Inhibition of TASK-1 led to significant depolarization in these cells. Knockdown of TASK-1 by siRNA significantly enhanced apoptosis and reduced proliferation in A549 cells, but not in weakly TASK-1 expressing NCI-H358 cells. Na+-coupled nutrient transport across the cell membrane is functionally coupled to the efflux of K+ via K+ channels, thus TASK-1 may potentially influence Na+-coupled nutrient transport. In contrast to TASK-1, which was not differentially expressed in lung cancer vs. normal lung tissue, we found the Na+-coupled nutrient transporters, SLC5A3, SLC5A6, and SLC38A1, transporters for myo-inositol, biotin and glutamine, respectively, to be significantly overexpressed in lung adenocarcinomas. In summary, we show for the first time that the TASK-1 channel regulates apoptosis and proliferation in a subset of NSCLC.
Assuntos
Apoptose , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Proliferação de Células , Neoplasias Pulmonares/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Humanos , Pulmão/metabolismo , Pulmão/patologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Proteínas do Tecido Nervoso/genética , Canais de Potássio de Domínios Poros em Tandem/genética , Interferência de RNA , RNA Interferente Pequeno/genéticaRESUMO
BACKGROUND: Prostaglandin (PG) D2 is an early-phase mediator in inflammation, but its action and the roles of the 2 D-type prostanoid receptors (DPs) DP1 and DP2 (also called chemoattractant receptor-homologous molecule expressed on T(H)2 cells) in regulating macrophages have not been elucidated to date. OBJECTIVE: We investigated the role of PGD2 receptors on primary human macrophages, as well as primary murine lung macrophages, and their ability to influence neutrophil action in vitro and in vivo. METHODS: In vitro studies, including migration, Ca(2+) flux, and cytokine secretion, were conducted with primary human monocyte-derived macrophages and neutrophils and freshly isolated murine alveolar and pulmonary interstitial macrophages. In vivo pulmonary inflammation was assessed in male BALB/c mice. RESULTS: Activation of DP1, DP2, or both receptors on human macrophages induced strong intracellular Ca(2+) flux, cytokine release, and migration of macrophages. In a murine model of LPS-induced pulmonary inflammation, activation of each PGD2 receptor resulted in aggravated airway neutrophilia, tissue myeloperoxidase activity, cytokine contents, and decreased lung compliance. Selective depletion of alveolar macrophages abolished the PGD2-enhanced inflammatory response. Activation of PGD2 receptors on human macrophages enhanced the migratory capacity and prolonged the survival of neutrophils in vitro. In human lung tissue specimens both DP1 and DP2 receptors were located on alveolar macrophages along with hematopoietic PGD synthase, the rate-limiting enzyme of PGD2 synthesis. CONCLUSION: For the first time, our results show that PGD2 markedly augments disease activity through its ability to enhance the proinflammatory actions of macrophages and subsequent neutrophil activation.
Assuntos
Pulmão/imunologia , Pulmão/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Infiltração de Neutrófilos/imunologia , Receptores Imunológicos/metabolismo , Receptores de Prostaglandina/metabolismo , Animais , Sinalização do Cálcio , Quimiotaxia de Leucócito/imunologia , Citocinas/biossíntese , Endotoxinas/efeitos adversos , Endotoxinas/imunologia , Expressão Gênica , Humanos , Mediadores da Inflamação/metabolismo , Pulmão/patologia , Lesão Pulmonar/etiologia , Lesão Pulmonar/metabolismo , Lesão Pulmonar/patologia , Macrófagos/efeitos dos fármacos , Camundongos , Neutrófilos/efeitos dos fármacos , Neutrófilos/imunologia , Neutrófilos/metabolismo , Prostaglandina D2/farmacologia , Receptores Imunológicos/genética , Receptores de Prostaglandina/genética , Síndrome do Desconforto Respiratório/etiologia , Síndrome do Desconforto Respiratório/metabolismo , Síndrome do Desconforto Respiratório/patologia , Fator de Necrose Tumoral alfa/biossínteseRESUMO
BACKGROUND: While respiratory bronchiolitis (RB) is a frequent histopathological finding in smoker's lungs, RB-associated interstitial lung disease (RB-ILD) remains a rare disease. OBJECTIVES: We analyzed how the histological finding of RB was associated with clinical information in a series of 684 consecutive surgical lung biopsies. METHODS: Retrospective analysis with delineation of clinical manifestations, smoking habits, pulmonary function test, and blood gas analysis in patients with RB in surgical lung biopsy. In 240 of these biopsies, RB was diagnosed, and in 146 of these cases a full clinical dataset was available. RESULTS: The final diagnosis of these 146 patients was consistent with RB-ILD (n = 18), pulmonary Langerhans cell histiocytosis (n = 7), various ILD (n = 9), spontaneous pneumothorax (n = 43), traumatic pneumothorax (n = 5), lung cancer (n = 41), various benign lung tumors (n = 8), and chronic pulmonary effusion (n = 15). Smoking history was positive in 93% of patients, 72% revealed centrilobular emphysema in their biopsy, and 58% described dyspnea as the main symptom. Amongst these diagnoses there were significant differences in age and smoking habits, but only small distinctions in pulmonary function test and blood gas analysis. Out of the patients with RB-ILD, 17% developed lung cancer in the later course. CONCLUSION: RB is strongly related to smoking, emphysema, and dyspnea and frequently associated with lung cancer. RB-ILD is a rare disease that may represent a considerable risk for lung cancer. Pulmonary function testing and blood gas analysis do not differ between RB-associated diseases. The finding of RB should prompt further diagnostic workup, and in case of RB-ILD, entail regular screening for lung cancer.
Assuntos
Adenocarcinoma/epidemiologia , Bronquiolite/epidemiologia , Carcinoma de Células Escamosas/epidemiologia , Histiocitose de Células de Langerhans/epidemiologia , Doenças Pulmonares Intersticiais/epidemiologia , Neoplasias Pulmonares/epidemiologia , Pulmão/patologia , Sistema de Registros , Adenocarcinoma/patologia , Adenocarcinoma/fisiopatologia , Adulto , Idoso , Áustria/epidemiologia , Gasometria , Bronquiolite/patologia , Bronquiolite/fisiopatologia , Carcinoma de Células Escamosas/patologia , Carcinoma de Células Escamosas/fisiopatologia , Dispneia/epidemiologia , Dispneia/fisiopatologia , Feminino , Histiocitose de Células de Langerhans/patologia , Histiocitose de Células de Langerhans/fisiopatologia , Humanos , Achados Incidentais , Pulmão/fisiopatologia , Doenças Pulmonares Intersticiais/patologia , Doenças Pulmonares Intersticiais/fisiopatologia , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/fisiopatologia , Macrófagos Alveolares/patologia , Masculino , Pessoa de Meia-Idade , Pneumotórax/epidemiologia , Pneumotórax/patologia , Pneumotórax/fisiopatologia , Enfisema Pulmonar/epidemiologia , Enfisema Pulmonar/patologia , Enfisema Pulmonar/fisiopatologia , Testes de Função Respiratória , Estudos Retrospectivos , Fumar/epidemiologia , Adulto JovemRESUMO
Alterations in extracellular matrix (ECM) have been implicated in the pathophysiology of pulmonary hypertension. Here, we have undertaken a compartment-specific study to elucidate the expression profile of collagens and their processing enzymes in donor and idiopathic pulmonary arterial hypertension (IPAH) pulmonary arteries. Predominant intimal, but also medial and perivascular, remodeling and reduced lumen diameter were detected in IPAH pulmonary arteries. Two-photon microscopy demonstrated accumulation of collagen fibers. Quantification of collagen in pulmonary arteries revealed collagen accumulation mainly in the intima of IPAH pulmonary arteries compared with donors. Laser capture-microdissected pulmonary artery profiles (intima+media and perivascular tissue) were analyzed by real-time PCR for ECM gene expression. In the intima+media of IPAH vessels, collagens (COL4A5, COL14A1, and COL18A1), matrix metalloproteinase (MMP) 19, and a disintegrin and metalloprotease (ADAM) 33 were higher expressed, whereas MMP10, ADAM17, TIMP1, and TIMP3 were less abundant. Localization of COLXVIII, its cleavage product endostatin, and MMP10, ADAM33, and TIMP1 was confirmed in pulmonary arteries by immunohistochemistry. ELISA for collagen XVIII/endostatin demonstrated significantly elevated plasma levels in IPAH patients compared with donors, whereas circulating MMP10, ADAM33, and TIMP1 levels were similar between the two groups. Endostatin levels were correlated with pulmonary arterial wedge pressure, and established prognostic markers of IPAH, right atrial pressure, cardiac index, 6-min walking distance, NH2-terminal pro-brain natriuretic peptide, and uric acid. Expression of unstudied collagens, MMPs, ADAMs, and TIMPs were found to be significantly altered in IPAH intima+media. Elevated levels of circulating collagen XVIII/endostatin are associated with markers of a poor prognosis.
Assuntos
Colágeno/biossíntese , Regulação da Expressão Gênica , Hipertensão Pulmonar/metabolismo , Artéria Pulmonar/metabolismo , Túnica Íntima/metabolismo , Biomarcadores/sangue , Endostatinas/sangue , Feminino , Humanos , Hipertensão Pulmonar/diagnóstico , Hipertensão Pulmonar/patologia , Masculino , Metaloproteases , Pessoa de Meia-Idade , Prognóstico , Artéria Pulmonar/patologia , Túnica Íntima/patologiaRESUMO
CONTEXT: Megakaryocytes are the "hallmark" of Philadelphia chromosome-negative myeloproliferative neoplasms, such as essential thrombocythemia, polycythemia vera, and primary myelofibrosis; their morphology in correlation with Janus kinase 2 (JAK2 V617F) mutation as well as clinical and laboratory parameters remains unknown. OBJECTIVE: To assess the morphology of megakaryocytes in bone marrow biopsies of patients with and without JAK2 V617F mutation. DESIGN: Assessment of morphologic features of megakaryocytes in 112 bone marrow biopsies (52 essential thrombocythemia, 38 polycythemia vera, and 22 primary myelofibrosis) and correlation with clinical and laboratory data. RESULTS: JAK2 V617F mutation was detected in 24 of 52 essential thrombocythemia cases (46.2%), 36 of 38 polycythemia vera cases (97.5%), and 14 of 22 primary myelofibrosis cases (63.6%). By investigating morphometric and clinical parameters using multivariate analysis, we found that higher hemoglobin concentration, higher white blood cell counts, and lower platelet counts were significantly associated with JAK2 V617F. Striking morphologic similarities were found between polycythemia vera JAK2 V617F and primary myelofibrosis JAK2 V617F concerning the localization and cytoplasmic size of megakaryocytes. Although polycythemia vera JAK2 V617F and essential thrombocythemia JAK2 V617F shared similarities in localization, distribution, and amount of megakaryocytes, morphology was different. Megakaryocytic morphology also differed between primary myelofibrosis JAK2 V617F and essential thrombocythemia JAK2 V617F. CONCLUSION: Our results indicate that primary myelofibrosis JAK2 V617F and polycythemia vera JAK2 V617F share pathogenetic pathways, resulting in morphologically similar megakaryocytes.
Assuntos
Janus Quinase 2/genética , Megacariócitos/patologia , Mutação/genética , Policitemia Vera/patologia , Mielofibrose Primária/patologia , Trombocitemia Essencial/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Biópsia , Medula Óssea/patologia , Feminino , Hemoglobinas/metabolismo , Humanos , Contagem de Leucócitos , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Contagem de Plaquetas , Policitemia Vera/genética , Mielofibrose Primária/genética , Trombocitemia Essencial/genéticaRESUMO
BACKGROUND: Hypoxia-induced genes are potential targets in cancer therapy. Responses to hypoxia have been extensively studied in vitro, however, they may differ in vivo due to the specific tumor microenvironment. In this study gene expression profiles were obtained from fresh human lung cancer tissue fragments cultured ex vivo under different oxygen concentrations in order to study responses to hypoxia in a model that mimics human lung cancer in vivo. METHODS: Non-small cell lung cancer (NSCLC) fragments from altogether 70 patients were maintained ex vivo in normoxia or hypoxia in short-term culture. Viability, apoptosis rates and tissue hypoxia were assessed. Gene expression profiles were studied using Affymetrix GeneChip 1.0 ST microarrays. RESULTS: Apoptosis rates were comparable in normoxia and hypoxia despite different oxygenation levels, suggesting adaptation of tumor cells to hypoxia. Gene expression profiles in hypoxic compared to normoxic fragments largely overlapped with published hypoxia-signatures. While most of these genes were up-regulated by hypoxia also in NSCLC cell lines, membrane metallo-endopeptidase (MME, neprilysin, CD10) expression was not increased in hypoxia in NSCLC cell lines, but in carcinoma-associated fibroblasts isolated from non-small cell lung cancers. High MME expression was significantly associated with poor overall survival in 342 NSCLC patients in a meta-analysis of published microarray datasets. CONCLUSIONS: The novel ex vivo model allowed for the first time to analyze hypoxia-regulated gene expression in preserved human lung cancer tissue. Gene expression profiles in human hypoxic lung cancer tissue overlapped with hypoxia-signatures from cancer cell lines, however, the elastase MME was identified as a novel hypoxia-induced gene in lung cancer. Due to the lack of hypoxia effects on MME expression in NSCLC cell lines in contrast to carcinoma-associated fibroblasts, a direct up-regulation of stroma fibroblast MME expression under hypoxia might contribute to enhanced aggressiveness of hypoxic cancers.
Assuntos
Biomarcadores Tumorais/metabolismo , Carcinoma Pulmonar de Células não Pequenas/enzimologia , Fibroblastos/enzimologia , Neoplasias Pulmonares/enzimologia , Neprilisina/metabolismo , Células Estromais/enzimologia , Apoptose , Biomarcadores Tumorais/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/mortalidade , Carcinoma Pulmonar de Células não Pequenas/patologia , Hipóxia Celular , Linhagem Celular Tumoral , Sobrevivência Celular , Fibroblastos/patologia , Perfilação da Expressão Gênica/métodos , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/mortalidade , Neoplasias Pulmonares/patologia , Neprilisina/genética , Análise de Sequência com Séries de Oligonucleotídeos , Células Estromais/patologia , Técnicas de Cultura de Tecidos , Regulação para CimaRESUMO
The pathology of pulmonary hypertension includes numerous abnormalities of the intima, media, and adventitia of the pulmonary vascular tree. A recently completed systematic analysis revealed high variability of morphologic measurements within a given pulmonary arterial hypertension (PAH) lung and among all PAH lungs, as well as distinct pathologic subphenotypes, and included a subset of lungs lacking intimal and medial remodeling. There was correlation between perivascular inflammation, remodeling, and hemodynamics. This article summarizes the pathologic features of the normal and abnormal pulmonary circulation, and correlations with animal models.
Assuntos
Hipertensão Pulmonar/patologia , Pulmão/patologia , Animais , Modelos Animais de Doenças , Hipertensão Pulmonar Primária Familiar , Hemodinâmica , Humanos , Hipertensão Pulmonar/fisiopatologia , Inflamação/patologia , Pulmão/irrigação sanguínea , Pulmão/fisiopatologiaRESUMO
OBJECTIVE: Docosahexaenoic acid (DHA), a member of n-3 polyunsaturated fatty acids (n-3 PUFA) is a potent regulator of molecular events implicated in cardiovascular health. In a previous study we found that Ca(2+)-dependent oxidative stress is the central and initial event responsible for induction of unfolded protein response (UPR), cell cycle arrest and apoptosis in DHA treated primary human smooth muscle cells isolated from small pulmonary artery (hPASMC). In the present study we examined the impact of heme oxygenase (HO)-1, induced by DHA, on DHA-induced oxidative stress, UPR, cell proliferation and apoptosis in hPASMC. METHODS & RESULTS: DHA led to a time- and concentration-dependent increase in HO-1 mRNA and protein levels in hPASMC. The DHA-induced HO-1 upregulation could be attenuated by preincubation of cells with a strong antioxidant Tempol or by siRNA-mediated depletion of nuclear factor erythroid 2-related factor-2 (Nrf2). In DHA-treated hPASMC, depletion of HO-1 by siRNA-mediated silencing resulted in increased levels of reactive oxygen species (ROS) and increased duration of UPR, the latter revealed by monitoring of spliced X-box binding protein 1 (XBP-1) variant. Moreover, HO-1 silencing augmented apoptosis in DHA-treated hPASMC as found by increased numbers of cleaved caspase-3-positive cells. HO-1 silencing did not affect proliferation of hPASMC exposed to DHA. CONCLUSION: Our results indicate that DHA-induced, ROS-dependent upregulation of HO-1 attenuates oxidative stress, UPR and apoptosis in DHA-treated hPASMC.
Assuntos
Ácidos Docosa-Hexaenoicos/metabolismo , Regulação Enzimológica da Expressão Gênica , Heme Oxigenase-1/metabolismo , Músculo Liso Vascular/efeitos dos fármacos , Antioxidantes/química , Apoptose , Caspase 3/metabolismo , Proliferação de Células , Sobrevivência Celular , Óxidos N-Cíclicos/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Relação Dose-Resposta a Droga , Inativação Gênica , Humanos , Músculo Liso Vascular/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo , Artéria Pulmonar/efeitos dos fármacos , Artéria Pulmonar/metabolismo , RNA Interferente Pequeno/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Fatores de Transcrição de Fator Regulador X , Marcadores de Spin , Fatores de Tempo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Resposta a Proteínas não Dobradas , Proteína 1 de Ligação a X-BoxRESUMO
Despite the importance of pulmonary veins in normal lung physiology and the pathobiology of pulmonary hypertension with left heart disease (PH-LHD), pulmonary veins remain largely understudied. Difficult to identify histologically, lung venous endothelium or smooth muscle cells display no unique characteristic functional and structural markers that distinguish them from pulmonary arteries. To address these challenges, we undertook a search for unique molecular markers in pulmonary veins. In addition, we addressed the expression pattern of a candidate molecular marker and analyzed the structural pattern of vascular remodeling of pulmonary veins in a rodent model of PH-LHD and in lung tissue of patients with PH-LHD obtained at time of placement on a left ventricular assist device. We detected urokinase plasminogen activator receptor (uPAR) expression preferentially in normal pulmonary veins of mice, rats, and human lungs. Expression of uPAR remained elevated in pulmonary veins of rats with PH-LHD; however, we also detected induction of uPAR expression in remodeled pulmonary arteries. These findings were validated in lungs of patients with PH-LHD. In selected patients with sequential lung biopsy at the time of removal of the left ventricular assist device, we present early data suggesting improvement in pulmonary hemodynamics and venous remodeling, indicating potential regression of venous remodeling in response to assist device treatment. Our data indicate that remodeling of pulmonary veins is an integral part of PH-LHD and that pulmonary veins share some key features present in remodeled yet not normotensive pulmonary arteries.
Assuntos
Endotélio Vascular/patologia , Cardiopatias/patologia , Hipertensão Pulmonar/patologia , Artéria Pulmonar/patologia , Veias Pulmonares/patologia , Adolescente , Adulto , Idoso , Animais , Western Blotting , Estudos de Casos e Controles , Proliferação de Células , Criança , Endotélio Vascular/metabolismo , Feminino , Imunofluorescência , Cardiopatias/complicações , Cardiopatias/metabolismo , Coração Auxiliar , Hemodinâmica , Humanos , Hipertensão Pulmonar/etiologia , Hipertensão Pulmonar/terapia , Técnicas Imunoenzimáticas , Microdissecção e Captura a Laser , Pulmão/irrigação sanguínea , Pulmão/metabolismo , Pulmão/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Estudos Prospectivos , Artéria Pulmonar/metabolismo , Veias Pulmonares/metabolismo , RNA Mensageiro/genética , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Adulto JovemRESUMO
BACKGROUND: Malignant mesothelioma is a highly aggressive tumor arising from mesothelial-lined surfaces, most often in the pleura cavities. Antifolates belong to the most effective cytotoxic drugs for malignant pleural mesothelioma (MPM) treatment. Pemetrexed is an antifolate inhibiting different folate pathway genes (thymidylate synthase [TS], dihydrofolate reductase, glycinamide ribonucleotide formyltransferase [GARFT], and aminoimidazole carboxamide ribonucleotide formyltransferase, [AICARFT]). Increased activity of pemetrexed occurs by folylpolyglutamate synthetase (FPGS), intracellular transport by reduced folate carrier (RFC). The aim of the study was to explore potential correlations between TS, GARFT, AICARFT, RFC, and FPGS levels in MPM and associations with clinical benefit from pemetrexed treatment. METHODS: Samples from 63 patients were tested using immunohistochemistry (IHC) and quantitative polymerase chain reaction(qPCR) for expression levels of TS, GARFT, AICARFT, RFC, and FPGS. Clinical data were evaluated to determine associations between efficacy of pemetrexed and enzyme expression levels. Evaluation of expression levels was done through TaqMan-based qPCR, and IHC was evaluated semiquantitatively by using the H-score. RESULTS: qPCR analysis showed no difference in expression pattern of GARFT and AICARFT. IHC analysis revealed a heterogeneous staining pattern for all the enzymes. No significant association was found between TS expression and survival or objective response of the tumors after pemetrexed treatment. FPGS (p = 0.0111) and RFC (p = 0.0088) mRNA expression levels were strongly associated with overall survival in these patients. CONCLUSIONS: Our results reveal that in pemetrexed-treated MPMs TS expression levels have no influence on patient outcome. Furthermore, GARFT and AICARFT were homogeneously expressed in the patient samples. Folate uptake mechanisms by RFC and activation by FPGS were associated with clinical benefit from pemetrexed treatment.
Assuntos
Antimetabólitos Antineoplásicos/uso terapêutico , Glutamatos/uso terapêutico , Guanina/análogos & derivados , Mesotelioma/enzimologia , Mesotelioma/terapia , Neoplasias Pleurais/enzimologia , Neoplasias Pleurais/terapia , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Expressão Gênica , Guanina/uso terapêutico , Humanos , Estimativa de Kaplan-Meier , Masculino , Mesotelioma/genética , Pessoa de Meia-Idade , Pemetrexede , Peptídeo Sintases/genética , Peptídeo Sintases/metabolismo , Fosforribosilaminoimidazolcarboxamida Formiltransferase/genética , Fosforribosilaminoimidazolcarboxamida Formiltransferase/metabolismo , Fosforribosilglicinamido Formiltransferase/genética , Fosforribosilglicinamido Formiltransferase/metabolismo , Neoplasias Pleurais/genética , Modelos de Riscos Proporcionais , RNA Mensageiro/metabolismo , Proteína Carregadora de Folato Reduzido/genética , Proteína Carregadora de Folato Reduzido/metabolismo , Timidilato Sintase/genética , Timidilato Sintase/metabolismoRESUMO
The potassium channel TWIK-related acid sensitive potassium (TASK)-1 channel, together with other potassium channels, controls the low resting tone of pulmonary arteries. The Src family tyrosine kinase (SrcTK) may control potassium channel function in human pulmonary artery smooth muscle cells (hPASMCs) in response to changes in oxygen tension and the clinical use of a SrcTK inhibitor has resulted in partly reversible pulmonary hypertension. This study aimed to determine the role of SrcTK in hypoxia-induced inhibition of potassium channels in hPASMCs. We show that SrcTK is co-localised with the TASK-1 channel. Inhibition of SrcTK decreases potassium current density and results in considerable depolarisation, while activation of SrcTK increases potassium current in patch-clamp recordings. Moderate hypoxia and the SrcTK inhibitor decrease the tyrosine phosphorylation state of the TASK-1 channel. Hypoxia also decreases the level of phospho-SrcTK (tyr419) and reduces the co-localisation of the TASK-1 channel and phospho-SrcTK. Corresponding to this, hypoxia reduces TASK-1 currents before but not after SrcTK inhibition and, in the isolated perfused mouse lung, SrcTK inhibitors increase pulmonary arterial pressure. We propose that the SrcTK is a crucial factor controlling potassium channels, acting as a cofactor for setting a negative resting membrane potential in hPASMCs and a low resting pulmonary vascular tone.
Assuntos
Canais de Potássio/fisiologia , Artéria Pulmonar/fisiologia , Quinases da Família src/fisiologia , Células Cultivadas , Humanos , Miócitos de Músculo Liso/metabolismo , Artéria Pulmonar/citologia , Artéria Pulmonar/enzimologiaAssuntos
Mutação em Linhagem Germinativa , Melanoma/genética , Mesotelioma/genética , Neoplasias Peritoneais/genética , Neoplasias Pleurais/genética , Neoplasias Cutâneas/genética , Proteínas Supressoras de Tumor/genética , Ubiquitina Tiolesterase/genética , Adulto , Feminino , Heterozigoto , Humanos , Masculino , Melanoma/patologia , Mesotelioma/patologia , Pessoa de Meia-Idade , Linhagem , Neoplasias Peritoneais/patologia , Neoplasias Pleurais/patologia , Fatores de Risco , Neoplasias Cutâneas/patologia , População Branca/genéticaRESUMO
Chronic thromboembolic pulmonary hypertension (CTEPH) is a rare disease with persistent thrombotic occlusion or stenosis of the large pulmonary arteries resulting in pulmonary hypertension. Surgical removal of the neointimal layer of these vessels together with the non-resolved thrombus consisting of organized collagen-rich fibrotic areas with partly recanalized regions is the treatment of choice (pulmonary endarterectomy, PEA). The present study investigates endothelial cells isolated from such material as well as factors present in the surgical PEA material, which may contribute to impairment of recanalization and thrombus non-resolution. We observed muscularized vessels and non-muscularized vessels in the PEA material. The isolated endothelial cells from the PEA material showed significantly different calcium homeostasis as compared to pulmonary artery endothelial cells (hPAECs) from normal controls. In the supernatant (ELISA) as well as on the tissue level (histochemical staining) of the PEA material, platelet factor 4 (PF4), collagen type I and interferon-gamma-inducible 10 kD protein (IP-10) were detected. CXCR3, the receptor for PF4 and IP-10, was particularly elevated in the distal parts of the PEA material as compared to human control lung (RT-PCR). PF4, collagen type I and IP-10 caused significant changes in calcium homeostasis and affected the cell proliferation, migration and vessel formation in hPAECs. The presence of angiostatic factors like PF4, collagen type I and IP-10, as recovered from the surgical PEA material from CTEPH patients, may lead to changes in calcium homeostasis and endothelial dysfunction.
Assuntos
Endarterectomia , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Hipertensão Pulmonar/metabolismo , Hipertensão Pulmonar/fisiopatologia , Cálcio/metabolismo , Movimento Celular/genética , Movimento Celular/fisiologia , Proliferação de Células , Células Cultivadas , Quimiocina CXCL10/metabolismo , Ensaio de Imunoadsorção Enzimática , Humanos , Imuno-Histoquímica , Receptores CXCR3/metabolismoRESUMO
RATIONALE: The impact of modern treatments of pulmonary arterial hypertension (PAH) on pulmonary vascular pathology remains unknown. OBJECTIVES: To assess the spectrum of pulmonary vascular remodeling in the modern era of PAH medication. METHODS: Assessment of pulmonary vascular remodeling and inflammation in 62 PAH and 28 control explanted lungs systematically sampled. MEASUREMENTS AND MAIN RESULTS: Intima and intima plus media fractional thicknesses of pulmonary arteries were increased in the PAH group versus the control lungs and correlated with pulmonary hemodynamic measurements. Despite a high variability of morphological measurements within a given PAH lung and among all PAH lungs, distinct pathological subphenotypes were detected in cohorts of PAH lungs. These included a subset of lungs lacking intima or, most prominently, media remodeling, which had similar numbers of profiles of plexiform lesions as those in lungs with more pronounced remodeling. Marked perivascular inflammation was present in a high number of PAH lungs and correlated with intima plus media remodeling. The number of profiles of plexiform lesions was significantly lower in lungs of male patients and those never treated with prostacyclin or its analogs. CONCLUSIONS: Our results indicate that multiple features of pulmonary vascular remodeling are present in patients treated with modern PAH therapies. Perivascular inflammation may have an important role in the processes of vascular remodeling, all of which may ultimately lead to increased pulmonary artery pressure. Moreover, our study provides a framework to interpret and design translational studies in PAH.
Assuntos
Hipertensão Pulmonar/patologia , Artéria Pulmonar/patologia , Adulto , Idoso , Remodelação das Vias Aéreas , Análise de Variância , Anti-Hipertensivos/uso terapêutico , Estudos de Coortes , Epoprostenol/uso terapêutico , Feminino , Humanos , Hipertensão Pulmonar/complicações , Hipertensão Pulmonar/tratamento farmacológico , Inflamação/complicações , Inflamação/patologia , Pulmão/irrigação sanguínea , Pulmão/ultraestrutura , Masculino , Pessoa de Meia-Idade , Neovascularização Patológica/patologia , Distribuição por SexoRESUMO
AIMS: Low-grade flat ductal intraepithelial neoplasia (DIN1a, flat epithelial atypia) is one of the earliest morphologically recognizable neoplastic lesions of the breast. Frequently, it occurs concomitantly with lobular intraepithelial neoplasia (LIN). We aimed to elucidate chromosomal aberrations in these early neoplastic breast lesions with the use of array comparative genomic hybridization analysis. METHODS AND RESULTS: Laser capture microdissection of 12 archival formalin-fixed, paraffin-embedded specimens harbouring foci of both DIN1a and LIN was performed. All analysed cases of DIN1a and LIN showed chromosomal gains and losses. The aberration encountered most often was loss of 16q, noted in seven DIN1a (70% of those successfully examined) and 10 LIN (91%) cases. The next most common alteration was a gain on 1q, noted in four DIN1a (40%) and seven LIN (64%) cases. CONCLUSIONS: The results show concurrent chromosomal aberrations of 1q gains and 16q losses in several cases with coexisting LIN and DIN1a. These aberrations are known to be common in low-grade invasive (ductal and lobular) carcinomas as well as in more advanced (conventional) types of low-grade ductal intraepithelial neoplasia (DIN) (low-grade ductal carcinoma in situ). Our results raise the possibility of similar molecular-genetic pathways in coexisting LIN and low-grade flat DIN.
Assuntos
Neoplasias da Mama/genética , Carcinoma Intraductal não Infiltrante/genética , Carcinoma Lobular/genética , Aberrações Cromossômicas , Neoplasias Primárias Múltiplas/genética , Neoplasias da Mama/patologia , Carcinoma Intraductal não Infiltrante/patologia , Carcinoma Lobular/patologia , Hibridização Genômica Comparativa , Feminino , Humanos , Neoplasias Primárias Múltiplas/patologiaAssuntos
Adenocarcinoma Mucinoso/secundário , Adenocarcinoma/secundário , Neoplasias do Colo/patologia , Mutação , Proteínas Proto-Oncogênicas/genética , Proteínas ras/genética , Adenocarcinoma/genética , Adenocarcinoma Mucinoso/genética , Células Clonais/patologia , Neoplasias do Colo/genética , Humanos , Linfonodos/patologia , Metástase Linfática , Masculino , Pessoa de Meia-Idade , Mosaicismo , Proteínas Proto-Oncogênicas p21(ras)RESUMO
CONTEXT: Formalin-fixed, paraffin-embedded tissue is the routine processing method for diagnostics practiced in pathology departments worldwide. OBJECTIVE: To determine the potential value of non-cross-linking, formalin-free tissue fixation for diagnostics in pathology and proteomic investigations. DESIGN: We tested 3 commercially available, formalin-free tissue fixatives-FineFIX, RCL2, and HOPE-in lung cancer specimens from 10 patients. The fixatives were evaluated for their effects on tissue morphology, protein recovery, and immunoreactivity for a selected panel of proteins differently expressed in lung cancer, using immunohistochemistry and Western blotting. RESULTS: Tumor-cell analysis with hematoxylin-eosin worked equally well for all tested fixatives when compared with the standard formalin-fixed, paraffin-embedded procedure. Movat pentachrome stains showed comparable results for the different matrices and cellular proteins analyzed. The RCL2 (P = .01) and HOPE fixatives (P = .03) improved protein recovery when compared with formalin-fixed, paraffin-embedded or frozen tissues. Our data clearly show that the fixatives evaluated influenced immunoreactivity to matched, formalin-fixed, paraffin-embedded lung cancer tissue. In particular, membrane-bound proteins, such as epidermal growth factor receptor EGFR, can be detected more efficiently by immunohistochemistry and Western blotting. CONCLUSION: We have demonstrated that formalin-free fixatives have the potential in routine pathology and research to replace formalin in histomorphology and protein preservation.