RESUMEN
UNLABELLED: Purpose/Aim of Study: The renin angiotensin system is involved in experimentally induced lung fibrosis. Angiotensin (ANG)-II is profibrotic. Angiotensin converting enzyme-2 (ACE-2) cleaves ANG-II and is thus protective. ACE-2 has recently been reported to be significantly decreased under hyperoxic conditions. Hyperoxia is linked to Bronchopulmonary Dysplasia and lung fibrosis. Fetal lung cells normally do not undergo fibrotic changes with physiologic hypoxemia. We hypothesized that hypoxia prior to hyperoxic exposure in fetal lung fibroblasts (IMR-90 cell line) might be protective by preventing ACE-2 downregulation. MATERIALS AND METHODS: IMR-90 cells were exposed to hypoxia (1%O2/99%N2) followed by hyperoxia (95%O2/5%CO2) or normoxia (21%O2) in vitro. Cells and culture media were recovered separately for assays of ACE-2, TNF-α-converting enzyme (TACE), αSmooth muscle actin (αSMA)-myofibroblast marker-, N-cadherin, and ß-catenin immunoreactive protein. RESULTS: ACE-2 significantly increased when IMR-90 were hypoxic prior to hyperoxic exposure with no recovery. In contrast to hyperoxia alone, ACE-2 did not decrease when IMR-90 were hypoxic prior to hyperoxic exposure with recovery. TACE/ADAM17 protein and mRNA were significantly decreased under these conditions. αSMA N-cadherin, and ß-catenin proteins were significantly decreased with or without normoxic recovery. CONCLUSIONS: Hypoxia prior to hyperoxic exposure of fetal lung fibroblasts prevented ACE-2 downregulation and decreased ADAM17/TACE protein and mRNA. αSMA, N-cadherin, and ß-catenin were also significantly decreased under these conditions.
Asunto(s)
Regulación hacia Abajo/fisiología , Fibroblastos/fisiología , Hiperoxia/fisiopatología , Hipoxia/fisiopatología , Pulmón/fisiología , Peptidil-Dipeptidasa A/metabolismo , Actinas/metabolismo , Angiotensina II/metabolismo , Enzima Convertidora de Angiotensina 2 , Cadherinas/metabolismo , Células Cultivadas , Fibroblastos/metabolismo , Humanos , Hiperoxia/metabolismo , Hipoxia/metabolismo , Pulmón/metabolismo , Fibrosis Pulmonar/metabolismo , Fibrosis Pulmonar/fisiopatología , Factor de Necrosis Tumoral alfa/metabolismo , beta Catenina/metabolismoRESUMEN
BACKGROUND: Angiotensin (ANG) II is involved in experimental hyperoxia-induced lung fibrosis. Angiotensin-converting enzyme-2 (ACE-2) degrades ANG II and is thus protective, but is downregulated in adult human and experimental lung fibrosis. Hyperoxia is a known cause of chronic fibrotic lung disease in neonates, but the role of ACE-2 in neonatal lung fibrosis is unknown. We hypothesized that ACE-2 in human fetal lung cells might be downregulated by hyperoxic gas. METHODS: Fetal human lung fibroblast IMR90 cells were exposed to hyperoxic (95% O2/5% CO2) or normoxic (21% O2/5% CO2) gas in vitro. Cells and culture media were recovered separately for assays of ACE-2 enzymatic activity, mRNA, and immunoreactive protein. RESULTS: Hyperoxia decreased ACE-2 immunoreactive protein and enzyme activity in IMR90 cells (both P < 0.01), but did not change ACE-2 mRNA. ACE-2 protein was increased in the cell supernatant, suggesting protease-mediated ectodomain shedding. TAPI-2, an inhibitor of TNF-α-converting enzyme (TACE/ADAM17), prevented both the decrease in cellular ACE-2 and the increase in soluble ACE-2 (both P < 0.05). CONCLUSION: These data show that ACE-2 is expressed in fetal human lung fibroblasts but is significantly decreased by hyperoxic gas. They also suggest that hyperoxia decreases ACE-2 through a shedding mechanism mediated by ADAM17/TACE.
Asunto(s)
Fibroblastos/metabolismo , Hiperoxia/metabolismo , Pulmón/embriología , Peptidil-Dipeptidasa A/metabolismo , Proteínas ADAM/metabolismo , Proteína ADAM17 , Angiotensina II/metabolismo , Enzima Convertidora de Angiotensina 2 , Dióxido de Carbono/metabolismo , Supervivencia Celular , Fibrosis/patología , Gases , Humanos , Ácidos Hidroxámicos/metabolismo , Hiperoxia/patología , Pulmón/citología , Oxígeno/química , ARN Mensajero/metabolismo , Factores de Tiempo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Single-molecule experimental methods have provided new insights into biomolecular function, dynamic disorder, and transient states that are all invisible to conventional measurements. A novel, nonfluorescent single-molecule technique involves attaching single molecules to single-walled carbon nanotube field-effective transistors (SWNT FETs). These ultrasensitive electronic devices provide long-duration, label-free monitoring of biomolecules and their dynamic motions. However, generalization of the SWNT FET technique first requires design rules that can predict the success and applicability of these devices. Here, we report on the transduction mechanism linking enzymatic processivity to electrical signal generation by a SWNT FET. The interaction between SWNT FETs and the enzyme lysozyme was systematically dissected using eight different lysozyme variants synthesized by protein engineering. The data prove that effective signal generation can be accomplished using a single charged amino acid, when appropriately located, providing a foundation to widely apply SWNT FET sensitivity to other biomolecular systems.
Asunto(s)
Muramidasa/química , Muramidasa/metabolismo , Nanotubos de Carbono/química , Ingeniería de Proteínas , Transducción de Señal , Modelos Moleculares , Transistores ElectrónicosRESUMEN
Earlier work showed that apoptosis of alveolar epithelial cells (AECs) in response to endogenous or xenobiotic factors is regulated by autocrine generation of angiotensin (ANG) II and its counterregulatory peptide ANG1-7. Mutations in surfactant protein C (SP-C) induce endoplasmic reticulum (ER) stress and apoptosis in AECs and cause lung fibrosis. This study tested the hypothesis that ER stress-induced apoptosis of AECs might also be regulated by the autocrine ANGII/ANG1-7 system of AECs. ER stress was induced in A549 cells or primary cultures of human AECs with the proteasome inhibitor MG132 or the SP-C BRICHOS domain mutant G100S. ER stress activated the ANGII-generating enzyme cathepsin D and simultaneously decreased the ANGII-degrading enzyme ACE-2, which normally generates the antiapoptotic peptide ANG1-7. TAPI-2, an inhibitor of ADAM17/TACE, significantly reduced both the activation of cathepsin D and the loss of ACE-2. Apoptosis of AECs induced by ER stress was measured by assays of mitochondrial function, JNK activation, caspase activation, and nuclear fragmentation. Apoptosis induced by either MG132 or the SP-C BRICHOS mutant G100S was significantly inhibited by the ANG receptor blocker saralasin and was completely abrogated by ANG1-7. Inhibition by ANG1-7 was blocked by the specific mas antagonist A779. These data show that ER stress-induced apoptosis is mediated by the autocrine ANGII/ANG1-7 system in human AECs and demonstrate effective blockade of SP-C mutation-induced apoptosis by ANG1-7. They also suggest that therapeutic strategies aimed at administering ANG1-7 or stimulating ACE-2 may hold potential for the management of ER stress-induced fibrotic lung disorders.
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Angiotensina I/farmacología , Antihipertensivos/farmacología , Apoptosis/efectos de los fármacos , Estrés del Retículo Endoplásmico/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Fragmentos de Péptidos/farmacología , Alveolos Pulmonares/efectos de los fármacos , Angiotensina II/farmacología , Antagonistas de Receptores de Angiotensina/farmacología , Antineoplásicos/farmacología , Comunicación Autocrina , Células Cultivadas , Células Epiteliales/patología , Humanos , Leupeptinas/farmacología , Alveolos Pulmonares/patología , Proteína C Asociada a Surfactante Pulmonar/antagonistas & inhibidores , Proteína C Asociada a Surfactante Pulmonar/genética , Proteína C Asociada a Surfactante Pulmonar/metabolismo , Receptores de Angiotensina/química , Transducción de Señal/efectos de los fármacos , Vasoconstrictores/farmacologíaRESUMEN
Alveolar epithelial type II cells, a major source of angiotensin-converting enzyme (ACE)-2 in the adult lung, are normally quiescent but actively proliferate in lung fibrosis and downregulate this protective enzyme. It was, therefore, hypothesised that ACE-2 expression might be related to cell cycle progression. To test this hypothesis, ACE-2 mRNA levels, protein levels and enzymatic activity were examined in fibrotic human lungs and in the alveolar epithelial cell lines A549 and MLE-12 studied at postconfluent (quiescent) versus subconfluent (proliferating) densities. ACE-2 mRNA, immunoreactive protein and enzymatic activity were all high in quiescent cells, but were severely downregulated or absent in actively proliferating cells. Upregulation of the enzyme in cells that were progressing to quiescence was completely inhibited by the transcription blocker actinomycin D or by SP600125, an inhibitor of c-Jun N-terminal kinase (JNK). In lung biopsy specimens obtained from patients with idiopathic pulmonary fibrosis, immunoreactive enzyme was absent in alveolar epithelia that were positive for proliferation markers, but was robustly expressed in alveolar epithelia devoid of proliferation markers. These data explain the loss of ACE-2 in lung fibrosis and demonstrate cell cycle-dependent regulation of this protective enzyme by a JNK-mediated transcriptional mechanism.
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Ciclo Celular , Fibrosis Pulmonar Idiopática/metabolismo , Fibrosis Pulmonar Idiopática/fisiopatología , Pulmón/patología , Peptidil-Dipeptidasa A/metabolismo , Enzima Convertidora de Angiotensina 2 , Animales , Apoptosis , Línea Celular Tumoral , Proliferación Celular , Regulación hacia Abajo , Citometría de Flujo , Regulación de la Expresión Génica , Humanos , Inmunohistoquímica , Pulmón/metabolismo , Ratones , Péptidos/química , Alveolos Pulmonares/metabolismo , ARN Mensajero/metabolismoRESUMEN
BACKGROUND: Single nucleotide polymorphisms (SNPs) in angiotensinogen (AGT) at positions -20 and -6 are associated with increased severity and progression of various fibrotic diseases. Our earlier work demonstrated that the progression of idiopathic pulmonary fibrosis (IPF) was associated with the A-6 allele. This study examined the hypothesis that the homozygous CC genotype at -20 and the AA genotype at -6 would confer worse measures of pulmonary function (measured by pulmonary function tests) in IPF. METHODS: Multiple logistic regression analysis was applied to a NIH Lung Tissue Research Consortium cohort and a Spanish cohort, while also adjusting for covariates to determine the effects of these SNPs on measures of pulmonary function. RESULTS: Analysis demonstrated that the CC genotype at -20 was strongly associated with reduced diffusing capacity in males in both cohorts (p = 0.0028 for LTRC and p = 0.017 for the Spanish cohort). In females, the AA genotype was significantly associated with lower FVC (p = 0.0082) and V alv (p = 0.022). In males, the haplotype CA at -20 and -6 in AGT was also strongly associated with reduced diffusing capacity in both cohorts. CONCLUSIONS: This study is the first to demonstrate an association of AGT polymorphisms (-20A > C and -6G > A) with lower measures of pulmonary function in IPF. It is also the first to relate the effect of gender in lung fibrosis with polymorphisms in AGT.
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Angiotensinógeno/genética , Fibrosis Pulmonar Idiopática/genética , Pulmón/fisiopatología , Polimorfismo de Nucleótido Simple , Regiones Promotoras Genéticas , Capacidad de Difusión Pulmonar/genética , Anciano , Distribución de Chi-Cuadrado , Femenino , Volumen Espiratorio Forzado/genética , Frecuencia de los Genes , Predisposición Genética a la Enfermedad , Haplotipos , Homocigoto , Humanos , Fibrosis Pulmonar Idiopática/fisiopatología , Funciones de Verosimilitud , Modelos Logísticos , Masculino , Persona de Mediana Edad , Fenotipo , Factores Sexuales , España , Estados Unidos , Capacidad Vital/genéticaRESUMEN
A 23-year-old woman was referred to the allergy and immunology clinic for recurrent abdominal, cutaneous and joint swelling and pain with a history of mucosal infections since childhood. Her history and clinical findings were suggestive of two rare and complex disorders, hereditary angioedema (HAE) and Ehlers-Danlos syndrome (EDS). Her recurrent episodes of abdominal and joint pain were initially misattributed to more common diagnoses such as esophagitis, depression and chronic pain syndrome. However, the coexistence of HAE and EDS likely contributed to a delay in diagnoses as the combination of these two rare but overlapping disorders is less understood by physicians. She had persistently low levels of C4 and C1-esterase inhibitor (C1-INH) with low to low-normal C1-esterase function, normal C1Q and no C1Q antibodies. In the setting of recurrent abdominal pain with cutaneous swelling, this supported the diagnosis of HAE type I. The increase in joint extensibility with recurrent shoulder subluxations since childhood was a manifestation of EDS. Although no known genetic mutations were identified for EDS, her diagnosis was confirmed by a geneticist based on her clinical phenotype. Before the diagnosis of HAE and EDS, our patient had at least 100 visits/year to the emergency department/hospitalisations for these recurrent symptoms. After starting on C1-INH replacement therapy, the frequency has decreased 10-fold. She also noted a 70% improvement in her quality of life. Familiarity with these rare disorders will assist healthcare providers in recognising HAE and EDS and include them as part of their differential diagnoses. Early diagnosis is important for a patient's well-being as both these chronic disorders have been associated with poor quality of life. Additionally, proper diagnoses will reduce healthcare costs by preventing unnecessary procedures due to misdiagnoses. Proper treatment will help to decrease hospitalisations and avoidance of life-threatening consequences (such as asphyxiation from fatal laryngeal attacks of HAE and rupture of aneurysms in EDS).
Asunto(s)
Dolor Abdominal/etiología , Angioedemas Hereditarios/complicaciones , Artralgia/etiología , Edema/etiología , Síndrome de Ehlers-Danlos/complicaciones , Femenino , Humanos , Recurrencia , Adulto JovenRESUMEN
CONTEXT: For many patients, end-of-life care (EOLC) wishes are unknown and are generally only brought up during healthcare crises. During such healthcare episodes, loved ones are often distraught, and as such, can find it difficult to focus on details surrounding the event. The best place for these discussions may be in non-acute settings including primary care clinics. The purpose of this study was to examine how well a sample of patients (N = 177) in three Michigan-based primary care teaching clinics thought they and their loved ones were prepared in terms of having their EOLC wishes known. METHODS: Prospective data were collected from three Internal Medicine teaching clinics in the Metro-Detroit area through an anonymous written survey addressing EOLC issues in a 16-item cross-sectional study. Respondents were adult patients at one of three participating primary care clinics. Perceived preparedness for EOLC was measured by: 1) possibly having had a prior EOLC discussion with a healthcare provider 2) having created an Advance Directive, such as a Durable Power of Attorney (DPOA) or Living Will with medical decision preferences, 3) reported preferences for quality versus quantity of life, and 4) preferences for CPR and other specific life-sustaining interventions. RESULTS: In this sample, 77 (43.5%) of 177 respondents had discussed their EOLC wishes with a provider. Regarding Advance Directives, 63/177 (35.6%) had established a DPOA, and 59/177 (22%) had made a Living Will. The majority of respondents preferred quality over quantity of life. The most difficult EOLC questions included the decision for CPR, tracheostomy, and PEG tube placement. CONCLUSIONS: Based on these results, EOLC discussions probably occur infrequently in the primary care or other healthcare settings. Most survey responses indicated that sample patients were unprepared concerning the details of future EOLC decisions. Engagement of such discussions should be a part of routine visits in the primary care clinic and should be re-visited when there is a change in a patient's health. Further larger-scale studies using validated surveys are required in this vitally important area of practice. Key Words: advance care planning, end-of-life care conversations, primary care.
RESUMEN
An established body of literature supports the hypothesis that activation of a local tissue angiotensin (ANG) system in the extravascular tissue compartment of the lungs is required for lung fibrogenesis. Transcriptional activation of the angiotensinogen (AGT) gene is believed to be a critical and necessary step in this activation. This paper summarizes the data in support of this theory and discusses transcriptional regulation of AGT, with an emphasis on lung AGT synthesis as a determinant of fibrosis severity. Genetic data linking AGT polymorphisms to the severity of disease in Idiopathic Pulmonary Fibrosis are also discussed.