RESUMEN
Glioblastoma is an aggressive brain cancer characterized by diffuse infiltration. Infiltrated glioma cells persist in the brain post-resection where they interact with glial cells and experience interstitial fluid flow. We use patient-derived glioma stem cells and human glial cells (i.e., astrocytes and microglia) to create a four-component 3D model of this environment informed by resected patient tumors. We examine metrics for invasion, proliferation, and putative stemness in the context of glial cells, fluid forces, and chemotherapies. While the responses are heterogeneous across seven patient-derived lines, interstitial flow significantly increases glioma cell proliferation and stemness while glial cells affect invasion and stemness, potentially related to CCL2 expression and differential activation. In a screen of six drugs, we find in vitro expression of putative stemness marker CD71, but not viability at drug IC50, to predict murine xenograft survival. We posit this patient-informed, infiltrative tumor model as a novel advance toward precision medicine in glioblastoma treatment.
RESUMEN
Objective: To investigate the effect of occupational factor exposures on carotid atherosclerosis (CAS) in steel workers. Methods: A frequency matched case-control study was conducted by age and factory proportion. A total of 1 033 workers with carotid atherosclerosis diagnosed by ultrasonography examination from February to June 2017 were selected as case group, and 1 033 workers without carotid atherosclerosis indicated by physical examination at the same time were selected as control group. The basic information of the workers, such as diet pattern, lifestyle, serum biochemical index and occupation history, were collected. The effects of occupational hazards on carotid atherosclerosis were analyzed by univariate and multivariate logistic regression analyses. The combined effects of various occupational hazards on carotid atherosclerosis were evaluated by environmental risk score (ERS). Results: High temperature, noise, occupational stress and night shift days increased the risk of CAS. With the increase of cumulative high temperature and noise exposure, occupational stress and night shift days, the risk of CAS increased (trend text: χ(2)=37.53, P<0.01; χ(2)=16.98, P<0.01; χ(2)=13.93, P<0.01; χ(2)=5.59, P<0.05). After adjustment of covariates, compared with P(20) group, the risk of carotid artery in P(40), P(60), P(80) and P(100) groups were as follows: high temperature 1.61 (1.19-2.18), 1.69 (1.25-2.30), 1.84 (1.36-2.49), 2.43 (1.77-3.34); noise 1.70 (1.15-2.52), 1.68 (1.20-2.35), 1.80 (1.34-2.42), 2.23 (1.53-3.26); occupational stress 1.39 (1.04- 1.86), 1.41 (1.06-1.89), 1.45(1.09-1.95), 1.48 (1.10-1.98); night shift days 1.58 (1.08-2.33), 1.66 (1.12-2.47), 1.55 (1.04-2.31), 1.76 (1.17-2.64). The results of the environmental risk score showed that the risk of carotid atherosclerosis increased with the increase of ERS (ERS trend text χ(2)=51.61, P<0.01); RCS results showed that there was a linear relationship between ERS and CAS in steel workers(P<0.01). Linear dose-response relationship existed between ERS and CAS (nonlinear test P>0.05). Conclusions: High temperature, noise, occupational stress and night shift days were related to carotid atherosclerosis. Linear dose-response relationship existed between ERS and CAS in steel workers.
Asunto(s)
Enfermedades de las Arterias Carótidas , Obreros Metalúrgicos , Enfermedades Profesionales , Exposición Profesional , Enfermedades de las Arterias Carótidas/epidemiología , Estudios de Casos y Controles , Humanos , Enfermedades Profesionales/epidemiología , Exposición Profesional/efectos adversos , Factores de RiesgoRESUMEN
Objective: To analyze the relationship between cumulative high temperature exposure and hypertension among steel workers. Methods: We conducted a survey among 7 660 production workers (7 023 males and 637 females) in a large steel mill during February-June 2017 and March-July 2018. Basic information, blood pressure, occupational history and high temperature data in workplace were collected through questionnaire survey, physical measurement and occupational exposure detection. Restrictive cubic spline model was used to analyze the dose-response relationship between cumulative high temperature exposure and hypertension, and the correlation between cumulative high temperature exposure and workers' hypertension was analyzed by logistic regression. Results: The median number of cumulative high temperature exposure was 626.56 â·year and 49.20% of workers were exposed to high temperature. The hypertension prevalence rate was 25.60% among the workers. According to the results of the spline model, the cumulative high temperature exposure was divided into three sections: <292.61, 292.61~<720.13, ≥720.13 â·year. Taking the exposure <292.61 â·year group as a reference, the risk of steel workers' hypertension in the 292.61~<720.13 â·year group and ≥720.13 â·year group is 1.44 and 2.17 times that of the reference group (P<0.05) . Conclusion: There was a nonlinear dose-response relationship between cumulative high temperature exposure and steelworkers' hypertension. With the increase of cumulative high temperature exposure, the risk of steelworkers' hypertension increases.
Asunto(s)
Hipertensión , Exposición Profesional , Presión Sanguínea , Femenino , Humanos , Hipertensión/epidemiología , Masculino , Acero , TemperaturaRESUMEN
Objective: To investigate the prevalence of pre-diabetes mellitus (PDM) and the impact of occupation-related factors on PDM, among workers from a steel company in Tangshan city, Hebei province. Methods: Clustering sampling method was used to select a steel company and to carry out occupational health-related physical checkup programs for eligible workers who had working in this company for longer than one year. The study began in February and ended up in June, 2017. Workers who were with FPG level as ≤6.9 mmol/L, and free from diabetes, were selected as the subjects for this study. Questionnaires were used and physical examinations and FPG testing conducted. Results: The total number of subjects in this study was 4 173, of which 2 648 appeared as pre-diabetic, with the prevalence rate as 63.4%. Increase of the PDM prevalence was in parallel with the length of service, among the workers. The risk for the pre-diabetes in those who worked more than 8 hours per day was 1.696 times higher than those who worked less than or equal to 8 h/d (95%CI:1.517-1.937). Compared with those workers without exposures to heat, noise or carbon monoxides, the proportion of pre-diabetes appeared higher in workers exposed to heat, noise or CO with OR=1.782 (95%CI: 1.205-2.636), 1.815 (95%CI: 1.209-2.794) and 1.653 (95%CI: 1.158-2.361), respectively. Risks for those who were exposed to heat or noise were higher than those who were free from exposure to any occupational hazards (OR=2.098, 95%CI: 1.296-3.397). Prevalence rates of pre-diabetes in those who were exposed to heat, noise or CO, were higher than that those who were not. Conclusion: Working hours and exposures to heat, noise or CO appeared as influential factors on PDM.
Asunto(s)
Metalurgia , Enfermedades Profesionales/epidemiología , Exposición Profesional/efectos adversos , Estado Prediabético/epidemiología , China/epidemiología , Humanos , Hierro , AceroRESUMEN
Objective: To investigate the association between occupational stress and carotid atherosclerosis among the workers in a steel plant. Methods: In October 2018, a total of 2947 workers from a steel plant, who underwent occupational health examination in the center for occupational health examination from March to May 2017, were selected as subjects. Job Content Questionnaire (JCQ) and Effort-Reward Imbalance (ERI) were used to investigate the job content and the degree of occupational stress. According to the results of carotid artery examination, the subjects were divided into normal carotid artery group with 2013 workers, increased carotid intima-media thickness (IMT) group with 277 workers, stable plaque group with 236 workers, and unstable plaque group with 421 workers. A unified questionnaire was distributed to each group, and related physical and biochemical examinations were performed. A multivariate unconditional logistic regression analysis was performed to investigate the risk factors for unstable plaque. Results: There were significant differences between the groups in sex, educational level, marital status, work in shifts, smoking, drinking, age, and working years (P<0.05) , while there was no significant difference in exercise between groups (P>0.05) . Based on the JCQ score, 761 (25.8%) had no stress, 959 (32.5%) had mild stress, 699 (23.7%) had moderate stress, and 528 (17.9%) had severe stress; based on the ERI score, 2526 (85.7%) had high effort and low reward and 421 (14.3%) did not have the high-effort and low-reward conditions. There was a significant difference in the composition of JCQ and ERI scores between groups (P<0.05) . Moderate stress (odds ratio [OR]=1.695) , severe stress (OR=5.443) , ERI (OR=7.391) , work in shift (OR=1.784) , old age (OR=1.009) , high systolic blood pressure (OR=1.105) , high fasting blood glucose (OR=1.212) , abnormal total cholesterol (OR=3.693) , abnormal apolipoprotein B (OR=39.215) , and abnormal high-sensitivity C-reactive protein (OR=1.632) were the risk factors for unstable plaque. Conclusion: Occupational stress may be involved in the development of carotid atherosclerosis.
Asunto(s)
Enfermedades de las Arterias Carótidas/epidemiología , Metalurgia , Estrés Laboral/epidemiología , Grosor Intima-Media Carotídeo , Estudios Transversales , Humanos , AceroRESUMEN
Objective: To improve the diagnosis and therapy of childhood pulmonary thromboembolism (PTE) by analyzing the clinical features of this rare condition. Methods: A total of 8 pediatric patients (4 males, 4 females) with PTE diagnosed in the Children's Hospital of Zhejiang University School of Medicine from March, 2014 to March, 2019 were enrolled. The clinical manifestation, laboratory results, imaging findings, diagnosis and treatment were summarized. Results: Among these 8 cases, aged from 9 hours to 14 years and 10 months. Fever was found in 4 cases, cough aggravation in 4, short of breath in 3, chest pain in 2, abdominal and back pain in one, hemoptysis in 2, cyanosis in 1, and edema of lower extremities in 2. Physical examination found decreased breath sound in 2 cases, phlegm rale in 3, and pleural friction rub in one. Pleural effusion was found in 5 cases by ultrasound. Plasma D-dimer increased in 6 cases (0.66-9.96 mg/L) and hypersensitive C-reactive protein elevated in 5 cases (10.78-78.00 mg/L). Chest enhanced CT showed pulmonary artery or venous filling defects, including pulmonary artery embolism in 7 cases and pulmonary vein embolism in one. The primary disease of these patients included Mycoplasma Pneumoniae pneumonia in 4 cases, nephritis in 2 and postoperative congenital heart disease in 2. Apart from one case who withdrew the treatment and was discharged, the other 7 patients received anticoagulant treatment had good outcome. Conclusions: For children with Mycoplasma pneumoniae pneumonia, immune disorders, long-term hormone therapy, cardiovascular invasive operation or other high-risk factors, PTE should be considered when fever, cough aggravation, short of breath, chest and back pain with pleural effusion are present. Chest enhanced CT scan should be performed as soon as possible, and anticoagulation should be started once the diagnosis is confirmed.
Asunto(s)
Neumonía por Mycoplasma/complicaciones , Arteria Pulmonar/diagnóstico por imagen , Embolia Pulmonar/diagnóstico , Adolescente , Dolor en el Pecho/diagnóstico por imagen , Niño , Femenino , Productos de Degradación de Fibrina-Fibrinógeno/metabolismo , Humanos , Masculino , Derrame Pleural , Neumonía por Mycoplasma/diagnóstico , Embolia Pulmonar/complicaciones , Tomografía Computarizada por Rayos X , Resultado del TratamientoRESUMEN
Objective: To screen sensitive indicators of renal injury in lead workers using benchmark dose method. Methods: Of the 486 subjects,116 did not occupationally contact to lead as a control. The blood lead was considered as exposure biomarker, while Uß2-MG and UNAG as effect biomarkers for renal injury. The BMD and BMDL of blood lead were estimated at the 10% benchmark response using BMDS Version 2.6. Results: There was statistical rise in blood lead between the lead group and control group (P<0.05) ; and the blood lead level was divided into four groups by quarterback spacing method, among which UNAG was statistically different (P<0.05) . There was an increased prevalence of abnormal rates of Uß2-MG and UNAG with increasing blood lead concentration (P<0.05) , after trend chi-square test. BMD and BMDL of UNAG and Uß2-MG were 602.784/431.838 µg/L and 130.398/100.981 µg/L caculated by Log-Probit model, respectively. Conclusions: Occupational lead exposure may cause kidney damage, and UNAG could be as a more sensitive marker for monitoring early renal injury than Uß2-MG.
Asunto(s)
Enfermedades Renales/inducido químicamente , Plomo/toxicidad , Enfermedades Profesionales/inducido químicamente , Exposición Profesional/efectos adversos , Adulto , Benchmarking , Biomarcadores/análisis , Humanos , Riñón , Plomo/sangre , Prevalencia , Microglobulina beta-2RESUMEN
OBJECTIVE: The correlation of vitamin D receptor (VDR) gene polymorphism and blood lead level had been in doult, which allowed us to write this article. METHODS: Relevant studies about the blood lead and VDR B/b gene polymorphism which published from 1979-2015, were searched in multiple bibliographic databases, such as: CNKI, Wanfang Database, PUBMED. Of the ten references selceted, three were divided into two groups which were classified as different researches, so there were thirteen studies in the meta-analysis. According to the level of blood lead, the studies were analyzed by three groups: normal group, low dose grou and high dose group. The analysis was performed by stata 12.0 software. RESULTS: The blood lead level of VDR B/b genotype was significantly difference in all the three groups (P<0.05) , but there were apparent heterogeneity between normal group and low dose group (P<0.05, I(2)=84.2%; P<0.05, I(2)=88.9%) , except the high dose group (P>0.05, I(2)=12.7%) ; after adjusted, all showed no heterogeneity, and the results were still the same. CONCLUSION: The genotype of VDR may be correlated with blood lead, and the levels of blood lead varied with different genetypes.
Asunto(s)
Receptores de Calcitriol/genética , Genotipo , Humanos , PlomoRESUMEN
Objective: To investigate the relationship between shift work and overweight/obesity in male steel workers. Methods: A questionnaire survey was conducted among the male steel workers selected during health examination in Tangshan Steel Company from March 2015 to March 2016. The relationship between shift work and overweight/obesity in the male steel workers were analyzed by using logistic regression model and restricted cubic splinemodel. Results: A total of 7 262 male steel workers were surveyed, the overall prevalence of overweight/obesitywas 64.5% (4 686/7 262), the overweight rate was 34.3% and the obesity rate was 30.2%, respectively. After adjusting for age, educational level and average family income level per month by multivariable logistic regression analysis, shift work was associated with overweight/obesity and obesity in the male steel workers. The OR was 1.19(95% CI: 1.05-1.35) and 1.15(95% CI: 1.00-1.32). Restricted cubic spline model analysis showed that the relationship between shift work years and overweight/obesity in the male steel workers was a nonlinear dose response one (nonlinear test χ2=7.43, P<0.05). Restricted cubic spline model analysis showed that the relationship between shift work years and obesity in the male steel workers was a nonlinear dose response one (nonlinear test χ2=10.48, P<0.05). Conclusion: Shift work was associated with overweight and obesity in the male steel workers, and shift work years and overweight/obesity had a nonlinear relationship.
Asunto(s)
Obesidad , Sobrepeso , Estudios Transversales , Humanos , Modelos Logísticos , Masculino , Prevalencia , Factores de Riesgo , Acero , Encuestas y Cuestionarios , Tolerancia al Trabajo ProgramadoRESUMEN
Maintaining the proper balance between cell apoptosis and proliferation is required for normal tissue homeostasis; when this balance is disrupted, disease such as pulmonary arterial hypertension (PAH) can result. Activity of K(+) channels plays a major role in regulating the pulmonary artery smooth muscle cell (PASMC) population in the pulmonary vasculature, as they are involved in cell apoptosis, survival and proliferation. PASMCs from PAH patients demonstrate many cellular abnormalities linked to K(+) channels, including decreased K(+) current, downregulated expression of various K(+) channels, and inhibited apoptosis. K(+) is the major intracellular cation, and the K(+) current is a major determinant of cell volume. Apoptotic volume decrease (AVD), an early hallmark and prerequisite of programmed cell death, is characterized by K(+) and Cl(-) efflux. In addition to its role in AVD, cytosolic K(+) can be inhibitory toward endogenous caspases and nucleases and can suppress mitochondrial cytochrome c release. In PASMC, K(+) channel activation accelerates AVD and enhances apoptosis, while K(+) channel inhibition decelerates AVD and inhibits apoptosis. Finally, inhibition of K(+) channels, by increasing cytosolic [Ca(2+)] as a result of membrane depolarization-mediated opening of voltage-dependent Ca(2+) channels, leads to PASMC contraction and proliferation. The goals of this review are twofold: (1) to elucidate the role of K(+) ions and K(+) channels in the proliferation and apoptosis of PASMC, with an emphasis on abnormal cell growth in human and animal models of PAH, and (2) to elaborate upon the targeting of K(+) flux pathways for pharmacological treatment of pulmonary vascular disease.
Asunto(s)
Apoptosis/fisiología , Proliferación Celular/efectos de los fármacos , Miocitos del Músculo Liso/fisiología , Canales de Potasio/fisiología , Arteria Pulmonar/fisiología , Animales , Apoptosis/efectos de los fármacos , Inhibidores de Caspasas , Caspasas/metabolismo , Humanos , Hipertensión Pulmonar/tratamiento farmacológico , Hipertensión Pulmonar/patología , Hipertensión Pulmonar/fisiopatología , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/patología , Potasio/fisiología , Canales de Potasio/efectos de los fármacos , Arteria Pulmonar/citologíaRESUMEN
A proper rate of programmed cell death or apoptosis is required to maintain normal tissue homeostasis. In disease states such as cancer and some forms of hypertension, apoptosis is blocked, resulting in hyperplasia. In neurodegenerative diseases, uncontrolled apoptosis leads to loss of brain tissue. The flow of ions in and out of the cell and its intracellular organelles is becoming increasingly linked to the generation of many of these diseased states. This review focuses on the transport of K(+) across the cell membrane and that of the mitochondria via integral K(+)-permeable channels. We describe the different types of K(+) channels that have been identified, and investigate the roles they play in controlling the different phases of apoptosis: early cell shrinkage, cytochrome c release, caspase activation, and DNA fragmentation. Attention is also given to K(+) channels on the inner mitochondrial membrane, whose activity may underlie anti- or pro-apoptotic mechanisms in neurons and cardiomyocytes.
Asunto(s)
Apoptosis/fisiología , Canales de Potasio/fisiología , Animales , Transporte Biológico/fisiología , Membrana Celular/metabolismo , Citocromos c/metabolismo , Humanos , Membranas Mitocondriales/metabolismo , Modelos Biológicos , Potasio/metabolismoRESUMEN
Activity of voltage-gated K+ (Kv) channels controls membrane potential (E(m)). Membrane depolarization due to blockade of K+ channels in mesenteric artery smooth muscle cells (MASMC) should increase cytoplasmic free Ca2+ concentration ([Ca2+]cyt) and cause vasoconstriction, which may subsequently reduce the mesenteric blood flow and inhibit the transportation of absorbed nutrients to the liver and adipose tissue. In this study, we characterized and compared the electrophysiological properties and molecular identities of Kv channels and examined the role of Kv channel function in regulating E(m) in MASMC and intestinal epithelial cells (IEC). MASMC and IEC functionally expressed multiple Kv channel alpha- and beta-subunits (Kv1.1, Kv1.2, Kv1.3, Kv1.4, Kv1.5, Kv2.1, Kv4.3, and Kv9.3, as well as Kvbeta1.1, Kvbeta2.1, and Kvbeta3), but only MASMC expressed voltage-dependent Ca2+ channels. The current density and the activation and inactivation kinetics of whole cell Kv currents were similar in MASMC and IEC. Extracellular application of 4-aminopyridine (4-AP), a Kv-channel blocker, reduced whole cell Kv currents and caused E(m) depolarization in both MASMC and IEC. The 4-AP-induced E(m) depolarization increased [Ca2+]cyt in MASMC and caused mesenteric vasoconstriction. Furthermore, ingestion of 4-AP significantly reduced the weight gain in rats. These results suggest that MASMC and IEC express multiple Kv channel alpha- and beta-subunits. The function of these Kv channels plays an important role in controlling E(m). The membrane depolarization-mediated increase in [Ca2+]cyt in MASMC and mesenteric vasoconstriction may inhibit transportation of absorbed nutrients via mesenteric circulation and limit weight gain.
Asunto(s)
4-Aminopiridina/farmacología , Depresores del Apetito/farmacología , Células Epiteliales/efectos de los fármacos , Intestinos/efectos de los fármacos , Arterias Mesentéricas/efectos de los fármacos , Músculo Liso Vascular/efectos de los fármacos , Bloqueadores de los Canales de Potasio , Animales , Peso Corporal/efectos de los fármacos , Calcio/metabolismo , Células Cultivadas , Electrofisiología , Células Epiteliales/ultraestructura , Intestinos/citología , Intestinos/ultraestructura , Contracción Isométrica/efectos de los fármacos , Potenciales de la Membrana/efectos de los fármacos , Técnicas de Placa-Clamp , Canales de Potasio/genética , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The balance between apoptosis and proliferation in pulmonary artery smooth muscle cells (PASMCs) is important in maintaining normal pulmonary vascular structure. Activity of voltage-gated K(+) (K(V)) channels has been demonstrated to regulate cell apoptosis and proliferation. Treatment of PASMCs with staurosporine (ST) induced apoptosis in PASMCs, augmented K(V) current [I(K(V))], and induced mitochondrial membrane depolarization. High K(+) (40 mM) negligibly affected the ST-induced mitochondrial membrane depolarization but inhibited the ST-induced I(K(V)) increase and apoptosis. Blockade of K(V) channels with 4-aminopyridine diminished I(K(V)) and markedly decreased the ST-mediated apoptosis. Furthermore, the ST-induced apoptosis was preceded by the increase in I(K(V)). These results indicate that ST induces PASMC apoptosis by activation of plasmalemmal K(V) channels and mitochondrial membrane depolarization. The increased I(K(V)) would result in an apoptotic volume decrease due to a loss of cytosolic K(+) and induce apoptosis. The mitochondrial membrane depolarization would cause cytochrome c release, activate the cytosolic caspases, and induce apoptosis. Inhibition of K(V) channels would thus attenuate PASMC apoptosis.
Asunto(s)
Apoptosis/fisiología , Mitocondrias/metabolismo , Músculo Liso Vascular/citología , Músculo Liso Vascular/metabolismo , Potasio/farmacocinética , Arteria Pulmonar/citología , 4-Aminopiridina/farmacología , Apoptosis/efectos de los fármacos , Células Cultivadas , Inhibidores Enzimáticos/farmacología , Colorantes Fluorescentes/farmacocinética , Humanos , Activación del Canal Iónico/fisiología , Potenciales de la Membrana/fisiología , Técnicas de Placa-Clamp , Bloqueadores de los Canales de Potasio , Canales de Potasio/fisiología , Rodamina 123/farmacocinética , Estaurosporina/farmacologíaRESUMEN
BACKGROUND: Activity of voltage-gated K(+) (K(v)) channels controls membrane potential (E(m)) that regulates cytosolic free Ca(2+) concentration ([Ca(2+)](cyt)) by regulating voltage-dependent Ca(2+) channel function. A rise in [Ca(2+)](cyt) in pulmonary artery smooth muscle cells (PASMCs) triggers vasoconstriction and stimulates PASMC proliferation. Whether c-Jun, a transcription factor that stimulates cell proliferation, affects K(v) channel activity in PASMCs was investigated. METHODS AND RESULTS: Infection of primary cultured PASMCs with an adenoviral vector expressing c-jun increased the protein level of c-Jun and reduced K(v) currents (I(K(V))) compared with control cells (infected with an empty adenovirus). Using single-cell reverse transcription-polymerase chain reaction, we observed that the mRNA level of Kv1.5 and the current density of I(K(V)) were both attenuated in c-jun-infected PASMCs compared with control cells and cells infected with antisense c-jun. Overexpression of c-Jun also upregulated protein expression of Kvbeta(2) and accelerated I(K(V)) inactivation. Furthermore, E(m) was more depolarized and [(3)H]thymidine incorporation was greater in PASMCs infected with c-jun than in control cells and cells infected with antisense c-jun. CONCLUSIONS: These results suggest that c-Jun-mediated PASMC proliferation is associated with a decrease in I(K(V)). The resultant membrane depolarization increases [Ca(2+)](cyt) and enhances PASMC growth.
Asunto(s)
Músculo Liso/efectos de los fármacos , Canales de Potasio/metabolismo , Proteínas Proto-Oncogénicas c-jun/farmacología , Adenoviridae/genética , Animales , División Celular/efectos de los fármacos , Expresión Génica , Técnicas de Transferencia de Gen , Vectores Genéticos , Músculo Liso/citología , Músculo Liso/metabolismo , Canales de Potasio/efectos de los fármacos , Proteínas Proto-Oncogénicas c-jun/genética , Arteria Pulmonar/citología , Arteria Pulmonar/efectos de los fármacos , Ratas , Ratas Sprague-DawleyRESUMEN
Cell shrinkage is an incipient hallmark of apoptosis in a variety of cell types. The apoptotic volume decrease has been demonstrated to attribute, in part, to K+ efflux; blockade of plasmalemmal K+ channels inhibits the apoptotic volume decrease and attenuates apoptosis. Using combined approaches of gene transfection, single-cell PCR, patch clamp, and fluorescence microscopy, we examined whether overexpression of Bcl-2, an anti-apoptotic oncoprotein, inhibits apoptosis in pulmonary artery smooth muscle cells (PASMC) by diminishing the activity of voltage-gated K+ (Kv) channels. A human bcl-2 gene was infected into primary cultured rat PASMC using an adenoviral vector. Overexpression of Bcl-2 significantly decreased the amplitude and current density of Kv currents (I(Kv)). In contrast, the apoptosis inducer staurosporine (ST) enhanced I(Kv). In bcl-2-infected cells, however, the ST-induced increase in I(Kv) was completely abolished, and the ST-induced apoptosis was significantly inhibited compared with cells infected with an empty adenovirus (-bcl-2). Blockade of Kv channels in control cells (-bcl-2) by 4-aminopyridine also inhibited the ST-induced increase in I(Kv) and apoptosis. Furthermore, overexpression of Bcl-2 accelerated the inactivation of I(Kv) and downregulated the mRNA expression of the pore-forming Kv channel alpha-subunits (Kv1.1, Kv1.5, and Kv2.1). These results suggest that inhibition of Kv channel activity may serve as an additional mechanism involved in the Bcl-2-mediated anti-apoptotic effect on vascular smooth muscle cells.
Asunto(s)
Apoptosis , Activación del Canal Iónico , Músculo Liso Vascular/citología , Canales de Potasio/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , 4-Aminopiridina/farmacología , Adenoviridae/genética , Adenoviridae/metabolismo , Animales , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Caspasas/metabolismo , Tamaño de la Célula/efectos de los fármacos , Supervivencia Celular , Células Cultivadas , Citoplasma/metabolismo , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Humanos , Immunoblotting , Pulmón/irrigación sanguínea , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Músculo Liso Vascular/efectos de los fármacos , Técnicas de Placa-Clamp , Bloqueadores de los Canales de Potasio , Canales de Potasio/genética , Proteínas Proto-Oncogénicas c-bcl-2/genética , Ratas , Estaurosporina/farmacologíaRESUMEN
Activity of K(+) channels regulates cytosolic free Ca(2+) concentration by controlling membrane potential. A rise in cytosolic free Ca(2+) concentration in pulmonary artery smooth muscle cells (PASMC) triggers pulmonary vasoconstriction and stimulates PASMC proliferation. Whether serum from children with pulmonary hypertension (PH) secondary to congenital cardiopulmonary diseases contains a factor(s) that inhibits K(+) channel function in PASMC was investigated using patch clamp techniques. PASMC isolated from normal subjects were cultured in media containing 5% serum from normotensive (NPH) or PH patients. Cell growth rate and the currents through voltage-gated K(+) channels were determined and compared between the cells treated with serum from NPH and PH patients. In the absence of growth factors, incubation of PASMC in media containing NPH serum for 48 h increased cell numbers by 2.5-fold, whereas incubation of the cells in media containing PH serum increased cell numbers by 4.5-fold (p < 0.001). Amplitude of whole-cell voltage-gated K(+) currents in NPH serum-treated cells (1119 +/- 222 pA at +80 mV, n = 43) was 3.5-fold greater than in PH serum-treated cells (323 +/- 34 pA, n = 43, p < 0.001). Consistently, membrane potential was much more depolarized in PASMC treated with PH serum (-28 +/- 2 mV, n = 29) than cells treated with NPH-serum (-47 +/- 2 mV, n = 28; p < 0.001). These results suggest that a circulating mitogenic agonist, which induces membrane depolarization by inhibiting voltage-gated K(+) channel activity in PASMC, may be produced or up-regulated in pediatric patients with secondary PH.
Asunto(s)
Sangre , Cardiopatías/congénito , Cardiopatías/complicaciones , Hipertensión Pulmonar/sangre , Músculo Liso Vascular/fisiopatología , Bloqueadores de los Canales de Potasio , Arteria Pulmonar/fisiopatología , División Celular , Células Cultivadas , Niño , Preescolar , Femenino , Humanos , Hipertensión Pulmonar/etiología , Hipertensión Pulmonar/fisiopatología , Lactante , Masculino , Músculo Liso Vascular/citología , Arteria Pulmonar/citologíaRESUMEN
Agonist-induced increases in cytosolic Ca(2+) concentration ([Ca(2+)](cyt)) in pulmonary artery (PA) smooth muscle cells (SMCs) consist of a transient Ca(2+) release from intracellular stores followed by a sustained Ca(2+) influx. Depletion of intracellular Ca(2+) stores triggers capacitative Ca(2+) entry (CCE), which contributes to the sustained increase in [Ca(2+)](cyt) and the refilling of Ca(2+) into the stores. In isolated PAs superfused with Ca(2+)-free solution, phenylephrine induced a transient contraction, apparently by a rise in [Ca(2+)](cyt) due to Ca(2+) release from the intracellular stores. The transient contraction lasted for 3-4 min until the Ca(2+) store was depleted. Restoration of extracellular Ca(2+) in the presence of phentolamine produced a contraction potentially due to a rise in [Ca(2+)](cyt) via CCE. The store-operated Ca(2+) channel blocker Ni(2+) reduced the store depletion-activated Ca(2+) currents, decreased CCE, and inhibited the CCE-mediated contraction. In single PASMCs, we identified, using RT-PCR, five transient receptor potential gene transcripts. These results suggest that CCE, potentially through transient receptor potential-encoded Ca(2+) channels, plays an important role in agonist-mediated PA contraction.