RESUMO
Although chronic progressive cardiovascular diseases such as atherosclerosis are often challenging to fully model in vitro, it has been shown that certain in vitro methods can effectively evaluate some aspects of disease progression. This has been demonstrated in in vitro and in vivo studies of endothelial cells that have illustrated the effects of nitric oxide (NO) production, filamentous actin (F-actin) formation, and cell and actin angle alignment on vascular function and homeostasis. Systems utilising shear flow have been established, in order to create a physiologically relevant environment for cells that require shear flow for homeostasis. Here, we investigated the use of a well-plate microfluidic system and associated devices (0-20dyn/cm²) to demonstrate applied shear effects on primary Human Aortic Endothelial Cells (HAECs). Changes in cell and actin alignment in the direction of flow, real-time production of NO and gross cell membrane shape changes in response to physiological shear flow were observed. These commercial systems have a range of potential applications, including within the consumer and pharmaceutical industries, thereby reducing the dependency on animal testing for regulatory safety assessments.
Assuntos
Aorta/citologia , Técnicas de Cultura de Células/instrumentação , Células Endoteliais/fisiologia , Dispositivos Lab-On-A-Chip , Resistência ao Cisalhamento , HumanosRESUMO
Dysfunction of the right ventricle (RV) is closely related to prognosis for patients with RV failure. Therefore, strategies to improve failing RV function are significant. In a mouse RV failure model, we previously reported that α1-adrenergic receptor (α1-AR) inotropic responses are increased. The present study determined the roles of both predominant cardiac α1-AR subtypes (α1A and α1B) in upregulated inotropy in failing RV. We used the mouse model of bleomycin-induced pulmonary fibrosis, pulmonary hypertension, and RV failure. We assessed the myocardial contractile response in vitro to stimulation of the α1A-subtype (using α1A-subtype-selective agonist A61603) and α1B-subtype [using α1A-subtype knockout mice and nonsubtype selective α1-AR agonist phenylephrine (PE)]. In wild-type nonfailing RV, a negative inotropic effect of α1-AR stimulation with PE (force decreased ≈50%) was switched to a positive inotropic effect (PIE) with bleomycin-induced RV injury. Upregulated inotropy in failing RV occurred with α1A-subtype stimulation (force increased ≈200%), but not with α1B-subtype stimulation (force decreased ≈50%). Upregulated inotropy mediated by the α1A-subtype involved increased activator Ca(2+) transients and increased phosphorylation of myosin regulatory light chain (a mediator of increased myofilament Ca(2+) sensitivity). In failing RV, the PIE elicited by the α1A-subtype was appreciably less when the α1A-subtype was stimulated in combination with the α1B-subtype, suggesting functional antagonism between α1A- and α1B-subtypes. In conclusion, upregulation of α1-AR inotropy in failing RV myocardium requires the α1A-subtype and is opposed by the α1B-subtype. The α1A subtype might be a therapeutic target to improve the function of the failing RV.
Assuntos
Insuficiência Cardíaca/metabolismo , Contração Miocárdica , Receptores Adrenérgicos alfa 1/metabolismo , Disfunção Ventricular Direita/metabolismo , Agonistas de Receptores Adrenérgicos alfa 1/farmacologia , Animais , Sinalização do Cálcio , Células Cultivadas , Insuficiência Cardíaca/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/fisiologia , Miosinas/metabolismo , Receptores Adrenérgicos alfa 1/classificação , Receptores Adrenérgicos alfa 1/genética , Disfunção Ventricular Direita/fisiopatologiaRESUMO
After myocardial infarction, a poorly contracting nonischemic border zone forms adjacent to the infarct. The cause of border zone dysfunction is unclear. The goal of this study was to determine the myofilament mechanisms involved in postinfarction border zone dysfunction. Two weeks after anteroapical infarction of sheep hearts, we studied in vitro isometric and isotonic contractions of demembranated myocardium from the infarct border zone and a zone remote from the infarct. Maximal force development (Fmax) of the border zone myocardium was reduced by 31 ± 2% versus the remote zone myocardium (n = 6/group, P < 0.0001). Decreased border zone Fmax was not due to a reduced content of contractile material, as assessed histologically, and from myosin content. Furthermore, decreased border zone Fmax did not involve altered cross-bridge kinetics, as assessed by muscle shortening velocity and force development kinetics. Decreased border zone Fmax was associated with decreased cross-bridge formation, as assessed from muscle stiffness in the absence of ATP where cross-bridge formation should be maximized (rigor stiffness was reduced 34 ± 6%, n = 5, P = 0.011 vs. the remote zone). Furthermore, the border zone myocardium had significantly reduced phosphorylation of myosin essential light chain (ELC; 41 ± 10%, n = 4, P < 0.05). However, for animals treated with doxycycline, an inhibitor of matrix metalloproteinases, rigor stiffness and ELC phosphorylation were not reduced in the border zone myocardium, suggesting that doxycycline had a protective effect. In conclusion, myofilament dysfunction contributes to postinfarction border zone dysfunction, myofilament dysfunction involves impaired cross-bridge formation and decreased ELC phosphorylation, and matrix metalloproteinase inhibition may be beneficial for limiting postinfarct border zone dysfunction.
Assuntos
Contração Miocárdica , Infarto do Miocárdio/fisiopatologia , Miofibrilas/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Doxiciclina/farmacologia , Masculino , Inibidores de Metaloproteinases de Matriz/farmacologia , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Miofibrilas/efeitos dos fármacos , Cadeias Leves de Miosina/metabolismo , Fosforilação , OvinosRESUMO
α1-Adrenergic receptors (α1-ARs) elicit a negative inotropic effect (NIE) in the mouse right ventricular (RV) myocardium but a positive inotropic effect (PIE) in the left ventricular (LV) myocardium. Effects on myofilament Ca(2+) sensitivity play a role, but effects on Ca(2+) handling could also contribute. We monitored the effects of α1-AR stimulation on contraction and Ca(2+) transients using single myocytes isolated from the RV or LV. Interestingly, for both the RV and LV, we found heterogeneous myocyte inotropic responses. α1-ARs mediated either a PIE or NIE, although RV myocytes had a greater proportion of cells manifesting a NIE (68%) compared with LV myocytes (36%). Stimulation of a single α1-AR subtype (α1A-ARs) with a subtype-selective agonist also elicited heterogeneous inotropic responses, suggesting that the heterogeneity arose from events downstream of the α1A-AR subtype. For RV and LV myocytes, an α1-AR-mediated PIE was associated with an increased Ca(2+) transient and a NIE was associated with a decreased Ca(2+) transient, suggesting a key role for Ca(2+) handling. For RV and LV myocytes, α1-AR-mediated decreases in the Ca(2+) transient were associated with increased Ca(2+) export from the cell and decreased Ca(2+) content of the sarcoplasmic reticulum. In contrast, for myocytes with α1-AR-induced increased Ca(2+) transients, sarcoplasmic reticulum Ca(2+) content was not increased, suggesting that other mechanisms contributed to the increased Ca(2+) transients. This study demonstrates the marked heterogeneity of LV and RV cellular inotropic responses to stimulation of α1-ARs and reveals a new aspect of biological heterogeneity among myocytes in the regulation of contraction.
Assuntos
Agonistas de Receptores Adrenérgicos alfa 1/farmacologia , Ventrículos do Coração/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Receptores Adrenérgicos alfa 1/metabolismo , Antagonistas de Receptores Adrenérgicos alfa 1/farmacologia , Animais , Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Ventrículos do Coração/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Contração Miocárdica/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Miofibrilas/metabolismo , Retículo Sarcoplasmático/metabolismoRESUMO
Atherosclerosis is a complex process involving progressive pathological events, including monocyte adhesion to the luminal endothelial surface. We have developed a functional in vitro adhesion assay using BioFlux microfluidic technology to investigate THP-1 (human acute monocytic leukaemia cell) monocyte adhesion to human aortic endothelial cells (HAECs). The effect of whole smoke conditioned media (WSCM) generated from University of Kentucky reference cigarette 3R4F, electronic cigarette vapour conditioned media (eVCM) from an electronic nicotine delivery system (ENDS) product (Vype ePen) and nicotine on monocyte adhesion to HAECs was evaluated. Endothelial monolayers were grown in microfluidic channels and exposed to 0-1500 ng/mL nicotine or nicotine equivalence of WSCM or eVCM for 24 h. Activated THP-1 cells were perfused through the channels and a perfusion, adhesion period and wash cycle performed four times with increasing adhesion period lengths (10, 20, 30 and 40 min). THP-1 cell adhesion was quantified by counting adherent cells. WSCM induced dose-dependent increases in monocyte adhesion compared to vehicle control. No such increases were observed for eVCM or nicotine. Adhesion regulation was linked to increased ICAM-1 protein expression. Staining of ICAM-1 in HAECs and CD11b (MAC-1) in THP-1 cells demonstrated adhesion molecule co-localisation in BioFlux plates. The ICAM-1 adhesion response to WSCM was downregulated by transfecting HAECs with ICAM-1 siRNA. We conclude that the BioFlux system is able to model human monocyte adhesion to primary human endothelial cells in vitro and WSCM drives the greatest increase in monocyte adhesion via a mechanism involving endothelial ICAM-1 expression.
Assuntos
Adesão Celular/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , Monócitos/efeitos dos fármacos , Nicotina/toxicidade , Fumaça/efeitos adversos , Aorta/citologia , Aorta/efeitos dos fármacos , Moléculas de Adesão Celular , Sistemas Eletrônicos de Liberação de Nicotina , Endotélio Vascular/citologia , Humanos , Molécula 1 de Adesão Intercelular , Microfluídica , Células THP-1 , Produtos do TabacoRESUMO
Chronic exposure to cigarette smoke can lead to endothelial dysfunction and potentially endothelial cell death. Here, we exposed Human Aortic Endothelial Cells (HAECs) to whole smoke conditioned media (WSCM) over a range of nicotine equivalence (n.e.) concentrations (0-8000â¯ng/mL n.e.). After 24â¯h, Neutral Red Uptake (NRU) and reduction of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) to formazan was determined for each exposure concentration and compared to control. IC50 values in the NRU assay were: 4582â¯ng/mL n.e. ± 1074, 4587â¯ng/mL n.e. ± 951, 4993â¯ng/mL n.e. ± 1239 and 4691â¯ng/mL n.e. ± 402 for four HAEC donors. IC50 values in the MTT assay were: 4885â¯ng/mL n.e. ± 1341, 4584â¯ng/mL n.e. ± 806, 5749â¯ng/mL n.e. ± 783 and 5228â¯ng/mL n.e. ± 593 for the four donors. To examine the mechanism responsible for WSCM-induced cytotoxicity in HAECs, flow cytometry using necrosis (Propidium Iodide) and apoptosis (Annexin V) markers were used. Annexin V-positive cell populations increased in a dose dependent manner while increases in PI-positive cell populations occurred at the highest doses of WSCM (5000-8000â¯ng/mL n.e.). Western blotting for cleaved caspase-3 confirmed that apoptosis occurs at >5000â¯ng/mL n.e. WSCM, coinciding with reduced HAEC survival.
Assuntos
Meios de Cultivo Condicionados/toxicidade , Células Endoteliais/efeitos dos fármacos , Nicotina/toxicidade , Fumaça/efeitos adversos , Aorta/citologia , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Células Endoteliais/metabolismo , Humanos , Produtos do TabacoRESUMO
Although epidemiological data and results from rodent studies support an inverse relationship between nicotine consumption and body weight, the molecular mechanisms are poorly understood. CD-1 mice were fed a basal diet or a basal diet containing low or high dose smokeless tobacco blend or high dose nicotine tartrate for 14 weeks. High dose tobacco blend and nicotine tartrate diets vs. basal diet reduced mouse body weight (16.3% and 19.7%, respectively), epididymal (67.6% and 72.5%, respectively) and brown adipose weight (42% and 38%, respectively), epididymal adipocyte size (46.4% and 41.4%, respectively), and brown adipose tissue lipid droplet abundance, with no elevation of adipose tissue inflammation. High dose tobacco blend and nicotine diets also increased mouse physical activity and decreased respiratory exchange ratio, suggesting that high dose nicotine intake induces adipose tissue triglyceride lipolysis to provide fatty acids as an energy source. Both low and high dose tobacco blend and nicotine diet feeding vs. basal diet increased plasma insulin levels (2.9, 3.6 and 4.3-fold, respectively) and improved blood glucose disposal without affecting insulin sensitivity. Feeding of the high dose tobacco blend or nicotine feeding in mice induces body weight loss likely by increasing physical activity and stimulating adipose tissue triglyceride lipolysis.
Assuntos
Adipócitos/efeitos dos fármacos , Nicotiana , Nicotina/farmacologia , Condicionamento Físico Animal , Redução de Peso/efeitos dos fármacos , Adipócitos/citologia , Tecido Adiposo/metabolismo , Animais , Glicemia/metabolismo , Tamanho Celular , Comportamento Alimentar , Insulina/sangue , Resistência à Insulina , Lipólise , Masculino , Camundongos , Triglicerídeos/metabolismoRESUMO
Trichloroethylene (TCE) is an organic solvent and common environmental contaminant. TCE exposure is associated with heart defects in humans and animal models. Primary metabolism of TCE in adult rodent models is by specific hepatic cytochrome P450 enzymes (Lash et al. in Environ Health Perspect 108:177-200, 2000). As association of TCE exposure with cardiac defects is in exposed embryos prior to normal liver development, we investigated metabolism of TCE in the early embryo. Developing chick embryos were dosed in ovo with environmentally relevant doses of TCE (8 and 800 ppb) and RNA was extracted from cardiac and extra-cardiac tissue (whole embryo without heart). Real-time PCR showed upregulation of CYP2H1 transcripts in response to TCE exposure in the heart. No detectable cytochrome expression was found in extra-cardiac tissue. As seen previously, the dose response was non-monotonic and 8 ppb elicited stronger upregulation than 800 ppb. Immunostaining for CYP2C subfamily expression confirmed protein expression and showed localization in both myocardium and endothelium. TCE exposure increased protein expression in both tissues. These data demonstrate that the earliest embryonic expression of phase I detoxification enzymes is in the developing heart. Expression of these CYPs is likely to be relevant to the susceptibility of the developing heart to environmental teratogens.
Assuntos
Sistema Enzimático do Citocromo P-450/biossíntese , Sistema Enzimático do Citocromo P-450/genética , Regulação Enzimológica da Expressão Gênica , Coração/efeitos dos fármacos , Coração/embriologia , Tricloroetileno/administração & dosagem , Animais , Embrião de Galinha , Relação Dose-Resposta a Droga , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Solventes/administração & dosagemRESUMO
Trichloroethylene is an organic solvent used as an industrial degreasing agent. Due to its widespread use and volatile nature, TCE is a common environmental contaminant. Trichloroethylene exposure has been implicated in the etiology of heart defects in human populations and animal models. Recent data suggest misregulation of Ca2+ homeostasis in H9c2 cardiomyocyte cell line after TCE exposure. We hypothesized that misregulation of Ca2+ homeostasis alters myocyte function and leads to changes in embryonic blood flow. In turn, changes in cardiac flow are known to cause cardiac malformations. To investigate this hypothesis, we dosed developing chick embryos in ovo with environmentally relevant doses of TCE (8 and 800 ppb). RNA was isolated from control and treated embryos at specific times in development for real-time PCR analysis of blood flow markers. Effects were observed on Endothelin-1 (ET-1), Nitric Oxide Synthase-3 (NOS-3) and Krüppel-like Factor 2 (KLF2) expression relative to TCE exposure and consistent with reduced flow. Further, we measured function in the developing heart after TCE exposure by isolating cardiomyocytes and measuring half-width of contraction and sarcomere lengths. These functional data showed a significant increase in half-width of contraction after TCE exposure. These data suggest that perturbation of cardiac function contributes to the etiology of congenital heart defects in TCE-exposed embryos.
Assuntos
Embrião não Mamífero/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Coração/efeitos dos fármacos , Solventes/toxicidade , Teratogênicos/toxicidade , Tricloroetileno/toxicidade , Animais , Biomarcadores/metabolismo , Velocidade do Fluxo Sanguíneo , Embrião de Galinha , Relação Dose-Resposta a Droga , Embrião não Mamífero/metabolismo , Endotelina-1/genética , Endotelina-1/metabolismo , Coração/embriologia , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/patologia , Miócitos Cardíacos/fisiologia , Óxido Nítrico Sintase Tipo III/genética , Óxido Nítrico Sintase Tipo III/metabolismo , Estresse MecânicoRESUMO
This study reports that subchronic exposure of Tuxedo, NY concentrated ambient particulates (CAPs) produces neuropathological damage in the brains of Apo E-deficient mice (Apo E-/-). These genetically modified mice are characterized by elevated levels of oxidative stress (OS) in the brain. Microscopic examination of coronal sections of the brain, immunocytochemically stained for dopamineric neurons, indicated that neurons from the substantia nigral nucleus compacta were significantly reduced by 29% in CAPs-exposed Apo E-/- mice relative to air-exposed Apo E-/- controls. In addition, statistically significant increases (p < .05) in immunocytochemically stained astrocytes were noted. The dopaminergic neurons of the nucleus compact are specifically targeted in Parkinson's disease. The present study expands the systems affected by particulate matter to include the brain, and supports an environmental role for the development of neurodegeneration in OS-susceptible individuals.