RESUMEN
Portal hypertension (PHT) is a common complication of liver cirrhosis and significantly increases morbidity and mortality. Abrogation of PHT using NSAIDs has demonstrated that prostacyclin (PGI(2)), a direct downstream metabolic product of cyclooxygenase (COX) activity, is an important mediator in the development of experimental and clinical PHT. However, the role of COX isoforms in PGI(2) biosynthesis and PHT is not fully understood. Prehepatic PHT was induced by portal vein ligation (PVL) in wild-type, COX-1(-/-), and COX-2(-/-) mice treated with and without COX-2 (NS398) or COX-1 (SC560) inhibitors. Hemodynamic measurements and PGI(2) biosynthesis were determined 1-7 days after PVL or sham surgery. Gene deletion or pharmacological inhibition of COX-1 or COX-2 attenuated but did not ameliorate PGI(2) biosynthesis after PVL or prevent PHT. In contrast, treatment of COX-1(-/-) mice with NS398 or COX-2(-/-) mice with SC560 restricted PGI(2) biosynthesis and abrogated the development of PHT following PVL. In conclusion, either COX-1 or COX-2 can mediate elevated PGI(2) biosynthesis and the development of experimental prehepatic PHT. Consequently, PGI(2) rather then COX-selective drugs are indicated in the treatment of PHT. Identification of additional target sites downstream of COX may benefit the >27,000 patients whom die annually from cirrhosis in the United States alone.
Asunto(s)
Ciclooxigenasa 1/metabolismo , Ciclooxigenasa 2/metabolismo , Epoprostenol/biosíntesis , Hipertensión Portal/metabolismo , Animales , Inhibidores de la Ciclooxigenasa/farmacología , Epoprostenol/química , Ratones , Ratones Noqueados , Estructura Molecular , Nitrobencenos , Isoformas de Proteínas/metabolismo , Pirazoles , SulfonamidasRESUMEN
AIMS: The excretion of low molecular weight heparin (LMWH) in breast milk was investigated in 15 lactating mothers after Caesarean section. METHODS: Blood and milk samples were collected before and 3-4 h after once daily routine subcutaneous injection of 2500 IU dalteparin. Anti-Xa activity was measured by an assay utilizing prolonged clotting times in plasma or breast milk as an index of LMWH activity. RESULTS: Plasma anti-Xa activities ranged from 0.074 to 0.308 IU ml(-1) of plasma. Anti-Xa activities in breast milk ranged from < 0.005-0.037 IU ml(-1) of milk. This is equivalent to a milk/plasma ratio of < 0.025-0.224. CONCLUSIONS: Therefore, it appears highly unlikely that puerperal thromboprophylaxis with LMWH has any clinically relevant effect on the nursing infant.
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Anticoagulantes/farmacocinética , Dalteparina/farmacocinética , Leche Humana/metabolismo , Anticoagulantes/administración & dosificación , Cesárea , Dalteparina/administración & dosificación , Factor Xa/metabolismo , Femenino , Humanos , Inyecciones Subcutáneas , Lactancia , Periodo Posparto , Factores de TiempoRESUMEN
OBJECTIVE: Chronic alcohol abuse is one of the major contributors to the onset and progression of hepatocellular carcinoma (HCC). We have previously identified increased expression and function of inhibitory guanine nucleotide regulatory proteins (Gi-proteins) in primary human and animal models of HCC. Stimulation of Gi-proteins in HCC stimulates cell mitogenesis, an effect not observed in hepatocytes. The aim of this study was to determine the effect of ethanol and ethanol metabolism on Gi-protein expression in an experimental model of HCC. DESIGN: Pharmacological agents that inhibit alcohol metabolism were used in conjunction with ethanol or ethanol metabolites. We were also able to assess the relative contribution of alcohol and acetaldehyde, the major metabolite of alcohol, on Gi-protein expression in HCC and hepatocytes. METHODS: These studies used the rat hepatic tumorigenic H4IIE cell line in conjunction with isolated rat hepatocytes. Cells were cultured in vitro and exposed to ethanol, ethanol in the presence of an alcohol dehydrogenase (ADH) inhibitor, or acetaldehyde for varying lengths of time. Ethanol metabolism and changes in Gi-protein expression were subsequently determined by assay. RESULTS: Exposure to ethanol alone led to significant dose and time dependent increases in Gialpha1/2 and Gialpha3 protein and mRNA expression in HCC cells. In contrast, ethanol failed to alter Gialpha1/2, and only moderately affected Gialpha3 protein expression in isolated cultured hepatocytes. Pretreatment of HCC cells and hepatocytes with 4-methyl pyrazole (4-MP, 10 microm) significantly inhibited alcohol metabolism. Treatment of HCC cells with 4-MP inhibited changes in Gi-protein expression following exposure to ethanol (25 mm, 24 h). In addition, the increased expression of Gi-proteins observed after exposure to ethanol in HCC were mimicked by direct exposure of HCC cells to acetaldehyde in a dose and time dependent manner. CONCLUSIONS: These data suggest that alcohol metabolites, not alcohol, lead to increased Gi-protein expression in HCC in vitro. Ethanol and ethanol metabolites, in contrast, fail to significantly alter Gialpha1/2 protein expression in hepatocytes. These data may have significant implications in HCC progression in vivo.
Asunto(s)
Alcohol Deshidrogenasa/antagonistas & inhibidores , Carcinoma Hepatocelular/enzimología , Etanol/efectos adversos , Proteínas de Unión al GTP/metabolismo , Neoplasias Hepáticas/enzimología , Animales , Northern Blotting , Western Blotting , Etanol/metabolismo , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Técnicas In Vitro , Modelos Animales , Ratas , Células Tumorales CultivadasRESUMEN
Right-lobe living donor liver transplantation has emerged as an alternative to cadaveric transplantation. An appreciation of the unique anatomy and behavior of the right lobe has emerged and has precipitated technical modifications. Living donors underwent right lobectomy, including preservation of significant inferior hepatic veins. The parenchyma was divided following a plane approximating the right border of the posterior two thirds of the midhepatic vein (MHV), but deviating anteriorly to include the distal one third of the MHV with the graft. Large venous tributaries from segment VIII were preserved. Anastomosis in the recipient was accomplished by means of complete cavoplasty. Significant inferior veins, tributaries to the MHV, and the distal portion of the MHV were reconstructed when technically possible. Forty-eight right-lobe resections and transplantations were performed in the manner described. There were no donor complications attributable to the technique. Forty-six of the 48 recipients are alive, and 44 of the 46 surviving patients have their original graft. Venous tributaries from segment VIII and/or the distal portion of the MHV were reconstructed in only 3 patients. Outflow obstruction was recognized intraoperatively in 2 patients; 1 patient had a caval web excised and the other patient required revision of the main anastomosis. Neither organ was lost. There were no other significant venous complications. The incidence of ascites was the same as that in recipients of whole organs. These methods of parenchymal transection and venous reconstruction resulted in a low rate of complications. The wide anastomosis and collateral pathways between the MHV and right hepatic vein seem to be more critical than reconstruction of tributaries from segment VIII or the distal MHV.
Asunto(s)
Hepatectomía/métodos , Trasplante de Hígado/métodos , Hígado/irrigación sanguínea , Donadores Vivos , Adulto , Anastomosis Quirúrgica , Femenino , Rechazo de Injerto , Supervivencia de Injerto , Venas Hepáticas/diagnóstico por imagen , Humanos , Hígado/anatomía & histología , Circulación Hepática , Trasplante de Hígado/mortalidad , Masculino , Pronóstico , Radiografía , Regeneración/fisiología , Estudios Retrospectivos , Tasa de Supervivencia , Resultado del TratamientoRESUMEN
Since oxidative DNA damage plays a role in experimental carcinogen-induced cancers, the purpose of the present study was to determine if hepatic oxidative DNA damage was increased in patients with HCC compared to patients with benign hepatic tumors or hepatic metastases (non-HCC) or to patients with end-stage alcoholic liver disease undergoing liver transplantation. Oxidative DNA damage was assessed by 8-hydroxy-2'-deoxyguanosine (8-OH-dG). Results showed that peritumoral 8-OH-dG was markedly increased in HCC (N= 51) (180 +/- 74 vs 32 +/- 58-OH-dG/10(6)dG for tumor, P < 0.005) in contrast to patients with non-HCC (N = 17), in whom the peritumoral 8-OH-dG did not differ from that in tumor (39 +/- 7 vs. 31 +/- 108-OH-dG/10(6)dG). Oxidative DNA damage can be both mutagenic and carcinogenic; our data suggested it will be important in future studies to determine the chronology of this type of liver injury relative to hepatocarcinogenesis.
Asunto(s)
Carcinoma Hepatocelular/genética , Daño del ADN , Desoxiguanosina/análogos & derivados , Neoplasias Hepáticas/genética , Hígado/metabolismo , 8-Hidroxi-2'-Desoxicoguanosina , Adulto , Carcinoma Hepatocelular/metabolismo , Humanos , Hepatopatías Alcohólicas/metabolismo , Neoplasias Hepáticas/metabolismoRESUMEN
Double hepatic arterial and portal venous branches are common anatomic variations of the isolated right hepatic lobe. Reconstruction of these vessels during transplantation can be challenging because of their small caliber, close proximity to other hilar structures, and abnormal alignment with the native vasculature. Practical techniques for the creation of these anastomoses would simplify the recipient surgery and might minimize the incidence of vascular complications. Alternative methods for management of these structures are summarized. The recipient's proper hepatic artery and its bifurcation are resected for use as an arterial Y-type graft. The donor arteries are individually anastomosed at the bifurcation of the recipient's hepatic artery at the back table. The free end of the Y graft is then replaced at the origin of the gastroduodenal artery using standard branch-patch technique. Reconstruction of a second donor portal branch is similarly facilitated by ex situ placement of a Y-type vascular conduit derived from the recipient's portal vein. Surgical management of these vessels and reconstruction of other hilar structures are noticeably less cumbersome. There have been no short-term vascular complications. The use of autologous vascular conduits with ex situ reconstruction facilitates management of double donor arterial and portal venous branches. The incidence of complications attributable to these methods is expected to be low.
Asunto(s)
Arteria Hepática/cirugía , Trasplante de Hígado/métodos , Donadores Vivos , Vena Porta/cirugía , Angiografía , Humanos , Vena Porta/diagnóstico por imagen , Complicaciones Posoperatorias , Análisis de Supervivencia , Resultado del TratamientoRESUMEN
Portal hypertension (PHT) is a common clinical syndrome associated with chronic liver diseases; it is characterized by a pathological increase in portal pressure. Pharmacotherapy for PHT is aimed at reducing both intrahepatic vascular tone and elevated splanchnic blood flow. Due to the altered hemodynamic profile in PHT, dramatic changes in mechanical forces, both pressure and flow, may play a pivotal role in controlling endothelial and vascular smooth muscle cell signaling, structure, and function in cirrhotics. Nitric oxide, prostacyclin, endothelial-derived contracting factors, and endothelial-derived hyperpolarizing factor are powerful vasoactive substances released from the endothelium in response to both humoral and mechanical stimuli that can profoundly affect both the function and structure of the underlying vascular smooth muscle. This review will examine the contributory role of hormonal- and mechanical force-induced changes in endothelial function and signaling and the consequence of these changes on the structural and functional response of the underlying vascular smooth muscle. It will focus on the pivotal role of hormonal and mechanical force-induced endothelial release of vasoactive substances in dictating the reactivity of the underlying vascular smooth muscle, i.e., whether hyporeactive or hyperreactive, and will examine the extent to which these substances may exert a protective and/or detrimental influence on the structure of the underlying vascular smooth muscle in both a normal hemodynamic environment and following hemodynamic perturbations typical of PHT and cirrhosis. Finally, it will discuss the intracellular processes that regulate the release/expression of these vasoactive substances and that control the transformation of this normally protective cell to one that may promote the development of vasculopathy in PHT.
Asunto(s)
Endotelio Vascular/fisiopatología , Hipertensión Portal/fisiopatología , Cirrosis Hepática/fisiopatología , Músculo Liso Vascular/fisiopatología , Animales , Factores Biológicos/metabolismo , Señalización del Calcio , Hipoxia de la Célula , Células Cultivadas , Endotelinas/metabolismo , Endotelio Vascular/metabolismo , Epoprostenol/metabolismo , Humanos , Hipertensión Portal/tratamiento farmacológico , Óxido Nítrico/metabolismo , Presión , Receptores de Superficie Celular/agonistas , Flujo Sanguíneo Regional/efectos de los fármacos , Circulación Esplácnica/efectos de los fármacos , Estrés Mecánico , Vasoconstricción , VasodilataciónRESUMEN
IFN-inducible protein-10 (IP-10/CXCL10) is a CXC chemokine that targets both T cells and NK cells. Elevation of IP-10 expression has been demonstrated in a number of human diseases, including chronic cirrhosis and biliary atresia. Cytokine-responsive gene-2 (Crg-2), the murine ortholog of IP-10, was induced following CCl(4) treatment of the hepatocyte-like cell line AML-12. Crg-2 expression was noted in vivo in multiple models of hepatic and bile duct injury, including bile duct ligation and CCl(4), D-galactosamine, and methylene dianiline toxic liver injuries. Induction of Crg-2 was also examined following two-thirds hepatectomy, a model that minimally injures the remaining liver, but that requires a large hepatic regenerative response. Crg-2 was induced in a biphasic fashion after two-thirds hepatectomy, preceding each known peak of hepatocyte DNA synthesis. Induction of Crg-2 was also observed in the kidney, gut, thymus, and spleen within 1 h of two-thirds hepatectomy. Characteristic of an immediate early gene, pretreatment of mice with the protein synthesis inhibitor cycloheximide before either two-thirds hepatectomy or CCl(4) injection led to Crg-2 superinduction. rIP-10 was demonstrated to have hepatocyte growth factor-inducing activity in vitro, but alone had no direct mitogenic effect on hepatocytes. Our data demonstrate that induction of Crg-2 occurs in several distinct models of liver injury and regeneration, and suggest a role for CRG-2/IP-10 in these processes.
Asunto(s)
Conductos Biliares/patología , Quimiocinas CXC/biosíntesis , Modelos Animales de Enfermedad , Regeneración Hepática/inmunología , Hígado/patología , Monocinas/biosíntesis , Animales , Conductos Biliares/inmunología , Tetracloruro de Carbono/toxicidad , Fraccionamiento Celular , Línea Celular , Células Cultivadas , Quimiocina CXCL10 , Quimiocinas CXC/fisiología , Regulación de la Expresión Génica/inmunología , Genes Inmediatos-Precoces , Hepatectomía , Factor de Crecimiento de Hepatocito/biosíntesis , Humanos , Ligadura , Hígado/efectos de los fármacos , Hígado/inmunología , Hígado/metabolismo , Cirrosis Hepática Biliar/inmunología , Fallo Hepático/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Mitógenos/biosíntesis , Mitógenos/fisiología , Monocinas/genética , Monocinas/fisiología , Factor de Necrosis Tumoral alfa/fisiología , Cicatrización de Heridas/inmunologíaRESUMEN
BACKGROUND: The endothelium may play a pivotal role in hemodynamic force-induced vascular remodeling. We investigated the role of endothelial cell (EC) plasminogen activator inhibitor-1 (PAI-1) in modulating flow-induced smooth muscle cell (SMC) migration. METHODS AND RESULTS: Human SMCs cocultured with or without human ECs were exposed to static (0 mL/min) or flow (26 mL/min; shear stress 23 dyne/cm(2)) conditions for 24 hours in a perfused capillary culture system. SMC migration was then assessed with a Transwell migration assay. In the absence but not in the presence of ECs, pulsatile flow significantly increased the migration of SMCs (264+/-26%) compared with SMCs under static conditions, concomitant with a 3- and 4-fold increase in PAI-1 mRNA and protein, respectively, in cocultured ECs. In the presence of PAI-1-/- ECs, flow increased wild-type SMC migration (226+/-25%), an effect that was reversed by exogenous PAI-1. To determine whether the antimigratory activity of PAI-1 was dependent primarily on inhibition of PAs or its association with vitronectin, experiments were conducted with PAI-1R (a mutant PAI-1 that binds to vitronectin but does not inhibit PA) and PAI-1K (a mutant that inhibits PA but has reduced affinity for vitronectin). PAI-1R inhibited both basal and flow-induced migration, whereas PAI-1K inhibited flow-induced migration in the absence of any effect on baseline migration. CONCLUSIONS: Flow-induced EC PAI-1 inhibits flow-induced SMC migration in vitro. EC PAI-1 expression may be one of the predominant mechanisms responsible for controlling the process of vascular remodeling.
Asunto(s)
Movimiento Celular/fisiología , Endotelio Vascular/fisiología , Músculo Liso Vascular/citología , Inhibidor 1 de Activador Plasminogénico/fisiología , Animales , Northern Blotting , Técnicas de Cultivo de Célula/métodos , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Técnicas de Cocultivo , Endotelio Vascular/química , Endotelio Vascular/citología , Eliminación de Gen , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Músculo Liso Vascular/efectos de los fármacos , Inhibidor 1 de Activador Plasminogénico/genética , Inhibidor 1 de Activador Plasminogénico/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Spontaneous massive retroperitoneal hemorrhage from an adrenal gland is a rare event. A thoughtful and meticulous approach to such a patient, with appropriate diagnostic studies, ICU and surgical care are essential for patient survival. In patients with active bleeding, angiographic embolization is a valuable adjunct to achieve hemostasis, to allow for further work-up of the adrenal tumor, and an improved subsequent oncologic resection. Hemodynamically unstable patients, however, may require supportive transfusions in the intensive care unit, potential embolization if deemed feasible, or urgent surgical exploration. If possible, however, the acute surgical removal of an adrenal tumor within a large retroperitoneal hematoma should be avoided, as under such conditions a proper oncologic resection may not be possible. The possibility of a pheochromocytoma must always be entertained. Early recognition and treatment of patients with presumed adrenal insufficiency may decrease patient morbidity and mortality.
Asunto(s)
Neoplasias de la Corteza Suprarrenal/complicaciones , Embolización Terapéutica , Hemorragia/terapia , Neoplasias de la Corteza Suprarrenal/diagnóstico , Neoplasias de la Corteza Suprarrenal/cirugía , Adulto , Femenino , Hemorragia/complicaciones , Hemorragia/patología , Humanos , Imagen por Resonancia MagnéticaRESUMEN
OBJECTIVE: To determine the outcomes associated with laparoscopic donor nephrectomy (LDN) performed as a 23-hour day surgery protocol. SUMMARY BACKGROUND DATA: Living donor renal transplantation is associated with immediate graft function and prolonged graft survival. The recent application of laparoscopic technology to living renal donation has refocused attention on the advantages of this donor source. LDN is associated with decreased donor pain, length of stay, time out of work, and opportunity costs. The authors hypothesized that LDN would be a viable 23-hour stay procedure. METHODS: All living donation procedures since May 1998 have been performed with LDN. The 23-hour protocol was initiated in January 1999. LDNs performed from May 1998 to December 1998 and traditional open donor nephrectomy procedures from May 1996 to May 1998 served as historical controls. The following variables were examined: donor sex, related versus nonrelated donation, operative time, blood loss, length of stay, time out of work, recipient and donor serum creatinine levels, hospital charges, and complications. RESULTS: The 23-hour LDN protocol was associated with high degrees of donor satisfaction. Thirty-six of the 41 donors were discharged within 23 hours; 1 was readmitted. Time out of work was equivalent to that of the control group; graft function was identical among all three comparison groups. Hospital charges were equivalent between the control group and the open nephrectomy group and were significantly decreased in the 23-hour group. CONCLUSIONS: Laparoscopic donor nephrectomy can be performed as a 23-hour stay procedure without incurring additional complications or donor dissatisfaction. By further decreasing opportunity costs and disincentives for donation, LDN may increase the number of potential donors appearing for evaluation.
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Trasplante de Riñón , Laparoscopía/métodos , Tiempo de Internación/estadística & datos numéricos , Donadores Vivos , Nefrectomía/métodos , Adulto , Procedimientos Quirúrgicos Ambulatorios , Estudios de Casos y Controles , Femenino , Precios de Hospital/estadística & datos numéricos , Humanos , Laparoscopía/estadística & datos numéricos , Donadores Vivos/psicología , Masculino , Factores de TiempoRESUMEN
Thrombin has recently been shown not only to exert procoagulant activities, but also to induce mitogenic responses of different cell types involved in wound healing via binding to and cleavage of the thrombin receptor. In order to further explore these aspects of thrombin function, human keratinocytes (HaCaT cell line) were examined for their potential mitogenic responsiveness to thrombin and for the dependency of this process on the expression of the high-affinity thrombin receptor. Quiescent keratinocytes were stimulated in the mitogenic assay with alpha-thrombin and the thrombin receptor activating peptides TRAP42-55 (SFLLRNPNDKYEPY) and TRAP42-46 (SFLLR). A strong induction of cell proliferation was noted with alpha-thrombin, TRAP42-55 and TRAP42-46, but not with the "scrambled" peptide (FSLLR). These findings confirm that keratinocytes express the thrombin receptor and that the sequence of the first two amino acids of the generated neo-N-terminus are important for the activation of the receptor. Using cDNA fragments of the 5' coding sequence of the receptor, Northern blot analysis confirmed that HaCaT keratinocytes express the thrombin receptor. Expression of the receptor was also detected on normal human keratinocytes by immunohistochemistry and in situ hybridization. These data demonstrate the expression and biologic function of the human thrombin receptor on human keratinocytes, suggesting that thrombin, among other mediators, plays an important part in the orchestration of epidermal growth and repair processes.
Asunto(s)
Queratinocitos/metabolismo , Receptores de Trombina/fisiología , Northern Blotting , División Celular , Células Cultivadas , Humanos , Inmunohistoquímica , Hibridación in Situ , Queratinocitos/efectos de los fármacos , Mitógenos/farmacología , Proteínas/farmacología , ARN Mensajero/análisis , Receptor PAR-1 , Receptores de Trombina/genética , Piel/metabolismo , Trombina/farmacologíaRESUMEN
Epidemiological studies demonstrate a significant protective effect of moderate alcohol consumption on the incidence of cardiovascular diseases which accounts for the majority of deaths in the Western world. In this review, possible mechanisms to explain the cardioprotective effect of ethanol are discussed. While the prevailing theory supported by a number of clinical and animal studies indicates that the ability of ethanol to elevate serum high-density lipoprotein (HDL) cholesterol levels is an important mechanism in ameliorating cardiovascular disease, other mechanisms whereby ethanol could exert its beneficial effect have been proposed. Namely, its ability to affect platelet function and endothelial cell and vascular smooth muscle cell function (In this review, the terms alcohol and ethanol are used interchangeably).
Asunto(s)
Enfermedad Coronaria/sangre , Etanol/farmacología , Lipoproteínas HDL/sangre , Músculo Liso Vascular/patología , Animales , División Celular/efectos de los fármacos , Humanos , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico Sintasa de Tipo III , Agregación Plaquetaria/efectos de los fármacosRESUMEN
Portal hypertension is associated with a wide range of pulmonary pathophysiologies, ranging from portopulmonary hypertension to hepatopulmonary syndrome. Although the clinical and pathological features of pulmonary dysfunction in this setting have been extensively characterized, the underlying biology is not well understood. Specifically, the role of mediators that regulate mesenteric vascular hemodynamics in portal hypertension, such as nitric oxide and endothelin, have not been studied in the lung. Using a rat model of prehepatic portal hypertension with preserved hepatic function, we examined pulmonary elaboration of endothelial nitric oxide synthase (NOS), inducible NOS, heme oxygenase- 1 (HO-1), heme oxygenase-2 (HO-2), endothelin-1 mRNA, and protein. In comparison to sham controls, portal hypertensive animals exhibited significantly increased pulmonary iNOS and HO-1 mRNA and protein. Cyclic GMP was significantly increased in portal hypertensive lung tissue, suggesting activation of guanylyl cyclase by the endproducts of iNOS and/or HO-1 activity. Using immunohistochemical analysis, iNOS expression was localized to the vascular endothelium, while HO-1 localized to bronchiolar epithelium and macrophages. These results suggest that production of nitric oxide and carbon monoxide may contribute to the pulmonary pathology associated with portal hypertension.
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Hemo Oxigenasa (Desciclizante)/metabolismo , Hipertensión Portal/enzimología , Pulmón/enzimología , Óxido Nítrico Sintasa/metabolismo , Animales , GMP Cíclico/metabolismo , Modelos Animales de Enfermedad , Endotelio Vascular/enzimología , Hemo-Oxigenasa 1 , Inmunohistoquímica , Masculino , Óxido Nítrico Sintasa de Tipo II , Proteínas/análisis , ARN Mensajero/análisis , Ratas , Ratas Sprague-Dawley , Regulación hacia ArribaRESUMEN
OBJECTIVE: Hepatocellular carcinoma (HCC) is associated with altered expression and function of inhibitory guanine nucleotide regulatory proteins (Gi-proteins). This study addresses the interaction between Gi-proteins and the extracellular regulated kinase (ERK) component of the mitogen activated protein kinase (MAPK) cascade in regulating mitogenesis in an experimental model of HCC. DESIGN: Pharmacological agents which selectively interact with specific target proteins involved in signal transduction through a Gi-MAPK pathway have recently become available. These agents in combination with scientific assays allow us to address the role of individual components of this cascade in the regulation of mitogenesis in HCC. METHODS: These studies were performed using rat hepatic tumorigenic cells (H4IIE) and isolated cultured hepatocytes in vitro in conjunction with pharmacological agents which interact with Gi-protein or MAPK components of intracellular signalling. RESULTS: Direct activation of Gi-proteins with mastoparan M7 (M7) significantly increased nuclear thymidine incorporation in hepatic tumorigenic H4IIE cells in a dose-dependent manner (10-1000 nM, n = 4, P < 0.05), an effect that was abolished by treatment with either pertussis toxin (PTx) or the selective mitogen-activated ERK-regulated kinase (MEK) inhibitor PD098059. In contrast, M7 inhibited nuclear thymidine incorporation in serum-stimulated isolated hepatocytes. ERK2 activity was then determined as the ability of immunoprecipitated ERK2 proteins to phosphorylate the ERK substrate myelin basic protein. These studies demonstrated a time- and dose-dependent increase in ERK2 activity in H4IIE cells following Gi-protein activation with M7, a maximal response being attained at 20 min. In contrast, M7 failed to significantly alter ERK2 activity in isolated cultured hepatocytes at any of the doses or time points assayed (10-5000 nM, 10-120 min). Gi-stimulated ERK activation was completely blocked in tumorigenic cells following treatment with PTx. CONCLUSIONS: These data demonstrate for the first time a Gi-linked MAPK cascade in experimental HCC, activation of which stimulates cellular mitogenesis.
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Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Carcinoma Hepatocelular/enzimología , Proteínas de Unión al GTP/metabolismo , Neoplasias Hepáticas Experimentales/enzimología , Animales , División Celular , Hígado/citología , Masculino , Fosforilación , Antígeno Nuclear de Célula en Proliferación , Ratas , Ratas Endogámicas ACI , Células Tumorales CultivadasRESUMEN
The objective of this study was to determine the effect of phenotype on pulse pressure-induced signaling and growth of vascular smooth muscle cells in vitro. Using a perfused transcapillary culture system, cells were exposed to increases in pulsatile flow and hence pulse pressure and maintained for 72 h before cells were harvested. Cell proliferation was determined by cell number, DNA synthesis, and proliferating cell nuclear antigen expression. Mitogen-activated protein kinase (MAPK) levels were determined by immunoblot and kinase activity by phosphorylation of myelin basic protein. Cell phenotype was determined by immunoblot and immunocytofluorescence using antisera specific for the differentiation markers alpha-actin, myosin, calponin, osteopontin, and phospholamban. In cells that highly expressed these differentiation markers, there was a significant increase in cell growth in response to chronic increases in pulse pressure without a significant change in MAPK activity in these cells. In contrast, in cells that weakly expressed SMC differentiation markers, there was a significant decrease in cell growth concomitant with a significant decrease in MAPK signaling in these cells. We conclude that SMC phenotype dictates the growth response of SMC to mechanical force in vitro.
Asunto(s)
Quinasas de Proteína Quinasa Activadas por Mitógenos , Proteínas Quinasas Activadas por Mitógenos , Músculo Liso Vascular/fisiología , Flujo Pulsátil , Transducción de Señal , Actinas/análisis , Animales , Proteínas de Unión al Calcio/análisis , Proteínas Quinasas Dependientes de Calcio-Calmodulina/biosíntesis , Bovinos , División Celular , ADN/biosíntesis , Estimulación Eléctrica , MAP Quinasa Quinasa 1 , MAP Quinasa Quinasa 2 , Masculino , Proteínas de Microfilamentos , Proteína Quinasa 1 Activada por Mitógenos , Proteína Quinasa 3 Activada por Mitógenos , Mitógenos , Músculo Liso Vascular/citología , Músculo Liso Vascular/metabolismo , Miosinas/análisis , Osteopontina , Fenotipo , Estimulación Física , Antígeno Nuclear de Célula en Proliferación/biosíntesis , Proteínas Serina-Treonina Quinasas/biosíntesis , Proteínas Tirosina Quinasas/biosíntesis , Ratas , Ratas Sprague-Dawley , Sialoglicoproteínas/análisis , CalponinasRESUMEN
BACKGROUND: Alcohol consumption protects against coronary heart disease by as yet unclear mechanisms. The aim of this study was to determine the effect of ethanol on vascular smooth muscle cell (SMC) migration which plays an important role in the pathogenesis of atherosclerosis. MATERIALS AND METHODS: Cultures of human SMC under static (no flow) or pulsatile flow conditions (perfused transcapillary culture system) were pretreated in the absence or presence of ethanol (EtOH) whereupon their random migration (chemokinesis) was assessed by Transwell assay. RESULTS: Ethanol pretreatment (24 h) dose dependently inhibited migration of HuSMC from static cultures with a maximal inhibition of 60.8 +/- 4.4% observed at 40-80 mM, in the absence of any effect on cell adhesion or cell viability as assessed by trypan blue exclusion. In HuSMC exposed to pulsatile flow (0.3 to 25 ml/min, 24 h), there was a flow-dependent increase in migration ranging from a 1.3 +/- 0.16- to 2.67 +/- 0.26-fold increase, compared to static cells, concomitant with a significant increase in urokinase-type plasminogen activator (uPA) mRNA levels. Ethanol pretreatment (20-80 mM, 24 h) dose dependently inhibited the flow-induced increase in SMC migration but did not affect uPA mRNA expression. CONCLUSIONS: The inhibitory effect of ethanol on basal and flow-stimulated SMC migration may be relevant to its cardiovascular effects in vivo.
Asunto(s)
Circulación Sanguínea/fisiología , Movimiento Celular/efectos de los fármacos , Movimiento Celular/fisiología , Etanol/farmacología , Músculo Liso Vascular/efectos de los fármacos , Adhesión Celular/efectos de los fármacos , Células Cultivadas , Humanos , Músculo Liso Vascular/citología , Músculo Liso Vascular/fisiología , Perfusión , Flujo Pulsátil , ARN Mensajero/metabolismo , Activador de Plasminógeno de Tipo Uroquinasa/genéticaRESUMEN
The aim of this study was to determine the effect of ethanol on endothelial nitric oxide synthase (eNOS), the enzyme responsible for the production of the important vasoactive agent nitric oxide. The effect of ethanol (0.8-160 mM) on both basal and flow-stimulated eNOS activity was determined using cultured bovine aortic endothelial cells (EC). In "static" EC ethanol dose-dependently increased basal eNOS activity with a maximum response (approximately 2.0-fold increase) achieved at 40 mM in the absence of any effect on cell viability or nitric oxide synthase protein expression. Pertussis toxin (PTX) pretreatment significantly inhibited the ethanol-induced increase in basal eNOS activity. EC exposed to steady laminar flow exhibited a flow- and time-dependent increase in eNOS activity. Ethanol significantly enhanced the laminar flow-induced eNOS response from 0.62 +/- 0.1 to 1.06 +/- 0. 06 pmol [14C]citrulline/mg/min, a response that was inhibited by PTX. PTX-catalyzed ribosylation of Gialpha substrates, an index of G-protein functional activity, was increased in laminar flow-exposed EC compared with static controls and was further enhanced by ethanol treatment. Likewise, EC exposed to low ( approximately 0.5 dynes/cm2) and high ( approximately 12 dynes/cm2) pulsatile flow demonstrated increased eNOS activity, an effect that was associated with increased PTX-catalyzed ribosylation of Gialpha substrates. Ethanol enhanced the low flow response in a PTX-sensitive manner. These data demonstrate a stimulatory effect of ethanol on basal and flow-stimulated eNOS activity, mediated in part by a mechanism involving a PTX-sensitive G protein.
Asunto(s)
Endotelio Vascular/fisiología , Etanol/farmacología , Proteínas de Unión al GTP/metabolismo , Óxido Nítrico Sintasa/metabolismo , Animales , Aorta , Bovinos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Activación Enzimática , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Proteínas de Unión al GTP/efectos de los fármacos , Cinética , Óxido Nítrico Sintasa de Tipo III , Toxina del Pertussis , Estrés Mecánico , Factores de Tiempo , Factores de Virulencia de Bordetella/farmacologíaRESUMEN
This study addresses the effect of sustained increased pulsatile flow on nitric oxide synthase (NOS) and cyclooxygenase (Cox) expression and activity in co-cultured endothelial cells (EC) and vascular smooth muscle cells (SMC). Using a perfused transcapillary co-culture system which permits the chronic exposure of cultured EC and SMC to physiological shear stresses, co-cultures were exposed to step-wise increases in flow up to: (i) 2 ml/min (low flow: 0.5 dyn/cm2): or (ii) 44 ml/min (high flow: 15 dyn/cm2) and maintained for 72 h before SMC and EC were harvested separately. There was no NOS activity or protein expression in co-cultured SMC under flow conditions. There was a significant increase in eNOS activity in co-cultured EC under high flow conditions, compared to low flow, which correlated with an increase in eNOS expression and mRNA levels. The flow-induced increase in eNOS activity was potentiated by indomethacin treatment, suggesting a modulatory role for a cyclooxygenase product. Prostacyclin levels in co-culture perfusate were significantly elevated under high flow conditions. While both co-cultured EC and SMC expressed cyclooxygenase (Cox-I and Cox-II), they were differentially regulated by pulsatile flow, EC Cox-I and Cox-II protein expression were both decreased. Indomethacin treatment increased the expression of both Cox-I and Cox-II in co-cultured SMC under high flow conditions. We conclude that sustained increases in pulsatile flow maintain elevated eNOS and Cox protein expression and activity in EC while decreasing Cox expression in co-cultured SMC. These data suggest that regulation of these pathways may contribute to flow-induced vascular remodeling in vivo.
Asunto(s)
Endotelio Vascular/metabolismo , Isoenzimas/metabolismo , Músculo Liso Vascular/metabolismo , Óxido Nítrico Sintasa/metabolismo , Prostaglandina-Endoperóxido Sintasas/metabolismo , Flujo Pulsátil/fisiología , Animales , Northern Blotting , Western Blotting , Bovinos , Células Cultivadas , Ciclooxigenasa 1 , Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa/farmacología , Inhibidores Enzimáticos/farmacología , Epoprostenol/metabolismo , Indometacina/farmacología , Proteínas de la Membrana , NG-Nitroarginina Metil Éster/farmacología , Perfusión , Prostaglandinas F/metabolismo , Radioinmunoensayo , RatasRESUMEN
BACKGROUND: Coronary endothelial dysfunction after brief ischemia-reperfusion (IR) remains a clinical problem. We investigated the role of heparin and N-acetylheparin, a nonanticoagulant heparin derivative, in modulating coronary endothelial function after IR injury, with an emphasis on defining the role of the nitric oxide (NO)-cGMP pathway in the heparin-mediated effect. METHODS AND RESULTS: Male mongrel dogs were surgically instrumented, and the effects of both bovine heparin and N-acetylheparin on coronary endothelial vasomotor function, expressed as percent change from baseline flow after acetylcholine challenge, were studied after 15 minutes of regional ischemia of the left anterior descending artery (LAD) followed by 120 minutes of reperfusion. In dogs treated with placebo (saline), coronary vasomotor function was significantly (P=0.03) decreased after 15 and 30 minutes of reperfusion (65+/-12% and 73+/-12%) compared with preischemia (103+/-6%). In contrast, the vasodilatory response to the endothelium-independent vasodilator sodium nitroprusside was maintained during reperfusion. Preischemic administration of both bovine heparin and N-acetylheparin (6.0 mg/kg IV) preserved coronary endothelial function throughout reperfusion. In a parallel group of dogs, nitrate/nitrite (NOx) and cGMP levels in the LAD were measured after treatment and during 15-minute reperfusion. Preischemic administration of N-acetylheparin caused a significant increase in basal NOx and cGMP levels compared with saline controls. Pretreatment with N-acetylheparin also caused a significant increase in NOx and cGMP levels in the LAD after 15 minutes of reperfusion compared with IR alone. CONCLUSIONS: These results suggest that heparin preserves coronary endothelial function after brief IR injury by a mechanism independent of its anticoagulant activity and that the effect of heparin may be mediated in part by activation of the NO-cGMP pathway.