RESUMEN
OBJECTIVE: We sought to test the hypothesis that turmeric-derived curcuminoids limit reperfusion brain injury in an experimental model of stroke via blockade of early microvascular inflammation during reperfusion. METHODS: Male Sprague Dawley rats subjected to MCAO/R were treated with turmeric-derived curcuminoids (vs. vehicle) 1 hour prior to reperfusion (300 mg/kg ip). Neutrophil adhesion to the cerebral microcirculation and measures of neutrophil and endothelial activation were assayed during early reperfusion (0-4 hours); cerebral infarct size, edema, and neurological function were assessed at 24 hours. Curcuminoid effects on TNFα-stimulated human brain microvascular endothelial cell (HBMVEC) were assessed. RESULTS: Early during reperfusion following MCAO, curcuminoid treatment decreased neutrophil rolling and adhesion to the cerebrovascular endothelium by 76% and 67% and prevented >50% of the fall in shear rate. The increased number and activation state (CD11b and ROS) of neutrophils were unchanged by curcuminoid treatment, while increased cerebral expression of TNFα and ICAM-1, a marker of endothelial activation, were blocked by >30%. Curcuminoids inhibited NF-κB activation and subsequent ICAM-1 gene expression in HBMVEC. CONCLUSION: Turmeric-derived curcuminoids limit reperfusion injury in stroke by preventing neutrophil adhesion to the cerebrovascular microcirculation and improving shear rate by targeting the endothelium.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Curcumina/farmacología , Endotelio Vascular/metabolismo , Activación Neutrófila/efectos de los fármacos , Neutrófilos/metabolismo , Daño por Reperfusión/metabolismo , Accidente Cerebrovascular/metabolismo , Animales , Antígeno CD11b/metabolismo , Células Cultivadas , Células Endoteliales/metabolismo , Células Endoteliales/patología , Endotelio Vascular/patología , Humanos , Rodamiento de Leucocito/efectos de los fármacos , Masculino , Neutrófilos/patología , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismo , Daño por Reperfusión/patología , Accidente Cerebrovascular/patologíaRESUMEN
The role of caspases in platelet function is not well understood. When platelets are activated, they express phosphatidylserine on the outer plasma membrane, form platelet microparticles, and aggregate (Pag). The aims of this study were to determine if caspases play a role in the platelet activation seen in type 2 diabetes. Diabetic rats (Zucker diabetic fatty) were treated with a broad-spectrum caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethyl ketone, in vivo and platelets were evaluated for phosphatidylserine expression, platelet microparticle formation, and Pag. We found a decreased phosphatidylserine exposure in zVAD-Zucker diabetic fatty rats compared to Zucker diabetic fatty-phosphate-buffered saline when activated with 20 micromol/l ADP. Zucker diabetic fatty rats treated with benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethyl ketone decreased platelet microparticle numbers compared to phosphate-buffered saline control Zucker diabetic fatty rats. Further, treatment with benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethyl ketone significantly decreased Pag. These results indicate that caspases play a role in platelet activation, suggesting a unique physiologic role of these proteases and perhaps the underlying mechanisms involved in the chronic platelet activation observed in type 2 diabetes.
Asunto(s)
Plaquetas/enzimología , Caspasas/fisiología , Activación Plaquetaria/fisiología , Clorometilcetonas de Aminoácidos/farmacología , Animales , Inhibidores de Caspasas , Diabetes Mellitus Tipo 2/sangre , Diabetes Mellitus Tipo 2/enzimología , Agregación Plaquetaria/fisiología , Ratas , Ratas ZuckerRESUMEN
Type 2 diabetes in humans is associated with a significant hypercoagulable state; however, the effects of this on stroke and cardiovascular disease are not completely understood. The genetic mutations in db/db and ob/ob mice produce metabolic abnormalities similar to type 2 diabetes, but little is known about their platelet or coagulation properties. The objective of this study was therefore to examine platelet function and coagulation in db/db and ob/ob mice to determine the degree of alteration induced by type 2 diabetes. Male db/db and ob/ob mice, 8-16 weeks old, and their respective genetic control mice were used for all experiments. To examine platelet function and coagulation, we measured ADP-induced whole blood aggregation at baseline and after inhibition with aspirin and fucoidan, whole blood coagulation by thromboelastography, and platelet CD61 expression by flow cytometry. Both db/db and ob/ob mice demonstrated significantly less ADP-induced whole blood aggregation compared with control mice (db/db mice, P < 0.001; ob/ob mice, P < 0.01). Aggregation was significantly inhibited with aspirin in all groups; however, fucoidan inhibited aggregation only in control mice. The db/db and ob/ob mice demonstrated significantly less maximal clot strength compared with control mice (P < 0.01), and ob/ob mice demonstrated premature clot fibrinolysis measured by thromboelastography. In conclusion, the db/db and ob/ob type 2 diabetes mouse models do not demonstrate a hypercoagulable state similar to humans with this disease. We caution their use for studying cardiovascular and cerebrovascular disease in the setting of type 2 diabetes.
Asunto(s)
Coagulación Sanguínea/fisiología , Diabetes Mellitus Tipo 2/sangre , Ratones Obesos/sangre , Agregación Plaquetaria/fisiología , Animales , Coagulación Sanguínea/genética , Diabetes Mellitus Tipo 2/genética , Modelos Animales de Enfermedad , Ratones , Agregación Plaquetaria/genética , Tromboelastografía , Trombofilia/sangreRESUMEN
Flow cytometry methods used to measure leukocyte function often entail sample preparation procedures that cause artifactual cell activation. To avoid leukocyte activation by isolation techniques, some preparation methods use fluorescent markers to discriminate leukocytes from erythrocytes in whole blood. One of these markers, laser dye styryl-751(LDS-751), has been used to distinguish leukocytes by staining nucleic acid, but has been found to stain other blood cells and dead cells indiscriminately. Thus, LDS-751 may not be an appropriate reagent for leukocyte identification in whole blood. Fixing samples with formaldehydes increases cell permeability and causes surface protein cross-linking that may alter staining of both intra- and extracellular markers. The degree of this sample alteration by formaldehyde fixation, however, remains in question. In addition, little is known about flow cytometry and sample preparation methods in mouse whole blood. The purpose of this study was to determine if labeling leukocytes with a monoclonal antibody specific to leukocyte common antigen (CD45) was superior to labeling with LDS-751 and to determine the effect of sample fixation on a mouse whole blood preparation for flow cytometry. Samples were incubated with CD16/CD32 Fc receptor blocker, and either 10 microg/ml LDS-751 or phosphate buffered saline (PBS). The samples were then fixed with paraformaldehyde or diluted with PBS followed by incubation with 5 microg/ml PerCP-conjugated anti-CD45, 5 microg/ml FITC-conjugated anti-CD11b, or 80 microM dichlorofluorescein diacetate. We found that samples labeled with LDS-751 demonstrated decreased fluorescence intensity for granulocyte CD11b expression and ROS production compared to samples labeled with anti-CD45. In addition, sample fixation decreased mean fluorescence intensity in samples labeled with either LDS-751 or anti-CD45. We conclude that labeling leukocytes with monoclonal antibody CD45 in a mouse whole blood preparation is preferable, as it provides improved measurement of leukocyte indices compared to LDS-751. Also, while sample fixation prior to antibody staining caused a decrease in overall fluorescence; it can be used to successfully identify extra-cellular markers.
Asunto(s)
Anticuerpos , Citometría de Flujo , Antígenos Comunes de Leucocito/inmunología , Leucocitos , Fijación del Tejido , Animales , Anticuerpos/sangre , Sitios de Unión de Anticuerpos , Antígeno CD11b/biosíntesis , Antígeno CD11b/sangre , Antígeno CD11b/genética , Separación Celular , Colorantes Fluorescentes/metabolismo , Granulocitos/inmunología , Granulocitos/metabolismo , Antígenos Comunes de Leucocito/sangre , Leucocitos/inmunología , Leucocitos/metabolismo , Ratones , Ratones Endogámicos C57BL , Compuestos Orgánicos/sangre , Especies Reactivas de Oxígeno/sangre , Coloración y EtiquetadoRESUMEN
Coronary ischemic events increase significantly following a "bad air" day. Ambient particulate matter (PM10) is the pollutant most strongly associated with these events. PM10 produces inflammatory injury to the lower airways. It is not clear, however, whether pulmonary inflammation translates to a systemic response. Lipopolysaccharide (LPS) is a proinflammatory molecule often associated with the coarse fraction of PM. It was hypothesized that PM>2.5 from coal plus LPS induce pulmonary inflammation leading to a systemic inflammatory response. Mice were intratracheally instilled with saline, PM (200 microg), PM + LPS10 (PM + 10 microg LPS), or PM + LPS100 (PM + 100 microg LPS). Eighteen hours later, histologic analysis was performed on lungs from each group. Pulmonary and systemic inflammation were assessed by measuring the proinflammatory cytokines tumor necrosis factor (TNF)-alpha and interleukin (IL)-6 in the pulmonary supernatant and plasma. In a follow-up study, the effects of LPS alone were assessed. Histologic analysis revealed a dose-dependent elevation in pulmonary inflammation with all treatments. Pulmonary TNF-alpha and IL-6 both increased significantly with PM + LPS100 treatment. Regarding plasma, TNF-alpha significantly increased in both PM + LPS10 and PM + LPS100 treatments. For plasma IL-6, all groups tended to rise with a significant increase in the PM + LPS100 group. The results of the follow-up study indicate that the responses to PM + LPS were not due to LPS alone. These results suggest that coarse coal fly ash PM>2.5 combined with LPS produced pulmonary and systemic inflammatory responses. The resulting low-level systemic inflammation may contribute to the increased severity of ischemic heart disease observed immediately following a bad air day.
Asunto(s)
Carbón Mineral/efectos adversos , Interleucina-6/sangre , Lipopolisacáridos/efectos adversos , Material Particulado/efectos adversos , Factor de Necrosis Tumoral alfa/sangre , Animales , Modelos Animales de Enfermedad , Polvo/inmunología , Interleucina-6/metabolismo , Recuento de Leucocitos , Lipopolisacáridos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Material Particulado/inmunología , Neumonía/inducido químicamente , Neumonía/inmunología , Neumonía/patología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Ischemic stroke and reperfusion (ISR) is associated with an inflammatory response characterized, in part, by the formation of leukocyte-platelet aggregates (LPA). Aggregate formation may amplify the immunologic and hemostatic functions of both cell types and thus exacerbate reperfusion injury after ischemic stroke. LPA formation in peripheral blood may also serve as a biomarker of the severity of injury. However, it is not fully known whether ISR causes LPA formation that can be detected in the peripheral blood. Therefore, the purpose of this study was to measure LPA in the peripheral blood after ISR using a rat model. The filament method was used to perform ISR. Blood was collected from the jugular vein before ischemia, after 4 hours of ischemia, and after 1 hour of reperfusion. Flow cytometry was used to quantify LPA in peripheral blood. Separate ISR groups were treated with tirofiban, a platelet GPIIb/IIIa inhibitor, and fucoidan, a selectin adhesion molecule inhibitor, and analyzed for LPA. Leukocyte CD11b expression and reactive oxygen species production were also analyzed to note the role of polymorphonuclear neutrophilic (PMN) activation on LPA formation. After ISR, LPA levels in peripheral blood were twice as large as preischemic levels. Both GPIIb/IIIa and selectin adhesion molecule inhibition (p < .05) decreased LPA to preischemic values. PMN CD11b expression was increased above baseline but did not differ between groups. Reactive oxygen species production did not differ between groups during reperfusion. These data suggest that ischemic stroke and reperfusion results in an increase in LPA that can be consistently measured in peripheral blood. LPA formation may be a useful biomarker and potential therapeutic target after ischemic stroke and reperfusion.
Asunto(s)
Plaquetas/inmunología , Isquemia Encefálica/complicaciones , Modelos Animales de Enfermedad , Leucocitos/inmunología , Daño por Reperfusión Miocárdica/sangre , Accidente Cerebrovascular/complicaciones , Tirosina/análogos & derivados , Análisis de Varianza , Animales , Anticoagulantes/uso terapéutico , Biomarcadores/sangre , Citometría de Flujo , Inflamación , Masculino , Daño por Reperfusión Miocárdica/etiología , Daño por Reperfusión Miocárdica/inmunología , Daño por Reperfusión Miocárdica/prevención & control , Activación Neutrófila , Neutrófilos/inmunología , Agregación Plaquetaria/inmunología , Inhibidores de Agregación Plaquetaria/uso terapéutico , Polisacáridos/uso terapéutico , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/inmunología , Índice de Severidad de la Enfermedad , Factores de Tiempo , Tirofibán , Tirosina/uso terapéuticoRESUMEN
It is known that inflammation affects the coagulation pathway, but the mechanisms are not clear. Because a persistent inflammatory condition is associated with several chronic diseases, including cardiovascular disorders, there is intense interest in determining if and how chronic inflammation contributes to a hypercoagulable state. One pathway by which inflammation interacts with coagulation is via monocyte binding and activation of coagulation Factor X (FX). Upon activation, monocytes express the alphaMbeta2 integrin CD11b/CD18, which has a binding site for the plasma protein FX. Binding is followed by the cleavage of FX into its activated form Xa(FXa) which, in turn, is responsible for the conversion of prothrombin to thrombin. To assess the contribution of this pathway, a straightforward assay in whole blood is needed for studies of inflammation-induced coagulation and thrombosis. The current assay for FXa binding requires isolation of the monocytes and measurement of bound FXa activity with a chromogenic substrate. Harvesting a sufficient number of monocytes for analysis requires a relatively large blood sample. In addition, it is known that the process of isolating neutrophils and monocytes from whole blood induces an upregulation of CD11b. Thus, the measurement process itself causes an artifact in receptor expression resulting in an overestimate of true state of monocyte activation and FX binding. To address these limitations, we developed a flow cytometric assay to directly measure the binding of FX to monocytes in whole blood. In this report we describe the methods of the procedure in detail and apply the procedure to demonstrate a significant increase of both monocyte CD11b expression and FXa binding when human blood samples were activated with the endotoxin, lipopolysaccharide in-vitro.
Asunto(s)
Factor X/metabolismo , Citometría de Flujo/métodos , Monocitos/metabolismo , Antígeno CD11b/análisis , HumanosRESUMEN
BACKGROUND: More than seventeen million Americans are afflicted with diabetes and these people have four times the rate of coronary heart disease (CHD) as non-diabetics. Furthermore, diabetic women have a 3.8 fold greater risk for CHD compared to diabetic men. Little is known why diabetic women are at an increased risk for CHD. It is possible that diabetic women with cardiovascular disease (CVD) have a greater inflammatory response resulting in an increased platelet neutrophil conjugate formation than diabetic men with CVD or non-diabetic women with CVD. This study tested the hypothesis that platelet-neutrophil conjugates, which are associated with several cardiovascular diseases, are increased in diabetic women with CVD compared to diabetic men with CVD and non-diabetic women with CVD. METHODS: Platelet-neutrophil conjugates were quantified by flow cytometry. The primary method is through direct binding of the neutrophil PSGL-1 receptor with P-selectin expressed on the platelet. RESULTS: In this study, we found when the blood was stimulated with PAF (platelet activating factor), diabetic women without CVD demonstrated an increase in platelet-neutrophil conjugates compared to diabetic women with CVD and non-diabetic women with CVD (% conjugates: 63.3 +/- 5.2 vs 46.8 +/- 4.3 vs 48.6 +/- 3.4, p < 0.05). The stimulation ratio was significantly increased in diabetic and non-diabetic women with CVD in comparison to diabetic men with CVD (ratio: 3.3 +/- 0.4 vs 3.3 +/- 0.3 vs 2.1 +/- 0.3, p < 0.05). CONCLUSION: These results suggest that platelets and neutrophils in diabetic women have a greater potential for activation compared to diabetic men and may contribute to thrombosis/inflammation and the greater severity of coronary heart disease observed in diabetic women as compared to diabetic men.
RESUMEN
Diabetics suffer from many complications including a prothrombotic condition. Activated platelet membrane provides an anchor, phosphatidylserine, for the attachment of the prothrombinase complex, which allows increased thrombin formation. This study aimed to further elucidate the interrelationship between coagulation proteins and activated platelets in type 2 diabetic blood. We found that there was a significant increase (30 x) in thrombin activity in the type 2 diabetic (ZDF) blood as compared to age-matched (ZL) controls (p<0.001). There was also a significant increase in the number of platelet microparticles in the type 2 diabetic rat compared to the lean control (p<0.001). Further, there were significant increases in caspase-3, -6, and -8 activities in the type 2 diabetic rats as compared to the lean controls (p<0.05). The combination of increased thrombin activity, increased PMP formation and increased caspase activity may contribute to the hypercoagulability of the diabetic blood. These results give more insight into the mechanisms underlying the interrelationship between diabetic platelets and coagulation proteins causing a prothrombotic condition in this patient population at increased risk from thromboembolic events.
Asunto(s)
Plaquetas/ultraestructura , Caspasas/metabolismo , Diabetes Mellitus Tipo 2/sangre , Trombina/metabolismo , Animales , Plaquetas/fisiología , Diabetes Mellitus Tipo 2/complicaciones , Integrina beta3/análisis , Activación Plaquetaria , Ratas , Ratas Mutantes , Trombofilia/sangre , Trombofilia/etiologíaRESUMEN
The incidence of myocardial infarction in patients who have the aquired immunodeficiency syndrome (AIDS) is increasing. However, no effective therapeutic agents have been discovered to reduce myocardial ischemia-reperfusion (I/R) injury in pathologies associated with AIDS. The aim of this study was to determine if infarct size is increased in murine AIDS after I/R injury and if I/R injury could be attenuated with vitamin E supplementation. Three groups of mice were studied: control, murine AIDS, and murine AIDS with vitamin E supplementation. Anesthetized mice were subjected to 30 min of left anterior descending coronary artery occlusion and 120 min of reperfusion. The hearts in mice that had murine AIDS had a larger infarct size compared to controls after I/R injury. Vitamin E supplementation significantly reduced infarct size and inhibited polymorphonuclear neutrophil (PMN) CD11b expression (p < 0.05). However, vitamin E supplementation did not affect PMN reactive oxygen species (ROS) production and platelet CD62p expression. These results suggest that the reduction of myocardial I/R injury with vitamin E supplementation may be the result of the inhibition of PMN CD11b expression. Vitamin E may be a promising prophylactic agent for the reduction of the severity of myocardial I/R injury in patients who have AIDS.
Asunto(s)
Síndrome de Inmunodeficiencia Adquirida/complicaciones , Síndrome de Inmunodeficiencia Adquirida/tratamiento farmacológico , Antioxidantes/farmacología , Isquemia Miocárdica/tratamiento farmacológico , Isquemia Miocárdica/etiología , Daño por Reperfusión Miocárdica/complicaciones , Daño por Reperfusión Miocárdica/tratamiento farmacológico , Vitamina E/farmacología , Síndrome de Inmunodeficiencia Adquirida/metabolismo , Adenosina Difosfato/farmacología , Animales , Antígeno CD11b/biosíntesis , Antígeno CD11b/efectos de los fármacos , Modelos Animales de Enfermedad , Femenino , Ventrículos Cardíacos/metabolismo , Ratones , Ratones Endogámicos C57BL , Modelos Cardiovasculares , Infarto del Miocardio/tratamiento farmacológico , Infarto del Miocardio/epidemiología , Infarto del Miocardio/etiología , Isquemia Miocárdica/epidemiología , Daño por Reperfusión Miocárdica/metabolismo , Miocardio/metabolismo , Miocardio/patología , N-Formilmetionina Leucil-Fenilalanina/farmacología , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , Selectina-P/biosíntesis , Selectina-P/efectos de los fármacos , Agregación Plaquetaria/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Factores de RiesgoRESUMEN
Diabetics have a much greater morbidity and mortality due to cardiovascular disease (CVD) than nondiabetics. Furthermore, diabetic women have a 3.8-fold greater risk for CVD compared to diabetic men. Inflammation is now considered a risk factor for CVD and it has been demonstrated that inflammation also plays a role in diabetes. One component of inflammation that has reported to be increased in patients with diabetes only and CVD only are proinflammatory cytokines, particularly interleukin-6 (IL-6), tumor necrosis factor (TNF-alpha), and interleukin-1 (IL-1beta). This study was performed to test the hypothesis that these proinflammatory cytokines were increased in women with CVD and further increased in diabetic women with CVD compared to nondiabetic women with CVD and healthy age-matched controls. We found that IL-6 was increased in diabetic women with CVD compared to healthy age-matched controls (1.41 = 0.48 to 0.33 +/- 0.06 pg/ml, P < .05). IL-6 was also increased in diabetic women without CVD compared to healthy age-matched controls, but not significantly (0.96 +/- 0.27 to 0.33 +/- 0.06 pg/ml). We found that TNF-alpha was increased in diabetic women with and without CVD compared to healthy age-matched controls, but not significantly (4.53 +/- 1.38 to 3.93 +/ -0.53 to 2.33 +/- 0.89 pg/ml). IL-1beta was not significantly different among any of the four groups of women. These results indicate that both IL-6 and TNF-alpha are chronically increased in diabetic women with and without CVD compared to nondiabetic women. The additive concentration of cytokines in diabetes and CVD suggests a common inflammatory state in both diabetes and CVD.
Asunto(s)
Enfermedades Cardiovasculares/sangre , Citocinas/sangre , Diabetes Mellitus Tipo 2/sangre , Angiopatías Diabéticas/sangre , Biomarcadores/sangre , Índice de Masa Corporal , Enfermedades Cardiovasculares/inmunología , Diabetes Mellitus Tipo 2/complicaciones , Diabetes Mellitus Tipo 2/inmunología , Angiopatías Diabéticas/epidemiología , Angiopatías Diabéticas/inmunología , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Inflamación , Interleucina-6/sangre , Persona de Mediana Edad , Factores de Riesgo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Cardiac ischemia is a serious complication of type 2 diabetes. However, the pathophysiology underlying the increased severity of myocardial ischemia in diabetes is not clear. This study tested the hypothesis that platelet adhesion protein expression is chronically increased in older type 2 diabetic patients with established ischemic heart disease (IHD) compared to age-matched, nondiabetic patients with IHD. We compared the chronic expression of two platelet adhesion proteins, P-selectin and GPIIb/IIIa, in whole blood and the platelet reactivity to an acute stimulus. We found that the expression of platelet P-selectin was chronically increased in the nondiabetic patients with IHD compared to normal subjects. P-selectin expression was further increased in the diabetic patients with IHD compared to the nondiabetic IHD patients (P<.05). The results were stratified to examine the potential effect of aspirin usage on adhesion protein expression. We found that the expression of the activated GPIIb/IIIa complex was significantly reduced in those diabetic cardiac patients who were taking aspirin (P<.05). These findings indicate that, in patients with IHD, platelet adhesion proteins are chronically expressed and that the level of expression is increased more in IHD patients with type 2 diabetes. This complication of diabetes may exacerbate thrombus formation during a recurrent event, increasing the severity of ischemic injury. The results give further support to the use of aspirin in type 2 diabetics with established cardiac disease.
Asunto(s)
Diabetes Mellitus Tipo 2/sangre , Diabetes Mellitus Tipo 2/complicaciones , Isquemia Miocárdica/sangre , Selectina-P/sangre , Activación Plaquetaria/fisiología , Adhesividad Plaquetaria , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/análisis , Biomarcadores/sangre , Citometría de Flujo , Humanos , Masculino , Selectina-P/efectos de los fármacos , Factor de Activación Plaquetaria/farmacología , Activación Plaquetaria/efectos de los fármacos , Adhesividad Plaquetaria/efectos de los fármacos , Valores de ReferenciaRESUMEN
Early intervention after acute ischemic stroke is essential to minimize brain cell injury. Although reperfusion of the ischemic brain is the treatment of choice for acute stroke, reperfusion itself may cause additional injury. The inflammatory cascade, characterized in part by early leukocyte interaction with endothelium, may contribute to this additional injury to blood vessels and surrounding brain tissue, extending the area of infarction. The selectin family of adhesion molecules mediates the initial, rolling and tethering of leukocytes to endothelium. P-selectin is rapidly expressed on ischemic endothelium in the brain vasculature, and L-selectin is expressed on leukocytes. Blocking the selectin-mediated tethering step may limit the inflammatory component of reperfusion injury in the brain. Fucoidin (FCN), a competitive inhibitor of P- and L-selectin, has been reported to decrease leukocyte accumulation during reperfusion of other organs. The effect of both leukocyte and endothelial selectin inhibition after cerebral ischemia and reperfusion has not been previously examined. The purpose of this study was to determine the effects of selectin adhesion molecule blockade on cerebral infarction size and neurological function after middle cerebral artery occlusion and reperfusion (MCAO-R) in the rat. MCAO was induced using the filament method. All animals were subjected to 4 h of MCAO and 24 h of reperfusion. After 24 h, brains were analyzed for size of infarction. Neurological function was assessed during stroke and 24 h after reperfusion. Two groups were studied, an untreated control group (n = 9) and a group treated with the selectin inhibitor, fucoidin (25 mg kg(-1)) (n = 9). We found that selectin blockade significantly reduced cerebral infarction size by 50% (p < 0.05) and improved neurological function (p < 0.05). In addition, a trend toward decreased cerebral edema was demonstrated with selectin inhibition. These results indicate that treatment of the blood and the endothelium with a selectin anti-inflammatory agent is protective after focal stroke and reperfusion in the rat.
Asunto(s)
Regulación hacia Abajo/efectos de los fármacos , Endotelio Vascular/metabolismo , Infarto de la Arteria Cerebral Media/sangre , Daño por Reperfusión/sangre , Selectinas/sangre , Animales , Endotelio Vascular/efectos de los fármacos , Infarto de la Arteria Cerebral Media/tratamiento farmacológico , Infarto de la Arteria Cerebral Media/metabolismo , Infarto de la Arteria Cerebral Media/patología , Recuento de Leucocitos , Masculino , Polisacáridos/farmacología , Polisacáridos/uso terapéutico , Ratas , Ratas Sprague-Dawley , Daño por Reperfusión/tratamiento farmacológico , Daño por Reperfusión/metabolismo , Daño por Reperfusión/patología , Selectinas/metabolismoRESUMEN
Blood is constantly in contact with a biological material, the blood vessel wall, without the need for anticoagulants to prevent clot formation on the vessel wall; however, man-made biomaterials require anticoagulants to prevent clot formation on the biomaterial. This study seeks to understand how some biomaterials elicit anticoagulant responses from endothelial cells (ECs), whereas others do not. Partial least squares regression analysis was used to correlate the activity of four relevant signaling molecules [extracellular signal-related kinase (ERK), c-Jun N-terminal kinase (JNK), Akt, and IκB kinase (IKK)] with human umbilical vein EC secretion of prostacyclin and clotting time of whole blood in contact with these cells. Prostacyclin secretion was increased when JNK activity (mean of all time-points) was elevated and IKK activity at 30 min was reduced. In addition, the clotting time, R-time measured by thromboelastography, was increased (reduced coagulability) when activity of both ERK and JNK (mean of all time-points) were increased and when Akt activity was increased at longer contact times (24-72 h after cell contact with material). Inhibition of each signaling molecule with subsequent testing for prostacyclin secretion and R-time confirmed the interrelationship between EC intracellular signaling and prostacyclin secretion. Generally, JNK inhibition decreased and IKK inhibition increased prostacyclin secretion. Inhibition of ERK or JNK generally increased coagulability, and Akt inhibition decreased the R-time of samples normally eliciting reduced coagulability. These findings increase our understanding of the signaling pathways involved in endothelial prostacyclin release and suggest targets for developing EC-seeded biomaterial surfaces that can minimize coagulation.
Asunto(s)
Epoprostenol/metabolismo , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Espacio Intracelular/metabolismo , Transducción de Señal , Animales , Humanos , Análisis de los Mínimos Cuadrados , Modelos Biológicos , Tromboelastografía , Tiempo de Coagulación de la Sangre TotalRESUMEN
Blood coagulation is the most significant complication of vascular biomaterials. A straightforward, sensitive, and standard measure of the compatibility of these materials with whole blood (hemocompatibility) is necessary to avoid coagulation. Current techniques used quantify only individual clotting components and are poor predictors of coagulation. The thromboelastograph (TEG) provides a measure of overall clot formation from whole blood. Although TEG is very common in clinical settings, its application to biomaterials is limited partly due to difficulty in sample preparation. In this protocol, whole blood samples are incubated with (1) biomaterials (tube with clamped ends) and (2) endothelial cells cultured on biomaterial surfaces (12-well plate) under controlled shearing conditions (10 rpm on rocker, at 37°C), and then the blood is transferred to the TEG to measure clot formation. TEG clearly discriminates among the R-times (time until initial clot formation) of expanded poly(tetrafluoroethylene), poly(urethane), and Tygon tubing. Marked differences in R-time are also seen when endothelial cells are cultured on various extracellular matrix proteins and proteoglycans. Thus, R-time provides a robust metric of overall thrombogenicity of biomaterials, and these procedures provide a standardized method for TEG to facilitate direct comparison among candidate biomaterials undergoing in-vitro testing.
Asunto(s)
Materiales Biocompatibles/química , Coagulación Sanguínea , Ensayo de Materiales , Politetrafluoroetileno/química , Poliuretanos/química , Tromboelastografía , Prótesis Vascular , Células Cultivadas , HumanosRESUMEN
OBJECTIVES: To determine the cardiovascular and hemostatic effects of fire suppression and postexposure active cooling. METHODS: Forty-four firefighters were evaluated before and after a 12-minute live-fire drill. Next, 50 firefighters performing the same drill were randomized to undergo postfire forearm immersion in 10 °C water or standard rehabilitation. RESULTS: In the first study, heart rate and core body temperature increased and serum C-reactive protein decreased but there were no significant changes in fibrinogen, sE-selectin, or sL-selectin. The second study demonstrated an increase in blood coagulability, leukocyte count, factors VIII and X, cortisol, and glucose, and a decrease in plasminogen and sP-selectin. Active cooling reduced mean core temperature, heart rate, and leukocyte count. CONCLUSIONS: Live-fire exposure increased core temperature, heart rate, coagulability, and leukocyte count; all except coagulability were reduced by active cooling.
Asunto(s)
Proteínas Sanguíneas/metabolismo , Crioterapia , Esfuerzo Físico/fisiología , Estrés Fisiológico , Adulto , Biomarcadores/sangre , Coagulación Sanguínea , Glucemia/metabolismo , Presión Sanguínea , Temperatura Corporal , Femenino , Incendios , Antebrazo , Frecuencia Cardíaca , Calor/efectos adversos , Humanos , Hidrocortisona/sangre , Inmersión , Recuento de Leucocitos , Masculino , Persona de Mediana Edad , Exposición Profesional , Selectina-P/sangre , Rehabilitación , Tromboelastografía , Factores de Tiempo , Adulto JovenRESUMEN
Recent studies indicate that not all diabetic subjects benefit from aspirin therapy. Our objective is to characterize diabetic subjects with aspirin resistance using urine thromboxane, and VerifyNow measures. Our results suggest that cardiovascular disease, microalbuminuria, poor diabetes control, and increased waist circumference help identify aspirin resistance in diabetes.
Asunto(s)
Aspirina/uso terapéutico , Angiopatías Diabéticas/prevención & control , Resistencia a Medicamentos , Inhibidores de Agregación Plaquetaria/uso terapéutico , Albuminuria/orina , Estudios de Cohortes , Angiopatías Diabéticas/sangre , Angiopatías Diabéticas/orina , Femenino , Hemoglobina Glucada/análisis , Humanos , Resistencia a la Insulina , Masculino , Persona de Mediana Edad , Nefelometría y Turbidimetría , Agregación Plaquetaria/efectos de los fármacos , Tromboxano B2/análogos & derivados , Tromboxano B2/orina , Circunferencia de la CinturaRESUMEN
Aspirin has lower antiplatelet activity in diabetic patients. Our aim is to study the roles of acute hyperglycemia and hyperlipidemia on aspirin function in diabetic subjects with and without cardiovascular disease. Using urine thromboxane (pg/mg creatinine) and VerifyNow (Aspirin Resistance Measures-ARU), we investigated diabetic subjects during a 2-hour glucose challenge (n = 49) or a 4-hour fat challenge (n = 11). All subjects were currently taking aspirin (81 or 325 mg). After fat ingestion, urine thromboxane increased in all subjects (Mean ± SE before: after) (1209 ± 336: 1552 ± 371, P = .01), while we noted a trend increase in VerifyNow measures (408 ± 8: 431 ± 18, P = .1). The response to glucose ingestion was variable. Diabetic subjects with cardiac disease and dyslipidemia increased thromboxane (1693 ± 364: 2799 ± 513, P < .05) and VerifyNow (457.6 ± 22.3: 527.1 ± 25.8, P < .05) measures after glucose. We conclude that saturated fat ingestion increases in vivo thromboxane production despite aspirin therapy.
RESUMEN
AIMS: Type 2 diabetes in humans is associated with hypercoaguability; however, little is known about platelet function in mouse models of type 2 diabetes used to study this disorder. Therefore, the purpose of this study was to examine platelet aggregation, coagulation, and markers of platelet activation in a diet-induced mouse model of type 2 diabetes. METHODS: Four week old, male, C57BL/6J mice were randomized to a standard chow or high fat (60% beef lard) diet for 4 months. To examine platelet function we measured ADP-induced whole blood aggregometry, whole blood coagulation by thromboelastography, tail bleeding times, platelet microparticle and platelet expression of p-selectin and platelet expression of CD61 by flow cytometry. RESULTS: Diabetic mice exhibited less aggregation (p<0.05), less coagulation (p<0.01), prolonged tail bleeding times (n.s.), and lower agonist stimulated platelet CD61 expression (p<0.001) compared to non-diabetic mice. There was no difference in platelet microparticle and platelet p-selectin expression. CONCLUSIONS: Diet-induced type 2 diabetic mouse do not demonstrate the hypercoagulability and platelet activation typically observed in humans with this disorder. More studies are warranted to further explore platelet function in this mouse model; to determine their applicability for studying these alterations in type 2 diabetes.
Asunto(s)
Coagulación Sanguínea/fisiología , Diabetes Mellitus/metabolismo , Activación Plaquetaria/fisiología , Animales , Diabetes Mellitus/inducido químicamente , Grasas de la Dieta/farmacología , Citometría de Flujo , Integrina beta3/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Selectina-P/metabolismo , Distribución Aleatoria , TromboelastografíaRESUMEN
The severity of ischaemic heart disease is markedly enhanced in type 2 diabetes. We recently reported that complement activation exacerbates I/R injury in the type 2 diabetic heart. The purpose of this study was to isolate and examine MBL pathway activation following I/R injury in the diabetic heart. ZLC and ZDF rats underwent 30 minutes of left coronary artery occlusion followed by 120 minutes of reperfusion. Two different groups of ZDF rats were treated with either FUT-175, a broad complement inhibitor, or P2D5, a monoclonal antibody raised against rat MBL-A. ZDF rats treated with FUT175 and P2D5 had significantly decreased myocardial infarct size, C3 deposition and neutrophil accumulation compared with untreated ZDF controls. Taken together, these findings indicate that the MBL pathway plays a key role in the severity of complement-mediated I/R injury in the type 2 diabetic heart.