RESUMEN
OBJECTIVE: Left ventricular assist device support produces a bleeding diathesis. Evidence suggests a major role for von Willebrand factor (vWF). We examined vWF metabolism in a preclinical model of short-term mechanical circulatory support. METHODS: In 25 calves (weight, 80-110 kg), the inflow/outflow graft of the Symphony Heart Assist System was sewn end-to-side to the carotid artery. Support was initiated (acute, n = 4; 1 week, n = 16; 2 weeks, n = 5). Acutely, carotid artery pressure and flow were measured to evaluate the hemodynamic changes near the anastomosis. At baseline and after ≤2 weeks of support, platelet aggregometry with adenosine 5'-diphosphate, collagen, and ristocetin was performed. Gel electrophoresis and wet immunoblotting qualitatively evaluated vWF multimers and quantified plasma ADAMTS-13, the vWF-cleaving protease. Carotid arterial rings near the anastomosis were studied with immunohistochemical staining for ADAMTS-13 and were cultured to quantify endothelial ADAMTS-13 production. Fluorescent resonance energy transfer was used to evaluate the enzymatic activity of ADAMTS-13 in the plasma and in supernatant from cultured carotid arterial rings. Plasma interleukin-6, which inhibits ADAMTS-13 activity, was measured using an enzyme-linked immunosorbent assay. RESULTS: During support, statistically significant (P < .05) changes in the carotid endothelium arterial hemodynamics were observed. The highest molecular weight vWF multimers were absent, and the vWF-ristocetin platelet aggregation pathway was significantly impaired. A modest but significant increase in plasma ADAMTS-13 protein and activity was observed. ADAMTS-13 decreased significantly in the carotid near the anastomosis but increased significantly in supernatant from cultured carotid arterial rings. The plasma interleukin-6 levels did not change significantly. CONCLUSIONS: Hemodynamic activation of vWF and increased plasma ADAMTS-13 activity may have reduced high-molecular-weight vWF multimers and thereby impaired the vWF-platelet aggregation pathway. Additional delineation of these pathways may improve management of left ventricular assist device-associated bleeding.
Asunto(s)
Arterias Carótidas/cirugía , Corazón Auxiliar , Agregación Plaquetaria , Factor de von Willebrand/metabolismo , Proteínas ADAM/sangre , Animales , Arterias Carótidas/metabolismo , Bovinos , Células Endoteliales/metabolismo , Corazón Auxiliar/efectos adversos , Hemodinámica , Hemorragia/sangre , Hemorragia/etiología , Interleucina-6/sangre , Masculino , Modelos Animales , Peso Molecular , Pruebas de Función Plaquetaria , Diseño de Prótesis , Factores de Tiempo , Técnicas de Cultivo de Tejidos , Función Ventricular IzquierdaRESUMEN
Lipid peroxidation products, such as 4-hydroxy-trans-2-nonenal (HNE), cause endothelial activation, and they increase the adhesion of the endothelium to circulating leukocytes. Nevertheless, the mechanisms underlying these effects remain unclear. We observed that in HNE-treated human umbilical vein endothelial cells, some of the protein-HNE adducts colocalize with the endoplasmic reticulum (ER) and that HNE forms covalent adducts with several ER chaperones that assist in protein folding. We also found that at concentrations that did not induce apoptosis or necrosis, HNE activated the unfolded protein response, leading to an increase in XBP-1 splicing, phosphorylation of protein kinase-like ER kinase and eukaryotic translation initiation factor 2α, and the induction of ATF3 and ATF4. This increase in eukaryotic translation initiation factor 2α phosphorylation was prevented by transfection with protein kinase-like ER kinase siRNA. Treatment with HNE increased the expression of the ER chaperones, GRP78 and HERP. Exposure to HNE led to a depletion of reduced glutathione and an increase in the production of reactive oxygen species (ROS); however, glutathione depletion and ROS production by tert-butyl-hydroperoxide did not trigger the unfolded protein response. Pretreatment with a chemical chaperone, phenylbutyric acid, or adenoviral transfection with ATF6 attenuated HNE-induced monocyte adhesion and IL-8 induction. Moreover, phenylbutyric acid and taurine-conjugated ursodeoxycholic acid attenuated HNE-induced leukocyte rolling and their firm adhesion to the endothelium in rat cremaster muscle. These data suggest that endothelial activation by HNE is mediated in part by ER stress, induced by mechanisms independent of ROS production or glutathione depletion. The induction of ER stress may be a significant cause of vascular inflammation induced by products of oxidized lipids.
Asunto(s)
Aldehídos/metabolismo , Aldehídos/farmacología , Estrés del Retículo Endoplásmico/efectos de los fármacos , Peroxidación de Lípido , Secuencia de Aminoácidos , Animales , Chaperón BiP del Retículo Endoplásmico , Endotelio/citología , Endotelio/efectos de los fármacos , Endotelio/metabolismo , Glutatión/metabolismo , Células Endoteliales de la Vena Umbilical Humana/citología , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Peroxidación de Lípido/efectos de los fármacos , Datos de Secuencia Molecular , Transporte de Proteínas/efectos de los fármacos , Proteínas/química , Proteínas/metabolismo , Respuesta de Proteína Desplegada/efectos de los fármacosRESUMEN
BACKGROUND: Acrolein is a dietary aldehyde that is present in high concentrations in alcoholic beverages and foods including cheese, donuts and coffee. It is also abundant in tobacco smoke, automobile exhaust and industrial waste and is generated in vivo during inflammation and oxidative stress. OBJECTIVES: The goal of this study was to examine the effects of dietary acrolein on atherosclerosis. METHODS: Eight-week-old male apoE-null mice were gavage-fed acrolein (2.5mg/kg/day) for 8 weeks. Atherosclerotic lesion formation and composition and plasma lipids and platelet factor 4 (PF4) levels were measured. Effects of acrolein and PF4 on endothelial cell function was measured in vitro. RESULTS: Acrolein feeding increased the concentration of cholesterol in the plasma. NMR analysis of the lipoproteins showed that acrolein feeding increased the abundance of small and medium VLDL particles. Acrolein feeding also increased atherosclerotic lesion formation in the aortic valve and the aortic arch. Immunohistochemical analysis showed increased macrophage accumulation in the lesions of acrolein-fed mice. Plasma PF4 levels and accumulation of PF4 in atherosclerotic lesions was increased in the acrolein-fed mice. Incubation of endothelial cells with the plasma of acrolein-fed mice augmented transmigration of monocytic cells, which was abolished by anti-PF4 antibody treatment. CONCLUSIONS: Dietary exposure to acrolein exacerbates atherosclerosis in apoE-null mice. Consumption of foods and beverages rich in unsaturated aldehydes such as acrolein may be a contributing factor to the progression of atherosclerotic lesions.
Asunto(s)
Acroleína/farmacología , Apolipoproteínas E/deficiencia , Aterosclerosis/patología , Acroleína/administración & dosificación , Administración Oral , Animales , Aterosclerosis/inducido químicamente , Colesterol , Dieta , Endotelio Vascular/efectos de los fármacos , Humanos , Masculino , Ratones , Ratones Noqueados , Factor Plaquetario 4/sangreRESUMEN
Acrolein is a common air pollutant that is present in high concentrations in wood, cotton, and tobacco smoke, automobile exhaust and industrial waste and emissions. Exposure to acrolein containing environmental pollutants such as tobacco smoke and automobile exhaust has been linked to the activation of the coagulation and hemostasis pathways and thereby to the predisposition of thrombotic events in human. To examine the effects of acrolein on platelets, adult male C57Bl/6 mice were subjected acute (5ppm for 6h) or sub-chronic (1ppm, 6h/day for 4days) acrolein inhalation exposures. The acute exposure to acrolein did not cause pulmonary inflammation and oxidative stress, dyslipidemia or induce liver damage or muscle injury. Platelet GSH levels in acrolein-exposed mice were comparable to controls, but acrolein-exposure increased the abundance of protein-acrolein adducts in platelets. Platelets isolated from mice, exposed to both acute and sub-chronic acrolein levels, showed increased ADP-induced platelet aggregation. Exposure to acrolein also led to an increase in the indices of platelet activation such as the formation of platelet-leukocyte aggregates in the blood, plasma PF4 levels, and increased platelet-fibrinogen binding. The bleeding time was decreased in acrolein exposed mice. Plasma levels of PF4 were also increased in mice exposed to environmental tobacco smoke. Similar to inhalation exposure, acrolein feeding to mice also increased platelet activation and established a pro-thrombotic state in mice. Together, our data suggest that acrolein is an important contributing factor to the pro-thrombotic risk in human exposure to pollutants such as tobacco smoke or automobile exhaust, or through dietary consumption.
Asunto(s)
Acroleína/toxicidad , Contaminantes Atmosféricos/toxicidad , Plaquetas/efectos de los fármacos , Exposición por Inhalación , Activación Plaquetaria/efectos de los fármacos , Animales , Coagulación Sanguínea/efectos de los fármacos , Plaquetas/metabolismo , Fibrinógeno/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Estrés Oxidativo/efectos de los fármacos , Agregación Plaquetaria/efectos de los fármacos , Contaminación por Humo de TabacoRESUMEN
Blood vessel loss and inflammation cause secondary degeneration following spinal cord injury. Angiopoietin-1 through the Tie2 receptor, and other ligands through alphavbeta3 integrin, promote endothelial cell survival during developmental or tumour angiogenesis. Here, daily intravenous injections with an alphavbeta3-binding peptide named C16 or an angiopoietin-1 mimetic following a spinal cord contusion at thoracic level 9 in mice rescued epicentre blood vessels, white matter and locomotor function, and reduced detrimental inflammation. Preserved vascularity and reduced inflammation correlated with improved outcomes. C16 and angiopoietin-1 reduced leukocyte transmigration in vitro. Growth factor receptors and integrins facilitate each others' function. Therefore, angiopoietin-1 and C16 were combined and the effects were additive, resulting in almost complete functional recovery. The treatment had lasting effects when started 4 h following injury and terminated after one week. These results identify alphavbeta3 integrin and the endothelial-selective angiopoietin-1 as vascular and inflammatory regulators that can be targeted in a clinically relevant manner for neuroprotection after central nervous system trauma.
Asunto(s)
Angiopoyetina 1/administración & dosificación , Integrina alfaVbeta3/administración & dosificación , Fármacos Neuroprotectores/administración & dosificación , Fragmentos de Péptidos/administración & dosificación , Traumatismos de la Médula Espinal/prevención & control , Médula Espinal/irrigación sanguínea , Médula Espinal/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Movimiento Celular/efectos de los fármacos , Movimiento Celular/fisiología , Quimioterapia Combinada , Femenino , Humanos , Inyecciones Intravenosas , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Recuperación de la Función/efectos de los fármacos , Recuperación de la Función/fisiología , Traumatismos de la Médula Espinal/fisiopatología , Vértebras Torácicas , Factores de TiempoRESUMEN
Exposure to arsenic-contaminated water has been shown to be associated with cardiovascular disease, especially atherosclerosis. We examined the effect of arsenic exposure on atherosclerotic lesion formation, lesion composition and nature in ApoE-/- mice. Early post-natal exposure (3-week-old mice exposed to 49 ppm arsenic as NaAsO(2) in drinking water for 7 weeks) increased the atherosclerotic lesion formation by 3- to 5-fold in the aortic valve and the aortic arch, without affecting plasma cholesterol. Exposure to arsenic for 13 weeks (3-week-old mice exposed to 1, 4.9 and 49 ppm arsenic as NaAsO(2) in drinking water) increased the lesion formation and macrophage accumulation in a dose-dependent manner. Temporal studies showed that continuous arsenic exposure significantly exacerbated the lesion formation throughout the aortic tree at 16 and 36 weeks of age. Withdrawal of arsenic for 12 weeks after an initial exposure for 21 weeks (to 3-week-old mice) significantly decreased lesion formation as compared with mice continuously exposed to arsenic. Similarly, adult exposure to 49 ppm arsenic for 24 weeks, starting at 12 weeks of age increased lesion formation by 2- to 3.6-fold in the aortic valve, the aortic arch and the abdominal aorta. Lesions of arsenic-exposed mice displayed a 1.8-fold increase in macrophage accumulation whereas smooth muscle cell and T-lymphocyte contents were not changed. Expression of pro-inflammatory chemokine MCP-1 and cytokine IL-6 and markers of oxidative stress, protein-HNE and protein-MDA adducts were markedly increased in lesions of arsenic-exposed mice. Plasma concentrations of MCP-1, IL-6 and MDA were also significantly elevated in arsenic-exposed mice. These data suggest that arsenic exposure increases oxidative stress, inflammation and atherosclerotic lesion formation.
Asunto(s)
Apolipoproteínas E/genética , Arsenitos/toxicidad , Aterosclerosis/inducido químicamente , Inflamación/inducido químicamente , Estrés Oxidativo/efectos de los fármacos , Compuestos de Sodio/toxicidad , Animales , Aorta Torácica/efectos de los fármacos , Aorta Torácica/metabolismo , Válvula Aórtica/efectos de los fármacos , Válvula Aórtica/metabolismo , Arsenitos/administración & dosificación , Relación Dosis-Respuesta a Droga , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/metabolismo , Compuestos de Sodio/administración & dosificación , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismo , Factores de TiempoRESUMEN
Spinal cord injury causes progressive secondary tissue degeneration, leaving many injured people with neurological disabilities. There are no satisfactory neuroprotective treatments. Protein tyrosine phosphatases inactivate neurotrophic factor receptors and downstream intracellular signaling molecules. Thus, we tested whether the peroxovanadium compound potassium bisperoxo(1,10-phenanthroline)oxovanadate (V) [bpV(phen)], a stable, potent and selective protein tyrosine phosphatase inhibitor, would be neuroprotective after a thoracic spinal cord contusion in adult rats. Intrathecal bpV(phen) infusions through a lumbar puncture rescued dorsal column sensory axons innervating the nucleus gracilis and white matter at the injury epicenter. At the most effective dose, essentially all of these axons and most of the white matter at the epicenter were spared (vs approximately 60% with control infusions). bpV(phen) treatments started 4 h after contusion were fully effective. This treatment greatly improved and normalized sensorimotor function in a grid-walking test and provided complete axonal protection over 6 weeks. The treatment rescued sensory-evoked potentials that disappeared after dorsal column transection. bpV(phen) affected early degenerative mechanisms, because the main effects were seen at 7 d and lasted beyond the treatment period. The neuroprotection appeared to be mediated by rescue of blood vessels. bpV(phen) reduced apoptosis of cultured endothelial cells. These results show that a small molecule, used in a clinically relevant manner, reduces loss of long-projecting axons, myelin, blood vessels, and function in a model relevant to the most common type of spinal cord injury in humans. They reveal a novel mechanism of spinal cord degeneration involving protein tyrosine phosphatases that can be targeted with therapeutic drugs.
Asunto(s)
Sistemas de Liberación de Medicamentos , Fármacos Neuroprotectores/uso terapéutico , Compuestos Organometálicos/uso terapéutico , Fenantrolinas/uso terapéutico , Proteínas Tirosina Fosfatasas/antagonistas & inhibidores , Traumatismos de la Médula Espinal/tratamiento farmacológico , Traumatismos de la Médula Espinal/enzimología , Animales , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Células Cultivadas , Modelos Animales de Enfermedad , Sistemas de Liberación de Medicamentos/métodos , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/patología , Femenino , Humanos , Mediadores de Inflamación/administración & dosificación , Mediadores de Inflamación/uso terapéutico , Fármacos Neuroprotectores/administración & dosificación , Compuestos Organometálicos/administración & dosificación , Fenantrolinas/administración & dosificación , Proteínas Tirosina Fosfatasas/metabolismo , Ratas , Ratas Sprague-Dawley , Traumatismos de la Médula Espinal/patología , Traumatismos de la Médula Espinal/fisiopatologíaRESUMEN
We examined the mechanism regulating intercellular cell adhesion molecule-1 (ICAM-1)-dependent monocyte transendothelial migration. Monocyte migration through endothelial cells expressing ICAM-1 alone was comparable to that of tumor necrosis factor-alpha-treated cells. Transmigration was reduced in ICAM-1 lacking the cytoplasmic tail and in tyrosine to alanine substitutions at Tyr-485 and Tyr-474. Tissue inhibitors of matrix metalloproteinases (TIMPs) -2 and -3 blocked transmigration, whereas TIMP-1 was ineffective. This profile suggested a role for membrane-type matrix metalloproteinases (MT-MMPs) in transmigration. Inhibitory antibodies and small interference RNA directed against MT1-MMP blocked transmigration, whereas overexpression of MT1-MMP in endothelial cells or monocytes promoted transmigration. MT1-MMP mediated the ectodomain cleavage of ICAM-1 that was blocked by TIMP-2 and -3. Overexpression of MT1-MMP rescued function in ICAM-1Y485A, and to a lesser extent in the cytoplasmic tail-deleted ICAM-1. In a binding assay, wild-type ICAM-1 bound to purified MT1-MMP while ICAM-1 mutants bound poorly. MT1-MMP co-localized with ICAM-1 at distinct structures in endothelial cells. MT1-MMP localization with cells expressing ICAM-1 mutations was reduced and diffused. These results indicate that the cytoplasmic tail of ICAM-1 regulates leukocyte transmigration through MT1-MMP interaction.
Asunto(s)
Regulación de la Expresión Génica , Molécula 1 de Adhesión Intercelular/biosíntesis , Metaloproteinasa 14 de la Matriz/fisiología , Monocitos/metabolismo , Adenoviridae/metabolismo , Aorta/metabolismo , Línea Celular , Movimiento Celular , Citoplasma/metabolismo , Células Endoteliales/citología , Humanos , Leucocitos/metabolismo , Modelos Biológicos , Mutación , Unión ProteicaRESUMEN
Consumption of arsenic contaminated drinking water has been linked to higher rates of coronary disease, stroke, and peripheral arterial disease. Recent evidence suggests that early life exposures may play a significant role in the onset of chronic adult diseases. To investigate the potential for in utero arsenic exposure to accelerate the onset of cardiovascular disease we exposed pregnant ApoE-knockout (ApoE(-/-)) mice to arsenic in their drinking water and examined the aortic trees of their male offspring for evidence of early disease 10 and 16 weeks after birth. Mice were maintained on normal chow after weaning. ApoE(-/-) mice are a commonly used model for atherogenesis and spontaneously develop atherosclerotic disease. Mice exposed to arsenic in utero showed a >2-fold increase in lesion formation in the aortic roots as well as the aortic arch compared to control mice at both 10 and 16 weeks of age. The mice exposed to arsenic also had a 20-40% decrease in total triglycerides, but no change in total cholesterol, phospholipids and total abundance of VLDL or HDL particles. Subfractionation of VLDL particles showed a decrease in large VLDL particles. In addition, the arsenic-exposed mice showed a vasorelaxation defect in response to acetylcholine suggesting disturbance of endothelial cell signalling. These results indicate that in utero arsenic exposure induces an early onset of atherosclerosis in ApoE(-/-) mice without a hyperlipidemic diet and support the hypothesis that in utero arsenic exposure may be atherogenic in humans.
Asunto(s)
Apolipoproteínas E/genética , Arsenitos/toxicidad , Aterosclerosis/inducido químicamente , Efectos Tardíos de la Exposición Prenatal , Compuestos de Sodio/toxicidad , Acetilcolina/farmacología , Administración Oral , Animales , Aorta Torácica/efectos de los fármacos , Aorta Torácica/metabolismo , Aorta Torácica/fisiopatología , Apolipoproteínas E/deficiencia , Arsenitos/administración & dosificación , Aterosclerosis/sangre , Aterosclerosis/fisiopatología , Inhibidores Enzimáticos/administración & dosificación , Inhibidores Enzimáticos/toxicidad , Femenino , Técnicas In Vitro , Lipoproteínas/sangre , Masculino , Ratones , Ratones Noqueados , Donantes de Óxido Nítrico/farmacología , Nitroprusiato/farmacología , Fenilefrina/farmacología , Fotomicrografía/métodos , Cloruro de Potasio/farmacología , Embarazo , Compuestos de Sodio/administración & dosificación , Vasoconstricción/efectos de los fármacos , Vasodilatación/efectos de los fármacos , DesteteRESUMEN
Ectodomain shedding has emerged as an important regulatory step in the function of transmembrane proteins. Intercellular adhesion molecule-1 (ICAM-1), an adhesion receptor that mediates inflammatory and immune responses, undergoes shedding in the presence of inflammatory mediators and phorbol 12-myristate 13-acetate (PMA). The shedding of ICAM-1 in ICAM-1-transfected 293 cells upon PMA stimulation and in endothelial cells upon tumor necrosis factor-alpha stimulation was blocked by metalloproteinase inhibitors, whereas serine protease inhibitors were ineffective. p-Aminophenylmercuric acetate, a mercuric compound that is known to activate matrix metalloproteinases, up-regulated ICAM-1 shedding. TIMP-3 (but not TIMP-1 or -2) effectively blocked cleavage. This profile suggests the involvement of the ADAM family of proteases in the cleavage of ICAM-1. The introduction of enzymatically active tumor necrosis factor-alpha-converting enzyme (TACE) into ICAM-1-expressing cells up-regulated cleavage. Small interfering RNA directed against TACE blocked ICAM-1 cleavage. ICAM-1 transfected into TACE-/- fibroblasts did not show increased shedding over constitutive levels in the presence of PMA, whereas cleavage did occur in ICAM-1-transfected TACE+/+ cells. These results indicate that ICAM-1 shedding is mediated by TACE. Blocking the shedding of ICAM-1 altered the cell adhesive function, as ICAM-1-mediated cell adhesion was up-regulated in the presence of TACE small interfering RNA and TIMP-3, but not TIMP-1. However, cleavage was found to occur at multiple sites within the stalk domain of ICAM-1, and numerous point mutations within the region did not affect cleavage, indicating that TACE-mediated cleavage of ICAM-1 may not be sequence-specific.
Asunto(s)
Proteínas ADAM/fisiología , Molécula 1 de Adhesión Intercelular/fisiología , Proteína ADAM17 , Animales , Sitios de Unión , Western Blotting , Adhesión Celular , Línea Celular , ADN Complementario/metabolismo , Ácido Edético/química , Ácido Egtácico/química , Fibroblastos/metabolismo , Humanos , Molécula 1 de Adhesión Intercelular/metabolismo , Ratones , Ratones Noqueados , Monocitos/metabolismo , Mutación , Mutación Puntual , Estructura Terciaria de Proteína , ARN Interferente Pequeño/metabolismo , Acetato de Tetradecanoilforbol/farmacología , Inhibidor Tisular de Metaloproteinasa-2/metabolismo , Inhibidor Tisular de Metaloproteinasa-3/metabolismo , Transfección , Regulación hacia ArribaRESUMEN
Elevated fibrinogen (Fg) concentration in blood is a high risk factor for many cardiovascular diseases. We hypothesize that Fg and its early degradation product, fragment D, may result in arterial constriction by binding endothelial intercellular adhesion molecule-1 (ICAM-1). The vasoconstriction induced by Fg and fragment D was studied in third- and second-order arterioles (3As and 2As, respectively) of Sprague-Dawley rat cremaster muscle in vivo, in aortic and femoral artery rings, and in the segments of first-order arterioles (1As) isolated from rat cremaster muscle. Intravascular infusion of Fg induced significant constriction of 3As and 2As (by 33.4 +/- 3.4 and 23.7 +/- 4.3%, respectively) in vivo and was abolished in the presence of the specific endothelin type A receptor blocker BQ-610. Fg and fragment D produced significant constriction of both aortic and femoral artery rings. Isolated 1As constricted in response to Fg (0.3 microM) and fragment D (3 microM) by 31 +/- 1.4 and 12 +/- 1.5%, respectively. Fluorescently labeled Fg and fragment D bound to the vascular wall, whereas albumin bound to a significantly lesser degree. The binding of Fg and fragment D to the arteriolar wall and constriction of aortic and femoral artery rings as well as isolated 1As were abolished in the presence of anti-Fg and anti-ICAM-1 antibodies. These results indicate that binding of Fg and fragment D to the vascular wall through ICAM-1 may contribute to the increased vascular tone and resistance that compromise circulation.
Asunto(s)
Aorta/efectos de los fármacos , Arteria Femoral/efectos de los fármacos , Productos de Degradación de Fibrina-Fibrinógeno/farmacología , Fibrinógeno/farmacología , Vasoconstricción/efectos de los fármacos , Animales , Aorta/fisiología , Arteriolas/efectos de los fármacos , Arteriolas/fisiología , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/fisiología , Arteria Femoral/fisiología , Técnicas In Vitro , Molécula 1 de Adhesión Intercelular/metabolismo , Masculino , Músculo Esquelético/irrigación sanguínea , Ratas , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiologíaRESUMEN
ICAM-1, a membrane-bound receptor, is released as soluble ICAM-1 in inflammatory diseases. To delineate mechanisms regulating ICAM-1 cleavage, studies were performed in endothelial cells (EC), human embryonic kidney (HEK)-293 cells transfected with wild-type (WT) ICAM-1, and ICAM-1 containing single tyrosine-to-alanine substitutions (Y474A, Y476A, and Y485A) in the cytoplasmic region. Tyrosine residues at 474 and 485 become phosphorylated upon ICAM-1 ligation and associate with signaling modules. Cleavage was assessed by using an antibody against the cytoplasmic tail of ICAM-1, which recognizes intact ICAM-1 and the 7-kDa membrane-bound fragment remaining after cleavage. Cleavage in HEK-293 WT cells was accelerated by phorbol ester PMA, whereas in EC it was induced by tumor necrosis factor-alpha. In both cell types, a 7-kDa ICAM-1 remnant was detected. Tyrosine phosphatase inhibitors dephostatin and sodium orthovanadate augmented cleavage. PD-98059 (MEK kinase inhibitor), geldanamycin and PP2 (Src kinase inhibitors), and wortmannin (phosphatidylinositol 3-kinase inhibitor) dose-dependently inhibited cleavage in both cell types. SB-203580 (p38 inhibitor) was more effective in EC, and D609 (PLC inhibitor) mostly affected cleavage in HEK-293 cells. Cleavage was drastically decreased in Y474A and Y485A, whereas it was marginally reduced in Y476A. Surprisingly, phosphorylation was not detectable on the 7-kDa fragment of ICAM-1. These results implicate distinct pathways in the cleavage process and suggest a preferred signal transmission route for ICAM-1 shedding in the two cell systems tested. Tyrosine residues Y474 and Y485 within the cytoplasmic sequence of ICAM-1 regulate the cleavage process.
Asunto(s)
Molécula 1 de Adhesión Intercelular/química , Transducción de Señal , Alanina/genética , Sustitución de Aminoácidos , Línea Celular , Citoplasma/metabolismo , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Inhibidores Enzimáticos/farmacología , Humanos , Molécula 1 de Adhesión Intercelular/efectos de los fármacos , Molécula 1 de Adhesión Intercelular/genética , Riñón/embriología , Fosforilación , Acetato de Tetradecanoilforbol/farmacología , Factor de Necrosis Tumoral alfa/farmacología , Tirosina/genéticaRESUMEN
Acrolein is a highly reactive aldehyde pollutant and an endogenous product of lipid peroxidation. Increased generation of, or exposures to, acrolein incites pulmonary and vascular injury. The effects of acrolein on the vasomotor responses of rat aortic rings were studied to understand its mechanism of action. Incubation with acrolein (10-100 microM) alone did not affect the resting tone of aortic vessels; however, a dose-dependent relaxation of phenylephrine-precontracted aortic rings was observed. Acrolein-induced relaxation was slow and time dependent and the extent of relaxation after 100 min of application was 44.7 +/- 4.1% (10 microM), 56.0 +/- 5.6% (20 microM), 61.0 +/- 7.9% (40 microM), and 96.1 +/- 2.1 (80 microM), respectively, versus 14.2 +/- 3.3% relaxation in the absence of acrolein. Acrolein-induced vasorelaxation was prevented by endothelial denudation and was abolished on pretreatment with the nitric oxide synthase inhibitor Nomega-nitro-L-arginine methyl ester, the guanylyl cyclase inhibitor 1H-[1,2,4]oxidazolo[4,3-a]quinoxaline-1-one, or the cyclooxygenase inhibitor indomethacin. Inhibition of K+ channels (by tetrabutylammonium) or Na+-K+-ATPase (by ouabain) did not significantly prevent acrolein-mediated vasorelaxation. Exposure to acrolein in the presence or absence of other compounds elicited slow wave vasomotor effect in 77% of aortic vessels versus 1.4% in control. Vasomotor responses were also studied on aortic rings prepared from rats fed 2 mg. kg-1. day-1 acrolein for 3 alternate days by oral gavage. These vessels developed a significantly lower contractile response to phenylephrine compared with controls. Together, these results indicate that acute acrolein exposure evokes delayed vasorelaxation due to a nitric oxide- and prostacyclin-dependent mechanism, whereas in vivo acrolein exposure compromises vessel contractility.
Asunto(s)
Acroleína/farmacología , Contaminantes Atmosféricos/farmacología , Aorta Torácica/efectos de los fármacos , Vasodilatación/efectos de los fármacos , Animales , Aorta Torácica/fisiología , Western Blotting , Epoprostenol/biosíntesis , Masculino , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa/análisis , Óxido Nítrico Sintasa de Tipo III , Fenilefrina/farmacología , Ratas , Ratas Sprague-Dawley , Vasoconstrictores/farmacologíaRESUMEN
tert-Butyl hydroperoxide (t-BOOH), a membrane permeant oxidant, elicits enhanced vasoconstriction of perfused kidney and mesenteric arterial beds isolated from DOCA-salt-induced hypertensive rats. We hypothesize that enhanced vasoconstriction to t-BOOH during DOCA-salt hypertension involves free radical species and decreases in the expression of the endogenous antioxidant enzyme, superoxide dismutase (SOD). t-BOOH (0.01-50 micromol) dose-dependently constricted the perfused kidney and mesenteric vascular beds (MVB) of rats. Infusion of tempol (100 microM), a free radical scavenger, reduced the constrictor responses from 116.70+/-16.65% to 57.45+/-9.25% (kidneys) and from 72.91+/-3.70% to 48.10+/-0.10% (mesenteric beds). t-BOOH-induced vasoconstriction of both vascular beds were also significantly reduced in DOCA-salt rats treated chronically (15 mg/kg ip, 3 weeks) with tempol (DOCA/TEMPOL). Catalase (500 IU) did not attenuate t-BOOH-induced responses in vascular beds of DOCA/TEMPOL rats. Western blot analyses showed significant reduction in Cu/Zn-SOD expression in DOCA-salt versus sham rats of both vascular preparations; SOD expressions were protected from down-regulation in DOCA/TEMPOL vascular beds. This study suggests that free radical species is involved in both t-BOOH-induced constrictions and in the down-regulation of SOD protein expressions during DOCA-salt hypertension.
Asunto(s)
Presión Sanguínea/efectos de los fármacos , Desoxicorticosterona , Hipertensión/fisiopatología , Vasoconstricción/efectos de los fármacos , terc-Butilhidroperóxido/farmacología , Animales , Presión Sanguínea/fisiología , Óxidos N-Cíclicos/uso terapéutico , Relación Dosis-Respuesta a Droga , Radicales Libres/metabolismo , Hipertensión/inducido químicamente , Hipertensión/tratamiento farmacológico , Hipertensión/enzimología , Masculino , Ratas , Ratas Sprague-Dawley , Marcadores de Spin , Superóxido Dismutasa/biosíntesis , Vasoconstricción/fisiologíaRESUMEN
The binding of plasma protein fibrinogen (Fg) to intercellular adhesion molecule-1 (ICAM-1) on endothelial cells mediates the attachment of leukocytes and platelets that may result in vascular occlusion. Fg:ICAM-1 interactions elicit an array of effects that could have implications in vascular pathology and inflammation. ICAM-1 expression is regulated during inflammation and upon Fg binding. The mechanistic model presented provides a framework to delineate the consequences of Fg binding to ICAM-1.