RESUMO
INTRODUCTION: We have previously shown that novel oxidation products of Bilirubin, called Bilirubin oxidation products (BOXes), are found in humans and animal models post subarachnoid hemorrhage. We have also proposed that BOXes may play a role in the pathogenesis and clinical complications post SAH. In this study we report on the direct toxicity effects of BOXes on rat brain. METHODS: Identical volumes of either vehicle (normal saline) or BOXes (30 µl of a 20 µM solution) were applied above the dura through a cranial window of young (approximately 7-13 weeks) and aged (approximately 12-18 months) adult male Sprague Dawley rats (Charles River, Wilmington, MA, USA). To determine the extent of BOX-mediated injury, histology and immunocytochemistry were performed at 1, 2, 4, and 7 days post-surgical application of BOXes. We assessed the area of stress gene induction of HSP25/27 and HSP32. Immunohistochemistry was performed using standard avidin-biotin techniques. A monoclonal antibody to HSP25/27 (StressGen, Victoria, British Columbia, Canada), a monoclonal antibody to HSP32/HO-1 (StressGen), and a polyclonal HSP 32/HO-1 antibody were used for the immunocytochemistry. RESULTS: A single dose of BOXes produced substantial increases in HSP25 and HO-1 in the aged rats at all early time points (≤4 days). After 7 days all groups were not significantly different than saline control. Young rats were resistant to BOXes effects compared to saline control with trends towards increased stress gene expression caused by BOXes that did not reach statistical significance. CONCLUSION: We conclude from these studies that BOXes have direct effects on stress gene expression of the cortex post single dose application and that this can be seen for several days with apparent resolution at about 7 days. If BOXes are produced at similar levels in patients, the latency and duration of some SAH complications are consistent with these results.
Assuntos
Envelhecimento , Antioxidantes/metabolismo , Bilirrubina/metabolismo , Encéfalo/metabolismo , Hemorragia Subaracnóidea/patologia , Análise de Variância , Animais , Antioxidantes/química , Bilirrubina/química , Encéfalo/efeitos dos fármacos , Modelos Animais de Doenças , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Choque Térmico HSP27/metabolismo , Heme Oxigenase (Desciclizante)/metabolismo , Heme Oxigenase-1/metabolismo , Masculino , Oxirredução , Ratos , Ratos Sprague-Dawley , Espectrofotometria/métodos , Fatores de TempoRESUMO
Apolipoprotein E (Apo E) is an important genetic risk factor for multiple neurological, vascular and cardiovascular diseases. Previously, we reported Apo E isoprotein-specific modulation of tissue plasminogen activator (tPA) using an in vitro blood-clotting assay. Here, we studied the conformational changes of Apo E2, E3 and E4 in the presence of tPA and vice versa using circular dichroism (CD) and dual polarization interferometry (DPI). We report isoprotein and state-specific intermolecular interactions between the Apo E isoforms and tPA. Apo E2 interaction with immobilized tPA leads to significant conformational changes which are not observed with Apo E3 or E4. Additionally, tPA induces changes in helicity of lipidated Apo E2 whereas no detectable changes were observed in Apo E3 or E4. The Tukey's test for interaction indicated a significant (P < 0.001) interaction between tPA and Apo E2 in the lipidated environment. These results may be important regarding the mechanism by which Apo E has isoprotein-specific effects on many biological processes and diseases involving blood clotting, proteolysis and perfusion.
Assuntos
Apolipoproteínas E/metabolismo , Ativador de Plasminogênio Tecidual/metabolismo , Humanos , Ligação ProteicaRESUMO
Apo E, and its respective isoforms, have been linked to outcome and survival in cerebral vascular and cardiovascular diseases. The effectiveness of intravenous tPA in patients with acute ischemic stroke appears to be enhanced in patients who have an Apo E2 phenotype. The ability of Apo E isoproteins (endogenous Apo E isoproteins or exogenous Apo E isoproteins) to modulate tPA-induced clot lysis in vitro was assessed using an in vitro clot assay system. Blood samples were obtained from 18-volunteers with three Apo E genotypes: E2, E3 and E4. tPA-induced clot lysis (0-4 microgram/ml tPA), was assessed in the presence or absence of supplemental Apo E2, E3 or E4 (9.8 microgram/ml). tPA-induced clot lysis was significantly (P equal or less than 0.0001) enhanced by supplementation with Apo E2 (EC50 0.20 0.06 microgram/ml) as compared to tPA alone (0.72 0.19). Apo E4 supplementation caused a significant (P < or = 0.05) inhibition of clot lysis (0.98 0.23), but there was no significant change caused by Apo E3. The genotype of the volunteer did not significantly affect the ability of the supplemental Apo E from modulating tPA-induced clot lysis. We conclude that the administration of Apo E isoproteins can modulate clot lysis in vitro. Our results suggest that the Apo E isoprotein may have an impact on clot dissolution and the effectiveness of thrombolytic therapy.
Assuntos
Apolipoproteínas E/fisiologia , Fibrinólise/fisiologia , Ativador de Plasminogênio Tecidual/fisiologia , Apolipoproteína E2 , Apolipoproteína E3 , Apolipoproteína E4 , Apolipoproteínas E/sangue , Apolipoproteínas E/genética , Apolipoproteínas E/farmacologia , Cromatografia em Camada Fina/métodos , Fibrinólise/efeitos dos fármacos , Fibrinólise/genética , Genótipo , Humanos , Mucosa Bucal/química , Isoformas de Proteínas/sangue , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiologia , Fatores de Tempo , Ativador de Plasminogênio Tecidual/sangue , Ativador de Plasminogênio Tecidual/genética , Ativador de Plasminogênio Tecidual/metabolismoRESUMO
OBJECT: Cerebral vasospasm is a common cause of morbidity and death following aneurysmal subarachnoid hemorrhage (SAH). Previous research has shown that bilirubin oxidation products (BOXes) are present in the cerebral spinal fluid in patients with SAH-induced cerebral vasospasm and can contribute to vasoconstriction and vasospasm in vitro and in vivo. The events leading to cerebral vasospasm are not understood; however, one component of the occlusion may be due to vascular remodeling. In this study the authors have investigated the actions of BOXes, okadaic acid ([OA], a phosphatase inhibitor), and phorbol-12 myristate-13 acetate ([PMA], a protein kinase activator) on vascular smooth-muscle cell (VSMC) morphology and metabolism. METHODS: Immunohistochemical analysis was performed to assess VSMC morphology and alpha-smooth-muscle actin (alphaSMA) distribution following the application of BOXes, OA, or PMA. Changes in the level of lactate dehydrogenase (LDH) release and oxidative metabolism were also measured. The BOXes, OA, or PMA caused VSMCs to change their shape and exhibit altered alphaSMA distribution. These treatments increased LDH release (p < 0.05), which is an index of increased cell stress. Oxidative metabolism significantly increased at low and high doses of BOXes, that is, 143 +/- 8.5% and 180 +/- 11.8%, respectively (p < 0.0001). Both PMA and OA also caused a significant increase in metabolism. CONCLUSIONS: The authors concluded that BOXes, OA, and PMA alter VSMC morphology and metabolic activity, events that have been observed during vascular remodeling. Although the mechanism remains unclear, the results indicate that BOXes may play a role in the vascular remodeling that occurs following aneurysmal SAH.