RESUMEN
Lipopolysaccharide (LPS)-induced (i.v. or i.c.v., 1.8 mg/kg) release of von Willebrand factor (vWF) was examined in spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats. SHR rats released significantly (P < 0.05) more vWF than WKY rats in response to LPS. LPS also inhibited factor VIII procoagulant activity (FVIII:c) which may indicate an increase in thrombin activity. Cultured cerebrovascular endothelial cells (EC) derived from both SHR and WKY rats, as well as human umbilical vein EC (HUVEC) cultures constitutively released vWF. Treatment with agonists including LPS, thrombin and tumor necrosis factor-alpha (TNF alpha) did not affect the in vitro secretion of vWF by cerebrovascular EC cultures but significantly upregulated vWF release by HUVEC cultures. Preincubation of cerebrovascular EC cultures with interleukin-1 (IL-1) +/- TNF alpha or co-culturing in the presence of LPS-activated syngeneic monocytes had no effect on vWF secretion. The findings demonstrate that conditions of hypertension may affect endothelial cells and make them more responsive to agonist stimulation and thereby increase secretion of vWF, an important factor in hemostasis as well as thrombosis. The capacity of LPS to significantly affect the in vivo secretion of vWF in SHR and WKY rats but not cultured cerebrovascular EC indicates that observed elevations in plasma vWF were not derived from cerebrovascular EC. It is suggested that hypertension may function as a risk factor for thrombotic stroke by influencing factors involved in coagulation processes, such as vWF and factor VIII:c.
Asunto(s)
Trastornos Cerebrovasculares/metabolismo , Factor VIII/agonistas , Factor VIII/metabolismo , Factor de von Willebrand/agonistas , Factor de von Willebrand/metabolismo , Animales , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Hipertensión/fisiopatología , Inyecciones Intravenosas , Inyecciones Intraventriculares , Interleucina-1/farmacología , Lipopolisacáridos/farmacología , Monocitos/efectos de los fármacos , Ratas , Ratas Endogámicas SHR , Ratas Endogámicas WKY , Factores de Riesgo , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
The amounts of tissue factor (TF) expressed by brain microvascular endothelial cells (BMECs) from normotensive Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR) were compared after stimulating the cells with different doses of lipopolysaccharide (LPS), thrombin, phorbol myristic acid (PMA), Ca(2+)-ionophore (A23187), or tumor necrosis factor (TNF) and interleukin-1 (IL-1). Treatment of cultured BMECs from WKY and SHR with all of these factors dose-dependently increased their total amount of TF; no substantive differences in the levels of enhanced TF expression were observed between WKY and SHR BMECs. We conclude that stimulated endothelium from rats with hypertension, a major stroke risk factor, is not hyperresponsive with respect to TF expression when compared to normotensive controls.
Asunto(s)
Encéfalo/efectos de los fármacos , Endotelio Vascular/efectos de los fármacos , Hipertensión/metabolismo , Tromboplastina/biosíntesis , Animales , Encéfalo/irrigación sanguínea , Encéfalo/metabolismo , Calcimicina/farmacología , Endotelio Vascular/metabolismo , Interleucina-1/farmacología , Lipopolisacáridos/toxicidad , Microcirculación/efectos de los fármacos , Ratas , Ratas Endogámicas SHR , Ratas Endogámicas WKY , Acetato de Tetradecanoilforbol/farmacología , Trombina/farmacología , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Microvascular endothelial cells from the adult rat brain were cultured on Matrigel and found to express many differentiated properties including secretion of prostacyclin (PGI2) and von Willebrand's factor (vWF). Brain microvascular endothelial cells (BMECs) were purified by dextran and percoll gradients after enzymatic treatment and cultured under various conditions. BMECs that were plated on Matrigel stained positively for factor VIII-related antigen and incorporated Di-I-acetylated low density lipoprotein, whereas BMEC plated on fibronectin, gelatin, or uncoated dishes did not express any of the above properties which are characteristic of endothelial cells. vWF was measured by a sensitive ELISA in the culture media of BMECs plated on different types of matrices. Specificity of the anti-human vWF antibodies for the rat vWF was verified by immunoabsorption on a solid phase, sodium dodecyl sulfate, and Western blot analysis. BMECs also secreted vWF into the culture media only when the cells were plated on Matrigel, and this secretion was augmented after a 6 h incubation with an interleukin-1 tumor necrosis factor-alpha mixture, but not by lipopolysaccharide. From different matrices tested, only Matrigel permitted the secretion of PGI2 by BMECs. Cells also proved to be sensitive to mechanical stimulation and became refractory to secretagogue if the mechanical stimulation was serially repeated. Under the best conditions, stimulation of the cells with bradykinin (1 microM) substantially increased PGI2 secretion. These data indicate that growth of BMECs on Matrigel in vitro permits the expression of classical endothelial cell markers in a manner similar to the behavior of these cells in situ.
Asunto(s)
Encéfalo/irrigación sanguínea , Endotelio Vascular/citología , Epoprostenol/metabolismo , Matriz Extracelular/fisiología , Lipoproteínas LDL/farmacocinética , Factor de von Willebrand/metabolismo , Acetilación , Animales , Anticuerpos/inmunología , Especificidad de Anticuerpos , Western Blotting , Bradiquinina/farmacología , Colágeno/análisis , Combinación de Medicamentos , Electroforesis en Gel de Poliacrilamida , Endotelio Vascular/metabolismo , Ensayo de Inmunoadsorción Enzimática , Matriz Extracelular/química , Fibronectinas/análisis , Gelatina/análisis , Interleucina-1/farmacología , Laminina/análisis , Microcirculación/metabolismo , Estimulación Física , Proteoglicanos/análisis , Ratas , Ratas Endogámicas , Factor de Necrosis Tumoral alfa/farmacología , Factor de von Willebrand/análisis , Factor de von Willebrand/inmunologíaRESUMEN
Rats produced more TNF activity in cerebrospinal fluid (CSF) than in blood after intracerebroventricular (i.c.v.) injection of lipopolysaccharide (LPS). After intravenous (i.v.) LPS, blood TNF levels exceeded CSF levels. Thus, brain cells appear to produce TNF in response to LPS. Rats with the stroke-risk factors hypertension or combined hypertension and genetic stroke-proneness produce more TNF in response to a provocative dose of LPS i.v. than control animals free of these risk factors. The possible relevance to stroke vulnerability is discussed.