RESUMO
The present study aimed to determine the role of Rho/Rho kinase (Rho/ROCK) phosphorylation on advanced glycation end products (AGEs)-induced morphological and functional changes in human dermal microvascular endothelial cells (HMVECs). HMVECs were respectively incubated with different concentrations of AGEs-modified human serum albumin (AGEs-HSA) for different time. In some other cases, HMVECs were pretreated with ROCK inhibitors (H-1152 or Y-27632). The morphological changes of F-actin cytoskeleton were visualized by rhodamine-phalloidin staining and the phosphorylation of Rho and ROCK were determined by Western blot. Endothelial monolayer permeability was assessed by measuring the flux of FITC-albumin across the endothelial cells. The results showed that the distribution of F-actin was significantly altered by AGEs-HSA in time and dose-dependent patterns. These effects were inhibited by ROCK inhibitors. The phosphorylation of Rho and RCOK was remarkably increased by AGEs-HSA treatment while total Rho and ROCK protein levels were not affected. The permeability of endothelial monolayer was dramatically increased by AGEs-HSA, and both ROCK inhibitors (H-1152 or Y-27632) attenuated these hyperpermeability responses. The results obtained suggest that the phosphorylation of Rho/ROCK plays an important role in AGEs-induced morphological and functional alterations in HMVECs.
Assuntos
Humanos , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina , Farmacologia , Citoesqueleto de Actina , Metabolismo , Actinas , Metabolismo , Amidas , Farmacologia , Células Endoteliais , Metabolismo , Endotélio Vascular , Biologia Celular , Fluoresceína-5-Isotiocianato , Metabolismo , Produtos Finais de Glicação Avançada , Farmacologia , Faloidina , Fosforilação , Piridinas , Farmacologia , Rodaminas , Albumina Sérica , Metabolismo , Farmacologia , Albumina Sérica Humana , Transdução de Sinais , Quinases Associadas a rho , MetabolismoRESUMO
<p><b>OBJECTIVE</b>To investigate the dose and time-dependent effects of lipopolysaccharide (LPS) on cytoskeletal F-acitn and G-actin reorganizations by visualizing their distribution and measuring their contents in human umbilical vein endothelial cell line ECV-304.</p><p><b>METHODS</b>F-actin was labeled with rhodamine-phalloidin and G-actin with deoxyribonuclease I (DNase I)conjugated with fluorescein isothiocyanate (FITC). Contents of cytoskeletal proteins were obtained by flow cytometry.</p><p><b>RESULTS</b>F-actin was mainly distributed peripherally in endothelial cells under normal conditions. LPS stimulation caused the formation of stress fibers and filopodia. G-actin was normally seen in perinuclear and nuclear areas in control ECV-304 cells. Under LPS stimulation, G-actin dots appeared in the cytoplasmic region. The actin disorganization was accompanied by the time- and dose- dependent decrease in F-actin pool and increase in G-actin pool.</p><p><b>CONCLUSIONS</b>LPS can induce characteristic morphological alterations of actin cytoskeleton and formation of intercellular gap in endothelial cells, accompanied by changes in F-actin and G-actin pools.</p>
Assuntos
Humanos , Actinas , Análise de Variância , Células Cultivadas , Desoxirribonuclease I , Relação Dose-Resposta a Droga , Células Endoteliais , Química , Escherichia coli , Fluoresceína-5-Isotiocianato , Corantes Fluorescentes , Lipopolissacarídeos , Farmacologia , Faloidina , Rodaminas , Veias Umbilicais , Biologia CelularRESUMO
<p><b>OBJECTIVE</b>To study the effect of cGMP-dependent protein kinase (PKG) on the pathogenesis of septic shock.</p><p><b>METHODS</b>Confluent endothelial cells were disintegrated and centrifugated to obtain cell lysates after being treated with LPS or PKG activator 8-Br-cGMP. PKG activity of lysates was measured with radioactive isotope label method in a reaction system of phosphorylation of specific substrate H2B by PKG, and the shape and the distribution of intracellular filamentous actin were detected by specific fluorescence staining. For the control study, the PKG specific inhibitor KT5823 was used to pretreat the endothelial cells before the administration of LPS or PKG activator 8-Br-cGMP.</p><p><b>RESULTS</b>Exposure to LPS for 5, 10, 30 and 60 minutes led to a rapid time-dependent increase in endothelial PKG activity (P < 0.01 compared to the blank) and the polar distribution of intracellular filamentous actin and preincubation with KT5823 abolished these effects. 8-Br-cGMP was similar to LPS.</p><p><b>CONCLUSIONS</b>The results suggested that LPS can mediate PKG activation and the stress variety of filamentous actin in the vascular endothelial cells, which probably induce the endothelial hyperpermeability after septic shock.</p>