P38 MAPK Regulates Vascular Endothelial Actin Cytoskeleton And Associated Tight Junction As Well As Permeability In Response To Coxsackievirus B₃

Objectives:The group B of Coxsackieviruses ( CVB) belongs to enterovirus of family picomavirus. There are six serotypes in CVB which are CVB_{1 to 6}, particularly CVB₃ are considered to be the most frequent cause of human viral heart disease. It is estimated more than 20% of cases of viral myocarditis are caused by CVB₃, and viral myocarditis can induce severe arrhythmia and sudden cardiac death and lead to the development of a chronic form of myocarditis, dilated cardiomyopathy, which is the second leading etiological cause of heart transplantation. CVB₃ infections are characterized by primary viral replication at the portal of entry, such as the nasopharynx and intestine, and then viruses into the bloodstream which is called viremia, via the blood during viremia, CVB₃ can invade several different organs which distant from the gut, such as heart, pancreatic islet and central nervous system and so on, causing myocarditis, pancretitis or viral meningitis.Viruses must either pass through the vascular endothelium by transcytosis or infection or be carried past the endothelial barrier by infected circulating cells, which can migrate into the target tissues. In fact, two such events must occur, since the virus must first enter into the circulation and then leave the circulation to gain access to the secondary sites of infection. Therefore, CVB₃ must induce endothelial barrier dysfunction during the process to get through the endothelial cells. Vascularendothelial cells have therefore a major role in viral tropism and disease pathogenesis. Which signal pathway may regulate the process? So the objectives of the study are:(1) To examine the contents and morphological changes of F-actin and ZO-1induced by CVB3.(2) To investigate the effects of CVB3 on viability and permeability of vascular endothelial cells.(3) To elucidate the role of p38 MAPK in modulation of CVB3-mediated cytoskeletal reorganization and hyperpermeability.Mehtods:(1) Double staining of F-actin and ZO-1 imaged by confocal microscopy.(2) Quantitative analysis of F-actin and ZO-1 proteins with flow cytometry.(3) Endothelial viability determined by MTT colorimetric assay.(4) Endothelial cell monolayers permeability measured by trans well. Results:(1) Time course of morphological changes of F-actin and ZO-1 in response toCVB3 were observed Smooth continuous staining for both F-actin and ZO-1 alongwith the intercellular borders of adjacent endothelial cells and the absence of intercellular gaps were seen in normal untreated cells. When endothelial cells exposed to CVB3 for 12 h, the F-actin and ZO-1 patterns displayed local discontinuities. The change becomes more significant by 24 h, CVB3-induced changes in F-actin organization, whereby the cells showed an increase and thinking of actin bundles and the formation of stress fibers. ZO-1 label was seen to consist of numerous sawtooth-shaped structures or short spurs that were often associated with the formation of intercellular gaps. It begins to reverse itself by 48 h of exposure to CYB3. This indicates that CVB3-mediated morphological changes may be time-dependent.(2) Time course of contents of F-actin and ZO-1 proteins in response to CVB3. We detected the contents of F-actin and ZO-1 proteins with flow cytometry and found that CVB3 lead to a strong increase of F-actin content compared with normal endothelial cells for a similar period. The amount of ZO-1 was not different in cellstreated with CVB3 compared with untreated normal cells for a similar period.(3) Time course of MTT of endothelial cells in response to CVB3. The absorbances were much lesser in CVB3 infected cells for a similar period. The results indicate CVB3 can decrease endothelial cells viability or proliferation.(4) Time course of Pa in response to CVB3. Endothelial cells monolayers exposed to CVB3 showed a significantly greater increase in permeability at 12, 24, and 48 hours compared with normal control monolayers.(5) The p38 MAPK inhibitor SB203580 alone did not affect the morphology of F-actin and ZO-1, basal F-actin and ZO-1 contents, the viability and permeability of normal endothelial cells.(6) Dose-dependent effect of SB2O358O on the morphological changes induced by CVB3. Addition of 0.25 umol/L, 2.5 umol/L, 25 umol/L and 50 fimol/L to pretreat ECs and then introduce CVB3, 0.25 umol/L SB203580 had no effect on attenuating CVB3-induced actin polymerization and ZO-1 redistribution, while 2.5umol/L SB203580 could partly reduced CVB3-induced formation of stress fiber and ZO-1 reorganization. 25 umol/L and 50 umol/L SB203580 could prevent CVB3 mediated morphological changes of F-actin and ZO-1. To investigate the effects of p38 MAP kinase inhibition on reversing CVB3-induced the morphological changes of endothelial cells, we introduced CVB3 first, followed SB203580 25 umol/L treated, and found inhibition of p38 MAPK by SB203580 reversed the changes of F-actin and ZO-1 induced by CVB3.(7) Dose-dependent effect of SB203580 on contents of CVB3 infected endothelial F-actin and ZO-1 proteins. 0.25 umol/L and 2.5 umol/L SB203580 could not completely prevent increased content of F-actin induced by CVB3, their contents of F-actin were still significantly higher than that of normal cells, while 25 umol/L and 50 umol/L SB203580 could prevent the elevated contents of F-actin mediated by CVB3. So the effect of SB203580 on F-actin protein content was dose dependent. 25 umol/L SB203580 could also reverse the elevated F-actin protein contents induced by CVB3 to normal value. SB203580 could not affect the amount of ZO-1 protein.(8) Dose-dependent effect of SB2O3580 on viability of CVB3 infectedendothelial cells. Only 25 umol/L and 50 umol/L SB203580 could prevent the decreased cell viability induced by CVB3. The cell viability was significantly restored by adding 25 umol/L SB203580 after exposure to CVB3.(9) Dose-dependent effect of SB203580 on permeability of CVB3 infected endothelial cells. We next examined the effect of p38 MAPK inhibition on endothelial permeability by pretreating endothelial monolayers with SB203580 30 min before CVB3 exposure. Only 25 umol/L and 50 umol/L SB203580 could reduce CVB3-induced endothelial hyperpermeability. 0.25 umol/L and 2.5 umol/L SB203580 had no significant effect on CVB3-induced permeability. The monolayer permeability was significantly restored by adding 25 umol/L SB20358O after exposure to CVB3. So the effect of SB203580 on permeability of CVB3 infected endothelial cells was dose dependent. Conclusions:CVB3 could induce F-actin and ZO-1 redistribution, increase contents of F-actin proteins and permeability of vascular endothelial cells, which might be the way for CVB3 to get through the endothelial cells. p38 MAPK activation could be involved in the pathogenesis of CVB3 infection in endothelial cells. SB203580 could reduce CVB3-induced actin polymerization and endothelial permeability.