| Background and objectiveThe vascular endothelial barrier dysfunction was a key initiatory factor in the genesis of atherosclerosis in vivo. The measure of endothelial permeability is a quantitative method to assay the endothelial barrier function. It was confirmed that changes in the F-actin cytoskeleton induced by exoteric stimuli were closely associated with increased endothelial permeability and endothelial dysfunction. Recently, a number of specific signaling molecules such as RhoA and serum response factor (SRF) were identified to modulate F-actin reorganization. Although changes in F-actin cytoskeleton regulated by different specific genes or various signal pathways in many cell types had been identified, it was amazing that how these signaling pathways orchestrated reacting on different exoteric stimuli in vascular endothelium and how they directly contributed to the vascular barrier dysfunction were not fully illustrated. In this study, we used different stimulation factors including serum-deprivation, TNF-alpha, LPS and PMA to mimic changes of cytoskeleton and permeability in HUVECs induced by different extrinsic stimuli in vitro and aimed to further investigate more about the molecular mechanism of change of cytoskeleton and endothelial permeability Methods 1. The RhoA, ROCK and SRF regulated the changes of cytoskeleton andpermeability induced by serum deprivation in HUVECs. (1) Endothelial permeability was measured with the Transwell chamber models and F-actin dynamics was detected using Rhodamine-phalloidine staining in HUVECs with serum deprivation. The activated RhoA and SRF expression was determinated by Western blot analysis in HUVECs with serum deprivation. (2) Constructing the recombinant retrovirus vectors of pLNCX-N19RhoA (dominate negative) and pLNCX-L63RhoA(constitutively active), transfecting them into Phoenix amphotropic 293 packaging cells by calcium phosphate/DNA coprecipitation to collect the supernatants viruses, then infecting the HUVECs with the retro viral supernatants and obtaining the single cell clone after the G418 selection. The activated RhoA expression was detected by pull down and Western blot analysis. The expression and distribution of SRF in normal and infected cells were evaluated by immunohistochemistry and Western blot in complete medium and in serum-deprived medium. The effect of F-actin polymerization was detected by Rhodamine-Phalloidine staining. (3) Y-27632 ( a Rho kinase inhibitor) and Latrunculin B (an actin depolymerization agent) were used to investigate the change of SRF and F-actin by Western blot and immunofluorescence in HUVECs with serum deprivation.2. The RhoA, ROCK and SRF regulated the changes of cytoskeleton and permeability induced by TNF-alpha, LPS and PMA in HUVECs.(1) Endothelial permeability was measured with the transwell chamber models and F-actin dynamics was detected using Rhodamine-phalloidin staining in HUVECs treated with TNF-alpha, LPS and PMA. (2) The expression of activated RhoA and SRF was determinated by Western blot analysis and the dynamic relationship of SRF and F-actin was investigated by Immunofluorescence in HUVECs treated with these three stimuli. (3) Pretreating HUVECs with Latrunculin B, the change of SRF and F-actin dynamics in HUVECs treated with these three stimuli were detected by Western blot and Immunofluorescence analysis (4)Western blot and Immunofluorescence analysis were used to observe the changes of SRF and F-actin dynamics both in N19RhoA- HUVECs and in HUVECs pretreated with Y-27632 after the treatment with the three stimuli.3. PKC-alpha and pERK regulated the changes of cytoskeleton and permeability in HUVECs induced by TNF-alpha and PMA.(1) PKC-alpha and pERK expression were detected by Western blot in HUVECs induced by the following stimuli: TNF-alpha, LPS, PMA and serum deprivation. (2) Pretreating HUVECs and N19RhoA-HUVECs with BIS II ( a PKC-alpha inhibitor), SRF and pERK expression was detected by Western blot induced by these four stimuli. (3)Western blot was used to analysis the SRF expression of N19RhoA-HUVECs pretreated with PD98059, an inhibitor of pERK. (4) Pretreating N19RhoA-HUVECs with BIS II or PD98059, immunofluorescence analysis was applied to investigate the SRF and F-actin dynamics induced by TNF-alpha and PMA. Results1. The RhoA-ROCK-SRF pathway took part in regulating the change of F-actin and endothelial permeability in HUVECs induced by serum deprivation .(1) The reorganization of F-actin cytoskeleton and increase of endothelial permeability were induced by serum deprivation in HUVECs. In HUVECs with serum deprivation, endothelial permeability increased and F-actin cytoskeleton reorganization were investigated obviously. In 2~3 d with serum depreviation, F-actin cytoskeleton polymerized and bundled to form large amount of stress fiber in the central and periphery portion of cells, simultaneously endothelial permeability increased obviously. In 4~5 d with serum deprivation, stress fiber depolymerized and endothelial permeability decreased slowly. The dynamics of F-actin kept closely consitent with the changes of endothelial permeability. (2) The activation of RhoA and SRF regulated the reorganization of F-actin cytoskeleton induced by serum deprivation. In HUVECs with serum deprivation, the expression of activated RhoA and SRF increased remarkably. In 2-3 d with serum depreviation, the enhancement of activated RhoA and total SRF expression was up to the maximum. In 4~5 d with serum deprivation, the expression of activated RhoA and total SRF showed a decreased trend, but the nuclear protein of SRF only detected in 2-3 d HUVECs with serum deprivation. (3)RhoA-ROCK-SRF regulated the reorganization of F-actin cytoskeleton induced by serum deprivation. To further investigate the relationship between SRF and RhoA protein, HUVECs were stably infected by retroviral vector pLNCX-L63RhoA or pLNCX-Nl9RhoA, the positive clone was obtained by G418 selection, immunohistochemistry analysis and Rhodamine-Phalloidine staining showedthat F-actin reorganized and bundled to form stress fiber in L63RhoA-HUVEC, and the expression of SRF was increased and localized in nuclei. It showed no obvious change in N19RhoA-HUVECs compared with the HUVECs. When cells treated with serum deprivation, there were no obvious change of F-actin dynamics in the N19RhoA-HUVECs and L63RhoA-HUVEC compared with the control groups which were cultured in serum condition. However, the expression and localization of SRF protein varied remarkably in HUVECs , N19RhoA-HUVECs and L63RhoA-HUVECs. The expression of SRF increased and localized in nuclei mainly in both HUVECs and L63RhoA-HUVECs in 2-3 d with serum deprivation. Although it was increased more obviously in N19RhoA-HUVECs, the localization of SRF was mainly in the perinuclear region, there was little expression in the nuclei. In 4-5 d serum-deprivated stimulation, the expression of SRF translocalized from the nuclei to the cytoplasm region in the HUVECs, compared with which was still localized in the nuclei in L63RhoA-HUVECs and in N19RhoA-HUVECs which was keeping in the perinuclear region. These results suggested that the activity of RhoA could promote the translocation of SRF into the nuclei and the formation of stress fiber , Inhibition of RhoA activity blocks the nuclear traffic of SRF and induced F-actin depolymerization. Pretreating HUVECs with Y-27632 to block the ROCK activation, SRF translocated from nuclei to cytoplasm, and stress fiber depolymerized in 2-3 d with serum deprivation. Pretreating HUVECs with Latrunculin B to inhibit the polymerization of F-actin, the extranuclear localization of SRF were observed in HUVECs in 2~3 d with serum deprivation. All of these results further indicated that the activity of SRF was closely associated with the F-actin dynamics and the RhoA-ROCK pathway participated in regulating the nuclear traffic of SRF protein and the reorganization of F-actin in HUVECs induced by serum deprivation . 2. RhoA-ROCK-SRF signal pathway took part in regulating the change of F-actin and endothelial permeability in HUVECs induced by TNF-alpha, LPS and PMA(1) The increase of endothelial permeability and the reorganization of F-actin were observed in HUVECs induced by the three stimuli. TNF-alpha, LPS and PMA induced endothelial permeability increase and F-actin reorganization in HUVECs. The significant effect doses and time points of different stimuliresulted in stress fiber formation and increased permeability were 100 ng/ml TNF alpha at 20min, 100 nmol/ L PMA at 20min, 100 ng/ml LPS at 6 hour in HUVECs. When the permeability in cells reached the maximum, it began to decline slowly accompanied by the depolymerization of F-actin followed with the prolonged time of stimulation (100 ng/ml TNF alpha at 120min, 100 nmol/ L PMA at 120min, 100 ng/ml LPS at 48 hour). (2) The activation of RhoA and SRF regulated the reorganization of F-actin cytoskeleton induced by TNF-alpha, LPS and PMA. To further study the molecular mechanism of F-actin reorganization, we found that in the HUVECs treated with these stimuli, the expression of activated RhoA increased , as well as stress fiber formation and the expression of SRF in nuclei also increased. With prolonged time of stimuli, the expression of RhoA decreased, mainly localized outside the nuclei accompanied by the depolymerization of F-actin. These results suggested that nuclear translocation of SRF was closely associated with F-actin reorganization in HUVECs treated with TNF-alpha, LPS and PMA. Pretreating cells with Latrunculin B, SRF redistributed outside the nuclei and F-actin depolymerized in cells treated with all the stimuli. It is strongly implicated that the activation of SRF with importing in nuclei induced by all stimuli plays a key role in the F-actin dynamics.(3)RhoA-ROCK-SRF was not the only pathway in regulating F-actin cytoskeleton reorganization induced by TNF-alpha, LPS and PMA. When inhibiting the RhoA activity in N19RhoA-HUVECs, we observed the SRF expression and F-actin dynamic induced by these three stimuli. Both the expression of SRF in nuclei and stress fiber formation were observed increased obviously in N19RhoA-HUVECs stimulated by 100 ng/ml TNF-alpha with 20 min and 100 nmol/ L PMA with 20 min. Reversely, in N19RhoA-HUVECs stimulated by 100 ng/ml LPS with 6 hour, the expression of SRF increased but mainly localized in cytoplasm accompanied by F-actin depolymerization. These results showed that, after RhoA activity was inhibited, the F-actin polymerization and nuclear translocalization of SRF still kept when cells were treated with TNF-alpha and PMA. When pretreating cells with Y-27632 to block the ROCK activity, similar results were obtained, which further identified that RhoA-ROCK signal pathway was the major but not the only pathway that controled the activation of SRF and F-actin dynamics in the HUVECs treated by TNF-alpha and PMA.3. PKC-alpha-pERK-SRF signal pathway participated in the change of F-actin and in HUVECs induced by TNF-alpha and PMA (1) Inhibition of RhoA activity altered the expression of PKC-alpha and pERK in HUVECs induced by TNF-alpha , PMA, LPS and serum deprivation. We detected the expression of PKC-alpha and pERK in HUVECs with four stimuli including TNF-alpha , PMA, LPS and serum deprivation. It was found that the expression of PKC-alpha was increased only in HUVECs stimulated by PMA and the expression of pERK was decreased only in HUVECs stimulated by serum deprivation. Then the expression of PKC-alpha and pERK in N19RhoA-HUVECs with four stimuli were observed and results showed that the expression of PKC-alpha was obviously increased in N19RhoA-HUVECs with all these four stimuli and the expression of pERK was also increased in N19RhoA-HUVECs treated with TNF-alpha and PMA accompanied by the activation of SRF and stress fiber formation in these cells.(2) PKC-alpha -pERK-SRF signal pathway participated in the change of F-actin and in HUVECs induced by TNF-alpha and PMA. The activation of SRF and F-actin polymerization also were investigated in N19RhoA-HUVECs treated with TNF-alpha and PMA could be completely blocked when cells were pretreated with the BIS II and PD98059. These results suggested that PKC-alpha-pERK signal pathway might be another pathway regulating the activity of SRF and F-actin reorganization. We also observed that PD98059 blocked the SRF activity via inhibiting its expression, unlike other regulator which controled its nuclear traffic. We found for the first time that the pivotal molecule regulating F-actin dynamics in endothelial cells isn't RhoA but SRF in this study and different stimuli active SRF with different style including regulating its nuclear traffic or regulating its expression. Conclusions1. These results show that RhoA-ROCK-SRF, as an important signal pathway, takes part in the F-actin dynamic and change of endothelial permeability induced by TNF-alpha, PMA, LPS and serum deprivation in HUVECs in vitro.2. PKC-alpha-pERK-SRF signal pathway is another signal pathway to play an important role in F-actin dynamic in HUVECs treated with TNF-alpha and PMA.3. These results indicate for the first time that the activation of SRF gene,...
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