The Role Of Cx43 In Regulation Of Vascular Leakage And Hyporeactivity After Shock And Its Mechanism

Shock is the most common complication of trauma, and the incidence and mortality are still high. Declined vascular function are presented in severe trauma or shock patients. It includes vascular hyporeactivity and vascular leakage. The decreased vascular reactivity can seriously affect the development and treatment of shock, and is one of the main causes of refractory hypotension and even death. Vascular barrier disfunction, namely vascular leakage, can lead to intravascular fluid extravasation and cause tissue edema and organ dysfunction after severe trauma, shock and sepsis.The present viewpoint for the mechanism of vascular hyporeactivity include: Receptor desensitization, Membrane hyperpolarization and Calcium desensitization. The mechanism for vascular leakage consists of two pathways: paracellular pathway and transcellular pathway. Both pathways play a major role in the vascular leakage in shock. A variety of cytokines such as vascular endothelial growth factor(VEGF), thrombin and TNF-α induced vascular hyperpermeability via these pathways.Gap junctions is an important vascular inter-cellular structure which mediates a variety of cell-cell transduction of the electrochemical signals involved in the regulation of vascular function. Cx43,as a major constitutive protein of gap junction, participates in the occurrence and development of many vascular diseases such as: Atherosclerosis, hypertension, etc. Our previous study found that Cx43 took part in regulation of vascular permeability after shock, but the mechanism is not clear. A recent study showed that Cx43 played a key role in vascular permeability in acute lung injury caused by the inhalation of acid. Whether or not Cx43 participates in regulation of vascular leakage after shock? And what is the mechanism? It is not clear.Studies have confirmed that Rho-kinase and PKC signaling pathway play a key role in the regulation of the cytoskeleton, causing cell contraction and cell migration, etc. Our previous study demonstrated Rho kinase and PKC pathway play an important role in the regulation of vascular reactivity after shock. Besides, study found that Rho kinase was involved in vascular leakage, for example, Rho A/Rock pathway mediated the increased endothelial permeability induced by thrombin. Osteopontin(OPN) as a signaling molecule was found to be involved in a variety of vascular pathological and physiological changes such as cell adhesion, angiogenesis and cell migration. For example, OPN mediated VEGF-induced cell migration by changing endothelial barrier function. OPN can activate FAK and ERK pathways, and these signaling pathways can lead to cell change in stiffness and cytoskeletal rearrangements. These physiological and pathological events indicate that OPN may have a close relationship to the vascular endothelial barrier function, but the mechanism is unclear.Based on these issues, using severe sepsis model and rat pulmonary vein endothelial cells and hemorrhagic shock and hypoxia treated vascular smooth muscle cells, we investigated the role of Cx43 in vascular leakage and vascular hyporeactivity after shock and the underling mechanisms.Main research contents and methods:Part one : the role of cx43 in regulation of vascular leakage after shock in rats and its mechanism1. Effect of Cx43 in regulation of vascular leakage after severe sepsis: with severe sepsis rats and pulmonary vein endothelial cells(VECs), the changes of Cx43 expression and vascular leakage were measured. The transfected VECs with Cx43 overexpressed and RNAi lentivirus were used to measure the infiltration rate of BSA and TER.2. The role of Rho kinase in regulation of vascular leakage by Cx43: with Cx43 overexpressed lentivirus transfected VECs, the changes of infiltration rate of FITC-BSA and TER after Rho kinase inhibitor Y-27632 treatment and the expression of Rho kinase were observed.3. The role OPN and tight junction proteins in regulation of vascular leakage by Cx43: with severe sepsis rats and VECs treated by LPS, the expression of zo-1,claudin-5,OPN and the vascular permeability were measured. With the VECs treated by LPS, the effect of OPN RNAi on vascular permeability and expression of zo-1and claudin-5 were observed. With VECs transfected by Cx43, expression of OPN and some transcription factor of OPN were measured.Part two: the effect of Cx43 on vascular hyporeactivity after shock and its mechanism.1. The role of Cx43 in regulation of vascular hyporeactivity by PDGF after shock and its relationship to PKC and Rho kinase: with hypoxia treated vascular ring, the effect of MEGJ inhibitor 18-αGA and Cx43 AODN on vascular reactivity and calcium sensitivity were observed. With vascular smooth muscle cells(VSMCs), the roles of Rho kinase and PKC in regulation of PDGF on vascular reactivity and the effect of Cx43 AODN on the activity of PKC and Rho kinase were observed.2. The role of Cx43 in regulation of BK on vascular hyporeactivity after shock and its mechanism.With hemorrhagic shock rats and hypoxia treated vascular ring and VSMCs, the effect of MEGJ inhibitor on vascular reactivity and the effect of Rho kinase and PKC in the regulation of BK on vascular reactivity were observed.Main resultsPart one : the role of cx43 in regulation of vascular leakage after shock in rats and its mechanism1. The role of Cx43 in vascular leakage after severe sepsisThe results showed that the expression of Cx43 was significantly increased after severe sepsis. It was positively correlated with vascular leakage changes. Variation in the expression of Cx43 induced vascular permeability changes. These results suggest that Cx43 participates in regulation of vascular leakage after shock.2. The role of Rho kinase-MLC20 pathway in regulation of vascular leakage by Cx43 and its mechanism.LPS and Cx43 significantly elevated the infiltfration rate of FITC-BSA and the phosphorylation of MLC20, reduced the TER values, and led to the stress fibers formation in endothelial cell. Rho kinase inhibitor Y-27632 blocked these changes. Cx43 over-expressed lentivirus could increase the expression of Rho kinase. These results suggest that Cx43 regulates the vascular leakage via Rho kinase-MLC20 pathway.3. The role OPN / tight junction proteins in regulation of vascular permeability by Cx43 and its mechanism.The expression of OPN was significantly increased and zo-1and claudin-5 were significantly decreased in severe septic rats and LPS treated entothelial cells. Over-expressed Cx43 significantly increased the expression of OPN. Tcf-4 and β-catenin RNAi inhibited Cx43 induced expression of OPN. OPN overexpresion induced the degradation of zo-1 and claudin-5. These results suggest that Cx43 regulation of vascular leakage is via Tcf-4/β-catenin-OPN/tight junction pathway.Part two: the role of Cx43 in regulation of vascular hyporeactivity after shock and its mechanism.1. Cx43 mediated the regulation of PDGF on vascular hyporeactivity and its relationship to PKC and Rho kinase.PDGF significantly improved the vascular reactivity after shock, MEGJ blocker 18-GA and Cx43 AODN inhibited the improvement of vascular reactivity by PDGF. PKC inhibitor Staurosporine and Rho kinase inhibitors Y-27632 significantly inhibited the vascular reactivity and calcium sensitivity. PDGF significantly increased the phosphorylation of the substrate of PKC(Peptag C) and Rho kinase(MYPT) in hypoxia –treated blood vessels, and Cx43 AODN significantly inhibited the phosphorylation vascular MYPT and Peptag C1. These results suggest that Cx43 mediates the regulation of vascular reacitivity by PDGF via Rho kinase and PKC pathway after shock.2. The role of Cx43 in regulation of vascular reactivity by BK and its mechanisms.High doses of BK significantly improved the vascular reactivity after shock, MEGJ blocker 18-GA and Cx43 AODN inhibited the improvement of vascular reactivity by BK. BK increased the phosphorylation of Cx43 at ser368 site and activated Rho kinase, PKC-α and PKC-ε. Rho kinase inhibitors Y-27632 and PKC inhibitor staurosporine blocked the improvement of vascular reactivity by BK. PKC and Rho kinase agonists could further improve the vascular reactivity of blood vessels to BK. This effect of BK was blocked by Cx43 AODN. These results suggest that Cx43 take part in regulation of vascular reactivity by BK via Rho kinase and PKC pathway.Conclusion1. Cx43 is involved in the regulation of vascular permeability after shock. One hand, Cx43 makes cytoskeletal changes and cell contraction, and increases the cell gap among endothelial cells by activating Rho kinase-MLC20 pathway; on the other hand, Cx43 regulates the expression of OPN by transcription factors Tcf-4/β-catenin and further inhibits the expression of zo-1 and claudin-5 which increase the gap of intercellular tight junctions.2. Cx43 takes part in regulation of vascular reactivity by PDGF and BK after shock. PDGF and BK activates PKC and Rho kinase pathway mainly via phosphorylation of the ser368 site of Cx43, and via this mechanism further improves the vascular calcium sensitivity and vascular reactivity.