Exchange Protein Directly Activated By CAMP Plays A Critical Role In Regulation Of Vascular Fibrinolysis

Objective:Endothelial cells?ECs?actively participate in balancing blood coagulation and fibrinolysis through complicated mechanisms,among which ANXA2 serves as important cell surface receptor in tissue plasminogen activators?PA?based fibrinolysis.It is noticeable that accumulating evidences have shown that the cAMP signaling pathway is relevant to the regulation of ANXA2 transport in ECs.Exchange protein activated by cAMP-1?Epac1?,which dominantly expressed in ECs.Thus we are seeking the potential targeting protein of cAMP-Epac1 signaling pathway in ECs.Taking advantage of our Epac1-null mice,and using biochemical and biomechanical assays,our study aimed at the critical role of Epac1 in maintaining vascular fibrinolytic function via regulating endothelial surface expression of ANXA2.Methods:1?The study of the fibrinolytic function in Epac1-null miceImmunohistochemistry?IHC?:Compare positive staining of fibrin deposition in lung,kidney and brain from Epac1-null mice with that in the sane tissue from wild-type mice.FeCl₃-induced thrombosis model:After applicating FeCl₃ on adventitia of carotid artery of Epac1-null and wild-type mice,the blood flows were detected by transonic probe and the values were recorded every minute.We compare maximal occlusion and maximal recovery of blood flow to indirectly reflect the ability to clear macrovascular thrombi of Epac1-null mice.2?The study of plasma ANXA2 variation in Epac1-null miceWe used enzyme linked immunosorbent assay?ELISA?to determine ANXA2concentration in plasma of Epac1-null mice compared with that of wild-type mice.3?The study to find out whether reintroduction of ANXA2 improves attenuatedfibrinolytic function in Epac1-null miceReintroduction of ANXA2 in circulation:reintroduce certain amount of recombinant ANXA2?rANXA2?through tail vein injection.We observe and record the change of carotid blood flow before and after rANXA2 injection.The blood flows were detected by transonic probe and the values were recorded every minute.We compare maximal occlusion and maximal blood flow recovery of rANXA2 pre-treated Epac1-null mice with PBS pre-treated Epac1-null mice.4?The effect of pharmacologically inhibition of Epac1 on the expression of cell surface ANXA2 in ECsHuman umbilical vein endothelial cells?HUVECs?were pretreated with Epac specific inhibitor 09?ESI09?as treatment group and dimethyl sulfoxide?DMSO?as vehicle group.Impermeable immmunofluorescence?IF?:Labeling by impermeable IF,quantify the expression of ANXA2 at ESI09-treated and DMSO-treated HUVECs cell surface by fluorescent densitometry function in Image J.Atomic force microscopy?AFM?:Anti-ANXA2 antibodies were immobilized on polystyrene spheres attached to a colloidal cantilever.This functionalized cantilever was approached into contact with the surface of a confluent monolayer of pretreated HUVECs.We measured the specific unbinding force during rupture of the interaction between the antigen?ANXA2?expressed at the apical surface of living HUVECs and the antibody coated AFM cantilever probe.Interactions between antibodies on the AFM cantilever and cell surface antigens cause large adhesion forces,which are quantified by the deflection signal during separation of the cantilever from the cell.Processed by Atomic J,adhesion works are calculated and compared between ESI09-treated HUVECs and DMSO-treated HUVECs,which indirectly represents the density of ANXA2 expressed in equal area at cell surface.5?The potential mechanism of Epac1 regulating ANXA2 in cell membrane compartment in ECsHuman umbilical vein endothelial cells?HUVECs?were pretreated with Epac specific inhibitor 09?ESI09?and dimethyl sulfoxide?DMSO?as vehicle.Post nuclear fractions?PNFs?and cell membrane fractions were obtained by differential centrifugation.Immunoprecipitation?IP?:S100A10 precipitates were acquired by incubating S100A10 antibodies with PNFs,followed by western blotting analysis and probed by anti-ANXA2 antibodies.The density was quantified by Image J.Results:1?Depletion of Epac1 results in fibrin deposition and fibrinolytic dysfunction invivoIHC showed that compared with that of wild-type mice,positive staining of fibrin deposition were found in lung,kidney and brain of Epac1-null mice.The study of FeCl₃-induced thrombosis model showed that after application of FeCl3 in Epac1-null mice,the recovery rate of decreased carotid blood flow was rather in smaller range than that of wild-type mice.The obstructive degree of carotid artery in Epac1-null mice was higher than that in wild-type mice.2?Depletion of Epac1 results in decreased ANXA2 in mice plasmaELISA quantitative detection showed reduced ANXA2 concentration in Epac1-null mice plasma.3?Reintroduce ANXA2 in circulation can restore the attenuated fibrinolytic function in Epac1-null miceEpac1-null mice showed attenuated fibrinolytic function in FeCl₃-induced thrombosis model.Reintroduction of recombinant ANXA2 can decrease the maximal occlusion in carotid artery and enhance the recovery rate of blood flow in Epac1-null mice.4?Pharmacologically functional suppression of Epac1 in HUVECs via ESI09decrease ANXA2 residing on EC apical surfacesQuantitative analysis of impermeable IF showed that markedly decreased expression of ANXA2 at cell surface of ESI09 treated HUVECs.Using an ANXA2 antibody-coated cantilever,lower adhesion works were measured on the surface of HUVECs exposed to ESI09 compared with that of HUVECs exposed to DMSO.5?Inhibition of Epac1 impedes ANXA2 association with S100A10 in ECsIP analysis showed that ESI09 treatment reduced associated ANXA2 in S100A10precipitates.Conclusion:Taking advantage of our Epac1-null mice,we found out that depletion of Epac1results in fibrin deposition and fibrinolytic dysfunction in vivo,which is similar pathogenesis discovered in ANXA2-null mice.Epac1 depletion results in reduced ANXA2 concentration in mice plasma.Insufficient fibrinolytic function in Epac1-null mice can be recovered by replenishing recombinant ANXA2 in circulation,which led us to probe potential correlation between cAMP-Epac1 and the levels of cell surface ANXA2.By pharmacologically functional suppression of Epac1 in HUVECs via ESI09,we show that endothelial apical surface expression of ANXA2 is markedly decreased in ESI09 treated HUVECs.These data associate Epac1 with dynamics of?ANXA2-S100A10?₂ in ECs and demonstrate the critical role of Epac1 in regulation of vascular fibrinolysis,which postulates a novel pathway and potential mechanism of the way ANXA2 and S100A10locolization on cell membrane compartment in order to exert specific functions.This study firstly link Epac1 to?ANXA2-S100A10?₂ complex and shed light to further developing Epac1 regulatory role in ECs.