Intermedin Promotes Vessel Fusion By Inducing VE-cadherin Accumulation At Potential Fusion Sites And To Achieve A Dynamic Balance Between VE-cadherin-complex Dissociation/Reconstitution

Vascular system is necessary for tumorigenesis and tumor metastasis.In theory,blocking the vascularization at the incipient state should maintain a dormancy state that without neoplasia or metastasis of tumors.The current use of anti-angiogenic cancer therapy has already been an important part of clinical cancer therapy.However,the anti-angiogenic cancer therapy is not always effective to patients,and the inevitable resistance to antiangiogenic therapy also been a limitation for clinical application.These restrictions motivating people to explore the underlying mechanisms of neovascularization which may be useful in the mainstream of developing novel and effective anticancer agents targeting vascularization.The process of angiogenesis which includes the following stages:(1)Endothelial cells(ECs)proliferation;(2)Vessel sprouting;(3)The vessels sprouts contacting with each other in a process called vessel fusion;(4)The lumen formation and vessel mature.It finally creates a closed vascular system to realize the perfusion function.The vessel fusion is a prerequisite for blood flow through the vessels.Up to now,just few studies have focused on the process of vessel fusion.Vessel fusion is a sequential process,it begins with approaching of adjacent sprouts.After the sprouts contact and anchor to each other,the anastomotic point would expand to enable interconnection of adjacent vessels.Studies have shown that endothelial cells serve as a “perfusionindependent” manager.Vascular endothelial cells masterminding those complex tasks by repopulating cells or rearranging the cell junctions.However,the underlying mechanisms of vessel fusion are still unclarified.Through control of cell-cell junction,endothelial cell regulates the vessel fusion process accurately.Thus,these ECs always act along with fusion process.So that,a typical regulator-vascular endothelial cadherin(VEC/ VE-Cadherin)-of vascular ECs which is an endothelial-specific transmembrane component of adherens junction could play an important role in vessel fusion.Previous studies showed,VEC could be recruited to the ECs interface,during the fusion of intersomitic vessels(ISV)and dorsal longitudinal anastomosing vessels(DLAV).Furthermore,VEC would be rearranged caused by endothelial cell migration and deformation when an ISV fuses to a DLAV in zebrafish.The down-regulating of VEC expression induces the abnormal fusion between ISV and DLAV.However,the definite mechanism in molecular should be further investigated.Intermedin(IMD)is a member of the calcitonin family.The widely expressing of IMD in tissues and organs reminds that it may be a vital regulator for organism.IMD has been reported to play roles in the maintenance of endothelial barrier integrity,the regulation of VEC activities and the angiogenesis process.Our previous studies showed IMD dramatically remodels the vasculature morphology into a hierarchical architecture that is well organized with relatively fewer sprouts,larger lumens,and more anastomosed vessels.Moreover,we verified IMD could induce the vessel fusion in an angiogenic factor-independent way.According to another research,IMD peptide even regulates the VEC behavior.Based on these studies,we hypothesized that IMD may induce the vascular fusion by fine-tuning the behavior of VEC.In the present study,we aim to explore whether IMD peptide alters the vessel perfusion and how IMD facilitates this process in three parts.First,to confirm the promotion of IMD-induced perfusion,the different models were performed;Second,to investigate the significance of VEC in IMDinduced perfusion;Last,clarifying the molecular mechanism underlying the role of IMD in regulating VEC activities.In the first part of this study,we established different models to represent vessel fusion process,including a three-dimensional in vitro angiogenic model(fibrin bead assay),a dynamic vascular pattern in zebrafish embryos,retinal vascular model,tumor model and skin transplantation model.These mammalian angiogenic models help to investigate the effect of IMD in physiological and pathological vasculature.Based on our data,we confirmed IMD promotes the vessel fusion in vitro and in vivo.In the second part,we investigated how IMD facilitates the perfusion process of vessels.We next found that IMD-induced perfusion relying on a two-step method:First,IMD facilitates vesicular VEC transport to potential contact points and induces ECs enter a “ready-to-anchor” state.Thus,the adjacent vessels can connect and anchor with each other,resulting in an increased chance of successful vessel fusion.Second,after the approaching sprouts anchor to each other,IMD induces continuous VEC phosphorylation and fine-tunes the behavior of VEC to achieve a dynamic balance between VEC complex dissociation and reconstitution.In order to widen the anastomotic point and improve blood perfusion.In the third part,we investigated how IMD directly regulates VEC activities by immunofluorescence analysis,site-directed mutagenesis technology and time-lapse microphotography.The experiments demonstrated IMD directly promotes VEC internalization from the cell surface by inducing persistent VEC-Y685 phosphorylation.The internalized phospho-VEC preferentially binds to Rab4 and Rab11,which can facilitate VEC vesicle transport in the cytoplasm and recycling back to cell-cell contact areas to reconstruct VEC complex.The active form of Rab4 or Rab11 is required for VEC transportation but may not be necessary for VEC cargo binding.The novelty of this study:To create a closed and hierarchical organized vascular system,angiogenic sprouts must meet and connect in a process called vessel fusion,which is a prerequisite for establishment of proper blood flow in nascent vessels.However,the molecular mechanisms underlying this process remain largely unknown.The focus of this study is to explain a novel mechanism of IMD-induced vascular fusion.This mechanism is consistent with the natural process of vascular fusion and is supported by our experimental data.Thus,the ability of IMD to promote vessel fusion may be useful for blood supply restoration after transplantation or during wound healing and for broadening of antiangiogenic drug screening via targeting of the vascular anastomotic process.