K_ATPActivation Facilitates The Proliferation,Migration And Angiogenesis Of Endothelial Progenitor Cells And The Underlying Mechanisms

Background and Objective:Pulmonary arterial hypertension?PAH?is a malignant pulmonary vascular disease,characterized by a progressive increase in pulmonary vascular resistance?PVR?and vascular remodeling,which contributes to right heart failure and death.In the absence of specific treatments,patients have a mean survival of 2.8 years.Specific therapies that have been approved for use to manage PAH include agents that have important vasoactive effects,modulating abnormalities in three main pathobiologic pathways for PAH:endothelin?ET?-1,prostacyclin?PGI2?,and nitric oxide?NO?.Otherwise,these options only improve symptoms and survival partially,cannot stop the development of PAH,reverse pulmonary vascular remodeling.PAH involves multiple clinical conditions,can complicate the majority of cardiovascular and respiratory diseases.Currently PAH research has become a hot global study.In recent years,pulmonary arterial endothelial cells?PAECs?dysfunction was thought to play a central and critical role in the initiation and development of PAH.Therefore,focusing on the protection of the PAECs function is an effective strategy to prevent PAH progress.Endothelial progenitor cells?EPCs?can be differentiated into endothelial cells?ECs?,repair and replace damaged or apoptosis ECs maintain the integrity of the endothelium,involved in blood vessel regeneration after birth.In PAH,studies showed that EPCs numbers declined and functions were impaired,EPCs transplantation had been proved to reverse vascular remodeling in many studies.Therefore,facilitation the function of EPCs could protect PAECs,reverse vascular remodeling and prevent the development of PAH.ATP-sensitive potassium channels(K_ATP),widely expressed in tissues and cells,participate in numerous cellular processes.Previous studies had shown that a variety of vasoactive stimuli,including H2S,CNP and VEGF can act through K_ATP to promote ECs function;Nicorandil?Nico?enhanced ECs proliferation,migration and angiogenesis in vitro and in vivo via direct activation of K_ATP.Potassium channel openers?KCOs?could alleviate the damage of hypoxia and monocrotaline on PAECs.Whether activation K_ATP can regulate the function of EPCs to facilitate the repair of injured pulmonary vascular would require further study.In this study,we tried to demonstrate the subunits of K_ATP expressed in EPCs isolated from adult peripheral blood,and investigate the influence of K_ATP activation on EPCs using a classic KCOs,Nico,which is widely used in clinic,and a novel KCOs Iptakalim?Ipt?.In addition,we studied the underlying mechanisms involved in the regulation of EPCs.Methods1.Isolated mono-nuclear cells from the adult peripheral blood were resuspended by EGM-2 medium.On average,after 12-28 days,colonies of EPCs were observed.The EPCs phenotype was confirmed by immunofluorescence:Adherent cells were incubated with DiL-labeled-acLDL and FITC-UEA-I,stained for PECAM-1?CD31?and vWF.The surface marker CD34?CD309?VEGFR2/KDR?,CD133 were analyzed on a FACS Canto ? Instrument.2.mRNA and protein levels of K_ATP expression were detected by RT-PCR and western blot in EPCs,as well as their K_ATP subunit.Immunofluorescence was used to further validate the subunits of K_ATP channels exist in EPCs.3.UsingNicorandil?Nico?and Iptakalim?Ipt?to evaluate the influence of K_ATP activation on EPCs,Gli as K_ATP blocker.5-ethynyl-2'-deoxyuridine?EdU?incorporation proliferation assays were performed to investigate the influence of K_ATP activation on proliferation of EPCs,transwell migration chamber assays to investigate cell migration and in vitro tube formation on matrigel plate to investigate cell angiogenesis.4.Effect of open K_ATP channel on the phosphorylation of CaMKII,Akt and eNOS in EPCs cells was determined by Western blotResults:1.Characterization of EPCs derived from peripheral blood:EPCs have been isolated and expanded ex vivo from adult peripheral blood.Most of the EPCs colonies appeared during 12-28 days.Cells have characteristic endothelial "cobblestone"morphology.We conducted several experiments to further verify the EPCs.We confirmed the expression of CD309?VEGFR2/KDR?,as 99.5%of the isolated cells expressed.However,CD34?3%?and CD 133?0.6%?were few as previously reported.Additionally,these cells displayed an endothelial phenotype:they incorporated Dil-ac-LDL and stained positive for FITC-UEA-I,as well as expressed PEC AM-1?CD31?and vWF.The results showed that obtained cells were endothelial colony-forming cells?ECFCs,late EPCs?,which were EPCs subpopulation.2.K_ATP subtype expressions in ECFCs:mRNA and protein expression of Kir6,1,Kir6.2,and SUR2b in EPCs.The results of three expressed subunits in isolated ECFCs samples were further confirmed by immunofluorescence.3.KCOs promote ECFCs proliferation,migration and angiogenesis:We found that the number of EdU-postive cells was markedly increased compared with the control group,KCOs improved ECFCs migration and tube formation also.K_ATP blocker Gli,a sulfonylurea-type molecule,did not significantly affect ECFCs function,but almost entirely suppressed the effects induced by KCOs.4.KCOs enhanced the phosphorylation of CaMK ?,Akt and eNOS in ECFCs.Pretreatment with Gil could abolish the enhancement induced by KCOs,however,no effect was observed on cells treated with Gli alone.Conclusions:1.K_ATP channels were expressed on ECFCs and composed of Kir?Kir6.1,Kir6.2?and SUR2b subunits.2.KCOs improved proliferation,migration and tube formation of ECFCs.KCOs may influence ECFCs function via intracellular Ca²⁺ signaling pathway activating downstream effectors such as CaMKII,Akt and eNOS.3.Our findings provide an effective target for ECFCs regulation,constituting a promising therapeutic option for PAH.