Clinical And Basic Experimental Research On The Effect Of MiR-126-3p In Vein Grafts Reendothdialization And Neointima Formation

BackgroundCoronary artery disease(CAD)is one of the world's leading causes of years of life lost due to premature death according to a recent report from World Health Organization(WHO).Coronary artery bypass grafting(CABG)and percutaneous coronary intervention(PCI)are the two main invasive methods for myocardial revascularization in patients with severe CAD,which are commonly adopted by clinicians.Although the drug-eluting stents(DES)was widely used,CABG remains the first choice of treatment in patients with left main coronary artery disease(LCMD),multiple arterial disease or occlusive arterial disease.Despite preferential choose of arterial grafts in clinical treatment,autogenous human saphenous vein(HSV)is still most frequently used type of conduit grafts in CABG.Revascularization with autologous HSV grafts is standard surgical therapy of all CABG surgery procedures.However,the benefits of CABG surgery remain limited by the long-term patency of the saphenous vein graft.During the first year after CABG surgery,up to 15%of HSV grafts occlude,between 1 and 6 years the attrition rate of graft is 1%to 2%per year,and between 6 and 10 years it is 4%per year.More than maybe 50%of HSV grafts will be occluded during the first ten years after CABG surgery,and this will cause recurrent symptoms,myocardial infarction(MI)or preoperative revascularization.A number of technical advances have been proposed over the past decade,such as off-pump CABG and no-touch HSV harvesting,however,the rates of vein graft failure remain largely similar.Currently,there is no effective treatment for vein graft disease.The most fundamental pathology process responsible for vein graft failure is neointimal hyperplasia,which occurs in reply to hemodynamic changes(blood pressure and turbulent flow)and vessel wall injury(distention and trauma during harvesting and implantation).Neointimal hyperplasia is a complicated,continuous pathophysiological process and endothelial cell(EC)injury and disfunction is the central mechanism in the initiation and progression of this process.Changes in endothelial monolayer may induce vascular inflammation,and stimulate subendothelial smooth muscle cells(SMCs)proliferation and their migration into neointima,and cause extracellular matrix(ECM)secretion increase,resulting in neointimal thickening and vessel wall remodeling,which contribute to vascular restenosis and occlusion.Many studies have been proved hat selective acceleration of reendothelialization after vascular injury was shown to attenuate neointimal thickening.Our previous study has been proved that accelerated endothelialization of vein graft could reduce neointimal formation in arterialized vein grafts in rats.It's commonly recognized and demonstrated that the damaged vessel wall is dependent on residual endothelial cell proliferation and migration to promote EC function recovery and reendothelialization,and then limiting neointimal thickening.Therefore,specific approaches to selectively accelerate endothelial function recovery by strengthening cell proliferative and migratory activity are promising treatment measures to further improve the long term patency of HSV grafts.MicroRNAs(mRNAs,miRs)are a newly discovered family of small,non-coding RNAs that regulate diverse cellular processes and cell death by binding to target mRNAs and induce their translational repression or degradation of their targets.Promising evidence in support of the role of miRNAs in cardiovascular function and disease has been shown in recently studies,and shown great potential applications in the biomedicine,clinical diagnosis.A lot of experimental results showed that the organism can regulate the expression of miRNAs in response to acute or chronic vascular injury,therefore changing of miRNA levels by specific tools may be a promising avenue for the possible treatment and prevention of patients with vascular diseases,including vein graft diseases.One popular miRNA,miR-126-3p,is the only EC-specific miRNA described to date.In the study miR-126-3p function of in mice and zebrafish,targeted deletion of miR-126-3p can result in endothelial dysfunction,loss of vessel integrity,imbalance of vascular homeostasis and defective angiogenesis.Other studies indicate that knockdown of miR-126-3p in mice caused partial embryonic lethality,leaky vessels,and hemorrhaging during embryonic development,due to vascular integrity damage and defects in EC proliferation,migration,and angiogenesis?Several groups have revealed that miR-126-3p acts as angiogenic miRNA in human umbilical vein endothelial cells(HUVECs)can facilitate cell proliferation,migration and angiogenesis via down-regulation of sprouty-related protein-1(SPRED-1)and phosphatidylinositol-3-kinase regulatory subunit 2(PIK3R2),which are known as the negative regulators of the vascular endothelial growth factor(VEGF)signaling pathway.Recent research demonstrated that ultrasound-mediated miR-126-3p delivery to chronic ischemic hindlimb muscle resulted in improved perfusion,vessel density,enhanced arteriolar formation,arteriolar formation,and neovessel maturation.Endothelial microparticle mediated transfer of miR-126-3p regulates human coronary artery endothelial cell migration and proliferation in vitro and reendothelialization in vivo.Contrast-enhanced ultrasound delivery miR-126-3p promoted angiogenesis and blood perfusion in preeclampsia placenta.In Jianzhong Hu et al's study,upregulation of miR-126-3p by intrathecal injections agomir-126-3p increase angiogenesis after spinal cord injury,which was concurrent with downregulation of the mRNA and protein levels of two validated target genes of miR-126-3p,SPREDI and PIK3R2.Clinical researches has implied that the expression of miR-126-3p levels in the serum of stable CAD patients is lower compared to normal people,and statistically analysis there are some relations and implicit coincidences between circulating miR-126-3p and fatal MI.Furthermore,expression of circulating miR-126-3p levels was lower in plasma of patients with type 2 diabetes than in healthy controls,and further analysis revealed that there was a relationship between miR-126-3p levels and diabetic vascular complications.All of these findings indicating that the endothelial enriched miR-126-3p may have a powerful vasculoprotective in the damage of blood vessel.In recent years,the roles of miR-126-3p has been discussed in many vascular diseases,however,no research to date has investigated the effect of miR-126-3p in the setting of vein graft disease.In this study,we assumed that miR-126-3p may play an important role in vein grafts disease of CABG,and upregulation of miR-126-3p by agomir may be used as an intervention to accelerate endothelialization of vein graft by promote endothelial cell proliferation and migration,thereby limit neointimal hyperplasia and vascular restenosis.This study can be divided into four parts:to investigate the role of miR-126-3p in the setting of CABG,we compared the expression levels of plasma circulating miR-126-3p in 10 healthy people and 10 patients with coronary artery heart disease before and after CABG(Part 1).To investigate the role of miR-126-3p in vein graft cells,we isolated primary human saphenous vein endothelial cells(HSVECs)and human saphenous vein smooth muscle cells(HSVSMCs)in human saphenous veins obtained from patients undergoing CABG(Part ?).To investigate the effect of miR-126-3p agomir in human saphenous vein grafts,we performed a well validated ex vivo organ culture model of HSV(Part III).We finally established a rat vein graft model,to investigate whether a single delivery of miR-126-3p agomir would be sufficient to reduce neointimal formation and luminal stenosis by enhancing reendothelialization in vein grafts(Part ?).Part IDynamic changes of circulating miR-126-3p in patients with coronary artery heart disease before and after surgery and its clinical significanceObjectiveRecent studies have found that circulating miR-126-3p levels are lower in the serum of stable CAD patients than normal people,and there are some association between circulating miR-126-3p and myocardial infarction(MI).Moreover,plasma loss of miR-126-3p in Type 2 Diabetes patients is associated with diabetic vascular complications.We aimed to detect the expression of plasma circulating miR-126-3p in healthy people and patients with coronary artery heart disease before and after surgery,and to investigate the role of miR-126-3p in the setting of vein graft disease,therefore to provide an evidence for further applications.MethodsIn this clinical study,we asked for 10 patients preparing CABG surgery and 10 healthy volunteers from urban and rural areas.There were no signifcant differences in the baseline characteristics(Age,Gender,BMI,Alcohol,Smoker,Family history,Hypertension,Diabetes)of the two groups.Peripheral blood samples(2 ml)were collected from the patients into EDTA tubes 1 day before and 1,3,7 and 14 days after surgery,and only one blood sample was collected from each person in healthy volunteers.The plasma circulating miR-126-3p in those people was detected using specific primers by RT-PCR.Meanwhile,we established an organ culture model of HSV to further validate the dynamic changes of miR-126-3p in neointimal formation of vein grafts.Discarded HSVs were obtained from patients receiving CABG surgery in our hospital and cultured for 7days and 14 days ex vivo.The expression of miR-126-3p in cultured HSVs rings were detected by RT-PCR.Results1.Using RT-PCR,we found that circulating miR-126-3p was significantly decreased in complex-serious CAD patients compared to normal people(P<0.05).2.Circulating miR-126-3p levels in patients undergoing CABG showed a brief rise in the perioperative period.It was increased at 1 day post-surgery,reached the peak level at 3 d post-surgery and then gradually declined to baseline at 14 d post-surgery(P<0.05).3.The peak of value of circulating miR-126-3p levels in patients undergoing CABG was still far belown the levels of miR-126-3p expression in healthy subjects(P<0.05).4.RT-PCR results of cultured HSVs rings showed that miR-126-3p levels in vein segments cultured for 7 day was increased(P<0.05)as compared with no cultured segments.However,there were made no significant differences in miR-126-3p levels between HSV segments cultured for 14 days and no cultured ones.The changing of miR-126-3p level in HSV was almost consistent with the finding in human plasma.Conclusions1.These data,for the first time,prove that circulating miR-126-3p may play a protective role in patients with complex-serious CAD.2.The dynamic change of miR-126-3p in plasma and HSV may be the compensatory mechanism by which maintains cell function in vein grafts,but this compensation is incomplete.3.Therapeutic upregulation of miR-126-3p level may be an efficient gene therapy to prevent vein graft disease,and whether this is true or is a chance finding requires further study.Part ?Effect of miR-126-3p overexpression on HSVECs and HSVSMCs proliferation and migration in vitroObjectivePrevious studies have demonstrated endothelial injury plays a critical role in the initiation and progression of a variety of proliferative vascular diseases,the delayed re-endothelialization as well as subendothelial SMC proliferation and migration stimulate by inflammation are the major pathophysiological events that lead to neointima formation and restenosis.It's commonly recognized that proliferative and migratory activity of ECs plays a central role in the promotion of reendothelialization and restoration of EC function.Several studies indicated that miR-126-3p plays a positive role in the function of HUVECs.Among the verified targets of miR-126-3p,PIK3R2 and SPRED 1 are known as the negative regulators of the VEGF signaling pathway to critically influence HUVEC proliferation and migration.However,the functions of miR-126-3p in HSVECs are completely unknown.Meanwhile,the side effects of dysregulated miR-126-3p expression in HSVSMC need to be addressed.Methods1.Discarded HSV segments were obtained from patients receiving CABG surgery,primary HSVECs and HSVSMCs were isolated and identified.Using Cy3-labeled miR-126-3p agomir,cell transfection efficiency was evaluated.2.Primary HSVECs were cultured and transfected with miR-126-3p agomir or antagomir to assess the in vitro effect of miR-126-3p on cell proliferation and migration.RT-PCR and Western-blots assays were carried out to measure the verified targets of miR-126-3p and the relevant signal pathways involved in endothelial cell proliferation and migration.3.Primary HSVSMCs were cultured and transfected with miR-126-3p to assess the in vitro effect of miR-126-3p overexpression on cell proliferation and migration.4.Inflammatory model of HSVSMCs was established to assess the in vitro effect of miR-126-3p overexpression on cell proliferation and migration induced by tumor necrosis factor-?(TNF-?).Results1.Primary HSVECs and HSVSMCs were successfully isolated and identified from patients undergoing CABG.100nmol agomir was selected as the best carrying concentration for the following studies.2.EdU incorporation assay revealed that overexpression of miR-126-3p by agomir resulted in increased proliferation,whereas downregulation of miR-126-3p had the opposite effect.The scratch wound assay and transwell migration assay revealed that overexpression miR-126-3p promoted HSVECs migration,whereas downregulation of miR-126-3p had the opposite effect.The mRNA and protein expression of PIK3R2 and SPRED-1 were decreased evidently when transfected with miR-126-3p agomir,and were significantly increased upon transfection with miR-126-3p antagomir Inhibitor.Overexpression miR-126-3p increased the phosphorylation levels of AKT and ERK1/2 in HSVECs,whereas downregulation of miR-126-3p had the opposite effect.3.EdU incorporation assay showed that miR-126 agomir did not promote HSVSMC proliferation.The scratch wound assay and transwell migration assay showed that miR-126 agomir did not promote HSVSMC migration.4.EdU incorporation assay and scratch wound assay and transwell migration assay revealed that miR-126-3p agomir at all concentrations tested 25-200 nmol did not affect HSVSMC proliferation or migration induced by TNF-a compared to control.Conclusions1.This is the first report that miR-126-3p exerts a vasculoprotective role in the setting of vein graft disease.The results suggested that miR-126-3p regulate HSVEC proliferation and migration by the mechanism of regulates ERK1/2 and AKT signaling pathway through SPRED-1 and PIK3R2 targeting.Meanwhile,miR-126-3p had no effect on HSVSMC proliferation and migration.2.Our study established miR-126-3p as a potential new target for the selective modulation of endothelialization.It is possible that manipulate miR-126-3p expression by agomir may be a viable gene strategy to modulate miR-126-3p functions in HSVECs and vein grafts.However,all that needs further in vivo confirmation.Part ?Overexpression of miR-126-3p by agomir inhibits neointima formation in HSV organ cultures ex vivoObjectiveIn light with our clinical findings and in vitro results that pointed towards the beneficial effects of miR-126-3p on HSVEC function,we wondered that upregulation of miR-126-3p by agomir could inhibit neointimal hyperplasia by accelerate reendothelialization in a well validated ex vivo organ culture model of HSV.MethodsFor ex vivo organ culture,leftover segments of HSV were obtained from patients undergoing CABG in our hospital.Each vein were made for four equal individual segments,one segment(uncultured)was immediately formalin-fixed and remaining segments were incubated in 30%FBS,in two of the three cultured segments,miR-126-3p agomir and miR-126-3p negative control agomir(NC agomir)were transfected for initial culture.The medium were changed every 2 days and the veins were embedded in paraffin,sectioned,and stained with hematoxylin-eosin(HE)to assess neointimal hyperplasia,and endothelial cell marker(CD31)to assess reendothelialization 14 days after culture.ResultsAfter 2 weeks in organ culture,we observed an increase in intimal thickness of cultured HSV segments compared to uncultured ones,which suggested that HSV ex vivo organ culture model was established.Morphometric analysis showed that neointima thickening was significantly attenuated in miR-126-3p agomir transfected veins than in NC agomir transfected veins and untreated veins(P both<0.05).Immunohistochemical staining of CD31 revealed that that treatment with miR-126-3p agomir increased the percentage of endothelial cell coverage at day 14 compared with the cultured controls,however,delivered with NC agomir did not exhibit this effect.ConclusionsThe results fully proved that upregulation of miR-126-3p by agomir could inhibit HSV neointimal hyperplasia and accelerate reendothelialization ex vivo,which provides a base for the next in vivo study.Part IVLocal delivery of miR-126-3p agomir inhabits intimal hyperplasia in rat vein grafts by promoting reendothelializationObjectiveDue to various limitations of ex vivo HSV model,we established a rat vein arterialization model and to test the efficiency and effectiveness of applying miR-126-3p agomir locally in vein graft in vivo.Methods1.We established a rat vein graft model using a "cuff' anastomotic technique,and measured the transfection efficiency using Cy3-labeled miR-126-3p agomir staining and western blot analysis.90 healthy rats are divided into four groups:normal vein group,vein graft group,NC agomir group,miR-126-3p agomir group.2.We performed vascular ultrasound examination to measure luminal diameter and blood flow in the distal anastomosis of vein grafts 4 weeks after surgery.3.We performed HE staining to measure neointimal thickness and Masson trichrome staining to detect collagen 28 days after surgery.We also performed?-smooth-muscle actin(a-SMA)immunostaining to identify VSMCs and immunohistochemistry for proliferating cell nuclear antigen(PCNA)to detect vascular proliferating cells 28 days after surgery.4.Evans Blue staining and CD34 immunofluorescence staining for vascular endothelial cell marker were used to assess endothelial recovery of vein graft 14 days after surgery.CD68 immunohistochemical staining was used to detect inflammatory cells(monocytes/macrophages).Results1.We successfully established rat vein grafting model.It was documented by fluorescent staining that local delivery of agomir was effective,long-lasting and had few side effects.Furthermore,western blot analysis indicated local delivery with miR-126-3p agomir resulted in significantly downregulated the protein levels of PIK3R2 and SPRED-1.2.The luminal diameter of vein grafts in miR-126-3p agomir treated group was much wider than in NC agomir group and vein graft group 4 weeks after surgery,and the peak-systolic velocity(PSV)was evidently higher in NC agomir group and vein graft group than in miR-126-3p agomir treated group,no significant difference was observed between the NC agomir group and vein graft group.3.HE staining revealed that treatment with miR-126-3p agomir dramatically reduced neointimal thickness,whereas treatment with NC agomir and no treatment,resulted in a dramatically increased neointima.Masson trichrome staining shows that the percentage of the neointima area occupied by blue-colored fibers(collagen)had no obviously difference between miR-126-3p agomir treated group and other control groups.Meanwhile,immunohistochemical staining of a-SMA demonstrated the presence of abundant SMCs localized to the neointima in vein grafts and the accumulation of positive-stained SMCs was markedly reduced in vein grafts treated with miR-126-3p agomir.PCNA staining revealed that it could induce a significant reduction in the PCNA-positive cells in the neointima after local delivery with miR-126-3p agomir.4.Luminal staining for Evans blue indicated that reendothelialization was significantly greater in the miR-126-3p agomir group in comparison with NC agomir group and vein graft group.CD34 immunofluorescence staining revealed the reendothelialization was significantly higher in the miR-126-3p agomir group tha NC agomir group and vein graft group at 14 days after surgery.CD6 immunohistochemical staining revealed that miR-126-3p agomir treatment resulted in a significant decrease in inflammatory cell infiltration compared with NC agomi group and vein graft group.Conclusions1.Our results proved that local delivery miR-126-3p agomir could accelerate reendothelialization after venous implantation and thereby attenuate vein grafts luminal stenosis and neointimal formation and improve blood flow.2.Our study suggested that miR-126-3p agomir possesses a potential clinical value in the prevention and treatment of autologous vein graft restenosis in CABG.