The Therapy With Cardiac-specific Hypoxia-regulated SDF-1 Single-plasmid Vigilant Vector In Mouse Model Of Myocardial Infarction

Stromal cell-derived factor 1 a (SDF-1a) is a homing factor that can cause stem cells to migrate from the bone marrow,which can react with its main receptor CXCR4.It can inhibit cell apoptosis,too.Although endogenous SDF-1a is secreted in the early phase of infarction in ischemic myocardium, the expression of myocardial SDF-1 is not effective for heart repair. SDF-1 has to be expressed in larger quantities and for a longer time.We try to build one single-plasmid vigilant vector.This vigilant vector has some features:high efficiency, hypoxia-regulated, vigilant,cardiac-specific.We evalute this vigilant system in cardiac myocytes. Furtherly, we evalute this vigilant system in mouse model of myocardial infarction.Another aim of this reserch is to evalute anti-apoptosis of SDF-1 and to find the inner mechanism.Objective SDF-1 has to be expressed in larger quantities and for a longer time for the treatment of myocardial infarction. A gene therapy approach using an SDF-1 a vector can increase SDF-1 expression and mobilize and direct stem cells to the ischemic myocardium. SDF-1 can serve as a heart-protective factor through its ability to activate phosphorylated-AKT and ERK1/2. The role of the Bcl-2-associated death (BAD) and phosphorylated-BAD (p-BAD) in this process, however, has not been determined. In addition,the detailed time course of a SDF-1 a vector adminastration remains unclear.Methods Contrust the p-CMV-hSDF-1 plasmids.H9C2 cells were transfected with the p-CMV-hSDF-1 plasmid under hypoxic stimuli. Real-time PCR and cell staining was used to confirm the transfection under hypoxic condition.Western blot analysis was used to determine the SDF-1 expression time course.In the SDF-1 peak expression,the conditioned medium was collected for further experiment.The SDF-1 conditioned medium in endogenous peak expression would be considered as control medium.Isolate mesenchymal stem cells (MSCs) and cardiac stem cells (CSCs).Under conditioned medium or control medium,MSCs and CSCs would be migrated. H2O2 was used to induce H9C2,MSCs and CSCs apoptosis.The apoptosis of H9C2,MSCs and CSCs was evaluated with conditioned medium or control medium.Western-blot,cell staining was to confirm reduction of dephosphorylation of p-Bad.Results The transfection efficiency was approximately 27.83%±2.61%. H9C2 cells transfected with the pCMV-hSDF-1 plasmid expressed 31-fold more h-SDF-1 than the control group (P=0.003563).Western blot analysis revealed that SDF-1 expression peaked from the third day to the 7th day after pCMV-hSDF-1 plasmid transfection. After treatment with conditioned medium, which was gathered on the third day, H9C2 cells, mesenchymal stem cells (MSCs) and cardiac stem cells (CSCs) were more resistant to hypoxic insult than control groups, based on the results of the Live/Dead cell viability test, morphological observation and trypan blue uptake test. Live/Dead cell viability test:in confocol laser scan,there were more green H9C2,MSCs,and CSCs cells in conditioned medium treatment groups than in control medium groups. Morphological observation:there were more round,bright H9C2,MSCs and CSCs in control groups than in conditioned medium. Trypan blue uptake test: MSCs (conditioned medium 84%±7% vs. control medium60%±2%, P< 0.01); CSCs (conditioned medium 82%±3% vs. control medium 62%±3%, P< 0.001); H9C2 (conditioned medium 89%±3% vs. control medium 65%±2%, P< 0.001).Both western blot and staining results confirmed the dephosphorylation reduction of p-BAD in H9C2s, MSCs and CSCs after being treated with SDF-1 conditioned medium.The conditioned medium made more MSCs migrate compared with control medium(56±4 vs.10±1, p< 0.001).Likewise, the conditioned medium made more CSCs migrate compared with control medium(34±11 vs.11±3, p< 0.001).Conclusions These findings indicate that, under hypoxic stimulation after SDF-1 gene delivery, the expression of SDF-1 peaks from the third day to the 7th and lasts longer than fourteen days. As a result, more MSCs and CSCs migrate into the hypoxic myocardium. In addition, treatment with SDF-1 conditioned medium causes H9C2s, MSCs and CSCs to become more resistant to hypoxic stimuli and to reduce dephosphorylation of the p-BAD proteins.Objective To construct one single-plasmid vigilant vector,which has special features:high-efficiency,vigilant,hypoxia-regulated,cardiac-specific.This system can meet the requirement of therapy in myocardial infarction.These features would be assessed in vitro and in vivo experiments.Another aim is to prove that SDF-1 can be anti-apoptosis in cardiac myocytes.The inner mechanism would be assessed.Methods After constructing the single-plasmid vigilant vector,this vigilant plasmid would be compared with double-plasmids vigilant vector under hypoxic stimuli.In addition,the on-set time of this single-plasmid vector would be evaluated. Furthermore,the hypoxia-regulated feature was appraised. Isolate and culture cardiac myocytes.The cardaic myocytes was transfected with single-vigilant plasmid. NaN3 was used to stimulate hypoxia insult. Cell apoptosis or death could be observed from cell morphology. Collect the used medium in the 5th day as conditioned medium.The cardiac myocytes would be divided into four groups:Control,AMD3100,AMD3100 with conditioned medium,conditioned medium.Phosphorylation of ERK1/2,p-38MAPK and Akt were detected by Westen blot. Then this single-plasmid was injected into myocardium in left ventricular apical.The animal experiment would be divided into four groups:MI,Sham+V,MI+V,Control.After 3 days,the left anterior descending coronary artery (LAD) was ligated as myocardial infarction model. 24 houres later,the SDF-1, p-ERK1/2, p-p38MAPK and p-Akt expression of MI or MI+V groups were detected by Western-blot and immunohistochemistry. TUNEL test was performed to assess cardiac myocytes apoptosis in vivo.7 days later, the echocardiographic assessment(EF%,LV mass and FS%) of myocardial infarction and heart function was performed.Masson staining was performed to identify collagen fibers.Results Under hypoxic stimuli,single-plasmid vigilant vector can express more 2.5 folds hSDF-1 gene than double-plasmids vigilant vector(p< 0.01).After 30 mins of hypoxia stimuli,SDF-1gene can be on-set.Compared with nomoxia,hypoxia stimuli can make more 8.6 foldes hSDF-1 be expressed in cardiac myocytes transfected with single vigilant vector.The transfection efficiency is about 55%±7%.The cardiac myocytes transfected with single-plasmid vigilant vector can be more resistant to hypoxic stimuli induced by NaN3.Compared with other groups starting dying in 3rd day and dying totally in 5th day.Part of these cardiac myocytes transfected with single plasmid would start to die in the 9th day.Most of them would die in eleven day.The conditioned medium can inhibit apoptosis of cardaic myocytes via increasing phosphorylation of Akt, p38MAPK and ERK1/2.After 24 houres,MI+V group has a stronger SDF-1,p-Akt,p-p38MAPK,p-ERK1/2 and p-Bad expression than MI group. MI+V group had less TUNEL positive cells than MI group(25±2 vs 43±8,P<0.01).The SDF-1 expression in MI+V group was stronger than liver organ.The results of echocardiographic assessment showed that MI+V group has better EF%,FS% and LV mass than MI group(EF%,36.68±5.21 VS 21.1±2.12, p<0.01; LV Mass,102.85±9.90 VS 151.90±9.08,p<0.01; FS%,17.42±2.22 VS 10.05±0.50,p< 0.01).Masson staining result showed that MI+V group has less collagen fibers.Conclusions The hypoxia-regulated single-plasmid vigilant vector has special features:high-efficiency,vigilant,hypoxia-regulated,cardiac-specific.In addition,SDF-1 can inhibit apoptosis of cardiac myocytes via increasing phosphorylation of Akt,p38MAPK and ERK1/2.