| Objective: To detect level and protein concentration of circulating microparticles(MPs)in chronic heart failure rats,and to investigate effect of MPs on proliferation ability,migration ability and apoptosis of human umbilical vein endothelial cells(HUVEC).Methods: SD rats were randomly divided into normal group(n=6),sham group(n=6)and heart failure group(n=15).A rat model of abdominal aortic constriction(AAC)-induced cardiac pressure overload was established.Firstly,place a rat in supine position on a surgery platform.Make an incision along the manubrium and midline of the abdomen with a scalpel.Identify and expose the position of the abdominal aorta and left renal arteries.Then,ligature the 7-gauge needle with abdominal aorta together at the point of 1 cm above the left renal artery.Finally,remove the needle carefully to achieve a constriction,and close the abdominal cavity.After the operation,observe the animal carefully until it regains sufficient consciousness.Sham group underwent laparotomy without ligation of abdominal aorta.Control group did not receive any treatment.The body weight(BW)of each group was measured on the day before surgery and at the end of 4 weeks,8 weeks,and 12 weeks after surgery.12 weeks after surgery,changes in cardiac structure and function were evaluated using echocardiography and HE staining.The detection indexes of echocardiography include left ventricular internal diameter at end-diastole(LVIDd),left ventricular internal dimension systole(LVIDs),left ventricular ejection fraction(LVEF)and left ventricular shortening fraction(LVFS).Expose and remove the rat heart,measure the heart weight(HW)and left ventricular weight(LVW),then make some pathological sections to observe the morphological changes of myocardial cells.Circulating MPs were isolated from rat blood by centrifugation.Blood collected from the common carotid artery was subjected to centrifugation at 1550×g for 20 min to obtain platelet-free plasma(PFP).Total MPs and Annexin ?(+)MPs were detected by flow cytometry,and the protein concentration in total MPs was measured by BCA.PFP was subjected to centrifugation at 15000×g for 45 minutes.The microparticle pellet was resuspended in sterile saline(vehicle)and stored at-80°C until further analysis.Supernatant(without MPs)after centrifugation was used as a supernatant control group.Human endothelial cell line(EA.hy926)was maintained in culture in vitro.Cells were treated for 24 h,36 h and 48 h by MPs or supernatants from three groups rats.Cell proliferation ability was detected by the MTT assay.Cell migration ability was evaluated by the scratch assay.Annexin V/PI double-staining was used to test apoptosis,and JC-1 dye was used to test changes in the mitochondrial membrane potential when measured by flow cytometry.Results: 1.After 12 weeks of ACC operation,the rats in the HF group showed marked signs of heart failure.At 1 day before surgery and at 4 weeks and 8 weeks after surgery,there was no significant difference in BW between the groups.At 12 weeks after surgery,the BW in HF group was significantly reduced compared with the normal group(P<0.01).2.Cardiac echocardiography results.The rats after ACC operation showed impaired ventricular function.Compared to normal rats,HF rats showed significant increase(P< 0.01)in LVIDs and LVIDd;and significant decrease(P<0.01)in LVEF and LVFS.3.Rat heart mass index results and Myocardial HE staining.The results showed that the HW/BW and LVW/BW in the HF group were significantly increased compared with the normal group(both P<0.01).The HE staining showed that myocardial cells of normal and sham rats showed normal morphology.The arrangement of myocardial cells was neat,and fiber content in myocardial matrix was at normal status.However,myocardia in HF rats showed severe edema and structural disorder,fibrosis formation in myocardial matrix,and inflammatory cell and erythrocyte infiltration were also observed.4.Flowcytometric detection of MPs in rats.We determined the levels of total circulating MPs and Annexin ? positive MPs.Compared with the normal group of(10.97±1.34)×104 events/?l,total MPs concentrations significantly elevated in HF rats with the value of(34.04±2.74)×104 events/?l(P<0.001).Annexin V(+)MPs(18.88 ± 1.48)× 104 events/?l also increased significantly in HF rats compare to normal rats(6.76 ± 0.95)× 104 events/?l,P<0.001),while total MPs and Annexin V(+)MPs in sham rats showed no statistical significance compare to normal rats.5.BCA assay for protein concentration in MPs.Results showed significantly increased protein concentration(295.37 ± 8.09 ?g/ml)in MPs in HF rats compared to normal(78.73 ± 19.07 ?g/ml)and sham rats(81.36 ± 7.50 ?g/ml,P<0.01).6.Effect of MPs on HUVEC proliferation.HUVEC was incubated with MPs from each group,then MTT assay was performed for testing proliferation ability of HUVEC.Cell viability in control group(HUVEC cultured with complete medium)was set as 100%.Results showed that there is no difference in viability when MPs or supernate was incubated with HUVEC for 24 h.At 36 h and 48 h,viability decreased significantly in HF MPs group compared to control group,with the value of 81.23±8.48% and 73.85±11.44%,respectively(P< 0.05).7.Effect of MPs on HUVEC migration ability.MPs or supernate in each group was used for scratch assay,and migration rate was calculated at different incubation time.The migration rate in control group was set as 100%,then the migration rates of other groups were calculated.Results showed that,significance decrease in migration rate was observed at 24 h in HF MPs group(88.64 ± 5.23%,P<0.05)compared to control group.After 36 h and 48 h,further decrease in migration rate of HF MPs was observed,with the value of 76.57 ± 4.47% and 76.57 ± 4.47%,respectively(P<0.01).8.Effect of MPs on apoptosis of HUVEC.HUVEC was incubated with MPs or supernate in each group for different time.Flowcytometry was used for apoptosis test by Annexin V/PI staining.Results showed that apoptosis rates had no difference between each group after incubation for 24 h.After incubated for 36 h,apoptosis rate in HF MPs-treated group increased significantly compared to control group,with the value of 10.66 ± 2.56% and 6.73 ± 0.78%(P<0.01).When incubated for 48 h,the apoptosis rate in control group was 8.08 ± 0.52%,while the rate in HF MPs-treated group increased significantly to 19.51 ± 2.03%(P<0.01).9.Effect of MPs on mitochondrial membrane potential of HUVEC.After 48 h incubation with MPs or supernate,HUVEC was stained by JC-1 and assessed by flowcytometry.Results showed that the depolarization ratio of mitochondrial in HF MPs group(4.88 ± 2.67%,P<0.01)increased significantly compared to control group(8.03 ± 0.83%)after 48 h incubation,suggesting that HF MPs induced marked decrease of mitochondrial potential in HUVEC.Conclusions: Circulating MPs in chronic HF rats showed increased MPs level and protein concentration,which significantly inhibited HUVEC proliferation and migration ability,also induced marked apoptosis of endothelial cells by decreasing mitochondria transmembrane potential. |
PDF Full Text Request