2.1 Animals and grouping
In current study, eight-week-old C57BL/6 wild-type male mice weighted about 25 g were selected to construct the myocardial infarction model by ligating the anterior descending branch of the heart. All mice were obtained from the SPF Experimental Animal Center of the Bone and Stem Cell Experimental Center of Nanjing Medical University Rearing. The policy was approved by the animal experiment ethics committee. Gene recombination method was used to prepare two kinds of protein including PTHrP (87-139) with nuclear localization sequence (NLS) and C-terminal, and PTHrP (1-84) without NLS or C-terminal.
The mice that finally survived were randomly divided into 4 groups, with 11 mice in each group. After opening the chest, the mice in the sham group were undergone needling of the anterior descending branch of the heart instead of ligation and on the next day injected with normal saline subcutaneously into the abdomen. The anterior descending branch of the mice in MI group was ligated to construct a myocardial infarction model, and normal saline was injected subcutaneously into the abdomen one day later. The mice in MI+ PTHrP (1-84) group were subjected to ligation of the anterior descending branch of the heart to construct a myocardial infarction model and subcutaneous injection of MI+PTHrP (1-84) into the abdomen 1 day later. The mice in the MI+PTHrP (87-139) group were subjected to ligation of the anterior descending branch of the heart to construct a myocardial infarction model and subcutaneous injection of MI+PTHrP (87-139) into the abdomen 1 day later. All mice were injected with a dose of 80 ug/kg9 once a day for 4 weeks for the following experiments.
2.2 Experimentation of PTHrP (1-84) and PTHrP (87-139) on HUVEC
The HUVEC (Chinese Academy of Sciences, Beijing, China) were storaged in Dulbecco's modified Eagle's medium (DMEM) , which is supplemented using 100 U/mL penicillin , 100 ug/mL streptomycin and 10% fetal bovine serum (FBS) in a cell incubator with 5% CO2 and 95% O2 at 37°C. In order to keep the medium fresh, it should be replaced at least once every two days. To study the effects of PTHrP (1-84) and PTHrP (87-139) on the survival of cells, HUVECs were cultured with the above two proteins (1 × 10-7 mmol/L) or bovine serum albumin (BSA) as control respectively for 24 hours and 48 hours, and then washed using phosphate-buffered saline (PBS) three times before following the subsequent assay.
Cell viability was detected by mitochondrial-dependent reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to formazan as described previously10. In general, 10-15uL of MTT solution was supplied to the cell supernatant before incubation (4 hours at 37°C). The medium was then discarded, and lysing cells with 2-propanol and solubilizing formazan. The absorbance of formazan was detected by a microplate reader at 570 nm (Bio-Rad Laboratories, Inc., Hercules, CA). Under normal conditions, the absorbance of formazan produced by untreated cells was taken as 100%.
2.3 Determination of heart function
High-frequency ultrasound imaging system (Vevo 2100, Visualsonics, Canada) was used to measure the left ventricular end diastolic diameter (LVEDD), left ventricular end systolic diameter (LVESD), left ventricular ejection fraction (LVEF) and left ventricular short axis shortening rate ( LVFS).Each parameter was measured for 3 times and the average value was recorded.
2.4 Histology and immunohistochemistry
Four weeks later, animals of each groups were anesthetized and then inoculated using 25 mL of PBS and 20 mL of phosphate-buffered formalin (PBF, 4%). After taken out, each hearts were first fixed in PBF for two days at 4°C; All samples were then embedded in paraffin. The heart tissues were cut into 5um sections. Immunostaining and Masson's trichrome staining (MTS) were performed. MTS was performed using a D026 Masson detection kit (Nanjing Bioengineering Institute, China) following the instructions provided by the manufacture. CD31 immunostaining (Abcam, Cambridge, England) in the granulation tissue at the border zone of MI was carried out, which is used to assess blood vessel density. The Vector ABC Vectastain Elite Kit (Vector Laboratories, Burlingame, CA) was used to visualize the brown colored reaction product, and the 3,3′-diaminobenzidine (DAB) substrate will be used. Stained vessels which are CD31 positive spot were detected in ten different fields per section under a light microscopy at x400 magnification (IPP 6.0, Media Cybernetics, Inc., Bethesda, MD).
2.5 Western-blot
Different kinds of proteins are extracted by different methods. The homogenized heart tissues and a standard method were used for total protein; Mem-PER™ Eukaryotic Membrane Protein Extraction Reagent Kit (Thermo, MA) was used for membrane protein following the manufacturer's instructions. The bicinchoninic acid assay (BCA) Protein Assay Kit was used to detect protein concentration. The proteins were transferred electrophoretically to nitrocellulose membranes (Millipore, Billerica, MA) after loading onto an sodium dodecyl sulfate polyacrylamidegel electrophoresis (SDS-PAGE) gel. The blocking method was done using 5% nonfat milk in PBS/Tween 20 for two hours at 37°C. All membranes were incubated overnight with the following primary antibody of the recommended dilution at 4°C: GAPDH (Abcam, Cambridge, England), VEGF (Abcam, Cambridge, England), VEGFR2 (Abcam, Cambridge, England). The membranes were washed subsequently, and further incubated with an horseradish peroxidase (HRP)-conjugated secondary antibody at 37°C for 1 hour. chemiluminescence system (Tanon, Beijing, China) was used to visualize the immunoreactive bands and the quantification was used by densitometry.
2.6 Statistical analysis
SPSS 21.0 software was used for statistical analysis. The measurement resources were expressed as mean±standard deviation. Single-factor analysis of variance and t test were used for comparison between groups, and P<0.05 indicated that the difference was statistically significant.