The Study On The Effect Of GLP-1 On Ang Ⅱ-induced Proliferation And Migration Of Vascular Smooth Muscle Cells

Objective:（1）.To investigate the effect of GLP-1 on the proliferation and migration of vascular smooth muscle cells induced by Ang Ⅱ;（2）.Further explore the molecular mechanism of the effect of GLP-1 on the proliferation and migration of vascular smooth muscle cells.Methods:（1）.RASMCs,originally derived from embryonic rat aortas,were purchased from the Shanghai Institute of Cell Biology,China.Cells were cultured in DMEM（Dulbecco’s modified Eagle’s medium）supplemented with FBS（fetal bovine serum）to a final concentration of 10%,and antibiotics at 37°C with 5%CO₂;（2）.Cell proliferation was measured by the MTT assay,which was performed according to published literature（Liu et al.2014）.Briefly,cells were seeded into 96-well plates（10⁴cells/well）,serum-starved and treated as described above for 24 h.Next,20μL of 5 mg/mL MTT was added to each well and incubated for 4 h.The medium was removed,and 150μL dimethyl sulfoxide was added to each well.The plates were incubated on a shaker at room temperature for 10 min,and the optical density（OD）was assayed at 570 nm on a microplate reader.The OD value was recorded,and the relative cell survival rates were calculated;（3）.The migration capacity of RASMCs was also characterized using a well-established in vitro scratch wound model.VSMCs were grown to confluence and then a 200μL sterile pipette tip was used to scratch the monolayer after serum starvation for 24 h.The scratch wound was allowed to heal for 24 h in the presence or absence of the indicated drug/chemical.Micrographs were captured for each sample at 0 and 24 h,and the capacity of VSMC migration was evaluated by measuring the width of the scratch wound at both time points using NIS-Elements software.（4）.RASMCs from each group were harvested after washing with ice-cold PBS and lysed using RIPA lysis buffer.Total proteins were loaded onto SDS-PAGE gels and transferred electrophoretically to PVDF membranes.Membranes were blocked with 5%skim milk for 2 h and incubated with primary antibodies overnight at 4°C.The primary antibodies used were as follows:p-MLC and MLC were diluted in 5%skim milk at a dilution of 1:500;cyclin D1 was diluted in Tris-buffered saline containing Tween-20（TBST）at a dilution of 1:1,000;RhoA and ROCK2 were diluted in TBST at 1:200;β-actin was diluted in TBST at 1:1,000.The membranes were washed and further incubated with a secondary antibody marked by horseradish peroxidase at room temperature for 2 h.Immunoreactive bands were visualized using enhanced chemiluminescence reagents.The level of each target protein was quantified and normalized to that ofβ-actin.（5）.All group values are expressed as means±standard deviation（SD）.GraphPad Prism 7 software was used to determine statistical significance.Differences between groups were analyzed by Student’s t test when two groups were compared,or by one-way analysis of variance when more than two groups were compared.Test results were considered significant at P<0.05.Results:（1）.GLP-1 inhibited Ang Ⅱ-induced proliferation of RASMCsThe effect of GLP-1 at various concentrations（5,10,20 nM）on the RASMC proliferation in response to Ang Ⅱ was determined.RASMC treat for 24 h with Ang Ⅱ showed a significant increase in cells proliferation compared to control group,and pretreatment with GLP-1 significantly attenuated Ang Ⅱ-induced proliferation in a concentration-dependent manner（P<0.05）.GLP-1 at a concentration of 20 nM significantly inhibited the proliferation of RASMC.Thus,this concentration was selected for further studies.To exclude the possible influence of GLP-1 on RASMC proliferation,GLP-1 at different concentrations were used to treat cells alone,which had no effect on the proliferation of RASMC.On the other hand,expression levels of cyclinD1 were also assessed.Stimulation with Ang Ⅱ for 24 h markedly increased the expression levels of cyclinD1 in RASMC and GLP-1 suppressed this increase（P<0.05）;（2）.GLP-1 inhibited Ang Ⅱ-induced migration of RASMCsThe migration capacity of RASMC was measured via wound-healing assay.The scratch wound assay indicated that incubation with Ang Ⅱ resulted in significantly more wound healing than control group.Pretreatment with GLP-1 was able to attenuate the effects of Ang Ⅱ on RASMC migration（P<0.05）.In addition,western blotting analysis showed that Ang Ⅱ treatment markedly increased the levels of p-MLC compared to the control.With the addition of GLP-1 and the increase in concentration,the phosphorylation level of MLC gradually decreased（P<0.05）Cell proliferation was measured by the MTT assay,which was performed according to published literature.Briefly,cells were seeded into 96-well plates（10⁴ cells/well）,serum-starved and treated as described above for 24 h.Next,20μL of 5 mg/mL MTT was added to each well and incubated for 4 h.The medium was removed,and 150μL dimethyl sulfoxide was added to each well.The plates were incubated on a shaker at room temperature for 10 min,and the optical density（OD）was assayed at 570 nm on a microplate reader.The OD value was recorded,and the relative cell survival rates were calculated;（3）.GLP-1 suppressed Ang Ⅱ-induced activation of RhoA/ROCK2 in RASMCs via cAMP/PKA pathwayAs key molecules in the regulation of cardiovascular function,Rho GTPase and its downstream effector ROCK are not only involved in the regulation of cell proliferation,but also that of migration.The role of GLP-1 on RhoA and ROCK2 expression in RASMC were examined.RhoA and ROCK2 expression were upregulated in RASMC treated with Ang Ⅱ,but RASMC pretreatment with GLP-1 significantly attenuated Ang Ⅱ-induced upregulation of RhoA and ROCK2 expression（P<0.05）.RASMC incubation with PKA inhibitor H89abrogated the effects caused by GLP-1 on RhoA or ROCK2 suppression（P<0.05）.Inhibition of RhoA and ROCK2 expression was also observed after pretreatment with exendin-4（Ex-4）or forskolin,and the effect of exendin-4 was also abrogated by H89（P<0.05）;（4）.GLP-1 inhibited Ang Ⅱ-induced proliferation of RASMCs through cAMP/PKA/RhoA/ROCK2 signal pathwayTo further investigate the mechanism involved in the positive effects of GLP-1 on RASMC,a ROCK2 selective inhibitor（Y-27632）was used in the experiment.Like GLP-1,exendin-4（Ex-4）,forskolin or Y277632 can respectively suppress the Ang Ⅱ-induced increases in the proliferation of RASMC.The inhibitory effect of GLP-1 and exendin-4 on Ang Ⅱ-induced RASMC proliferation was significantly obliterated by incubation with H89.Furthermore,combination of GLP-1 and Y-27632 further attenuated RASMC proliferation（P<0.05）.In line with these above changes in MTT assay,the expression levels of cyclinD1assessed by western blotting was suppressed by GLP-1,exendin-4,forskolin,or Y-27632,and the effect of GLP-1 and exendin-4 was eliminated by H89（P<0.05）;（5）.GLP-1 inhibited Ang Ⅱ-induced migration of RASMC through cAMP/PKA/RhoA/ROCK2 signal pathwayIn terms of intervention of cell migration,both Y-27632,forskolin,exendin-4 and GLP-1 inhibited Ang Ⅱ-induced migration,and the inhibitory effect of GLP-1 or exendin-4 on migration was abolished by H89（P<0.05）.In addition,western blotting analysis showed that Y-27632 or GLP-1 significantly suppressed the elevated levels of p-MLC induced by Ang Ⅱ treatment,and H89 abrogated the GLP-1-induced suppression of Ang Ⅱ-induced phosphorylation levels of MLC（P<0.05）.