The Effect Of Sphingosine-1-phosphate Receptors In AGE-induced Proliferation And Migration Of Vascular Smooth Muscle Cells

With the development of economy and the improvement of people's living standards,environment and diet have been changed remarkably in recent decades.The incidences of cardiovascular and cerebrovascular diseases are increasing significantly.The effect of various metabolic diseases,such as obesity,diabetes and metabolic syndrome are becoming more and more prominent on inducing the development of those cardiovascular and cerebrovascular diseases.The consequences of diabetic vascular complications cause morbidity and even mortality to the patient,and bring heavy burden to the patient,patient's family and the society.Vascular remodeling is one of the important pathologic basis of diabetic vascular complications.The elucidation of its pathogenesis would help to understand the development of diabetic vascular complications,to provide evidences for early diagnosis and prognosis,and to explore new therapeutic target for the treatment as well.Tissue remodeling is actually a response process of tissue injury and repair,when this repair process is disturbance or excessive,these responses can lead to tissue fibrosis.Fibrosis results from the excessive deposition of extracellular matrix components by myofibroblasts after injury.However,the most significant characteristic of vascular remodeling is the excessive proliferation and migration of vascular smooth muscle cells,which will result in obvious thickening of the arterial muscle layer(tunica media),luminal stenosis,and even lead to arteriosclerosis.The role of sphingosine-1-phosphate(SIP)in vascular remodeling has been noticed in recent years.Produced by phosphorylation of sphingosine,SIP is a bioactive sphingolipid in plasma.SIP is an important balance substance in plasma and mostly it works as a second messenger in cell signal.But it can also work as the first messenger by binding with membrane receptors and participate in the regulation of cellular differentiation,migration,proliferation,and survival,and are involved in angiogenesis,immune and allergic responses.Studies have confirmed that SIP plays an important role in wound healing and fibrosis after tissue injury.Through its receptors and down-stream signaling,SIP regulates endothelial barrier function,modulates fibroblast migration and myofibroblast differentiation.Reports have shown that the serum content of S1P were increased in patients with diabetes and in diabetic animal model,indicating that S1P may participate in the initiation and progression of diabetes and its complications.It is still unknown whether S1P receptors plays a role in the proliferation and migration of vascular smooth muscle cells during the development of diabetic vascular remodeling.Objects:This study was first aimed to investigate the role of advanced glycation end products(AGEs)in inducing the proliferation,migration of vascular smooth muscle cells(VSMCs).The effects of different SIP receptors(S1PRs),such as S1PR1 and S1PR2,were then explored in AGE-induced VSMC proliferation and migration.Methods:Human umbilical artery smooth muscle cells(HUASMCs)were used in this study.AGE-modified bovine senum albumin(AGE-BSA)was made by incubating bovine serum albumin(BSA)in PBS with D-glucose for eight weeks in a sterile environment at 37?.AGE-BSA was filtered before usage.The proliferation of HUASMCs was measured using CCK-8 kit.Scratch wound healing assay and transwell migration assay were used to test HUASMC migration ability.Western blot was applied to detect the expression of S1PR1 and S1PR2.Results were presented as means ± SD.Data was analyzed by one-way analysis of variance using SPSS 13.0 software package.P-values less than 0.05 were considered significant.Results:1.BSA and AGE-BSA promote the proliferation and migration of HUASMCs.(1)BSA and AGE-BSA promote the proliferation of HUASMCsThe results of CCK-8 experiment showed that there is significant difference between different groups(F=5.434,P=0.045).Compared with control group,OD value in BSA-treated group and AGE-BSA-treated group were both increased,the differences were statistically significant(P<0.05).Compared with BSA-treated group,AGE-BSA treatment was obviously more effective on promoting HUASMC proliferation(P<0.05).These results show that BSA can promote proliferation of VSMCs,which is consistent with clinical and experimental observations that the exposure of smooth muscle cells to plasma after injury of endothelial cells leads to excessive proliferation of smooth muscle cells,resulting in vascular remodeling and arterial stiffness.The effect of AGE-BSA on promoting smooth muscle cell proliferation is more prominent.(2)BSA and AGE-BSA promote the migration of HUASMCsWound healing assay results showed that there is significant difference between different groups(F=16.35,P=0.006).Compared with control group,the recovery percentage(migration distance)of BSA-treated and AGE-BSA-treated group were both increased obviously(P<0.05).Compared with BSA-treated group,AGE-BSA treatment enhanced the recovery of wound more apparently,with statistical significance(P<0.05).Transwell migration assay of HUASMCs revealed similar results.These results demonstrate that BSA itself is effective in increasing vascular smooth muscle cell migration,which is also consistent with clinical and experimental observations that the exposure of smooth muscle cells to plasma after injury of endothelial cells initiate the migration of smooth muscle cells.The effect of AGE-BSA on promoting smooth muscle cell migration is more prominent.2.The influence of S1PR1 activity on HUASMC proliferation and migration induced by BSA and AGE-BSA(1)The inhibition of S1PR1 attenuated HUASMC proliferation and migrationHUASMCs were pretreated with VPC,an inhibitors of S1PR1,and then were stimulated with BSA or AGE-BSA.The results of CCK-8 assay showed that there was significant difference between different groups(F=20.75,P=0.004).Compared with BSA group,OD value of VPC+BSA group was obviously decreased(P<0.05).Compared with AGE-BSA group,OD value in VPC+AGE-BSA group were also significantly decreased(P<0.05).And compared with control group,VPC itself could significantly inhibit smooth muscle cell proliferation.Wound healing assay results also showed that there was significant difference between different groups(F=12.55,P=0.002).Compared with BSA group,the percentage of migration distance in VPC+BSA group was decreased obviously(P<0.05).Compared with AGE-BSA group,the recovery percentage in VPC+AGE-BSA group was markedly reduced(P<0.05).And compared with control group,VPC itself can significantly inhibit the migration of smooth muscle cells.Transwell migration assay of HUASMCs revealed similar results.The results demonstrate that inhibition of S1PR1 activity can prevent BSA and AGE-BSA-induced excessive proliferation and migration of smooth muscle cells,indicating that S1PR1 activation is involved in mediating the effects of BSA and AGE-BSA on enhancing VSMC proliferation and migration.(2)The activation of S1PR1 enhanced HUASMC proliferation and migrationHUASMCs was pretreated with a S1PR1 agonist,SEW,and then were stimulated with or without BSA or AGE-BSA.The results of CCK-8 showed that there was significant difference between different groups(F=4.388,P=0.0419).Compared with BSA group,OD value of SEW+BSA group was obviously increased(P<0.05).Compared with AGE-BSA group,OD value was no obviously increased in SEW+AGE-BSA group.Compared with blank control,SEW alone could increased OD value significantly.Wound healing assay results showed that there was significant difference between different groups(F=12.22,P=0.002).Compared with BSA group,the percentage of migration distance of SEW+BSA group was increased obviously(P<0.05).Compared with AGE-BSA group,the percentage of migration distance in SEW+AGE-BSA group was not changes obviously.Compared with blank control,SEW alone could increased the migration distance significantly.Transwell migration assay of HUASMCs revealed similar results.The above results demonstrated that,the activation of S1PR1 is capable in enhancing the proliferation and migration of smooth muscle cells.While BSA induced the proliferation and migration through S1PR1,the addition of S1PR1 agonist could further strengthen this effect.However,the results shows no differences between AGE-BSA and SEW+AGE-BSA group in VSMC proliferation and migration,indicating that AGE-BSA would have activated S1PR1 intensively,the addition of S1PR1 agonist could no longer exert further activation.3.The effect of S1PR2 activity on HUASMC proliferation and migration induced by BSA and AGE-BSAHUASMCs were pretreated with JTE,an inhibitors of S1PR2,and then were stimulated with BSA or AGE-BSA.The results of CCK-8 assay showed that there was significant difference between different groups(F=19.62,P=0.0005).Compared with BSA group,OD value of JTE+BSA group was decreased obviously(P<0.05).Compared with AGE-BSA group,OD value of JTE+AGE-BSA group was also significantly decreased(P<0.05).JTE itself could also attenuated the proliferation of HUASMCs.Wound healing assay results showed that there was significant difference between different groups(F=10.35,P=0.004).Compared with BSA group,the percentage of migration distance of JTE+BSA group was decreased obviously(P<0.05).Compared with AGE-BSA group,the changes of percentage of migration distance in JTE+AGE-BSA group was also obvious.JTE itself could also attenuated the migration of HUASMCs.The results demonstrated that the inhibition of S1PR2 attenuated BSA and AGE-BSA-induced proliferation and migration of vascular smooth muscle cells,indicating the involvement of S1PR2 activation in promoting smooth muscle cell proliferation and migration.3.The effect of AGE-BSA on S1PRl and S1PR2 protein expression in HUASMCs(1)Different concentrations of AGE-BSA stimulation does not affect the expression of S1PR1 and S1PR2 in HUASMCs.Using different concentration of AGE-BSA(12.5,25,50,100 ?gg/ml),respectively,to stimulate HUASMCs for 24 h,and with BSA(100 ?g/ml)as control,we detected S1PR1 and S1PR2 expression by Western Blot.The results showed no significant difference between different groups(P>0.05).The results demonstrated that SIPR1 and S1PR2 protein expression were not affected by increasing dose of AGE-BSA treatment.(2)Different time of AGE-BSA stimulation does not affect the expression of S1PR1 and S1PR2 in HUASMCsAGE-BSA(100 ?g/ml)was used to stimulate HUASMCs for 12,24,48,72 h,respectively,with BSA(24 h in 100 ?g/ml)as control.The protein expression of S1PR1 and S1PR2 were detected by Western Blot.The results showed that there was no significant differences between different groups(P>0.05).The results demonstrated that S1PR1 and S1PR2 protein expression were not affected by increasing treatment time of AGE-BSA.The results demonstrate that BSA itself has the role of promoting vascular smooth muscle cell proliferation and migration,which conform to the clinical and experimental phenomenon that the exposure of vascular smooth muscle cells to the plasma after endothelial cell injury leads to excessive proliferation and migration.AGE-BSA is more effective on promoting smooth muscle cell proliferation and migration.The inhibition of S1PR1 attenuated BSA and AGE-BSA-induced smooth muscle cell proliferation and migration.The activation of S1PR1 further enhanced BSA and AGE-BSA-induced smooth muscle cell proliferation and migration.These data suggests that S1PR1 is involved in mediating the effect of BSA and AGE-BSA on promoting smooth muscle cell proliferation and migration,and AGE-BSA exerts stronger effect on S1PR1 activation.The inhibition of S1PR2 also attenuated BSA and AGE-BSA-induced smooth muscle cell proliferation and migration,suggesting that S1PR2 is involved in mediating the effect of BSA and AGE-BSA on promoting smooth muscle cell proliferation and migration.BSA and AGE-BSA stimulation did not change the expression of S1PR1 and S1PR2 in HUASMCs.Conclusion:1.The BSA and AGE-BSA can promote the proliferation and migration of vascular smooth muscle cell,and AGE-BSA is more effective.2.S1PR1 and S1PR2 activation can mediate the effects of BSA and AGE-BSA on promoting proliferation and migration of smooth muscle cells.The activating effect of AGE-BSA on S1PR1 and S1PR2 might be more prominent.3.BSA and AGE-BSA does not affect the expression of S1PR1 and S1PR2 in vascular smooth muscle cells.