| Cardiovascular disease(CVD)has become the primary key factor that threatens people's lives and health.About stenotic CVD,the most widely used method for clinical treatment is drug eluting stent(DES)in the current.However,after performing the therapeutic function in the human body,DES will permanently stay as a foreign body and cause a series of foreign body reactions,especially the occurrence of stent thrombosis.Therefore,bioresorbable stent(BRS)based on degradable materials has attracted the more and more attention in the world.Poly-L-lactic acid(PLLA)stent is the first one that enters clinical use due to its good histocompatibility and biodegradability.However,clinical studies have shown that serious complications after the PLLA stent implantation.Thus,investigating the causes of vascular stenosis accompanying the degradation of PLLA stent will be the key to optimize the future design of polymeric vascular stents.As a basic substrate for synthesis of PLLA,lactic acid(LA)is the primary degradation product of PLLA.At the same time,LA is also an intriguing regulator of cell function and can produce different physiological or pathological effects on cells and tissues.The local accumulation of LA during PLLA degradation can stimulate blood vessels to cause pathological responses,which would imply that LA may be involved in the occurrence of vascular stenosis after PLLA stent implantation.Therefore,this study proposes a scientific hypothesis:"LA produced by PLLA degradation may be a key pathogenic factor leading to vascular stenosis after PLLA stent implantation".Around this scientific hypothesis,the research can mainly draw the following five conclusions:1.PLLA stent leads to severe in-stent stenosisThe stented vessels at 3 and 6 month after PLLA and BMS stents were implanted into the carotid artery of New Zealand white rabbits were taken for histomorphological analysis.Hematoxylin and eosin(H&E)staining results showed that the vessel stenosis rate in PLLA stent group was much higher than the BMS group.Masson staining also showed that the degree of fibrosis in the carotid stent segment of rabbits was significantly increased after PLLA stent implantation.At the same time,SM22?,a gene related to vascular smooth muscle cells(SMCs),was subjected to immunohistochemical staining.The results showed that SM22?was abundantly expressed in the stented vessels,especially around the stent filaments aggregated by PLLA degradation product,which may imply that the role of PLLA degradation product in the process of in-stent stenosis.2.PLLA degradation product LA induces vascular fibrosisLA was used to locally stimulate the abdominal aorta of rats in this study.Masson staining showed that the abdominal aorta blood vessels of the rats after LA treatment significantly increased fibrosis compared with the control group.The immunohistochemical staining results also showed the fiber-related gene OPN was expressed significantly higher in the LA-treated group than the control group.In addition,q PCR was also used to detect the expression of the fiber-related gene collagen-1,Collagen-3,MMP2 and MMP9 in the blood vessels after LA treatment and consistently increased in the LA treatment group.These results indicate that LA can induce vascular fibrosis,and LA-induced vascular fibrosis may be the cause of in-stent stenosis after PLLA stent implantation.3.LA-induced endothelial-to-mesenchymal transition(EndMT)directly may be the cause of vascular fibrosisAccumulating evidences suggest that EndMT is a critical reason for inducing vascular fibrosis.The enface staining results showed that the expression of endothelial marker v WF in the intima gradually decreased with time after LA stimulation,while mesenchymal marker SM22?was co-expressed with v WF in the intima and gradually increased with time.Section staining results showed that SM22?was abundantly expressed in the intima after LA stimulation,and the proportion of SM22?~+cells co-expressed with v WF was about 70%.Immunohistochemical staining also showed a consistent trend.These results indicate that LA can induce EndMT in vivo.Furthermore,human umbilical vein endothelial cells(HUVECs)were selected to verify whether LA could induce EndMT in vitro.The results showed that the morphology of HUVECs changed from cobblestone or elliptical shape to long strips after LA stimulation,LA could down-regulate the expression of endothelial markers and up-regulate the expression of mesenchymal markers,and the gene expression profile of HUVECs after LA stimulation was different with the control group tending to the TGF-?1 treatment group.In addition,this study also excluded the effect of LA-induced p H reduction on EndMT.These results indicate that LA can also induce EndMT in vitro.4.LA may mediate EndMT by stimulating TGF-?1 signal through receptor GPR81The expressions of receptor G-protein-coupled receptor 81(GPR81)and TGF-?1signaling pathway-related proteins were significantly up-regulated after LA stimulation.After silencing the expression of GPR81 or TGF-?1 signal combined with LA stimulation,the expressions of endothelial markers and mesenchymal markers did not change compared with the control group.These results indicate that GPR81 and TGF-?1 signal are involved in mediating LA-induced EndMT.TGF-?1 and specific protein-1(Sp-1)which was a TGF-?1 transcription factor were activated after LA stimulation,but the expressions did not change after GPR81was knocked down.After inhibiting the expression of Sp-1 combined with LA stimulation,the expressions of endothelial markers and mesenchymal markers did not change compared with the control group.These results indicate that Sp-1 is involved in the regulation of LA-activated TGF-?1.The possible mechanism for LA-induced EndMT is that when LA comes into contact with endothelial cells(ECs),the LA receptor GPR81 is activated first and then activates transcription factor Sp-1,then Sp-1regulates the expression of TGF-?1,thus promoting EndMT.5.Changes in cell mechanics may be one of the mechanisms mediated LA-induced EndMTLA could significantly enhance the migration and contraction capabilities of HUVECs.Furthermore,LA could also promote cytoskeletal rearrangement of HUVECs,while inhibitingcytoskeletal rearrangement could block LA-induced EndMT.These results suggest that changes in cell mechanics may also be one of the mechanisms involved in mediating LA-induced EndMT.In summary,the research fully confirms the role of LA in the occurrence of vascular stenosis during PLLA stent implantation and stent degradation.The reason of LA affecting the occurrence of in-stent stenosis after PLLA stent implantation may be that LA induces EndMT leading to vascular fibrosis.The possible mechanism of LA-induced EndMT is that LA stimulates TGF-?1 signal through the receptor GPR81.This project explores the cause of in-stent stenosis after PLLA stent implantation from the perspective of the accumulation of LA,the degradation product of PLLA.It provides a new point for the study of the cause of severe complications in the late stage after degradable vascular stent implantation,and also provides new theoretical support for the optimal design and application of degradable vascular stent based on poly-lactic acid in the future. |
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