Biological Mechanism Of Arsenic Trioxide For Promoting Vascular Endothelialization And Inhibiting In-stent Restenosis

Cardiovascular diseases are the leading causes for morbidity and mortality worldwide.Drug-eluting metal stents(DES),as the main treatment for stenosis of cardiovascular disease,significantly reduce the incidence of in-stent restenosis.However,the drugs(rapamycin,paclitaxel,etc.)carried by DES can also delay the endothelialization at the implantation site,causing adverse events such as in-stent thrombosis and late thrombosis.Therefore,it will be an ideal strategy to develop�endothelium-friendly�DES,which can not only inhibit in-stent restenosis but also promote the re-endothelialization.Clinical evaluation of arsenic trioxide-eluting stents(AES),a drug-eluting stent developed by Beijing AmsinoMed Medical Co.,Ltd.and Ge Junbo's research team,has shown that these stents can effectively inhibit in-stent restenosis and significantly decrease late thrombosis.The low rate of late thrombosis suggests that AES has a role in promoting endothelial repair.Thus,we hypothesize that arsenic trioxide(ATO)may function as an endothelium-friendly anti-restenosis drug.To verify this hypothesis,we performed the following studies and findings:1.AES is an endothelial-friendly DESAES was compared with bare metal stents,polymer-coated metal stents,rapamycin-eluting stents(RES)to study the safety and effectiveness of AES.The results of morphology observation,blood compatibility and biocompatibility evaluation and histopathological analysis showed that the drug coating on AES stent was uniform and intact,the blood compatibility was good and there was no obvious adverse effect on the main organs like heart,liver,spleen,lung,kidney.The results of platelet adhesion and pathological section staining showed that ATO could effectively inhibit platelet adhesion,and significantly reduce the occurrence of in-stent restenosis,and its performance was even better than that of RES.Scanning electron microscopy(SEM)was used to observe the vascular reendothelialization at the stent site of RES and AES 1 week post implantation.We found that the reendothelialization rate of AES was faster than RES.The en face staining of RES and AES with endothelium-specific marker?WF showed the typical cobblestone morphology of?WF~+cells in AES groups,and that the number of?WF~+cells in AES groups were nearly ten times higher than that in the RES groups.Then,the number of proliferative cells in endothelial repair were analyzed by?WF and Ki67(cell proliferation marker)double immunofluorescence staining.The results showed that a large number of ECs in the innermost layer of neointima in AES group were proliferating compared with RES group,which had only a small number of proliferating ECs.It is concluded that AES can significantly promote the process of vascular endothelialization,indicating that AES is an endothelium-friendly DES.2.ATO plays endothelial-friendly role by affecting mitochondrial.In vitro cultures of the primary porcine coronary artery endothelial cells(PCAECs)and primary porcine coronary artery smooth muscle cells(PCASMCs)were used to further confirm our conclusion that�AES is an endothelium friendly drug-eluting stent�.The cell viability of ECs and SMCs was measured after treatment with drugs that are commonly used in DES,such as rapamycin,paclitaxel,pravastatin,hirudin,heparin and ATO.We found that only ATO could inhibit the viability of SMCs and promote the viability of ECs at the concentration of 2-6?mol/L.JC-1 dye was applied to assay mitochondrial membrane potential,and our results showed that ATO also had unique effects on early apoptosis of ECs and SMCs.It is concluded that ATO can maintain or even enhance the activity of ECs while promoting SMCs apoptosis.Next,we evaluated the effects of ATO on diverse PCAECs functions,including cell proliferation,cell migration,ECs tubular formation,permeability and monocyte adhesion.The results showed that 2?mol/L of ATO could promote the proliferation,migration and angiogenesis of ECs,as well as enhance the permeability barrier of ECs and inhibit the adhesion of monocyte THP-1.These in vitro results further confirmed the endothelial friendly effect of ATO.We also analyzed the effect of ATO on the gene expression of ECs and SMCs by transcriptome sequencing.Our RNA-seq data showed an enrichement of diffentially expressed genes in metabolic pathways in ATO-treated ECs and SMCs.As a major metabolic compartment,mitochondrial structure and function were significantly affected after ATO treatment by MitoTracker mitochondrial tracer probe and intracellular ATP production assay.Our results showed that 2?mol/L ATO could increase the number and enhance structural integrity of ECs mitochondria and promote the production of ATP,while the effect on SMCs was opposite.Taken together,we conclude that ATO plays an endothelium-friendly role,probably by enhancing mitochondrial structural integrity and promoting ATP production.3.YAP-mediated SMCs phenotypic transformation is the key to ATO inhibiting restenosisWe then focused on synthetic and contractile SMCs in vitro,specifically on the effects of ATO on cell viability,phenotypic transformation and cytomechanice of SMCs by transcriptome sequencing,immunofluorescence staining,Western blotting and atomic force microscopy.Our results showed that ATO could inhibit the proliferation of synthetic SMCs,promote their apoptosis,enhance actin filament assembly,increase Young's modulus and increase the expression of contractile phenotype protein.In addition,immunofluorescence and immunohistochemical staining of rabbit carotid artery slices after stent implantation further confirmed that ATO could increase SMCs transformation to contractile phenotype.Thus,both in vitro and in vivo studies confirm that ATO can promote the transformation of synthetic SMCs into contractile type.Immunofluorescence staining of Yes-associated protein(YAP)and SMCs phenotypic protein was performed in rabbit carotid artery specimens after stents implantation.We found that compared with other stents,AES inhibited the YAP nuclear localization at the early stage of implantation(1 week),thereby regulating the differentiation(1 month)and maturation of SMCs in neointima(3 months).Immunofluorescence double staining of cytoskeleton F-actin and phosphorylated YAP(p-YAP)in vitro showed that the fluorescence intensity of p-YAP increased with the increase of ATO concentration,and the distribution of p-YAP was closely related to the arrangement of cytoskeleton.Subsequently,the relationship between p-YAP and SMCs phenotype was studied by Western blot.The results showed that ATO could increase the expression of SMCs contractile protein SM22?and p-YAP protein at the same time.The expression of SM22?did not change significantly when YAP inhibitor only changed the total amount of YAP but had no significant effect on the expression of p-YAP.The addition of ATO still increased the expression of p-YAP and increased the expression of SM22 alpha.We conclude that the expression of SM22?is positively correlated with the amount of p-YAP,and ATO promotes the transformation of SMCs from synthetic to contractile depending partly on the YAP pathway.To sum up,this study applied genomic,cell and molecular biology,pathophysiology and cell biomechanics to confirm the endothelium-friendly function of ATO in promoting endothelialization and inducing phenotypic transformation of SMCs to inhibit restenosis.The structural integrity of mitochondria and ATP production of ECs could be the determinants for the differential responses of ECs and SMCs to ATO.YAP signaling partly mediates ATO-induced SMCs'transformation from synthetic to contractile phenotypes.The in vivo and in vitro results are consistent in showing of the endothelialium-friendly role of ATO.Our study offers insights into how ATO inhibits in-stent restenosis while promotes vascular re-endothelialization,and could provide avenues to the optimal design of ATO-eluting stents.