Effects Of PIP Targeting LOX-1 Eluting Stents On In-stent Restenosis In Rat Abdominal Aorta Stenting Models

Currently available drug-eluting stents (DES) partially exert the beneficial effects on the prevention of restenosis. However, currently available DES not only inhibited resnosis but also impaired re-endothelialization, which increased the incidence of late stent thrombosis (LST). The focal nature of restenosis makes it a highly attractive target for locally delivered gene silencing agents that may separate therapeutic benefits from unwanted side effects by selectively suppressing the pathogenic genes. Pyrrole-Imidazole polyamide (PIP) is a novel gene silencer that can be readily designed and synthesized to target any gene. Contrary to traditional gene-silenceing agents, PIP is resistant to nucleases and can enter into the nuclei of target cells without any particular delivery system. Moreover, PIP has a low molecular weight, a high efficiency of cellular uptake and bioavailability, and is hydrophobic and lipophilic, making it pharmacokinetically suitable for loading onto the metal stent.Interventional procedures induced vascular injury could stimulate vascular smooth muscle cells (VSMCs) to proliferate and migrate from the media into the neointima, thus promoting neointimal hyperplasia (NIH), which is one of the major pathophysiologic process responsible for restenosis. The activation of lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) and the positive feedback between LOX-1 and oxidative stress played a key role in VSMCs proliferation and migration and neointimal formation. Thererfore, the present study aimed to evaluate whether PIP targeting LOX-1 could reduce LOX-1expression, inbibit ox-LDL induced VSMCs proliferation and migration and decrease NIH after vascular injury without imparing re-endothelialization. The present study was composed of two parts: Part I Effects of PIP targeting LOX-1 on ox-LDL induced VSMCs proliferation and migration; Part II Effects of PIP targeting LOX-1 eluting stents on on in-stent restenosis and re-endothelialization in rat abdominal aorta. Aim The present study was designed to synthesize pyrrole-Imidazole polyamide (PIP) targeting rat lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) gene promoter and investigate potential effect of PIP on oxidized low-density lipoprotein (ox-LDL) induced vascular smooth muscle cells (VSMCs) proliferation and migration. Methods HEK293 cells were transfected with recombinant rat LOX-1 promoter plasmids to determine AP-1 binding sites. PIP targeting rat LOX-1 were synthesized according to side-by-side pairing of pyrrole and imidazole in the minor groove of DNA. VSMCs were cultured in vitro and identified by?-SMA immunoflurescence staining. All VSMCs were assigned to four groups (n=6 per group): Control group, Control+ox-LDL (10?g/ml) group, Control+ox-LDL+PIP (10^-6mmol/L) group, Control+ox-LDL+mismatch PIP (10^-6mmol/L) group. The expression of LOX-1 protein was determined by Western blot. The proliferation of VSMCs was determined by cell count, MTT metabolism measuring assay. The migration of VSMCs was observed by Transwell Cell Migration Assay.Results Enzyme digestion of collagenase I was applied for the culture of normal VSMCs, which showed the typical pattern“Valley-peak”.?-SMA was abundant in the VSMCs by immunoflurescence staining. ox-LDL at lower concentrations of 10ug/ml could obviously trigger VSMCs cells number proliferation, increase VSMCs MTT metabolism and promote VSMCs migration (P>0.01). PIP targeting rat LOX-1 promoter could significantly inhibit the ox-LDL induced LOX-1 upregulation. Besides, PIP targeting rat LOX-1 promoter (10^-6mmol/L) could significantly inhibit VSMCs cells number proliferation, MTT metabolism increase and VSMCs migration (P>0.01). Mismatch PIP had no such effects.Conclusions PIP targeting LOX-1 could reduce LOX-1 expression, inbibit ox-LDL induced VSMCs proliferation and migration. Therefore, PIP targeting LOX-1 as a feasible gene silencing agent for the prevention of in-stent restenosis of the coronary artery warrants further investigations Aim The present study was designed to produce PIP targeting LOX-1 eluting stents and to evaluate the potential effects of them on in-stent restenosis and re-endothelialization in rat abdominal aorta stenting models.Methods 2.0×9.0mm PIP targeting LOX-1 eluting stents were developed with a concentration of 1.0?g/mm2 PIP. PIP release kinetics in stents was analysized by high performance liquid chromatography (HPLC) in vitro and in vivo. Rat abdominal aorta stenting models were established. Rats were assigned to four groups (n=10 for each group): Control group, Bare-metal stent group, PIP-eluting stent group and Mismatch PIP-eluting stent group. After 14 days, some aortas were harvested, fixed, embedded and cut, and a small amount of sample was used for HPLC analysis. Then, re-endothelialization indices were determined by scanning electron microscopy (n=3). After 28 days, other aortas (n=6) were harvested, rapidly excised and cleaned of connective tissue, and a small amount of sample was used for HPLC analysis. The other samples were divided into three parts: One segment was immediately frozen and stored at -80°C. Another segment was blotted dry and weighed. The third segment was fixed, embedded in methylmethacrylate. The expression of LOX-1, NADPH p22^phox, NADPH p47^phox will be determined by quantitative real-time PCR and Westernblot. The oxidative stress in the aorta will be determined by measuring aortic MDA levels. Sections in methylmethacrylate will be cut into 5-?m thickness and stained with hematoxylin-eosin.The area of neointimal hyperplasia (NIH) ,the rate of restenosis, and the injury and inflammation scores were measured.Results In vitro pharmacological release kinetics showed that PIP eluting stents showed almost complete release of the PIP with in 24h. In vivo pharmacological release kinetics demonstrated that trace amount of PIP was still remained in the local aorta tissues. Rat abdominal aorta stenting models were successfully established. Scanning electron microscopy showed that re-endothelialization was nearly complete in all the three stents groups (n=3) at 14days after stents implantation. The expression of LOX-1, NADPH p22^phox, NADPH p47^phox and the vascular MDA levels was significantly increased in BMS group compared with control group at 28 days after stents implantation (P<0.05). The increased LOX-1, NADPH p22^phox, NADPH p47^phox expression and vascular MDA levels was significantly inhibited in PIP eluting stent group compared to BMS group (P<0.05). All the three stents groups showed lower inflammation score. However, the area of NIH and the rate of restenosis was significantly lower in PIP eluting stent group compared to BMS group (P<0.05). The area of NIH and the rate of restenosis was similar in mismatch PIP eluting stent group compared to BMS group (P>0.05)Conclusions PIP targeting LOX-1 eluting stents could decrease in-stent restenosis (ISR) without imparing re-endothelialization by silencing LOX-1 and lowering oxidative stress. These results indicated that PIP targeting LOX-1 is considered to be a feasible gene silencing agent used the next generation of DES for the prevention of in-stent restenosis of the coronary artery.