Study Of New ARB Inhibiting In-stent Restenosis And Characterization Of ILKAPs

BackgroudPercutaneous coronary intervention (PCI) is an important way of coronary heart disease (CHD). However restenosis after PCI procedure remains an important issue. Neointimal hyperplasia is the leading mechanism of in-stent restenosis which is mainly related to proliferation and migration of vascular smooth muscle cells (VSMCs) and production of abundant extracellular matrix (ECM). Angiotensin receptor blockade (ARBs) might have the ability to reduce the rates of in-stent restenosis. Drug-eluting stent (DES), such as sirolimus eluting stent (SES) and paclitaxel eluting stent (PES), is the main method to prevent restenosis after PCI. ARB is a new potential agent for DES, because it can inhibit proliferation and not affect endothelialization process. On the other hand, combination of gene therapy and DES might be a potential method for preventing restenosis after PCI. However, the factors that might prevent gene therapy still were not clarified yet. Sp100was identified as a potential role which inhibits therapeutic gene expression. To clarify its interacting proteins might provide clues to improve gene therapy.ObjectiveIn order to improve the DES, we aimed to compare valsartan and telmisartan on human aortic smooth muscle cells (HASMCs) proliferation in vitro and try to explore its possible mechanism. Further, we wanted to investigate the difference of inhibiting intima proliferation between telmisartan, valsartan and sirolimus eluting stent. To seek the potential gene therapy method that might be combined with DES, we identified the interacting proteins of SP100and characterized ILKAPs.Material and MethodsHASMCs were cultivated with angⅡ, valsartan, telmisartan and GW9662. Cell proliferation was measured by CCK-8. Type Ⅰ and Ⅲ collagen secretion were measured by ELISA assay. AT1and AT2receptor protein expression were detected by Western blot. Type I and type Ⅲ collagen mRNA expression were measured by Real-Time PCR.Telmisartan eluting stent, valsartan eluting stent, sirolimus eluting stent and polymer eluting stent were randomized to be implanted in twenty eight experimental swines. All the swines were sacrificed at the end of1st week,1st month and3rd month, respectively. Coronary angiography and OCT were used to evaluate the coronary artery before they were sacrificed. Pathological examination was completed after they were sacrificed.PlexA-sp100as a bait was used to screen a premade pb42AD based fetal cDNA library. The positive clones were analysed by on-line bioinformatics. Two ILKAP isoforms were indentified. The ILKAPS were further characterized by MTC based expression pattern analysis, subcellular location assay. The effects of ILKAPs on cell apoptosis and cell cycle also were determined. ResultsAngiotensin Ⅱ prompted HASMCs proliferation and telmisartan was superior to valsartan inhibiting HASMCs proliferation induced by angiotensin Ⅱ.Telmisartan directly inhibited HASMCs proliferation depending on the dose but Valsartan did not have the same result.Telmisartan was superior to valsartan up-regulating AT2receptor, and both of them down-regulated AT1receptor.Both telmisartan and valsartan inhibited collagen typel and type3gene mRNA expression and telmisartan was more aggressive inhibiting type3gene mRNA expression.Five swines were sacrificed at the end of1st week and13stents were followed up by CAG and OCT. Diameter stenosis of TES,VES,SES and PES was lower than BMS.There was no obvious difference in MLD measured by CAG and intima thickness and area stenosis measured by OCT between TES,VES,SES and BMS groups.Twelve swines were prepared to be killed at the end of the1st month after stenting but one died1week after stenting.Twenty nine stents were followed by OCT and CAG. There was obvious difference in MLD and diameter stenosis measured by CAG between TES, VES, SES and PES groups. MLD of TES, VES, SES were higher than PES and diameter stenosis lower than PES. MLD of TES, VES were higher than SES and diameter stenosis lower than SES. MLD of TES was higher than VES and diameter stenosis lower than VES. New intima thickness and area stenosis measured by OCT of TES, VES and SES were lower than PES. New intima thickness and area stenosis of TES a VES were higher than SES and TES lower than VES.Eleven swines were sacrified at the end of3rd month after stenting and31stents were implanted and followed up by CAG and OCT. There was obvious difference in MLD and diameter stenosis measured by CAG between TES, VES, SES and PES groups. MLD of TES, VES, SES were higher than PES and diameter stenosis lower than PES. MLD of TES, VES were higher than SES and diameter stenosis lower than SES. MLD of TES was higher than VES and diameter stenosis lower than VES. New intima thickness and area stenosis measured by OCT of TES, VES and SES were lower than PES. New intima thickness and area stenosis of TES a VES were higher than SES and TES lower than VES.Sixty four interacting clones of Sp100were isolated. SUMO1and CDC like kinase were isolated as expected as priously study. A novel isoform of ILKAP was identified as a Sp100interacting protein, which suggest a novel sp100pathway related to local extracellular signatures. Further, we indentified three ILKAP isofroms (ILKAPs). ILKAPs negative regulates cell growth and this activity was associated to its catalytic activity.ConclusionsTelmisartan is superior to valsartan inhibiting HASMCs proliferation, collagen type3gene expression and up regulating AT2receptor.TES inhibiting intima proliferation is similar to SES and better than VES.Sp100play a role in regulating the gene therapy. ILKAPs were the interacting protein of sp10and ILK, which might involve in regulating the local signals.