Identificación de nuevos biomarcadores genéticos y mecanismos moleculares de la reestenosis tras revascularización vía angioplastia: Papel del factor de transcripción NF-Y

Restenosis is the major limitation of atherosclerotic vessels revascularization procedure via angioplasty percutaneous coronary intervention (APCI). Restenosis, a disease that occurs during first 4-12 months after APCI, has a high economic and clinical impact due to the necessity of repeat restenotic vessel revascularization via APCI or coronary artery bypass grafting. Nowadays, APCI is performed via the implantation of a metallic prosthesis called stent. These devices improves the safety of the procedure through reducing restenosis rates compared to conventional APCI (15-30% vs 25-50%) by avoiding negative remodeling of the target vessels. The recently introduction of drug eluting stents (DES) has supposed a revolution of interventional cardiology by reducing restenosis rates (60-80%) compared to conventional stents. DES deliver lipophilic drugs (i.e sirolimus or paclitaxel) locally that block cell cycle, inhibit cell proliferation and reduce restenosis rates. Restenosis is a hiperproliferative disease initiated by mechanically-induced endothelial damage due to stent deployment. Endothelial damage triggers an inflammatory response that activates vascular smooth muscle cells (VSMC) to a “synthetic” phenotype characterized by high proliferative and migratory response and elevated extracellular matrix components production. These events end up with the neointimal lesion development and vessel re-occlusion. The proper process development requires inflammation resolution and complete re-endothelization of affected lesion. In this sense, unselective inhibition of cell proliferation by DES dampers re-endothelization causing an increase in the risk to suffer late thrombosis (a feature of these stents). Therefore, considering the socioeconomic impact of restenosis and drawbacks associated to use of DES respect to BMS appears to be necessary identify biomarkers of the risk of restenosis in order to stratify patients to develop a patient-tailored therapy. During last years, increasing evidence suggest the association of genetic polymorphisms with the risk to suffer complex diseases. Severals studies demonstrate that single nucleotide polymorphisms (SNPS) located in different human genes are associated with increased in-stent restenosis (ISR). Interestingly, Van tiel and colleagues demonstrated that -838AA individuals (SNP -838(C>A) within the p27Kip1 gene) show reduced ISR risk probably due to reduced promoter activity. The goal of this work is to identify SNPs in cell cycle regulatory genes that predict ISR risk after coronary stent deployment. Our analysis of a cohort of 284 patients undergoing coronary angioplasty and stent deployment showed that SNPs rs350099 (-957[T/C]), rs350104 (-475[T/C]), rs164390 (+102[G/T]) y rs875459 (+7010[G/T]) (located in CCNB1 gene, codifying cyclin B1 protein), rs2282411 (+7733[T/C], located in CCNA1 gen, codifying cyclin A1) and rs733590 polymorphism (-1284[T/C]), located in CDKN1A gene, codifying p21Cip1 protein) are associated with increased ISR risk. Based on its location in CCNB1 promoter region, analysis of -957[T/C], (-475[T/C]) y (+102[G/T]) polymorphisms in additional 715 patients cohort also showed significant association with increased ISR risk. Moreover, risk allelic variants of -957[T/C], (-475[T/C]) y (+102[G/T]) polymorphisms are associated with increased reporter gene expression through NF-Y, AP-1 and SP1 transcription factors, respectively. High risk alleles containing haplotype is associated with augmented cyclin mRNA levels in peripheral blood lymphocytes and increased ISR risk. The identification of NF-Y transcription factor, known controller of cell cycle regulation gene transcription (cyclin B1 included), led us to investigate its role in the context of vasculo-proliferative diseases. By using molecular and expression studies in cultured cells, animal models and human tissues we demonstrate that platelet-derived growth factor-BB (PDGF-BB), a main inductor of neointimal lesion development, induces in rat and human VSMCs the recruitment of NF-Y to the CCNB1 promoter and augments both CCNB1 mRNA expression and cell proliferation through extracellular signal-regulated kinase 1/2 (Erk1/2) and Akt activation. We find co-expression of NF-Y and cyclin B1 in proliferative regions of murine atherosclerotic plaques and mechanically-induced lesions, which correlates with higher binding of NF-Y to target sequences in the CCNB1 promoter. Moreover, adenovirus-mediated overexpression of a NF-YA dominant-negative mutant inhibits PDGF-BB-induced CCNB1 expression and VSMC proliferation in vitro and neointimal lesion formation in a mouse model of femoral artery injury. We also detect NF-Y expression and DNA-binding activity in human neointimal lesions. In summary, we have identified NF-Y as a key positive regulator of cyclin B1 expression, VSMC proliferation and neointimal lesion formation. We have also found overexpression and activation of NF-Y in restenotic and atherosclerotic lesions, both in animal models and human samples. Moreover, we have identify polymorphic variants of rs350099 (-975T/C), rs350104 (-475 T/C) y rs164390 (+102G/T) within human CCNB1 promoter region that are associated with increased risk of ISR and generates novel NF-Y, AP-1 and SP1 DNA-binding sites, respectively. In fact, high risk haplotype is associated with increased cyclin B1 mRNA levels and augmented ISR risk. In addition, we also found significant association of rs2282411 and rs733590 polymorphisms with increased ISR risk. Therefore, our studies suggest that individuals carrying -957TT/-475CC/+102GG genotype show increased NF-Y, AP-1 and SP1 recruitment contributing through higher cyclin B1 levels and VSMC proliferation to increased ISR risk after coronary stent deployment.