Abstract
BackgroundNeointimal formation plays an important role in the pathogenesis of coronary restenosis after percutaneous coronary intervention (PCI), especially in patients with diabetes mellitus. Recently, some studies have shown that 5-ethynyl-2'-deoxyuridine (EdU) incorporation can serve as a novel alternative to the 5-bromo-2'-deoxyuridine (BrdU) antibody detection method for detection of DNA synthesis in regenerating avian cochlea, chick embryo and the adult nervous system. However, few studies have been performed to assess the suitability of EdU for detecting DNA synthesis in vascular neointima.MethodsThe carotid artery balloon injury model was established in Goto-Kakizaki (GK) and Wistar rats. A Cell-LightTM EdU Kit was used to detect EdU-labeled cell nuclei of common carotid arteries at day 7 after catheter balloon injury. Different methods of injecting EdU were tested. The protein levels of proliferating cell nuclear antigen (PCNA) and p-Akt (Ser473), as well as the mRNA levels of PCNA were evaluated by Western blotting and quantitative real-time PCR (qRT-PCR), respectively. Immunohistochemical staining was also employed to visualize PCNA-positive cells.ResultsAt day 7 after catheter balloon injury, far more EdU-positive and PCNA-positive cells were observed in GK rats. When comparing groups that received different EdU doses, it was found that the percentage of EdU-positive cells at a dose of 100 mg/kg body weight was than at doses of 25 mg/kg and 50 mg/kg. The number of positive cells was significantly higher in the repeated injection group compared to the single injection group. Further, after balloon injury DNA synthesis in GK rats was more notable than in Wistar rats. Neointimal formation in GK rats was more obvious than in Wistar rats. The protein levels of PCNA and p-Akt (Ser473) and the mRNA levels of PCNA were increased in injured rats as compared to uninjured rats, and were significantly higher in GK rats than in Wistar rats.ConclusionBy intraperitoneal injections of EdU at a dose of 100 mg/kg three times, EdU incorporation can detect carotid arterial DNA synthesis caused by neointimal formation in GK rats and Wistar rats at day 7 after balloon injury by the EdU click reaction quickly and effectively. Moreover, more obvious DNA synthesis in the vascular neointima could be observed in GK rats than in Wistar rats.
Highlights
Neointimal formation plays an important role in the pathogenesis of coronary restenosis after percutaneous coronary intervention (PCI), especially in patients with diabetes mellitus
Drug eluting stents are considered by many to be the standard of care for patients with diabetes undergoing PCI; analysis of diabetic subgroup in the SIRIUS (SIRolImUS-coated Bx Velocity balloon-expandable stent in the treatment of patients with de novo coronary artery lesions) trial demonstrated that absolute late loss of lumen and restenosis remains higher in patients with diabetes receiving stents eluting sirolimus [4]
EdU staining for DNA synthesis The percentage of EdU-positive cells in GK and Wistar rats using different doses of EdU was studied
Summary
Neointimal formation plays an important role in the pathogenesis of coronary restenosis after percutaneous coronary intervention (PCI), especially in patients with diabetes mellitus. Restenosis remains the major limitation for long-term success after percutaneous coronary intervention (PCI) even in the drug eluting stent era, especially in patients with diabetes mellitus [1,2]. Angiographic restenosis rates of 15.2% have been demonstrated following successful drug-eluting stenting among patients with diabetes in a study in Germany [3]. Observation of VSMCs proliferation and neointimal formation is an important method for studying restenosis after vascular injury. Detecting DNA synthesis in the vascular wall can indirectly reflect the VSMC proliferation in vivo
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