Attenuated retinoblastoma gene product and associated E2F/retinoblastoma imbalance in anastomotic intimal hyperplasia

Objective: The retinoblastoma gene product is a cell cycle control protein that when inhibited allows cell proliferation to progress by releasing E2F. Retinoblastoma manipulation has been attempted to prevent intimal hyperplasia (IH) in injured native vessels by arresting vascular smooth muscle cell proliferation. However, no studies have identified the role, if any, of retinoblastoma in anastomotic IH formation after prosthetic arterial grafting. The goal of this study was to describe the relation of retinoblastoma and E2F to anastomotic IH with analyzing retinoblastoma/E2F levels, retinoblastoma phosphorylation, and transcription of retinoblastoma and E2F in prosthetic arterial grafting. Methods: Six-mm-diameter expanded polytetrafluoroethylene carotid interposition grafts (n = 12) were implanted in 25-kg mongrel dogs. The intervening arterial segments were harvested as controls. The distal anastomoses were harvested at 14 and 30 days after implantation for immunoblot, messenger RNA (mRNA), and immunohistochemistry analyses. Tissue homogenate was separated with sodium dodecylsulfate-polyacrylamide gel electrophoresis and probed with antibody to total retinoblastoma, phosphorylated retinoblastoma at serine 795, serine 780, and serine 807/811, and E2F-1. Bands at each time point were quantitated and compared with control artery (n = 12). Each lane was standardized with reprobing with antibody to β-tubulin. Immunohistochemistry was performed with antibody to retinoblastoma. Retinoblastoma and E2F mRNA expression levels in anastomotic IH and control artery were analyzed with an oligonucleotide microarray. Results: Total retinoblastoma, from immunoblot analysis, was decreased at the 14-day and 30-day distal anastomoses by 35.7% and 33.6%, respectively, compared with control (P