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 beta-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 <.01). Furthermore, retinoblastoma at these time points was unphosphorylated at phosphorylation sites serine 795, serine 780, and serine 807/811. E2F-1 levels at 14 days and 30 days were unchanged compared with control. Positive staining for retinoblastoma was seen in endothelial and vascular smooth muscle cell from control, 14-day, and 30-day tissue. A qualitative decrease appeared to be seen in retinoblastoma in the neointima at 14 and 30 days compared with the native wall. No differential expression of retinoblastoma and E2F mRNA was seen in anastomotic IH compared with control.


CONCLUSIONS
This study showed that total retinoblastoma levels are decreased and E2F-1 levels remain unchanged in anastomotic IH. Attenuated retinoblastoma is a novel concept to anastomotic IH after prosthetic arterial grafting. Retinoblastoma/E2F imbalance may not be the result of transcriptional regulation and may increase unbound E2F to promote cell proliferation. Hypophosphorylation of remaining retinoblastoma may minimize uncontrolled proliferation by preventing further increases in unbound E2F. Therefore, retinoblastoma/E2F imbalance may lead to the early but limited increase in cell proliferation seen after prosthetic arterial grafting and appears to contribute to the development and progression of anastomotic IH.
Sivamurthy
Nayan
N
Department of Surgery, Beth Israel Deaconess Medical Center, Harvard Institutes of Medicine, 4 Blackfan Circle, Boston, MA 02115, USA.
Stone
David H
DH
LoGerfo
Frank W
FW
Quist
William C
WC
D000714:Anastomosis, Surgical; D000818:Animals; D001807:Blood Vessel Prosthesis; D018797:Cell Cycle Proteins / Q000378:metabolism; D004268:DNA-Binding Proteins; D004285:Dogs; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; D006965:Hyperplasia / Q000473:pathology; D015151:Immunoblotting; D007150:Immunohistochemistry; D012333:RNA, Messenger / Q000235:genetics; D016160:Retinoblastoma Protein / Q000378:metabolism*; D013997:Time Factors; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic; D017539:Tunica Intima / Q000473:pathology
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; D012333:RNA, Messenger; D016160:Retinoblastoma Protein; D014157:Transcription Factors