P53 gene transfer to the injured rat carotid artery promotes apoptosis

Abstract

In a previous study we have demonstrated significant reduction of intimal hyperplasia after adenovirus-mediated gene transfer of p53 protein to the injured rat carotid artery. The purpose of this study was to elucidate whether apoptosis is one of the mechanisms responsible for this reduction. Apoptosis, a physiologic cell death process that stabilizes cell numbers in tissues, can be independently induced by p53. In vivo gene transfer was used in isolated segments of balloon-injured rat carotid arteries. Genetically modified adenovirus encoding for wild-type p53 protein (AdWTp53) was applied at 8 x 10(10) plaque-forming units/mL. Control rats received either adenovirus null at the same concentration or balloon injury alone. Arteries were harvested at 24 and 48 hours after the procedure. Apoptosis was detected in tissue sections by in situ terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assay. Specimens were graded either negative or positive less than 10%, 10% to 20%, 20% to 30%, or greater than 30% according to the number of apoptotic cells in the medial or intimal layer per high power field. Specimens were also subjected to DNA agarose gel electrophoresis and transmission electron microscopy. With the TUNEL assay no apoptosis was visualized at 24 and 48 hours in the controls (n = 5 in each group), whereas in the AdWTp53 groups (n = 5 in each) all specimens presented apoptosis (P < .05, AdWTp53 vs controls). The average grade of apoptotic cells detected in the medial layer in the AdWTp53 groups was less than 10% to 20% at 24 hours and 20% to 30% at 48 hours. The DNA agarose gel electrophoresis failed to detect a DNA laddering pattern, characteristic of apoptosis. Electron microscopy revealed morphologic changes typical of apoptosis in the treated group, whereas specimens from control group did not reveal any apoptotic features. At 48 hours after balloon injury alone, no apoptosis was observed in the vessel wall. However, when p53 gene was transferred, apoptosis was visualized in all specimens with greater intensity at 48 hours after injury. Promotion of apoptosis may play a key role in the mechanism by which p53 gene decreases intimal hyperplasia.