Transfer of Wild-Type p53 Gene Effectively Inhibits Vascular Smooth Muscle Cell Proliferation In Vitro and In Vivo

From the Department of Pathology I (Y.Y., Y.N., K. Sueishi) and Surgery II (Y.Y., K.K., K. Sugimachi), Faculty of Medicine, Kyushu University 60, Fukuoka, Japan; the Molecular Hepatology Laboratory (S.T.), Massachusetts General Hospital Cancer Center, Harvard Medical School, Charlestown; and the Division of Cellular Genetics (Y.K.), Institute for Molecular and Cellular Biology, Osaka University, Suita, Japan.

Correspondence to Yoshikazu Yonemitsu, MD, PhD, Ion Transport Unit, National Heart and Lung Institute, Imperial College School of Medicine, Emmanuel Kaye Building, Manresa Road, London SW3 6LR, UK.

Abstract—Wild-type p53 (wt-p53), a key protein in cell cycle regulation, inactivates the G₁ cyclins through direct activation of p21^{Waf-1/Cip-1/Sdi-1}. Persistent vascular smooth muscle cell (VSMC) proliferation following vascular interventions hinders the benefits of these therapeutics. Using the hemagglutinating virus of Japan/liposome–mediated gene transfer method, we examined the inhibitory effect of overexpression of exogenous wt-p53 on VSMC proliferation in vitro and in vivo. We assessed the proliferative activity of human p53 cDNA–transduced bovine VSMCs by DNA synthesis assay, flow cytometry, and cell proliferation assay. p53 gene transfer reduced thymidine incorporation of VSMCs stimulated by platelet-derived growth factor-BB (P<.001). The p53-transduced VSMCs underwent synthetic phase depletion (mean, 8.02% versus 33.7% of control; P<.001) and transient G₂/M accumulation 2 days after gene transfection, and in almost all cells, G₁ arrest occurred (mean, 92.6% versus 79.3% of control; P<.001) 5 days later. The wt-p53 gene transfection also inhibited the VSMC proliferation (P<.001) with no detectable induction of apoptosis. Cell death of p53-transduced VSMCs was induced only by additional treatment with an apoptosis-stimulating reagent, doxorubicin. The verification of apoptosis was made by DNA ladder, flow cytometry, and electron microscopy. In vivo transfection of p53 cDNA inhibited neointimal formation after balloon injury in rabbit carotid arteries, without apoptotic stimuli (P<.01). Thus, overexpression of the p53 gene in the injured arterial wall inhibits the proliferation of VSMCs in vitro and in vivo. This novel concept, including not only exogenous but also endogenous p53 overexpression in the vessel wall, may be one approach worth exploring in the treatment of patients with restenosis occurring after vascular interventions.

Key Words: wild-type p53 • cell cycle arrest • hemagglutinating virus of Japan/liposome • smooth muscle • apoptosis

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