Abstract 5891: Inhibition of MDM2 by Nutlin-3 Stabilizes p53 and Attenuates Neointimal Hyperplasia via Suppression of Vascular Proliferation and Inflammation
Introduction: The tumor suppressor p53 plays a protective role in vascular proliferative diseases. p53 expression is negatively regulated by MDM2. Antagonizing MDM2 by Nutlin-3 stabilizes p53 and attenuates cell proliferation. However, the effect of Nutlin-3 on vascular proliferative diseases has not been investigated. We examined the effect of Nutlin-3 on vascular smooth muscle cells (VSMCs) and neointimal hyperplasia.
Methods and Results: Western blot analysis showed that Nutlin-3 upregulated p53 and its downstream p21 in VSMCs. Nutlin-3 dose-dependently inhibited platelet-derived growth factor (PDGF)-stimulated incorporation of [3H]-thymidine (p<0.001) in VSMCs. Flow cytometric analysis revealed that Nutlin-3 induced cell cycle arrest (85% of reduction in the percentage of S-phase vs PDGF, p<0.05) but did not affect apoptosis in VSMCs. Norhtern blot analysis showed that Nutlin-3 suppressed PDGF-induced expression of interleukin-6, monocyte chemoattractant protein-1, and vascular cell adhesion molecule-1, and gel mobility shift assay revealed that Nutlin-3 inhibited NF-κB activation. These effects depend on p53 because Nutlin-3 failed to show any effects in p53-deficient VSMCs. To examine the effect of Nutlin-3 in vivo, we performed vascular injury by insertion of wire into femoral arteries in C57BL/6J mice (10-week-old), and delivered Nutlin-3 (5 mg/kg/day) by osmotic minipumps (Control: n=9, Nutlin-3: n=7). Nutlin-3 attenuated neointimal hyperplasia at 28 days after vascular injury (intima/media ratio 1.84±0.31 vs control 3.00±0.34, p<0.05). Vascular cell proliferation analyzed by BrdU incorporation was decreased at 14 but not 28 days after injury in Nutlin-3-administered mice. Increased apoptosis was not observed at both 14 and 28 days after injury by TUNEL assay.
Conclusions: Nutlin-3 inhibits VSMC proliferation via cell cycle arrest and inflammatory cytokine expression via NF-κB suppression, and also attenuated neointimal hyperplasia after vascular injury without enhanced apoptosis. Nutlin-3 is capable of inhibiting inflammation and proliferation in VSMCs without inducing apoptosis which may destabilize lesion, and thereby may have a unique therapeutic potential against vascular proliferative diseases.