Controlled release of small interfering RNA targeting midkine attenuates intimal hyperplasia in vein grafts

ObjectiveIntimal hyperplasia is a major obstacle to patency after vein grafting. Despite of a diverse array of trials to prevent it, a satisfactory therapeutic strategy for clinical use has not been established. However, sufficient inhibition of early stages of intimal hyperplasia may prevent this long-term progressive disease. Midkine (MK) is a heparin-binding growth factor that was originally discovered as the product of a retinoic acid-responsive gene. We previously demonstrated that MK-deficient mice exhibit a striking reduction of neointima formation in a restenosis model, which is reversed on systemic MK administration. In this study, we evaluated a strategy of using small interfering RNA (siRNA) targeting MK as a therapy for vein graft failure.MethodsWe first made a highly effective siRNA to rabbit MK. Jugular vein-to-carotid artery interposition vein grafts, which are applied to a low flow condition, were made in Japanese white rabbits. Small interfering RNA mixed with atelocollagen was administrated to the external wall of grafted veins. Cy3-conjugated stabilized siRNA was used to confirm its stability and successful transfer into the vein graft wall. Neointimal hyperplasia was evaluated 4 weeks after the operation. The proliferation index and leukocyte infiltration were determined.ResultsMK expression was induced and reached the maximum level 7 days after operation. Fluorescence of Cy3-labeled siRNA could be detected in the graft wall even 7 days after operation. Knockdown of the gradually increasing expression was achieved by perivascular application of siRNA using atelocollagen. The intima-media ratio and the intima thickness at 28 days after grafting were both reduced >90% by this treatment compared with controls. This phenomenon was preceded by significant reductions of inflammatory cell recruitment to the vessel walls and subsequent cell proliferation in MK siRNA-treated grafts.ConclusionsThese results suggest that midkine is a candidate molecular target for preventing vein graft failure. Furthermore, for clinical applications of siRNA, a single intraoperative atelocollagen-based nonviral delivery method could be a reliable approach to achieve maximal function of siRNA in vivo. This strategy may be a useful and practical form of gene therapy against human vein graft failure.

Clinical RelevanceIntimal hyperplasia is a major obstacle to patency after vein grafting. Despite a diverse array of trials to prevent it, a satisfactory therapeutic strategy for clinical use has not been established. However, sufficient inhibition of early stages of intimal hyperplasia may prevent this long-term progressive disease. Here, we report almost complete suppression of intimal hyperplasia. Knockdown of growth factor midkine expression was achieved by the perivascular application of small interfering RNA (siRNA) using atelocollagen. Our data indicate that the combination of siRNA and its controlled release could be a therapeutic strategy for vein graft failure and that midkine is a potent molecular target for the prevention of intimal hyperplasia.