J. Johnson1, T. C. Bodewes1, S. Muralidharan1, M. Auster1, M. Contreras1, F. W. LoGerfo1, L. Pradhan-Nabzdyk1 1Beth Israel Deaconess Medical Center,Vascular Surgery, Harvard Medical School,Boston, MA, USA
Introduction: Long-term patency rates following vascular interventions remain poor due to the formation of intimal hyperplasia (IH). Currently, no preventive treatment is available to attenuate long-term failure. This study evaluates the ability to inhibit expression of Thrombospondin-2 (TSP-2) and Myristoylated Alanine-Rich C-Kinase Substrate (MARCKS), the IH-associated proteins identified by our group, using commercially available siRNAs in a rat model of IH.
Methods: One of the carotid arteries of Wistar rats were denuded using a 2F Fogarty balloon catheter. Following denudation, the artery was transfected intraluminally for 15 minutes with one of the following treatments: saline, transfection reagent Polyethylenimine (PEI), or PEI + siRNA(s) (non-targeting, siTSP-2, siMARCKS, or siTSP-2 + siMARCKS) (n=6). Denuded and non-denuded contralateral arteries were harvested at 24 hours or 21 days for analysis. TSP-2 and MARCKS gene expression were determined using q-RT-PCR. TSP-2 and MARCKS protein expression and cell proliferation were analyzed using immunohistochemistry. mRNA expression is presented as fold change over the control (either non-denuded untreated or denuded saline treated) . Protein expression is presented as an arbitrary score (1 to 5). Cell proliferation is presented as no. of Ki+ cells/mm2.
Results: Data are presented in Table 1. Statistical significance was measured by comparing denuded saline group vs. non-denuded untreated group to validate the effect of arterial injury on target gene expression and cellular proliferation (represented by †). Data in the denuded treatment groups were compared to denuded saline group to measure the effect of the treatment on denudation-related target gene expression and cellular proliferation (represented by *). Data are expressed as Mean±SD. * = P < 0.05.
Conclusion: The carotid artery balloon injury in rats up-regulated the expression of TSP-2 and MARCKS proteins, previously identified in large animal models of IH and respective siRNAs reduced their expression. This model can serve to investigate siRNA-based therapies targeting proteins for IH prevention. Preliminary results suggest that combination siRNA treatment targeting IH-associated proteins could be a feasible approach to curtail IH.
