Michelle Martin, Nicholas D. Andersen, MD, Thomas S. Monahan, MD, Junaid Y. Malek, MD, Christiane Ferran, MD, PhD, Frank W. LoGerfo, MD
Beth Israel Deaconess Medical Center- Department of Vascular Surgery Research, Boston, MA
PURPOSE OF STUDY
Intimal hyperplasia (IH) limits the patency of all cardiovascular bypass grafts. The myristoylated alanine-rich C kinase substrate (MARCKS), a key protein kinase C (PKC) substrate, is upregulated in canine models of IH. The role of MARCKS in IH is explored and characterized as a target for RNA interference-mediated gene therapy in human saphenous vein (HSV).
HSV was distended luminally at 120 mmHg for 10 minutes with 25 Ámol/L Cy5, control or MARCKS small interfering RNA (siRNA). Transfected vein segments were cultured in media with 100 Ámol/L bromodeoxyuridine (BrdU). Confocal microscopy was used to confirm siRNA delivery 24 hours after transfection with Cy5-siRNA. MARCKS knockdown was assessed in whole vein homogenates by qRT-PCR and Western blot. Cultured vein segments were stained with an anti-BrdU antibody to quantify proliferating medial and neointimal nuclei. Neointimal formation was quantified using Verhoeff-van Gieson (VVG) staining to delineate the internal elastic lamina.
SUMMARY OF RESULTS:
Confocal micrographs demonstrated global siRNA delivery after transfection with Cy5-siRNA. Transfection with MARCKS siRNA reduced MARCKS mRNA levels by 36% (P<0.05) at day 2 and MARCKS protein levels by 44% (P<0.05) at day 5. MARCKS silencing resulted in a 60% reduction in proliferating vascular smooth muscle cell (VSMC) nuclei (P<0.05) and a 41% reduction in neointimal formation at day 5 (P<0.05).
MARCKS silencing by siRNA reduced VSMC proliferation and neointimal formation in cultured HSV. This investigation supports a role for MARCKS and the PKC pathway in the pathogenesis of IH and identifies a promising new target and therapeutic modality for the prevention of IH.