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MARCKS Silencing Inhibits Vascular Smooth Muscle Cell Proliferation and Neointimal Formation In Human Saphenous Vein

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).

METHODS USED:
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).

CONCLUSIONS:
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.

 

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