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Research Paper

Enhancing Endothelial Cell Retention on ePTFE Constructs by siRNA-Mediated SHP-1 Gene Silencing

[+] Author and Article Information
Brandon J. Tefft

Department of Biomedical Engineering, Northwestern University, 2145 Sheridan Road, Tech E310, Evanston, IL 60208btefft@northwestern.edu

Adrian M. Kopacz

Department of Mechanical Engineering, Northwestern University, 2145 Sheridan Road, Tech B224, Evanston, IL 60208a-kopacz@northwestern.edu

Wing Kam Liu

Department of Mechanical Engineering, Northwestern University, 2145 Sheridan Road, Tech B224, Evanston, IL 60208w-liu@northwestern.edu

Shu Q. Liu

Department of Biomedical Engineering, Northwestern University, 2145 Sheridan Road, Tech E310, Evanston, IL 60208sliu@northwestern.edu

J. Nanotechnol. Eng. Med 2(1), 011007 (Feb 04, 2011) (6 pages) doi:10.1115/1.4003273 History: Received September 29, 2010; Revised December 12, 2010; Published February 04, 2011; Online February 04, 2011

Polymeric vascular grafts hold great promise for vascular reconstruction, but the lack of endothelial cells renders these grafts susceptible to intimal hyperplasia and restenosis, precluding widespread clinical applications. The purpose of this study is to establish a stable endothelium on expanded polytetrafluoroethylene (ePTFE) membrane by small interfering RNA (siRNA)-induced suppression of the cell adhesion inhibitor SH2 domain-containing protein tyrosine phosphatase-1 (SHP-1). Human umbilical vein endothelial cells (HUVECs) were treated with scrambled siRNA as a control or SHP-1 specific siRNA. Treated cells were seeded onto fibronectin-coated ePTFE scaffolds and exposed to a physiological range of pulsatile fluid shear stresses for 1 h in a variable-width parallel plate flow chamber. Retention of cells was measured and compared between various shear stress levels and between groups treated with scrambled siRNA and SHP-1 specific siRNA. HUVECs seeded on ePTFE membrane exhibited shear stress-dependent retention. Exposure to physiological shear stress (10dyn/cm2) induced a reduction in the retention of scrambled siRNA treated cells from 100% to 85% at 1 h. Increased shear stress (20dyn/cm2) further reduced retention of scrambled siRNA treated cells to 55% at 1 h. SHP-1 knockdown mediated by siRNA enhanced endothelial cell retention from approximately 60% to 85% after 1 h of exposure to average shear stresses in the range of 1530dyn/cm2. This study demonstrates that siRNA-mediated gene silencing may be an effective strategy for improving the retention of endothelial cells within vascular grafts.

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Copyright © 2011 by American Society of Mechanical Engineers
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Figures

Grahic Jump Location
Figure 1

A schematic demonstrating siRNA transfection of HUVECs, seeding of transfected cells onto an ePTFE construct within a parallel plate flow chamber, and exposure of the cell-seeded constructs to fluid shear stress. The arrow beside the flow chamber represents flow direction.

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Figure 2

Endothelial cell retention on fibronectin-coated ePTFE after exposure to 15 dyn/cm2 of average fluid shear stress for 1 h. (A) Control cells treated with scrambled siRNA. (B) cells treated with SHP-1 specific siRNA. White represents cell nuclei. Scale bars are 150 μm.

Grahic Jump Location
Figure 3

Endothelial cell retention on fibronectin-coated ePTFE after exposure to a range of fluid shear stresses for 1 h. ∗ indicates p<0.05. n=8 for each data point. Data shown as mean±SEM.

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