174. Post-Deployment Modifications of Stent with Endothelial Cells
Authors: Ilia Fishbein, Ivan S. Alferiev, Richard F. Adamo, David T. Guerrero, Yanqing Tang, Michael Chorny, Robert J. Levy
Journal: Molecular Therapy (2016)

Accelerated restoration of a functional endothelial layer in the injured arteries following stent angioplasty has been pursued over the past 20 years as an approach for preventing uncontrolled expansion of neointimal tissue and subsequent in-stent restenosis. To achieve enhanced re-growth of endothelium over the denuded arterial segments, strategies based on the pre-procedure seeding of stents with syngeneic/autologous endothelial cells (EC) and protocols involving stent surface modification to expedite capture and proliferation of circulating endothelial progenitor cells (EPC) have been explored. The main hurdle for the clinical translation of these "pro-healing" stents is the impediment in establishing an integral EC/EPC monolayer arrangement immediately after stent deployment, which is paramount for aborting platelet attachment to the exposed sub-endothelium and preventing inflammatory cell recruitment. The current work investigates a novel practical way to attain coverage of stent struts with syngeneic/autologous EC simultaneously with stent implantation. Stainless steel surfaces of tubing and stents were consecutively modified with 1% polyallylamine bisphosphonate, 4 mM sulfo NHS-LC-LC-biotin and 100 µg/ml avidin. Control samples were prepared omitting avidin attachment step. Rat aortic endothelial cells (RAEC) were surface modified in suspension with 1 mM sulfo NHS-LC-LC-biotin for 10 min, followed by pelleting and resuspension in PBS. Stainless steel tubing samples (avidin-coated or control; n=3 for both) were placed in succession inside 2 separate loops of polypropylene tubing that were mounted on a Chandler loop apparatus that models continuous blood flow. RAEC were resuspended in 20 ml of DMEM (2.5×105 EC/ml) and 10 ml of EC suspension was used to fill each loop. After 45 min of biotinylated RAEC recirculation through the avidin-modified and control steel samples (25 dynes/cm2, 37°C), the samples were removed, fixed in 4% PFA, inverted and stained with Hoechst 33258. The density of fluorescent nuclei associated with avidin-modified and control stainless steel surface was determined by fluorescence microscopy, demonstrating a 50-fold higher attachment density of biotinylated EC on avidin-modified surfaces. Firefly luciferase expressing RAEC (via lentiviral transduction) were surface modified with 1 mM sulfo NHS-LC-LC-biotin and intraluminally delivered (2×105 cells) into temporally isolated segments of rat carotid arteries immediately after deployment of PAB/sulfo NHS-LC-LC-biotin/avidin modified (N=4) or PAB only modified control stents (N=4). The animals underwent bioluminescent imaging 2 days post-stent deployment showing a 14.5-fold higher luminescent signal in animals treated with avidin-modified, than with control stents, demonstrating preferential attachment of biotinylated EC to the avidin-modified steel substrate and attesting to the viability of the delivered cells. Post-deployment binding of biotinylated EC to the surface of the avidin-modified stent can present a viable method of stent re-endothelialization and may have a therapeutic value in the prevention of in-stent restenosis and thrombosis.