Sarah T Hsiao1,2, Tim Spencer3, Luke Boldock4,2, Svenja Dannewitz Prosseda1,2, Ioannis Xanthis1,2, Francesco J Tovar-Lopez5, Heleen M M Van Beusekom6, Ramzi Y Khamis7, Nicolas Foin8, Neil Bowden1,2, Adil Hussain1,2, Alex Rothman1,2, Victoria Ridger1,2, Ian Halliday3, Cecile Perrault4,2, Julian Gunn1,2, Paul C Evans9,2,10. 1. Department of Infection, Immunity and Cardiovascular Disease, University of Sheffield, Sheffield S10 2RX, UK. 2. INSIGNEO Institute of In Silico Medicine, University of Sheffield, Sheffield S10 2RX, UK. 3. Materials and Engineering Research Institute, Sheffield Hallam University, Sheffield S1 4RF, UK. 4. Department of Mechanical Engineering, University of Sheffield, Sheffield S10 2RX, UK. 5. School of Electrical and Computer Engineering, RMIT University, Melbourne VIC 3001, Australia. 6. Department of Cardiology, ERASMUS MC, Rotterdam 3015 CE, the Netherlands. 7. Faculty of Medicine, National Heart and Lung Institute, Imperial College London WI2 0HS, UK. 8. National Heart Centre, Singapore 169609. 9. Department of Infection, Immunity and Cardiovascular Disease, University of Sheffield, Sheffield S10 2RX, UK paul.evans@sheffield.ac.uk. 10. Bateson Centre, University of Sheffield, Sheffield S10 2RX, UK.
Abstract
AIMS: Stent deployment causes endothelial cells (EC) denudation, which promotes in-stent restenosis and thrombosis. Thus endothelial regrowth in stented arteries is an important therapeutic goal. Stent struts modify local hemodynamics, however the effects of flow perturbation on EC injury and repair are incompletely understood. By studying the effects of stent struts on flow and EC migration, we identified an intervention that promotes endothelial repair in stented arteries. METHODS AND RESULTS: In vitro and in vivo models were developed to monitor endothelialization under flow and the influence of stent struts. A 2D parallel-plate flow chamber with 100 μm ridges arranged perpendicular to the flow was used. Live cell imaging coupled to computational fluid dynamic simulations revealed that EC migrate in the direction of flow upstream from the ridges but subsequently accumulate downstream from ridges at sites of bidirectional flow. The mechanism of EC trapping by bidirectional flow involved reduced migratory polarity associated with altered actin dynamics. Inhibition of Rho-associated protein kinase (ROCK) enhanced endothelialization of ridged surfaces by promoting migratory polarity under bidirectional flow (P < 0.01). To more closely mimic the in vivo situation, we cultured EC on the inner surface of polydimethylsiloxane tubing containing Coroflex Blue stents (65 μm struts) and monitored migration. ROCK inhibition significantly enhanced EC accumulation downstream from struts under flow (P < 0.05). We investigated the effects of ROCK inhibition on re-endothelialization in vivo using a porcine model of EC denudation and stent placement. En face staining and confocal microscopy revealed that inhibition of ROCK using fasudil (30 mg/day via osmotic minipump) significantly increased re-endothelialization of stented carotid arteries (P < 0.05). CONCLUSIONS: Stent struts delay endothelial repair by generating localized bidirectional flow which traps migrating EC. ROCK inhibitors accelerate endothelial repair of stented arteries by enhancing EC polarity and migration through regions of bidirectional flow.
AIMS: Stent deployment causes endothelial cells (EC) denudation, which promotes in-stent restenosis and thrombosis. Thus endothelial regrowth in stented arteries is an important therapeutic goal. Stent struts modify local hemodynamics, however the effects of flow perturbation on EC injury and repair are incompletely understood. By studying the effects of stent struts on flow and EC migration, we identified an intervention that promotes endothelial repair in stented arteries. METHODS AND RESULTS: In vitro and in vivo models were developed to monitor endothelialization under flow and the influence of stent struts. A 2D parallel-plate flow chamber with 100 μm ridges arranged perpendicular to the flow was used. Live cell imaging coupled to computational fluid dynamic simulations revealed that EC migrate in the direction of flow upstream from the ridges but subsequently accumulate downstream from ridges at sites of bidirectional flow. The mechanism of EC trapping by bidirectional flow involved reduced migratory polarity associated with altered actin dynamics. Inhibition of Rho-associated protein kinase (ROCK) enhanced endothelialization of ridged surfaces by promoting migratory polarity under bidirectional flow (P < 0.01). To more closely mimic the in vivo situation, we cultured EC on the inner surface of polydimethylsiloxane tubing containing Coroflex Blue stents (65 μm struts) and monitored migration. ROCK inhibition significantly enhanced EC accumulation downstream from struts under flow (P < 0.05). We investigated the effects of ROCK inhibition on re-endothelialization in vivo using a porcine model of EC denudation and stent placement. En face staining and confocal microscopy revealed that inhibition of ROCK using fasudil (30 mg/day via osmotic minipump) significantly increased re-endothelialization of stented carotid arteries (P < 0.05). CONCLUSIONS: Stent struts delay endothelial repair by generating localized bidirectional flow which traps migrating EC. ROCK inhibitors accelerate endothelial repair of stented arteries by enhancing EC polarity and migration through regions of bidirectional flow.
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