The fate of an endothelium layer after preconditioning.
BACKGROUND
A strategy in minimizing thrombotic events of vascular constructs is to seed the luminal surface with autologous endothelial cells (ECs). The task of seeding ECs can be achieved via bioreactors, which induce mechanical forces (shear stress, strain, pressure) onto the ECs. Although bioreactors can achieve a confluent layer of ECs in vitro, their acute response to blood remains unclear. Moreover, the necessary mechanical conditions that will increase EC adhesion and function remain unclear. We hypothesize that preconditioning seeded endothelium under physiological flow will enhance their retention and function.


OBJECTIVE
To determine the role of varying preconditioning protocols on seeded ECs in vitro and in vivo.


METHODS
Scaffolds derived from decelluarized arteries seeded with autologous ECs were preconditioned for 9 days. Three specific protocols, low steady shear stress (SS), high SS, and cyclic SS were investigated. After preconditioning, the seeded grafts were exposed to 15 minutes of blood via an ex vivo arteriovenous shunt model or alternately an in vivo arteriovenous bypass graft model.


RESULTS
The shunt model demonstrated ECs remained intact for all conditions. In the arteriovenous bypass model, only the cyclic preconditioned grafts remained intact, maintained morphology, and resisted the attachment of circulating blood elements such as platelets, red blood cells, and leukocytes. Western blotting analysis demonstrated an increase in the protein expression of eNOS and prostaglandin I synthase for the cyclic high shear stress-conditioned cells relative to cells conditioned with high shear stress alone.


CONCLUSIONS
Cyclic preconditioning has been shown here to increase the ECs ability to resist blood flow-induced shear stress and the attachment of circulating blood elements, key attributes in minimizing thrombotic events. These studies may ultimately establish protocols for the formation of a more durable endothelial monolayer that may be useful in the context of small vessel arterial reconstruction.
Yazdani
Saami K
SK
Wake Forest Institute for Regenerative Medicine, Wake Forest University School of Medicine, Winson-Salem, NC, USA. syazdani@cvpath.org
Tillman
Bryan W
BW
Berry
Joel L
JL
Soker
Shay
S
Geary
Randolph L
RL
D000818:Animals; D001166:Arteriovenous Shunt, Surgical; D019149:Bioreactors; D001807:Blood Vessel Prosthesis; D019917:Blood Vessel Prosthesis Implantation; D015153:Blotting, Western; D002339:Carotid Arteries / Q000166:cytology* / Q000637:transplantation / Q000648:ultrastructure; D002448:Cell Adhesion; D018929:Cell Culture Techniques; D049109:Cell Proliferation; D002478:Cells, Cultured; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D042783:Endothelial Cells / Q000378:metabolism / Q000502:physiology* / Q000637:transplantation / Q000648:ultrastructure; D005260:Female; D019746:Intramolecular Oxidoreductases / Q000378:metabolism; D007601:Jugular Veins / Q000601:surgery; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism; D011474:Prosthesis Design; D011673:Pulsatile Flow; D012756:Sheep; D033581:Stem Cell Transplantation; D013234:Stem Cells / Q000378:metabolism / Q000502:physiology* / Q000648:ultrastructure; D013314:Stress, Mechanical; D013552:Swine; D013997:Time Factors; D023822:Tissue Engineering / Q000295:instrumentation / Q000379:methods; D054457:Tissue Scaffolds
D003577:Cytochrome P-450 Enzyme System; D052250:Nitric Oxide Synthase Type III; D019746:Intramolecular Oxidoreductases; C022461:prostacyclin synthetase