Thrombotic Responses of Endothelial Outgrowth Cells to Protein-Coated Surfaces

Abstract

There is significant clinical need for viable small-diameter vascular grafts. While there are many graft biomaterials in development, few have been clinically successful. Evaluation of grafts with a clinically relevant model is needed to drive development. This work examined extracellular matrix coatings on the thrombotic phenotype of endothelial outgrowth cells (EOCs). EOCs were tested on flat plates and tubular grafts. Flat plate studies examined collagen I, collagen IV, fibronectin and α-elastin coatings. EOCs attached or proliferated more readily on collagen I and fibronectin surfaces as determined by total DNA. The production of activated protein C (APC) by EOCs was also dependent on the surface coating, with collagen I and fibronectin displaying a higher activity than both collagen IV and α-elastin on flat plate studies. Based on these results, only collagen I and fibronectin coatings were tested on expanded polytetrafluoroethylene (ePTFE) in the ex vivo model. Tubular samples showed significantly greater tissue factor pathway inhibitor gene expression on collagen I than on fibronectin. Platelet adhesion was not significantly different, but EOCs on collagen I produced significantly lower APC than on fibronectin, suggesting that differences exist between the flat plate and tubular cultures. Overall, while the hemostatic phenotype of EOCs displayed some differences, cell responses were largely independent of the matrix coating. EOCs adhered strongly to both fibronectin- and collagen-I-coated ePTFE grafts under ex vivo (100 ml/min) flow conditions suggesting the usefulness of this clinically relevant cell source, testing modality, and shunt model for future work examining biomaterials and cell conditioning before implantation.

In vitro and ex vivo hemocompatibility of off-the-shelf modified poly(vinyl alcohol) vascular grafts

“…ePTFE vascular grafts (W.L. Gore) were constructed as described previously to ensure a uniform transition between the graft and the silicone tubing [27] and used as a clinical, negative control. Similarly, the ePTFE grafts were coated with 1 mg ml −1 equine collagen type I (Chrono-log Corp.) for use as a positive control.…”

“…Chronic baboon femoral arteriovenous shunts were performed as described previously [27]. In brief, autologous platelets were labeled with 111-Indium prior to device testing and reinfused into the animal.…”

Endothelial Outgrowth Cells: Function and Performance in Vascular Grafts

“…58 Intimal hyperplasia after vascular injury can also be reduced by activated Protein C. 62,63 Activated Protein C acts to inhibit inflammation-induced EC apoptosis and permeability, thereby maintaining functional EC regulation of underlying smooth muscle cells. 64 Similar to ECs, EOCs can activate Protein C. 46,65 To enhance Protein C activation, EOCs were engineered to overexpress thrombomodulin via adenoviral transfection. Although in vitro Protein C activation was increased, 47 thrombomodulin overexpression by EOCs seeded onto an ePTFE graft did not significantly reduce intimal hyperplasia or thrombus formation compared with non-transfected EOCs in a rat model.…”

“…36 Studies of EOCs seeded onto ePTFE coated with either collagen I or fibronectin indicated that EOCs had a more robust adhesion and proliferation potential on either of these proteins than on aelastin or collagen IV. 65 With either collagen I or fibronectin coatings, EOCs formed a confluent monolayer and were able to remain adherent at supraphysiological shear stresses. 65 The mechanical properties, particularly the elasticity, of polyurethanes have stimulated investigation into their utility as vascular graft materials.…”

“…65 With either collagen I or fibronectin coatings, EOCs formed a confluent monolayer and were able to remain adherent at supraphysiological shear stresses. 65 The mechanical properties, particularly the elasticity, of polyurethanes have stimulated investigation into their utility as vascular graft materials. The harmful degradation products of polyurethanes have eliminated their use in long-term implants.…”