Abstract
Several lifesaving procedures, including hemodialysis, femoropopliteal bypass, and renal artery replacement, require functional vascular access in the form of vascular grafts. While autologous grafts are preferred, many surgeries will use expanded polytetrafluoroethylene (ePTFE), a medical-grade polymer. These synthetic grafts are sufficient replacements when autologous options are unavailable. The grafts often lack regenerative properties congruent to those found in native blood vessels due to an absent endothelial lining. Without regenerative properties, these grafts will experience high rates of thrombosis, causing graft occlusion resulting in a decrease or complete loss of graft patency. Consequently, additional procedures are required for graft replacement and reinterventions that increase medical costs and predispose patients to higher infection rates. Our research focused on modifying the commercially available ePTFE graft surface with a novel peptide ligand to promote in situ endothelialization for a long-lasting vascular graft.
Previous research in Dr. Wang’s lab at UC Davis has identified a peptide ligand, LXW7, that can be chemically synthesized to bind to the surface of the ePTFE graft. Research has confirmed that this ligand holds high affinity binding to endothelial progenitor cells, weak binding to platelets, and no binding to inflammatory cells, such as monocytes. We have found that circulating endothelial progenitor cells (EPCs) and endothelial cells (ECs) will attach to this graft and can promote cell division and migration, allowing them to cover the luminal surface of the graft, creating a “living” self-renewable endothelium. Creating a layer of living functional endothelium may reduce thrombosis, infection, and stenosis, and prolong graft patency.
Our proposed project aimed to confirm the effectiveness of the LXW7-modified ePTFE graft in capturing EPCs in three different models, in vitro, ex vivo, and in vivo. We originally modified bare-ePTFE grafts with LXW7. However, we transitioned to modifying heparin-bonded ePTFE grafts with LXW7 for technical and clinical feasibility reasons.
The in vitro static cell seeding assay used green fluorescent protein (GFP) labeled EPCs seeded on LXW7-modified heparin-bonded ePTFE grafts. A fluorescent microscope examined cell attachment on the luminal surface and showed no visual difference between LXW7 modified and unmodified grafts. The LXW7-modified heparin-bonded ePTFE graft was then evaluated using the normothermic machine perfusion (NMP) model to confirm EPC binding under physiological circulating conditions. NMP systems are typically used for organ transplants as they mimic physiologically and clinically relevant conditions. Therefore, this study established a novel graft attachment model. After the LXW7 modified heparin–bonded ePTFE graft was attached to the machine for two hours, results showed only EPCs attached to the grafts, confirming cell attachment under clinically relevant conditions. Lastly, a rat carotid artery interposition graft model was used to determine endothelialization and graft patency in a small translational in vivo model. This model investigated LXW7 bare-ePTFE grafts only. After explantation, gross images of the medial cross-section of the graft were taken and ImageJ was used to measure the cross-sectional area of occlusion. Every rat at the one-week time points displayed patent grafts, while the two-week and six-week grafts displayed increasing occlusion percentages. LXW7 proved to hold strong binding affinity to circulating EPCs, via cell attachment to LXW7-modified grafts, but further in vivo evaluations need to be performed to evaluate in situ endothelialization on the graft’s luminal surface and graft patency effects.