Capture of endothelial cells under flow using immobilized vascular endothelial growth factor

Abstract

We demonstrate the ability of immobilized vascular endothelial growth factor (VEGF) to capture endothelial cells (EC) with high specificity under fluid flow. To this end, we engineered a surface consisting of heparin bound to poly-L-lysine to permit immobilization of VEGF through the C-terminal heparin-binding domain. The immobilized growth factor retained its biological activity as shown by proliferation of EC and prolonged activation of KDR signaling. Using a microfluidic device we assessed the ability to capture EC under a range of shear stresses from low (0.5 dyne/cm2) to physiological (15 dyne/cm2). Capture was significant for all shear stresses tested. Immobilized VEGF was highly selective for EC as evidenced by significant capture of human umbilical vein and ovine pulmonary artery EC but no capture of human dermal fibroblasts, human hair follicle derived mesenchymal stem cells, or mouse fibroblasts. Further, VEGF could capture EC from mixtures with non-EC under low and high shear conditions as well as from complex fluids like whole human blood under high shear. Our findings may have far reaching implications, as they suggest that VEGF could be used to promote endothelialization of vascular grafts or neovascularization of implanted tissues by rare but continuously circulating EC.

Successful endothelialization and remodeling of a cell-free small-diameter arterial graft in a large animal model

“…Bioactive, recombinant VEGF isoform 165 was produced and purified as previously described[33]. Briefly, bacteria strain Escherichia coli BL21-DE3-pLysis containing pGEX-VEGF, encoding for a thrombin cleavable glutathione-s-tranferase (GST) tag followed by the VEGF-165 gene, was induced with 1mM isopropyl β-D-1-thiogalactopyranoside for protein production for 4–6hr at 37°C and 300rpm.…”

“…The biological activity of recombinant VEGF was assessed using a standard cell proliferation assay as previously described[33]. Briefly, human umbilical vein endothelial cells (HUVECs) were seeded onto a 96-well plate with varying concentrations of recombinant VEGF or commercial (control) VEGF (Cell Signaling).…”

Adding Functions to Biomaterial Surfaces through Protein Incorporation

“…Prolonged, low level release or delivery of VEGF, similar to BMP-2, leads to improved bone formation and the two growth factors have been successfully used in combination to achieve improved, sometimes synergistic effects on osteogenesis. [197] In surface endothelialization applications, tethered VEGF captures VEGF receptor-bearing endothelial cells to improve endothelialization of vascular grafts, [198] albeit with a reported lower efficiency than CD34-binding antibodies. [199] Collagen occurs in elongated fibrils and is the main structural protein of connective tissues.…”

Cell-free vascular grafts: Recent developments and clinical potential

“…These grafts exhibited high patency rates in the high flow environment of ovine carotid arteries and produced fully endothelialized, functional arteries as soon as one month post-implantation 50 . Unlike antibodies which have also been applied to capture EPCs from the host bloodstream 51,52 , VEGF enhances the survival, proliferation and cell spreading of captured progenitors 53–56 , making it a more attractive option for functionalizing TEBVs.…”

“…Interestingly, two in vivo studies, reported that immobilized heparin was insufficient in maintaining long term patency (>1 month) and required the presence of VEGF, which was bound via the heparin binding domain 50,60 . Our laboratory recently demonstrated the ability of heparin-bound, surface immobilized VEGF to capture endothelial cells (EC) under flow in vitro 56 . Subsequently, we employed this strategy to immobilize VEGF on the surface of a natural biomaterial, namely Small Intestinal Submucosa (SIS) to engineer a cell free vascular graft.…”