Evaluation of late outgrowth endothelial progenitor cell and umbilical vein endothelial cell responses to thromboresistant collagen‐mimetic hydrogels

Abstract

Bioactive coatings which support the adhesion of late-outgrowth peripheral blood endothelial progenitor cells (EOCs) are actively being investigated as a means to promote rapid endothelialization of "off-the-shelf," small-caliber arterial graft prostheses following implantation. In the present work, we evaluated the behavior of EOCs on thromboresistant graft coatings based on the collagen-mimetic protein Scl2-2 and poly(ethylene glycol) (PEG) diacrylate. Specifically, the attachment, proliferation, migration, and phenotype of EOCs on PEG-Scl2-2 hydrogels were evaluated as a function of Scl2-2 concentration (4, 8, and 12 mg/mL) relative to human umbilical vein endothelial cells (HUVECs). Results demonstrate the ability of each PEG-Scl2-2 hydrogel formulation to support EOC and HUVEC adhesion, proliferation, and spreading. However, only the 8 and 12 mg/mL PEG-Scl2-2 hydrogels were able to support stable EOC and HUVEC confluence. These PEG-Scl2-2 formulations were, therefore, selected for evaluation of their impact on EOC and HUVEC phenotype relative to PEG-collagen hydrogels. Cumulatively, both gene and protein level data indicated that 8 mg/mL PEG-Scl2-2 hydrogels supported similar or improved levels of EOC maturation relative to PEG-collagen controls based on evaluation of CD34, VEGFR2, PECAM-1, and VE-Cadherin. The 8 mg/mL PEG-Scl2-2 hydrogels also appeared to support similar or improved levels of EOC homeostatic marker expression relative to PEG-collagen hydrogels based on von Willebrand factor, collagen IV, NOS3, thrombomodulin, and E-selectin assessment. Combined, the present results indicate that PEG-Scl2-2 hydrogels warrant further investigation as "off-the-shelf" graft coatings. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1712-1724, 2017.

Collagen-Mimetic Proteins with Tunable Integrin Binding Sites for Vascular Graft Coatings

“…To generate DC2-containing hydrogel coatings, acrylate-derivatized proteins were combined with PEG-diacrylate (3.4 kDa; Laysan Bio) in PBS supplemented with 20 mM acetic acid to achieve final concentration of 8 mg/mL protein and 100 mg/mL PEG, respectively. This protein concentration was selected based on our previous data showing that PEG-DC2-1X coatings containing 8 mg/mL DC2-1X were able to support EOC attachment, migration, and confluence without the protein precipitation/loss occasionally observed at higher DC2 concentrations . Hydrogel precursor solutions were then cross-linked in 0.5 mm thick rectangular glass molds in the presence of 1 mg/mL Irgacure 2959 (Sigma) by 6 min exposure to longwave UV light (∼6 mW/cm 2 , 365 nm).…”

“…The EOCs (“late outgrowth” EPCs) utilized in the present study were previously isolated from blood collected from a 60 year old female CAD patient through a protocol approved by the Duke University Institutional Review Board. ,, The isolated EOCs were positive for cell surface markers CD31 and CD105 and negative for CD133, CD14, and CD45. These and other CAD EOCs have been shown to have similar proliferative capacity, surface marker expression, and response to physiological shear as EOCs derived from healthy patients .…”

“…Previous in vitro studies have shown that DC2-1X maintains the triple helical structure and low platelet aggregation of Scl2-1 . Furthermore, DC2-1X coatings – DC2-1X conjugated within a poly­(ethylene glycol) diacrylate (PEG) hydrogel base–have been shown to enable EC and EOC attachment, migration, and differentiation under static conditions. ,,− Nonetheless, even coatings containing the highest DC2-1X concentrations that could be stably incorporated were associated with reduced EC/EOC attachment in comparison to native collagen. − Moreover, the retention, migration, and differentiation of adherent EOCs on DC2-1X coatings has not been assessed in a physiologically relevant manner in which cells are subjected to shear flow.…”

“…The objectives of the present work were therefore: (1) to modify DC2-1X toward improving the levels of initial EOC attachment supported by the DC2 coatings and to demonstrate this improvement in a manner allowing comparison with our previous work (24 h static culture) and (2) to evaluate the capacity of these DC2 protein variants to support the retention, migration, and differentiation of adherent EOCs under physiologically relevant shear stress conditions in vitro. Since cell attachment is a function of both ligand concentration − and ligand spacing, , both the number of IBS (ligand concentration) and the distance between IBS (ligand spacing) were modified utilizing a series of single point mutations.…”

General Study and Gene Expression Profiling of Endotheliocytes Cultivated on Electrospun Materials

“…To study the endothelization of 3D matrices, several types of cells can be used, including primary endothelial cells obtained from arterial endothelium (human coronary artery endothelial cells, human aortic endothelial cells, human pulmonary artery endothelial cells), microvascular endothelial cells (human dermal microvasculature endothelial cells, human pulmonary microvasculature endothelial cells, human brain microvasculature endothelial cell), circulating endothelial progenitor cells or human umbilical vein endothelial cells, as well as transformed endothelial cells [21][22][23][24]. Use of human over animal endothelial cells is advisable because of repeatedly observed inadequacy of animal experimental models, convenience of data analysis and direct applicability of the results [25].…”