Research Article: Static spatial growth restriction micropatterning of endothelial colony forming cells influences their morphology and gene expression

Date Published: June 12, 2019

Publisher: Public Library of Science

Author(s): Matthew W. Hagen, Monica T. Hinds, Alain-Pierre Gadeau.

http://doi.org/10.1371/journal.pone.0218197

Abstract

Endothelialization of small diameter synthetic vascular grafts is a potential solution to the thrombosis and intimal hyperplasia that plague current devices. Endothelial colony forming cells, which are blood-derived and similar to mature endothelial cells, are a potential cell source. Anisotropic spatial growth restriction micropatterning has been previously shown to affect the morphology and function of mature endothelial cells in a manner similar to unidirectional fluid shear stress. To date, endothelial colony forming cells have not been successfully micropatterned. This study addresses the hypothesis that micropatterning of endothelial colony forming cells will induce morphological elongation, cytoskeletal alignment, and changes in immunogenic and thrombogenic–related gene expression.

Spatially growth restrictive test surfaces with 25 μm-wide lanes alternating between collagen-I and a blocking polymer were created using microfluidics. Case-matched endothelial colony forming cells and control mature carotid endothelial cells were statically cultured on either micropatterned or non-patterned surfaces. Cell elongation was quantified using shape index. Using confocal microscopy, cytoskeletal alignment was visualized and density and apoptotic rate were determined. Gene expression was measured using quantitative PCR to measure KLF-2, eNOS, VCAM-1, and vWF.

Endothelial colony forming cells were successfully micropatterned for up to 50 hours. Micropatterned cells displayed elongation and actin alignment. Micropatterning increased the packing densities of both cell types, but did not affect apoptotic rate, which was lower in endothelial colony forming cells. KLF-2 gene expression was increased in micropatterned relative to non-patterned endothelial colony forming cells after 50 hours. No significant differences were seen in the other genes tested.

Endothelial colony forming cells can be durably micropatterned using spatial growth restriction. Micropatterning has a significant effect on the gross and subcellular morphologies of both cell types. Further study is required to fully understand the effect of micropatterning on endothelial colony forming cell gene expression.

Partial Text

Durable artificial small-diameter synthetic vascular grafts for the treatment of vascular occlusions represent a critical unmet need in modern medicine. Autologous grafting is the current clinical standard for arterial bypass procedures, however limited availability and donor site complications make a substantial portion of the potential patient population ineligible for autologous vessel harvesting [1]. Given that currently available synthetic biomaterials including expanded polytetrafluoroethylene (ePTFE) show limited patency at diameters less than 6 mm, this leaves many patients without viable alternatives [2,3]. One potential solution to this problem is the use of biomaterials endothelialized in vitro prior to implantation. The pre-implant establishment of a functional endothelium is capable of limiting the thrombosis and neointimal hyperplasia which limit patency and lead to graft failure.

To our knowledge, this study represents the first successful demonstration that ECFC morphology and gene expression can be manipulated in static culture. While the ECFC literature contains conflicting reports on the degree of similarity between ECFCs and mature ECs [5], in our non-human primate model, ECFCs are remarkably similar to ECs in morphology and function [8,14,33,34]. Our earlier studies revealed similar expression levels of the genes and cell surface proteins that regulate monocyte adhesion, tight junction formation, and platelet activation between ECs and ECFCs both basally and following TNF-α stimulation [27]. We furthermore previously showed similar performances of EC and ECFC-seeded vascular grafts in our ex vivo shunt model of platelet and fibrin accumulation [27]. However, ECs and ECFCs do have a few notable differences: ECFCs have a much lower basal level of eNOS gene expression [27], and are capable of higher rates of proliferation and extracellular matrix remodeling in culture than ECs [25,34]. We have hypothesized that this latter difference is the primary reason that a prior attempt to micropattern ECFCs was not successful: the use of BSA to block ECFC expansion between patterned lanes was not sufficient to overcome the enhanced proliferation and remodeling ability of these cells [25]. Through the use of an increased concentration of Pluronic, a previously validated [20] food-grade hydrophobic polymer, we have created the conditions for durably elongated ECFCs under static culture conditions for longer periods of time.

Small-diameter artificial vascular grafts remain a critical unmet need. As interest in ECFCs as a potential tool for tissue engineered grafts grows, this study supports the use of this cell type for in vitro device endothelialization, particularly on micropatterned devices. These results demonstrate that ECFCs can be morphologically manipulated in the absence of fluid shear stress using spatial growth restriction micropatterning. Micropatterned ECFCs showed robust and lasting changes in gross and subcellular morphology analogous to that shown previously and repeated here with mature ECs. While the gene expression results shown here were largely unaffected by micropatterning, it should be noted that this study supports the use of ECFCs as equivalent to mature ECs in regulating neointima formation through the expression of adhesion molecules.

 

Source:

http://doi.org/10.1371/journal.pone.0218197