Effect of endothelium mimicking self-assembled nanomatrices on cell adhesion and spreading of human endothelial cells and smooth muscle cells

Abstract

The goal of this study is to develop unique native endothelium mimicking nanomatrices and evaluate their effects on adhesion and spreading of human umbilical vein endothelial cells (HUVECs) and aortic smooth muscle cells (AoSMCs). These nanomatrices were developed by self-assembly of peptide amphiphiles (PAs) through a solvent evaporation technique. Three PAs, one containing the Tyr-Ile-Gly-Ser-Arg (YIGSR) ligand, second containing the Val-Ala-Pro-Gly (VAPG) ligand, and a third without cell adhesive ligands were developed. Cell adhesion and spreading were evaluated by a PicoGreen-DNA assay and Live/Dead assay respectively. Our results show that PA-YIGSR significantly enhances HUVEC adhesion (26704±2708) spreading (84 ±8%), and proliferation (50 ±2%) when compared to other PAs. PA-VAPG and PA-YIGSR showed significantly greater AoSMC adhesion when compared to PA-S. PA-VAPG also showed significantly greater spreading of AoSMCs (63 ±11%) when compared with other PAs. Also, all the PAs showed significantly reduced platelet adhesion when compared with collagen I (control). These findings would facilitate the development of novel vascular grafts, heart valves, and cell based therapies for cardiovascular diseases.

Introduction

Tissue engineering solutions to cardiovascular diseases have become highly attractive due to paucity in conventional methods of treatments and the enormous burden on medical budgets in the western world. For perspective, over 500,000 coronary bypass grafts are performed per annum in the world [1]. In some cases, these procedures are performed with autologous mammary arteries or saphenous veins. However, due to shortage of supplies, synthetic vascular grafts have been widely used. There are several synthetic vascular grafts available in the market, but limitations exist for these grafts as well. The usage of synthetic materials commonly results in graft failure, especially in small diameter blood vessels, due to thrombosis and the lack of re-endothelialization, followed by restenosis and intimal hyperplasia. Thus, it is imperative to develop a tissue engineered vascular graft by mimicking the native endothelium to provide the best possible environment, thereby enhancing clinical patency.

The success of a tissue engineered graft depends largely on its ability to replicate the microenvironments in the native tissue. Despite recent advances, the generation of a functional microvasculature remains elusive [2,3]. Several types of scaffolds have been used, ranging from synthetic materials, such as Dacron [4], to natural ECM proteins, such as collagen [5]. Most scaffolds, however, result in failure due to the early onset of thrombosis, especially at the small diameter level [6].

To develop tissue engineered vessels, the critical components needed include a functional endothelium, a collagenous network for mechanical strength, and an elastin network to recreate the mechanical flexibility of native blood vessels [7]. Endothelial cells (ECs) and smooth muscle cells (SMCs) form the cellular components of a blood vessel. Therefore, to adequately aid the functional development of engineered vessels, the scaffolds should interact with the cells and improve their adhesion and survival, thus promoting integration into the surrounding tissue. The scaffold should provide the cells with their native environment by mimicking the natural ECM. This homing of cells leads to their secretion of essential proteins, such as collagen and elastin. The formation of a confluent endothelial cell layer is essential for developing an artificial vessel, as it provides an antithrombogenic surface [7]. However, ECs are susceptible to shear forces and scaffold adaptations are needed, such as entities that provide the cells with an adhesive moiety. Various techniques have been attempted to induce endothelial cell attachment and retention, including plasma treatment of scaffolds [8] and inclusion of peptides for cell adhesion [9]. Several scaffolds have been modified by the addition of Arg-Gly-Asp (RGD), a general cell adhesive ligand derived from fibronectin. However, RGD also promotes the attachment of platelets, which can lead to thrombosis [10]. Thus, the incorporation of a general cell adhesive moiety, such as RGD, proves counterproductive. Tyr-Ile-Gly-Ser-Arg (YIGSR) is a laminin derived peptide that has shown affinity for endothelial cells [11]. Laminin is a major component of the basement membrane that comprises the native vasculature for endothelial cells. YIGSR has been found to selectively enhance the endothelialization of polyurethaneurea scaffolds, without platelet adhesion [12]. Thus, the incorporation of YIGSR into scaffolds for vascular tissue engineering offers a promising step in the right direction.

Similarly, to aid the adhesion, growth, and proliferation of SMCs, scaffolds should provide the same type of natural environment. In vivo, SMCs are found in the medial layer of the blood vessel. An elastin derived peptide sequence, Val-Ala-Pro-Gly (VAPG) [13], which is a quantitative marker for elastins, has been shown to specifically enhance the adhesion and growth of vascular SMCs [14].

Altogether, the goal of this study is to evaluate the effect of novel endothelium mimicking selfassembled peptide amphiphile (PA) nanomatrices. PAs typically consist of alkyl chains attached to short peptides [15], and they can assemble into a variety of shapes, depending on dimensions, charge, and environment [16]. Conical shaped PAs with a bulky head group self assemble into cylindrical micelles with the PAs stacked in form of β-sheets parallel to the long axes, packed radially within the nanofibers, as the hydrophilic peptide segments extend outward towards the surface [15,17]. The PAs developed for this study consist of a hydrophobic alkyl (palmityl) tail coupled to a matrix metalloprotease-2 (MMP-2) enzyme degradable site, Gly-Thr-Ala-Gly-Leu-Ile-Gly-Gln (GTAGLIGQ), and a cell adhesive ligand. The alkyl tail constitutes the hydrophobic component of the molecule and is responsible for driving the self assembly of the PAs into a nanofibrous matrix with the cell adhesive ligands presented on the outer surface. The presence of the MMP-2 sensitive site within the PA molecules is designed to allow matrix remodeling, as cells grow and constitutively secrete MMP-2. MMP-2 mediated degradation of PAs and their remodeling has been studied in 3D networks [17]. The cell adhesive ligands used in this study are YIGSR, which is a known endothelial cell adhesive moiety, and VAPG, which is known to elicit a favorable response from SMCs. Therefore, the two PAs synthesized are C16-GTAGLIGQYIGSR (PA-YIGSR) and C16-GTAGLIGQVAPG (PA-VAPG). A third PA was synthesized without any cell adhesive ligands, C16-GTAGLIGQS (PA-S), to serve as a control. Thus, the incorporation of these cell adhesive sequences and enzyme degradable sites into the nanomatrices potentially provide cells with native endothelium mimicking environments, which are tailored to meet their needs for survival, growth, and integration into host tissue.