The Effect of Functionalized Self-Assembling Peptide Scaffolds on Human Aortic Endothelial Cell Function

ARTICLE IN PRESS Biomaterials 26 (2005) 3341–3351 www.elsevier.com/locate/biomaterials The effect of functionalized self-assembling peptide scaffolds on human aortic endothelial cell function Elsa Genove´ a, Colette Shenb, Shuguang Zhanga,c, Carlos E. Seminoa,c,Ã aCenter for Biomedical Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA bHarvard University, Cambridge, MA, 02138, USA cBiotechnology Process Engineering Center, Massachusetts Institute of Technology, Cambridge, MA 02139, USA Received 2 February 2004; accepted 10 August 2004 Available online 13 October 2004 Abstract A class of designed self-assembling peptide nanofiber scaffolds with more than 99% water content has been shown to be a good biological material for cell culture. Here, we report the functionalization of one of these peptide scaffolds, RAD16-I (AcN–RADARADARADARADA–CONH2), by direct solid phase synthesis extension at the amino terminal with three short- sequence motifs. These motifs are present in two major protein components of the basement membrane, laminin 1 (YIGSR, RYVVLPR) and collagen IV (TAGSCLRKFSTM). These motifs have been previously shown to promote specific biological activities including endothelial cell adhesion, spreading, and tubular formation. Therefore, the generic functionalized peptide developed was AcN–X–GG-RADARADARADARADA–CONH2 with each motif represented by ‘‘X’’. We show in this work that these tailor-made peptide scaffolds enhance the formation of confluent cell monolayers of human aortic endothelial cells (HAEC) in culture. Moreover, additional assays designed to evaluate endothelial cell function showed that HAEC monolayers obtained on these scaffolds not only maintained LDL uptake activity but also enhanced nitric oxide release and elevated laminin 1 and collagen IV deposition. These results suggest that this new scaffold provide a better physiological substrate for endothelial cell culture and suggest its further application for biomedical research, cancer biology and regenerative biology. r 2004 Elsevier Ltd. All rights reserved. Keywords: Self-assembly; Biomimetic material; Cell proliferation; Extracellular matrix; Endotheliar monolayer 1. Introduction and biopolymers [3–8]. The basement membrane is a three-dimensional network composed mainly of lami- We previously reported a self-assembling peptide nins and collagens [9–12]. The basement membrane is nanofiber scaffold [1] that provides a good support not only important as a structural component, support- material for cell culture [2]. However, self-assembling ing cell attachment and providing boundaries between peptides can be modified with known functional peptide tissues and organs, but it also gives to the cells an motifs from proteins of the basement membrane to instructive microenvironment that modulates their promote specific cellular responses in similar way as function. For instance, it is well established that the other investigators have previously modified polymers maintenance and function of endothelial cells are critically influenced by their interaction with the base- ÃCorresponding author. Center for Biomedical Engineering, Mas- ment membrane [4,13–20]. Short sequences present in sachusetts Institute of Technology, 77 Massachusetts Avenue, Cam- basement membrane proteins have been identified to bridge, MA 02139, USA. Tel.: +1-617-258-0249; fax: +1-617-258- participate in series of important biological functions, 5239. E-mail addresses: [email protected] (S. Zhang), [email protected] including cell migration and morphogenesis [4,13–20]. (C.E. Semino). Laminin 1 is a heteroprotein that is formed by three 0142-9612/$ - see front matter r 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2004.08.012 ARTICLE IN PRESS 3342 E. Genove´ et al. / Biomaterials 26 (2005) 3341–3351 different polypeptide subunits, a1; b1; and g1; which synthetic polymers, polyethylene terephthalate (PET) assemble into a cross-like structure and that interacts as and poly-(tetrafluoroethylene) (PTFE) [28]. The addi- a connector or bridge between the cells and the tion of these sequences has enhanced cell adhesion, cell extracellular matrix through several integrin and non- spreading rate, and focal contact formation of human integrin receptors [11]. Two sequences present in the umbilical vein endothelial cells (HUVEC) cultured in laminin-1 b1 chain, YIGSR and RYVVLPR, have been these polymers that naturally possess very low-adhesive shown to promote cell adhesion, cell migration, and properties [28]. Similar results have been reported with endothelial cell tubular formation [3–6]. endothelial cells after immobilizing the RGD peptide in Type IV collagen, the other major component of the synthetic polymers such as polyurethane (PEU) [29], basement membrane, forms a network structure that polyvinyl alcohol (PVA) [30], and polyvinyl amine involves its interaction with other components of the (PVAm) [31]. Recently, a class of self-assembling basement membrane, including laminin, nidogen, and peptide exposing the bioactive peptide IKVAV from heparan sulphate proteoglycan [21–23]. Collagen IV laminin has been described to form nanofiber scaffolds molecules are composed of two a1(IV) chains and one that can be used to select rapid differentiation of neural a2(IV) chains and can interact with cells indirectly progenitor cells into neurons [32,33]. through laminins by low affinity interactions [12,21] or In this work, we report the functionalization of the by strong interactions mediated by nidogen, a glyco- peptide scaffold RAD16-I (AcN–RADARADARA- protein of about 150 KDa, which bind tightly to laminin DARADA–CONH2) through direct solid phase [22,23]. The peptide TAGSCLRKFSTM from the synthesis extension at the amino terminal with three a1(IV) chain of collagen IV was found to bind short-sequence motifs from two major proteins of the specifically to heparin in a dose-dependent manner basement membrane, laminin 1 (YIGSR, RYVVLPR) [7,8] and to promote adhesion and spreading of bovine and collagen IV (TAGSCLRKFSTM). We showed that aortic endothelial cells [8]. Finally, in presence of these tailor-made scaffolds increased the formation of heparin or chondroitin/dermatan sulphate glycosami- confluent cell monolayers with cobblestone phenotype noglycan side chains, the binding of endothelial cells of human aortic endothelial cells (HAEC) in culture to the peptide TAGSCLRKFSTM was inhibited, with respect to non-modified peptide scaffolds. In suggesting that the peptide is a crucial domain involved parallel experiments consisting of culture HAEC on in the determination of basement membrane ultra- other substrate as controls including collagen I gels or structure, by connecting collagen IV with proteoglycans Matrigel (natural basement membrane), similar mono- [11]. layer formation was observed, suggesting that the We selected self-assembling peptide nanofiber scaf- functionalized peptide scaffolds may be considered like folds to recreate a basement membrane analog. These synthetic basement membrane analogs. Moreover, peptides have a b-sheet structure and can self-assemble additional assays designed to evaluate endothelial cell into a network of interweaving nanofibers of 10 nm function indicated that HAEC monolayers obtained on diameter, forming hydrogel scaffolds with pores these scaffolds not only maintain LDL uptake activity 5–200 nm in diameter and over 99% water content but also enhanced nitric oxide release and elevated [1,2]. These peptide scaffolds have been shown to laminin 1 and collagen IV deposition. These results support attachment, growth, maintenance, and differ- suggest that HAEC cultured on these new functionalized entiation of a variety of mammalian cells [2,24–27]. scaffolds enhanced the endothelial cell phenotype and For example, it supported differentiation of rat PC12 promoted basement membrane deposition, which is a cells and functional synapse formation of rat primary physiological prerequisite for successful culture and hippocampal neurons [24], promoted bovine chondro- expansion of endothelial cells. cytes to developcartilage constructs in vitro [25], and sustained differentiation of rat liver progenitor cells into functional hepatocyte-like cells [26].In 2. Materials and methods addition, the scaffold provides a new way of isolation of migrating hippocampal neural stem cells from 2.1. Materials organotypic cultures through a cell entrapment techni- que [27]. All peptide sequences used in this work were obtained Previous investigators have studied the biological from SynPepCorporation ( www.synpep.com, Dublin, activity of the short peptide sequences Arg–Gly–Asp CA) and dissolved in deionized water at final concen- (RGD), which is present in fibronectin and other tration of 0.5% (5 mg/ml) and sonicated for 20 min extracellular matrix proteins and is critical for cell (aquasonic, model 50 T, VWR, PA). Peptide hydrogel adhesion, and Thy–Ile–Gly–Ser–Arg (YIGSR), which is formation was developed on top of a cell chamber insert present in laminin 1 and is important for cell function, (10 mm diameter, 0.5 cm2 area, pore size=0.2 mm, cat# after immobilizing them to glass surfaces and in two 136935, Nalge Nunc International, IL), see below. ARTICLE IN PRESS E. Genove´ et al. / Biomaterials 26 (2005) 3341–3351 3343 2.2. Circular dichroism (DC) b Circular dichroism (CD) spectra were gathered on an Aviv model 202 spectropolarimeter. Samples were prepared by diluting stock peptide solutions in water (0.5%) to a working concentration of 25

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