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Human VE-cadherin fusion protein as an artificial extracellular matrix enhancing the proliferation and differentiation functions of endothelial cell Ke Xu, Qizhi Shuai, Xiaoning Li, Yan Zhang, Chao Gao, Lei Cao, Feifei Hu, Toshihiro Akaike, Jianxi Wang, Zhongwei Gu, and Jun Yang Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.5b01467 • Publication Date (Web): 09 Feb 2016 Downloaded from http://pubs.acs.org on February 11, 2016
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Human VE-cadherin fusion protein as an artificial extracellular matrix enhancing the proliferation and differentiation functions of endothelial cell
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Ke Xu1, Qizhi Shuai1, Xiaoning Li1, Yan Zhang1, Chao Gao1, Lei Cao1, Feifei Hu1,Toshihiro
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Akaike2, Jian-xi Wang3, Zhongwei Gu3, Jun Yang1#
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(1The Key Laboratory of Bioactive Materials, Ministry of Education, College of Life Science,
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Nankai University, Tianjin, 300071, China;
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2Biomaterials Center for Regenerative Medical Engineering, Foundation for Advancement of
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International Science, Tsukuba, Japan;
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3National Engineering Research Center for Biomaterials, Sichuan University, Chengdu, 610064,
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China ;)
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#Corresponding author.
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Prof. Jun Yang, The Key Laboratory of Bioactive Materials, Ministry of Education, College of life
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science, Nankai University, Tianjin, 300071, China
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94 Weijin Road, Nankai District, Tianjin, China
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E-mail address:
[email protected] 29
Tel& Fax: +0086 22 23498038 1
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ABSTRACT
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In an attempt to enhance endothelial cell (EC) capture and promote the
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vascularization of engineered tissue, we biosynthesized and characterized the
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recombinant fusion protein consisting of human vascular endothelial-cadherin
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extracellular domain and immunoglobulin IgG Fc region (hVE-cad-Fc) to serve as a
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bioartificial extracellular matrix (ECM). The hVE-cad-Fc protein naturally formed
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homodimers and was used to construct hVE-cad-Fc matrix by stably adsorbing on
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polystyrene (PS) plates. Atomic Force Microscop (AFM) assay showed uniform
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hVE-cad-Fc distribution with nano-rod topography. The hVE-cad-Fc matrix markedly
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promoted human umbilical vein endothelial cells (HUVECs) adhesion and
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proliferation, with fibroblastoid morphology. Additionally, the hVE-cad-Fc matrix
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improved HUVECs migration, vWF expression and NO release, which are closely
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related to vascularization. Furthermore, the hVE-cad-Fc matrix activated endogenous
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VE-cadherin/β-catenin proteins, and effectively triggered the intracellular signals such
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as F-actin stress fiber, p-FAK, AKT and Bcl-2. Taken together, hVE-cad-Fc could be a
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promising bioartificial matrix to promote vascularization in tissue engineering.
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KEYWORDS: bioartificial extracellular matrix; fusion protein; VE-cadherin;
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vascularization.
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1. Introduction
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Tissue engineering holds enormous potential to replace or restore the function of
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damaged or diseased tissue.1 While significant progress has been achieved in seeding
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cells on polymer scaffolds, the inability to timely develop a sufficient and functional
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blood vessel system mainly limits the growing of thick, 3-dimensional viable
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engineered tissue in vitro survival and in vivo integration.2 Different tissue
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engineering vascularization strategies have included material-based and cell-based
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vascularization. These approaches have shown that scaffold surface modifications can
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resist nonspecific protein or cell adhesion and improve endothelial-specific adhesion
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to form an endothelial monolayer on the scaffolds. Consequently, there has been an
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increasing interest in exploring biomaterials as bioartificial ECM for modifying the
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surfaces of tissue engineering scaffolds and rapidly inducing endothelialization and
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vascularization.3
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In the last decades, ECM proteins (collagen, fibronectin and laminin),4
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glycosaminoglycans (hyaluronic acid),5 ECM-derived peptides (RGDS, YIGSR and
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REDV)6,7,8 and soluble signaling proteins (EGF, VEGF and FGF)9-11 have been
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frequently used to coat the surfaces of various biomaterials by chemical conjugating
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or physical adsorbing to augment their interaction with endothelial cells and
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accelerate endothelialization on the scaffolds. More recently, with large scale
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purification at high yields, new biological materials composed of recombinant
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proteins are now being developed to create well-defined, multifunctional matrix using
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common genetic engineering.12 Several recombinant proteins have been employed to
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engineer bioartificial ECM, including structural proteins, growth factors and adhesion
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proteins that are fused with ECM protein binding domain or immunoglobin G (IgG)
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Fc domain.13-15 Fc-fusion proteins, which comprise an IgG Fc region linked
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genetically to the target protein, have shown great promise as chimeric proteins with
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both functional integrity and long-term stability.16,17 In a Fc-fusion protein, the Fc
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domain can bind directly to the scaffold surface via hydrophobic interactions,
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allowing the fused target protein to stretch out and interact with a suitable partner.18 In
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our previous work, multiple Fc-fusion proteins, including epithelial (E)-cadherin-Fc, 3
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neuronal (N)-cadherin-Fc and cell growth factor-Fc, were established and used as
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bioartificial ECM for biomaterial surface modification and shown to successfully
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provide a biofunctional extracellular microenvironment for regulating cell
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behavior.14,19-22
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In an attempt to improve endothelialization and vascularization rates within
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tissue engineering scaffolds, this study focused on designing a novel vascular
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endothelial (VE)-cadherin-Fc fusion protein to enable the construction of the
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bioactive interface via biomaterial surface modification. Cadherins are a large family
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of transmembrane proteins with more than thirty members. VE-cadherin, an
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endothelial specific cell-cell adhesion molecule, stabilizes endothelial junctions
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through the homophilic binding of VE-cadherin extracellular regions.23 The adherens
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junctions play a pivotal role in endothelial cell adhesion, proliferation, migration and
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apoptosis, as well as the formation and maturation of the endothelium during
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vascularization.23,24 Additionally, the adherens junctions, which are protein complexes,
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contain large numbers of intracellular proteins that anchor to the actin cytoskeleton
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and activate a series of intercellular signals.25 Given our previous work on E/N-cad-Fc
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fusion proteins for culturing stem cells, we hypothesized that the extracellular domain
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of VE-cadherin would be an ideal candidate enabling the development of a novel
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multifunctional bioartificial ECM able to mimic adherens junctions and regulate
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endothelial cell-to-cell, cell-ECM and cell-cell growth factor interactions.
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In this study, hVE-cad-Fc was biosynthesized and applied to the surface
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modification of biomaterials as a novel ECM. To begin, the hVE-cad-Fc protein was
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identified and characterized via immobilization on polystyrene (PS) plates. Next, we
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investigated the abilities of the hVE-cad-Fc matrix to promote adhesion, proliferation,
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migration and differentiation in human umbilical vein endothelial cells (HUVECs) in
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vitro.
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2. Materials and methods
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2.1. Construction and expression of the fusion protein hVE-cad-Fc
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VE-cadherin extracellular domains from HUVECs were amplified by PCR to 4
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generate cDNA. The specific primer pair used for amplification was as follows:
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5’-CCGGATATCATGCAGAGGCTCATGATGCTCC-3’
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GCCGCTCTGGGCGGCCATATC-3’. The amplified VE-cadherin cDNA was
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inserted into the EcoRV-NotӀ site of the pcDNA3.1 vector, which contained the
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human IgG Fc domain (a gift from the Akaike research group). 293-6E cells were
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grown in serum-free FreeStyleTM 293 Expression Medium (Life Technologies,
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Carlsbad, CA, USA). The recombinant plasmid encoding hVE-cad-Fc was transiently
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transfected into 293-6E cell suspension. At day 6, cell culture supernatants were
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collected and used for purification, with target proteins captured on a Protein A CIP 5
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ml column (GenScript, Cat.No.L00433), followed by a second SEC purification step.
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The purified protein was analyzed by SDS-PAGE and Western blotting using 20 µL of
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sample, with primary goat anti-human IgG-HRP utilized (GenScript, Cat. No.
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A00166).
and
5’-AAGCG
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2.2 Preparation of hVE-cad-Fc-, Collagen- and Poly-L-lysine- coated plates
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The purified hVE-cad-Fc protein was diluted in PBS at different concentrations
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and incubated on polystyrene (PS) plates. After 2 h incubation at 37 °C，the surface
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was washed three times with PBS and incubated with 1.0% bovine serum albumin
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(BSA) for 1 h to block nonspecific interactions. To prepare the control groups, PS
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plates were treated with 1% Collagen Ӏ (BD, USA), 5µg/ml of poly-L-lysine (PLL)
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(ScienCell, USA) or tissue culture treated polystyrene (TC-PS) plates (Corning
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Corporation) for 2 h at 37 °C.
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2.3 Assessment of hVE-cad-Fc immobilization onto PS plates
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The quantities of immobilized hVE-cad-Fc on the plates were determined by
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ELISA assay. Briefly, the coated plates were first incubated with 1.0 % (w/v) BSA for
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1 h to block nonspecific interactions and then incubated with rabbit anti-VE-cadherin
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antibody (1:1000, Cell Signaling Technology) for 2 h at 37 °C. After washing with
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PBS, the plates were incubated with HRP-labeled goat anti-rabbit IgG (H+L)
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antibody for an additional 2 h, followed by the addition of TMB solution, which was 5
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added for 15 min at 37 °C in darkness. Stop buffer was added, and the absorbance was
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measured at 450 nm.
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The amount of immobilized hVE-cad-Fc was calculated basing on the
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quantification of the hVE-cad-Fc in the solution before and after immobilization with
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a VE-cadherin ELISA kit (BD, USA).
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For the water contact angle (WCA) analysis, 5 µl water was dripped onto the
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hVE-cad-Fc-coated plates (5µg/ml) and other plates and measurements were obtained
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using an optical contact angle meter (Dataphysics Inc, OCA20). For the adsorption
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stability evaluation, PBS and culture medium were added to the hVE-cad-Fc-coated
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plates (5µg/ml) for one week and then immobilized hVE-cad-Fc was measured by
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ELISA assay. To examine the topography of the hVE-cad-Fc-coated (5µg/ml) surface,
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the samples were characterized by atomic force microscope (AFM; NTEGRA Prima,
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NT-MDT). The modified surface was dried with nitrogen for 1 h and the results were
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analyzed using AFM nasoscope V7.2 version.
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2.4 Cell culture HUVECs and endothelial cell medium were obtained from the ScienCell
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Corporation (USA). Human vascular smooth muscle cells (HVSMCs; ScienCell, USA)
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were cultured in DMEM-F12 supplemented with 10 % (v/v) fetal bovine serum.
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2.5 HUVECs adhesion and proliferation assay
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The HUVECs were seeded into a 96-well plate at a density of 1×104 cells/well.
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After incubation for 4 h, 24 h or 48 h, the supernatants were removed and the cells
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were washed three times with PBS. Cells were incubated with CCK-8 at 37 °C for 4 h
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and read on a micro-plate reader (Biotech, MQX200) at an absorbance of 450nm. For
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inhibition experiments, cells were pre-cultured with anti-VE-cadherin antibody (Cell
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Signaling Technology) at 37 °C for 30 min and seeded onto Collagen, PLL or
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hVE-cad-Fc-coated (5µg/ml) plates. Non-adherent cells were removed after 4 h by
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rinsing with PBS and the remaining HUVECs were quantified using a CCK-8 assay.
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Calcium-dependent adhesion was evaluated by incubating cells in EDTA, EGTA and 6
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MgSO4 at final concentrations of 5 mM for 30 min prior to the CCK-8 assay.
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2.6 Nitric oxide release assay
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Supernatants were collected from different substrate surfaces after HUVECs
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culturing and nitric oxide (NO) concentrations were determined using Total Nitric
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Oxide Assay Kit (Beyotime, China) according to the manufacturer's instructions;
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absorbance was measured at 540 nm (n=3).
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2.7 Cell migration assay
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Cell suspensions were seeded onto 24 well AggreWellTM plates (Stem Cell, USA)
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and then centrifuged at 1000 rpm for 5 min. The cells were subsequently cultured for
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16 h to form 3D HUVECs aggregates. The cell aggregates were then quickly
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transferred onto hVE-cad-Fc, Collagen, PLL or TC-PS matrices. After 4 h, cellular
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spreading and crawling from the aggregates were observed by microscopy.
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2.7 Western blotting analysis
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Total protein was extracted by adding lysis buffer [10 mM Tris (pH 7.4), 150 mM
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NaCl, 1% Triton X -100, 1% sodium deoxycholate, 0.1% SDS, 10 mM EDTA and
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protease inhibitor cocktail] to the cell cultures while on ice. The cells were disrupted
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with the assistance of a cell scraper and lysates were centrifuged at 13,000 rpm for 10
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min at 4 °C. The supernatants were collected and protein concentrations were
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determined using BCA assay (Beyotime, China). Samples were then separated by
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electrophoresis using ~8 – 12% SDS-polyacrylamide gels, transferred onto
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polyvinylidene difluoride membranes (Roche, USA) and incubated with one of the
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following primary antibodies: rabbit anti-VE-cadherin antibody (1:1000, Cell
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Signaling Technology), anti-rabbit von Willebrand factor antibody (1:2000, Abcam),
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rabbit anti-Phospho-Akt (Ser473) antibody (1:1000, Cell Signaling Technology),
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rabbit anti-Bcl-2 antibody (1:1000, Gene Tex), rabbit anti-Phospho-FAK antibody
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(Tyr397) (1:1000, Cell Signaling Technology), rabbit anti-β-actin antibody (1:1000,
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Cell Signaling Technology) or mouse anti-α/β/p120-catenin antibody (1:1000, BD 7
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Transduction Laboratories). The membranes were then incubated with HRP-labeled
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goat anti-rabbit IgG (H+L) antibody (1:1000, Beyotime) and washed with PBS. All
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bands were quantified using Quantity One v.4.62 software.
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2.8 Cell cytoskeleton staining
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After 4h, 24h or 48h of culture, the cells were fixed with 4 % (v/v)
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paraformaldehyde for 10 min and incubated with 0.1% (v/v) Triton X-100 for 10 min
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at 37 °C. Cells were then blocked with 1.0% (w/v) BSA for 30 min at 37 °C, stained
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with FITC-phalloidin/propidium iodide and the nuclei were counterstained with DAPI.
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Staining was assessed using a confocal laser scanning microscope (TCS SP8, Leica).
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2.9 Immunofluorescence staining
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Cells were fixed with 4% (v/v) paraformaldehyde for 10 min and then blocked
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with 1% (w/v) BSA for 30 min at 37 °C. The samples were then incubated with
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primary rabbit VE-cadherin antibody (1:100, Abcam), washed with PBS and
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subsequently incubated with goat anti-rabbit antibody conjugated with Alexa
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Fluorophore 488 and 594 (Invitrogen) at room temperature for 1 h. All samples were
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examined by confocal laser scanning microscopy (TCS SP8, Leica).
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2.10 Immunoprecipitation
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HUVECs were lysed in lysis buffer [1% Triton X-100, Tris-HCl (pH 7.5), 100
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mM NaF and 1.0 % NP-40] and cleared by centrifugation at 12,000 rpm for 10 min at
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4 °C. The supernatants were collected, incubated with FAK-specific antibody (1:100,
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Abcam) for 6 h and then protein A/G plus agarose beads (Beyotime, China) for 2 h at
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4 °C. The immunocomplexes were washed three times, boiled in 1x sample loading
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buffer and electrophoresed on sodium dodecyl sulfate-polyacrylamide gels for protein
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analysis.
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2.11 Statistical analyses GraphPad Prism Version 5.0 software for windows (GraphPad, San Diego, CA) 8
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was used for statistical analysis. Data analysis was performed by one-way analysis of
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variance (ANOVA). The minimum significance level was set at * p < 0.05 and **p
