regulation of endothelial cell function by grgdsp peptide grafted on interpenetrating polymers

Regulation of endothelial cell function by GRGDSPpeptide grafted on interpenetrating polymers

Shyam Patel,1 Jonathan Tsang,1 Gregory M. Harbers,1,2 Kevin E. Healy,1,3 Song Li11Department of Bioengineering, University of California at Berkeley, Berkeley, California 947202Department of Biomedical Engineering, Northwestern University, Evanston, Illinois 602083Department of Materials Science and Engineering, University of California at Berkeley, Berkeley,California 94720-1760

Received 3 April 2006; revised 17 November 2006; accepted 5 February 2007Published online 23 April 2007 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jbm.a.31320

Abstract: Vascular endothelium plays an important rolein preventing thrombogenesis. Bioactive molecules suchas fibronectin-derived peptide Gly-Arg-Gly-Asp-Ser-Pro(GRGDSP) can be used to modify the surface of cardiovas-cular implants such as vascular grafts to promote endotheli-alization. Here we conjugated GRGDSP peptide to the non-fouling surface of an interpenetrating polymer network(IPN), and investigated the effects of the immobilizedGRGDSP molecules on EC functions under static and flowconditions at well-defined GRGDSP surface densities (*0 to3 pmol/cm2). EC adhesion and spreading increased withGRGDSP surface density, reached a plateau at 1.5 pmol/cm2,and increased further beyond 2.8 pmol/cm2. Cell adhesionand spreading on GRGDSP induced two waves of extracel-lular signal-regulated kinase (ERK) activation, and 0.2pmol/cm2 density of GRGDSP was sufficient to activateERK. EC proliferation rate was not sensitive to GRGDSPsurface density, suggesting that cell spreading at low-den-sity of GRGDSP is sufficient to maintain EC proliferation.

EC migration on lower-density GRGDSP-IPN surfaces wasfaster under static condition. With the increase of GRGDSPdensity, the speed and persistence of EC migration droppedquickly (0.2–0.8 pmol/cm2) and reached a plateau, followedby a slower and gradual decrease (1.5–3.0 pmol/cm2). Thesedata suggest that the changes of EC functions were moresensitive to the increase of GRGDSP density at lower range.Under flow condition with shear stress at 12 dyn/cm2, ECmigration was inhibited on GRGDSP-IPN surfaces, whichmay be attributed to the assembly of large focal adhesionsinduced by shear stress, suggesting a catch-bond character-istic for RGD-integrin binding. This study provides arational base for surface engineering of cardiovascularimplants. � 2007 Wiley Periodicals, Inc. J Biomed Mater Res83A: 423–433, 2007

Key words: endothelial cell; RGD peptide; interpenetratingpolymer network; adhesion; migration; proliferation; shearstress

INTRODUCTION

Vascular endothelial cells (EC) form the inner lin-ing of the blood vessels. ECs play important roles inthe homeostasis and functions of the blood vessels,e.g., resisting platelet adhesion and thrombogenesis,regulating the permeability of the vessel wall, andcontrolling vascular tone. Cardiovascular implantssuch as stents, vascular grafts, and valve grafts oftenillicit a foreign body reaction from the host.1,2

Although various polymeric and protein coatingshave been developed and researched to prevent celland protein adhesion, endothelialization of theseimplants is widely regarded as the most reliablestrategy for long-term implant patency.3 Endothelial-ization of cardiovascular implants avoids the directcontact of implants with the blood components andthus prevents thrombogenic reactions and the prolif-eration of smooth muscle cells.

Endothelialization of vascular grafts can either beperformed before implantation by seeding ECin vitro or by promoting host EC recruitment afterimplantation.4,5 However, EC adhesion and retentionrates are very low on materials that are commonlyused for vascular grafts such as polytetrafluoroethyl-ene (PTFE). Therefore, to increase EC adhesion, theblood-contacting surfaces of cardiovascular deviceshave been coated with adhesive molecules spanningfrom the glycoprotein fibronectin (FN)6 to monoclonal

Correspondence to: S. Li; e-mail: [email protected] grant sponsor: Whitaker Foundation; contract

grant number: RG-01-0210Contract grant sponsor: National Institute of Health;

contract grant number: NIH AR43187

' 2007 Wiley Periodicals, Inc.

antibodies against CD34 to capture circulating endo-thelial progenitor cells.5 FN binds to many extracel-lular matrix (ECM) proteins such as fibrin, collagenand gelatin, and is a crucial substrate for cell migra-tion during wound healing.7–9 The major cell bind-ing domain of FN, Arg-Gly-Asp (RGD) or Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP), has been widely used topromote cell adhesion on a variety of substrates.10

Synthetic peptides containing the RGD sequence canbe covalently attached to various polymers and met-als.11,12 Studies have shown that immobilized RGDpeptides improve EC adhesion and retention on pol-ymeric surfaces such as expanded PTFE and polyur-ethanes.13,14 RGD peptides can also be coupled withantithrombotic agents such as heparin or insertedwithin engineered proteins to create surfaces thatresist thrombosis and encourage endothelializa-tion.15,16 The versatility and resilience of the RGDpeptide has made it attractive for surface modifica-tion applications. For example, RGD peptide derivedfrom bone sialoprotein, in either linear or cyclicform, has been used to modify surfaces for osteo-blast adhesion and differentiation.17 The flankingsequence, length, and the structure of RGD peptidesmay affect RGD activity for cell binding and func-tions.18,19

Although the potential of RGD to promote endo-thelialization of cardiovascular implants has beenwidely researched, these studies have focused onRGD’s ability to promote cell adhesion on implantsurfaces and cell retention under fluid shearstress.20,21 In order for RGD to serve as a functionalmolecule for cardiovascular applications, it needs tonot only promote EC adhesion to blood contactingsurfaces of implants, but also enhance EC prolifera-tion and migration.

In this study, we fully characterized the ability ofimmobilized FN-derived GRGDSP peptide to pro-mote EC adhesion, proliferation and migration andthe dependence of these cell functions on GRGDSPsurface density. EC migration on GRGDSP-coatedsurfaces under flow conditions was also examined.The substrate used in this study was tissue culturepolystyrene modified with a covalently bound inter-penetrating polymer network (IPN) coating. Thecoating was originally developed to prevent proteinand cell adhesion to implant surfaces.22–24 The IPNis an ultra-thin hydrogel consisting of a crosslinkedpoly(acrylamide) layer with an interpenetrating poly(ethlylene glycol-co-acrylic acid) layer. The poly(acrylamide) layer and poly(ethylene glycol) (PEG)molecules create a hydrophilic surface that resistsboth protein and cell adhesion for long periods oftime (e.g. 760 days). Once grafted with peptidesmimicking the binding domains of ECM proteins thesurfaces become bioactive.25 Activation of this IPNwith the covalent grafting of GRGDSP enabled us to

study very specific interactions between the immobi-lized GRGDSP molecules and EC cultured on the IPNsurface.

MATERIALS AND METHODS

IPN and peptide conjugation

The major steps of IPN-peptide conjugation have beenpublished previously.26 In this study, we used a modifiedprotocol (e.g., polymerization time, UV exposure, chemicalincubation time, peptide coupling buffer, etc.), whichresulted in slightly different IPN properties and conjuga-tion efficiencies compared with what was previously done.The surfaces of 24-well and 96 well tissue-culture polysty-rene plates (Becton Dickinson, Franklin Lakes, NJ) and tis-sue-culture polystyrene slides (Nalge Nunc Int., Rochester,NY) were activated with oxygen plasma at 100 watts and500 mTorr for 5 min. Acrylamide monomer (Polysciences,Warrington, PA), N,N0-methylenebisacrylamide (Poly-sciences) and the photoinitiator [3-(3,4-dimethyl-9-oxo-9H-thioxanthen-2-yloxy)-2-hydroxypropyl]trimethylammoniumchloride (QTX) (Sigma-Aldrich, St. Louis, MO) were dis-solved in 97% ultrapure water, 3% isopropyl alcohol (IPA)solution. The acrylamide solution was pipetted intoplasma treated wells of 24-well plates and on slides andplaced in the dark for 8 min to allow for adsorption ofmonomers to the surfaces. The solution was polymerizedby placing the plates and slides on an Ultra-Lum UVB-40UV transilluminator (300 nm wavelength) (Ultra-Lum, Par-amount, CA) for 6–8 min at 408C. After polymerization,excess solution and gel were aspirated and the surfaceswere washed vigorously with ultrapure water followed bysonication for 5 min. To form the complete IPN, a 50:50water: IPA solution containing PEG 1000 monomethylethermonomethylmethacrylate (Polysciences), N,N0-methylenebi-sacrylamide, acrylic acid, and QTX was pipetted onto theacrylamide grafted TCPS surfaces. After 8 min incubationin the dark, the surfaces were exposed to UV for 10–12min. Subsequently, the surfaces were washed in the samemanner as before.

The IPN surface was further modified by grafting di-amino-PEG (MW 3400, Nektar Therapeutics, Huntsville,Alabama) onto the IPN’s acrylic acid molecules usingstandard carbodiimide chemistry. Briefly, di-amino-PEG,N-hydroxysulfosuccinimide (Sulfo-NHS) (Pierce, Rockford,IL), and [1-ethyl-3-(3-dimethylaminopropyl)carbodiimidehydrochloride] (EDC) (Pierce) were dissolved in 0.5M 2-morpholinoethanesulfonic acid (MES) buffer. The solutionpH was adjusted to 7.0 and pipetted onto the IPN surfaces.The samples were incubated with the di-amino-PEG solu-tion for 1 h at room temperature. Following di-amino-PEGgrafting, the heterobifunctional crosslinker sulfosuccini-midyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate(sulfo-SMCC) (Pierce) was used to graft the peptideCGGGRGDSP (GRGDSP) (American Peptide, Sunnyvale,CA) onto the IPN surfaces. The surfaces were first incu-bated with sulfo-SMCC dissolved in 50 mM sodium boratebuffer (pH 8.0) for 30 min at room temperature. Subse-quently, IPN surfaces were incubated overnight in phos-phate buffered saline (PBS) solution (pH 7.4) at various

424 PATEL ET AL.

Journal of Biomedical Materials Research Part A DOI 10.1002/jbm.a

GRGDSP concentrations (0–20 lM). Upon peptide grafting,the plates and slides were washed with PBS three times,sterilized with 70% ethanol and stored dry at 48C.

Quantitative measurement of peptidesurface density

To quantify peptide density on the IPN surface, a CGGpeptide linker (same as that for GRGDSP peptide) conju-gated with fluorescein isothiocyanate (FITC) (AmericanPeptide) was used. Various amounts of CGG-FITC wereimmobilized on IPN modified 96-well plates using thesame protocol as described above. The fluorescence inten-sity was measured using a Bio-Tek FLx800 fluorescenceplate reader (Bio-Tek, Winooski, Vermont). To correlatethe pixel intensity with the surface density, CGG-FITC sol-utions of various concentrations (0.001–0.1 lM) were spot-ted onto 96-well IPN plates and the fluorescence intensitywas measured with the Bio-Tek FLx800 plate reader. Thesurface density at each spot was calculated, and the rela-tionship between the peptide surface density and fluores-cence signal was plotted after curve fitting. The standardcurve was then used to correlate peptide surface densitywith input concentration of peptide solutions for the IPNsurface.

Cell culture

Bovine aortic endothelial cells (BAECs) were harvestedfrom calf aorta by treating the luminal surfaces of the aortabriefly with collagenase as described previously.27 BAECwere cultured and expanded in Dulbecco’s modifiedEagle’s medium (DMEM) (Invitrogen, Carlsbad, CA) sup-plemented with 10% fetal bovine serum (FBS) (Invitrogen),4 mM L-glutamine, and penicillin (100 units/mL)-strepto-mycin (100 lg/mL) (complete medium).

Cell adhesion assay

BAEC were seeded onto GRGDSP-IPN surfaces in 24-well plates (300,000 cells/well) in DMEM supplementedwith 1% FBS, 4 mM L-glutamine, and penicillin (100 units/mL)-streptomycin (100 lg/mL) (low-serum medium), andincubated at 378C in a humidified incubator with 5% CO2

overnight. After incubation, the surfaces were washedwith PBS three times to remove dead and nonadherentcells. Adherent cells were fixed with 4% paraformaldehyde(Sigma-Aldrich) in PBS solution. After permeabilization ofcell membranes with 0.5% Triton-X 100 solution (Sigma-Aldrich), the cell nuclei were stained with 10 lg/mL pro-pidium iodide (PI) (Sigma-Aldrich) in PBS solution for 10min. Nuclei of adherent BAEC were visualized with aNikon TE300 inverted fluorescence microscope and aHamamatsu Orca100 cooled CCD monochrome camera.Digital images were captured with C-Imaging System soft-ware (Compix, Cranberry Township, PA) and cell adhe-sion was quantified using Scion Image.

Analysis of protein activity

Western blotting was performed to determine the phos-phorylation levels of extracellular signal-regulated kinase(ERK) in BAEC after adhesion to GRGDSP. BAEC wereseeded on GRGDSP-IPN surfaces in 24-well plates (300,000cells/well) in low-serum medium for various lengths oftime. BAEC kept in suspension (on IPN surfaces with noGRGDSP) were used as a negative control. The sampleswere washed with PBS and adherent cells were lysed withlysis buffer supplemented with 1 mM Na3VO4, 1 mM phe-nylmethylsulfonylfluoride (PMSF) and 100 lg/mL leupep-tin (Sigma-Aldrich). After centrifugation for 10 min at13,400 g, the collected protein lysates were quantifiedusing DC protein assay (Bio-Rad, Hercules, CA). The pro-teins in the lysates were then separated by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE)and blotted onto nitrocellulose membranes. After blockingwith 3% nonfat milk for 1 h, the membranes were probedwith antiphosphorylated ERK (p-ERK)(Thr202/Tyr204)mouse monoclonal IgG (1:500 dilution, Cell Signaling, Bev-erly, MA) antibody for 2 h. The membranes were thenincubated with antimouse IgG conjugated with horserad-ish peroxidase (1:1000 dilution, Santa Cruz Biotechnology,Santa Cruz, CA) for 1 h. After thorough washing, themembranes were incubated with ECL solution (AmershamBiosciences, Piscataway, NJ) for 1 min. Kodak Biomax MRfilms were exposed to the ECL treated membranes for 15and then 30 s. The exposed films were developed with aKodak X-OMAT 1000A film processor, scanned and savedas TIFF images for further analysis.

Cell proliferation analysis

BAEC were seeded onto GRGDSP-IPN surfaces in 24-well plates (300,000 cells/well) in low-serum medium, andincubated overnight in a humidified incubator. The follow-ing day, 10 lM bromodeoxyuridine (BrdU) (Amersham)was added to the media, and cells were returned to the in-cubator for 1 h. After incubation, the surfaces werewashed with PBS three times and the cells were fixed with4% paraformaldehyde. Cell membranes and nuclei werepermeabilized with 50% methanol treatment followed by0.5% Triton X-100 treatment. Cellular DNA was then dena-tured by incubating samples in 2N HCl at 378C. Followingpermeabilization, the samples were incubated with mouseanti-BrdU IgG (0.25 lg/mL in PBS) (Becton Dickinson, SanJose, CA) for 1.5 h at 378C. Subsequently, the sampleswere incubated with donkey antimouse FITC conjugatedIgG (14 lg/mL in PBS) (Jackson Immunoresearch, WestGrove, PA) for 1 h at 378C. Finally, the cell nuclei werecounterstained with 10 lg/mL PI in PBS for 10 min. BrdUstaining and PI counterstaining were visualized andimaged using the same microscope/software setup asmentioned above.

Analysis of cell migration

BAEC were seeded onto the GRGDSP-IPN surfaces ofslides (40,000 cells/slide) in low-serum medium. The slides

EC FUNCTION ON IPN-LINKED GRGDSP PEPTIDE 425


were incubated in the incubator for 2 h to allow for cellattachment and spreading. After 2 h, the slides weresecured onto a square plastic dish with adhesive tape, andplaced on the scanning stage of a Nikon TE300 invertedmicroscope to monitor cell migration. The microscope waskept in a temperature and humidity controlled environ-ment to maintain temperature at 378C and ventilated with95% humidified air and 5% CO2. Fifteen fields wereselected for each slide and cell migration was monitoredusing a scanning stage and C-Imaging Software with imag-ing cycle intervals of 20 min. The images for each fieldwere exported as AVI movie files. The movie files wereanalyzed with Dynamic Image Analysis Software (DIAS)(Solltech, Oakdale, IA) to analyze individual cell migra-tion. Cells that came in contact with other cells or did notmigrate were excluded from the final analysis. Imagesfrom the cell migration samples (2 h after initial cell seed-ing) were also used to assess cell spreading area usingScion Image (Scion, Frederick, MD).

Analysis of cell migration under flow

To study migration in a more physiologically relevantsetting, we examined EC migration on GRGDSP-IPN surfa-ces under fluid flow. BAEC were seeded on the GRGDSP-IPN surfaces of slides (40,000 cells/slide) in low-serum me-dium and incubated for 2 h to allow cell attachment andspreading. After 2 h, the slides were loaded onto a parallelflow chamber as described previously.28 Briefly, a slidewith BAEC was mounted in a rectangular flow channel(0.025 cm in height, 1.0 cm in width, and 5.0 cm in length)created by sandwiching a silicone gasket between the glassslide and an acrylic plate. The channel had an inlet and anoutlet for perfusing the cultured cells with medium. Lami-nar shear stress was generated by using a MasterFlex peri-staltic pump (Cole-Parmer Instrument Company, VernonHills, IL) and a damping reservoir. Shear stress was calcu-lated by the following equation: s ¼ 6lQ/Wh2, where s isshear stress, l is viscosity of the medium (0.0084 poise), Qis the flow rate across the flow chamber, and W and h arethe width and height of the chamber respectively. All ex-periments were conducted with shear stress at 12 dyn/cm2,which is the median level of shear stress in arteries and hasbeen shown to regulate EC gene expression, migration, andother functions.29,30 During the flow experiments, the sys-tem was kept at 378C in a constant temperature hood, andcirculating medium was ventilated with 95% air and 5%CO2. The flow chamber was mounted onto the NikonTE300 microscope scanning stage. Cell migration was thentracked and analyzed using the same microscopy andimaging setup as for static migration samples.

Immunostaining and confocal microscopy

In addition to time-lapse microscopy, the cells on theGRGDSP-IPN slides were stained for vinculin to compareBAEC focal adhesions (FAs) between static and flowtreated samples. BAEC were seeded on the GRGDSP-IPNmodified surfaces of slides at the same conditions asabove. One set of slides was subjected to 12 dyn/cm2

shear stress for 2 h while another set was kept under staticcondition. Subsequently, BAEC on both sets of slides werefixed with 4% paraformaldehyde and permeabilized with0.5% Triton-X100 in PBS. The slides were incubated withmouse antivinculin IgG monoclonal antibody (Sigma) (1:50dilution) for 1.5 h and FITC conjugated donkey antimouseIgG (14 lg/mL in PBS) for 1 h. The vinculin staining wasvisualized using a Leica confocal microscope and digitalimages were captured with Leica Confocal Imaging Soft-ware (Leica Microsystems AG, Germany).

Statistical analysis

For statistical analysis, the experiments were run in trip-licate and significant differences were detected usingANOVA and Holm’s t-test. Curve fitting and regressionanalysis were performed by using Excel and CurveExpertsoftware.

RESULTS

Quantication of peptide surface density

We first quantified the surface density of peptidesconjugated on the IPN surface. Sulfo-SMCC acti-vated IPN surfaces were incubated with various con-centrations of CGG-FITC and the densities of conju-gated peptide were measured using a fluorescenceplate reader. As shown in Figure 1, the surface den-sity of the conjugated peptide increased with theincrease of peptide concentration (0–20 lM) in the

Figure 1. Quantification of peptide surface density. Pep-tide density was measured by conjugating a CGG-FITCpeptide onto IPN surfaces and measuring the fluorescencesignal using a Biotek FLx800 plate reader. To correlate flu-orescence intensity of the conjugated peptide with surfacedensity a standards curve was generated by spotting vary-ing concentrations (0.001–0.1 lM) of CGG-FITC peptideand measuring fluorescence intensities with the platereader. Surface density of the conjugated peptide was plot-ted as a function of the input solution concentration of thepeptide. The experimental data were fitted with an expo-nential curve.

426 PATEL ET AL.


solution used for the conjugation step. The relation-ship could be fitted with an exponential functionthat gradually approached a plateau. The peptidesurface density ranged between 0.01 and 3 pmol/cm2 for this study. These results suggest that we canachieve well-defined peptide density with our exper-imental condition. Since both RGD and FITC wereconjugated to IPN surfaces through a CGG peptidelinker, the results from CGG-FITC conjugation experi-ments should represent those using CGGGRGDSPpeptide. The conjugation efficiency of the peptideswas different from that in previous work17 due to themodification of the experimental conditions (seeMethods section).

Cell adhesion and spreading

We then determined the effects of peptide densityon cell adhesion and spreading as shown in Figures2 and 3. Cell adhesion and spreading were observed

on surfaces where the GRGDSP density was at least0.2 pmol/cm2. The control IPN surfaces, withoutgrafted GRGDSP, prevented cell adhesion. Cell adhe-sion on surfaces with less than 0.2 pmol/cm2

GRGDSP was negligible (data not shown). Cell adhe-sion and spreading on the GRGDSP-IPN surfacesincreased with the surface density of GRGDSP,reached a plateau at medium GRGDSP density(1.5–2.8 pmol/cm2), and increased further. On the0.2 pmol/cm2 GRGDSP-IPN surface, BAEC adhesionand spreading were significantly less than that on allother GRGDSP-IPN surfaces (p < 0.05). BAEC adhe-sion on the 3 pmol/cm2 GRGDSP-IPN surface wassignificantly higher than on all other surfaces (p <0.05), with as much as five times higher cell adhe-sion when compared with 0.2 pmol/cm2 GRGDSP-IPN surfaces. These results suggest that cell adhesionand spreading increase with the density of GRGDSPwithin the range of 0.2–3 pmol/cm2 with anincrease-plateau-increase pattern.

Figure 2. BAEC adhesion on GRGDSP-grafted IPN surfa-ces. BAEC were cultured on GRGDSP-IPN surfaces in 24-well plates overnight. Adherent cells were fixed andstained for nuclei with propidium iodide. (A) Fluorescenceimage of nuclei in adhered BAEC on 0.2 pmol/cm2

GRGDSP-IPN surface. Scale bar ¼ 50 lm. (B) Fluorescenceimage of nuclei in adhered BAEC on 3.0 pmol/cm2

GRGDSP-IPN surface. (C) Statistical analysis of the num-ber of BAEC adhered on IPN surfaces grafted with differ-ent GRGDSP densities (0.2, 0.8, 1.5, 2.1, 2.8, and 3.0 pmol/cm2). The experimental data were fitted with a polynomialcurve. * Significant difference (p < 0.05) in comparison toall other samples.

Figure 3. BAEC spreading on GRGDSP-IPN surfaces.BAEC were cultured on GRGDSP-IPN surfaces in 24-wellplates for 2 h. BAEC spreading was imaged using phasecontrast microscopy. Representative images of adheredand spread BAEC on 0.2 pmol/cm2 GRGDSP-IPN (A) and3.0 pmol/cm2 GRGDSP-IPN (B) surfaces are shown. Scalebar ¼ 50 lm. (C) Statistical analysis of BAEC spreadingarea on IPN surfaces grafted with varying densities ofGRGDSP peptide (0.2, 0.8, 1.5, 2.1, 2.8, and 3.0 pmol/cm2).The experimental data were fitted with a polynomialcurve. * Significant difference in comparison to all othersamples (p < 0.05). # Significant difference (p < 0.05) incomparison to 3.0 pmol/cm2 GRGDSP-IPN sample.



Protein activity

Since ERK has been shown to mediate cell adhe-sion-induced signaling and regulate multiple cellfunctions, we determined the activation of ERK byusing the antibody against p-ERK(Thr202/Tyr204). Atime course of p-ERK is shown in Figure 4(A). Uponcell adhesion, p-ERK increased with an early (5 min)and a late (30 min) peak, possibly due to initialintegrin-RGD binding and cell spreading respec-tively. To determine the effect of GRGDSP densityon ERK activation, we measured ERK activity at

5 min after cell adhesion. As shown in Figure 4(B),ERK activity increased at the lowest GRGDSP den-sity (0.2 pmol/cm2) that allowed cell adhesion, andplateaued beyond this density.

Cell proliferation

BAEC proliferation rate was measured as a per-centage of nuclei that incorporated BrdU in anygiven field of adhered BAEC on GRGDSP-IPN surfa-ces. As shown in Figure 5, BAEC proliferated atapproximately the same rate on all GRGDSP-IPNsurfaces regardless of GRGDSP surface density. Onaverage, 25–30% of the total BAEC on each surfacewere proliferating based on BrdU incorporation rate.There were no statistical differences in proliferationrate between the cells on different surface densitiesof GRGDSP, suggesting that cell spreading for*500 lm2

at 0.2 pmol/cm2 GRGDSP surface density was suffi-cient to support EC proliferation.

EC migration under static condition

The effect of GRGDSP density on the migration ofBAEC on GRGDSP-IPN surfaces was determined byusing time-lapse microscopy. Migration path analysisrevealed that cells were motile on IPN surfaces withdifferent GRGDSP densities, and representative migra-tion paths are shown in Figure 6(A,B). ECmigration onthe 0.2 pmol/cm2 RGD surface was significantly fastercompared with higher density surfaces, suggestingthat integrin-RGD binding is not a limiting factor forcell migration under this condition. The dependenceof migration speed on GRGDSP density includedthree phases: a quick decrease (0.2–0.8 pmol/cm2), aplateau region (0.8–1.5 pmol/cm2) and a slower and

Figure 4. The activation of signaling molecules byGRGDSP-IPN surfaces. (A) BAEC were kept in suspension(S) or seeded on 2.1 pmol/cm2 GRGDSP-IPN surfaces forvarious lengths of time, and lysed for ERK phosphoryla-tion assay. The amount of ERK was detected by re-probingthe same membrane with an antibody against ERK2. Statis-tical analysis of ERK activation from three independentexperiments is shown in the lower panel. * Significant dif-ference (p < 0.05) in comparison to suspension samples (t¼ 0). (B) BAEC were kept in suspension or seeded on pep-tide-grafted IPN with different densities of GRGDSP (0.2,0.8, 1.5, 2.1, 2.8, and 3.0 pmol/cm2) for 5 min, and the pro-tein lysates were used for immunoblotting analysis forERK phosphorylation. The amount of ERK was detectedby re-probing the same membrane with an antibodyagainst ERK2. Statistical analysis of ERK activation fromthree independent experiments is shown in the lowerpanel. * Significant difference (p < 0.05) in comparison tosuspension samples on IPN surfaces without GRGDSPpeptide (no adhesion).

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gradual decrease (1.5–3 pmol/cm2). These resultssuggest that higher density of GRGDSP may increaseEC adhesion but decrease EC motility and the rateof decrease in migration is dependent on the rangeof GRGDSP density. Interestingly, the migration per-sistence showed a similar trend to that of migrationspeed, suggesting that the time interval for persistentmigration almost remained a constant for cell migra-tion on different GRGDSP densities.

EC migration under flow condition

To study EC migration on GRGDSP-coated surfa-ces under fluid flow, the migration of BAEC in the

flow chamber was tracked for 10 h using time-lapsemicroscopy. Although cell membrane ruffling wasobserved, individual BAEC failed to migrate effec-tively on both the 0.2 and 2.1 pmol/cm2 GRGDSP-IPN surfaces under flow condition with shear stressat 12 dyn/cm2. The cell paths revealed largely sta-tionary cells for the entire duration of the experi-ment (Fig. 7).

We postulated that shear stress induced the as-sembly and strengthening of cell adhesions onGRGDSP, thus retarding cell motility. Therefore, weperformed immunostaining of vinculin and confocalmicroscopy to determine the amount and size of FAson GRGDSP-coated surfaces. As shown in Figure 8,shear stress induced lamellipodial protrusion in theflow direction. When compared with the static con-trol, the cells subjected to shear stress showed more

Figure 5. Proliferation rate of BAEC on GRGDSP-IPNsurfaces. BAEC were cultured overnight on GRGDSP-IPNsurfaces (0.2, 0.8, 1.5, 2.1, 2.8, and 3.0 pmol/cm2). The sam-ples were then incubated with BrdU containing media for1 h. After 1 h incubation, BAEC were fixed, permeabilizedand stained for BrdU incorporation. Propidium iodide wasused to stain the nuclei of all adherent cells. BAEC prolif-eration rate was determined as the percentage of BrdUincorporated cells within any given field. (A) Representa-tive fluorescence image of BAEC nuclei with incorporatedBrdU. (B) Representative fluorescence image of all adher-ent BAEC nuclei stained with propidium iodide. (C) Statis-tical analysis of EC proliferation rate represented as a per-centage of proliferating cells in a given field. Scale bar ¼50 lm.

Figure 6. EC migration on GRGDSP-IPN surfaces understatic condition. BAEC were cultured on GRGDSP-IPNsurfaces (0.2, 0.8, 1.5, 2.1, 2.8, and 3.0 pmol/cm2) andallowed to adhere for 2 h. After 2 h incubation, time-lapsephase contrast microscopy was used to track cell migrationfor 10 h with images taken at 20-min intervals. Migrationdata was exported as a movie file and analyzed usingDIAS software. Representative migration paths of BAECon 0.2 pmol/cm2 (in A) and 3.0 pmol/cm2 (in B)GRGDSP-IPN surfaces. Scale bar ¼ 50 lm. (C) Statisticalanalysis of BAEC migration speed under static conditionson GRGDSP-IPN surfaces. * Significant difference (p <0.05) compared with all other samples. #p < 0.05 in com-parison to 3.0 pmol/cm2 GRGDSP-IPN samples. (D) Statis-tical analysis of persistence of BAEC migration under staticconditions on GRGDSP-IPN surfaces. * Significant differ-ence (p < 0.05) compared with 0.8 and 3.0 pmol/cm2

GRGDSP-IPN samples. # Significant difference (p < 0.05)in comparison to 3.0 pmol/cm2 GRGDSP-IPN samples.



vinculin staining, with a increased number of largeFAs, suggesting that shear stress increases the as-sembly of FAs that may inhibit cell migration andthat RGD-integrin binding may have a catch-bondcharacteristic.

DISCUSSION

The nonfouling IPN surface allowed us to studyspecific interactions of integrins and graftedGRGDSP ligand, and determine the effects ofGRGDSP surface density on EC adhesion, spreading,proliferation, and migration. The presentation ofGRGDSP ligands was achieved by using the hetero-bifunctional crosslinker, sulfo-SMCC. Once graftedto the di-amino-PEG molecules via the NHS-ester,the free maleimide on sulfo-SMCC specificallyreacted with the sulfhydryl containing residue, Cys,at the N-terminus of the GRGDSP peptide. This spe-cific interaction ensured the same orientation for allGRGDSP peptides on the IPN surface. The densitiesof GRGDSP in this study cover a wide range from alow density that barely supports cell adhesion to ahigh density that saturates the surface bindingcapacity and allows sufficient cell spreading.

Our results suggest that the density of GRGDSPpeptide at 0.2 pmol/cm2 is the lowest threshold forcell adhesion and spreading because further titrationof the peptides was not sufficient for cell adhesionand spreading (data not shown). If we assume thatthe peptides distribute uniformly across the surface,

0.2 pmol/cm2 is equivalent to the density with pepti-des about 30 nm apart on the surface. Interestingly,a recent study using nanostructured surfaces withequally spaced gold nanodots provided exact mea-surements of minimal densities required for cellattachment.31 As each gold nanodot (<8) was graftedwith RGD peptides, it could only bind to one integ-rin molecule. This study suggested that the distancebetween 58 and 73 nm was the length scale forintegrin clustering and activation and was necessaryfor cell attachment. Our data fit closely with thisrange of RGD peptide densities, although there maybe variation in peptide clusters and distribution onIPN surfaces.

Since GRGDSP is the main cell-binding domain inFN, we used it as a model to test the dependence of

Figure 7. BAEC migration under shear stress onGRGDSP-IPN surfaces. BAEC were cultured on GRGDSP-IPN slides with 0.2 and 2.1 pmol/cm2 GRGDSP density.The slides were incubated for 2 h to allow cells to adhereand spread. After 2 h incubation, the slides were loadedonto flow chambers and subjected to shear stress at 12dyn/cm2. BAEC migration was tracked using time-lapsephase microscopy for 10 h with images taken at 20-minintervals. Representative migration paths of BAEC on 0.2pmol/cm2 (A) and 2.1 pmol/cm2 (B) GRGDSP-IPN slidesare shown here. Arrow indicates direction of fluid flow.Scale bar ¼ 50 lm.

Figure 8. Effect of shear stress on FAs on the GRGDSP-IPN surface. BAEC were cultured on 2.1 pmol/cm2

GRGDSP-IPN slides. After 2-h incubation, one slide wasloaded into a parallel plate flow chamber and subjected toshear stress at 12 dyn/cm2 for 2 h. The other slide was keptin static conditions for 2 h. BAEC on both slides were fixed,permeabilized and stained for vinculin. Fluorescenceimages were taken with a Leica confocal microscope. Rep-resentative images of BAEC FAs on samples kept in staticconditions (A) and on samples exposed to shear stress (B).Arrow indicates the FAs in lamellipodial protrusion in theflow direction. Scale bar ¼ 25 lm. (C) Quantitative analysisof FAs. The number and sizes of FAs in each cell (n ¼ 5 foreach sample) were quantified using Scion Image software.The FAs were grouped into two categories according to thesize. Small FAs had area less than 100 pixel2 (12 lm2), andlarge FAs had area larger than 100 pixel2. * Significant dif-ference (p < 0.05) compared with static samples.

430 PATEL ET AL.


cell functions on the peptide density. However, sinceBAEC has many integrin subunits such as av, a5, a1,a2, a6, b1, and b3 (data not shown), the effect ofRGD on EC functions may be mediated by multipleintegrins, e.g., a5b1 and avb3. Future studies usingmore selective peptides can be performed to differ-entiate the role of each integrin molecule in EC func-tions.

A linear peptide bound to a surface may be sub-jected to changes such as enzymatic cleavage andconformation changes, which may result in thechanges of peptide density and activity on surfacesduring long-term experiments, e.g., proliferation andmigration experiments. These changes at nano scaleawait further characterization. However, when wemonitored cell migration for 10 h, we did not detectsignificant change of migration speed with time, sug-gesting the density of peptides on IPN surfaces maynot have significant changes or may not have signifi-cant effects on EC functions within our experimentaltime frame.

Our data showed that both cell adhesion andspreading increased with the surface density ofligands (0.2–1.5 pmol/cm2), reached a plateau (1.5–2.8 pmol/cm2), and increased further beyond 2.8pmol/cm2 (Figs. 2 and 3). The underlying mecha-nism is not known. One explanation is that the firstphase of increase is related to the increase of RGDpeptide density and that the second phase ofincrease may be related to the extensive clustering ofRGD peptides. At the GRGDSP densities that sup-ported cell adhesion, no significant differences inBAEC proliferation rate were detected (Fig. 5). It islikely that even a low level of cell spreading (>500lm2) is sufficient to activate the essential signalingpathways required for cell proliferation.

At the molecular level, a low density of GRGDSPpeptide (0.2 pmol/cm2) was sufficient to activateERK (Fig. 4), suggesting that integrin-mediated sig-naling is activated if the density of GRGDSP peptideis enough to support cell adhesion and spreading.The level of ERK activation may be limited to thedensity and availability of cell surface integrin recep-tors and/or the kinetics of ERK activation by integ-rins. Interestingly, we detected two waves of ERKactivation by GRGDSP peptide. The exact mecha-nism of this two-phase activation is not clear. It ispossibly related to initial integrin-RGD binding andcell spreading respectively.

Previous studies have shown that the density ofECM proteins controls the level of integrin-ECM ad-hesive interaction and modulates cell migrationspeed. At low adhesiveness, the cell cannot formstrong and stable adhesions at its leading edge togenerate traction force; at high adhesiveness, the cellcannot break the cell-ECM adhesions at the trailingedge. Therefore, cell migration shows a maximum

speed at intermediate levels of adhesiveness.32–35 ECmigration on FN shows a biphasic dependence onFN surface density under both static and flow condi-tion.36 However, BAEC migration on GRGDSP-IPNsurfaces was faster at low density (0.2 pmol/cm2)(Fig. 6), which is different from that on full-lengthFN, suggesting that the cell-binding domains in FNother than GRGDSP also contribute significantly toEC migration. Although the cells were unable tospread as well on the lower GRGDSP density IPNsurfaces, they were far more motile on these surfa-ces, suggesting that cell detachment at the rear,rather than cell adhesion at the front, is the limitingfactor for cell migration on GRGDSP peptide-modi-fied surface. Consistent with this concept, the de-crease of migration speed and persistence with theincrease of peptide density correlates inversely withan increase of cell attachment and spreading (Figs. 2,3, and 6). For example, the decrease of the migrationspeed had three phases: a quick decrease (0.2–0.8pmol/cm2), a plateau region (0.8–1.5 pmol/cm2),and a slower and gradual decrease (1.5–3 pmol/cm2),while cell attachment and spreading had increase-plateau-increase pattern. The decrease of migrationspeed within the lower range of peptide densityhad a much greater slope [Fig. 6(C)] than that withinthe higher range of peptide density, suggesting thatcell migration was more sensitive to the changes inpeptide density within the lower range.

EC are constantly exposed to shear stress in vivo.Shear stress induces EC cytoskeletal remodeling,activates signal transduction, and promotes ECmigration.30,37 Various studies have shown that pre-treatment of ECs in vascular grafts with shear stresscan help retain the integrity of the EC monolayer invascular grafts upon implantation.38–40 A potentialmethod of endothelialization on vascular implants isto recruit native ECs after implantation. For such sit-uations, EC migration on implant surfaces under theinfluence of shear stress is necessary. However, ourstudy showed that BAEC were unable to migrate onthe GRGDSP-IPN surfaces when subjected to shearstress (Fig. 7). To rule out effects of ligand density,we performed flow migration studies on peptide-modified IPN surfaces treated with two differentGRGDSP concentrations: 0.2 and 2.1 pmol/cm2. Onboth surfaces, the cells behaved similarly withobserved membrane ruffling but lack of cell migra-tion. In contrast, BAEC were capable of migrating onthese same surfaces under static conditions. Vinculinstaining indicated that BAECs increased the numberof large FAs on the GRGDSP-IPN surface whenexposed to flow, suggesting that the applied shearstress increased EC attachment, and therefore re-tarded EC migration under shear stress. The increasein FAs is also a possible explanation for better ECretention on GRGDSP coated implant surfaces under



shear stress conditions. In this sense, RGD-integrinbinding is a catch-bond interaction. Regardless of themechanism, these data point to potential limitationsof grafting a single GRGDSP-containing peptide to asurface to promote endothelization.

Our previous studies have shown that BAEC canmigrate efficiently on FN under flow condition.41 Wedid observe a transient increase of FAs followed bythe disassembly of FAs when ECs were subjected toshear stress. It is likely that the transient increase ofFAs is mediated by the RGD domain in FN. Sincethe cells were able to migrate on FN coated surfacesbut not on GRGDSP grafted surfaces, it is likely thateither different forms of RGD peptides or other cellbinding domains on the FN molecule mediate ECmigration under shear stress. For example, RGDpeptides with different flanking sequences, differentpeptide lengths, and structure (linear vs. cyclic) mayhave different activity in mediating cell adhesionand functions.17,19 Furthermore, FN has multiplecell-binding domains for integrins and nonintegrinreceptors on the cell surface.42–44 Besides the pri-mary interaction of GRGDSP with integrin b1 oravb3, other integrins such as a4b1 can bind to Arg-Glu-Asp-Val (REDV) and Leu-Asp-Val (LDV)domains in the type III connecting strand region.Recently, Pro-His-Ser-Arg-Asn (PHSRN) sequence inIII9 region of FN has been identified as a synergysite that cooperates with the GRGDSP sequence inmediating cell adhesion and migration,45–47 andPHSRN peptide dramatically accelerates epithelialwound healing in vivo.48 Aside from FN-integrininteraction, FN binds to transmembrane heparin sul-fate proteoglycan (e.g., syndecan-4) at the cell sur-face via motifs in repeats FN12-14 such as Pro-Arg-Ala-Arg-Ile (PRARI), which act in concert with FN-integrin binding to stimulate the formation ofFAs.49–51 Although cell binding domains such asREDV and PHSRN are much less effective in sup-porting EC adhesion than GRGDSP peptide, howthese cell-binding domains synergize with GRGDSPto promote EC migration under flow remain to bedetermined in a future study.

This study showed the effects of GRGDSP densityon EC adhesion, spreading and proliferation andthat low levels of GRGDSP density allowed more ef-ficient EC migration. We also revealed the highersensitivity of EC adhesion, spreading, and migrationwithin the lower range of RGD peptide density andthe three phases of peptide density dependency.However, the GRGDSP peptide used in this studywas not effective in mediating EC migration underflow condition. Other cell binding domains found ineither FN or other matrix proteins are needed for ef-ficient EC migration under flow condition. Thisstudy advances our understanding of the effects ofimmobilized GRGDSP peptide on EC function and

provides a rational framework for surface engineer-ing of cardiovascular implants.

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