Dynamics of endothelial cell responses to laminar shear stress on

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Bio-interactions and Biocompatibility

Dynamics of endothelial cell responses to laminar shear stress on surfaces functionalized with fibronectin-derived peptides Corinne Annette Hoesli, Catherine Tremblay, Pierre-Marc Juneau, Mariève D. Boulanger, Ariane V Beland, Si Da Ling, Bruno Gaillet, Carl Duchesne, Jean Ruel, Gaetan Laroche, and Alain Garnier ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.8b00774 • Publication Date (Web): 11 Oct 2018 Downloaded from http://pubs.acs.org on October 23, 2018

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ACS Biomaterials Science & Engineering

Dynamics of endothelial cell responses to laminar shear stress on surfaces functionalized with fibronectin-derived peptides 1*

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Corinne A. Hoesli , Catherine Tremblay , Pierre-Marc Juneau , Mariève D. Boulanger 1

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135

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, Ariane V.

Beland , Si Da Ling , Bruno Gaillet , Carl Duchesne , Jean Ruel , Gaétan Laroche , Alain Garnier 1

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Department of Chemical Engineering, Faculty of Engineering, Wong Building, 3610 University Street, McGill University, Montréal, Québec, H3A 0C5, Canada

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Département de génie mécanique, Faculté des sciences et de génie, Université Laval, pavillon AdrienPouliot, 1065 avenue de la Médecine, Québec (Québec), G1V 0A6, Canada 3

Département de génie chimique, Faculté des sciences et de génie, Université Laval, pavillon AdrienPouliot, 1065 avenue de la Médecine, Québec (Québec), G1V 0A6 Canada 4

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PROTEO Research Center, Québec, Canada

Centre de Recherche sur les Matériaux Avancés, Département de génie des mines, de la métallurgie et des matériaux, Université Laval, Québec, Canada

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Centre de recherche du CHU de Québec, Hôpital Saint-François d’Assise, 10 rue de l’Espinay, bureau E0-165, Québec (Québec), G1L 3L5, Canada

*Corresponding author. e-mail: [email protected]

ACS Paragon Plus Environment 1

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ABSTRACT

Surface endothelialization could improve the long-term performance of vascular grafts and stents. We previously demonstrated that aerosol-generated fibronectin-derived peptide micropatterns consisting of GRGDS spots over a WQPPRARI background increase endothelial cell yields in static cultures. We developed a novel fluorophore-tagged RGD peptide (RGD-TAMRA) to visualize cell-surface interactions in real-time. Here, we studied the dynamics of endothelial cell response to laminar flow on these peptide2

functionalized surfaces. Endothelial cells were exposed to 22 dyn/cm wall shear stress while acquiring time-lapse images. Cell surface coverage and cell alignment were quantified by undecimated wavelet transform multivariate image analysis. Similar to gelatin-coated surfaces, surfaces with uniform RGDTAMRA distribution led to cell retention and rapid cell alignment (˜63% of the final cell alignment reached within 1.5 hours), contrary to the micropatterned surfaces. The RGD-TAMRA peptide is a promising candidate for endothelial cell retention under flow while the spray-based micropatterned surfaces are more promising for static cultures.

KEYWORDS : endothelial cells, shear stress, live cell imaging, peptides, surface modification


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INTRODUCTION 1

Up to 1 out of 3 deaths worldwide are caused by cardiovascular disease . Over 50 000 coronary revascularization procedures (comprising coronary artery bypass surgeries and percutaneous coronary 2

3

interventions) are performed in Canada each year , and this number increases to >1 million in the US . The main complications after these interventions are restenosis and thrombosis, which could be prevented by the endothelialization of the vascular substitutes placed into contact with the circulation. Several strategies have been investigated to achieve the endothelialization of vascular substitutes such as stents, vascular grafts or vascular scaffolds. For example, endothelial cells can be expanded in vitro 4

and seeded on vascular grafts prior to transplantation . The surface adsorption or conjugation of extracellular matrix-derived proteins or peptides has been investigated to promote endothelial cell adhesion to vascular substitutes both in vitro and in vivo. These proteins or peptides may be micropatterned to mimic the anisotropic distribution of cell ligands in the normal endothelial cell 5

extracellular matrix, or to favor endothelial cell alignment . Micropatterns of covalently grafted extracellular matrix-derived peptide or protein micropatterns have been applied to polytetrafluoroethylene 6-8

9

(PTFE) , polyethylene terephthalate and other polymers

10

via techniques such as microcontact printing,

inkjet printing and photolithography. We previously developed an aerosol-based technique to generate surface-conjugated peptide micropatterns on aminated surfaces via a bifunctional linking arm that reacts 7

with thiol groups (e.g. on Cys) and primary amines . The main advantage of the aerosol micropatterning techniques is the simplicity and versatility of spraying nozzles. Sprayed patterns can more readily be applied to the lumen of existing vascular grafts and stents than more uniform and controlled patterns obtained by printing, stamping or other common patterning techniques. However, understanding the effects of the patterns on cell-surface interactions becomes more challenging than for well-controlled pattern sizes and geometries such as stripes. In previous studies, we applied the aerosol-based technique to micropattern the fibronectin-derived 6-8, 11

GRGDS and WQPPRARI peptides onto aminated PTFE

and glass substrates. In static cultures,

significantly higher yields of bovine aortic endothelial cells were obtained on surfaces with micropatterns 7

consisting of ~10 µm diameter GRGDS spots with WQPPRARI peptides covering the remaining surface . We also found that these aerosol-based micropatterns, similar to 300 µm x 300 µm checkered patterns,



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significantly increased the yields of human saphenous vein endothelial cells (HSVECs) after 9 days of 2

expansion. In the presence of flow (15 dyn/cm for 6 hours), the mean focal adhesion size was higher on surfaces with peptide micropatterns compared to surfaces with uniform peptide distribution, although the 6

extent of actin filament alignment was similar on both surfaces . Probing the mechanism by which the peptide patterns affected cell growth in static cultures and cell alignment under flow was challenging due to the opaque PTFE surfaces used and difficulty in localizing the peptide patterns on the surfaces. To study the local interactions between endothelial cells and the peptide micropatterns using live cell 12

imaging, we previously designed a fluorophore-labelled RGD peptide (referred to as “RGD-TAMRA”) . This peptide consisted in the GRGDS sequence added to the C-terminus of a CGKG sequence used for surface conjugation and fluorophore labelling. The TAMRA fluorophore was linked to the lysine side chain of this peptide via a 3-subunit length polyethylene glycol chain used as a spacer to avoid steric hindrance between the fluorophore and the cell adhesion motif. In static conditions, the RGD-TAMRA peptide significantly accelerated HSVEC spreading kinetics compared to the GRGDS peptide. On surfaces micropatterned with RGD-TAMRA and WQPPRARI using the aerosol technique, the cell shape was strongly affected by the location of patterns in the cell vicinity, as cells pseudopods anchored to the RGD12

TAMRA spots via focal adhesions . However, the effects of the RGD-TAMRA peptide and of the micropatterns were not probed under flow, which would better reflect the intended in vivo use of these novel biomaterials.

The response of endothelial cells to mechanical stimuli including shear stress is a key determinant of vascular homeostasis. In appropriate test conditions, the shear stress levels found in coronary arteries 2

2

(15 dyn/cm to 30 dyn/cm ) lead to endothelial cell alignment in the direction of flow, reduced endothelial cell proliferation rates, increased expression of anti-inflammatory molecules and atheroprotective effects

13-14

. The culture conditions that impact this process include cell confluency, the composition of the

test medium, the composition of the test surface, as well as the time and geometry-dependent flow 15

profile . Focal adhesions act as mediators in both outside-in and inside-out shear stress-dependent mechanotransduction pathways

15-16

. The endothelial cell adhesion substrate profoundly affects 17

endothelial cell responses to laminar flow. For example, we

and others

18-19

have previously shown that a


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simple change in the surface conjugation strategy of fibronectin may dramatically alter endothelial cell adhesion to the surface, as well as the response of the cells to shear stress. For HSVECs seeded on alternating WQPPRARI and GRGDS stripes of 50 µm to 100 µm width oriented in the direction of flow, we observed more rapid stress fiber alignment in the direction of flow than on surfaces with uniform peptide 9

distribution . On more confined 15 µm width fibronectin stripes, Wu et al. observed reduced numbers of stress fibers, a lack of focal adhesion kinase phosphorylation and increased rates of apoptosis compared 10

to wider stripes . When flow was applied parallel to the stripes but not perpendicular to the stripes, apoptosis was reduced.

The fluorophore-tagged RGD-TAMRA peptide offers the opportunity to better understand the effect of the aerosol-based peptide micropatterns on endothelial cell adhesion and alignment under flow in real-time. In the current study, we report the dynamic effects of the RGD-TAMRA:WQPPRARI peptide micropatterns on endothelial cells exposed to flow. To monitor cell-surface interactions in real-time, we first developed a multi-well parallel-plate flow chamber system suitable for live cell imaging. To fully exploit the potential of this live cell imaging system, we adapted an undecimated wavelet transform multivariate image analysis method

20

to quantify cell alignment kinetics under flow. We then used this

platform to quantify endothelial cell retention, cell alignment kinetics as well as focal adhesion formation on the peptide micropatterned surfaces. The results from this study confirm RGD-TAMRA as a promising candidate for further testing in vascular applications, while endothelial cells were poorly retained on micropatterned surfaces or WQPPRARI alone. The multi-well flow chamber, live cell imaging and textural image analysis platform developed in this study could be applied to further study the dynamics of endothelial cell retention and alignment under flow on a variety of surfaces.

EXPERIMENTAL SECTION Materials and Methods Peptides The CWQPPRARI (referred to as “WQPPRARI”) peptide was produced by Thermo Fisher Scientific (Waltham, MA), whereas the RGD-TAMRA peptide was produced by Anaspec (Fremont, CA). The RGD-



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12

TAMRA peptide has been previously described . The sequence of the RGD-TAMRA peptide is CGK(PEG3-TAMRA)-GGRGDS-NH2, where PEG3 represents a three unit-long ethylene glycol spacer grafted onto the side chain of a lysine residue, TAMRA represents the 5-carboxytetramethylrhodamine fluorophore, and -NH2 represents C-terminal amidation of the peptide. The RGD-TAMRA peptide was solubilized at 2 mM in dimethyl sulfoxide (Sigma-Aldrich, St. Louis, MO) and kept frozen until use. Immediately prior to use, peptide solutions were reconstituted at 20 µM in a solution containing 7.5% glycerol added to 0.2 µm-filtered 10 mM citrate (Sigma-Aldrich) buffer at pH 7.4.

Surface conjugation and micropatterning Aminoalkylsilane-treated glass slides (Silane slides, Electron Microscopy Sciences, Hatfield, PA) were 12

cut to 3.0 cm length and functionalized as previously described . For static controls, the test surfaces were cut to ~1 cm length to fit into 6-well plates. Unless otherwise mentioned, all incubations were performed with 50 rpm agitation at room temperature in the dark in humidified chambers. The glass surfaces were first reacted for 2 h with 600 µL/slide of a 3 mg/mL suspension of sulfo-succinimidyl-4-(pmaleimidophenyl)-butyrate (S-SMPB, Thermo Fisher Scientific, Waltham, MA) in 0.2 µm-filtered calciumfree phosphate buffered saline (PBS, Thermo Fisher Scientific). To minimize reaction volumes, the circumference of each slide was lined with Teflon tape to maintain the solution on the slide surface. The slides were rinsed in distilled deionized water (ddH2O), air-dried and stored overnight in the dark before reacting with peptides. For uniform peptide treatments, the slides were immerged in solutions containing either 20 µM RGD-TAMRA, 20 µM WQPPRARI, or a 35:65 mixture of the two peptides containing 7 µM RGD-TAMRA and 13 µM WQPPRARI. The micropatterned surfaces were obtained as previously described

11-12

. The aerosol-based micropatterning apparatus consisted in a x-y computer-controlled table

(Velmex, Bloomfiel, NY) placed 21 cm below a 5 cm long 22 G needle connected to a 250 µL glass syringe (Gastight No 1725, Hamilton Company, Reno, NV) vertically mounted on a syringe pump (780100C, Cole-Parmer Vernon Hills, IL). The needle was introduced into an air atomizing nozzle (SU12SS, Spraying Systems Co., Wheaton, IL) with coaxial air flow at 30 psig. During peptide spraying, the x-y table was moved at 15.8 mm/s over 15 cm x-axis distances with 0.5 cm y-axis increments to pattern the entire slide surfaces. The syringe pump rate was set to 500 µL/h to obtain ~35% slide surface


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coverage

12

. The peptide solution droplets were reacted without agitation in a humidified chamber for 3 h.

The micropatterned slides were then rinsed in PBS and reacted for 3 h in a 20 µM WQPPRARI solution. All slides were rinsed in PBS and stored for at most 1 month in filtered PBS before use. Gelatin-coated controls were obtained by incubating the glass surfaces for 20 min at room temperature in a filtersterilized 0.2% gelatin solution prepared in PBS. The gelatin-coated surfaces were then rinsed in PBS, rinsed in filter-sterilized ddH2O, allowed to dry and used immediately for experiments.

Multi-well flow chamber system design and validation A custom flow chamber system was designed and fabricated to enable live cell imaging in 4 parallelplate flow chambers. The system consisted in two polycarbonate plates holding in place 4 glass test surfaces functionalized with peptides or coated with gelatin. The system was designed to provide fully 2

developed laminar flow over a 1.13 cm imaging surface area in each chamber with a uniform wall shear 2

stress ranging from 0.2 dyn/cm2 to 40 dyn/cm . A detailed description of the flow chamber design, fabrication and validation including rheological measurements as well as particle imaging velocimetry methods and results are provided in the Supplementary Information (Error! Reference source not found.).

Flow chamber circulation loop The flow chamber and circulation loops were assembled according to Figure 1 and Figure 2 respectively. A commercial 4-channel pulsation dampener acting as a bubble trap (BioSurface Technologies Corporation, Bozeman, MT) was placed at the peristaltic pump outlet. During overnight culture at low wall shear stress, the pump used was a Masterflex RK-7543-02 fixed speed (2 rpm) drive with two Masterflex L/S two-channel Easy-Load II pump heads, using L/S 13 BPT tubing in the pump line (all from Cole-Parmer, Vernon Hills, IL). The next day, the pump drive was changed to a Masterflex L/S, EW-77521-40 drive, and the tubing to LS 25 BPT tubing (all from Cole-Parmer). The tubing used in the rest of the circulation loop was 2.2 mm ID Pharmed BPT tubing, except for 3/32” ID platinum-cured silicone tubing used for gas exchange at the medium reservoir outlet and 1/16” ID C-flex tubing used to connect the flow circuit to the imaging chamber (all from Cole-Parmer). The tubing was connected to the



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chamber via RN connectors (5117K81, McMaster-Carr) sealed by silicone O-rings (9396K13, McMasterCarr). For cell culture experiments requiring aseptic culture conditions, the polycarbonate plates and the peptide-functionalized glass surfaces were sterilized with 95% ethanol (Commercial Alcohols, Markham ON). The circulation loop, medium reservoirs, pulsation dampener, RN connectors and O-rings were autoclaved. The RN connectors were autoclaved with a small length of dead-end tubing placed on the connector outlet to facilitate aseptic handling. After sterilization, the Viton O-rings followed by the glass test surfaces were placed into the carbonate plates using autoclaved forceps. The two carbonate plates, the metal plates and the mounting brackets were then assembled using stainless steel screws and bolts (4-40UNC x ¾), followed by aseptically inserting the RN connectors. The assembled chamber was kept overnight to avoid rare instances where glass slides that were misplaced fissured overnight. The next day, the flow chambers were connected to the circulation loop. Warm degassed medium was added to the medium reservoirs, and the chamber and circulation loop were mounted onto an automated inverted microscope (IX81, Olympus, Tokyo, Japan) with environmental control provided by an in-house made cage incubator. The medium reservoirs and silicone lines were placed inside a zip-lock bag acting as an aerator supplied with 300 mL/min humidified gas consisting of a mixture of 95% air and 5% CO2. The mounted flow system was placed on the incubated microscope stage until the temperature equilibrated at 37°C. All of the flow lines were then filled with medium before starting the experiment. The medium reservoirs initially contained 50 mL medium, with ~35 mL remaining in the reservoirs after filling the flow circuit.

Figure 1. Design of the 4 flow chamber live cell imaging system. (A) overall schematic


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representation, (B) cross-sectional view of one of the chambers, 1) metal plates, 2) top plate, 3) support bracket, 4) Viton O-rings, 5) glass test surface, 6) bottom plate and (C) photograph of the assembled flow chamber system.

Figure 2. Circulation loop for the live cell imaging experiments. The dotted box represents the incubation chamber surrounding the microscope. The direction of flow was from the injection port side to the medium recirculation or waste lines.

Medium preparation The M199 + 20% FBS complete medium consisted in M199 basal medium supplemented with 20% FBS, 50 units/mL heparin, 10 ng/mL basic fibroblast growth factor (bFGF, amino acids 10-155), 50 units/mL penicillin and 50 µg/mL streptomycin (all from Thermo Fisher Scientific). The serum-free EGM-2 medium consisted in EBM-2 and Bulletkit supplements (Lonza, Basel, Switzerland), except that the serum supplied in the kit was replaced by 0.4% bovine serum albumin (BSA, Sigma-Aldrich A9647), 1.0 mg/ml recombinant human insulin, 0.55 mg/ml human transferrin and 0.5 µg/ml sodium selenite (100X diluted ITS supplement, Thermo Fisher Scientific). Antibiotics (50 units/mL penicillin and 50 µg/ml streptomycin from Thermo Fisher Scientific) were also added.

HSVEC isolation and maintenance Healthy saphenous vein segments removed during varicose vein stripping surgeries were obtained with



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the informed consent of donors at the CHU de Québec, Saint-François d’Assise Hospital in Québec city. Vein segments were stored in Hank’s balanced salt solution (Thermo Fisher Scientific) until HSVEC isolation. Cells were harvested by rinsing the vein in PBS, introducing 1 mg/mL collagenase type 1A solution (Sigma-Aldrich) into the vein and incubating for 15 min at 37°C. The collected cells were harvested and maintained in complete M199 + 20% FBS medium. The cells were maintained in tissue culture treated t-flasks (BD Biosciences, Franklin Lakes, NJ) coated for 20 min with 0.2% gelatin (type A from porcine skin, Sigma-Aldrich). Cells were frozen in 90% FBS, 10% DMSO freezing medium after the second passage. All experiments were performed with cultures containing >90% von Willebrand factor (vWF) positive cells, used at passage 5 or 6.

NB4 cell maintenance NB4 cells were a kind gift of Prof. Richard Leask, Department of Chemical Engineering, McGill University. NB4 cells were thawed and maintained at 500,000 cells/mL in RPMI medium with ATCC modifications (Thermo Fisher Scientific) supplemented with 10% FBS (Hyclone Laboratories, Logan, UT), 50 units/ml penicillin and 50 µg/ml streptomycin (both from Thermo Fisher Scientific). All-trans retinoic acid (ATRA) (Sigma) at a concentration of 10 µM was added to the cell suspension 72 h prior to starting perfusion.

Real-time imaging of endothelial cells under flow Prior to live cell imaging experiments, the flow chamber system was placed on the stage of an Olympus IX81 inverted microscope with incubated stage as shown in Figure 2. The medium used for a given experiment was circulated through the flow system at 37°C for at least 1 h prior to cell seeding to allow pH and temperature equilibration. HSVECs were trypsinized, washed in serum-containing complete medium, and then re-suspended at 600 000 cells/mL in complete medium or in serum-free EGM-2 2

medium to obtain a seeding density of 30 000 cells/cm . The HSVEC suspension was then introduced into the flow chambers through the cell injection ports. The HSVEC injection was performed by (1) closing the inlet line to the chamber upstream from the injection port, (2) closing the line to the medium reservoir and opening the line to the waste container, (3) connecting an empty syringe to the port, (4) aspirating


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medium into the syringe to remove air in the injection line, (5) connecting a new syringe containing a wellmixed cell suspension, (6) injecting the cells. After HSVEC seeding, the cells were left to adhere for 2 h in static conditions by closing the outlet lines of the flow chambers. After 2 h, the non-adhered HSVECs were removed by circulating 5 mL medium to waste at 0.8 mL/min, and then the waste line was closed to start re-circulation between the medium reservoir and the flow chamber. The HSVECs were maintained 2

overnight at low perfusion rates (0.2 dyn/cm wall shear stress). The next day, the perfusion rate was 2

increased to obtain 5 dyn/cm wall shear stress for 5 to 20 min to test for leaks before increasing the flow 2

2

rate to obtain 22 dyn/cm wall shear stress. After 6 h at 22 dyn/cm , the cell nuclei were stained with Hoechst by momentarily stopping the perfusion, infusing 2.8 µg/mL Hoechst in the corresponding medium (Thermo Fisher Scientific), and re-starting the perfusion for 30 min. During the entire duration of the experiment, between 25 and 81 phase contrast images were acquired in each chamber at 5 min to 30 min time intervals using 10X or 20X phase contrast objectives. In addition, fluorescence imaging was performed for the same imaging areas before seeding cells and after adding Hoechst to visualize the peptide micropatterns and Hoechst-labeled nuclei. At the end of the experiment, the flow chamber was rapidly removed from the microscope and flow circuit, dismantled, and the cells on the test surfaces were immediately fixed in 3.7% formaldehyde (VWR International, Radnor, PA) in PBS for 20 min at room temperature and stored in PBS.

NB4 adhesion to endothelialized surfaces under flow with live cell imaging HSVECs were seeded onto peptide-functionalized surfaces in serum-free EGM-2 medium, left to 2

adhere for 2 hours in static conditions, perfused overnight at 0.2 dyn/cm wall shear stress and then 2

-

perfused for 6 h at 22 dyn/cm wall shear stress as described above. NB4 cells were stained with 5 X 10 7

o

M calcein AM dye (Thermo Fisher Scientific) for 30 minutes protected from light at 37 C and 5% CO2. 6

The NB4 cells were then washed twice and re-suspended in serum-free EGM-2 medium at 2.5 x 10 2

cells/mL concentration. After 6 hours perfusion of the HSVECs at 22 dyn/cm , the flow was momentarily stopped to allow for the collection of 10 mL medium from each medium reservoir using a syringe. Media samples were used for cytokine quantification as described below. Immediately after, 10 mL of the NB4 cell suspension were added to each medium reservoir for a final circulating NB4 cell concentration of



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2

500,000 cells/mL. The perfusion was re-started for 1 h at 2 dyn/cm wall-shear stress. Phase contrast images at 4 positions per chamber were acquired every minute using the 10X objective at 5 ms exposure time. Following the 1 h circulation of NB4 cell suspension, surfaces with endothelial cells and NB4 cells were fixed using a 3.7 % paraformaldehyde solution (Millipore Sigma). After the experiment, 10 images per chamber were taken by fluorescence imaging to visualized adhered calcein stained cells. NB4 cell adhesion was quantified by visually comparing phase contrast images captured before NB4 cell injection and 10 min after injection. Fluorescence imaging was used to confirm that circulating cells quantified were NB4 cells (FITC+ cells).

Quantification of cytokines in circulating media 2

Media samples collected after 6 h HSVEC exposure to flow at 22 dyn/cm wall shear stress as described above were centrifuged for 10 min at 1000 x g and kept frozen at -20°C until analysis. The 10 mL samples represent 20% of the total liquid volume present in the flow loop. Supernatants were used to quantify interleukin-6 (IL-6) and interleukin-11 (IL-11) concentrations by enzyme-linked immunosorbent assays (ELISAs) using commercial kits (Abcam, Cambridge, MA) according to the manufacturer’s instructions.

HSVEC immunostaining and image analysis Fixed HSVEC samples were permeabilized for 15 min with 0.1% Triton X (VWR) in PBS. After rinsing in PBS, the slides were blocked for 15 min using serum-free protein block (Dako, Glostrup, Denmark) and antibodies against human vinculin (mouse anti-vinculin, Sigma-Aldrich) or human vWF (rabbit anti-vWF, Sigma-Aldrich) diluted 1:200 in Antibody diluent (Dako) were added. After 1 h at 37°C (anti-vinculin) or 2 h at room temperature (vWF), the slides were washed in PBS and incubated for 1 h in the dark with goat anti-mouse Alexa 488 (for vinculin) or goat anti-rabbit Alexa 568 (for vWF) secondary antibodies (both from Thermo Fisher Scientific) diluted 1:200 in Antibody diluent. The slides were then stained for 1 h at 37°C with TRITC-phalloidin (Sigma-Aldrich) diluted 1:250 in PBS before washing in PBS and ddH20, followed by staining for 10 min with 1 µg/mL DAPI (Sigma-Aldrich) diluted in ddH2O. The slides were washed with ddH2O and PBS, mounted using Vectashield and imaged on an Olympus BX51 microscope.


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Fluorescent images were analyzed using CellProfiler freeware

to identify cell nuclei, RGD-TAMRA

spots, focal adhesions and actin filaments. These objects were identified by correcting the image illumination, enhancing speckles in the case of the focal adhesions, and applying the “Robust background” CellProfiler thresholding method. To quantify actin filament alignment and the number of focal adhesions, >50 images of stained cells captured with a 60X objective were analyzed, whereas the micropatterned spots were analyzed by quantifying >1000 spots in images captured with a 4X objective. Actin filament alignment was quantified by determining the mean and standard deviation of the main axis of the actin filament objects. Note that all results are shown with the direction of flow from left to right on images.

Wavelet transform multivariate image analysis and cell alignment dynamics The cell confluency (surface coverage) and cell alignment in time-lapse phase contrast images were quantified by undecimated wavelet transform - multivariate image analysis (UWT-MIA) as previously described

20, 22

. The undecimated wavelet transform performs space-frequency decomposition of the 2D

image signals by convoluting the signals with a dilated mother wavelet at different scales and along different orientations within the image. The result of the image convolution is a so-called wavelet detail coefficients matrix (wavelet plane) representing how well the wavelet matches the signal at a given scale and position in the image. We applied the continuous Gabor wavelet transform to the image, which shape was found to match roughly the cells shape. For each image, wavelet planes (collection of wavelet detail coefficients for each pixel of the image) were generated for different combinations of wavelet scales and orientations. The information in these wavelet planes was then reduced to two principal components (PC1-PC2) using multivariate image analysis. Pixels were categorized as background pixels, cellular pixels associated with non-elongated cells or pixels associated with horizontally elongated cells by their localization in a PC1-PC2 score scatter plot (known as a “score density histogram” in the chemometrics field). Background pixels were selected by manually delimiting a region near the origin of the PC1-PC2 score density histogram. A larger region centered on the origin was delimited to select pixels characteristic of non-elongated cells (using control images). The remaining pixels were categorized as characteristic of horizontal elongation (low PC1 score and low PC2 score) or vertical elongation (high



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PC1 score and high PC2 score).In the density score histograms shown in the results, the white dotted ellipse represents pixels associated with the image background and non-elongated cells, while the pixels outside the ellipse in quadrants 2 and 4 (Q2 and Q4) are associated with horizontally elongated cells. Control images of non-aligned or horizontally-aligned cells selected visually were used to validate this approach. Moreover, the results obtained by multivariate image analysis were compared to cell alignment values based on the mean cosine angle of cell contours traced manually (see Supporting Information). Following image analysis, cell alignment dynamics were quantified based on the characteristic equation for first-order stable responses to a single step change in an input variable X (Equation 1): = ∆ 1 − ⁄

(1)

where Y’(t) is the change in the response variable Y, or Y(t)-Y(0), KP is the gain, ∆X is the value of the step change in the input variable, t is the response time after the step change, and τ is the time constant. In this case, X represents the wall shear stress experienced by cells in the imaging area and Y is the fraction of horizontally elongated cell pixels normalized to the total number of elongated cell pixels at time t after the step change. To simplify the notation, the units of Y are reported as a dimensionless percentage (horizontal cell pixels / 100 elongated cell pixels). The step change used in most experiments 2

was a change from 0.22 dyn/cm wall shear stress during overnight culture in the flow chamber to 22.5 2

2

dyn/cm applied for 6 hours on the next day, resulting in a ∆X value of 22.3 dyn/cm . The value of Y(0) was determined from the average value of Y measured at each acquisition time during overnight culture 2

at 0.22 dyn/cm wall shear stress. The value of Y at equilibrium was taken as the average values of Y measured over the last 4 time points. The value of KP was determined from the value of Y’(t)/∆X at equilibrium, where ∆Y was equal to Y at equilibrium, minus Y(0). The value of τ was determined by least squares fitting of Y’(t) as a function of the response time t or from the slope of a plot of ln(1-Y’(t)/(KP∆X)) as a function of t.

Statistics Two-way comparisons between samples relied on Student’s t-tests. Unless otherwise mentioned, results represent the average ± standard error of the mean of 3 experiments. For cell culture experiments, each experiment was performed with cells from a different donor. Multiple group means were compared


Page 15 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


by ANOVA using the cell donor as a blocking variable, followed by applying the Tukey-Kramer honestly significant difference test. When analysing cell alignment dynamics, the value of τ was determined using linear regression analysis of ln(1-Y’(t)/(KP∆X)) as a function of the response time. Analysis of variance was used to determine standard error and the p-values of the linear model and of parameter estimates. All statistical analyses were performed using JMP software (SAS Institute, Cary, NC). Results were considered statistically significant at p50 microscope stage positions per chamber approximately every 20 min. When imaging at a single stage position at higher acquisition frequencies, both adherent and circulating cells could be visualized (Error! Reference source not found. in the Supporting Information). Phase contrast imaging is a non-intrusive method to monitor live cell morphology. To quantify the surface area covered by cells, we applied a previously



Page 16 of 49

23

described cell segmentation algorithm based on range filters . This first step quantified cell retention on the surfaces as a function of time. To quantify morphological parameters in time-lapse images associated 20, 22

with cell alignment, we applied textural image analysis using UWT-MIA

. The general principle of this

method is to generate wavelets that fit the pixel intensity signal in the image. Since the wavelets have an orientation, the wavelet fitting is repeated at different angles. For cells aligned in a given orientation, wavelets with characteristic frequencies and amplitudes can be identified by the subsequent multivariate image analysis. For example, endothelial cells aligned in the direction of flow would be expected to be well-represented by a wave with relatively constant frequency in the flow direction, since the cell length should be relatively constant. In the direction perpendicular to flow, a higher frequency (lower cell width than length) would be expected. We previously successfully applied this approach to quantify cell growth kinetics and to extract morphological features including local cell alignment in myoblast cultures

20, 22

.

To determine whether UWT-MIA could be used to quantify endothelial cell alignment under flow, we applied this analysis to endothelial cells exposed to different flow and medium conditions. Three sets of control images were chosen manually to represent the three main situations encountered in the flow experiments: (1) cells after adhesion but before exposure to flow, (2) cells exposed to flow but without clear alignment observed on non-optimal surfaces and (3) aligned cells adhered to surfaces. Each pixel in the original images was scored according to the principal components calculated by multivariate image analysis (Figure 3), creating one score density histogram per image. A novel approach to quantify the cell alignment score was developed: a gate was created around the pixels clustered at the center of the score density histogram. Based on control images, these pixels were associated with non-elongated cells. The remaining regions in quadrants 1 to 4, minus this central region, represented pixels associated with 20

elongated cells based on the control image data set. As was demonstrated with myoblasts , pixels with lower PC1 values were associated with bright cell contours or other bright regions such as non-adhered cells, whereas higher PC1 values represented cytoplasmic cellular regions. The PC2 values were indicative of horizontal or vertical orientation. Pixels that had similar PC1 and PC2 scores (either low or high) were characteristic of horizontally aligned cells. As expected, most pixels were clustered in the central region for non-aligned adhered cells prior to flow exposure (Figure 3 left-hand panel). Cells that detached under flow without clear directional alignment had low PC1 scores, without bias towards low or


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high PC2 scores (Figure 3 central panel). On the other hand, a clear shift towards Q2 and Q4 (i.e. a positive correlation between PC1 and PC2 scores) was observed for cells that aligned under flow (Figure 3 right-hand panel). These results indicate that UWT-MIA and time-lapse phase contrast imaging can provide a non-invasive method of quantifying endothelial cell alignment dynamics in response to flow.

Figure 3. Score density histograms obtained by UWT-MIA of sample phase contrast images. In the score density histogram, each point represents the PC1 and PC2 score of a single pixel from the phase contrast image above. Note that the PC1 and PC2 scores were scaled, rounded and binned. The origin of the score density histograms was set to the top left corner, and the scale represents bins between 0 and 256. Higher colour intensities represent higher number of pixels clustered in the same PC1-PC2 bins. The central region represents pixels associated with the image background or with non-elongated cells.

Endothelial cell retention and alignment on functionalized surfaces with serum Next, the potential of the flow chamber system and image analysis approach to determine cell alignment kinetics under flow were assessed. The cell alignment dynamics quantified by UWT-MIA were also compared to a standard method based on cell segmentation and orientation analysis. HSVECs were



Page 18 of 49

seeded onto gelatin-coated aminoalkylsilane glass slides inserted into the flow chamber and allowed to adhere under static conditions for 2 hours in serum-containing complete medium. The cells were then 2

perfused at 0.2 dyn/cm wall shear stress overnight to obtain a confluent monolayer. The next day, the 2

flow was increased to obtain 5 dyn/cm wall shear stress for 30 min, followed by an increase to 2

22 dyn/cm wall shear stress for 6 h. No change in confluency or cell number was measured during flow exposure. Although cell monolayers appeared confluent after shear stress exposure in phase contrast imaging of live cells (Figure 3 and Error! Reference source not found. in the Supporting Information), gaps were observed between cell edges in immunofluorescence for both static and dynamic cultures. This observation could be due to partial cell detachment during flow chamber disassembly prior to fixing (~10 minutes), or this could represent a lack of direct cell-cell contacts on the gelatin-coated glass test surface. Incomplete confluency may lead to more heterogeneous mechanical load distribution within cells 24

and alter mechanosensing pathways when compared to confluent monolayers . Nevertheless, at the end of the experiments, the HSVECs and actin filaments were aligned in the direction of flow as expected, contrary to controls maintained in static conditions (Figure 4A and B; Error! Reference source not found. in the Supporting Information). Focal adhesions formed mainly at the leading and trailing edge of the cells, with larger focal adhesions forming downstream, based on qualitative observations (Figure 4B). The extent of cell alignment in the direction of flow (fraction of pixels characteristic of horizontal alignment) was determined as a function of time (Figure 4C). The kinetics of cell alignment followed the trend expected for the step response of a first-order system without significant time delay (Figure 4C). On 2

2

gelatin-coated surfaces with a step change from 0.2 dyn/cm to 22 dyn/cm , the gain value was 0.6% ± -1

0.1% dyn cm

2

and the time constant was 1.5 ± 0.1 hours. The time constants obtained by the

conventional cell segmentation method were not significantly different from these values. Compared to UWT-MIA, the variability of the results obtained by cell segmentation and mean cosine determination was significantly higher both between images for a given time point and between time points, as can be seen by comparing Figure 4 to Figure S4 (in Supporting Information).


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2

Figure 4. HSVEC response to 22 dyn/cm wall shear stress exposure on gelatin-coated surfaces. Actin filaments (phalloidin, red), focal adhesions (vinculin, green) and nuclei (DAPI, blue) in static cultures 2

(A) or after overnight slow perfusion culture followed by 6 hours of exposure to 22 dyn/cm wall shear stress (B). Scale bar: 50 µm. (C) Proportion of horizontal pixels as a function of time. The continuous black line represents the cell alignment response to the step change in wall shear stress modeled using Equation 1. The error bars represent the standard error of the mean of 25 images captured at each time point in a single chamber.



Page 20 of 49

Using the image analysis and modeling approach developed with gelatin-coated surfaces, the effects of peptide-functionalized surfaces on HSVEC retention and alignment under flow were tested. Test surfaces included RGD-TAMRA:WQPPRARI peptide micropatterns, consisting in 9 ± 1 µm RGD-TAMRA spots covering 35% of the surface, with the rest of the surface functionalized with WQPPRARI peptides, as 12

previously described . Surfaces functionalized with RGD-TAMRA alone, with WQPPRARI alone, or with a 35:65 mixture of these two peptides without micropatterning were included as controls. Similar to the 2

gelatin-coated surfaces, the cells attained confluency after overnight perfusion at 0.2 dyn/cm wall shear 2

stress, and remained nearly confluent after 6 hours of exposure to 22 dyn/cm wall shear stress (Figure 5). No significant differences in cell alignment kinetics were noted compared to the gelatin-coated surfaces. Moreover, no correlation between the location of vinculin-positive focal adhesions and the RGDTAMRA micropatterns was observed. Together, these observations suggested that the surface-grafted peptides did not significantly affect HSVEC adhesion, retention or cell alignment under flow in the presence of serum. One likely explanation for this observation was that the proteins from the serum obscured the effects of the surface-grafted peptides, potentially via surface adsorption of subsets of serum proteins.


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Figure 5. HSVEC alignment and retention on peptide-modified surfaces in serum-containing



Page 22 of 49

medium. (A) Actin filaments (phalloidin, white), focal adhesions (vinculin, green) and nuclei (DAPI, blue) 2

after overnight slow perfusion culture followed by 6 hours of exposure to 22 dyn/cm wall shear stress. RGD-TAMRA micropatterns are shown in red on the “35:65 patterns” surfaces, but the uniform red images are omitted from the “RGD-TAMRA” and “35:65 mix” surfaces for clarity. Scale bar: 50 µm. (B) Cell retention under flow based on cell enumeration in phase contrast images before and after the 22 2

dyn/cm wall shear stress exposure step. No statistically significant difference before and after flow, or between surface types.

Cell retention and alignment on functionalized surfaces in serum-free medium To determine the effects of the peptide-functionalized surfaces on endothelial cell responses to flow in defined chemical conditions, the kinetics of HSVEC retention and alignment under flow were studied in serum-free medium. This medium was previously developed to study the effects of the same peptide 12

micropatterns on cell adhesion and expansion in static conditions . Contrary to the studies conducted in the presence of serum, significant differences in the level of surface endothelialization (Figure 6) and cell alignment kinetics (Figure 7) were observed between the test surfaces. The cell density was quantified 2

before and after exposure to 22 dyn/cm wall shear stress, initiated after overnight cell adhesion and 2

2

expansion at 0.2 dyn/cm wall shear stress. Compared to the 30 000 cells/cm seeding density, cells expanded by approximately 50% on all surfaces (Figure 6A). However, the surface area covered by cells was significantly lower on the WQPPRARI and the micropatterned surfaces compared to the surfaces with uniform RGD-TAMRA distribution (100% or 35% RGD-TAMRA), even before initiating high levels of 2

flow (Figure 6B). After exposure to 22 dyn/cm wall shear stress for 6 hours, no significant changes in cell density were observed on the surfaces with uniform RGD-TAMRA distribution. On the other hand, only 11% ± 3% of the cells were retained on the WQPPRARI surfaces after flow exposure, and 32% ± 20% on the micropatterned surfaces. These results were also reflected by the quantification of the fraction of the surface covered by cells (Figure 7B). During overnight culture, the confluency observed on the WQPPRARI and micropatterned surfaces was significantly lower than on the RGD-TAMRA and the 35:65 mix surfaces (Figure 6B). Since this was not reflected by lower cell numbers (Figure 6A), this result can be interpreted as reduced cell spreading on the WQPPRARI and on the micropatterned


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surfaces. This can also be observed qualitatively when visualizing the time-lapse videos of the cells on these surfaces compared to the RGD-TAMRA and the mixed surfaces (Movies S3-S6 in the Supporting Information). After flow exposure, a progressive loss in cell surface coverage was observed particularly on the WQPPRARI surfaces (Figure 6B), as expected from the cell enumeration results (Figure 6A).

Figure 6. HSVEC retention under flow on the peptide-functionalized surfaces in serum-free 2

2

medium. The wall shear stress was 0.2 dyn/cm up to 9.5 hours, 5 dyn/cm up to 10 hours and 22 2

dyn/cm thereafter. (A) Cell retention under flow based on cell enumeration in phase contrast images 2

before and after the 22 dyn/cm wall shear stress exposure step. *p < 0.05 compared to the same surface before flow, or to the RGD-TAMRA and 35:65 mix surface after flow. (B) Fraction of the surface area covered by cells as a function of time. *p < 0.05 based on MANOVA of the last 4 time points.



Page 24 of 49

Figure 7. HSVEC alignment under flow and leukocyte-like cell adhesion on the peptidefunctionalized surfaces in serum-free medium. (A) Actin filaments (phalloidin, white), focal adhesions (vinculin, green) and nuclei (DAPI, blue) after overnight slow perfusion culture followed by 6 hours of 2

exposure to 22 dyn/cm wall shear stress. RGD-TAMRA micropatterns are shown in red on the “35:65 patterns” surfaces, but the uniform red images are omitted from the “RGD-TAMRA” and “35:65 mix” surfaces for clarity. Scale bar: 50 µm. (B) Kinetics of cell alignment in response to the step change in wall 2

shear stress. (C) Adhesion of ATRA-treated NB4 cells to surfaces and HSVECs under 2 dyn/cm wall shear stress applied for 10 min after endothelial cell alignment. *p < 0.05 based on MANOVA of the last 4 time points (B) or based on ANOVA followed by a Tukey-Kramer HSD post-hoc test (C).

The WQPPRARI and the micropatterned surfaces also were not as conducive to cell alignment and cytoskeletal reorganisation in the direction of flow as the surfaces with uniform RGD-TAMRA distribution (Figure 7). In all serum-free conditions (Figure 7), based on qualitative observations, focal adhesions were more difficult to identify and quantify than in serum-containing conditions due to a decrease in focal adhesion size and an increase in cytosolic vinculin staining. A reduction in focal adhesion size in serumfree medium is consistent with the role of serum stimulation in Rho-mediated cell contractility and focal


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25

adhesion assembly . Although a cell alignment response was measured on all surfaces, the extent of cell alignment quantified by wavelet transform multivariate image analysis was significantly lower on the WQPPRARI and on the micropatterned surfaces. The actin filament alignment in the direction of flow was apparent on all surfaces, but cell elongation and overall filament alignment appeared to be more extensive on the surfaces with uniform RGD-TAMRA distribution. The gain values for horizontal cell alignment on the RGD-TAMRA, WQPPRARI, 35:65 mix or the micropatterned surfaces were respectively -1

2

-1

2

-1

2

0.62% ± 0.07% dyn cm , -0.03% ± 0.02% dyn cm , 0.6% ± 0.1% dyn cm and 0.2% ± 0.2% -1

2

dyn cm . The gain values for the RGD-TAMRA and the uniform mix surfaces were significantly higher than for the WQPPRARI surface. The time constant for the RGD-TAMRA (1.4 ± 0.6 h) and the 35:65 mix (1.5 ± 0.6 h) surfaces were similar to the gelatin-coated surfaces. The time constants were more elevated on the micropatterned surfaces and on the WQPPRARI surfaces, but could not be reliably determined as the response did not reach equilibrium. Overall, the response dynamics for the RGD-TAMRA and the 35:65 mix surfaces were not significantly different from the response dynamics on the gelatin-coated surfaces.

Effect of peptide-functionalized surfaces on the inflammatory profile of endothelial cells Healthy endothelial cells exposed to unidirectional arterial levels of laminar shear stress are expected to express anti-inflammatory molecules such as cytokines (e.g. IL-11) that inhibit leukocyte adhesion and 26

decreased levels of pro-inflammatory cytokines (e.g. IL-6) . The effect of peptide-functionalized surfaces on the inflammatory profile of the endothelial cells was assessed by quantifying cytokine concentrations in the circulating medium as well as NB4 cell adhesion to the HSVECs (Figure 7C). After 6 h exposure to 22 2

dyn/cm wall shear stress in serum-free medium, 3.2 ± 0.6 pg/mL IL-6 concentrations were detected in the circulating medium, which was significantly higher than the background signal detected in initial media samples. No significant differences in IL-6 concentrations were observed between different peptide-functionalized surfaces. Also, no significant IL-11 production was detected by the HSVECs on any of the surfaces. Next, ATRA-treated NB4 cells were circulated over the surfaces with adhered HSVECs as a model of leukocyte adhesion to endothelium, as previously described

27-28

. The

promyelocytic NB4 cell lines undergoes morphological and functional maturation in the presence of



29

Page 26 of 49

30

ATRA , with increased propensity to adhere to activated endothelium . Within 10 min of circulation, significantly higher numbers of NB4 cells adhered to the micropatterned surfaces than the RGD-TAMRA surfaces (Figure 7C). This observation is consistent with the higher surface coverage and lower IL-6 production by HSVECs on the RGD-TAMRA surfaces than on the micropatterned surfaces prior to NB4 infusion (i.e. after shear stress exposure). The number of adhered NB4 cells on the micropatterned surfaces was similar to the 35:65 mix surfaces. Overall, surfaces with 100% RGD-TAMRA led to high levels of endothelial cell retention under flow in both serum-containing and serum-free conditions, were conducive to cell alignment, and displayed the lowest extent of NB4 leukocyte-like cell adhesion. Even in the absence of serum, the surfaces with uniform RGD-TAMRA distribution (100% RGD-TAMRA or 35:65 RGD-TAMRA:WQPPRARI mixture) led to similar endothelial cell retention and alignment under flow as gelatin-coated surfaces. The NB4 cell adhesion tests suggest that 100% RGD-TAMRA may be preferable to 35% RGD-TAMRA mixed with WQPPRARI to reduce leukocyte adhesion. No significant synergies between the RGD-TAMRA and the WQPPRARI peptides were observed, and micropatterning was detrimental to endothelial cell retention under flow.

Discussion Promoting the re-endothelialization of vascular substitutes such as stents and vascular grafts is thought to be key to achieve long-term patency at the stent or graft site. Grafting extracellular matrix-derived peptides to the luminal surfaces of these scaffolds could potentially favor endothelial progenitor cell 7-8, 11-12

adhesion, endothelial cell expansion and/or endothelial cell function under flow. We

and others

31-33

have described several surface functionalization techniques that can be applied to common stent and TM

vascular graft materials such as expanded polytetrafluoroethylene (ePTFE, or Teflon ). Stents 34

functionalization with cyclic RGD peptides significantly accelerated endothelialization rates in rabbit 35

porcine

and

implants. The surface grafting of RGD onto small-diameter ePTFE vascular grafts also 36

enhanced endothelial cell adhesion . In a small preliminary non-randomized study, RGD-functionalized 37

ePTFE stents appeared to be safe and efficacious for the treatment of stenosed saphenous vein grafts . However, the vast majority of promising in vitro studies of peptide-functionalized biomaterials for vascular


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applications have not been translated into clinical or even pre-clinical animal models. This translational gap may in part be due to the lack of medium to high-throughput in vitro screening platforms allowing endothelial cell culture in flow conditions reaching the shear stress experienced by endothelial cells in vivo. Live cell imaging is a powerful tool to study the dynamic responses of cells to stimuli such as shear stress. The most commonly used live cell imaging platforms used to study cell responses to flow are parallel-plate flow chambers and cone-and-plate systems. The cone-and-plate systems can be used to 2

apply wall shear stress over a broad range (0 to >200 dyn/cm ). On the other hand, the parallel-plate flow systems are more amenable to higher-throughput experiments. Multiplexed parallel-plate live cell imaging experiments are typically performed using custom microfluidic systems or using commercially available flow chamber systems. Due to the relatively small dimensions of these systems, the area over which fully 38-39

developed laminar flow and uniform levels of wall shear stress are observed can be restrictive

.

Testing surfaces with micropatterned peptides or proteins in pre-mounted commercial systems can also be problematic. Commonly used microfabrication techniques apply high curing temperatures that may damage surface-grafted peptides or proteins. We designed a parallel-plate flow chamber system that can 2

2

2

be used to apply 0.2 dyn/cm to 22 dyn/cm wall shear stress over a 1.13 cm imaging area in 4 chambers with independent flow paths. Based on the chamber design, even higher wall shear stress 2

values (up to 40 dyn/cm ) could be applied, although these conditions were not tested experimentally. The system was designed to fit into 96-well plate holders commonly used on inverted automated fluorescent microscopes. This system is highly adaptable: different test surfaces, different cell types or different wall shear stress levels can be applied in each chamber. The flow chamber dimensions were designed to accommodate standard glass slides cut to 30 mm length to facilitate implementation by other laboratories. These glass slide inserts could potentially be replaced by supports with surface properties closer to polymers used in vascular applications. For example, polymer films could be deposited (e.g. polylactic acid coatings) or simply attached (e.g. fluorinated ethylene propylene films as an optically clear model of fluoropolymer surfaces) to the glass inserts. In our experience, inserts with similar dimensions (30 mm x 25 mm x 1 mm) can be successfully used in the flow chamber system if edges are smooth and the material rigidity is similar to glass. Softer, thinner or thicker inserts could be suitable by adapting the



Page 28 of 49

O-ring dimensions or compressibility. To quantify cell alignment dynamics, UWT-MIA was applied to the time-lapse phase contrast images acquired using this multi-chamber live cell imaging system. An advantage of this approach over ellipse-fitting algorithms typically used to quantify cell alignment

40

is that cell segmentation is not required.

Cell segmentation algorithms are more prone to error in the presence of artefacts (e.g. bubbles) or for images of varying cell confluency, as was the case for the different surfaces tested. In our hands, the variability of measurements obtained via UWT-MIA was also much smaller than cell alignment quantified through manual cell segmentation followed by ellipse-fitting. When comparing UWT-MIA (Figure 4) to manual cell segmentation followed by ellipse-fitting (Figure S4), the variability between time points as well as the variability between images (error bars shown in the figures) was significantly lower using UWT-MIA. By performing a principal component analysis of the textural image information extracted from the wavelet transform approach, a characteristic “principal component signature” of horizontally aligned cells was identified. This principal component profile was used to quantify cell alignment as a function of time in response to shear stress exposure. The response kinetics of the cells were characterized using a first-order step response model. This empirical model was selected to provide a means to compare cell alignment dynamics on the functionalized surfaces using two parameters: the gain constant and the time constant. The gain constant as defined in this work is proportional to the maximum change in cell alignment that can be observed on a given surface, under a given level of shear stress. The time constant reflects the rate at which cells reach this maximum change in alignment, with elevated time constants representing a slower response. Few existing mechanistic models are available to predict cell alignment 41

dynamics ,

and

these

models

do

not

take

into

account

the

interplay between

different

mechanotransduction pathways involved in cell responses to shear stress. The first order step response model is consistent with the notion that the stimuli leading to cell alignment (e.g. hydrodynamic force 42

applied to the nucleus) decrease in amplitude as cells become more aligned , while forces hindering 43

further spreading (e.g. membrane tension) increase , eventually leading to a net null driving force for further cell alignment. -1

2

Based on the first order step response model, the gain value was ~0.6% dyn cm and the time constant was ~1.5 hours for conditions where cell alignment was observed. In other words, the fraction of


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horizontally aligned cell pixels reached ~63% of the final alignment response within 1.5 hours. As expected, this response time was more elevated than the ~40 min response time that can be estimated 2

44

from experimental data obtained at 68 dyn/cm wall shear stress . The application of control theory to endothelial cell responses to flow could provide a simple and predictive means of assessing the effect of shear stress on cell biology. Future work could investigate the predictive power of this type of analysis by applying other types of transient shear stress changes rather than a simple step change. State equations 45

and control theory have been previously applied to model cardiac blood flow and pressure . To our knowledge, this type of analysis has not yet been applied to model the dynamic changes in endothelial cell alignment in response to changes in shear stress. It would also be interesting to apply this type of 46

analysis to the kinetics of cytoskeleton remodeling following shear stress exposure . The performance of the peptide-functionalized surfaces in the presence of flow was different from the performance of the same surfaces in previous static culture experiments. In static conditions, we previously found that GRGDS:WQPPRARI micropatterns promoted bovine endothelial cell expansion on 7

12

PTFE and did not alter total HSVEC yields on glass surfaces . Conversely, the current study suggests that uniform RGD-TAMRA distribution favours HSVEC yields under arterial levels of wall shear stress. The reduction in cell retention and alignment under flow observed in the current experiment on the micropatterned and WQPPRARI-functionalized surfaces was only apparent in the absence of serum. The adsorption of proteins (e.g. fibronectin) present in serum onto the peptide-modified surfaces may have masked the peptide effects. To limit this masking effect, we conducted subsequent studies in fully defined serum-free medium. Although serum-free conditions do not reflect the in vivo situation, this approach allows to delineate the effects of the peptides from the effects of undefined media components adsorbing onto the surfaces. Once promising peptide combinations have been identified using screening platforms such as the current live cell imaging system, these peptides could be applied to clinically relevant polymers such as PTFE or polylactic acid in physiologically-relevant conditions in the presence of human serum, platelets and/or other blood components. To avoid or reduce the masking of the peptides by adsorbed serum proteins, longer linking arms or surface modifications that reduce protein adsorption could be applied in the peptide grafting strategy. Under flow in serum-free conditions, the micropatterned surfaces led to cell losses and reduced cell



Page 30 of 49

alignment compared to surfaces with uniform RGD-TAMRA distribution (Figure 6 and Figure 7). These results were mirrored by the poor cell retention and cell alignment observed on the surfaces functionalized with WQPPRARI alone. This peptide was originally selected to promote haptotatic cell 47-48

migration

. Yet, in static serum-free conditions, the peptide micropatterns were previously shown to

lead to similar rates of cell spreading and focal adhesion kinase phosphorylation as surfaces with uniform 12

RGD-TAMRA distribution . It is unlikely that serum starvation alone explains the cell losses on the micropatterned surfaces: in static serum-free cultures, HSVECs expansion was similar on the 12

micropatterned surfaces and on 35:65 mix RGD-TAMRA:WQPPRARI . Interestingly, during cell adhesion in serum-free static conditions, focal adhesions were clearly co-localized with RGD-TAMRA 12

12

spots , but not after overnight culture

or after 6 hours of exposure to flow (Figure 7). The improved cell

retention on the RGD-TAMRA and 35:65 mix RGD-TAMRA:WQPPRARI surfaces may thus not be directly related to focal adhesion formation during flow exposure. One notable difference between the micropatterned as well as the WQPPRARI surfaces and the surfaces with uniform peptide distribution was the increased level of cell spreading associated with uniform RGD-TAMRA distribution, even prior to flow exposure. No significant difference in cell number was observed after cell adhesion and overnight 2

culture at 0.2 dyn/cm wall shear stress, but the surface area covered by the cells was significantly lower on the micropatterned and WQPPRARI surfaces compared to the surfaces with uniform RGD-TAMRA distribution. Previous studies have shown that the elongation of endothelial cells in the absence of flow increased collagen IV and fibronectin deposition by the cells. Future experiments could investigate whether the lower level of cell spreading on the micropatterned surfaces prior to flow exposure reduced the levels of extracellular matrix protein secretion by the cells. It would be particularly interesting to study 49

extracellular matrix protein production by the cells on the different surfaces in serum-free conditions . The effect of the micropatterned surfaces in mechanotransduction pathways required for endothelial cell adaptation to flow could also be investigated. For example, the micropatterns may lead to cytoskeletal protein or integrin distributions that hinder the polarization events required for cell alignment in response to flow. Different micropattern densities or geometries could be investigated. Stripes of RGD and 9-10

WQPPRARI peptides were previously shown to improve cell retention and alignment under flow

.

However, compared to the aerosol technique, it would be more challenging to apply such patterns to


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commercially available tubular vascular substitutes such as stents or vascular prostheses. Contrary to the micropatterned surfaces, surfaces with uniform RGD-TAMRA distribution led to similar extent of endothelial cell retention and alignment as gelatin, even in the absence of serum. The surfaces functionalized with RGD-TAMRA alone also led to reduced leukocyte adhesion compared to the micropatterned surfaces. Although these results should be interpreted in the test conditions applied (glass surfaces, serum-free medium, unidirectional laminar flow), the RGD-TAMRA peptide is an outstanding candidate for future in vitro or in vivo studies. For fundamental studies of endothelial cell responses to flow, the RGD-TAMRA functionalized surfaces and serum-free medium described here provide a platform with completely defined chemical conditions to study endothelial cell biology. For clinical applications, peptides with similar structure and distance between the surface and the RGD moiety but without the fluorophore (e.g. CGKGGRGDS) could be of interest. Combined with UWT-MIA, the multi-well flow chamber system is a powerful platform to investigate the effect of surface modifications on the time-dependent effects of flow on endothelial cells. This platform will be used to identify improved surface functionalization strategies favouring endothelial cell retention and 9

alignment under arterial levels of wall shear stress. Based on the current and past work, linear micropatterns that promote cell elongation in the direction of flow may be preferable to the random spraybased patterns. However, the spray-based micropatterning approach is highly advantageous from a manufacturing perspective: these types of patterns could readily be applied to commercially available vascular grafts or stents using a simple spraying nozzle. The simplicity of the spraying technique and the potential to apply spray-based micropatterns to vascular biomaterials that are already in clinical practice could significantly reduce the regulatory burden for clinical translation. To improve the performance of the 50

spray-based peptide micropatterns, different peptide combinations, ligand densities , linking arms surface topographies

52

51

or

could be investigated. Importantly, the RGD-TAMRA peptide functionalized

surfaces, as well as surfaces functionalized with a 35:65 mixture of RGD-TAMRA:WQPPRARI led to similar rates of cell retention and cell alignment under flow as gelatin-coated surfaces. The peptide 2

surface density determined previously was ~0.2 peptides/nm on the surfaces with RGD-TAMRA alone, 2

and hence ~0.07 RGD-TAMRA peptides/nm on the 35:65 RGD-TAMRA:WQPPRARI mix surfaces. The RGD-TAMRA peptide and further modifications of this peptide are therefore excellent candidates for



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further surface modification optimization efforts.

CONCLUSIONS The objective of this work was to investigate the effect of surface-grafted peptides, including micropatterned peptides, on endothelial cells exposed to arterial levels of wall shear stress. The surface and time-dependent effects of shear stress on primary human endothelial cells were studied using a custom multi-well flow chamber with live cell imaging. Wavelet transform multivariate image analysis was applied to time-lapse phase contrast images taken over the course of ~24 hours, during which the wall 2

2

shear stress was increased from 0.2 dyn/cm to 22 dyn/cm . This non-intrusive imaging approach was used to determine the response time of endothelial cells to shear stress exposure. This response time was ~1.5 hours both on gelatin-coated surfaces as well as surfaces functionalized with the fluorophoretagged RGD-TAMRA peptide, or with a 35:65 mixture of RGD-TAMRA with the fibronectin-derived WQPPRARI peptide. While these surfaces led to elevated levels of cell retention, micropatterned surfaces consisting of RGD-TAMRA spots covering 35% of the surface over a WQPPRARI background led to significant endothelial cell losses under flow in serum-free medium. In static serum-free conditions, the same micropatterned surfaces previously did not significantly reduce endothelial cell spreading rates or cell expansion. In the presence of flow, no significant colocalization of focal adhesions with RGD12

TAMRA spots were observed on the micropatterns, contrary to observations in static conditions . These results highlight the importance of testing the effects of surface functionalization strategies in the presence of physiologically relevant levels of wall shear stress. The RGD-TAMRA peptide is a promising candidate to visualize the effects of peptide micropatterns on endothelial cells exposed to flow. The live cell imaging and image analysis methods described in this study provide a quantitative and simple method to study the kinetics of endothelial cell responses to shear stress. Our results also highlight the importance of screening the effects of surface modifications on endothelialization under flow rather than in static conditions, which may not be predictive of endothelial cell behaviour under flow. Together, these methods enable the multi-factorial screening and optimisation of peptide micropatterning strategies applied to vascular biomaterials.


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AUTHOR INFORMATION Corresponding Author *Corinne A. Hoesli, Department of Chemical Engineering, McGill University, 3610 University Street, Montreal, QC, Canada, H3A 0C5

Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding Sources

This work was supported by the Canadian Institutes of Health Research (MOP 142285; CH and GL), by a See the Potential postdoctoral fellowship from Pfizer Ltd and Pfizer Canada Inc. and the Canadian Stem Cell Network (CH), by a non-restricted grant from Pfizer Ltd (CH, AG and GL) and by the Canada Foundation for Innovation (CD, CH). CH is the recipient of the Canada Research Chair in Cellular Therapy Bioprocess Engineering. This work was also supported by the Centre Québécois sur les Matériaux Fonctionnels and the Quebec Network for cell and tissue therapies –ThéCell (a thematic network funded by the Fonds de recherche du Québec–Santé). ACKNOWLEDGMENT We thank Ian Askill at Aspire Biotech for personal handling of our chamber for vapor polishing. We also thank Stephanie Fernandez for rheometry training and Dr. Richard Leask for providing access to the rheometer.

SUPPORTING INFORMATION AVAILABLE

Flow chamber design and validation. This file provides a more detailed description of the materials, methods and results related to the flow chamber design, fabrication and validation. 2

Movie S1. Real-time time-lapse imaging of adhered and flowing endothelial cells (9 dyn/cm wall shear


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stress; scale bar: 100 µm). Movie S2. Cell adhesion and alignment under flow on gelatin-coated surfaces in serum-containing medium (scale bar: 100 µm). Movie S3. Cell adhesion and alignment under flow on RGD-TAMRA functionalized surfaces in serumfree medium (scale bar: 100 µm). Movie S4. Cell adhesion and alignment under flow on the WQPPRARI surfaces in serum-free medium (scale bar: 100 µm). Movie S5. Cell adhesion and alignment under flow on the 35:65 RGD-TAMRA:WQPPRARI mix surfaces in serum-free medium (scale bar: 100 µm). Movie S6. Cell adhesion and alignment under flow on the 35:65 RGD-TAMRA:WQPPRARI micropatterned surfaces in serum-free medium (scale bar: 100 µm).

ABBREVIATIONS ANOVA, analysis of variance; HSVEC, human saphenous vein endothelial cell; MANOVA, multivariate ANOVA; PC, principal component; PTFE, polytetrafluoroethylene; RGD-TAMRA, RGD-TAMRA, CGK(PEG3-TAMRA)-GGRGDS-NH2; UWT-MIA, undecimated wavelet transform-multivariate image analysis.



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TABLE OF CONTENT FIGURE (For Table of Contents Use Only)



Figure 1. Design of the 4 flow chamber live cell imaging system. (A) overall schematic representation, (B) cross-sectional view of one of the chambers, 1) metal plates, 2) top plate, 3) support bracket, 4) Viton Orings, 5) glass test surface, 6) bottom plate and (C) photograph of the assembled flow chamber system. 62x45mm (300 x 300 DPI)

ACS Paragon Plus Environment

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Figure 2. Circulation loop for the live cell imaging experiments. The dotted box represents the incubation chamber surrounding the microscope. The direction of flow was from the injection port side to the medium recirculation or waste lines. 62x47mm (300 x 300 DPI)



Figure 3. Score density histograms obtained by UWT-MIA of sample phase contrast images. In the score density histogram, each point represents the PC1 and PC2 score of a single pixel from the phase contrast image above. Note that the PC1 and PC2 scores were scaled, rounded and binned. The origin of the score density histograms was set to the top left corner, and the scale represents bins between 0 and 256. Higher colour intensities represent higher number of pixels clustered in the same PC1-PC2 bins. The central region represents pixels associated with the image background or with non-elongated cells. 105x67mm (300 x 300 DPI)


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Figure 4. HSVEC response to 22 dyn/cm2 wall shear stress exposure on gelatin-coated surfaces. Actin filaments (phalloidin, red), focal adhesions (vinculin, green) and nuclei (DAPI, blue) in static cultures (A) or after overnight slow perfusion culture followed by 6 hours of exposure to 22 dyn/cm2 wall shear stress (B). Scale bar: 50 μm. (C) Proportion of horizontal pixels as a function of time. The continuous black line represents the cell alignment response to the step change in wall shear stress modeled using Equation 1. The error bars represent the standard error of the mean of 25 images captured at each time point in a single chamber. 162x160mm (300 x 300 DPI)



Figure 5. HSVEC alignment and retention on peptide-modified surfaces in serum-containing medium. (A) Actin filaments (phalloidin, white), focal adhesions (vinculin, green) and nuclei (DAPI, blue) after overnight slow perfusion culture followed by 6 hours of exposure to 22 dyn/cm2 wall shear stress. RGD TAMRA micropatterns are shown in red on the “35:65 patterns” surfaces, but the uniform red images are omitted from the “RGD-TAMRA” and “35:65 mix” surfaces for clarity. Scale bar: 50 μm. (B) Cell retention under flow based on cell enumeration in phase contrast images before and after the 22 dyn/cm2 wall shear stress exposure step. No statistically significant difference before and after flow, or between surface types. 216x287mm (300 x 300 DPI)


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Figure 6. HSVEC retention under flow on the peptide-functionalized surfaces in serum-free medium. The wall shear stress was 0.2 dyn/cm2 up to 9.5 hours, 5 dyn/cm2 up to 10 hours and 22 dyn/cm2 thereafter. (A) Cell retention under flow based on cell enumeration in phase contrast images before and after the 22 dyn/cm2 wall shear stress exposure step. *p < 0.05 compared to the same surface before flow, or to the RGD-TAMRA and 35:65 mix surface after flow. (B) Fraction of the surface area covered by cells as a function of time. *p < 0.05 based on MANOVA of the last 4 time points. 110x147mm (600 x 600 DPI)



Figure 7. HSVEC alignment under flow and leukocyte-like cell adhesion on the peptide-functionalized surfaces in serum-free medium. (A) Actin filaments (phalloidin, white), focal adhesions (vinculin, green) and nuclei (DAPI, blue) after overnight slow perfusion culture followed by 6 hours of exposure to 22 dyn/cm2 wall shear stress. RGD TAMRA micropatterns are shown in red on the “35:65 patterns” surfaces, but the uniform red images are omitted from the “RGD-TAMRA” and “35:65 mix” surfaces for clarity. Scale bar: 50 μm. (B) Kinetics of cell alignment in response to the step change in wall shear stress. (C) Adhesion of ATRAtreated NB4 cells to surfaces and HSVECs under 2 dyn/cm2 wall shear stress applied for 10 min after endothelial cell alignment. *p < 0.05 based on MANOVA of the last 4 time points (B) or based on ANOVA followed by a Tukey-Kramer HSD post-hoc test (C). 91x51mm (600 x 600 DPI)


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Table of content graphic 35x14mm (300 x 300 DPI)


Dynamics of endothelial cell responses to laminar shear stress on

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