Collagen-Mimetic Proteins with Tunable Integrin Binding Sites for

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Tissue Engineering and Regenerative Medicine

Collagen-Mimetic Proteins with Tunable Integrin Binding Sites for Vascular Graft Coatings Juan Felipe Diaz-Quiroz, Patricia Diaz-Rodriguez, Joshua D. Erndt-Marino, Viviana Guiza, Bailey Balouch, Tyler Graf, William M. Reichert, Brooke Russell, Magnus Hook, and Mariah S. Hahn ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.8b00070 • Publication Date (Web): 13 Jun 2018 Downloaded from http://pubs.acs.org on June 16, 2018

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ACS Biomaterials Science & Engineering

Collagen-Mimetic Proteins with Tunable Integrin Binding Sites for Vascular Graft Coatings Juan Felipe Diaz Quiroz1*, Patricia Diaz Rodriguez1*, Josh D. Erndt-Marino1, Viviana Guiza1, Bailey Balouch1, Tyler Graf1, William M. Reichert2, Brooke Russell3, Magnus Höök3 and Mariah S. Hahn1

1. Department of Biomedical Engineering, Rensselaer Polytechnic Institute, Troy, NY, USA. 2. Department of Biomedical Engineering, Duke University, Durham, North Carolina, USA. 3. Institute of Biosciences and Technology, Texas A&M Health Science Center, College Station, Texas, USA. * - These authors contributed equally to this work.

*Contact Author: Mariah S. Hahn, Ph.D. Professor of Biomedical Engineering, Rensselaer Polytechnic Institute Tel: 518-276-2236 Email: [email protected]

Keywords: vascular grafts, endothelial precursor cells, cell migration, shear stress, endothelial differentiation

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ABSTRACT Achieving graft endothelialization following implantation continues to be a challenge in the development of “off-the-shelf,” small-caliber, arterial prostheses. Coating grafts with biomolecules to support the retention, migration, and differentiation of adherent endothelial precursor cells (EPCs) is a promising approach toward improving graft endothelialization. Designer Collagen Scl2-2 with 1 integrin binding site per strand (DC2-1X) is a Streptococcus pyogenes-derived, collagen-like protein that has previously been evaluated as a graft coating due to its ability to resist platelet aggregation and to promote attachment and migration of “late outgrowth” EPCs (EOCs). However, these prior assessments were performed in the absence of physiological shear. In addition, although DC2-1X coatings supported increased migration rates relative to native collagen coatings, EOC attachment and spreading remained inferior to collagen controls at all DC2-1X concentrations assayed. Thus, the objectives of the present work were: 1) to improve EOC attachment on DC2 coatings by modulating the number and spacing of DC2 integrin binding sites (IBS) and 2) to evaluate the retention, migration, and differentiation of adherent EOCs under physiological shear stress. Using single point mutations, three novel DC2 variants were generated containing either two IBS (DC2-2X) or three IBS (DC2-3X1 and DC23X2) per strand. After initial evaluation of the potential of each DC2 variant to support increased EOC attachment relative to DC2-1X, DC2-2X and DC2-3X1 coatings were further assessed under physiological shear for their capacity to promote EOC retention, migration, and differentiation relative to DC2-1X and collagen controls. An increase in the number of IBS from 1 to 3 significantly improved EOC retention on DC2 coatings while also supporting increased average migration rates. Moreover, EOCs on DC2-3X1 coatings showed increased gene-level expression of intermediate endothelial cell differentiation markers relative to collagen. Overall,


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the current results suggest that DC2-3X1 warrants further investigation as a vascular graft coating.



1. INTRODUCTION Approximately 400,000 coronary artery bypass grafts surgeries are performed each year in the United States1-2. Although autologous mammary artery or saphenous vein grafts remain the standard of care, synthetic prostheses are frequently used in cases for which healthy autologous donor tissue is unavailable3-6. However, current synthetic grafts suffer from limited long-term patency in small caliber graft applications, due in part to associated platelet activation and intimal hyperplasia7-8. As such, rapid endothelialization of “off-the-shelf” synthetic prostheses is believed to be necessary for improving their longer-term success as small-caliber grafts4-5, 9. Development of bioactive luminal coatings to promote endothelialization of synthetic grafts by circulating endothelial precursor cells (EPCs) is therefore an area of active research1019.

Although EPCs are relatively scarce within the blood stream, “late outgrowth” EPCs

(hereafter referred to as EOCs) can be isolated and expanded ex vivo at high yields20-21. EOCs have also been shown to express normal endothelial cell (EC) surface markers20-21, to exhibit typical EC functions22

23-25

, and to be capable of endothelializing vessels following injection in

an in vivo rabbit model26. Furthermore, recent studies have demonstrated that EOCs isolated from patients with coronary artery disease (CAD) exhibit similar expansion capacity, surface marker expression, and EC-like responses to shear flow as EOCs from healthy individuals27-28. Thus, patient-specific EOCs could potentially be isolated from CAD patients receiving “off-theshelf” vascular grafts, expanded in vitro, and subsequently injected to populate the implanted grafts. A graft coating designed to support EOC-based endothelialization must allow for the retention, migration, and differentiation of adherent EOCs. Natural polymers such as fibronectin,


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fibrin, or collagen have previously been investigated as synthetic graft coatings with moderate success in vitro and in various animal models5, 29. Although these proteins support the formation of a confluent endothelium, they also present significant obstacles for use as “off-the-shelf” graft coatings. For instance, each of these biopolymers is known to support platelet activation and aggregration30-31 and, depending on the source, can be immunogenic31. Furthermore, recombinant human collagen and fibrin can currently only be produced in mammalian cells due to needed post-translational modifications. This requirement substantially increases the production costs associated with these recombinant proteins32. The discovery of Streptococcus pyogenes collagen-like proteins33-34 has opened the possibility for a novel collagen-mimetic protein source that does not require specialized mammalian post-translational modifications to achieve a native triple helical structure35-36. In particular, Streptococcal Collagen-Like protein Scl2.28 (termed Scl2-1) contains the GXY repeats associated with native collagen, resists platelet aggregation, and has been shown to be non-immunogenic in SJL/J and Arc mice37-38. This protein is also unique in that it also contains no known cytokine binding or cell adhesion motifs39, enabling only desired cell-protein interactions to be programmed into the protein structure via site-directed mutagenesis. Recently, our team introduced the integrin binding site (IBS) GFPGER into the Scl2-1 sequence, generating the modified protein DC2-1X (Designer Collagen Scl2-2 containing 1 IBS per strand of the triple helix). GFPGER is a structural analog of GFOGER (O; hydroxyproline), an adhesion motif in native collagen that is recognized by α2 and α1 integrins16, 40 but which does not induce platelet activation41. Previous in vitro studies have shown that DC2-1X maintains the triple helical structure and low platelet aggregation of Scl2-140. Furthermore, DC2-1X coatings – DC2-1X conjugated



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within a poly(ethylene glycol) diacrylate (PEG) hydrogel base – have been shown to enable EC and EOC attachment, migration, and differentiation under static conditions38-39,

42-44

.

Nonetheless, even coatings containing the highest DC2-1X concentrations that could be stably incorporated were associated with reduced EC/EOC attachment in comparison to native collagen42-44. Moreover, the retention, migration, and differentiation of adherent EOCs on DC21X coatings has not been assessed in a physiologically relevant manner in which cells are subjected to shear flow. The objectives of the present work were therefore: 1) to modify DC2-1X towards improving the levels of initial EOC attachment supported by the DC2 coatings and to demonstrate this improvement in a manner allowing comparison with our previous work (24 h static culture)44 and 2) to evaluate the capacity of these DC2 protein variants to support the retention, migration, and differentiation of adherent EOCs under physiologically relevant shear stress conditions in vitro. Since cell attachment is a function both ligand concentration45-48 and ligand spacing47-48, both the number of IBS (ligand concentration) and the distance between IBS (ligand spacing) were modified utilizing a series of single point mutations. After generating, expressing, and characterizing several variants of DC2-1X, an initial screening was performed to identify modifications that improved initial EOC attachment relative to the parent protein. Thereafter, selected DC2 variants were compared for their ability to support the retention, migration, and differentiation of adherent EOCs under shear stress conditions relative to native collagen. Overall, results from this study will allow identification of a DC2 variant demonstrating potential for further development as a bioactive coating for synthetic vascular grafts.


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2. MATERIALS AND METHODS

2.1 Addition of Integrin Binding Sites DC2-1X – a variant of Scl2-1 containing a single GFPGER IBS39 – was used as a template to introduce 1 or 2 additional IBS into the protein structure. The introduction of additional IBS was achieved via single amino acid mutations using a QuickChange II sitedirected mutagenesis kit (Agilent Technologies). Primers to introduce mutations were designed following the recommendations of the kit manufacturer. Via this method, three DC2-1X variants were engineered: 1) DC2-2X, containing a second IBS per protein strand located 57 amino acids downstream of the first IBS, 2) DC2-3X1, containing the 2 IBS associated with DC2-2X as well as a third IBS 48 amino acids downstream of the second IBS, and 3) DC2-3X2, containing the 2 IBS associated with DC2-2X as well as a third IBS 123 amino acids downstream of the second IBS (Figure 1A, Table S1). Appropriate insertion of additional IBS was confirmed by gene sequencing (Genewiz, NJ).

2.2 DC2 Protein Expression and Characterization Recombinant expression of each DC2 variant was conducted as described previously33. Briefly, starter cultures of Escherichia coli BL21 (Novagen) transformed with pCold plasmids containing the sequence of the desired protein were grown overnight at 37 °C. Cultures were



then added to 1.5 L of Luria Broth (LB) and cultured at 37 °C in a shaker at 180 rpm. Cell growth was monitored every hour until the plateau phase was reached. Cultures were then cooled to 18 °C, and protein expression was induced by adding IPTG to a 0.5 mM final concentration. Bacterial cultures were then maintained at 18 °C in a shaker at 225 rpm for 16-20 h, after which the cultures were centrifuged, and protein was extracted by sonication. Released protein was separated by affinity chromatography (HisTrap HP, GE Healthcare) followed by a HiTrap Q column (GE Healthcare). Isolated proteins were then dialyzed and lyophilized, after which sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) was used to qualitatively assess protein purity. As previously noted, DC2-1X proteins contain the GXY-repeats characteristic of native collagen and spontaneously assemble into triple-helical structures. To confirm that the modified daughter proteins retained the capacity to take on a triple helical conformation, native versus denaturing electrophoresis as well as circular dichroism analyses were conducted as described below.

2.2.1

Native and Denaturalized Protein Electrophoresis Gels: Ten percent polyacrylamide gels

were prepared with a final concentration of 0.05 % SDS. For non-denaturing conditions, each DC2 protein was dissolved in 6 % glycerol and directly loaded into the gel. For denaturing conditions, each dissolved DC2 protein was heated to 95 °C for 10 min in the presence of 2 % SDS and 1 % β-mercaptoethanol before loading. Electrophoresis was carried out using nondenaturing buffer at 150 V for 2 h at 4 °C. Gels were subsequently stained with Coomassie Blue and protein size was evaluated using protein standard.


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2.2.2

Circular Dichroism: Collagen-mimetic proteins were dissolved in phosphate buffered

saline (PBS) at a concentration of 0.5 mg/ml. Protein concentration was then confirmed by UV spectrophotometry and subsequently adjusted to 0.3 mg/ml. Ellipticity data were collected for each sample at 25 °C over a wavelength range from 250 nm to 200 nm at 0.2-nm intervals using a Jasco 815 CD Spectrometer and a 0.1 cm light path. For each spectrum, the contribution from the buffer was subtracted.

2.3 Acrylate-derivatization of DC2 Variants and Collagen Although DC2 proteins possess the triple helical structure of native collagen, they lack the capacity of collagen to assemble into physically crosslinked hydrogel networks. To generate stable coatings, DC2 proteins were therefore first acrylate-derivatized and subsequently covalently conjugated within PEG hydrogels. Briefly, each DC2 variant was reacted with acryloyl-PEG-succinimidyl valerate (ACRL-PEG-SVA, 3.4 kDa; Laysan Bio) at a molar ratio of 1:6 for 2 h in PBS. The resulting ACRL-PEG-DC2 products were then immediately used for the fabrication of PEG-DC2 coatings. As a control, rat tail collagen I (Life Technologies) was also reacted with ACRL-PEG-SVA for 2 h at a 1:6 molar ratio in 50 mM sodium bicarbonate buffer, pH 8.549, after which the product was dialyzed against double deionized water for 48 h. Following purification, the acrylate-derivatized collagen was lyophilized and stored at -40 ºC until use.

2.4 Hydrogel Fabrication To generate DC2-containing hydrogel coatings, acrylate-derivatized proteins were combined with PEG (3.4 kDa; Laysan Bio) in PBS supplemented with 20 mM acetic acid to



achieve final concentration of 8 mg/mL protein and 100 mg/mL PEG, respectively. This protein concentration was selected based on our previous data showing that PEG-DC2-1X coatings containing 8 mg/mL DC2-1X were able to support EOC attachment, migration, and confluence without the protein precipitation/loss occasionally observed at higher DC2 concentrations44. Hydrogel precursor solutions were then crosslinked in 0.5 mm thick rectangular glass molds in the presence of 1 mg/mL Irgacure 2959 (Sigma) by 6 min exposure to longwave UV light (~6 mW/cm2, 365 nm). Resulting hydrogel slabs were sterilized by immersion in 70 % ethanol for 1.5 h and subsequently washed with a series of graded ethanol solutions for 20 min each (70 %, 50 %, 20 % and 0 % in PBS). For static cell attachment screening, 6 mm discs were cut from each hydrogel slab using a biopsy punch and transferred to 48 well plates. For cell retention, migration, and phenotypic analyses, 30 mm diameter discs were cut from each slab and attached to individual wells of a 6 well plate using surgical adhesive (Silastic, Down Corning). All discs were immersed overnight in PBS containing 1% PSA (PSA: 10,000 U/mL penicillin, 10,000 mg/L streptomycin and 25 mg/L amphotericin; Mediatech) prior to cell seeding.

2.5 Cell Culture The EOCs (“late outgrowth” EPCs) utilized in the present study were previously isolated from blood collected from a 60 year old female CAD patient28 through a protocol approved by the Duke University Institutional Review Board27-28, 44. The isolated EOCs were positive for cell surface markers CD31 and CD105 and negative for CD133, CD14, and CD45. These and other CAD EOCs have been shown to have similar proliferative capacity, surface marker expression, and response to physiological shear as EOCs derived from healthy patients28. Furthermore, prior


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characterization of these EOCs has indicated that gender is not a significant variable impacting their isolation, proliferative behavior, or surface marker expression28. For the current work, the cryopreserved donor EOCs were thawed and expanded under standard conditions in Growth Medium (GM: EBM-2 medium (Lonza) supplemented with EGM-2 SingleQuots, 10% fetal bovine serum (FBS; Altanta Biologicals), and 1% PSA (Mediatech)). Media was changed every other day, and cells were passaged at 70-80% confluence. EOCs were used at passage 7 for all experiments.

2.6 Initial Screening of EOC Attachment under Static Conditions For static cell attachment assessment, sterilized 6 mm hydrogel discs were washed twice for 20 min with PBS and immersed in GM. Harvested EOCs were then seeded on each coating at ~1x104 cells/cm2 and cultured for 24 h at 37 °C under static conditions. Attached cells were then formalin-fixed and stained with rhodamine phalloidin (Life Technologies) to allow for cytoskeletal visualization. For six discs per coating type, phalloidin stained cells were imaged in 5 randomly selected regions per disc using a Zeiss Axiovert 200M fluorescence microscope. Initial cell attachment on each DC2 coating was calculated from these images as the number cells per cm2 in comparison to the known seeding density and normalized by the corresponding cell attachment on the collagen controls.

2.7 EOC Retention and Migration under Shear Flow For cell retention and migration assays, EOCs were pre-labeled via exposure to 2 µM Green-CMFDA (Thermo-Fisher Scientific) per manufacture instructions to allow for cell tracking. Sterilized hydrogel discs were immersed in GM and Green-CMFDA labeled EOCs



were harvested and seeded at a surface density of ~1x104 cells/cm2. Cells were then allowed to attach and spread for 24 h at 37 °C under static conditions, after which a laminar flow shear stress (τ) of ~7 dynes/cm2 was initiated using a rotating custom cone spindle (30 mm diameter, 0.5° angle) controlled by a step motor (DMX-J-SA, Arcus Technology). This value of τ was selected to be within the range of arterial shear stresses experienced in vivo50 51-52. Images of the cells in five different regions of each hydrogel disc were collected at 5 min intervals for a period of 1 h under constant shear stress utilizing a Zeiss LSM 510 Meta/Multiphoton NLO microscope equipped with an environmental chamber to maintain cells at 37 °C. Time series image alignment was carried out using ImageJ software, and the average cell migration speed for EOCs separated by greater than ~100 µm was then determined using the Image J Chemotaxis and Migration Tool 2.0 (Ibidi ®). EOC retention was calculated as the percentage of initially attached cells remaining in each imaged region following 1 h of flow. At least three independent retention and migration experiments were performed for each coating formulation.

2.8 Evaluation of EOC Differentiation To evaluate the capacity of the various DC2 coatings to support EOC differentiation relative to collagen controls, EOCs were seeded onto 30 mm hydrogel discs (n = 3-4 per formulation) at ~1x104 cells/cm2. Cells were then allowed to attach and spread for 24 h at 37 °C under static conditions, after which cells were subjected to laminar shear stress (~7 dynes/cm2) for 2 h at 37 °C. Immediately following cessation of flow, cells were briefly rinsed with PBS and then immersed in lysis buffer from the Dynabeads mRNA direct kit (Ambion, Life Technologies). PolyA-mRNA was extracted from the lysates following manufacturer instructions. Gene expression was quantified using qPCR verified primers (OriGene; Table S2),


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the SuperScript III Platinum One-Step qRT-PCR kit, and a StepOne Real-Time PCR System (Life Technologies). Gene expression was calculated using the ∆∆Ct method, with RPL-32 serving as the reference gene and collagen coatings serving as the reference control. Markers selected for analyses were chosen to represent early (CD34, VEGFR253-54), intermediate (EphrinB2 and EphB455-57), and late (vWF, PECAM-1, VE-Cadherin53) stages of EC differentiation. Melting temperature analysis was performed for each reaction product to verify the presence of the appropriate amplicon.

2.9 Statistical Analyses All data are shown as mean + the standard error of the mean. To determine significant differences among the DC2 and collagen coatings, one-way ANOVA was conducted with a Tukey’s multiple comparison post-hoc test. All statistical analyses were performed using SPSS software (IBM) with significance established at p < 0.05.

3. RESULTS 3.1 DC2 Variant Generation and Characterization Using single point mutations and DC2-1X as a template, 3 novel DC2 daughter proteins were engineered containing either two (DC2-2X) or three IBS (DC2-3X1 and DC2-3X2) (Figure 1A, Table S1). While all proteins were designed with conserved locations between the first and second IBS (57 amino acid spacing (~18.8 nm)), DC2-3X1 was designed to display a third IBS 48 amino acids (~15.8 nm) downstream from the second IBS. In contrast, DC2-3X2 was designed to display a third IBS 123 amino acids (~40.6 nm) downstream of the second IBS



(Figure 1A, Table S1). The latter two protein variants were developed to allow for the impact of ligand number versus ligand spacing/steric factors to be assessed. Downstream spacing was approximated based on 0.33 nm/residue associated with the coiled strands comprising native collagen I. To ensure that sequence modifications did not alter the capacity of the daughter proteins to take on a triple helical conformation, the extent of protein migration under native versus denaturing gel electrophoresis was compared and circular dichroism was performed. Gel electrophoresis results indicated that inclusion of additional IBS in DC2-2X, DC2-3X1, and DC2-3X2 did not inhibit the ability of individual protein strands to associate into trimers (Figure 1B). Specifically, each DC2 variant showed a band at ~34 kDa, corresponding to a monomeric strand under denaturing conditions, and a band at ~120 kDa corresponding to a trimeric grouping under native conditions. In addition, each of the DC2 variants showed a peak in ellipticity at 220 nm (Figure 1C), indicating the presence of a triple helical configuration40. Cumulatively, these data suggest that the inclusion of additional IBS did not affect the capability of the proteins to establish a collagen-like triple helical conformation. 3.2

EOC Attachment Screen to Identify Promising DC2 Variants To identify DC2 variants warranting further testing under physiological shear, an initial

cell attachment screen was conducted under static conditions. In brief, EOCs were seeded onto the surface of various DC2 coatings, and the capacity of the introduced IBS to support improved EOC attachment relative to DC2-1X was evaluated following 24 h of culture44. As shown in Figure 2A-D, initial cell attachment and spreading was dependent on the number and location of IBS present in the DC2 protein variant. Qualitatively, the extent of cell spreading on DC2-3X1


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coatings appeared to be greater than the remaining DC2 variants, although all DC2 coatings were associated with reduced EOC spreading relative to collagen controls (Figure 2A-E). When IBS spacing was maintained at ~16-18 nm (DC2-2X and DC2-3X1), quantification revealed a gradual increase in initial cell attachment with the increasing IBS number (Figure 2F). Specifically, DC2-2X coatings supported increased initial EOC attachment relative to DC21X (~1.4 fold, p = 0.016), and DC2-3X1 coatings demonstrated significantly greater attachment relative to both DC2-1X and DC2-2X coatings (~1.9 and ~1.4 fold, respectively; p < 0.006). Moreover, the initial cell attachment on DC2-3X1 coatings was statistically indistinguishable from collagen controls (p = 0.10; Figure 2F). In contrast, increasing the spacing between the second and third IBS from to ~40.6 nm (in DC2-3X2) resulted in a loss of the improvement in initial cell attachment observed with increasing IBS number between DC2-2X to DC2-3X1. In particular, initial cell attachment on DC2-3X2 coatings was not significantly increased relative to DC2-1X or DC2-2X coatings (p > 0.40). Furthermore, the initial EOC attachment supported by DC2-3X2 coatings was significantly lower than both DC2-3X1 coatings and collagen controls (p < 0.005). Combined, these results indicate that initial EOC attachment is dependent on both the number and spacing of IBS within the DC2 proteins. Based on these static cell attachment measures, only DC2-1X, DC2-2X, and DC2-3X1 were further evaluated in subsequent retention, migration, and differentiation studies. 3.3

Effect of the Incorporation of Additional IBS on EOC Retention and Migration An ideal vascular graft coating should present an EOC-substratum adhesiveness that

allows EOCs to remain attached to the graft surface under physiological flow conditions while retaining their capacity to migrate to populate the graft surface. Thus, DC2-1X, DC2-2X, and



DC2-3X1 coatings were further evaluated for their ability to support EOC retention (an indirect metric of cell-substrate adhesion strength) and migration under physiological shear. For these studies, adherent, fluorescently-labelled EOCs were subjected for 1 h to constant shear stress (~7 dynes/cm2) using a rotating cone with fluorescence monitoring at 5 min intervals to allow for assessment of average migration speed. Cell retention was quantified as the percentage of initial cells that remained attached to each surface following 1 h of shear flow relative to collagen controls. In terms of cell adhesion strength, although all DC2 variants were associated with decreased EOC retention compared to collagen controls (p < 0.013; Figure 3A), the introduction of additional IBS sites led to improved cell retention relative to DC2-1X. In particular, DC2-3X1 coatings displayed a higher percent EOC retention than DC2-1X coatings (~1.3 fold, p = 0.013). Similarly, DC2-2X coatings appeared to support increased retention relative to DC2-1X coatings (~1.2 fold), although this difference fell below statistical significance (p = 0.056). Average cell migration speed on the various DC2 coatings also showed a dependence on IBS number, with EOCs on DC2-2X (~1.3 fold, p < 0.005) and DC2-3X1 (~1.3 fold, p < 0.005) coatings exhibiting significantly higher average migration speeds relative to DC2-1X controls (Figure 3B). However, in contrast to cell retention trends, all DC2 coatings were associated with significantly greater average EOC migration speeds than collagen controls (p < 0.001). Cumulatively, these results suggest a biphasic relationship between EOC retention and average migration speed. Namely, as cell-substrate adhesion strength (retention) increased from DC2-1X to DC2-2X to DC2-3X1 to collagen, the average migration rate initially increased from DC2-1X to DC2-2X but then plateaued between DC2-2X and DC2-3X1 before decreasing between DC23X1 and collagen.


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3.4 Effect of the Incorporation of Additional IBS on EOC Differentiation A coating intended to enable EOC-based graft endothelialization must also support EOC differentiation toward an EC-fate. To gain insight into the capacity increasing DC2 IBS number to promote EC differentiation by adherent EOCs, the gene-level expression of several EC markers by EOCs on each of the DC2 coatings was compared to collagen, a control known to support EC commitment and maturation. Toward this goal, EOC expression of early (CD34, VEGFR253-54; Figure 4A), intermediate (EphrinB2 and EphB455-57; Figure 4A), and late (vWF, PECAM-1, VE-Cadherin53; Figure 4B) EC markers was evaluated by RT-qPCR for each of the DC2 coatings following 2 h exposure to ~7 dynes/cm2 shear stress. EOC expression of CD34 was relatively consistent across the DC2 coatings, with CD34 levels being significantly lower on DC2-1X (~2.3 fold; p = 0.028) and DC2-3X1 (~2.2 fold; p = 0.023) coatings relative to collagen controls (Figure 4A). Expression of VEGFR2 also appeared to be largely independent of DC2 IBS number, although EOC VEGFR2 expression was significantly higher on all DC2 variants (p < 0.001) relative to collagen coatings (Figure 4A). Although both CD34 and VEGFR2 are expressed by EOCs undergoing early EC differentiation, the expression of CD34 is known to decrease with increasing EC commitment, while the expression of VEGFR2 increases53. As such, these early marker results suggest that the DC2 variants each stimulate EOCs to undergo more rapid EC lineage progression relative to collagen. Analysis of intermediate markers EphrinB2 and EphB4 appeared to support this interpretation of the early marker data. Specifically, expression of both EphrinB2 and EphB4 was significantly increased on all DC2 coatings relative to collagen controls (p < 0.001; Figure 4A). Although EphrinB2 is commonly used as arterial marker and EphB4 as a venous marker55-



57

, the expression of both EphB4 and its ligand EphrinB2 has been shown to be required at early

stages of vasculogenesis for the proper formation and maturation of both arteries and veins55, 58. However, in contrast to the early markers CD34 and VEGFR2, EOC expression of the intermediate EC markers EphrinB2 and EphB4 displayed a dependence on DC2 IBS number (Figure 4A). Specifically, EphrinB2 expression was significantly greater on DC2-1X coatings than on DC2-2X and DC2-3X1 coatings (p < 0.001), and EphB4 expression was ~1.2 fold higher on DC2-1X coatings relative to DC2-3X1 coatings (p = 0.050). That said, none of the late phenotypic markers investigated (VE-cadherin, PECAM-1, and vWF) showed significant differences between DC2 and collagen formulations (Figure 4B).

4. DISCUSSION In the present study, three novel variants of the collagen-mimetic DC2-1X protein were generated toward improving the capacity of DC2-based coatings to support EOC-based graft endothelialization. These DC2-1X daughter proteins were engineered to contain 1 or 2 additional GFPGER IBS relative to the parent protein at defined spacings. Following sequence-based confirmation of IBS introduction at desired positions within the DC2-1X structure, the ability of each of the resulting protein variants (DC2-2X, DC2-3X1, DC2-3X2) to spontaneously assemble into triple-helical structures previously described for DC2-1X40,39 was confirmed (Figure 1). Assessments of initial EOC attachment to statically cultured DC2 coatings revealed a monotonic increase in cell attachment with increasing IBS number when IBS spacing was maintained at ~16-18 nm (Figure 2). Furthermore, the initial EOC attachment supported by DC2-3X1 coatings was statistically indistinguishable from collagen controls. These data are


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consistent not only with data indicating that each strand of collagen I presents 3-4 different α1β1 and α2β1 IBS59-60, but also with more general reports demonstrating enhanced cell attachment with increasing ligand concentration38, 42. That said, the extent of EOC spreading on collagen coatings qualitatively exceeded that supported by each of the examined DC2 variants, likely reflecting the additional binding sites beyond α1β1 and α2β1 provided by native collagen. In terms of IBS spacing, even though DC2-3X1 and DC2-3X2 each presented 3 IBS per strand, the increase in EOC attachment observed between DC2-1X and DC2-3X1 (where the IBS spacing was maintained at ~16-18 nm) was lost for DC2-3X2 (where the spacing between the second and third IBS was increased to ~40.6 nm). These results are consistent with reports indicating that increases in cell attachment due to increases ligand number are highly dependent on ligand spacing61-63. For instance, Koo et al. demonstrated that cells exhibited marked decreases in integrin-dependent adhesion when average RGD spacing increased from 14 nm to 25 nm62. Due to the triple helical nature of the DC2 proteins, the differences in initial cell attachment observed between DC2-3X1 and DC2-3X2 coatings could also be due in part to steric factors. Based on these static cell attachment measures, only DC2-1X, DC2-2X, and DC2-3X1 were evaluated in subsequent retention, migration, and differentiation analyses conducted under physiological shear stress conditions. Overall, the EOC retention and migration data were consistent with the biphasic relationship between cell-substrate adhesion strength and average migration speed (Figure 5) frequently reported in literature45-46. A balance between adhesion strength and migration rate is necessary for achieving rapid graft endothelialization following implantation. DC2-3X1 appears to be the most promising DC2 variant in term of achieving this balance, with DC2-3X1 coatings displaying significantly improved cell-substrate adhesion



strength relative to DC2-1X coatings as well as increased average EOC migration speed relative to the more adhesive collagen controls To further evaluate the DC2 variants, initial assessment of the capacity of each DC2 to promote EOC differentiation toward an EC-fate was evaluated relative to collagen, which is known to support EC commitment and maturation64-65. Following 2 h of ~7 dynes/cm2 shear flow, EOCs on DC2 coatings were compared to collagen coating controls in terms of gene expression of early, intermediate, and late EC differentiation markers. Cumulatively, EOCs on each of the DC2 variants displayed a gene expression pattern relative to native collagen indicative of more rapid EC lineage progression on the DC2 coatings (early: ↓CD34, ↑VEGFR2; intermediate: ↑EphrinB2, ↑EphB4; late: ↔PECAM, ↔vWF, ↔VE-Cadherin). The lack of differences between the DC2 variants and collagen in terms of EOC expression of late EC differentiation markers may be due in part to the selected 2 h time frame of flow conditioning prior to phenotypic assessment. The gene expression patterns associated with the various DC2 coatings is largely consistent with our previous static differentiation data for the same donor EOCs on DC2-1X coatings relative to collagen coatings44. Specifically, we observed that EOCs seeded on DC2-1X coatings displayed the following gene expression signature relative to collagen controls: (early: ↓CD34, ↑VEGFR2; intermediate: ↑EphrinB2:EphB4, late: ↓vWF, ↔VE-Cadherin)44. However, care must be exercised in conducting a more extensive comparison between these two data sets for a number of reasons, including differences in the collagen concentrations between the 2 studies (previous: 4 mg/mL and current: 8 mg/mL) as well as differences in EOC culture conditions.


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What are potential mechanism(s) for the more rapid EC lineage progression observed on DC2 coatings relative to collagen controls? Cell differentiation has been demonstrated to be influenced by substrate stiffness, substrate ligand density66-69, and substrate ligand type70. Differences in the present study are unlikely to be due to stiffness, as we previously have shown that the stiffness of PEG-DC2 coatings of the composition used herein is determined by the molecular weight and concentration of PEG43, both of which remain consistent across the DC2 variants and controls. Furthermore, when substrate ligand type was held constant across the DC2 variants), an increase in substrate ligand density (i.e. IBS number) had modest impact on the overall gene expression pattern (slight changes in Ephrin B2 and EphB4). In terms of substrate ligand type, while DC2-3X1 and collagen I proteins contains similar per strand α1β1 and α2β1 IBS, collagen I possesses binding sites for other integrin receptors as well as binding sites for non-integrin receptors, such as Discordin Domain Receptors (DDRs). These additional receptors can compete with α1β1 and α2β1 integrins in the regulation of cell function60. Thus, the specificity of DC2 proteins for α1β1 and α2β integrins may underlie the apparent increase in the rate of EOC progression toward and EC-like fate. However, further work would be required to definitively decouple the contributions of substrate ligand density versus ligand type on EOC differentiation on DC2 versus collagen coatings. Several limitations to the present study require comment. First, EOC behaviors were only investigated at one level of physiological shear stress – when in vivo they will experience a range of shear stresses. Even though the response of EOCs to varying levels of shear stress has been extensively studied by others70-74, assessing the impact of DC2 IBS site number on EOC responses to increasing shear stress is an important area for future work. Furthermore, the



capacity of DC2 proteins to support the formation of a functional, confluent endothelium that is stable under long-term exposure to physiological stresses will need to be evaluated.

5. CONCLUSIONS In the present work, we have shown that increasing the number of GFPGER IBS in the collagen-mimetic protein DC2-1X to generate the daughter protein DC2-3X1 improves initial EOC attachment to levels comparable to collagen controls. Moreover, the DC2-3X1 coatings also promoted higher average EOC retention under physiological shear stress relative to DC2-1X coatings while also promoting increased average migration speeds in comparison to both DC21X and collagen coatings. Furthermore, all DC2 variants, including DC2-3X1, appeared to support more rapid EOC lineage progression toward an EC-fate than native collagen. Overall, the current results suggest that DC2-3X1 warrants further investigation for use in vascular graft coatings.

6. ACKNOWLEDGEMENTS The authors acknowledge the NIH NIBIB (R01 EB013297) and NSF DMR (0955259) for funding.


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FIGURE LEGENDS

Figure 1. (A) DC-2 protein design for the inclusion of 1 or 2 extra integrin binding sites on DC2-1X. (B) Native and denatured protein electrophoresis gels of the designed DC2 proteins. (C) Circular dichroism of the template protein (DC2-1X) and new designed proteins (DC2-2X, DC2-3X1 and DC2-3X2).

Figure 2. Representative rhodamine phalloidin images of EOCs attached to (A) DC2-1X, (B) DC2-2X, (C) DC2-3X1, (D) DC2-3X2, (E) collagen after 24 hours of seeding. The scale bar equals 50 µm and applies to all images. (F) Quantification of the obtained cell attachment relative to PEG-collagen for the designed proteins. ‘$’ denotes a significant difference relative to the PEG-DC2-1X. ‘*’ denotes a significant difference relative to the PEG-DC2-2X. ‘#’ denotes a significant difference relative to the PEG-DC2-3X1. ‘&’ denotes a significant difference relative to the PEG-DC2-3X1.

Figure 3. (A) Relative cell retention and (B) normalized migration speed of EOCs seeded onto the PEG-DC2 and PEG-collagen hydrogels subjected to a shear stress of approximately 7 dynes/cm2 for 1 hour at 37˚C. ‘$’ denotes a significant difference relative to the PEG-DC2-1X. ‘*’ denotes a significant difference relative to the PEG-DC2-2X. ‘#’ denotes a significant difference relative to the PEG-DC2-3X1.

Figure 4. Relative gene expression of EOCs seeded onto PEG-DC2 or PEG-collagen hydrogels after 2 hour of shear stress for (A) early and intermediate endothelial differentiation markers



(CD34, VEGFR2, EphrinB2 and EphB4) and (B) late differentiation markers (VE-Cadherin, VWF, PECAM-1). ‘$’ denotes a significant difference relative to the PEG-DC2-1X. ‘*’ denotes a significant difference relative to the PEG-DC2-2X. ‘#’ denotes a significant difference relative to the PEG-DC2-3X1.

Figure 5. Summary of retention (indirect metric for adhesiveness) and migration data superimposed on a chart displaying the biphasic relationship between cell migration and cell substratum-adhesiveness or ligand density. Increasing cell-substratum adhesiveness46 or ligand density46, 48 induces an increment in cell migration rates with up to a maximum, after which, further increments cause a decrease in cell migration.


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FOR TABLE OF CONTENTS ONLY

Collagen-Mimetic Proteins with Tunable Integrin Binding Sites for Vascular Graft Coatings Juan Felipe Diaz Quiroz, Patricia Diaz Rodriguez, Josh D. Erndt-Marino, Viviana Guiza, Bailey Balouch, Tyler Graf, William M. Reichert, Brooke Russell, Magnus Höök, and Mariah S. Hahn



Figure 1. (A) DC-2 protein design for the inclusion of 1 or 2 extra integrin binding sites on DC2-1X. (B) Native and denatured protein electrophoresis gels of the designed DC2 proteins. (C) Circular dichroism of the template protein (DC2-1X) and new designed proteins (DC2-2X, DC2-3X1 and DC2-3X2). 104x66mm (300 x 300 DPI)


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Figure 2. Representative rhodamine phalloidin images of EOCs attached to (A) DC2-1X, (B) DC2-2X, (C) DC2-3X1, (D) DC2-3X2, (E) collagen after 24 hours of seeding. The scale bar equals 50 µm and applies to all images. (F) Quantification of the obtained cell attachment relative to PEG-collagen for the designed proteins. ‘$’ denotes a significant difference relative to the PEG-DC2-1X. ‘*’ denotes a significant difference relative to the PEG-DC2-2X. ‘#’ denotes a significant difference relative to the PEG-DC2-3X1. ‘&’ denotes a significant difference relative to the PEG-DC2-3X1. 68x86mm (300 x 300 DPI)



Figure 3. (A) Relative cell retention and (B) normalized migration speed of EOCs seeded onto the PEG-DC2 and PEG-collagen hydrogels subjected to a shear stress of approximately 7 dynes/cm2 for 1 hour at 37˚C. ‘$’ denotes a significant difference relative to the PEG-DC2-1X. ‘*’ denotes a significant difference relative to the PEG-DC2-2X. ‘#’ denotes a significant difference relative to the PEG-DC2-3X1. 42x16mm (300 x 300 DPI)


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Figure 4. Relative gene expression of EOCs seeded onto PEG-DC2 or PEG-collagen hydrogels after 2 hour of shear stress for (A) early and intermediate endothelial differentiation markers (CD34, VEGFR2, EphrinB2 and EphB4) and (B) late differentiation markers (VE-Cadherin, VWF, PECAM-1). ‘$’ denotes a significant difference relative to the PEG-DC2-1X. ‘*’ denotes a significant difference relative to the PEG-DC2-2X. ‘#’ denotes a significant difference relative to the PEG-DC2-3X1. 49x21mm (300 x 300 DPI)



Figure 5. Summary of retention (indirect metric for adhesiveness) and migration data superimposed on a chart displaying the biphasic relationship between cell migration and cell substratum-adhesiveness or ligand density. Increasing cell-substratum adhesiveness46 or ligand density46, 48 induces an increment in cell migration rates with up to a maximum, after which, further increments cause a decrease in cell migration. Figure adapted from Charras et al.48 64x57mm (300 x 300 DPI)


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Collagen-Mimetic Proteins with Tunable Integrin Binding Sites for

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