Improved Endothelial Function of Endothelial Cell Monolayer on the

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Improved endothelial function of endothelial cell monolayer on the soft polyelectrolyte multilayer film with matrix-bound VEGF Hao Chang, Mi Hu, He Zhang, Kefeng Ren, Bochao Li, Huan Li, Limei Wang, Wenxi Lei, and Jian Ji ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b01870 • Publication Date (Web): 25 May 2016 Downloaded from http://pubs.acs.org on June 5, 2016

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ACS Applied Materials & Interfaces

Improved endothelial function of endothelial cell monolayer on the soft polyelectrolyte multilayer film with matrix-bound VEGF Hao Chang, Mi Hu, He Zhang, Ke-feng Ren*, Bo-chao Li, Huan Li, Li-mei Wang, Wen-xi Lei, and Jian Ji*

MOE Key Laboratory of Macromolecule Synthesis and Functionalization, Department of Polymer Science and Engineering, Zhejiang University, Hangzhou 310027, China.

KEYWORDS: endothelialization, endothelial function, stiffness, polyelectrolyte multilayer film, vascular endothelial growth factor

ACS Paragon Plus Environment

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ABSTRACT

Endothelialization on the vascular implants is of great importance for prevention of undesired post-implantation symptoms. However, endothelial dysfunction of regenerated endothelial cell (EC) monolayer has been frequently observed, leading to severe complications, such as neointimal hyperplasia, late-thrombosis, and neoatherosclerosis. It has significantly impeded long-term success of the therapy. So far, very little attention has been paid on endothelial function of EC monolayer. Bioinspired by microenvironment of endothelium in blood vessel, this study described a soft polyelectrolyte multilayer film (PEM) through layer-bylayer assembly of poly(L-lysine) (PLL) and hyaluronan (HA). The (PLL/HA) PEM was chemically crosslinked and further incorporated with vascular endothelial growth factor. It demonstrated that this approach could promote EC adhesion and proliferation, further inducing formation of EC monolayer. Further, improved endothelial function of the EC monolayer was achieved as shown with the tighter integrity, higher production of nitric oxide and expression level of endothelial function related genes, compared to EC monolayers on traditional substrates with high stiffness (e.g. glass, tissue culture polystyrene and stainless steel). Our findings highlighted the influence of substrate stiffness on endothelial function of EC monolayer, giving a new strategy in surface design of vascular implants.


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1. Introduction Coronary artery disease (CAD) is a leading cause of death and mortality worldwide, necessitating a great number of implantation surgeries by applying vascular implanting devices, such as vessel bypass grafts, stents and heart valves.1-3 However, the implantations usually accompany acute complications, such as neointimal hyperplasia, thrombosis and sustained inflammation, which impede the sustainable treatments of CAD.4, 5 These severe complications are initiated by intravascular injury and endothelial denudation during process of implantation.6 Endothelium, which is composed of endothelial cell (EC) monolayer, lies on the surface of intima and dynamically regulates homeostasis of blood vessel via its diverse functions, including anti-coagulation, anti-thrombosis, and production of nitric oxide (NO).7 Therefore, promoting endothelialization on the surface of vascular implants has been considered as an efficient strategy for reducing undesired post-implantation effects.8-10 Currently a lot of attention and efforts has been made to promote EC adhesion, migration and proliferation on the surfaces of materials through surfaces modification techniques, such as immobilization of biomolecules (e.g. extracellular matrix (ECM) proteins, peptides and gene),1118

which has received a great number of success in achievement of endothelialization. However,

endothelial dysfunction of regenerated endothelium has been frequently observed, which is associated with severe complications, such as (very) late-thrombosis and neoatherosclerosis.7, 19 So far, little attention has been paid on endothelial function of EC monolayer. Actually, complete formation of EC monolayer is not synonymous with fully restoring its endothelial function.7, 20, 21 For instance, Van Beusekom et al. found endothelium formed on stent surfaces lost its original phenotype and function, in comparison to normal endothelium in vivo.22 Nakazawa et al. applied CD34-antibody-immobilized stent to achieve rapid-endothelialization, but endothelial function


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remained impaired, leading to limited effect against restenosis.23 Therefore, improvement of endothelial function of regenerated endothelium is a critical determinant for the long-term vascular health after implantation, which remains as a great challenge. In natural blood vessels, stiffness of matrix that endothelium resides on is very low, inspired by which one could envision that a soft substrate would be an inherent requirement for endothelium and its functions. Interestingly, studies have revealed the relationship between mechanical property of substrates and behavior of EC monolayer.24-27 For instance, it was demonstrated that substrate stiffening promoted EC monolayer disruption and neutrophil transmigration.24,

25

Our group has recently demonstrated that soft substrate favored the

maintenance of EC phenotype in EC monolayer.28 Based on these findings, we hypothesized that development of a soft coating on traditional stiff substrate would be beneficial to endothelial function of EC monolayer. Poly(L-lysine)/hyaluronan (PLL/HA) polyelectrolyte multilayer film (PEM) based on layerby-layer assembly technique has been widely applied in the field of biomaterials and tissue engineering. This film can be easily deposited on many types of surfaces to impart these surfaces new chemical and physical properties. The stiffness of the (PLL/HA) PEM can be regulated by tuning the degree of chemical crosslinking, therefore this PEM offers an ideal platform for studying influence of stiffness on cell behavior.29, 30 The (PLL/HA) PEM is an exponentiallygrowing layer-by-layer film, leading to the thickness of several microns.31 This thick PEM can be used as an efficient reservoir for growth factors. Herein, (PLL/HA) PEM with low crosslinking degree (soft PEM) was employed and was further modified with vascular endothelial growth factor (VEGF) (Figure 1). Vascular endothelial growth factor (VEGF) is a very important biomacromolecules for endothelium regeneration and has been widely used to


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functionalize surfaces for promoting EC growth.11, 32, 33 Adhesion and proliferation of ECs and smooth muscle cells (SMCs) were investigated. After formation of EC monolayer, endothelial function was evaluated by characterizing morphology, integrity, NO release and expression of function related genes.

2. Experiments 2.1.(PLL/HA) film preparation and crosslinking Poly(L-lysine) (PLL, Mw 30,000 - 70,000) and polyethylenimine (PEI, branched, Mw 25,000) were purchased from Sigma (USA). Hyaluronate acid (HA, Mw 351 - 600 kDa) was purchased from Lifecore Biomedical (USA). PLL (0.5 mg/mL) and HA (1 mg/mL) were dissolved in a Hepes-NaCl buffer (20 mM Hepes at pH 7.4,150 mM NaCl). The (PLL/HA)12 films (where 12 stands for the number of layer pairs) was prepared as previously described.30 Briefly, Φ14 mm glass coverslips were dipped in the PLL solution for 8 min. After being rinsed with NaCl solution (150 mM) three times, the coverslips were dipped in the HA solution for 8 min. The coverslips were then rinsed again as described above. The sequence was repeated 12 times. For 6-well plates, films were fabricated starting with a first layer of PEI at 3 mg/mL in Hepes-NaCl buffer. The (PLL/HA)12 films were crosslinked for 18 h at 4 oC by using a crosslinking solution containing 1-ethyl-3-(3-dimethylamino-propyl) carbodiimide (EDC, Aladdin, China) (30 mg/mL) and N-hydrosulfosuccinimide (sulfo-NHS, Aladdin, China) (11 mg/mL) in NaCl (150 mM, pH = 5.5) (Here, the crosslinker directly was named as EDC30). After crosslinking, the films were rinsed by Hepes-NaCl buffer at least 8 times (30 min each time). The crosslinked (PLL/HA)12 film was named hereafter PEM.


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2.2. Incorporation of VEGF in PEM Recombinant human VEGF165 was purchased from PeproTech (100-20, USA) and reconstituted in eppendorf tubes according to provider protocol. The storage concentration of VEGF was 1 mg/mL in Mili-Q water deionized water (18 MR, Milli-Q Ultrapure Water System, Millipore) The loading process of VEGF in PEM was similar to previous reported method for bone morphogenetic protein (BMP-2) loading.34 Briefly, VEGF was diluted to desired initial concentrations by HCl solution (1 mM, pH = 3.0). PEM was pre-equilibrated for 30 min in the HCl solution and then a volume of 50 µL for 14 mm glass coverslips or 0.6 mL for 6-well plates of VEGF at desired concentration was deposited onto PEM and left to adsorb overnight at 4 °C. Hepes-NaCl buffer was added onto PEM and left at room temperature for 15 min and the washing process was repeated for 7 times in order to keep only matrix-bound VEGF.35 PEMs with matrix-bound VEGF by initial VEGF concentration at 5, 10, 20, 50, 100 and 200 µg/mL were named hereafter PEM@bV5, PEM@bV10 PEM@bV20, PEM@bV50, PEM@bV100, and PEM@bV200, respectively. To quantify amount of VEGF incorporated in PEM, concentration of VEGF in the remaining solution after loading and in combined washing solution was measured by using an enzymelinked immunosorbent assay (ELISA) kit, as recommended by the manufacturer (Boster Bioengineering, China). The density of matrix-bound VEGF in PEM was calculated from the difference between the initial and remaining amount of VEGF.14 For visualizing the VEGF loaded in PEM, samples were blocked with 0.1% bovine serum albumin (BSA, Sangon, China) solution for 1 h and incubated with polyclonal rabbit anti-human VEGF165 antibody (1:50,


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NRPB25, Hangzhou Neuropeptide Biological Science and Technology, China) for 45 min at 37 o

C. After rinsing for three times in tris-buffered saline (TBS), samples were incubated with

Alexa Fluor 488-conjugated goat anti-rabbit IgG antibody (1:400, Invitrogen, USA) in TBS with 0.1% BSA for 30 min. After washing three times with TBS, all samples were mounted onto glass slides with antifade reagent (ProLong gold, Invitrogen) and viewed under an Axiovert 200 M microscope (Axio-vert 200M, Zeiss, Germany) using 10 × objectives.

2.3. Cell culture ECs (human umbilical vein endothelial cells, 8000) and SMCs (human umbilical artery smooth muscle cells, 8030) were purchased from Sciencell (USA). ECs and SMCs were cultured in Endothelial Cell Medium (ECM, 1001, Sciencell) and Smooth Muscle Cell Medium (SMCM, 1101, Sciencell), respectively, in petri dish at 37°C and 5% CO2. The culture medium was changed every 3 days and ECs or SMCs at 80-90% confluence were used for further cell experiments. The ECs and SMCs used for experiments were between 3-8 passages.

2.4. Cell adhesion assay ECs or SMCs were seeded on PEM, PEM+sVEGF (In this condition, 150 ng/mL VEGF was added into cell culture media), PEM@bV5, PEM@bV10 PEM@bV20, PEM@bV50, PEM@bV100, PEM@bV200 and glass at a density of 2 × 104 cells/cm2. After 6 h of culture, cells were fixed in 4% paraformaldehyde in PBS for 15 min and permeabilized in TBS containing 0.1% Triton X-100 (T8787, Sigma, USA) for 10 min. After rinsing three times with


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TBS, the slides were blocked with 0.1% BSA in TBS for 1 h. The cells were then incubated with rhodamine-phalloidin (1:500, Sigma, P1951) and monoclonal rabbit anti-vinculin (1:400, Sigma, V9131) in TBS with 0.1% BSA for 50 min. After rinsing for three times in TBS, the samples were incubated with Alexa Fluor 488-conjugated goat anti-rabbit IgG antibody (1:400) in TBS with 0.1% BSA for 30 min. Finally, the nuclei were stained with DAPI (1:100, Sigma, D8417). All the samples were mounted onto glass slides with ProLong® Gold antifade reagent and viewed under an Axiovert 200 M microscope using 10 × or 100 × objectives. The fluorescence images were analyzed with Image J software (v 1.48, NIH, Bethesda). For VEGF receptor 2 (VEGFR2) inhibitor assay, ECs or SMCs were pre-treated by VEGFR2 inhibitor SU4312 (5 µM, Sigma, USA) for 10 min and then seeded on PEM@bV20, PEM@bV100, and glass at a density of 2 × 104 cells/cm2. After 2 h of culture, cells were fixed and then were stained with rhodaminephalloidin and DAPI

2.5. Cell proliferaion assay ECs and SMCs were seeded on PEM, PEM+sVEGF (In this condition, 150 ng/mL VEGF was added into cell culture media), PEM@bV5, PEM@bV10, PEM@bV20, PEM@bV50, PEM@bV100, PEM@bV200 and glass at a density of 2.5 × 104 cells/cm2, respectively. After 1 day and 3 days, cells were fixed and then were stained with rhodamine-phalloidin and DAPI as described in cell adhesion assay.

2.6. Morphology and integrity of EC monolayer


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PEM@bV20 were selected as studying group. Glass, tissue culture polystyrene (TCPS) and stainless steel (SS) were modified by one bilayer of PEM and VEGF with initial concentration at 20 µg/mL, named hereafter Glass@bV20, TCPS@bV20, SS@bV20, respectively. The amount of VEGF bound on Glass@bV20, TCPS@bV20 and SS@bV20 was also calculated by ELISA according to Section 2.2. ECs were seeded on PEM@bV20, Glass@bV20, TCPS@bV20 and SS@bV20, at a density of 5 × 104 cells/cm2. The media was changed every 2 days. After reaching confluence, cells continued being cultured for 2 days and media was changed every day. Then EC monolayers were obtained. For evaluating integrity of EC monolayer, 2 U/mL thrombin (T6884, Sigma) was added into cell media for 15 min after formation of EC monolayer. EC monolayers were stained with rhodamine-phalloidin (1:500), mouse anti-human CD31 (1:200, P8590, Sigma, USA), goat anti-mouse Alexa Fluor 488 (1:400) and DAPI according to the process described in 2.3. All samples were mounted onto glass slides with antifade reagent and viewed under an Axiovert 200 M microscope using 10 × or 100 × objectives. Gap area of EC monolayer was calculated by Image J software from at least 8 images per sample and then normalized to the gap area of EC monolayer formed on SS.

2.7. NO release of EC monolayer Cells continued being cultured for 2 days after formation of EC monolayers. The culture media was collected and concentration of NO was measured by applying NO assay kit (Boster Bio-engineering, China) according to protocol provided by manufacturer.15

2.8. Expression of endothelial function related genes by RT-qPCR


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Expression of endothelial function related genes, namely, endothelial nitric oxide synthase (eNOS), Collagen IV (α1), Fibronectin (Fn), platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31), VE-cadherin/CD144, and biglycan were analyzed by real-time quantitative PCR (RT-qPCR) assay. ECs were seeded on PEM@bV20, Glass@bV20, TCPS@bV20 and SS@bV20 in 6-well plates, at a density of 5 × 105 cells/cm2. The media was changed every 2 days. After reaching confluence, cells continued being cultured for 2 days and media was changed every day. RNA was extracted from ECs after the ECs layer formation by using the TRIzol Reagent (Haogene Biotech., China). Extracted and purified RNA (500 ng) was reverse transcribed into cDNA using the 1st-Strand cDNA Synthesis Kit (Haogene Biotech., China). Generated cDNA was used as a template to perform standard PCR analysis using Power SYBR® Master Mix (Invitrogen). The primer sequences for human eNOS were: forward 5’CCGAGTCCTCACCGCCTTCT-3’, reverse 5’-GGTAACATCGCCGCAGACAAA-3’, product size

142

bp.

The

primer

sequences

CCTGGCACTTCTGGTCAGCAAC-3’,

reverse

for

human

Fn

were:

forward

5’-

5’-CCTACATTCGGCGGGTATGGTC-3’,

product size 133 bp. The primer sequences for human PECAM-1/CD31 were: forward 5’CACCTCCAGCCAACTTCACCAT-3’, reverse 5’-CACTGTCCGACTTTGAGGCTATCT-3’, product size 90 bp. The primer sequences for human Collagen IV(α1) were: forward 5’CCACAGGGACCACCAGGACAAA-3’, reverse 5’-GGGTTTCCAGGGTAGCCAGATG-3’, product size 104 bp. The primer sequences for human Biglycan were: forward 5’GCGGGAACCCACTGGAGAACA-3’, reverse 5’-CGATGGCCTGGATTTTGTTGTGGTC-3’, product size 162 bp. The primer sequences for human CD144/VE-cadherin were: forward 5’CCAAGCCCTACCAGCCCAAAGT-3’, reverse 5’-GCCGTGTTATCGTGATTATCCGTGA3’, product size 163 bp. The housekeeping genes (18S rRNA) were forward 5’-


10

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GACTCAACACGGGAAACCTCAC-3’,

reverse

5’-CCAGACAAATCGCTCCACCAAC-3’

product size 122 bp. PCR products were detected by Real-Time PCR Detection Systems (CFX384, Bio-Rad, USA).

2.9. Statistical analysis All data were obtained from at least 3 independent experiments with at least three parallel samples per condition in each experiment and expressed as mean ± standard deviation (SD). Statistical significance was assessed with ANOVA and Student’s t test and a probability value of p < 0.05 is considered as statistically significant.

3. Results 3.1. EC adhesion Amount of VEGF incorporated in PEM was regulated depending on initial VEGF concentration and distributed in PEM homogeneously (Figure S1 and S2). EC adhesion was firstly investigated. Corresponding EC morphology was shown in Figure 2A. ECs hardly adhered on PEM and PEM+sVEGF (Figure S3), displaying round and poorly spread morphology. They began to spread on PEM@bV5. Adhesion and spread of ECs were gradually improved along with increasing amount of incorporated VEGF. Furthermore, cell density and spreading area were quantified (Figure 2B and C). Both adherent density and spreading area increased along with increasing amount of VEGF in PEM. After PEM@bV20, adherent density and spreading


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area did not significantly change with amount of VEGF in PEM. These results indicated matrixbound VEGF obviously promoted EC adhesion and spread on PEM.

3.2 SMC adhesion Corresponding SMC morphology was shown Figure 3A. SMCs hardly adhered on PEM and PEM+sVEGF (Figure S3), displaying round and poorly spread, which was similar to EC adhesion and spread under same condition. There were very little and round SMCs even on PEM@bV5, PEM@bV10 and PEM@bV20. Adhesion and spread of SMCs were gradually improved when initial VEGF concentration reached 50 µg/mL. Furthermore, cell density and spreading area were quantified (Figure 3B and C). There were scarce adherent SMCs on PEM, PEM+sVEGF, PEM@bV5, PEM@bV10 and PEM@bV20. After initial VEGF concentration reached 50 µg/mL, adherent density and spreading area increased along with increasing amount of VEGF in PEM. These results indicated matrix-bound VEGF obviously improved SMC adhesion and spread on PEM.

3.3 Proliferation of ECs and SMCs Furthermore, proliferation of ECs and SMCs for 3 days was evaluated. Morphology of ECs was shown in Figure 4A. ECs on PEM@bV20 and PEM@bV100 aggregated together after 1 day while ECs on glass showed dispersive distribution. ECs on all substrates proliferated and gradually reached confluence after 3 days. Noticeably, ECs on PEM@bV20 and PEM@bV100 displayed elongated spindle morphology and aggregated tightly, while ECs on glass showed


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round morphology. Conversely, SMCs on PEM@bV20 and PEM@bV100 did not proliferate after 3 days, although SMCs adhered and spread well on PEM@bV100 after 1 day (Figure 4B). It might be because low stiffness leaded to insufficient spread of SMC which further inhibited normal cell replication and transcription.36 SMCs on glass proliferated to a confluence. Then proliferation of ECs and SMCs was quantified. Density of ECs on PEM@bV20 and PEM@bV100 significantly increased after 3 days, while density of SMCs on same types of substrates decreased (Figure 5). The data indicated competitive growth of ECs over SMCs was achieved by incorporating VEGF into PEM with low stiffness.

3.4. Morphology of EC monolayer Morphology of EC monolayers formed on PEM@bV20, Glass@bV20, TCPS@bV20 and SS@bV20 was checked by staining with CD31 (green) and DAPI (blue). The fluorescent images after merging two color channels were showed in Figure 6. All types of substrates were fully covered by integrated EC monolayers which were homogenous and confluent. In addition, distribution of CD31 at EC borders was continuous and clear on all substrates. ECs with EC monolayer on PEM@bV20 were regularly aligned and elongated. In contrast, ECs within EC monolayer on Glass@bV20, TCPS@bV20 and SS@bV20 showed round shape and there was no obvious alignment and orientation of ECs.

3.5 Integrity of EC monolayers


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Thrombin, which is an inflammatory mediator produced by injured endothelium, can disturb the barrier function of endothelium.37 Before treating with thrombin, EC monolayers on all types of substrates were integrated with continuous distribution of CD31 at EC borders (Figure 7A). After treatment with thrombin, EC monolayer on PEM@bV20 still kept integrated and ECs connected tightly with neighbors. Conversely, stress fibers appeared and evident gaps (referred by white arrows) were observed within EC monolayers on Glass@bV20, TCPS@bV20 and SS@bV20, accompanying with disappearance of CD31 distribution at EC borders. Then normalized gap area was quantified after treatment with thrombin. The gap area within EC monolayer on PEM@bV20 were much smaller than gap area within EC monolayers on three controls and there was no significant difference in gap area among EC monolayers on three controls (Figure 7B). These results suggested EC monolayer on PEM@bV20 showed better integrity, compared to EC monolayers on Glass@bV20, TCPS@bV20 and SS@bV20.

3.6 NO release of EC monolayers As shown in Figure 8, concentration of NO released per cell in culture media was 6.34 ± 1.03 ×10-4 µM on PEM@bV20, 2.04 ± 0.40 ×10-4 µM on Glass@bV20, 1.85 ± 0.55 ×10-4 µM on TCPS@bV20, and 1.97 ± 0.52 ×10-4 µM on SS@bV20, respectively. The data indicated EC monolayer on PEM@bV20 displayed higher NO production ability, compared to EC monolayers on Glass@bV20, TCPS@bV20 and SS@bV20.

3.7 Expression of endothelial function related genes


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Furthermore, expression of endothelial function related genes was tested by RT-qPCR. As shown in Figure 9, expression level of these genes were all greatly down-regulated on Glass@bV20, TCPS@bV20 and SS@bV20 vs. PEM@bV20, including Collagen IV (0.23 ± 0.03, 0.23 ± 0.04, 0.18 ± 0.04 vs. 0.99 ± 0.09, normalized to PEM@bV20), Fn (0.45 ± 0.03, 0.42 ± 0.02, 0.41 ± 0.04 vs. 1.00 ± 0.07), PECAM-1/CD31 (0.41 ± 0.03, 0.37 ± 0.03, 0.37 ± 0.03 vs. 0.90 ± 0.06), VE-Cadherin/CD144 (0.50 ± 0.04, 0.44 ± 0.01, 0.38 ± 0.04 vs. 0.97 ± 0.08), eNOS (0.49 ± 0.04, 0.37 ± 0.02, 0.41 ± 0.05 vs. 0.86 ± 0.12) and biglycan (0.30 ± 0.01, 0.32 ± 0.05, 0.30 ±0.05 vs. 0.84 ± 0.09). The data suggested EC monolayer on PEM@bV20 showed higher expression level of endothelial function related genes, compared to EC monolayers on Glass@bV20, TCPS@bV20 and SS@bV20.

4. Discussion In terms of cardiovascular implants, regeneration of a healthy and functional endothelium is extremely desirable for prevention of post-implanting complications.10 Currently, researchers mainly focus on designing materials for promotion of endothelialization. In fact, improvement of endothelial function of regenerated endothelium is another essential requirement for the longterm health of blood vessel after implantation,7,

15, 16, 38

which, however, has received little

attention. Stiffness of ECM is known as a powerful stimulus capable of controlling cell fate.39, 40 It has been demonstrated that substrate stiffness has profound influence on endothelial function and substrates with high stiffness easily lead to dysfunction of EC monolayer.25, 26 This stiffnessinduced endothelial dysfunction also happens in pathogenesis of atherosclerosis caused by agerelated intimal stiffening.41, 42 In addition, the matrix of endothelium in vivo is much softer than


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traditional implant materials which have extremely high stiffness. From the biomimetic stand point, designing a soft substrate should thus be a natural demand for endothelium to exert its healthy endothelial function. (PLL/HA) PEM has been widely applied to mimic ECM in the field of tissue engineering.43 The stiffness of (PLL/HA) PEM can be tuned by modulating the degree of chemical crosslinking.29,

30

Herein, (PLL/HA)12 PEM with EDC30 crosslinking was applied as soft

substrate. Its Young’ modulus is ~200 kPa (Table S1),30 which is similar to the stiffness of artery.44 VEGF was bound to PEM in order to promote formation of EC monolayer. Matrixbound VEGF did not influence the stiffness and surface zeta potential of PEM (Table S1). Since both ECs and SMCs were involved in a competitive growth during endothelialization,15, 16, 45 adhesion and proliferation of both ECs and SMCs were evaluated to check the formation process of EC monolayer. The naive PEM did not support EC adhesion (Figure 2), which hampered the formation of EC monolayer. It might result from the low stiffness of PEM as previous reported.29, 30 VEGF has been reported that it can promote EC adhesion and proliferation after being bound to surface.46 Thus we incorporated VEGF into PEM in order to achieve efficient formation of EC monolayer on soft PEM. Adhesion of both ECs and SMCs was remarkably improved after incorporation of VEGF into PEM (Figure 2 and 3), which was also demonstrated by evaluation of focal adhesions (FAs) (Figure S4 and S5). This phenomenon was similar to previous report by Picart et al. and they thought it might be attributed to the cross-talks between growth factor receptors signaling and cell-adhesion receptors signaling.34 Since both EC and SMC can express the VEGF receptor,47, 48 the inhibitor of VEGFR2, SU4312, was then applied to pretreat cells, resulting in remarkable decrease in adherent density and spreading area of ECs and SMCs (Figure S6). We thus speculated the VEGFR2 might contribute to EC well adhesion


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on the soft PEM after incorporation of VEGF, which is corresponded with a recent study by Andreadis et al.32 Interestingly, both adhesion of ECs and SMCs on PEM depended on the amount of incorporated VEGF (Figure 2 and 3). It can be easily found that on PEM@bV20, number of ECs reached to the maximum value compared with the number of adherent EC on the glass (positive control). However, number of SMCs was still little, which means SMCs need much higher VEGF densities for adhesion. This might be attributed to difference in sensitivity to VEGF between ECs and SMCs. This discrepancy leaded to achievement of EC competitive growth over SMC on PEM@bV20 (Figure 4 and 5). It has demonstrated that enhancement of EC competitive growth over SMC favors the formation of a pure and confluent EC monolayer13,

15, 16, 18

.

Therefore, PEM@bV20 can efficiently facilitate formation of EC monolayer. Then formed EC monolayer on PEM@bV20 was evaluated. Glass@bV20, TCPS@bV20 and SS@bV20 were selected as controls which had much higher surface stiffness (over 700 MPa) and similar surface zeta potential compared with PEM@bV20 (Table S1). Morphology of EC monolayer on PEM@bV20 was aligned and elongated, differing from these on Glass@bV20, TCPS@bV20 and SS@bV20 (Figure 6). This phenomenon was probably attributed to the combinative influence of soft substrate and VEGF, which was corresponded to previous studies.49,

50

Aligned and elongated ECs might be resistant to inflammatory reactions while

nonaligned or cuboidal ECs seem to induce atherosclerosis.51, 52 Therefore, a fully confluent EC monolayer with in vivo-like EC alignment and elongation was obtained on PEM@bV20, which might be more capable of maintaining vascular homeostasis.45 Our recent study found soft substrate helped ECs keep their phenotype.28 Previous studies reported the integrity of EC monolayer was influenced by stiffness.24,

26, 42

Inspired from these studies, we envisioned


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reducing substrate stiffness might be an ideal strategy for promotion of endothelial function. In fact, endothelial function should be evaluated in terms of its integrity, formed cell junction, expression of antithrombotic molecules and NO production.7 Therefore, we applied the soft PEM as a platform to investigate whether soft substrate was able to favor improvement of endothelial function. Endothelium in vivo keep compact barrier integrity to be resistant to undesirable external stimuli, which mostly relies on tight intercellular junctions.53 EC monolayer formed on PEM@bV20 maintained better barrier integrity compared to EC monolayers on three controls (Figure 7). This result was consistent with other studies on integrity of EC monolayers responding to substrate stiffness.25,

54

Substrates with high stiffness (e.g. Glass@bV20,

TCPS@bV20 and SS@bV20) increase cell-substrate interaction and weaken cell-to-cell adherence,55,

56

which results in easy separation of neighboring ECs by thrombin. Then

permeability of endothelium increased, which further exacerbates the inflammatory response that leads to atherothrombosis.57 Production of NO, is one of the most significant functions of endothelium.58 The EC monolayer on PEM@bV20 showed higher NO production ability (Figure 8). Finally, expression of several genes was tested by RT-qPCR. These genes were associated with important endothelial functions. Thereinto, collagen IV and Fn are key structural extracellular matrix (ECM) molecules and their production reflects the ECM remodeling ability of endothelium.59 VE-cadherin/CD144 is an important junction component.7 PECAM-1/CD31 is an antithrombotic molecule.60 Biglycan plays an important role in inhibiting SMC growth and migration.59 All of genes expression level was much higher when EC monolayer resided on PEM@bV20 than on control groups (Figure. 9). Of note, high expression of VE-cadherin/CD144 on the PEM@bV20 reflected tight cell junctions which is a critical determinant of endothelial


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integrity. It thus was consistent with the result of integrity evaluation (Figure 7). In addition, result of eNOS expression was consistent with the result of NO release (Figure 8). Altogether, EC monolayer on PEM@bV20 was more competent, as demonstrated by compact integrity, tightly formed cell-to-cell junctions, increased expression of antithrombotic molecules and NO production, compared with the EC monolayers on Glass@bV20, TCPS@bV20 and SS@bV20. Of note, the amount of VEGF bound to PEM@bV20 was ~0.70 µg/cm2 (Figure S1), which was higher than amount of VEGF bound to Glass@bV20, TCPS@bV20 and SS@bV20 (~0.3 µg/cm2) (Figure S7). In order to investigate contribution of VEGF to endothelial function, we evaluated the EC monolayers formed on Glass (which was also modified by one bilayer (PLL/HA) and on Glass@bV20. The data suggested that VEGF improved NO production, expression of eNOS, VE-cadherin/CD144 and PECAM-1/CD31,61 but did not impact the morphology, integrity, expression of collagen IV, Fn and biglycan, as shown from Figure S8 to S11. Regardless of difference in amount of VEGF, the low stiffness of PEM@bV20 is an indispensable factor contributing on improvement of endothelial function, especially on achieving in vivo-like EC morphology, improving integrity, up-regulating expression of collagen IV, Fn and biglycan. Therefore, our results highlighted the benefit of soft substrate to endothelial function. Endothelium is quite different from single ECs. Increase substrate stiffness can promote EC adhesion and proliferation because high stiffness favors ECs maintain normal cell replication and transcription.36

62

However, subsequently formed EC monolayer needs low

stiffness to keep high endothelial function. Endothelium mainly relies on cell-cell connection to exert its function.53 It is because high stiffness can increase of cell contractility and decrease of cell-cell interaction, which leading to poor and sensitive integrity of EC monolayer.54, 63 Overall,


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our results suggested the soft PEM with matrix-bound VEGF efficiently improved endothelial function of regenerated EC monolayer.

5. Conclusions In this study, a low stiffness (PLL/HA)12 PEM with incorporated VEGF was developed. EC monolayer efficiently formed on the PEM due to promotion of competitive growth of ECs over SMCs by the matrix-bound VEGF. In addition, the endothelial function of as-formed EC monolayer was greatly improved, which highlights the benefit of soft substrates to endothelial function. Taken together, this study provides insights into the influence of substrate stiffness on EC monolayer and endothelial function, which is of great potential for a new design strategy of surface modification in the field of vascular implants.

ASSOCIATED CONTENT Supporting Information Available Experiments of Young’s modulus test by Atomic Force Microscope and zeta potential measurements. The summary of Young’s modulus and surface zeta potentials of related substrates was shown in Table S1. Amount of VEGF incorporated in PEM are shown in Figure S1. Fluorescent images of PEM with matrix-bound VEGF stained with VEGF antibody are shown in Figure S2. Morphology of ECs and SMCs stained by F-actin after 6 h culture on PEM with supplement of 150 ng/mL VEGF in media are shown in Figure S3. Immunofluorescent images of ECs and SMCs stained by vinculin are shown in Figure S4 and S5. The result of


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VEGFR2 inhibitor assay is shown in Figure S6. Amount of VEGF bound to Glass@bV20, TCPS@bV20 and SS@bV20 is shown in Figure S7. The evaluations of EC monolayer and endothelial function on Glass and Glass@bV20 are shown in Figure S8-S11. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author E-mail: [email protected]; [email protected]; Fax: +86-571-87953729; Tel: +86-571-87953729.

ACKNOWLEDGMENT This research was supported by Zhejiang Provincial Natural Science Foundation of China under Grant No. LR15E030002, the Key Science Technology Innovation Team of Zhejiang Province (no. 2013TD02), the National Natural Science Foundation of China (51333005, 21374095), the National Basic Research Program of China (2011CB606203), Research Fund for the Doctoral Program of Higher Education of China (20120101130013), and International Science & Technology Cooperation Program of China (2014DFG52320). We thank Prof. Bo Song from Soochow University, China, for his kindly help in the AFM experiments. We thank Prof. Zhikang Xu from Zhejiang University, China, for his kindly help in the surface zetapotential tests.


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List of Figures

Figure 1. Illustration of promotion of endothelial cell (EC) competitive growth over smooth muscle cells and improvement of endothelial function of regenerated EC monolayer on the lowcrosslinked (PLL/HA) polyelectrolyte multilayer film (PEM) with matrix-bound VEGF.


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Figure 2. Adhesion of ECs after 6 h culture on PEM, PEM@bV5, PEM@bV10 PEM@bV20, PEM@bV50, PEM@bV100, PEM@bV200 and glass. (A) Fluorescent images of EC morphology stained by F-actin. (B) Density and (C) spreading area of ECs was quantified after 6 h culture. At least 200 cells were counted for each condition. N = 3 parallel samples per group. The data are representative of three independent experiments, mean ± SD, * p < 0.05. The scale bar is 200 µm.


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Figure 3. Adhesion of SMCs after 6 h culture on PEM, PEM@bV5, PEM@bV10 PEM@bV20, PEM@bV50, PEM@bV100, PEM@bV200 and glass. (A) Fluorescent images of EC morphology stained by F-actin. (B) Density and (C) spreading area of SMCs was quantified after 6 h culture. At least 200 cells were counted for each condition. N = 3 parallel samples per group. The data are representative of three independent experiments, mean ± SD, * p < 0.05. The scale bar is 200 µm.


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Figure 4. Proliferation of (A) ECs and (B) SMCs after 1 day and 3 days on PEM@bV20, PEM@bV100 and glass. Morphology of ECs and SMCs was observed by staining their actin cytoskeleton. The scale bar is 200 µm.


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Figure 5. Density of (A) ECs and (B) SMCs quantified after 1 day and 3 days on PEM@bV20, PEM@bV100 and glass. N = 3 parallel samples per group. At least 8 images were analyzed per sample. The data are representative of three independent experiments.


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Figure 6. Morphology of EC monolayer formed on PEM@bV20, Glass@bV20, TCPS@bV20 and SS@bV20. EC monolayer was stained by CD31 (green) and nuclei (blue). The immunofluorescent images were obtained by merging two color channels. The scale bar is 200 µm.


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Figure 7. Integrity of EC monolayer formed on PEM@bV20, Glass@bV20, TCPS@bV20 and SS@bV20. EC monolayer was treated with 2 U/mL thrombin for 15min before fixing. EC monolayer was stained by CD31 (green), F-actin (red) and nuclei (blue). (A) The immunofluorescent images were obtained by merging three color channels. (B) Quantitative data of gap area appeared in EC monolayers formed on PEM@bV20, Glass@bV20, TCPS@bV20 and SS@bV20 after treatment of thrombin, which was normalized to gap area of EC monolayer on SS@bV20. N = 3 parallel samples per group. At least 8 images were analyzed per sample. The data are representative of three independent experiments, mean ± SD, * p < 0.05, ns: no significant difference. The scale bar is 50 µm.


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Figure 8. Concentration of NO released in culture media per EC from EC monolayers formed on PEM@bV20, Glass@bV20, TCPS@bV20 and SS@bV20. N = 3 parallel samples per group. The data are representative of three independent experiments, mean ± SD, * p < 0.05.


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Figure 9. Normalized expression of endothelial function related genes of EC monolayers formed on PEM@bV20, Glass@bV20, TCPS@bV20 and SS@bV20. Each type of gene expression was detected by RT-qPCR and normalized to that on PEM@bV20. N = 3 parallel samples per group. The data are representative of three independent experiments, mean ± SD, * p < 0.05.


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TOC Graphic


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Improved Endothelial Function of Endothelial Cell Monolayer on the

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