Effect of Polyelectrolyte Film Stiffness on Endothelial Cells During

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Effect of polyelectrolyte film stiffness on endothelial cells during endothelial-to-mesenchymal transition He Zhang, Hao Chang, Limei Wang, Kefeng Ren, M. Cristina L. Martins, Mário A. Barbosa, and Jian Ji Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.5b01057 • Publication Date (Web): 19 Oct 2015 Downloaded from http://pubs.acs.org on October 22, 2015

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Effect of polyelectrolyte film stiffness on endothelial cells during endothelial-to-mesenchymal transition He Zhang a, Hao Chang a, Li-mei Wang a, Ke-feng Ren a,b,*, M. Cristina L. Martins c,d,e, Mário A. Barbosa d,e, Jian Ji a,* a.

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

b.

State Key Laboratory of Molecular Engineering of Polymers (Fudan University), China.

c.

i3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Portugal.

d.

INEB - Instituto de Engenharia Biomédica, Universidade do Porto, Rua do Campo Alegre, 823, 4150-180, Portugal.

e.

ICBAS - Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto, Portugal.

KEYWORDS: endothelial-to-mesenchymal transition, endothelial cells, layer-by-layer assembly, substrate stiffness, vascular implants.

ACS Paragon Plus Environment

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ABSTRACT

Endothelial-to-mesenchymal transition (EndMT), during which endothelial cells (ECs) transdifferentiate into mesenchymal phenotype, plays a key role in the development of vascular implant complications such as endothelium dysfunction and in-stent restenosis. Substrate stiffness has been confirmed as a key factor to influence EC behaviors; however, so far, the relationship between substrate stiffness and EndMT has been rarely studied. Here, ECs were cultured on the (poly(L-lysine)/hyaluronate acid) (PLL/HA) multilayer films with controlled stiffness for two weeks, and their EndMT behaviors were studied. We demonstrated that ECs lost their markers (vWf and CD31) in a stiffness-dependent manner even without supplement of growth factors, and the softer film favored the maintaining of EC phenotype. Further, induced by transforming growth factor β1 (TGF-β1), ECs underwent EndMT as characterized by losing their typical cobblestone morphology and markers, and gaining smooth muscle cell markers (αsmooth muscle actin and calponin). Interestingly, stronger EndMT was observed when ECs were cultured on the stiffer film. Collectively, our findings suggest that substrate stiffness has significant effects on EndMT, and a softer substrate is beneficial to ECs by keeping their phenotype and inhibiting EndMT, which presents a new strategy for surface design of vascular implant materials.


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1. Introduction Vascular implants such as stents, vascular grafts and heart valves, are of great significance in treating vascular diseases. However, severe complications such as in-stent restenosis and thrombosis remain happening, leading to implant failures and even patients’ death.1-3 It has been recognized that an intact endothelium is essential to prevent these complications.2, 4-6 Hence, great efforts have been made to promote the rapid re-endothelialization on implant surfaces.6-11 The promoted growth of endothelial cells (ECs) and rapid EC coverage on the implanted materials, however, may not be enough to guarantee a long-term healthy endothelium. Recent findings from both experimental and clinical studies have demonstrated that, although there was a fully coverage of ECs, abnormal endothelial functions on implants were detected, resulting in high risk issues such as late stent thrombosis and neo-atherosclerosis.12-14 Considering the complex biological responses after vascular device implantations, the interactions between implanted materials and formed endothelium is poorly investigated, though.15 Endothelial-to-mesenchymal transition (EndMT) is an important EC biological behavior. Undergoing EndMT, ECs would lose their original cell-cell junction and endothelial specific markers, such as CD31 (PECAM-1), and gain a mesenchymal phenotype by expressing mesenchymal markers or smooth muscle cell (SMC) markers, such as α-smooth muscle actin (αSMA) and calponin,16-19

which was referred to as myofibroblast lineage19,

20

or smooth

muscle-like cells.16-18 Although EndMT is a critical process in early embryonic cardiac development, tissue regeneration, and wound healing,19, 21 it also contributes to the development of cardiovascular diseases and endothelium dysfunction after vascular device implantations.22-25 For instance, ECs that undergo EndMT are important source for myofibroblasts which contribute to in-stent restenosis and vascular fibrosis.20 EndMT is regulated by signaling pathways


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mediated by cytokines, and transforming growth factor β1 (TGF-β1) plays the most important role.16,

18, 26, 27

Especially, in the case of vascular stent implantation, the level of TGF-β1 is

significantly up-regulated after implantation,20, 28 and the enhanced EndMT induced by overexpressed TGF-β1 can result in inhibition of endothelial regeneration24,

29

and formation of

neointima.16, 23, 30, 31 As an essential pathological process of many diseases, EndMT has been extensively studied from the biological aspects, such as its signaling pathways and molecular mechanisms.27, 31-33 Still, little is understood about the relationship between implant materials and EndMT process. Surface stiffness is a key material property, and it strongly affects cell-material interactions and almost all cell behaviors such as adhesion, proliferation and differentiation.34-37 Importantly, researchers have also found that substrate stiffness has profound influence on TGF-β1 and its bioactivity on cells.38 For instance, latent TGF-β1 stress activation and some TGF-β1-induced cell differentiations require a stiffer matrix to take place, such as differentiations of stem cells and hepatic stellate cells.39, 40 Nevertheless, the effect of material stiffness on EndMT remains poorly investigated, yet. Meanwhile, worldwide used vascular implants, e.g. stainless steel stents, have highly rigid surfaces compared to soft tissues in the body, which do raise a concern about the relationship between material stiffness and ECs during EndMT process. In this study, we constructed polyelectrolyte multilayer films with controlled stiffness through layer-by-layer (LBL) assembly of poly(L-lysine) (PLL) and hyaluronate acid (HA) (Scheme 1).41-45 ECs were cultured on the (PLL/HA) films for up to two weeks. EC density, morphology and cellular marker expressions were characterized. Furthermore, EndMT in ECs were induced by TGF-β1 stimulation, and the influence of film stiffness on ECs and EndMT were analyzed and discussed.


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2. Experiments 2.1 Materials Poly(L-lysine) (PLL, Mw 30000-70000), polyethylenimine (PEI, branched, Mw 25,000), Triton X-100 and 4,6-diamidina-2-phenylin (DAPI) were purchased from Sigma-Aldrich (USA). Hyaluronate acid (HA, Mw 351-600 kDa) was purchased from Lifecore Biomedical (USA). N[2-hydroxyethyl] piperazine-N’-[2-ethanesulfonic acid] (HEPEs, free acid, high purity grade), phosphate buffered saline (PBS), bovine serum albumin (BSA) and tris-buffered saline (TBS) were obtained from Sangon Biotech (Shanghai, China). 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-Hydroxysulfosuccinimide sodium salt (sulfo-NHS) were purchased from Aladdin (Shanghai, China). Human recombinant transforming growth factor β1 (TGF-β1) was purchased from PeproTech (USA). Endothelial Cell Medium was purchased from ScienCell Research Laboratories (USA). RPMI 1640 Medium, penicillin and streptomycin (P/S) and 0.25% trypsin–EDTA solution were purchased from Genom Biomedicaltech (Hangzhou, China). Fetal bovine serum (FBS) was obtained from Hyclone (USA). All solutions were prepared using deionized water (18 MΩ, Milli-Q Ultrapure Water System, Millipore).

2.2 The (PLL/HA) multilayer films The (PLL/HA) multilayer films with different stiffness were prepared according to the method described previously.41 Briefly, glass coverslips (Φ=14 mm) were cleaned with Piranha solution


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[7:3 (v%) 98% H2SO4 : 30% H2O2)] for 20 min, rinsed with water, and then cleaned in 1:1:5 (v%) 30% H2O2 : 25% NH3 : H2O mixture at 60 ºC for 30 min. PEI (3 mg/mL), HA (1 mg/mL), and PLL (0.5 mg/mL) were dissolved in HEPES-NaCl buffer solution (20 mM HEPES, 0.15 M NaCl, pH 7.4). Prior to LBL assembling, substrates were modified with an anchoring PEI layer for 30 min. Then the substrates were immersed in HA solution for 8 min, rinsed three times with NaCl (0.15 M, pH 6.4), and submerged in PLL solution for 8 min. This cycle was repeated until [PEI-HA-(PLL/HA)11] films were finished. Films were crosslinked for 18 h at 4 ºC with EDC at various final concentrations (30, 70, 100 and 150 mg/mL) and sulfo-NHS at 11 mg/mL, both dissolved in NaCl (0.15 M, pH 5.5). Finally, films were extensively rinsed with HEPES-NaCl buffer solution. Films crosslinked with 30, 70, 100 and 150 mg/mL EDC are respectively referred to as EDC30, EDC70, EDC100 and EDC150 in this study. To measure stiffness of the (PLL/HA) films, silicon wafers coated with EDC30, EDC70, EDC100 and EDC150 films were characterized by Atomic Force Microscope (AFM, multimode 8, Bruker). The probe (SNL-10, Bruker) had a nominal spring constant of 0.06 N/m. The samples were immersed in a drop of 0.15 M NaCl aqueous solution, and the tests were performed in liquid environment. For each sample, measurements were obtained at eight different sites, and the data were analysed by NanoScope Analysis software using the Sneddon model as modulus fit model. The fitting range was from 30 % - 90 %.

2.3 HUVECs isolation and culture Human umbilical vein endothelial cells (HUVECs) were isolated fresh from umbilical cords as the protocols described previously with local ethical committee approvals.46 HUVECs were used


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for experiments between 3 and 8 passages. ECs were cultured in complete medium, namely Endothelial Cell Medium (ECM, ScienCell Research Laboratories, Cat No. 1001, USA) supplemented with 5% FBS, endothelial cell growth factor supplements (ECGS) and penicillin/streptomycin, at 37ºC in a humidified atmosphere containing 5% CO2. Culture medium was changed every 3 days. ECs with 90% confluence were harvested by using trypsin–EDTA, and passaged or seeded onto the substrates for further experiments.

2.4 Inducing EndMT of ECs ECs were seeded onto bare glass coverslips, glass coverslips coated with EDC30, EDC70, EDC100 and EDC150 films in 24-well plates at a density of 50,000 cells/cm2 (60,000 cells/cm2 in multilayered films-coated 6-well plates for real-time quantitative PCR analysis), and were allowed to grow for 1 to 2 days in the complete medium (ECM) to reach 80-90% confluence. Then the culture medium was changed. In our control groups, cells were cultured in control medium (CM) comprised of RPMI 1640 supplemented with 20% FBS and 1% penicillin/streptomycin (without either exogenous TGF-β1 or ECGS). In order to simulate the TGF-β1-induced EndMT, the medium was replaced by mesenchymal differentiation medium (MDM) comprised of RPMI 1640 supplemented with 20% FBS, 1% penicillin/streptomycin and 5 ng/mL TGF-β1. Cells were cultured for additional 1, 7 and 14 days.

2.5 Analysis of cell numbers, morphologies and cell viability


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On day 1, 7 and 14, the morphology of cells on different substrate were observed in phase contrast microscopy by using a 10× objective under an Olympus DP72 (Olympus, Japan) inverted microscope. The analysis of cell number was performed by either counting the cells in the phase contrast images or counting the nuclei in the DAPI-labeling images, using the cell counter plugin of ImageJ software (v 1.44p, NIH). At least 100 cells of each group were analyzed with ImageJ to determine the cell morphologies, which were characterized by circularity (4π(area/perimeter2)). The closer the circularity is to 1, the cell morphology is more similar to a circle, while a value close to 0 corresponds to cells with a very low area and high perimeter.47 The EC viability was characterized by standard MTT assay. Briefly, ECs were seeded in EDC70, EDC100 and EDC150-coated 96-well plates at the same density of that in 24-well plates and were allowed to grow for another 24 h. Then the culture medium was changed into CM or MDM. On day 1, 7 and 14, the wells were washed with PBS and each well was treated with the mixture of 100 µL medium and 20 µL MTT (5 mg/mL in PBS) at 37 °C for 4 h. Then the medium was carefully removed and 150 µL of dimethyl sulfoxide (DMSO) was added to each well. The plates were further incubated at 37 °C for 5 min, and then the absorbance at 570 nm was measured by a microplate reader (MODEL 550, Bio Rad). The relative cell viability (%) = the absorption of the well / the average absorption of EDC70 wells × 100%.

2.6 Immunofluorescence assay Immunofluorescence staining was performed to detect cell adhesion, spreading, the expression of endothelial specific markers, and that of mesenchymal markers (also SMC markers


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herein). Rabbit anti-human von Willebrand factor (vWF) polyclonal antibody (Sigma, USA, 1:300 v/v), mouse anti-human CD31 polyclonal antibody (Sigma, USA, 1:200 v/v), mouse antihuman α-smooth muscle actin (αSMA) monoclonal antibody (Sigma, USA, 1:400 v/v), mouse anti-human calponin monoclonal antibody (Sigma, USA, 1:200 v/v) and mouse anti-human vinculin monoclonal antibody (Sigma, USA, 1:300 v/v) were employed as primary antibodies. Alexa Fluor 568-conjugated goat anti-rabbit IgG antibody (Invitrogen, USA) and Alexa Fluor 488-conjugated goat anti-mouse IgG antibody (Invitrogen, USA) were used as secondary antibodies. On day 14, the samples were rinsed with PBS, fixed with 4% paraformaldehyde, treated with 0.2% Triton X-100 in TBS, and blocked with 0.1% BSA in TBS. Then the samples were incubated with the chosen primary antibody solution (in TBS with 0.1% BSA), washed with TBS and incubated with corresponding secondary antibody solution (in TBS with 0.1% BSA). For F-actin staining, samples were incubated with phalloidin-tetramethyl-rhodamine B isothiocyanate (phalloidin-TRITC, Sigma, USA, 1:400 v/v) in TBS. Finally, cell nuclei were stained with DAPI and samples were washed with TBS. Samples were then mounted onto glass coverslips by using ProLong® Gold antifade reagent (Invitrogen, USA). Fluorescent images were taken with an Olympus DP72 inverted microscope. To determine the expression of vWF and CD31, the fluorescence intensity of the images was measured using ImageJ, and the results were calculated as mean fluorescence intensity per unit area of each cell in each image.

2.7 Real-time quantitative PCR (RT-qPCR) analysis To further quantify the expression of endothelial as well as mesenchymal (also SMC) marker molecules in cells on substrates with different stiffness, real-time quantitative PCR (RT-qPCR)


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was carried out by widely accepted methods. Briefly, total RNA from the sample cells on day 14 was extracted using Trizol reagent (Invitrogen, USA) according to the manufacturer’s instructions and quantified with a biophotometer (Eppendorf, Germany). The primer sequences for human

vWF were:

forward

5’-GTGTCCACCGAAGCACCATCT-3’,

reverse 5'-

CCCATCACGGCATCCTCCAT-3', product size 94 bp. The primer sequences for human CD31 were:

forward

5’-CACCTCCAGCCAACTTCACCAT-3’,

reverse

5’-

CACTGTCCGACTTTGAGGCTATCT-3’, product size 90 bp. The primer sequences for human αSMA

were:

forward

5’-CTAGCACCCAGCACCATGAAGA-3’,

reverse

5’-

GCCAGGATGGAGCCACCGAT-3’, product size 83 bp. The primer sequences for human calponin

were:

forward

5’-GCAGTCCACCCTCCTGGCTTT-3’,

reverse

5’-

GCGTACTTCACTCCCACGTTCA-3’, product size 83 bp. The primer sequences for the housekeeping genes (18S rRNA) were: forward 5’-GACTCAACACGGGAAACCTCAC-3’, reverse 5’-CCAGACAAATCGCTCCACCAAC-3’, product size 122 bp. RT-qPCR was performed with iQ SYBR green PCR Master Mix (Bio-Rad, CA, USA), and the samples were handled in One iCycler iQ5 (Bio-Rad, CA, USA) as the following steps: initial denaturation at 95 ºC for 10 min, 45 cycles of 94 ºC for 5 s and 62 ºC for 20 s. CT (threshold cycle) values were calculated with the iQ5 optical system software. To determine the relative expression ratio of genes, a mathematical model previously described in detail was applied.48 The relative gene expression values were calculated against the rate of 18S, and shown as 2△Ct×106.

2.8 Statistical analysis


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The experiments in this study were all independently repeated at least three times. The data were presented as mean ± standard deviation (SD). To compare the data obtained from different samples under identical experimental conditions, One-way ANOVA and Student's t test were used. p values of less than 0.05 were considered to be statistically significant.

3. Results 3.1. Multilayer films and endothelial cell growth The (PLL/HA) multilayer films with controlled stiffness were employed as cell culture substrate because this kind of film has been well characterized and extensively used as a model platform for investigation of stiffness roles in cell behaviors. It is convenient to generate films with various crosslinking degrees by simply controlling the concentrations of crosslinking reagents (EDC and sulfo-NHS),41-44 and these films do not require any extracellular matrix protein pre-coating.43 In this study, we regulated the EDC concentrations to 30, 70, 100 and 150 mg/mL for crosslinking. As measured by AFM (Figure S1), the Young’s modulus of EDC30, EDC70, EDC100 and EDC150 was 196±41, 317±30, 431±39 and 491±63 kPa, respectively, which was at same level as the previous studies on the same kind of (PLL/HA) films.41, 44 The stiffness of the films grew with increasing crosslinking degree, indicating that films with different stiffness were successfully generated. Besides, some studies suggest that variations in other film properties such as roughness and hydrophilia are not significantly changed in this range of crosslinking degree.41, 42 The cell adhesion and proliferation on the (PLL/HA) films were firstly investigated. ECs were cultured in complete ECM on EDC30, EDC70, EDC100 and EDC150. As shown in Figure 1,


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ECs attached on all the films except EDC30. The density of ECs on EDC70 was slightly lower than that on EDC100 and EDC150 at the beginning, but after two days the cell number of EDC70 was close to EDC100 and EDC150 (Figure S2). On day 4, few cells existed on EDC30. At the meantime, cells on EDC70, EDC100 and EDC150 proliferated to confluence, similarly to that on bare glass. This result is quite in accordance with previous study.41, 43 EDC70, EDC100 and EDC150 are able to support the adhesion and the proliferation of ECs, and are suitable substrates for further study. Whereas, EDC30 was too soft for cell adhesion, and was not used afterwards.

3.2. ECs in response to film stiffness Culturing ECs in a medium containing TGF-β1 is a typical way to induce EndMT.16, 18 Before studying the EndMT behavior of ECs with TGF-β1 stimulation, ECs were cultured in the control medium (CM) without either soluble TGF-β1 or endothelial cell growth factor supplements, for understanding the role of stiffness alone in cell growth and phenotype. As the phase-contrast images show (Figure 2A-I), ECs could be cultured in CM for as long as 14 days. Neither proliferative behavior nor abrupt decline in EC densities was observed on EDC70, EDC100 or EDC150, and ECs demonstrated their cobblestone morphology. The cell densities and cell viability on all films were decreasing slowly (Figure 2J, K), but there was no significant change in cell morphology (Figure 2L). There was no significant difference in ECs among EDC70, EDC100 and EDC150. To further investigate the EC phenotype, endothelial markers (vWF and CD31) and smooth muscle markers (αSMA and calponin) of cells on day 14 were immunostained, as shown in


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Figure 3. The ECs on EDC70 clearly expressed the endothelial markers vWF and CD31. However, remarkably, decreases of both vWF and CD31 were seen with increasing substrate stiffness (Figure 3A-F). In all conditions, no obvious smooth muscle markers were observed (Figure 3G-L). Further quantitative analysis confirmed that the immunofluorescence intensity of endothelial markers reduced with increasing film stiffness (Figure 3M, N). All the data suggest that ECs tended to lose their original phenotype without growth factor supplements, while the softer substrate is favorable to maintaining cell phenotype.

3.3. EndMT behaviors on films with different stiffness To investigate the effect of substrate stiffness on TGF-β1-induced EndMT, ECs were cultured in mesenchymal differentiation medium (MDM) containing 5 ng/mL TGF-β1 on different films for 14 days. One can clearly observe from the phase-contrast images (Figure 4A-I) that cell densities on all substrates quickly dropped with time, resulting in both relatively low cell density and low confluence on day 14. Figure 4J shows the relevant quantitative data. Compared with day 1, the cell densities on day 14 dropped by 80.3±2.0%, 81.7±1.7% and 82.6±3.3% on EDC70, EDC100 and EDC150, respectively, and this decrease was stiffness-independent. This trend was further confirmed by cell viability assay (Figure 4K) which shows that a lot of ECs died during the incubation with TGF-β1. There were also changes in cell morphology as time prolonged. Most of the ECs on EDC70 maintained their native cobblestone-like morphology with very few cells changing their shapes. With increasing film stiffness, more ECs lost their cobblestone-like shape, and some of the cells even showed an elongated, spindle-shape morphology, as shown in Figure 4H and 4I. On day 14, the circularity of cells on EDC70, EDC100 and EDC150 decreased


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successively (Figure 4L), proving the elongating of the cells with increasing stiffness. Our data suggest that the TGF-β1 led to a high mortality of ECs regardless of film stiffness, while the change in morphology was stiffness-dependent. The loss of native endothelial morphology was stronger for cells on stiffer films. The EndMT behavior of ECs was further characterized by immunofluorescence assay. Cells were again stained for endothelial markers (vWF and CD31) and smooth muscle markers (αSMA and calponin) (Figure 5). There were still apparent vWF and CD31 expressions in most of the cells on EDC70, while the expressions of vWF and CD31 dropped sharply on EDC100 and EDC150 (Figure 5A-F). The expressions of SMC markers in cells cultured on EDC70 were seen in very few cells (Figure 5G, J), while in the case of EDC100 and EDC150, clear smooth muscle markers in most of the cells were observed (Figure 5H, I, K, L). Figure 6 shows the quantitative ratio of positive cells for each marker. As for endothelial markers, the ratio of vWF-positive cells decreased with increasing film stiffness; while over 75% of the cells were CD31-positive without difference among EDC70, EDC100 and EDC150 (Figure 6A, B). Surprisingly, almost all the cells on stiffer EDC100 and EDC150 expressed the two SMC markers, while on softer EDC70, only 14.5±6.1% and 30.0±24.4% of ECs had detectable protein expression levels of αSMA and calponin, respectively (Figure 6C, D). To further quantitatively measure EndMT behaviors on the level of gene expression, RTqPCR was performed on day 14 with data shown in Figure 7. Compared with cells on softer EDC70, down-regulation of the gene expressions of the two EC markers and significant upregulation of that of the two SMC markers took place in cells on stiffer EDC100 and EDC150. This trend was quite in accordance with the immunofluorescence results. There was no very significant difference in those gene expressions between EDC100 and EDC150. Of note,


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although most of the ECs on all substrates were found to be CD31-positive as described above (Figure 6B), RT-qPCR shows that their CD31 gene expression ratio varied significantly in response to film stiffness (Figure 7B). The CD31 expression ratio of cells on EDC100 and EDC150 was 82.6±2.0% and 72.8±2.1% lower than that on EDC70, respectively. The data suggest that with stimulation of soluble TGF-β1, ECs exhibited notable SMC marker expressions on the stiffer films; while on the softer film, SMC marker expressions were inhibited, and the maintained EC marker expressions were observed.

4. Discussion In the current study, we investigated the effect of substrate stiffness on EndMT in ECs by using (PLL/HA) films. We have fabricated the (PLL/HA) films with various stiffness Then, the EndMT behavior of ECs in response to substrate stiffness was characterized by cell density, morphology and cellular marker expressions. Previous studies reported that ECs undergoing EndMT tend to exhibit decreased EC markers such as vWF and CD31, and increased intermediate mesenchymal markers such as vimentin and Twist,30,

33, 49

and even SMC or

myofibroblast markers such as αSMA and calponin.16-19 Herein, considering the role of EndMT in vascular implant complications, the two SMC markers were chosen to be characterized as the transdifferentiation markers. In this study, the TGF-β1 in the culture medium and substrate stiffness are two factors which might influence EndMT on their own, since substrate stiffness have been reported to influence some differentiations even without growth factor stimulation.37, 40 Hence, in order to investigate whether stiffness itself would affect the transdifferentiation of ECs without the stimulation of


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TGF-β1, ECs were cultured on different substrates in control medium (CM) supplemented only with 20% FBS, without either soluble TGF-β1 or endothelial cell growth factor supplements (ECGS). It is surprising and interesting to see the significant decrease of EC marker expression (both vWF and CD31) with increasing film stiffness. It is known that ECs should be cultured in specific endothelial culture medium (ECM) containing ECGS, which is comprised of some endothelial cell growth factors, such as vascular endothelial growth factor (VEGF) and fibroblast growth factors (FGF) family members. These growth factors are essential to support EC growth and sustain EC phenotype.23,

30, 50

Studies have shown that if cultured in a culture medium

deprived of FGF for 3 to 4 weeks, ECs can lose their normal phenotype and undergo EndMT, even without the stimulation of TGF-β1.17 Hence, the phenomenon in CM on the stiff films can be attributed to the lack of the EC growth factors, consistent with the results on stiff plastic culture dishes in the previous studies. The loss of EC markers without strong expressions of SMC markers or obvious morphology change on day 14 suggests that those cells might be undergoing an early stage of EndMT, importantly, which was significantly reduced with decreasing substrate stiffness. Hence, even without exogenous TGF-β1, the substrate stiffness alone has effects on ECs, and a softer substrate favors maintaining of EC phenotype. Afterward, the effect of film stiffness on EndMT under the stimulation of TGF-β1 was studied. The mesenchymal differentiation medium (MDM) that we used containing exogenous TGF-β1 has been reported extensively to induce EndMT in vitro. For instance, Arciniegasa incubated ECs in 1 ng/mL TGF-β1 for 5 days and found ECs transdifferentiated into a smooth muscle-like phenotype characterized by a significant decrease of Factor VIII-related antigen (FVIII) and an up-regulation of αSMA.16 In another study, ECs not only lost their cobblestone morphology, expressed smooth muscle protein 22α and αSMA, but also gained SMC functions


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such as contractile behavior even indistinguishable from genuine vascular SMCs, after being cultured in MDM for 21 days.18 They also observed remarkably high cell mortality, which was due to TGF-β1-induced apoptosis on ECs. In our study, we interestingly found that with decreasing stiffness (from EDC150 to EDC70), the TGF-β1-induced EndMT significantly reduced, and relatively high EC marker expressions and inhibited SMC marker expressions were observed in the condition of softer film. Notably, we found that the immunofluorescence of CD31 was observed in the majority of ECs, suggesting that, when undergoing EndMT, ECs did not completely lose their CD31 expression, which was in consistent with previous studies.18, 26 This analysis on CD31-positive cell ratios only shows whether the ECs had the corresponding protein expressions, and did not provide information about the level of expressions. Although there was no significant difference in the CD31-positive cell ratios among the EDC70, EDC100 and EDC150 group (Figure 6B), further RT-qPCR experiments proved that the quantitative gene expression ratios of CD31 varied distinctly in response to film stiffness (Figure 7B), suggesting that film stiffness did have effects on the level of CD31 expression. EndMT is a very important biology process of ECs characterized by loss of EC phenotype and gain of mesenchymal phenotype, and in some cases cell would express SMC markers, such as αSMA, calponin and SM22α.16-19 EndMT is not only a critical process of early embryonic cardiac development, tissue regeneration and wound healing,19, 21 but also serves as a central process in many pathologies such as cardiac fibrosis,27,

31, 33

cancer fibrosis26 and in-stent

restenosis.31, 51 TGF-β1 plays a key role in inducing EndMT,16, 19, 29, 32, 33, 52 but the connections between the mechanical cues and TGF-β1-induced EndMT is still poorly understood. Our findings in the present study suggest that reducing in substrate stiffness can inhibit the EndMT process, while ECs undergo stronger EndMT with increasing substrate stiffness. Although the


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detailed biological mechanism behind these phenomena is still unknown, the interplay between cell tension and substrate stiffness may play a key role. From a perspective of cell-material interaction, it is well known that cells who have attached onto a material surface internally generate mechanical tension, which is resisted by substrate stiffness. Therefore, stiffness strongly affects cell mechanical tension and further influences related cellular events as well. For example, mesenchymal stem cells,37, 39 portal fibroblasts53 and hepatic stellate cells40 have all been reported to transdifferentiate into smooth muscle-like cells or myofibroblasts only on stiffer substrates. Notably, enhanced EndMT was reported by mechanical stretch forces.54, 55 Thus, it is believed that increased stiffness of the surrounding matrix is essential to provide mechanical stimulus for cell activation, which can be further amplified by chemical signals, such as Smad for TGF-β1.32, 40 Last but not the least, recent studies show that EndMT and some other TGF-β1 functions require cooperative activation of Rho pathway,56-59 which has been proved to play a significant role in cell response to substrate rigidity.34, 35 In sum, these studies suggest profound connections among substrate stiffness, cell tension and TGF-β1-induced EndMT. Different from previous studies that focus on promoting adhesion and growth of ECs or rapid EC coverage on materials, our study pays attention to EndMT that may influence long-term endothelium health. Concerning the up-regulation of TGF-β1 after vascular device implantation, ultrahigh stiffness of traditional implant materials (e.g. metal and poly(ethylene terephthalate)) may enhance the risk of EndMT which further results in long-term dysfunction of endothelium and severe complications (e.g. late thrombosis and neo-atherosclerosis). Our study highlights the benefit of surface with low stiffness, and we have proved that the softer substrate is in favor of inhibiting EndMT and maintaining EC phenotype. Thus, surface stiffness should be taking into consideration for surface design of vascular implant materials.


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5. Conclusions In this study, we investigated the EndMT behavior of endothelial cells on substrates with different stiffness by using (PLL/HA) multilayer films. ECs tended to undergo EndMT and lost their endothelial phenotype with increasing substrate stiffness, especially in the condition of TGF-β1 stimulation. A reduction in stiffness significantly inhibited EndMT development and favored maintaining of endothelial phenotype. Our findings indicate that substrate stiffness is a key property to affect EC behaviors and EndMT, and a strategy of “soft surface” for vascular implants may be a promising way for better protection of endothelium.

Supporting Information Available Young’s modulus of the EDC30, EDC70, EDC100 and EDC150 films, corresponding quantitative analysis of EC density for Figure 1, and corresponding DAPI-labeling images for Figure 2 and Figure 4 are shown in the supporting information. 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


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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), International Science & Technology Cooperation Program of China (2014DFG52320), and State Key Laboratory of Molecular Engineering of Polymers (Fudan University) (K2015-10). We thank Miss. Antalya Ho-Shui-Ling from CNRS 5628, France, for her help in the cell experiments. We thank Prof. Bo Song, Miss. Yijun Xia and Miss. Yajun Zhang from Soochow University, China, for their kindly help in the AFM experiments.

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Figure captions Scheme 1. Schematic representation of the effect of substrate stiffness on EndMT by using the PLL/HA polyelectrolyte multilayer films with controlled stiffness.

Figure 1. Adhesion and growth of ECs on glass (A, F, K), EDC30 (B, G, L), EDC70 (C, H, M), EDC100 (D, I, N) and EDC150 (E, J, O) at 18 h (A-E), 2 d (F-J) and 4 d (K-O). The scale bar is 200 µm.

Figure 2. Images of EC morphology on films with different stiffness. ECs were cultured in control medium without TGF-β1 for 1 (A, B, C), 7 (D, E, F) and 14 days (G, H, I), on EDC70 (A, D, G), EDC100 (B, E, H) and EDC150 (C, F, I). The scale bar is 200 µm. Cell density (J), cell viability (K) and cell circularity (L) of ECs were analyzed.

Figure 3. Immunofluorescence images of ECs cultured for 14 days. ECs were cultured in control medium without TGF-β1 on EDC70 (A, D, G, J), EDC100 (B, E, H, K) and EDC150 (C, F, I, L). Two endothelial markers, vWF (red in A, B, C) and CD31 (green in D, E, F), two SMC markers, αSMA (green in G, H, I) and calponin (green in J, K, L), and nuclei (blue) were stained. The scale bar is 200 µm. Normalized fluorescence intensity of vWF (M) and CD31 (N) per cell area was calculated (*P < 0.05).


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Figure 4. Morphology images of EC on different films cultured with stimulation of TGF-β1 (AI). ECs were cultured for 1 (A, B, C), 7 (D, E, F) and 14 days (G, H, I), on EDC70 (A, D, G), EDC100 (B, E, H) and EDC150 (C, F, I). The scale bar is 200 µm. Cell density (J), cell viability (K) and cell circularity (L) of ECs were analyzed (*P < 0.05).

Figure 5. Immunofluorescence images of ECs cultured for 14 days with stimulation of TGF-β1, on EDC70 (A, D, G, J), EDC100 (B, E, H, K) and EDC150 (C, F, I, L). Two endothelial markers, vWF (red in A, B, C) and CD31 (green in D, E, F), two SMC markers, αSMA (green in G, H, I) and calponin (green in J, K, L), and nuclei (blue) were stained. The scale bar is 200 µm.

Figure 6. Quantitative ratios of positive cells for each marker after the 14 day culture. The ratio of vWF-positive (A), CD31-positive (B), αSMA-positive (C) and calponin-positive (D) cells were calculated (*P < 0.05).

Figure 7. Gene expressions of vWF (A), CD31 (B), αSMA (C) and calponin (D) of ECs cultured on different substrates for 14 days with stimulation of TGF-β1 (*P < 0.05).


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List of Figures

Scheme 1. Schematic representation of the effect of substrate stiffness on EndMT by using the PLL/HA polyelectrolyte multilayer films with controlled stiffness.


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Figure 1. Adhesion and growth of ECs on glass (A, F, K), EDC30 (B, G, L), EDC70 (C, H, M), EDC100 (D, I, N) and EDC150 (E, J, O) at 18 h (A-E), 2 d (F-J) and 4 d (K-O). The scale bar is 200 µm.


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Figure 2. Images of EC morphology on films with different stiffness. ECs were cultured in control medium without TGF-β1 for 1 (A, B, C), 7 (D, E, F) and 14 days (G, H, I), on EDC70 (A, D, G), EDC100 (B, E, H) and EDC150 (C, F, I). The scale bar is 200 µm. Cell density (J), cell viability (K)and cell circularity (L) of ECs were analyzed.


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Figure 3. Immunofluorescence images of ECs cultured for 14 days. ECs were cultured in control medium without TGF-β1 on EDC70 (A, D, G, J), EDC100 (B, E, H, K) and EDC150 (C, F, I, L). Two endothelial markers, vWF (red in A, B, C) and CD31 (green in D, E, F), two SMC markers, αSMA (green in G, H, I) and calponin (green in J, K, L), and nuclei (blue) were stained. The scale bar is 200 µm. Normalized fluorescence intensity of vWF (M) and CD31 (N) per cell area was calculated (*P < 0.05).


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Figure 4. Morphology images of EC on different films cultured with stimulation of TGF-β1 (AI). ECs were cultured for 1 (A, B, C), 7 (D, E, F) and 14 days (G, H, I), on EDC70 (A, D, G), EDC100 (B, E, H) and EDC150 (C, F, I). The scale bar is 200 µm. Cell density (J), cell viability (K) and cell circularity (L) of ECs were analyzed (*P < 0.05).


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Figure 5. Immunofluorescence images of ECs cultured for 14 days with stimulation of TGF-β1, on EDC70 (A, D, G, J), EDC100 (B, E, H, K) and EDC150 (C, F, I, L). Two endothelial markers, vWF (red in A, B, C) and CD31 (green in D, E, F), two SMC markers, αSMA (green in G, H, I) and calponin (green in J, K, L), and nuclei (blue) were stained. The scale bar is 200 µm.


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Figure 6. Quantitative ratios of positive cells for each marker after the 14 day culture. The ratio of vWF-positive (A), CD31-positive (B), αSMA-positive (C) and calponin-positive (D) cells were calculated (*P < 0.05).


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Figure 7. Gene expressions of vWF (A), CD31 (B), αSMA (C) and calponin (D) of ECs cultured on different substrates for 14 days with stimulation of TGF-β1 (*P < 0.05).


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TOC Graphic


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Effect of Polyelectrolyte Film Stiffness on Endothelial Cells During

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