Dynamic Behavior of Vitronectin at the CellâMaterial Interface - ACS

Article pubs.acs.org/journal/abseba

Dynamic Behavior of Vitronectin at the Cell−Material Interface Georgi Toromanov,† Dencho Gugutkov,† Johan Gustavsson,† Josep Planell,†,‡ Manuel Salmerón-Sánchez,§ and George Altankov*,†,‡,∥ †

Institute for Bioengineering of Catalonia, Barcelona 08028, Spain Centro de Investigación Biomédica en Red en Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Zaragoza 50018, Spain § School of Engineering/Division of Biomedical Engineering, University of Glasgow, Glasgow G12 8QQ, United Kingdom ∥ Institució Catalana de Recerca i Estudis Avancats (ICREA), Barcelona 08010, Spain ‡

S Supporting Information *

ABSTRACT: Considering that vitronectin (VN) can promote both cell adhesion and matrix degradation, it is likely to play a dual role at the cell-biomaterial interface. In this paper we therefore describe details of the dynamic interplay between matrix adhesion and pericellular proteolysis in endothelial cells adhered to glass model substratum. Initially we show that coating concentration determines protein organization at the surface. When the protein coating density approached saturation (63 ng cm−2), VN spontaneously organized itself in multimeric aggregates at the surface (30−50 nm in diameter). At subsaturation protein density (17 ng cm−2) VN molecules were present predominantly as single entities, indicating that a minimum coating density was required for VN multimerization. By fluorescent visualization of surface-associated VN in different ways, we provide the first evidence of significant proteolytic remodelling of VN by endothelial cells (HUVECs) at the sites of αv integrin clusters. The degree of proteolysis was estimated using a novel approach relying on dequenching of FITC-labeled VN upon proteolytic activity, showing that about one-third of the surface-associated VN was proteolytically altered by adhering HUVECs. In addition, we demonstrate that HUVECs can internalize surface-associated VN and deposit it in a linear pattern along longitudinal actin filaments. Deposited VN was partly colocalized with urokinase receptors. Taken altogether, we elucidate the complex and dynamic behavior of VN during initial cell−biomaterials interactions, the equilibrium if which could have a significant impact on the biocompatibility of any blood contacting implants. KEYWORDS: cell adhesion, vitronectin matrix, pericellular proteolysis, protein adsorption, AFM

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specifically bind VN,7 namely: αvβ1, αvβ3, αIIbβ3, and αvβ5, where αvβ3 and αvβ5 seem to be the most important ones.15,16 VN has also been proposed as a molecular link between cell adhesion machinery and proteinase cascades.9,17,18 The latter fact is based on the unique properties of immobilized VN to promote adhesion mediated by integrins via the RGD sequence,19 to stabilize plasminogen activator inhibitor-1 (PAI-1)20 and to bind urokinase-type plasminogen activator (uPA).21 Binding of VN to αv integrins results in recruitment of uPA receptor (uPAR)22 that controls conversion of plasminogen to plasmin.23,24 Although uPA acts mainly to dissolve fibrin clots, it can also activate the complement system as well as collagenases that are able to cleave various components of the provisional ECM, such as collagens, fibronectin, thrombospondin and von Willebrand factor.9 All those facts lead to the assumption that VN is a potent

INTRODUCTION

Vitronectin (VN) is the major cell adhesive protein in serumcontaining cell cultures,1 and it belongs to the group of adhesive glycoproteins that play important roles in cell−biomaterials interaction.2,3 Together with fibronectin (FN), it readily adsorbs at the interface of biomaterials, where competing with other plasma proteins, it promotes cell adhesion and spreading even at very low concentrations.2,4 First described as serum spreading factor5 or S-protein,6 VN is now defined as a multifunctional 70 kDa protein that is a basic component of both the extracellular matrix (ECM) and blood plasma.1,7,8 It is involved in various physiological processes, including regulation of the blood clotting cascade,9,10 immunomodulation,1,4 and embryonic stem cell differentiation.11 It has also been associated with various pathologies, such as atherosclerotic plaque formation,12 abnormalities in the fibrinolytic system,9 cancer progression, and metastasis.13 Cell attachment to VN is an integrin-dependent process.14 From the known 24 integrin heterodimers, at least four © XXXX American Chemical Society

Received: March 24, 2015 Accepted: September 14, 2015

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DOI: 10.1021/acsbiomaterials.5b00147 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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ACS Biomaterials Science & Engineering

Atomic Force Microscopy. For atomic force microscopy (AFM) studies, VN (1 and 10 μg mL−1) was adsorbed onto clean glass coverslips for 30 min at room temperature, and then washed with ultrapure water and dried under nitrogen flow. AFM was performed in tapping mode in air (NanoScope III, Digital Instruments, Veeco, UK) using Si-cantilevers with force constant of 2.8 N/m and resonance frequency of 75 kHz. The phase signal was set to zero at a frequency 5−10% lower than the resonance one. The drive amplitude was 200 mV and the amplitude set point was 1.4 V. The ratio between amplitude set point and free amplitude was kept at 0.7. Cells. Human umbilical vein endothelial cells (HUVECs; PromoCell) were cultured in a humidified CO2 incubator at 37 °C using endothelial cell growth medium supplemented with 0.4% ECGS/H, 2% fetal calf serum and 0.1 ng mL−1 epidermal growth factor (PromoCell). For experiments, cells were harvested with trypsin/EDTA that was subsequently inactivated by addition of fetal bovine serum. Importantly, trypsinized cells were washed twice with serum-free culture medium to remove any traces of serum proteins before seeded onto VN or FITCVN coated glass samples placed in standard 24-well tissue culture plates (1.5 × 104 cells cm−2). As specified below, different incubation times were used depending on the purpose of the experiment. Cellular Remodeling of Vitronectin. Cellular remodeling of adsorbed VN was investigated morphologically. Samples coated with VN or FITC-VN were seeded with cells as described above and incubated for 5 h in serum-free medium before washed with PBS and fixed with 4% paraformaldehyde. Whereas FITC-VN samples were mounted and viewed directly, VN-coated samples were first permeablized with 0.5% Triton X-100 for 5 min and thereafter immunostained either with primary monoclonal or polyclonal anti-VN antibodies (Sigma V7881 and Abcam ab78875, respectively). Secondary antibodies were goat antimouse AlexaFluor 555 (Invitrogen) or goat antirabbit AlexaFluor 488 (Invitrogen), respectively. Co-localization of Vitronectin with Other Proteins. Colocalization between VN and fibronectin, αv-integrins, uPAR, and actin, respectively, was studied after 5 h of incubation in serum-free medium. In all cases, VN was visualized with the polyclonal antibody detailed above. The other proteins were detected using monoclonal antifibronectin (Sigma, F0916), polyclonal anti-uPAR (Antibodies Online, Cat No ABIN223158) or antiαv (Chemicon, mab1980) antibodies, respectively. For VN, the secondary antibody was goat antirabbit AlexaFluor 555, while for uPAR and FN we used goat antimouse AlexaFluor 488. For visualization of the actin cytoskeleton, FITC-Phalloidin (Invitrogen) diluted in PBS (1:100) was used. All antibodies were applied in preoptimized dilutions for 30 min at 37 °C, followed by washing with 1% albumin in PBS. The samples were finally washed with dH2O, mounted with Mowiol, and imaged with an inverted fluorescent microscope (Nikon Eclipse E800). Quantification of Pericellular Proteolysis of Vitronectin. During pilot studies we had observed that the fluorescent yield of FITC-VN was greatly increased if exposed to trypsin. This indicated selfquenching of fluorescence, presumably as a consequence of the protein being labeled with excess dye. We thus speculated that if trypsin-induced protein degradation can induce dequenching, then also cellular proteolysis could do the same? To test that hypothesis, we first coated glass coverslips with 10 μg mL−1 FITC-VN as described above. Cells were grown on these substrates for 5 h in serum-free medium, before exposed to 0.2 M NaOH for protein extraction. Fluorescent yields of NaOH-extracts obtained from samples with cells were compared to the fluorescent yield of NaOH-extracts from FITC-VN coated samples without cells (negative control, i.e., no proteolysis), and also to FITCVN coated samples that had been exposed to trypsin for 30 min (see Figure 5). The latter condition, was considered as our positive control as we assumed that 100% of the protein was degraded by trypsin and consequently could yield the maximal fluorescent yield. Visualization of Secreted Vitronectin Matrix. To follow endogenous secretion of VN by adhering cells, as well as its subsequent organization on the substratum as provisional matrix, we seeded HUVECs on samples coated with native type IV collagen (Abcam), 50 μg mL−1 dissolved in 0.1 M sodium acetate (pH 4.5). Cells were fixed

regulator of pericellular proteolysis - the physiological mechanism for removal of excess ECM in vivo.25,26 VN further has distinguished importance for endothelial cells physiology.9,10,18 Interestingly, αv integrins are abundantly expressed on endothelial cells27 implying on their strong affinity to bind VN, though they are also expressed on platelets, lymphocytes and monocytes.8 Moreover, it has been proposed that VN plays a distinct role in the matrix remodeling that occurs at the vessel wall as an adaptation to pressure or injury, and which subsequently leads to neovascularization.28 In addition, VN is considered a potent cell adhesion mediator at the luminal surface of vascular grafts, and therefore coating with VN comprises a strategy for improving the endothelization of implants29 although it may also attract pathogens such as bacteria30 or provoke inflammatory reaction.13,31 It must also be considered that VN could strongly influence the fate of implants as it triggers the enzymatic degradation of provisional ECM25,32 to prevent fibrosis33 and to enable migration of adjacent cells.26,32 Despite the fact that VN promote cell adhesion, migration, and matrix remodeling, the fate of this multifunctional protein at biomaterials interfaces is only partly understood. Therefore, here we describe the surface adsorption of VN molecules to a glass biomaterial model surface, and their subsequent evolution following exposure to primary human umilical endothelial cells (HUVECs).

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MATERIALS AND METHODS

Protein Adsorption. Glass coverslips (Fisher Scientific, 12 mm diameter) were cleaned in an ultrasonic bath for 10 min in a 1:1 mixture of 2-propanol and tetrahydrofuran. They were then immersed in Piranha solution (7:3 molar ratio of H2SO4 and H2O2) for 30 min followed by copious rinsing with Milli-Q water before air-dried in a chemical fume hood. The samples were thereafter coated with either human plasma VN (Sigma V8379) or fluorescently labeled VN (see below) diluted in PBS. Protein coatings at different concentrations were done at 37 °C. After 30 min, the coating solution was removed and samples were rinsed with PBS three times. Preparation of FITC-Labeled Vitronectin. To label the VN with fluoroisothiocyanate (FITC), the protein was initially dissolved in 0.1 M bicarbonate buffer (pH 9.0) at a concentration of 0.5 mg mL−1. Ten microliters of FITC dissolved in DMSO (10 mg mL−1) was then added per milliliter of protein solution. The mixture was incubated in the dark for 2 h at room temperature, and thereafter passed through a SephadexG25 column pre-equilibrated with PBS. We determined the concentration of the product, hereafter referred to as FITC-VN, by absorbance measurements at 280 nm (A280) and by using Beer− Lambert’s law. The standard compensation for the additional absorbance caused by the fluorochrome itself was applied by using the established correction factor (CF) of 0.3 for FITC at its maximal wavelength (494 nm). Thus, CVN = (A280 − CFA494)/εpercent where εpercent is the extinction coefficient of VN (13.8) for a solution of 1% w/v (i.e., 10 mg/mL). To obtain the CVN in units of mg mL−1, εpercent was divided by a factor of 10. After the concentration was determined, the labeled protein was stored at −20 °C until used for experiments. Quantification of Protein Adsorption. Fluorescence photometry was used to quantify protein adsorption using previously described protocols.34 Briefly, glass coverslips coated with FITC-VN (0.5−40 μg mL−1) were rinsed with PBS and any adsorbed protein was thereafter extracted from the substrate with 0.5 mL of 0.2N NaOH for 2 h at room temperature. We assume that this procedure removes all protein from the substrate since no fluorescence was detected at the surface after extraction. The fluorescence intensity of the extracted protein was measured with a fluorometer (Horiba Jobin Yvon; λex = 488 nm, λem = 530 nm). Known concentrations of FITC-VN in 0.2N NaOH ranging from 0.01 to 1.0 μg mL−1 were used to produce a calibration curve (Figure S1) and to calculate the amount of adsorbed protein per surface area. B


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ACS Biomaterials Science & Engineering and VN was visualized at distinct time intervals during the 5 h long incubation period using monoclonal anti-VN antibody (see above). Internalization of Adsorbed Vitronectin. Evidences for cellular internalization of VN was obtained by first seeding HUVECs on FITCVN-coated samples (10 μg mL−1) and incubating them in serum-free medium for 5 h at 37 °C. Thereafter, the cells were detached using trypsin/EDTA to remove all extracellular proteins, and then washed twice with PBS before replating them onto collagen IV-coated glass samples (prepared as above). After another 2 h of incubation, the cells were fixed, mounted in Mowiol and internalization of FITC-VN was detected using an inverted fluorescence microscope. Statistics. StatGraphics Plus (StatGraphics) software employing ANOVA test was used to determine statistically significant differences between groups (P < 0.05). Each data point represents mean ± standard deviation from at least three independent experiments. Nonlinear curve fittings was performed using the Solver function in Microsoft Excel for Mac 2011 (version 14.3.9).

We moreover observed that about half of the VN spontaneously desorbed after 5 h of incubation in serum-free medium (Figure 2). As a consequence, a detectable amount of FITC-VN was present in the supernatant after 5 h.

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RESULTS Adsorption of Vitronectin onto Glass. To quantify VN adsorption to the substratum, we extracted FITC-VN that had been adsorbed onto clean glass coverslips at varying coating concentrations using NaOH. The obtained data (Figure 1A)

Figure 2. Amount of substratum-extracted fluorescent signal immediately after (0 h) and 5 h following FITC-VN adsorption (10 μg/mL) in the absence of cells.

Cellular Influence on Adsorbed Vitronectin. Figure 3 shows representative images of the behavior of adsorbed VN

Figure 3. Morphological view of VN upon contact with endothelial cells, as viewed by direct labeling of VN with (A) FITC, (B) polyclonal antiVN antibody, or (C) monoclonal anti-VN antibody. Scale bars represent 20 μm.

after 5 h of incubation in the presence of adhering endothelial cells. Three different methods of protein visualization were employed. First, direct labeling of VN with FITC indicated limited cellular VN reorganization and in an irregular pattern (Figure 3A). Nevertheless, diffuse accumulation of fluorescent signal at cell borders was often observed, suggesting proteolytic alteration of VN beneath adhering cells. Second, the polyclonal anti-VN antibody only resulted in slight accumulation of VN around cell borders (Figure 3B). Finally, and in great contrast to both previous cases, visualization of VN using the monoclonal antibody (Sigma V7881) revealed a pattern of dark “foot prints” along the cell perimeter (Figure 3C), indicating pericellular proteolysis. The areas of concentrated pericellular proteolytic activity coincided very well with αv-integrin clusters in focal adhesions (Figure 4). Relative Quantification of Cell-Induced Proteolysis of Vitronectin. In an attempt to estimate the proteolytic cleavage of VN observed with the monoclonal antibody (Figure 3C), we initially measured the amount of released FITC-VN into the medium after 5 h of incubation in absence and presence of cells. However, no significant difference was observed between the two conditions. In contrast, the fluorescent yield of NaOH-extracted FITC-VN increased about five times if the labeled protein had been in contact with adherent cells (middle bar, Figure 5) as compared to the negative control (left bar, extracted FITC-VN in

Figure 1. Vitronectin adsorption onto glass at different coating concentrations. (A) Fluorescent signal Following extraction of FITCVN from the substrate using NaOH (left y-axis) was used to calculate the amount of adsorbed protein (right y-axis). (B) Representative AFM images of VN on glass at different coating concentrations (0, 1, and 10 μg/mL, respectively). Scale bars represent 200 nm.

were successfully fitted to the Langmuir isotherm (R2 = 0.95), which was used to estimate complete saturation (89 ng adsorbed protein per cm2). AFM data (Figure 1B) revealed that the VN coating concentration influenced how the protein assembled on the substrate. At 10 μg mL−1 coating concentration (63 ng cm−2), protein adsorption occurred nonstochastically as VN tended to aggregate in globules sized from 30 to 50 nm. In contrast, at lower concentration (1 μg mL−1), features sized similar to single protein molecules (3−11 nm)35,36 clearly prevailed among sparsely appearing aggregates. Neither aggregates nor single molecular features were observed on noncoated substrates. C


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cells harvested directly from the flask onto collagen type IV substrata using serum-free medium, and subsequently visualized VN with monoclonal anti-VN showing no cross-reactivity with other ECM proteins according to the manufacturer as well as our own control experiments (not shown). Interestingly, accumulation of VN at the cell border was apparent already within 30 min from cell seeding, and the protein was gradually organized in a fibril-like pattern as the incubation proceeded (Figure 6).

Figure 6. Secreted VN organization starts with the accumulation of protein at the cell borders (30 min) followed by gradual organization in fibril-like pattern within 2 h, which is pronouncedly expressed after 5 h of incubation. Scale bar represent 20 μm.

Co-localization of Secreted Vitronectin with Other Cellular Proteins. Although VN is a nonfibrillar protein, VN deposited by HUVECs resembled much the well-known organization of FN matrix fibrils.37,38 However, we did not find any evidence of colocalization between VN and FN fibrils (Figure 7, left column), but VN was rather transiently associated

Figure 4. Co-localization of proteolytically altered VN (viewed with monoclonal antibody) with αv integrin clusters in focal adhesions. Scale bars represent 20 μm.

Figure 5. Substratum-extracted fluorescent signal from samples coated with FITC-VN. NaOH-extracts were obtained from samples with or without cells, and compared to FITC-VN treated with trypsin.

Figure 7. Fate of VN secreted by cells grown on collagen type IV-coated substrata. Co-localizaton of VN with other proteins was investigated for fibronectin (left column), actin (middle column), and uPAR (right column). Scale bars represent 20 μm.

absence of cells). Trypsin-induced proteolysis (i.e., positive control) was however remarkedly more efficient in dequenching the FITC-VN than cell-induced proteolysis given the experimental conditions (right bar, Figure 5). Fate of Secreted Vitronectin. Considering that we observed slight accumulation of VN around cell borders when using the polyclonal antibody (Figure 3B), we wanted to know whether HUVECs secreted VN. To be able to distinguish newly secreted VN from preadsorbed one, we plated trypsin-treated

with longitudinal actin fibers (Figure 7, middle column). Given that the components of the serine protease family such as plasmin associate with αv-integrin4 and that uPAR is an important receptor involved in the regulation of plasminogen activation,39 we also wanted to know wheter uPAR associated with secreted VN. As shown in Figure 7 (right column), partial colocalization between secreted VN and uPAR was observed. D


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ACS Biomaterials Science & Engineering Evidence for Internalization of Surface-Associated Vitronectin. HUVECs do not synthesize VN,9 but as shown in Figures 6 and 7 they readily deposit it on the substratum. One possible explanation for that observation could be that VN undergoes transcytosis,40 i.e., the protein is translocated to the substratum from another origin. For example, it is well documented that VN from serum or exogenous VN can be internalized by cells.39 Considering that internalization of VN may be an important cellular process for reorganization of VN at the cell-biomaterial interface, we transferred HUVECs cultured on FITC-VN coated substrata to a VN-free environment in order to observe the cellular capacity to internalize surface-associated FITC-VN. In Figure 8, we show how HUVECs cultured on collagen type IV contain fluorescent intracellular vesicles whose only origin could be surface-associated FITC-VN.

Although VN is mixed with other proteins in physiological conditions, we choose to study the single protein condition in order to simplify the system and to avoid competing mechanisms such as the Vroman effect. The obtained quantitative data was successfully fit to the Langmuir adsorption isotherm showing that VN adsorption onto glass saturated at approximately 90 ng cm−2, which is considerably less than what has been previously reported for VN adsorption onto polystyrene (∼400 ng cm−2).2,43 We attribute the low protein adsorption to the intrinsic material properties. For example, we have previously shown that VN adsorption is much lower on hydrophilic surfaces than hydrophobic ones at the same concentration of the adsorbing solution.44 In this context, one has to recall that we used Piranha-treated glass, which is extremely hydrophilic. Although this study relied on the use of bare glass coverslips, and considering that such samples are much rougher than mica glass or the native oxide layer of silicon wafers, the AFM images of adsorbed protein clearly indicated that beyond a certain coating concentration (≥10 μg mL−1, i.e., approaching saturation), VN adsorbed nonstochastically as globular aggregates sized between 30 and 50 nm, which is much greater than the estimated size of individual VN molecules.35,36 These aggregates are believed to be representative of the multimerization of single VN molecules that occurs in the blood.1,4 During the multimerization process in vivo, VN monomers (75 kDa) undergo irreversible conformational changes that renders the molecule more biologically active by exposing its heparin and plasminogen binding sites20,21 as the molecules multimerize into a larger structure of 800−1200 kDa.45 Our observations correlate well with other studies that show that VN tends to unfold upon adsorption on solid substrata,9 and it adds evidence for multimerization of VN at the biomaterials interface.45 Yet it should be noted that aggregates were seen only at relatively high VN coating concentration, indicating the importance of initial molecular density for the multimerization process to initiate. Further studies are required to learn more about the formation and evolution of these aggregates as they form. Cellular Remodelling of VN. Regarding the cellular interaction with adsorbed VN, and although it has been suggested that the multimeric form of VN exposes the RGD sequence better than the monomeric form, 7 very low concentration of VN (0.1−1 μg mL−1) is sufficient to induce optimal cell adhesion of various cell types.46 Yet, many blood contacting devices, including small diameter vascular grafts, stents, and heart valves, commonly suffer from insufficient ingrowth of endothelial cells, which in turn often result in restenosis and accelerated device failure.47,48 Among many possible explanations, one could be poor long-term interaction with VN, as has been demonstrated in vitro where ECMlocalized PAI-1 dissociates cell−ECM contacts by interfering with the uPAR signaling and with the focal adhesion turnover.49 We therefore anticipated that the dual role of VN in cell adhesion and coagulation cascades9,17,18,22 could cause a dynamic interplay between matrix adhesions and pericellular proteolysis with important consequences for the cellular response at the vessel wall. To address the nature of such interplay, we decided to follow the fate of adsorbed VN in the presence of endothelial cells in vitro. Surprisingly, we observed only minor cellular influence on the adsorbed protein when using FITC-VN or immunofluorescence with a polyclonal antibody against VN (Figure 3). That behavior stands in great contrast to the pronounced activity that HUVECs show toward other adsorbed matrix proteins such as fibronectin, 37 fibrinogen,50 and

Figure 8. Internalization of adsorbed VN was visible as a dotlike pattern of fluorescent vesicles around the cell nucleus. Scale bar represent 20 μm.

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DISCUSSION Although VN plays a vital role in endothelial cells physiology,19 data on its dynamic behavior after adsorption onto biomaterial interfaces, including its remodelling by adjacent endothelial cells, are generally missing. Our approach to address that issue was to first investigate how and to what extent VN would adsorb onto a model substratum, and then to follow the fate of the adsorbed protein in the presence of cells. The choice of glass as a model surface is particularly motivtated by its use as a golden standard for cell adhesion studies, whereas HUVEC represents a welldescribed cell system that importanly do not synthesize VN itself, a property that could corrupt the experimental evaluations. Protein Adsorption. Although it is well-known that VN readily adsorbs to glass and different polymeric surfaces,2,4,41 methods to monitor VN adsorption, e.g., direct radiolabeling,2 Western blot,41 ELISA,2,42 and AFM,35 have mainly been used to describe relative differences in protein adsorption between surfaces or among other plasma proteins.2,42 Quantitative data describing total amount of protein, concentration-dependent curves, etc., has rarely been reported. To give an improved overall view of the adsorption process, we therefore used both a qualitative (AFM) and a quantitative approach (i.e., extraction of surface-associated FITC-VN) to follow VN adsorption to glass. E


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ACS Biomaterials Science & Engineering interestingly also type IV collagen, which undergo fibril-like reorganization even being a nonfibrilar protein.34,51 To our view, surface remodeling of adsorbed matrix proteins is a consequence of the balance between protein deposition by adhering cells (involving also mechanical protein (re)organization on the substratum34,37,50,51) and protein degradation via pericellular proteolysis.25,34,51 The former process reflects the organization of provisional ECM,37,50,52 whereas the latter represents the physiological mechanism for the removal of excess ECM.25 Although both mechanisms may have critical impact on the biocompatibility of cardiovascular implants, little is known about cellular remodeling of VN. Here, we provide data that clearly indicate limited reorganization of VN by HUVECs, and are believed to be due to strong interaction between the protein and the substratum, thus preventing any mechanical VN reorganization by cells. Another aspect that has to be considered is that VN, apart from most other matrix proteins, has no binding site for fibronectin that can promote fibril-like reorganization of the substratum-associated protein.34,50,51 That fact also corroborates with our data for absent colocalization of secreted VN with FN matrix fibrils (Figure 7, left column). Interestingly, we found a completely different pattern of adsorbed VN when the protein was visualized with the monoclonal anti-VN antibody (Figure 3C). In that case, dark focalized zones appeared beneath cells indicating proteolytic activity by HUVECs. Areas of proteolytic activity were often located close to where αv-integrins clustered in the focal adhesion complexes (Figure 4). Since VN interacts with a variety of ligands from the coagulation cascade, including serine proteases and their inhibitors,22,53 it is not surprising that the protein might undergo proteolysis. As visualization of proteolytic activity was only possible with the monoclonal anti-VN antibody, we conclude that the monoclonal antibody specifically targets a protease-sensitive region of the VN molecule, and that most of the proteolytically altered VN remained on the substratum since cleaved epitopes were detected by the polyclonal antibody. That conclusion was further strengthened by the observation that fluorescent intensity from FITC-VN in the supernatant did not increase in samples undergoing cell-induced proteolysis (not shown). Finally, and in an attempt to quantify the observed pericellular proteolytic activity, we once again employed FITC-VN. Quantification of proteolytic activity became possible because FITC-VN was quenched (i.e., its fluorescence was partly inhibited because of autoabsorbance from neighboring tightly packed FITC molecules) as judged by the sharp increase in fluorescence observed if FITC-VN (soluble as well as adsorbed protein) was treated with trypsin (Figure 5). Quenching of FITC-VN thus provided an opportunity to use it as a sensitive system to measure proteolysis where trypsin-treated samples were considered as positive control (i.e., 100% proteolysis). That approach revealed that a significant amount (about one-third) of the adsorbed VN was proteolytically altered by adhering endothelial cells in the present culture conditions. To the best of our knowledge, this is the first study showing that adsorbed VN undergoes significant proteolytic remodeling by endothelial cells at the material interface. Late VN Matrix. Although synthesis of VN is restricted to hepatocytes,9 already early on in our studies we suspected that VN was secreted by HUVECs as judged by protein accumulation around the cell perimeter (Figure 3B). We therefore decided to culture cells on collagen substrata for 5 h and then stained for VN in the late matrix produced by cells (Figure 7, top row). To our

surprise, HUVECs not only secreted VN but also organized it in a distinct linear pattern resembling fibronectin matrix fibrils. Such linear organization of VN matrix around endothelial cells has not been described before, but it typical for other matrix proteins.34,50,51 We initially supposed a supporting role of secreted FN for VN organization as FN is able to translocate surrounding proteins during fibrilogenesis.50,51 However, no colocalization between VN and FN was observed (Figure 7). Instead we found extensive co-organization of secreted VN with well-developed actin stress fibers, implying that VN organization is a cell-dependent process that requires successful cell spreading. We further show that VN organization develops as the cell spread (Figure 6), suggesting a trans-membrane association between VN and the cytoskeleton, presumably via αv-integrin receptors. Such lateral association could go outside the focal adhesion complexes (where VN is proteolytically altered), in a process resembling the generation of fibrillar adhesions.38 Given that surface-associated VN is known to accumulate in ternary complexes with uPA and uPAR in focal contacts of endothelial cells,49 we also wanted to investigate whether secreted VN accumulated together with uPAR. Indeed, secreted VN colocalize with some of the uPAR in the pericellular matrix (Figure 7, right column), which could allow for urokinasemediated proteolysis to occur.54 This raises the question whether VN is sufficiently protected in such an aggressive environment? Indeed, the ternary uPA/uPAR/VN complex might be important for the enzymatic remodeling of ECM that promote cell locomotion55 and invasiveness of cancer cells.9,22 However, the binding of active PAI-1 to VN near its N-terminal somatomedin B domain suggests that VN may also protect matrix proteins against cellular proteases.9,22,23 As for the origin of secreted VN it should once again be emphasized that HUVECs do not synthesize VN9 by themselves. However, endothelial cells have capacity to internalize VN from serum.40 Although the levels of soluble extracellular VN in vivo are controlled mainly by fibroblasts via receptor-mediated endocytosis,56 endothelial cells are also involved to some extent, presumably via an αvβ5 integrin-mediated mechanism.39 HUVECs in particular are able to internalize VN within a VNthrombin-antithrombin ternary complex leading to its deposition into the subendothelial matrix via a process known as trancytosis.40 Here, we show that secretion of VN starts almost immediately after successful cell adhesion and thus before new synthesis of protein occur (Figure 6). With other words, VN present in the late matrix must have its origin as preinternalized VN. In our system, the only reasonable source of VN would be the serum contained in the culture medium used during cell maintenance (i.e., before any of the presented experiments were initiated), which is easily recognized by both the less specific polyclonal antibody (giving some cross reactivity with other ECM proteins) and much better by the monoclonal one. Why the monoclonal VN antibody better discerns the linear patterns of the VN matrix than the polyclonal antibody remains at this stage still unclear to us. Considering that internalization of VN may be an important cellular process for reorganization of VN at the cell-biomaterial interface, we finally demonstrate that HUVECs are perfectly able to internalize surface-associated VN (Figure 8). However, it should be mentioned that internalization of adsorbed VN by cells was observed only at high coating concentration (10 μg mL−1). That observation fits well with the fact that only multimeric VN can enter this pathway.56 It is important to emphasize that the fluorescent signal associated with internalized FITC-VN is F


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(2) Underwood, P. A.; Steele, J. G.; Dalton, B. A. Effect of polystyrene surface chemistry on the biological activity of solid phase fibronectin and vitronectin, analysed by monoclonal antibodies. J. Cell Sci. 1993, 104, 793−803. (3) Wilson, C. J.; Clegg, R. E.; Leavesley, D. I.; Pearcy, M. J. Mediation of biomaterial-cell interactions by adsorbed proteins: a review. Tissue Eng. 2005, 11, 1−18. (4) Preissner, K. T. Structure and biological role of vitronectin. Annu. Rev. Cell Biol. 1991, 7, 275−310. (5) Barnes, D. W.; Silnutzer, J.; See, C.; Shaffer, M. Characterization of human serum spreading factor with monoclonal antibodies. Proc. Natl. Acad. Sci. U. S. A. 1983, 80, 1362−1366. (6) Jenne, D.; Stanley, K. K. Molecular cloning of S-protein, a link between complement, coagulation and cell-substrate adhesion. EMBO J. 1985, 4, 3153−3157. (7) Felding-Habermann, B.; Cheresh, D. Vitronectin and its receptors. Curr. Opin. Cell Biol. 1993, 5, 864−868. (8) Reheman, A.; Gross, P.; Yang, H.; Chen, P.; Allen, D.; Leytin, V.; Freedman, J.; Ni, H. Vitronectin stabilizes thrombi and vessel occlusion but plays a dual role in platelet aggregation. J. Thromb. Haemostasis 2005, 3, 875−883. (9) Seiffert, D. Constitutive and regulated expression of vitronectin. Histol. Histopathol. 1997, 12, 787−797. (10) Kjøller, L.; Kanse, S.; Kirkegaard, T.; Rodenburg, K.; Rønne, E.; Goodman, S.; Preissner, K.; Ossowski, L.; Andreasen, P. Plasminogen activator inhibitor-1 represses integrin- and vitronectin-mediated cell migration independently of its function as an inhibitor of plasminogen activation. Exp. Cell Res. 1997, 232, 420−429. (11) Gil, J.; Woo, D.; Shim, J.; Kim, S.; You, H.; Park, S.; Paek, S.; Kim, S.; Kim, J. Vitronectin promotes oligodendrocyte differentiation during neurogenesis of human embryonic stem cells. FEBS Lett. 2009, 583, 561−567. (12) Van Aken, B.; Seiffert, D.; Thinnes, T.; Loskutoff, D. Localization of vitronectin in the normal and atherosclerotic human vessel wall. Histochem. Cell Biol. 1997, 107, 313−320. (13) Edwards, S.; Lalor, P.; Tuncer, C.; Adams, D. Vitronectin in human hepatic tumors contributes to the recruitment of lymphocytes in an apha v beta 3 - independent manner. Br. J. Cancer 2006, 95, 1545− 1554. (14) Pytela, R.; Piershbacher, M.; Ruoslahti, E. A 125/115-kDa cell surface receptor specific for vitronectin interacts with the arginineglysine-aspartic acid adhesion sequence derived from fibronectin. Proc. Natl. Acad. Sci. U. S. A. 1985, 82, 5766−5770. (15) Smith, J.; Vestal, D.; Irwin, S.; Burke, T.; Cheresh, D. Purification and functional characterization of integrin alpha v beta 5. An adhesion receptor for vitronectin. J. Biol. Chem. 1990, 265, 11008−11013. (16) Horton, M. The alpha v beta 3 integrin ‘vitronectin receptor’. Int. J. Biochem. Cell Biol. 1997, 29, 721−725. (17) Stefansson, S.; Lawrence, D. The serpin PAI-1 inhibits cell migration by blocking integrin alpha V beta 3 binding to vitronectin. Nature 1996, 383, 441−443. (18) Kanse, S.; Kost, C.; Wilhelm, O.; Andreasen, P.; Preissner, K. The urokinase receptor is a major vitronectin-binding protein on endothelial cells. Exp. Cell Res. 1996, 224, 344−353. (19) Preissner, K.; Seiffert, D. Role of vitronectin and its receptors in haemostasis and vascular remodeling. Thromb. Res. 1998, 89, 1−21. (20) Mimuro, J.; Loskutoff, D. Binding of type 1 plasminogen activator inhibitor to the extracellular matrix of cultured endothelial cells. J. Biol. Chem. 1989, 264, 5058−5063. (21) Moser, T. L.; Enghild, J. J.; Pizzo, S. V.; Stack, M. S. Specific Binding of Urinary-type Plasminogen Activator (u-PA) to Vitronectin and its Role in Mediating u-PA-Dependent Adhesion of U937 Cells. Biochem. J. 1995, 307, 867−873. (22) Madsen, C.; Sidenius, N. The interaction between urokinase receptor and vitronectin in cell adhesion and sinaling. Eur. J. Cell Biol. 2008, 87, 617−629. (23) Kost, C.; Stuber, W.; Erlich, H.; Pannekoek, H.; Preissner, K. Mapping of Binding Sites for Heparin, Plasminogen Activator Inhibitor 1, and Plasminogen to Vitronectin s Heparin binding Region Reveals a

negligible compared to pericellular proteolysis presented in Figure 5, and thus does not perturb that data. In summary, these morphological evidence suggest that trancytosis can be an important mechanism for the remodelling of VN at biointerfaces.

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CONCLUSIONS This study provides new insights on the fate of adsorbed VN at the cell-material interface. We show that HUVECs do not reorganize adsorbed protein, but they readily degrade it at their focal contact complexes. Protein degradation was further quantified via FITC-VN dequenching, showing that a signficant portion of the adsorbed protein was proteolytically altered already within a few hours. We also show that HUVECs internalize and further secret and spatially organize VN in a linear pattern along with the actin cytoskeleton. Taken altogether, the dynamic remodeling of the extracellular microenvironment with respect to vitronectin is of greatest importance for successful endothelization of biomaterials, and thus of relevance for any blood contacting device.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsbiomaterials.5b00147. Figure S1 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +34 93 403 97 09. Fax: +34 93 403 98 73. Author Contributions

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

This work was supported by CIBER-BBN (Spain) and the European Commission through the FP7 Industry-Academia Partnerships and Pathways (IAPP) project FIBROGELNET and the EuroNanoMed project STRUCTGEL. The valuable support of the project MAT2012-38359-C03-03 HEALINSYNERGY, funded by Spanish Ministry of Science and Innovation is also acknowledged. Notes

The authors declare no competing financial interest.

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ABBREVIATIONS AFM, atomic force microscopy BSA, bovine serum albumin ECM, extracellular matrix FITC, fluorescein isothiocyanate FN, fibronectin HUVECs, human umbilical vein endothelial cells PAI-1, plasminogen activator inhibitor-1 PBS, phosphate buffered saline uPA, urokinase-type plasminogen VN, vitronectin

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REFERENCES

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