Dermatopontin promotes adhesion, spreading and migration of cardiac fibroblasts in vitro

Matrix Biology 32 (2013) 23–31

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Dermatopontin promotes adhesion, spreading and migration of cardiac fibroblasts in vitro Xiaoyan Liu 1, Liukun Meng 1, Qiang Shi, Shenghua Liu, Chuanjue Cui, Shengshou Hu ⁎, Yingjie Wei ⁎ State Key Laboratory of Cardiovascular Disease, National Center for Cardiovascular Disease and Fuwai Hospital, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing, PR China

a r t i c l e

i n f o

Article history: Received 15 July 2012 Received in revised form 27 November 2012 Accepted 28 November 2012 Keywords: dermatopontin (DPT) myocardial infarction ventricular remodeling

a b s t r a c t Dermatopontin (DPT), an extracellular matrix (ECM) protein, has been previously shown to be upregulated in the infarct zone of experimentally induced myocardial infarction (MI) rats. However, the accurate role that DPT exerts in the ventricular remodeling process after MI remains poorly understood. In this study, we evaluated the expression pattern of DPT mRNA and protein as well as its secretion in cultured neonatal rat cardiomyocytes (CMs) and cardiac fibroblasts (CFs) under conditions of hypoxia and serum deprivation (hypoxia/SD). Further, we tested the possible roles of DPT in CFs adhesion, spreading, migration and proliferation, which greatly promote the ventricular remodeling process after MI. Results showed that hypoxia/SD stimulated DPT expression and secretion in CMs and CFs and that DPT promoted adhesion, spreading and migration of CFs whereas had no effect on CFs proliferation. In addition, functional blocking antibodies specific for integrin α3 and β1 significantly reduced CFs adhesion and migration that DPT induced, suggesting that integrin α3β1 is at least one receptor for CFs adhesion and migration to DPT. These results implicated that DPT participates in the ventricular remodeling process after MI and may act as a potential therapeutic target for ventricular remodeling. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Ventricular remodeling starts as a compensatory response of the heart to the dramatically changed hemodynamic status resulted from myocardial infarction (MI); however, long-term ventricular remodeling unavoidably results in decompensatory conditions and finally congestive heart failure (HF) (Tiyyagura and Pinney, 2006). During the geometric change of the ventricle, cardiomyocytes (CMs) experience death or apoptosis in the infarct region and adaptive hypertrophy in non-infarct region. Meanwhile, another important cell type of the heart, cardiac fibroblasts (CFs), migrate into the ischemic area, proliferate, secrete various cytokines and growth factors and alter extracellular matrix turnover. Although many genes and Abbreviations: DPT, dermatopontin; ECM, extracellular matrix; MI, myocardial infarction; CMs, cardiomyocytes; CFs, cardiac fibroblasts; hypoxia/SD, hypoxia and serum deprivation; HF, heart failure; DMEM, dulbecco's modified Eagle's medium; FBS, fetal bovine serum; TRITC-phalloidin, Tetramethyl rhodamine B isothiocyanate conjugated phalloidin; Fn, fibronection; real time RT-PCR, real-time quantitative reverse transcription polymerase chain reaction; RT, room temperature; HRP, horseradish peroxidase; GAPDH, Glyceraldehyde-3-phosphate dehydrogenase; PBS, phosphate-buffered saline; BrdU, 5-bromo-2′-deoxyuridine; DAPI, 4′, 6-diamidino-2-phenylindole; HSPG, heparan sulfate proteoglycan. ⁎ Corresponding authors at: State Key Laboratory of Cardiovascular Disease, National Center for Cardiovascular Disease and Fuwai Hospital, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing 100037, PR China. Tel.: +86 10 88398494; fax: +86 10 88396050. E-mail address: [email protected] (Y. Wei). 1 These authors contributed equally to this work. 0945-053X/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.matbio.2012.11.014

proteins have been implicated in the ventricular remodeling process, the accurate mechanisms of ventricular remodeling have not been completely unraveled. Thus, searching for novel targets involved in the pathophysiological course is still necessary and urgent in this field nowadays. Dermatopontin (DPT) is a non-collagenous extracellular matrix (ECM) protein originally discovered in dermis and subsequently detected in various tissues including the heart (MacBeath et al., 1993; Arslan et al., 2005; Li et al., 2009). Using gene microarray technology, we observed a significant increase of DPT mRNA in human failing hearts compared with non-failing control hearts (Wei et al., 2008). Its multifunctional effects in cell adhesion, proliferation and matrix assembly have been explored in different cell types (Lewandowska et al., 1991; MacBeath et al., 1993; Tzen and Huang, 2004; Takeuchi et al., 2006; Okamoto et al., 2010; Yamatoji et al., 2012). Furthermore, a previous study presented that DPT mRNA level was significantly elevated in the infarct zone of experimentally induced MI rats in a time-dependent manner, which was roughly paralleled with that of decorin and type I collagen (Takemoto et al., 2002). Thus, we speculate that DPT exerts an important role in the pathogenesis of ventricular remodeling after MI. In this study, we examined the expression pattern of DPT mRNA and protein and its secretion in CMs and CFs under conditions of Hypoxia/SD, and further investigated the effects of DPT on CFs adhesion, spreading, migration and proliferation which are important aspects of ventricular remodeling in vitro. Moreover, receptors for CFs adhesion and migration to DPT were also investigated.

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2. Results 2.1. Effects of hypoxia/SD on DPT expression and secretion in CMs and CFs After intervals of 0, 6, 12 and 24 h, the changes of DPT mRNA and protein levels were examined in CMs and CFs treated with hypoxia/SD by real time RT-PCR and Western blotting analysis, respectively, while the amount of DPT secreted in cell culture supernatants was assessed by enzyme-linked immunosorbent assay (ELISA). As demonstrated in

Fig. 1A and D DPT mRNA level in CMs and CFs cultured in hypoxia/SD conditions significantly increased at 6 h and approximately reached a plateau at 12 h which lasted until 24 h, and the maximum value of DPT mRNA level at 12 h is 1.82 fold in CMs and 3.5 fold in CFs of that at 0 h serving as control group. Corresponding to the alteration in DPT mRNA level, DPT protein level in CMs and CFs as well as the amount of DPT in cell culture supernatants of CMs and CFs under hypoxia/SD conditions also significantly elevated at 6 h and almost achieved a maximum at 12 h which lasted until 24 h (Fig. 1B–C and E–F).

Fig. 1. Hypoxia/SD induced DPT expression and secretion in CMs and CFs. CMs and CFs were treated by hypoxia/SD for different time intervals and harvested for real time RT-PCR and Western blotting analysis, meanwhile, cell culture supernatants were collected for ELISA. A. Real time RT-PCR analysis of DPT mRNA expression in CMs. B. Western blotting analysis of DPT protein expression in CMs. Upper panel: representative immunoblotting image. Lower panel: pooled data of densitometric scanning. C. ELISA of DPT secretion in cell culture supernatants of CMs. D. Real time RT-PCR analysis of DPT mRNA expression in CFs. E. Western blotting analysis of DPT protein expression in CFs. Upper panel: representative immunoblotting image. Lower panel: pooled data of densitometric scanning. F. ELISA of DPT secretion in cell culture supernatants of CFs. Data are expressed as mean ± SD, *P b 0.05, **Pb 0.01 versus control (0 h).

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2.2. Effects of DPT on CFs adhesion and spreading Previous studies reported that DPT promotes the adhesion of Balb/c 3T3 cells, dermal fibroblasts and epidermal keratinocytes; however, whether DPT promotes the adhesion of CFs has never been reported. Using a solid-phase cell adhesion assay, we found that DPT promoted the adhesion of CFs in a dose-dependent manner and reached a maximum plateau at a concentration of 20 μg/ml (Fig. 2A–B). Receptors for CFs adhesion to DPT were determined using EDTA (5 mM), peptides GRGDSP (400 μM) and GRGESP (400 μM), heparin (10 μg/ml) and heparinase III (0.2 IU/ml). Adhesion of CFs to DPT was completely inhibited by EDTA and peptide GRGDSP, both of which were concentration-dependent (Fig. 2C), whereas, it was not affected by control peptide GRGESP, heparin and heparinase III (data not shown). These results suggested that receptors of DPT on CFs belonged to the integrin family. Since integrin α3β1 was reported to be a receptor for DPT on epidermal keratinocytes (Okamoto et al., 2010) and it is expressed on the cell surface of CFs (Manso et al., 2009), we tested the ability of neutralizing antibodies specific for integrin α3 and integrin β1 to block the adhesion of CFs to DPT. Results showed that, both integrin α3 and integrin β1 neutralizing antibodies inhibited the adhesion of CFs to DPT in a dose-dependent manner and the inhibition was most effective when they were simultaneously used. These results demonstrated that, integrin α3β1 was at least one receptor for CFs adhesion to DPT. Then, we tested whether CFs adhered to DPT could also spread on it and formed focal adhesions by immunofluorescent staining with

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Tetramethyl rhodamine B isothiocyanate conjugated phalloidin (TRITC-phalloidin) and an anti-vinculin antibody. We observed that CFs in wells coated with DPT formed well organized actin filaments and focal adhesions containing vinculin (Fig. 3). 2.3. Effects of DPT on CFs migration Next, we tested whether DPT promoted CFs migration in addition to adhesion using transwell cell culture chambers with 8-μm pore size polycarbonate membranes. As illustrated in Fig. 4A, the number of CFs that migrated through the 8-μm pores in polycarbonate membrane showed a mounting trend with the amounts of DPT added into the lower compartments of the chambers increasing, which suggested that DPT induced CFs migration in a dosedependent manner. The number of migrated CFs reached a plateau at the concentration of 10 μg/ml. These data suggested that DPT served as a chemotaxin to facilitate CFs migration. Furthermore, the chemotactic effect of 10 μg/ml DPT on CFs was significantly decreased by 10 μg/ml anti-integrin α3 and 10 μg/ml anti-integrin β1 blocking antibodies, and when the two antibodies were used together they almost eliminated the chemotactic effect of DPT (Fig. 4B), suggesting that integrin α3β1 was one receptor for DPT induced CFs migration. 2.4. Effects of DPT on Fn-induced and FBS-induced CFs migration Subsequently, we tested the effects of DPT on 10 μg/ml Fn-induced and 2% FBS-induced CFs migration. The concentrations of Fn and

Fig. 2. DPT promoted CFs adhesion via integrin α3β1. Wells of 96-well plates were coated with increasing concentrations of DPT and CFs adhesion assay were performed as described in experimental procedures. For inhibiting studies, cells were preincubated with inhibitors at 37 °C for 20 min before plating on a constant concentration (20 μg/ml) of DPT coated wells. A. DPT promoted CFs adhesion in a dose-dependent manner. B. Crystal violet staining of adhered CFs on increasing amounts of DPT (×200). DPT concentrations were: (B-1) 1 μg/ml, (B-2) 2 μg/ml, (B-3) 5 μg/ml, (B-4) 10 μg/ml, (B-5) 20 μg/ml, (B-6) 50 μg/ml. C. EDTA and peptide GRGDSP inhibited CFs adhesion in a concentrationdependent manner. D. Functional blocking antibodies to integrin α3 and β1 suppressed the adhesion of CFs to DPT in a dose-dependent manner. Data are expressed as mean±SD, *Pb 0.05, **Pb 0.01 versus control.

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Fig. 3. Immunofluorescent staining of actin and vinculin in CFs adhered to DPT. CFs were allowed to adhere to 20 μg/ml DPT coated wells and immunofluorescent staining of actin (red) and vinculin (a marker of focal adhesion, green) were performed. CFs adhered to DPT showed well organized actin stress fibers and focal adhesions containing vinculin. Nuclei were counterstained with DAPI (blue). Bar, 50 μm.

FBS used were determined according to preliminary studies. As expected, with the amounts of DPT added into the upper compartment of the chamber increasing, an inverse tendency in the number of migrated CFs was observed. Finally, we can conclude that, as shown in Fig. 5A and C, DPT significantly inhibited CFs migrating towards 10 μg/ml Fn and 2% FBS in a dose-dependent manner and progressively achieved the maximal inhibition activity at 10 μg/ml DPT both for Fn and 2% FBS. Similarly, the inhibitory effect of DPT on Fn-induced or FBS-induced CFs migration was dramatically weakened by integrin α3 or integrin β1 blocking antibodies, and the most obvious impact was achieved when both antibodies were used, which blocked nearly all the inhibiting effect of DPT (Fig. 5B and D). 2.5. Effect of DPT on CFs proliferation In order to accurately determine the role of DPT in the process of CFs proliferation, 5-bromo-2′-deoxyuridine (BrdU) incorporation assay was performed to investigate the effect of the increasing concentrations of DPT and the prolonged DPT treating time on CFs proliferation. Although wide range of concentrations and time gradients were examined, no significant difference was found in the number of CFs between control group and DPT treated group (data not shown). 3. Discussion The present study was designed to determine the expression pattern of DPT mRNA and protein as well as it's secretion in CMs

and CFs under hypoxia/SD conditions, and to determine the roles of DPT in CFs adhesion, spreading, migration and proliferation which are important aspects of ventricular remodeling. We demonstrated that: (1) The expression (mRNA and protein levels) and secretion of DPT were significantly elevated in conditions of hypoxia/SD in the major cell types of the heart (CFs and CMs); (2) DPT promoted adhesion, spreading and migration of CFs whereas had no effect on CFs proliferation. (3) Integrin α3β1 was at least one receptor for CFs adhesion and migration to DPT. DPT, an ECM component, has been reported to participate in many pathological conditions. Its mRNA and protein expression have been detected in human and murine hearts (Takemoto et al., 2002; Okamoto and Fujiwara, 2006; Gabrielsen et al., 2007), and DPT was also found to be secreted by fibroblasts (Superti-Furga et al., 1993). Interestingly, in experimental acute MI rats, DPT mRNA was elevated in the infarct zone of the heart in a time-dependent manner and in parallel with that of decorin and collagen I, suggesting that DPT contributed to ECM remodeling by interacting with decorin and collagen I (Takemoto et al., 2002). Furthermore, by means of global mRNA profiling, DPT mRNA expression was found to be elevated in human chronic ischemic myocardium (Gabrielsen et al., 2007), and our previous study showed significant elevation of DPT mRNA in human failing hearts compared with non-failing control hearts (Wei et al., 2008). The foregoing research implicated DPT to be a feasible candidate in the ventricular remodeling due to many pathological conditions, while the adhesion, migration and proliferation of CFs have been implicated to play a predominant role in ventricular remodeling induced by cardiac ischemia or infarction (Baudino et al., 2006; Porter and Turner, 2009; Souders et al., 2009). So it is necessary to explore

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Fig. 4. DPT induced CFs migration via integrin α3β1. The lower compartments of the chambers were added with different concentrations of DPT and the upper compartments of the chambers were seeded with 5 × 104 CFs, followed by incubating in an incubator for 24 h. In inhibiting experiments, the lower compartments of the chambers were added with a constant concentration of DPT (10 μg/ml) and CFs were preincubated with blocking antibodies to integrin α3 (10 μg/ml) or β1 (10 μg/ml) or both antibodies at 37 °C for 20 min prior to being seeded. A. DPT induced CFs migration in a concentration-dependent manner. Upper panel: representative images of CFs migrated to the underside of the membrane. Lower panel: quantitative analysis of migrated cells per high field (×400). B. The chemotactic effect of DPT was inhibited by anti-integrin α3 and anti-integrin β1 neutralizing antibodies. Mean ± SD, **P b 0.01 versus control. Bar, 50 μm.

the potential effects of DPT on CFs adhesion, migration and proliferation, which are significant aspects of ventricular remodeling induced by cardiac ischemia or myocardial infarction. Ischemia is the main cause of myocardial infarction, thus, in the present study, we used hypoxia/SD conditions to mimic the in vivo

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conditions of ischemic myocardium and found that DPT mRNA and protein levels as well as its secretion were elevated both in CMs and CFs in a time-dependent manner (Fig. 1) under conditions of hypoxia/SD. Our in vitro results are in accordance with in vivo reports that DPT mRNA was elevated in chronic ischemic myocardium and in infarct zone of experimental acute MI rats (Takemoto et al., 2002; Gabrielsen et al., 2007). However, to our knowledge, the accurate role of DPT in the pathogenesis of ventricular remodeling after MI or during HF has largely not yet been elucidated. Subsequently, we examined the effects of DPT on CFs adhesion, spreading, migration and proliferation. Cells adhering to and spreading on ECM components such as fibronectin, collagen and laminin play critical roles in cell morphology, migration, proliferation, differentiation and survival. Using solid phase cell adhesion assay and immunofluorescent staining for F-actin and vinculin, we demonstrated that DPT facilitated CFs adhesion in a dose-dependent manner and CFs adhered to DPT also formed actin filaments and focal adhesions including vinculin (Fig. 2 A–B and Fig. 3). Our present observations are in general agreement with previous findings that DPT promotes adhesion and spreading of dermal fibroblasts and epidermal keratinocytes (Lewandowska et al., 1991; Okamoto et al., 2010). Integrins and heparan sulfate proteoglycans (HSPGs) on cell surface are essential for adhesion of cells to the ECM and can coordinate the interactions between ECM components and cells (Bernfield et al., 1999), therefore, we tested the potential of integrins and HSPGs to be the receptor of DPT on CFs by using EDTA, peptides GRGDSP and GRGESP, heparin, heparinase III and special functional blocking antibodies. Our results demonstrated that integrin α3β1 was at least one receptor for CFs adhesion to DPT (Fig. 2 C–D). Migration of CFs from the infarction margin to the infarct zone facilitates repair of the damaged ECM in the primary ventricular remodeling and exerts a compensative response to sustain the normal cardiac function after cardiac infarction, however, sustained ventricular remodeling will eventually lead to decompensation that incurs progressive, decompensated HF. Thus, it is requisite to study the effect of DPT on the migration activity of CFs. Using transwell cell culture chamber assays, we found that DPT stimulated CFs migration when added into the lower compartments of the chambers and inhibited CFs migration towards 2% FBS or 10 μg/ml Fn when added into the upper compartments of the chambers, both in a dosedependent manner. Moreover, these effects of DPT were all inhibited by functional blocking antibodies specific for integrin α3 and integrin β1 (Figs. 4–5), which interestingly expounded that DPT served as a chemotaxin for CFs migration via integrin α3β1. Recently, Yamatoji et al. demonstrated that DPT over-expressed H1 and Sa3 cells exhibited decreased invasiveness compared with control cells using a similar transwell system (Yamatoji et al., 2012). The apparently contradictory findings implied that the extracellular DPT served as a chemotaxin for CFs migration and the over-expressed intracellular DPT for invasiveness assay perhaps exert their influences on cellular migration/invasion through distinct mechanisms, such as different receptors and different signaling pathways, of course, the diverse function of DPT on different cell types should be taken into account for the reasonable explanation of the foregoing discrepancy. CFs proliferation is another important aspect in the ventricular remodeling process after cardiac ischemia or infarction. Previous researchers have identified that proliferation of CFs in the marginal region of cardiac infarction and the subsequent migration of CFs into the infarction area are involved in the ventricular remodeling process (Porter and Turner, 2009). So we tested the effects of DPT on CFs proliferation by Brdu incorporation assay. However, no effects with statistic significance of DPT on CFs proliferation were finally detected. These results achieved here confirmed that the pro-migratory effect of DPT on CFs was not attributed to its proliferative effects. In conclusion, our studies on DPT presented in this research suggest the following hypothetical model for DPT in ventricular remodeling

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Fig. 5. DPT inhibited Fn-induced and FBS-induced CFs migration and the inhibiting effect were weakened by integrin α3 and integrin β1 blocking antibodies. Fifty thousand CFs were preincubated with different concentrations of DPT at 37 °C for 20 min and seeded into the upper compartments of the transwell tissue culture chambers. The lower compartments of the chambers were added with 2% FBS or 10 μg/ml Fn as chemotactic factors. In inhibiting experiments, CFs were incubated with blocking antibodies against α3 (10 μg/ml) or β1 integrin (10 μg/ml) or both antibodies at 37 °C for 20 min before being incubated with a constant concentration of DPT (10 μg/ml) and added to the upper compartment of the chambers. A. DPT inhibited 2% FBS-induced CFs migration in a dose-dependent manner. Upper panel: representative images of CFs migrated to the underside of the membrane. Lower panel: quantitative analysis of migrated cells per high field (×400). B. The effect of DPT inhibiting 2% FBS-induced CFs migration was reduced by integrin α3 and β1 blocking antibodies. C. DPT inhibited 10 μg/ml Fn-induced CFs migration in a dose-dependent manner. Upper panel: representative images of CFs migrated to the underside of the membrane. Lower panel: quantitative analysis of migrated cells per high field (×400). D. The effect of DPT inhibiting 10 μg/ml Fn-induced CFs migration was reduced by integrin α3 and β1 blocking antibodies. Mean ± SD, **Pb 0.01 versus control. Bar, 50 μm.

after myocardial injury or infarction: ischemic injury stimulates the release of DPT from CMs and CFs into blood circulation and extracellular environment, then extracellular DPT interacting with integrins facilitates CFs adhesion and spreading and promotes migration of CFs into the injured area, meanwhile, myofibroblasts secrete various

bioactive molecules including ECM components, cytokines, vasoactive peptides and growth factors to participate in the ventricular remodeling process. Briefly, extracellular DPT may be a novel therapeutic target for ventricular remodeling after cardiac injury or infarction.

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4. Experimental procedures 4.1. Cell culture CMs and CFs isolated from ventricles of 1–3 days old Sprague–Dawley (SD) rats as previously described were cultured in DMEM supplemented with 10% FBS and penicillin–streptomycin (100 IU/ml and 100 mg/ml, respectively) and incubated at 37 °C in a humidified atmosphere containing 5% CO2 and 95% air (Lu et al., 2010). CMs of primary cultures and CFs of the second or third passage were utilized for the subsequent experiment tests. The experimental protocol of CMs and CFs isolation from neonate SD rats was approved by the Ethics Committee on Animal Study of Fuwai Hospital and in compliance with the guidelines of “Regulation to the Care and Use of Experimental Animals” (1996) of the Beijing Council on Animal Care and the Guide for the Care and Use of Laboratory Animals (NIH Publication 85-23, revised 1996). 4.2. Cell treatment When cultured CFs reached subconfluent status and CMs started beating, they were treated with hypoxia/SD conditions generated by washing cells in serum free DMEM and incubating in a sealed hypoxic GENbox jar fitted with a catalyst to scavenge free oxygen as previously described (Zhu et al., 2006). After treated with hypoxia/SD for 0, 6, 12 and 24 h, they were harvested for real-time quantitative reverse transcription polymerase chain reaction (real time RT-PCR) and Western blotting analysis. Meanwhile, cell culture supernatants were collected and centrifuged at 1000 g for 15 min, after which they were stored at −80 °C until use. 4.3. Real time RT-PCR analysis of DPT mRNA Total RNA was extracted from CMs and CFs after exposure to hypoxia/SD for different time intervals using Trizol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer's instructions and protocols described previously (Fortunati et al., 2010). 1 μg of total RNA was used to synthesize the first strand cDNA with an AMV Reverse Transcriptase Kit (Promega, Madison, WI, USA). 1% agarose gel electrophoresis was performed to examine the integrity of total RNA and the RNA was quantified according to the OD260 value measured by an UV spectrophotometer. Quantification of DPT transcript levels was determined by real time PCR with SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA) and specific primers for DPT and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) which served as an internal control in an Applied Biosystems 7300 Fast Real-Time PCR System (Foster City, CA, USA). Primers of DPT and GAPDH used in this work were as follows: DPT: 5′-AGGGCTCTGACAGACAGTGGAACTA-3′ and 5′-ACTGACTCGAAGTA A CGGCTT TGG-3′; GAPDH: 5′-GGCACAGTCAAGGC TGAG AATG-3′ and 5′-ATGGTGGTGAAGACGCCAGTA-3′. 4.4. Western blotting analysis of DPT protein Total proteins from hypoxia/SD stimulated CMs and CFs were prepared and Western blotting was performed as previously described (Shi et al., 2011). Briefly, cultured cells were lysed with lysis buffer and centrifuged at 13,000 g for 10 min under 4 °C, then supernatants were collected and the protein concentrations were determined by the BCA Protein Assay. Subsequently, equal amounts of protein (40 μg) from each sample were applied to 4–12% gradient SDS-PAGE and transferred onto nitrocellulose membranes by semi-dry transfer followed by blockage with 5% skimmed milk for 1 h at room temperature (RT) and incubation overnight with anti-DPT (Proteintech Group, Chicago, IL, USA) or anti-β-tubulin primary antibody (Santa Cruz, CA, USA) at 4 °C. Then, the membranes were rinsed with Tris Buffered

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Saline containing 1‰ tween 20 and incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (Santa Cruz, CA, USA) for 1 h at RT. Signals were detected using an enhanced chemiluminescence detection kit and quantified by a Bio-Rad GS700 densitometer with Quantity One software. 4.5. ELISA of DPT in cell culture supernatants Secretion of DPT in cell culture supernatants of CMs and CFs with or without hypoxia/SD treatment (0 h, 6 h, 12 h and 24 h) was assessed by ELISA using a commercially available kit (Bogoo, Shanghai, China) according to the manufacture's protocols. All standards and samples were assayed in duplicate. 4.6. CFs adhesion assays Cell adhesion assays were performed as previously reported with minor modifications (Hozumi et al., 2006; Okamoto et al., 2010). Briefly, plates of 96 wells were coated overnight with different concentrations of recombinant mouse DPT (R&D Systems, Minneapolis, MN, USA) (2, 5, 10, 20 and 50 μg/ml), Fn (Merk KgaA, Darmstadt, Germany) (2, 5, 10, 20 and 50 μg/ml) or 1% (wt/vol) bovine serum albumin (BSA) which served as control group in 50 μl of phosphate-buffered saline (PBS) at 4 °C, then the non-binding sites in the plate bottoms were blocked with 1% BSA for 1 h at RT followed by washing three times with PBS, finally an almost equal protein coating efficiency could be achieved (Hozumi et al., 2006). Rat CFs were detached with 0.025% trypsin-EDTA for less than 1 min at 37 °C, then trypsin was discarded and the cells were resuspended in DMEM containing 10% FBS and restored in an incubator at 37 °C for 20 min. After that, cells were washed twice with serum free DMEM and used for adhesion assays. Thirty thousand cells (100 μl) were added into each well and incubated in an incubator for 60 min at 37 °C, after which the wells were washed once with warm PBS, fixed with 1% glutaraldehyde for 30 min, stained with 0.1% crystal violet for 1 h and photographed using an inverted phase contrast microscope (Olympus IX70, Tokyo, Japan). Subsequently, the wells were rinsed three times with PBS and dried. Finally, cells were washed with 100 μl 0.1% triton X-100 for 1 h and the absorbance at 595 nm was measured with a microplate reader. Each sample was assayed in triplicate, and cells attached to 1% BSA were subtracted from all measurements. For experiments investigating the receptors for CFs adhering to DPT, the cells were preincubated with EDTA, peptide GRGDSP or GRGDSE (GL Biochem, Shanghai, China), heparin, heparinase III (Sigma-Aldrich, St. Louis, MO), functional blocking antibodies for integrin α3 (Santa Cruz, CA, USA) or integrin β1 (BD Biosciences, Oxnard, CA) for 20 min at 37 °C before plating, and then incubated for 60 min at 37 °C in an incubator and measured as described above. 4.7. Immunofluorescent staining For actin and vinculin staining, glass cover slips in plates of 24 wells were coated overnight with DPT (20 μg/ml) at 4 °C and blocked with 1% BSA for 1 h at RT. CFs were detached and recovered as described for the adhesion assays. After the cells were washed twice with serum free DMEM, 2 × 10 4 cells/400 μl were added to each well and incubated for 150 min at 37 °C in a humidified atmosphere of 5% CO 2. After the non-adherent cells were rinsed off by warm PBS, adherent cells were fixed by 4% paraformaldehyde for 15 min, permeabilized with 0.3% Triton X-100 for 10 min and blocked by 1% BSA for 1 h. Primary antibodies to vinculin (1:400; Abcam, Cambridge, MA) was applied at 4 °C overnight. Then, the cells were stained with fluorescein isothiocyanate (FITC)-conjugated anti-mouse secondary antibody (1:500; Jackson ImmunoResearch Laboratories) at 37 °C for 1 h, after which F-actin was visualized by 5 μg/ml TRITC-phalloidin (Promega Madison, WI, USA) at 37 °C for 1 h and the nuclei were

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detected with 4′, 6-diamidino-2-phenylindole (DAPI). Finally, the cells were washed three times with PBS by a 5 min interval and examined by a Nikon EZ-C1 FreeViewer 3.00.502 fluorescent confocal microscope.

ANOVA. Comparisons between two groups were evaluated using Student's t-test. P values less than 0.05 were considered to be statistically significant.

4.8. CFs migration assays

Contributors

Cell migration assays were performed using Transwell cell culture chambers with 8-μm pore size polycarbonate membrane as described previously (Liaw et al., 1994). Briefly, cells were prepared as described for adhesion assays and after CFs were washed twice with serum free DMEM, 5 × 10 4 cells in 100 μl of serum free DMEM were seeded into the upper chambers. The lower compartments were loaded with 600 μl serum free DMEM supplemented with different concentrations of DPT (0, 5, 10 and 20 μg/ml; 0 μg/ml was designed as the control group). After incubation for 24 h at 37 °C in a tissue culture incubator, non-migrated cells on the upper surface of the membranes were rubbed off with a moist cotton swab. Migrated cells attached onto the lower surface of the membrane were fixed in 4% paraformaldehyde for 15 min, stained with crystal violet for 1 h and photographed using a light microscope (Olympus BX61, Tokyo, Japan). Migration was quantified by counting the migrated cells in 10 random high power fields (× 400) for each membrane. For inhibition studies, CFs were preincubated with anti-integrin α3 (10 μg/ml) or anti-integrin β1 (10 μg/ml) blocking antibody or both antibodies at 37 °C for 20 min before seeding and the lower compartments were loaded with 600 μl serum free DMEM supplemented with 10 μg/ml DPT. Then, the cells were incubated and the migrated cells were calculated as described above. Furthermore, the effects of DPT on CFs migration induced by 10 μg/ml Fn or 2% FBS (vol/vol) were assessed. Concisely, 5×104 CFs suspended in 100 μl serum free DMEM supplemented with different concentrations of DPT (0, 5, 10 and 20 μg/ml; 0 μg/ml was designed as the control group) were added into the upper wells of the transwell cell culture chambers after incubation at 37 °C for 20 min, then serum free DMEM supplemented with 600 μl of 10 μg/ml Fn or DMEM supplemented with 2% FBS serving as chemoattractants was loaded into the lower wells of the chambers. In inhibiting experiments, CFs were incubated with neutralizing antibodies against integrin α3 (10 μg/ml) or integrin β1 (10 μg/ml) or both antibodies at 37 °C for 20 min before being incubated with 10 μg/ml DPT and added to the upper wells of the transwell cell culture chambers. After incubation in a humidified incubator at 37 °C for 6 h, migrated cells were fixed, stained, photographed, and counted as described above.

Xiaoyan Liu: design of the study, undertaking the majority of the lab work, drafting and revision of the paper. Liukun Meng: design of the study, revision of the paper. Qiang Shi: undertaking part of the lab work. Shenghua Liu: undertaking part of the lab work. Chuanjue Cui: undertaking part of the lab work. Yingjie Wei: design of the study, revision of the paper. Shengshou Hu: design of the study, revision of the paper. All authors approved the final draft for publication.

4.9. CFs proliferation assays CFs proliferation was evaluated by BrdU incorporation assay with a colorimetric BrdU Cell Proliferation ELISA kit (Calbiochem, Gibbstown, NJ, USA) according to the manufacture's specifications. Briefly, CFs were seeded in 96-well microplates at a density of 5000 cells/well and allowed to adhere overnight, thereafter, they were serum starved for 24 h and treated with DPT at doses of 0, 2, 5, 10, 20 and 50 μg/ml (0 μg/ml was designed as the control group) with different time intervals of 12, 24, 48 and 72 h. After that, 10 μl BrdU was added into the cell culture medium and CFs were reincubated for another 6 h followed by fixing with Fixing Solution for 30 min. Then, CFs were incubated with anti-BrdU antibody for 90 min. Finally, the immune complex was detected by substrate reaction and quantified by measuring the absorbance at 450 nm using a microplate reader (Bio Rad, Mode 680, Tokyo, Japan). 4.10. Statistical analysis Data are expressed as mean ± SD and were analyzed using the statistical package SPSS 13.0. All experiments were repeated at least three times. Differences among groups were tested by one-way

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