Direct intercellular communications dominate the interaction between adipose-derived MSCs and myofibroblasts against cardiac fibrosis
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The onset of cardiac fibrosis post myocardial infarction greatly impairs the function of heart. Recent advances of cell transplantation showed great benefits to restore myocardial function, among which the mesenchymal stem cells (MSCs) has gained much attention. However, the underlying cellular mechanisms of MSC therapy are still not fully understood. Although paracrine effects of MSCs on residual cardiomyocytes have been discussed, the amelioration of fibrosis was rarely studied as the hostile environment cannot support the survival of most cell populations and impairs the diffusion of soluble factors. Here in order to decipher the potential mechanism of MSC therapy for cardiac fibrosis, we investigated the interplay between MSCs and cardiac myofibroblasts (mFBs) using interactive co-culture method, with comparison to paracrine approaches, namely treatment by MSC conditioned medium and gap co-culture method. Various fibrotic features of mFBs were analyzed and the most prominent anti-fibrosis effects were always obtained using direct co-culture that allowed cell-to-cell contacts. Hepatocyte growth factor (HGF), a well-known anti-fibrosis factor, was demonstrated to be a major contributor for MSCs’ anti-fibrosis function. Moreover, physical contacts and tube-like structures between MSCs and mFBs were observed by live cell imaging and TEM which demonstrate the direct cellular interactions.
KEYWORDScardiac fibrosis stem cell therapy adipose-derived mesenchymal stem cells myofibroblasts cell-to-cell contact anti-fibrosis
alpha-smooth muscle actin
hepatocyte growth factor
magnetic resonance imaging
mesenchymal stem cells
transforming growth factor-beta 1
Myocardial infarction accounts for over 40% of cardiovascular diseases (CVD) related human mortality, as reported by the World Health Organization (WHO, 2013). Cardiac fibrosis, the most eminent pathological feature post infarction (van den Borne et al., 2010; Fan et al., 2012; Weber et al., 2013), would form a scattering area of fibrosis scars that generate tonic contraction forces and interfere with the original conduction property of healthy myocardium, thus greatly impairs the physiological functions (i.e. blood pumping) of normal heart (Rohr, 2009; Weber et al., 2013). Histological characterization revealed that an excessive deposition of fibrillar collagen was accumulated within the infarcted myocardium and an altered cell population which was positive for alpha-smooth muscle actin (α-SMA) replaced the lost cardiomyocytes, namely myofibroblasts (mFBs) (Rohr, 2009). mFBs were believed to mainly originate from interstitial cardiac fibroblasts (Brown et al., 2005; Czubryt, 2012) which are responsible for the stability of extracellular matrix in heart and its dynamic balance with cardiomyocytes (Fan et al., 2012; Weber et al., 2013). The transition from interstitial fibroblasts to mFBs has been demonstrated to be initiated by transforming growth factor-beta 1 (TGF-β1) secreted by both immunocytes and necrotic cardiomyocytes (Brown et al., 2005; Rohr, 2009; Czubryt, 2012; Weber et al., 2013).
The current treatments for myocardial infarction, including the administration of thrombolytic drugs (e.g. streptokinase, urokinase and alteplase) (Tomasevic et al., 2008; Minami et al., 2010; Sehestedt et al., 2011; Lyngbaek et al., 2012; Juarez-Herrera and Jerjes-Sanchez, 2013; Lippi et al., 2013), the implantation of vascular stents (Armstrong, 2006; Stefanini and Windecker, 2012; Tokushige et al., 2013; Zhang et al., 2013) and bypass operation (Favaloro, 1969, 1971) mainly concentrated on improving the hemodynamics thus restoring the blood supply to infarcted myocardium. Nonetheless, the efficacy of these conventional therapies was limited as they could hardly compensate for the massive loss of necrotic cardiomyocytes, which account for approximately 25% of total cardiomyocytes during a single episode of infarction (Deutsch et al., 2013). Given the limited regenerative capacity of adult human heart, effective therapies should focus on the remuscularization of the diseased heart (Deutsch et al., 2013). Therefore, transplantation of autologous cells into the diseased heart has shown to be a reasonable and effective therapeutic strategy. During the past decade, multiple cell types, including endothelial progenitors (Asahara et al., 1997) and mesenchymal stem cells (MSCs, either derived from bone marrow or adipose tissue) (Miyahara et al., 2006; Emmert et al., 2013; Blocki et al., 2015; Li and Zhang, 2015) have shown therapeutic effects (shrunken area of fibrotic scar and thickened ventricle wall post-infarction) on cardiac fibrosis, among which the utility of MSCs has reached phase II or III clinical trials due to their ease of access and consistent outcome (Sheridan, 2013). However, the exact behavior of MSCs post-transplantation in vivo was barely known and the exact underlying cellular mechanisms were not fully understood. Although the development of advanced imaging techniques, e.g. magnetic resonance imaging (MRI) has been used to track the stem cells post transplantation (Drey et al., 2013; Emmert et al., 2013), the intercellular activity between MSCs and host tissue cells remained difficult to monitor. In addition, the paracrine activity of MSCs was reported to activate the quiescent cardiac progenitor cells or stimulate the residual cardiomyocytes to re-enter proliferative phase (Ranganath et al., 2012). However, few of these studies have provided solid explanation on how MSCs attenuate the fibrosis condition within the infarcted myocardium, since the hostile avascular and hypoxic environment could not support cell survival and factor diffusion to help remuscularization of the diseased heart.
As the main ‘architect’ of cardiac fibrosis, the interplay between cardiac mFBs and MSCs should be investigated to provide a better understanding for cardiac fibrosis therapy. Some studies showed that the conditioned medium from MSC culture (Ohnishi et al., 2007; Mias et al., 2009; Wang et al., 2011; Mao et al., 2013) could inhibit the proliferation, the expression of α-SMA and collagen production of mFBs, and the secretion of matrix metalloproteinases (MMPs) by fibroblasts was elevated as well. Nonetheless, the treatment of conditioned medium in vitro cannot fully recreate the condition of cell therapy in vivo where multiple intercellular activities were involved. For instance, Cselenyák et al. observed a significant dependency of cell-to-cell contact for MSC therapy to rescue the cardiomyocytes from cell death using an in vitro ischemia model, whereas MSCs cultured in inserts, the conventional format for paracrine studies, cannot exert similar beneficial effect (Cselenyak et al., 2010). Besides, Plotnikov et al. discovered that specific cellular contacts, namely nanotubes, were formed within the co-culture of MSCs with cardiomyocytes (Plotnikov et al., 2008). Therefore, the direct cell-to-cell contact, or so-called intercellular communication, would be a dominant factor for proper therapeutic benefit of MSCs to cardiac fibrosis.
To elucidate the dominant role of intercellular communications for MSC therapy, the direct cell co-culture model using adipose-derived MSCs and cardiac mFBs was introduced in vitro and multiple pathological features of mFBs, including cellular viability, biomarker expression (α-SMA, collagen), cellular contractility and motility were analyzed in the model, which were compared to the treatment of MSC conditioned medium in parallel. An engineered microfluidic-based co-culture platform was fabricated to further assess the importance of direct cell-to-cell communication for MSCs’ anti-fibrotic therapy. The system comprised two chambers separated by varying distances of cell-repellent gap, which would only allow communication by paracrine factors and no direct contact could be formed.
RESULTS AND DISCUSSION
Viability of mFBs during co-culture and comparison with treatment using MSC conditioned medium
Conditioned medium harvested from normal MSC cultures was used to incubate mFBs for 48 h as a conventional paracrine method, resulting in decreased viability of mFBs relative to controls (Fig. 1D). However, the viability reduction was not comparable to direct co-culture manner, indicating that specific stimuli from microenvironment (cell-to-cell communications) would be important for MSCs exerting better therapeutic effect. Indeed, it has been reported that preconditioning for MSCs would serve as a stress environment and enable better regenerative effects in various diseases (Haque et al., 2013). For example, the application of hypoxic preconditioning during culture would enhance the survival rate and therapeutic potential of MSCs in treating brain (Chang et al., 2013) or liver (Yu et al., 2013) injuries. Here mFBs culture recapitulated the cardiac fibrosis environment and thus served as the stress preconditioning for MSCs.
Phenotypic and functional analysis of mFBs during co-culture
Besides the phenotypic alteration of cells, mFBs could also exert strong contractile ability which could be characterized by the extent of collagen gel contraction in vitro. Gels populated by co-cultured mFBs remained relatively larger in size compared with those populated with pure mFBs (Fig. 2E, marked by ‘-’), and mFB-populated collagen gels would contract independently of different comprising number of mFBs (data not shown), implying that the contractile property of mFBs was greatly inhibited by MSCs. As a member of MSC secretome, HGF has been demonstrated to be an effective inhibitor for fibrosis condition. Collagen gel lattice remained much bigger in size than control group with the addition of exogenous HGF (Fig. 2D), and the HGF antibody could block the inhibitory effect of MSCs in co-culture without affecting the original contractile ability of mFBs (Fig. 2E).
Therefore, it was demonstrated here that co-culturing with MSCs could drive the mFBs return to a quiescent dedifferentiated state where some of pathological characteristics (both phenotypic and functional), e.g. α-SMA expression and collagen gel contraction ability, were significantly alleviated. HGF played an important role for enhancing the anti-fibrosis activity of MSCs. Moreover, MSCs themselves were reported to have strong contractile ability in mono-culture (Sumanasinghe et al., 2009; Espagnolle et al., 2014) and similar results were also obtained in this study (Fig. S1), implying that specific microenvironment was essential to stimulate the therapeutic potential of MSCs.
Comparison of mFBs dedifferentiation during co-culture between direct contact mode and gap mode
Dynamic interaction between MSCs and mFBs
Directional migration property of mFBs during co-culture
Though no inducible agent was added inside the empty chamber, cells would still move towards the opposite due to limited growth space. However, the proliferation of mFBs was mostly inhibited by MSCs as above investigated. Importantly, the expression of α-SMA, the major cytoskeletal actin that mFBs depend on to contract and migrate, was also proved to be greatly reduced due to the co-culture. Thus mFBs in co-culture could hardly migrate for long distance in the engineered system.
Formation of cell-to-cell contacts during co-culture
In this study, we employed an in vitro interactive cell co-culture model to demonstrate that adipose-derived MSCs could ameliorate the key pathological characteristics of cardiac fibrosis mediated by mFBs mainly in a direct cell-to-cell contact manner, namely direct intercellular communication. Consistent with previous studies, we obtained the inhibitory effects of conditioned medium from MSCs on the viability and α-SMA expression of mFBs, however, the effects were more significant in the direct co-culture system. Hepatocyte growth factor (HGF), one of the known factors that reduce fibrosis in multiple organs, was demonstrated to be a major contributor for MSCs’ anti-fibrosis function. Moreover, using the engineered co-culture platform, the paracrine activity was found to be dependent on the distance between the two cell populations and be efficient at a narrow gap width (500 μm). Proper therapeutic benefits of MSCs to myocardial infarction should be based on the amelioration of fibrosis condition, in order to provide a favorable microenvironment for cell survival and factor diffusion. The study here, for the first time, elucidated that MSCs could exert better therapeutic effects by directly communicating with pathogenic cells (i.e. mFBs in cardiac fibrosis). We anticipated that this study could provide novel insight and more precise guidance for cell therapy based on MSCs, e.g. to introduce MSCs sufficiently close to the disease foci. Systematic investigations into anti-fibrosis molecules that were involved during the therapy process were necessary to provide better understanding of the underlying molecular mechanisms for the regenerative capacity of MSCs and could lead to establishment of novel therapeutic approaches.
MATERIALS AND METHODS
Polyethylene glycol (PEG, MW 258), 3-Trimethoxysilyl-propyl-methacrylate (TMSPMA), and Octadecyltrichlorosilane (OTS) were purchased from Sigma-Aldrich (St. Louis, MO). The photo initiator, 2-hydroxy-1-[4-(hydroxyethoxy)-phenyl]-2-methyl-1-pro-panone (Irgacure D2959), was purchased from Insight High Technology Co. LTD (China). CellTiter-Blue cell viability assay kit (alamar blue) was purchased from Promega (Wisconsin, USA).
All animal experiments were approved by the Animal Ethics Committee of the Center of Biomedical Analysis, Tsinghua University. Cardiac fibroblasts were derived from hearts of Sprague-Dawley rats (male, 80–100 g) as previously described (Santiago et al., 2010) except some modifications (Zhao et al., 2014). Briefly, left ventricle tissue was excised, washed with HBSS (Wisent, Canada) and cut into pieces of ~1 mm3. Enzymatic digestion using Collagenase Type II (100 U, Gibco) and trypsin–EDTA (0.125%, Wisent, Canada) was employed to dissociate the tissue pieces, which were agitated at 37°C for 40~60 min. Supernatant was collected every 10 min into a conical tube and digestion was neutralized with 1/10 volume of fetal bovine serum (Wisent, Canada). Finally, the cells were centrifuged at 1,200 rpm for 5 min, resuspended in fresh medium (DMEM containing 10% FBS) and plated into 25 cm2 culture flasks. Non-adherent cells were discarded after 60 min. The rest of cells were propagated 4~5 passages, which would be induced spontaneously into mFBs as reported (Santiago et al., 2010).
MSCs were isolated from human adipose tissues obtained from patients undergoing liposuction operation as previously reported (Li et al., 2010). After isolation, the MSCs were expanded in growth medium (BIOWIT, China) and incubated at 37°C in a humidified environment containing 5% CO2. The isolated MSCs were positive for CD29, CD44, CD105 and Flk-1 while negative for CD31, CD34, CD45 and HLA-DR, as described earlier (Cao et al., 2005).
For direct co-culture of MSCs with mFBs, cells were mixed in ratios ranging from 1:0.01~1:1 (mFBs:MSCs) and plated in a density of 10,000/cm2 (Rahmat et al., 2013). Co-culture medium was composed by half of fibroblast medium (DMEM containing 10% FBS) and the other half of MSC growth medium. To prepare conditioned medium of adipose-derived MSCs, 90% confluent MSCs (less than passage 5) were washed with PBS and then exposed in fresh co-culture medium for 24 h (Yang et al., 2013). The medium was then collected and filtered through a 0.22 μm filter unit (Millipore) before use. All analyses were conducted 48 h post co-culture or treatment in conditioned medium.
Cell viability analysis
Viability was assayed using CellTiter-Blue® cell viability assay kit and performed as per manufacturer’s instructions. For viability assay, MSCs in the co-culture were inactivated by 10 μg/mL mitomycin C (Dalian Meilun Biology Technology Co., Ltd, China) for 3 h before being mixed with mFBs. Cells were plated in 48-well plate with constant number of mFBs and an equal number of inactivated MSCs used in the co-culture among each ratio were plated in individual wells for background elimination. Before the assay, culture medium was discarded. Alamar blue solution prepared in fresh medium was added to each well and incubated at 37°C. Fluorescence values were read by microplate reader (Molecular Devices, USA) with excitation at 560 nm and emission at 590 nm. All values were normalized to the mFB mono cultures.
Cell apoptosis detection kit (Beyotime, China) was used to verify that the mFBs were viable in co-culture. All procedures were accomplished as per the instructions. Cells treated with 500 μmol/L H2O2 for 2 h were used as positive control.
Samples were prepared by culturing cells on circular coverslips, then fixed with absolute methanol at −20°C for 10 min and blocked with 5% w/v bovine serum albumin (biotechnology grade, Wisent). Primary antibody against α-SMA (rabbit, Abcam) was diluted at 1:500 and incubated with samples overnight at 4°C. This was followed by 1 h incubation with Alexa Fluor 488 goat anti-rabbit IgG at room temperature. Hoechst 33324 (1:4000, Invitrogen) was used to stain cell nuclei. Fluorescence observation was performed on Nikon Eclipse Ti-S microscope (Nikon, Japan). α-SMA fluorescence intensity was analyzed using ImageJ software (NIH, USA) according to software manual. Briefly, all the fluorescent images were taken with identical optical settings (i.e. exposure time and binning numbers). Images were then imported into ImageJ and more than 3 different ROIs in the image were selected for pixel intensity quantification.
Sirius Red collagen detection kit (Chondrex, Inc., USA) was used to quantify the amount of collagen as per manufacturer’s instructions. Similarly, an equal number of inactivated MSCs used in the co-culture among each ratio were plated in individual wells for background elimination. Briefly, samples were fixed and incubated with Sirius Red solutions for 30 min at room temperature, and eluted using extraction buffer provided in the kit. The absorbance of the extracted solution was read at 540 nm by microplate reader.
Total proteins were extracted from cultured cells using RIPA lysing buffer (Beyotime, China), according to the manufacturer’s protocol. Proteins were separated by 10%–12% SDS/PAGE gels and transferred to PVDF membrane (Millipore), which was then blocked by 5% (w/v) nonfat dry milk in TBS-Tween (0.2%) for 1 h. Membranes were probed with rabbit anti-rat α-SMA (1:400, Abcam) overnight at 4°C. After several washes in TBS-Tween, membranes were incubated with goat anti-rabbit HRP-conjugated secondary antibody (1:2000, ZSGB-BIO, China). The subsequent visualization was performed using SuperSignal West Pico Chemiluminescent Substrate (Thermo) by the ChemiDoc XRS+ with image Lab software (Bio-Rad).
Collagen contraction assay
Time-lapse recording was performed to monitor the dynamic interaction between MSCs and mFBs. Culture plate was mounted in a 5% CO2 filled incubation chamber (Nikon, Japan). Cells were labeled with CellTracker (green for mFB and red for MSCs, Invitrogen) before imaging. Pictures were taken at 35 min interval using inverted Fluorescent Microscope (Ti-U series, Nikon, Japan) and cellular movement tracking was realized using ImageJ software equipped with an open access manual tracking plugin.
Fabrication of compartmentalized microfluidic devices
The device was fabricated using PDMS stamps by conventional soft-lithography technique. The molds were fabricated using UV cross-linkable polyethylene glycol (PEG, Mw = 258, Sigma) through pre-designed photomask. Briefly, 1 wt% photo initiator (Irgacure D2959) was dissolved in PEG258 solution, which was then pipetted onto 3-(trimethoxysilyl)propyl methacrylate (TMSPMA) coated glass slides and exposed to UV light (OmniCure SERIES 1500, 27.6 mW/cm2, Canada) for 18~25 s. Then, the patterned molds were immersed into ethanol to remove any unreacted precursors for 5 min and air dried. Afterwards, PDMS prepolymer solution was poured onto the molds and polymerized at 70°C for 8 h.
Device for investigating directional cell migration was composed of two isolated chambers (2 cm long 150 μm high) interconnected by narrow channels (1 mm long 0 μm high). Device for investigating gap mode co-culture was composed of two separated chambers in the same size as above mentioned despite that they were separated by cell-repellent gaps in a range of distances.
For cell seeding in migration device, PDMS was removed from the molds and stamped on clean glass slides through thermal binding after 2 min of plasma treatment. The slides were then functionalized with 0.1% gelatin solution for cell adherence. Cells were seeded into one chamber at a density of 1 × 104 cells/cm2. To prevent cell leakage, the interconnected micro-channel was filled with 2 mg/mL collagen gel prior to cell seeding.
For gap mode co-culture, PDMS stamp was pressed onto OTS-treated glass slides which would provide a cell-repellent surface between separated chambers, followed by plasma treatment that enabled protein adsorption in cell growing chambers. MSCs and mFBs were seeded into each chamber and allowed to adhere before stamp removal, leaving separated cell patterns on the slide. Co-culture medium was then added on top of the slide, covering both patterns and incubated for 48 h.
Transmission electron microscopy (TEM)
For the TEM analysis, cells were immersed in 2.5% glutaraldehyde (VASE, China), fixed in 1% OsO4, dehydrated using 70% EtOH containing 2% uranyl acetate and embedded in Epon 812 (Fluka, USA). After Epon polymerized, the samples were removed from the culture dish and cut into ultra-thin (7 μm) sections using a Leica EM UC6. The sections were stained with lead citrate and examined using H-7650B microscope (Hitachi, Japan).
Quantitative data were plotted as the mean ± standard deviation. Statistical analysis was performed using Student’s unpaired two-way t-tests and ANOVA analysis. Differences were considered to be statistically significant when P < 0.05.
We thank Professor Kexin Yuan at Tsinghua University for his kind assistance in animal experiments. This work is financially supported by the National Natural Science Foundation of China (Grant Nos. 81171474, 51273106, 51461165302 and 11372243), the Beijing Municipal Natural Science Foundation (Grant No: 157142090) and International Science & Technology Cooperation Program of China (2013DFG02930).
COMPLIANCE WITH ETHICAL STANDARDS
Xiaokang Li, Hui Zhao, Chunxiao Qi, Yang Zeng, Feng Xu and Yanan Du declare that they have no conflict of interest. All institutional and national guidelines for the care and use of laboratory animals were followed.
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