TISSUE-SPECIFIC STEM CELLS Resistin Promotes Angiogenesis in Endothelial Progenitor Cells Through Inhibition of MicroRNA206: Potential Implications for Rheumatoid Arthritis CHEN-MING SU,a CHIN-JUNG HSU,b,c CHUN-HAO TSAI,b,d CHUN-YIN HUANG,d,e SHIH-WEI WANG,f CHIH-HSIN TANGa,g,h Key Words. Resistin • Endothelial progenitor cell • Rheumatoid arthritis • Collagen-induced arthritis • MicroRNA

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Graduate Institute of Basic Medical Science, cSchool of Chinese Medicine, College of Chinese Medicine, dGraduate Institute of Clinical Medical Science, gDepartment of Pharmacology, School of Medicine, and bDepartment of Orthopedic Surgery, China Medical University, Taichung, Taiwan; eDepartment of Orthopedic Surgery, China Medical University Beigang Hospital, Yun-Lin County, Taiwan; fDepartment of Medicine, Mackay Medical College, New Taipei City, Taiwan; hDepartment of Biotechnology, College of Health Science, Asia University, Taichung, Taiwan Correspondence: Chih-Hsin Tang, Ph.D., Graduate Institute of Basic Medical Science, China Medical University, No. 91, Hsueh-Shih Road, Taichung, Taiwan. Telephone: 1886-422052121 Ext. 7726; Fax: 1886-4-22333641; e-mail: [email protected] Received September 19, 2014; accepted for publication March 11, 2015; first published online in STEM CELLS EXPRESS April 7, 2015. C AlphaMed Press V

1066-5099/2014/$30.00/0 http://dx.doi.org/ 10.1002/stem.2024

ABSTRACT Endothelial progenitor cells (EPCs) promote angiogenesis and are therefore key contributors to a wide variety of angiogenesis-related autoimmune diseases such as rheumatoid arthritis (RA). However, the signaling mechanisms through which these progenitor cells influence RA pathogenesis remain unknown. The aim of this study was to examine whether resistin plays a role in the pathogenesis of and angiogenesis associated with RA by circulating EPCs. We found that levels of resistin in synovial fluid and tissue from patients with RA and from mice with collageninduced arthritis were overexpressed and promoted the homing of EPCs into the synovium, thereby inducing angiogenesis. EPCs isolated from healthy donors were used to investigate the signal transduction pathway underlying EPC migration and tube formation after treatment with resistin. We found that resistin directly induced a significant increase in expression of vascular endothelial growth factor (VEGF) in EPCs. We also found that the expression of microRNA-206 (miR-206) was negatively correlated with the expression of resistin during EPC-mediated angiogenesis. Notably, the increased expression of VEGF was associated with decreased binding of miR-206 to the VEGF-A 30 untranslated region through protein kinase C delta-dependent AMPactivated protein kinase signaling pathway. Moreover, blockade of resistin reduced EPC homing into synovial fluid and angiogenesis in vivo. Taken together, our study is the first to demonstrate that resistin promotes EPCs homing into the synovium during RA angiogenesis via a signal transduction pathway that involves VEGF expression in primary EPCs. These findings provide support for resistin as a therapeutic target for the patients with RA. STEM CELLS 2015;33:2243–2255

INTRODUCTION Rheumatoid arthritis (RA), one of the most common autoimmune diseases, is characterized by the infiltration of proinflammatory cytokines into synovial fluid leading to pannus formation and joint destruction [1]. Proangiogenic cells such as endothelial stem cells are crucial to the development and progression of RA as they promote the creation of angiogenic ventilation shafts which consequently cause synovitis by forming new capillaries in the joints [2, 3]. Bone marrow-derived endothelial stem cells are characterized by their ability to selfrenew and develop into intermediate stem cell–endothelial progenitor cells (EPCs) [4]. EPCs possess cellular subpopulations with different functional capacities, including proliferation, migration, and recruitment in response to angiogenesis. Circulating EPCs, a cell

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population characterized by the CD1331/ CD341/VEGFR21 phenotype, are mobilized from the bone marrow into the bloodstream and are involved in neovascularization by becoming incorporated within neovessels [5–7]. Although EPCs are critical components of the earliest phases of tumor neovascularization [8], it is still unclear whether EPCs are involved in the initiation of RA by causing deterioration in the joints. Resistin, a small cysteine-rich adiposederived peptide hormone, is specifically expressed and secreted by adipocytes and is linked to both inflammation [9, 10] and insulin resistance [11]. Previous research has shown that increased serum resistin levels in patients with insulin resistance correspond to increased levels of protein kinase C delta (PKC-d) signaling [12, 13]. A number of studies have demonstrated that resistin levels in serum and synovial fluid are elevated in patients with RA C AlphaMed Press 2015 V

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[14–16] and that resistin levels might correlate with markers of inflammation. Thus, elevated resistin may be a predictor of RA. Increased resistin expression has been shown to be correlated with increased expression of vascular endothelial growth factor (VEGF), one of the most important angiogenic switch molecules [17]. Accumulating evidence has revealed that VEGF is regulated by microRNAs (miRNAs) at multiple levels during angiogenesis [18, 19]. These miRNAs are small noncoding RNA molecules that map to intronic locations within protein-coding genes and function in RNA silencing and posttranslational regulation of gene transcription. Some miRNAs have been shown to be possible effectors of autoimmune diseases and to be involved in both physiological and pathological angiogenesis, as they regulate several genes that participate in both functions [20, 21]. However, miRNAs have hundreds of putative targets, and it is challenging to determine the physiological functions of individual miRNA-target interactions. A previous study showed that chemokine-induced angiogenesis is an early and critical event in RA pathogenesis [2]. However, the role resistin plays in the development of and angiogenesis associated with autoimmune diseases such as RA remains unclear. In this study, we investigated whether highly expressed resistin in patients with RA upregulates the function of human circulating EPCs in RA-associated degeneration and explored the signaling mechanisms involved in this process. We conclude by discussing the possible significance of our results.

MATERIALS

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METHODS

Materials Anti-mouse and anti-rabbit horseradish peroxidase-conjugated IgG and rabbit polyclonal antibodies specific for b-actin, PKCd, phosphorylated PKC-d, AMP-activated protein kinase a1 (AMPKa1), phosphorylated AMPKa1, CD31, and CD133 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, http://www.scbt.com). A mouse monoclonal antibody and enzyme immunoassay kit specific for resistin were purchased from R&D Systems (Minneapolis, MN, http://www.rndsystems. com). Recombinant human resistin was purchased from PeproTech (Rocky Hill, NJ, http://www.peprotech.com). Antiresistin neutralized antibody was purchased from Fisher Scientific (Thermo Fisher Scientific, NY, http://www.fisherscientific. com). ON-TARGETplus small interfering RNA (siRNA) targeting VEGF, PKC-d, and AMPKa1 and ON-TARGETplus nontargeting siRNA (control) were purchased from Dharmacon Research (Lafayette, CO). Synthesized sequences of a human miRNA206 (miR-206) mimic, a miR-206 inhibitor, and a negative control miRNA were purchased from GeneDireX (Las Vegas, NV). Customized miRNA array primers were purchased from System Biology Ireland (Galway, Ireland). Rottlerin, Compound C, and adenosine-9-b-D-arabino-furanoside (Ara A) were purchased from Calbiochem (San Diego, CA, http://www.emdbiosciences.com). All other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, http://www.sigmaaldrich.com).

Human Synovial Fluids and Tissues We obtained approval for this study from the local ethics committee, and all subjects gave written informed consent to participate. Abnormal synovial fluids and synovial tissues were C AlphaMed Press 2015 V

obtained from 10 patients (aged 40–60 years) during total knee arthroplasty for RA, and normal synovial fluids and tissues were obtained from four patients during arthroscopy for trauma/joint derangement. The protocol for this study was approved by the Institutional Review Board of the China Medical University Hospital (DMR-103-059), and informed consent was obtained from each donor. Primary synovial fibroblasts from RA patients and normal patients were isolated, cultured, and characterized as previously described [22]. Briefly, human primary synovial fibroblasts were isolated in a solution of II collagenase for 16–18 hours and then filtered through 70-mm nylon filters. The cells from passages 3–9 were used for the experiments.

Isolation of Human Circulating EPCs EPCs were derived from healthy donors who gave informed consent before enrollment, and ethical approval was granted by the Institutional Review Board of Mackay Medical College (New Taipei City, Taiwan; reference number: P1000002). EPCs were isolated and purified as previously described [23, 24]. Briefly, mononuclear cells were isolated from peripheral blood (80 mL) using the Ficoll-Paque plus (Amersham Biosciences, Uppsala, Sweden, http://www.amersham.com) centrifugation method. Then, EPCs were separated from the isolated mononuclear cells using the MicroBead Kit and the MACS Cell Separation System (all from Miltenyi Biotec, Bergisch Gladbach, Germany, http://www.miltenyibiotec.com). EPCs were maintained and characterized as follows: briefly, EPCs were seeded on gelatin-coated dishes containing MV2 medium, SupplementMix (PromoCell, Heidelberg, Germany, http://www.promocell.com) and 20% nonheat-inactivated defined fetal bovine serum (FBS; HyClone, Logan, UT, http://www.hyclone.com) and incubated for 3 days; the medium was changed every 3 days. For characterization, labeled EPCs were examined and characterized with CD341/CD1331/VEGFR21 antibodies by using a FACSCalibur flowcytometer and CellQuest software (both from BD Biosciences, San Jose, CA, http://www.bdbiosciences.com) [25, 26].

Collagen-Induced Arthritis Mouse Model Male C57BL/6J mice (aged 8–10 weeks) were purchased from the National Laboratory Animal Centre in Taipei. Mice were maintained under conditions consistent with the guidelines established by the Animal Care Committee of the China Medical University. A collagen-induced arthritis (CIA) mouse model was established according to published protocols [27]. Briefly, 0.1 mL of an emulsion containing 100 mg of bovine type II collagen (CII; Sigma) dissolved at a concentration of 2 mg/mL in 0.1 M acetic acid and complete Freund’s adjuvant was injected intradermally into the base of the tail. Two weeks after the primary immunization, a booster injection of 100 mg CII dissolved and emulsified 1:1 with incomplete Freund’s adjuvant was administered into the hind leg. The incidence of arthritis in the CIA mice was very high within 6 weeks after the first immunization, and 95% of the mice developed severe arthritis. The clinical severity of arthritis in each knee was measured in a blinded manner with a plethysmometer (Marsap, Mumbai, India, http://www.marsap.com) once weekly for 4 weeks. Upon sacrifice on day 42, the phalanges and knee joints were removed immediately and fixed in 4% paraformaldehyde for microcomputed tomography (micro-CT) analysis. Micro-CT scanning of the STEM CELLS

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knees was performed using an in vivo micro-CT scanner (Skyscan 1176; Bruker, Kontich, Belgium, http://www.skyscan.be) at 9 mm resolution and with a rotation step of 0.30 per image. Scanning was done at 50 keV of X-ray voltage, 500 mA of current, and 885 ms of exposure time/image with a 0.5 mm aluminum filter. Reconstruction of sections was carried out with GPU-based scanner software (NRecon, Bruker). In addition, the grayscale was based on the Hounsfield unit, and the validated calcium standards were scanned as the density reference. The threedimensional microstructural volumes from the micro-CT scans were analyzed using Skyscan software (CTAn, Bruker). To evaluate bone focal erosion in the knee joint, quantification of volumetric bone mineral density (BMD) was performed in bone areas defined by manually drawn regions of interest (8 mm per 913 slices from the proximal calcaneus to the metatarsals).

according to the manufacturer’s recommendations. Real-time quantitative polymerase chain reaction (qPCR) analysis was carried out with the KAPA SYBR FAST qPCR Kit (Applied Biosystems, Foster City, CA, http://www.appliedbiosystems.com). The cycling conditions were as follows: polymerase activation for 10 minutes at 95 C followed by 40 cycles at 95 C for 15 seconds and 60 C for 60 seconds. Relative normalization of gene expression was performed using endogenous GAPDH as the internal control for mRNA or snRNA U6 as the internal control for miRNA. Relative expression levels were calculated using the comparative threshold cycle (CT), which is the number of the cycle at which the transcript was detected [28]. All target gene primers were purchased from Applied Biosystems and are listed in Supporting Information Table S1.

EPC Tube Formation

Human primary EPCs were cultured in 24-well plates, treated with resistin, and then incubated in a humidified incubator at 37 C for 24 hours. To examine the downstream signaling pathways involved in resistin treatment, cells were pretreated with various inhibitors for 30 minutes before resistin (3 ng/mL) was added. After incubation, the supernatant was collected as conditioned medium (CM) and stored at 280 C until the assay was performed. VEGF in the CM was assayed using a VEGF-A enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems) according to the procedure described by the manufacturer.

Matrigel (BD Biosciences, Bedford, MA) was dissolved at 4 C, added to 48-well plates at 100 mL per well, and incubated at 37 C for 30 minutes. EPCs (2 3 104 per 100 mL) were resuspended in MV2 serum-free medium with the indicated concentration of resistin and then added to the wells. VEGF (50 ng/mL) and phosphate buffered saline (PBS) were used as the positive and negative control, respectively. After 6–8 hours of incubation at 37 C, EPC tube formation was assessed by microscopy, and each well was photographed at 3200 magnification under a light microscope. The number of tube branches and the total tube length were calculated and quantified using MacBiophotonics Image J software.

Immunohistochemistry Paraffin-embedded plug sections were prepared, mounted on silane-coated slides, deparaffinized in xylene, rehydrated in a graded alcohol series, and washed in deionized water. After antigen retrieval (boiling in a microwave for 30 minutes in 10 mM sodium citrate, pH 6.0), the intrinsic peroxidase activity was blocked by incubation with 3% hydrogen peroxide. Nonspecific antibody-binding sites were blocked using 3% bovine serum albumin (BSA) in PBS. Plug sections were then incubated with appropriately diluted primary rabbit polyclonal anti-mouse resistin, VEGF, CD31, or CD133 antibodies (Abcam, MA, http://www.abcam.com) at 4 C overnight. After three washes in PBS, the secondary antibody (biotin-labeled goat anti-rabbit IgG) was applied for 1 hour at room temperature. Staining was detected with 3,30 -diaminobenzidine tetrahydrochloride, and the sections were then counterstained with hematoxylin and eosin and observed under a light microscope. Some specimens were also stained with Safranin O-fast Green for bone erosion.

Quantification of mRNA and miRNA by Real-Time Quantitative Polymerase Chain Reaction Amplification Total RNA was extracted from EPCs with a TRIzol kit (MDBio, Taipei, Taiwan, http://0800072222.tw), and miRNAs were quantified using the Mir-XTM miRNA First Strand Synthesis Kit (Clontech Laboratories, Palo Alto, CA, http://www.clontech. com), as per the manufacturers’ protocols. The RNA quality and yield of each total RNA sample was determined via A260 measurements using a NanovueTM Spectrophotometer (GE Healthcare, WI, http://www.gehealthcare.com). Complementary DNA was derived from 1 lg of total RNA using an MMLV RT kit (Invitrogen, CA, http://www.invitrogen.com),

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Enzyme-Linked Immunosorbent Assay

Transwell Migration Assay All EPC migration assays were performed using Transwell inserts (8 lm pore size; Costar, NY, http://www.corning.com) in 24-well plates. EPCs were pretreated with the indicated concentrations of inhibitors (Rottlerin, Compound C, or Ara A; Sigma) for 30 minutes or transfected with various siRNAs for 16–18 hours; then, the cells were treated with resistin for another 24 hours. EPCs (5 3 103 in 200 lL of medium with 10% FBS) were then seeded in the upper chamber, and 300 lL of the same medium containing the appropriate concentration of resistin was placed in the lower chamber. Cells on the upper side of the Transwell membrane were examined and counted under a microscope. Each experiment was performed in triplicate and was repeated at least three times.

Western Blot Analysis Proteins were resolved using sodium dodecyl sulfatepolyacrylamide gel electrophoresis and transferred to Immobilon polyvinyldifluoride membranes (Millipore, MA, http:// www.millipore.com). The membranes were blocked with 4% BSA for 1 hour at room temperature and then probed with rabbit antibodies against human phosphorylated PKC-d, PKC-d, phosphorylated AMPKa1, or AMPKa1, (1:1,000) for 1 hour at room temperature. After three washes, the blots were incubated with a donkey anti-rabbit peroxidase-conjugated secondary antibody (1:1,000) for 1 hour at room temperature. The blots were then developed via enhanced chemiluminescence and visualized using a Fujifilm LAS-3000 chemiluminescence detection system (Fujifilm, Tokyo, Japan, http://www. fujifilm.com).

Plasmid Construction and Luciferase Assays The 30 -untranslated region (30 -UTR) of human VEGF-A contains a miR-206 binding site. A DNA fragment containing VEGF 30 C AlphaMed Press 2015 V

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UTR (wt-VEGF 30 -UTR) and a fragment containing a version of the VEGF 30 -UTR in which the miR-206 binding site had been mutated (mut-VEGF 30 -UTR) were purchased from Invitrogen. Each fragment was subcloned into the luciferase reporter vec-

tor pGL2-Control (Promega, Madison, WI, http://www.promega.com), upstream of the vector’s promoter. All constructs were confirmed by sequencing using the Applied Biosystems 3730xl DNA Analyzer (Thermo Fisher Scientific, NY). EPCs,

Figure 1. C AlphaMed Press 2015 V

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Su, Hsu, Tsai et al. grown to 80% confluence, were transiently cotransfected with a luciferase reporter construct (wt-VEGF 30 -UTR or mut-VEGF 30 -UTR) and the miR-206 mimic, the miR-206 inhibitor, or the control miRNA using Lipofectamine 2000 according to the manufacturer’s instructions (Invitrogen). After treatment with resistin for 24 hours, luminescence from treated EPCs lysates was measured using a microplate luminometer. Luciferase activity was normalized to that of the cotransfected bgalactosidase expression vector.

Chick Chorioallantoic Membrane Assay In vivo angiogenetic activity was determined using a chick chorioallantoic membrane (CAM) assay as previously described [29]. Briefly, 5-day-old fertilized chick embryos (6 eggs per group) were incubated at 37 C in an 80% humidified atmosphere. On developmental day 8, PBS (vehicle) or resistin was resuspended in Matrigel and placed onto the CAMs for another 3 days. The CAMs were then examined by microscopy and photographed. Angiogenesis was quantified by counting the number of blood vessel branches. All animal work was performed in accordance with a protocol approved by the China Medical University (Taichung, Taiwan) Institutional Animal Care and Use Committee.

In Vivo Matrigel Plug Assay The Matrigel plug angiogenesis assay was performed as previously described [30]. Thirty 4-week-old male nude mice were randomized into three groups and subcutaneously injected with 0.15 mL Matrigel containing PBS (vehicle) or resistin (3 ng/mL). On day 7, the Matrigel plugs were harvested; some were fixed with 3% paraformaldehyde for at least 2 days and then embedded in paraffin and subsequently processed for immunostaining of VEGF, CD31, and CD133; others were evaluated by Drabkin’s method (Drabkin’s Reagent Kit; Sigma) to quantify the hemoglobin content.

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RESULTS Resistin is Highly Expressed in Synovial Fluid and Tissues of RA Patients Resistin has been shown to be associated with the overexpression of proinflammatory cytokines such as interleukin (IL)-6, tumor necrosis factor (TNF)-a, and IL-1b in the progression of RA [31, 32]. Thus, we first examined whether resistin is highly expressed in patients with RA. We found that resistin protein and resistin mRNA levels were significantly higher in synovial fluids and tissues from patients with RA than in healthy patients (Fig. 1A, 1B). Resistin protein levels were also significantly higher in primary synovial fibroblasts from RA patients than in normal synovial fibroblasts (Fig. 1C). A previous study demonstrated preferential homing of EPCs to the inflamed synovium [33]. Therefore, we conducted a migration assay combined with antiresistin antibody to investigate the homing abilities of EPC in synovial fluids from patients with and without RA following resistin treatment (Fig. 1D) and found that the observed effects in RA synovial fluid were attributable to highly expressed resistin. In addition, we examined whether resistin had similar functions in the CIA mice, one of the most commonly used animal models for evaluating the efficacy of RA therapies [34]. We found that CIA mice had significantly greater bone erosion and worse BMD in the knee joints than mice in the control group on day 42 (Fig. 1E, 1F). Safranin O staining also showed significantly greater bone erosion in knee joints of CIA mice (Fig. 1G). Double immunofluorescent staining of the synovium with EPC markers CD34 and CD133 (Fig. 1H) and immunostaining of tissue sections for resistin, EPC markers (CD133 and VEGFR2), and vessels markers (CD31 and VEGF) revealed increased expression in CIA mice (Fig. 1I). These observations suggest that EPC infiltration and VEGF production are involved in arthritic development in CIA mice. Taken together, these data demonstrate that resistin plays a significant role in angiogenesis during the arthritic process.

Statistical Analysis All quantified results were calculated using GraphPad Prism 5.0 software and are presented as the mean 6 SD of at least three experiments. Statistical comparison of two groups was performed using the Student’s t test. Statistical comparisons of more than two groups were performed using two-factor analysis of variance with the Bonferroni post hoc test or the Mann–Whitney U test, as appropriate. In all cases, p < .05 was considered statistically significant.

Resistin Promotes VEGF-Dependent EPC Migration and Tube Formation Since VEGF is one of the most potential angiogenic mediators and induces the formation of new capillaries from pre-existing vessels, angiogenesis may occur and progress in RA pathogenesis [2, 35]. Although resistin has been shown to directly enhance angiogenesis in numerous diseases [17, 36], the mechanism by which it promotes angiogenesis and EPC

Figure 1. Resistin is highly expressed in synovial fluid and tissue from patients with RA and from mice with CIA. (A, B): Synovial fluid or tissue from 10 patients with RA and NL synovial fluid from four healthy patients undergoing arthroscopy for trauma/joint derangement were measured for levels of resistin protein or mRNA by enzyme-linked immunosorbent assay or real-time quantitative polymerase chain reaction, respectively. (C): NL primary cells and human synovial fibroblasts from RA patients were studied for resistin expression by Western blot. (D): Circulating EPCs isolated from human peripheral blood were examined in a migration assay using RA or NL synovial fluids with or without resistin neutralizing antibody. (E): Representative microcomputed tomography images of the knees of mice with CIA on day 42 (F, femur; T, tibia; P, patella; M, meniscus). Scale bar 5 1 mm. (F): Quantification of volumetric BMD from femur and tibia. (G): Knee joint sections were stained with hematoxylin and eosin and safranin O. Scale bar 5 10 mm (H): Tissue sections of synovium from control and CIA mice were detected with CD34 (CD34-positive cells, red) and CD133 (CD133-positive cells, green) by using double immunofluorescent staining; DAPI (blue) was used for nuclear staining. Arrows indicate tri-fluorescent tagged EPCs in the synovium. Scale bar 5 5 mm. (I): Knee joint tissue sections from control and CIA mice were immunohistochemically stained for resistin, CD133, VEGFR2, CD31, and VEGF. Arrows indicate dye-tagged targets in the synovium of the CIA mice. Scale bar 5 5 mm and 3 mm (inset). Bars show the mean 6 SEM from three individual experiments; *, p < .05 versus NL (A, B, and D) or control (F); #, p < .05 versus RA fluids (D). Abbreviations: BMD, bone mineral density; CIA, collagen-induced arthritis; DAPI, 40 ,6-diamidino-2-phenylindole; EPC, endothelial progenitor cell; F, femur; M, meniscus; NL, normal patients; P, patella; RA, rheumatoid arthritis; T, tibia; VEGF, vascular endothelial growth factor; VEGFR2, VEGF receptor 2.

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Figure 2. Resistin promotes VEGF-dependent EPC migration and tube formation. (A): Treatment of EPCs with resistin (resistin; 0.03–3 ng/mL) for 24 hours induced VEGF mRNA expression in a concentration-dependent manner. (B): Resistin stimulation increased VEGF protein expression in a concentration-dependent manner, as shown by enzyme-linked immunosorbent assay. (C, D): Transfection with siRNA targeting VEGF or with control siRNA for 16–18 hours abolished the resistin-induced increase in EPC migration and tube formation. (E): The number of tube branches and the total tube length were calculated and quantified. Bars show the mean 6 SEM from three individual experiments. *, p < .05 versus control; #, p < .05 versus resistin (3 ng/mL) (C, E). Abbreviations: EPC, endothelial progenitor cell; siRNA, small interfering RNA; VEGF, vascular endothelial growth factor.

homing is not clear. Therefore, we used circulating EPCs to investigate whether resistin plays a role in the expression of VEGF. Treatment of EPCs with resistin (0.03–3.0 ng/mL) for 24 hours induced VEGF mRNA expression in a concentrationdependent manner (Fig. 2A). Resistin stimulation also led to a concentration-dependent increase in VEGF protein expression (Fig. 2B) and induced a concentration-dependent increase in EPC migration and tube formation (Fig. 2C–2E). These results indicate that activation of EPC migration and tube formation is critical for resistin-mediated VEGF expression. In contrast, transfection with VEGF siRNA notably abolished resistinincreased EPC migration and tube formation (Fig. 2C–2E). Taken together, these data suggest that resistin facilitates VEGF-dependent EPC migration and tube formation. C AlphaMed Press 2015 V

The Effect of Resistin on EPCs is Mediated by the PKCd/AMPK Signaling Pathway Previous research has shown that increased serum resistin levels are associated with increased PKC-d signaling, which is involved in angiogenesis [13, 37, 38]. To verify the involvement of PKC-d in the response of EPCs to resistin, PKC-d phosphorylation was examined in EPCs after resistin stimulation (Fig. 3A). We then pretreated EPCs with the PKC-d inhibitor Rottlerin or transfected cells with PKC-d siRNA or control siRNA. We found that both Rottlerin and the PKC-d siRNA abolished resistin-induced VEGF production (Fig. 3B, 3C) as well as EPC migration and tube formation (Fig. 3D–3F). A previous study showed that PKC-d is upstream of AMPK and is required for the activation of AMPK in phenylephrine STEM CELLS

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Figure 3. The PKC-d signaling pathway is involved in resistin-induced EPC migration and tube formation. (A): EPCs were incubated with resistin (3 ng/mL) for the indicated time intervals, and then PKC-d phosphorylation was analyzed by Western blot. (B): The effect of siRNA targeting PKC-d (2 mM) on EPCs versus that of control siRNA, as analyzed by Western blot (upper graph). The upregulation of VEGF mRNA in response to resistin was reversed by pretreatment with the PKC-d inhibitor Rottlerin or PKC-d siRNA, as shown by real-time quantitative polymerase chain reaction (lower graph). (C): Pretreatment with Rottlerin or transfection with PKC-d siRNA abolished resistin-induced VEGF production, as shown by the enzyme-linked immunosorbent assay. (D–F): Resistin-promoted EPC migration and tube formation were reduced by pretreatment with Rottlerin or transfection with PKC-d siRNA. (E): Quantification of tube formation. Data represent mean 6 SEM from three individual experiments. *, p < .05 versus control; #, p < .05 versus resistin alone or control siRNA (B–D, F). Abbreviations: EPC, endothelial progenitor cell; PKC-d, protein kinase C delta; VEGF, vascular endothelial growth factor; siRNA, small interfering RNA.

preconditioning [39]. In this study, we found that resistin stimulation enhanced AMPK phosphorylation in a timedependent manner (Fig. 4A). We also examined whether AMPK is a downstream effector of PKC-d. Pretreatment with a PKC-d inhibitor or PKC-d siRNA abolished resistin-promoted AMPK phosphorylation (Fig. 4B). As shown in Figure 4C and 4D, levels of VEGF mRNA and VEGF protein were reduced in

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cells pretreated with the AMPK inhibitors Ara A and Compound C and in cells that underwent transfection with AMPKa1 siRNA. To ascertain whether resistin activated EPC migration and tube formation via the AMPK signaling pathway, we used AMPK inhibitors or AMPKa1 siRNA. The results show that AMPK activation is required for the EPC response to resistin (Fig. 4E–4G). Taken together, these results indicate C AlphaMed Press 2015 V

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Figure 4. The role of AMPKa1 in resistin-induced EPC migration and tube formation. (A): EPCs were incubated with resistin (3 ng/mL) for the indicated time intervals, and then phosphorylated AMPKa1 was measured by Western blot. (B): EPCs were pretreated with Rottlerin for 30 minutes or transfected with siRNA targeting PKC-d for 16–18 hours and then stimulated with resistin; subsequently, phosphorylated AMPKa1 was measured by Western blot. (C): EPCs were transfected with AMPKa1 siRNA or control siRNA (2 mM), and then the level of AMPKa1 protein expression was measured by Western blot (upper graph). EPCs were pretreated with the AMPK inhibitors Ara A or Compound C or transfected with AMPKa1 siRNA and stimulated with resistin; then, levels of VEGF mRNA were assessed by real-time quantitative polymerase chain reaction (lower graph). (D): ELISA assay indicated that resistin-mediated VEGF production was abolished by pretreatment with Ara A or Compound C or transfection with AMPKa1 siRNA. (E–G): Resistin-induced EPC migration and tube formation were reduced by pretreatment with Ara A and Compound C or transfection with AMPKa1 siRNA, as shown by quantification of tube formation (G). Data represent mean 6 SEM from three individual experiments. *, p < .05 versus control; #, p < .05 versus resistin alone or control siRNA (C–E, G). Abbreviations: AMPKa1, AMP-activated protein kinase a1; Ara A, adenosine-9-b-D-arabino-furanoside; EPC, endothelial progenitor cell; PKCd, protein kinase C delta; VEGF, vascular endothelial growth factor; siRNA, small interfering RNA.

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Figure 5. Resistin induces EPC migration and tube formation by downregulating miR-206 expression via the PKC-d/AMPK-signaling pathway. (A): EPCs were incubated with resistin (1–3 ng/mL) for 24 hours, and then miR-206 expression was examined by real-time quantitative polymerase chain reaction (qPCR). (B–D): EPCs were transfected with an miR-206 mimic (10 nM), an miR-206 inhibitor (10 nM), or a NC miR (10 nM) followed by resistin treatment; then, VEGF expression was evaluated by Western blot (B), qPCR (C), and enzyme-linked immunosorbent assay (D). (E–G): Resistin-induced EPC migration and tube formation were attenuated by transfection with a miR-206 mimic and significantly enhanced by transfection with an miR-206 inhibitor. (H): EPCs were transfected with indicated luciferase plasmids for 24 hours and then treated with resistin, after which the relative luciferase activity was measured. (I): Pretreatment with Rottlerin, Compound C, or Ara A or transfection with siRNA targeting either PKC-d or AMPK reversed the resistin-mediated downregulation of miR-206 expression in EPCs. Data represent mean 6 SEM from three individual experiments. *, p < .05 versus control; #, p < .05 versus resistin alone or NC miR (C–E, G). Abbreviations: AMPKa1, AMP activated protein kinase alpha 1; EPC, endothelial progenitor cells; miR-206, miRNA 206; miRNA, microRNA; NC, negative control; PKC d, protein kinase C delta; siRNA, small interfering RNA; UTR, untranslated region; VEGF, vascular endothelial growth factor.

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that resistin increases VEGF expression and EPC migration and tube formation via activation of the PKC-d-dependent AMPK signaling pathway.

Resistin Induces EPC Migration and Tube Formation by Downregulating miR-206 via the PKC-d/AMPK Signaling Pathway Recently, miRNAs have been shown to be powerful regulators in autoimmune diseases; however, the targets of most miRNAs remain unknown and the roles of miRNAs in biological processes are not clearly understood [40]. We used a customized miRNome microRNA Profilers Kit, which contains 384 human miRNAs and found that approximately one-fifth of the miRNAs examined were influenced by resistin. To specifically study miRNAs affected by resistin, we selected miRNAs for which expression changed by at least twofold in the presence of resistin. We found that eight of the miRNAs were upregulated and three were significantly downregulated when EPCs were treated with resistin (Supporting Information Fig. S1). Next, we integrated the results of the miRNAs array with three online computational algorithms (TargetScan, PicTar, and miRanda) to identify conserved miRNAs that targeted VEGF-A and found 1 candidate miRNA: miR-206. We then evaluated the miR-206 expression profile in EPCs. Treatment of human circulating EPCs with resistin resulted in a significant, concentration-dependent decrease in miR-206 levels (Fig. 5A). To determine whether miR-206 directly regulates VEGF, we transiently transfected EPCs with a miR-206 mimic, a miR-206 inhibitor, or a negative control miRNA and found that transfection with the miR-206 mimic inhibited the induction of VEGF mRNA and VEGF protein in EPCs that had been exposed to resistin. In contrast, transfection with the miR-206 inhibitor increased the expression of VEGF mRNA and VEGF protein in response to resistin (Fig. 5B–5D). In addition, resistin-induced EPC migration and tube formation were attenuated by the miR206 mimic but were largely enhanced by the miR-206 inhibitor (Fig. 5E–5G). To further investigate the specificity for which miR206 targets VEGF 30 -UTR, we transfected two firefly luciferase reporter plasmids, wt-VEGF 30 -UTR and mut-VEGF 30 -UTR, into EPCs and then analyzed cell extracts for luciferase activity. The results showed that resistin notably increased luciferase activity in cells transfected with the wt-VEGF 30 -UTR luciferase reporter plasmid but not in those transfected with the mut-VEGF 30 -UTR luciferase reporter plasmid (Fig. 5H), suggesting that resistin promotes the expression of the VEGF 30 -UTR in the absence of the miR-206 binding site. Furthermore, pretreatment with Rottlerin, Compound C, or Ara A or transfection with siRNAs against PKC-d or AMPK reversed the resistin-mediated downregulation of miR206 expression in EPCs (Fig. 5I), indicating that the PKC-d/AMPKdependent regulation of miR-206 expression is involved in the resistin-induced signaling pathway. These data therefore show that resistin downregulated the expression of miR-206 and then promoted VEGF expression and EPC migration as well as tube formation via the PKC-d/AMPK signaling pathway.

Resistin is Associated with EPC Angiogenesis In Vivo To determine the effects of resistin on VEGF expression and EPC tube formation in vivo, we investigated angiogenesis using the CAM assay. PBS (vehicle) or resistin (3 ng/mL) were resuspended in Matrigel and then placed onto the surface of the CAMs for clear observation. Interestingly, CAMs incubated with C AlphaMed Press 2015 V

Resistin Induces EPCs Migration and Tube Formation

resistin had significantly more new capillaries than CAMs incubated with PBS (Fig. 6A). Quantification of the vessels at the surface of the CAMs is shown in Figure 6B. To further confirm that EPCs are recruited by resistin in vivo, we subcutaneously injected 0.15 mL Matrigel containing PBS or resistin (3 ng/mL) into the flanks of nude mice and found that the Matrigel plugs containing resistin resulted in a greater degree of microvessel formation than plugs containing PBS (Fig. 6C). In addition, we quantified the hemoglobin content of the Matrigel plugs using Drabkin’s method (Fig. 6D). When we evaluated paraffinembedded plug sections by immunohistochemistry, we found that levels of VEGF, CD31, and CD133 were higher in resistintreated Matrigel plugs than in Matrigel plugs containing PBS (Fig. 6E). To further demonstrate the role of resistin in RA angiogenesis, we tested the effect of resistin on tube formation by exposing synovial fluid from RA patients and control subjects to blockade of resistin (Fig. 6F, 6G) and found that resistin played a crucial role in RA angiogenesis in vitro. In addition, we investigated in synovial fluid from RA patients with or without exposure to blockade of resistin using the CAM assay and Matrigel plug assay, respectively (Fig. 6H, 6J) and quantified vessels count and hemoglobin content, respectively (Fig. 6I, 6K). We found that blockade of resistin attenuated angiogenesis in RA synovial fluid in vivo, indicating that highly expressed resistin plays an important role in RA angiogenesis. Taken together, the results suggest that resistin significantly promotes EPC migration and tube formation in vivo, implicating that resistin is a crucial modulator of the pathogenesis of RA.

DISCUSSION RA is characterized by peripheral symmetrical polysynovitis associated with the accumulation of body fat in patients with RA [41]. The abnormal accumulation of adipose tissue in rheumatoid joints might implicate certain factors in the regulation of peripheral polysynovitis in RA, as both bone marrow and white adipose tissues secrete a wide variety of proteins known as adipokines that participate in the regulation of processes involved in metabolism, immunity, and inflammation [42]. Previous studies have characterized resistin as a potent proinflammatory adipokine that appears to have a critical role in RA pathogenesis [9, 43]. In our study, we found that high levels of resistin in patients with RA drive the function of circulating EPCs during RA processes. EPCs are associated with the induction and modulation of VEGF during RA pathogenesis [44–46], and angiogenesis has recently become a possible therapeutic target for patients with the disease [47, 48]. As widespread pattern, it is not yet clear however, whether circulating EPCs are associated with the regulation and mechanistic function of resistin in RA pathogenesis. Several studies have demonstrated that EPCs are connected with abnormal angiogenesis via the induction and modulation of VEGF during RA pathogenesis [44–46]. In our study, there are limitations of the present study that EPCs from RA patients are too rare and hard to acquire. However, we were only able to provide indirect evidence of the role EPCs play in the pathogenesis of RA because we used EPCs derived from mononuclear cells that had been isolated from peripheral blood of healthy patients rather than EPCs from RA patients. In addition, irrespective of whether resistin plays a STEM CELLS

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Figure 6. Resistin is associated with endothelial progenitor cell-mediated angiogenesis in vivo. (A): Chick chorioallantoic membrane (CAM) assay using 5-day-old fertilized chick embryos (6 eggs per group): PBS (vehicle) or resistin (3 ng/mL) were resuspended in Matrigel and placed onto the CAMs, which were allowed to develop for another 3 days. The CAMs were then examined by microscopy and photographed. Scale bar 5 2 mm. (B): Vessels on the CAMs were calculated and quantified. (C, D): Matrigel plugs containing PBS (vehicle) and resistin (3 ng/mL) were subcutaneously injected into the flanks of nude mice. After 7 days, the plugs were photographed (C) and hemoglobin levels were quantified and normalized to those obtained with PBS (D). (E): Specimens from the plugs were immunostained with antibodies against VEGF, CD31, and CD133. Scale bar 5 5 mm. (F): Micrographs of tube formation by endothelial progenitor cells in normal (NL) and RA fluid. (G): The number of tube branches and the total tube length were calculated and quantified. (H): CAM assay using 5-day-old fertilized chick embryos (6 eggs per group): NL fluid, RA fluid, or RA fluid plus resistin neutralized antibody was resuspended in Matrigel and placed onto the CAMs, which were allowed to develop for another 3 days. The CAMs were then examined by microscopy and photographed. Scale bar 5 2 mm. (I): Vessels on the CAMs were calculated and quantified. (J): After 7 days incubation, Matrigel plugs containing NL fluid, RA fluid, or RA fluid plus resistin neutralized antibody were photographed. (K): Matrigel plugs were quantified with hemoglobin levels and normalized to those obtained with NL fluid. Data represent mean 6 SEM from three individual experiments. *, p < .05 versus vehicle (B and D) or NL fluids (G, I, and K); #, p < .05 versus RA fluids (G, I, and K). Abbreviations: NL, normal patients; RA, rheumatoid arthritis; VEGF, vascular endothelial growth factor.

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key role in inflammatory or neovascular-signaling pathways, angiogenesis as a target of therapeutic intervention in RA has become a current possibility [47, 48]. On the other hand, although it is possible to observe most of the histopathological characteristics of RA in CIA mice [49], EPC infiltration through the synovial membrane in CIA mice has not been reported previously. Our study is the first to identify the role resistin plays in promoting the homing of EPCs to the synovium and the subsequent production of VEGF during angiogenesis in RA. The VEGF-related activation of PKC-d has been previously observed, and PKC-d may regulate a number of pathways required for angiogenesis [50, 51]. Although different isoforms of PKC have been shown to regulate various cellular molecular responses [52], Gliki et al. [53] found that Rottlerin was not specific for PKC-d. Here, we additionally used PKC-d siRNA to confirm PKC-d function in EPCs. Our findings indicate that one of the earliest steps in sprouting angiogenesis requires the PKC-d and the downstream AMPK signaling pathway in resistin-promoted EPC migration and tube formation. As miRNAs are able to modulate target gene translation via binding to the 30 -UTR of mRNAs and thus affecting multiple protein-encoding genes at the posttranscriptional level, they are implicated in the control of a wide range of biological functions [54]. Therefore, we investigated whether miRNAs were involved in angiogenesis following resistin stimulation of EPCs. We first found that of 384 human miRNAs in a customized microRNA Profiler Kit, approximately one-fifth were influenced by resistin in EPCs. We subsequently identified 11 miRNAs that underwent at least a twofold change in expression in response to resistin. Both clinical and experimental studies have shown that miRNAs appear to negatively regulate angiogenic events, including neovascularization and VEGF production [55, 56]. Therefore, we looked for miRNAs that were predicted by multiple algorithms (TargetScan, miRDB, and PicTar) to downregulate VEGF-A, and identified miR-206 as a possible candidate. Recent research has provided growing evidence for the importance of miR-206 in development and proliferation of adenocarcinoma and lipid metabolism [57, 58]. Carlos et al. reported that miR-1/miR-206 negatively regulated angiogenesis in zebrafish [59]. However, a role for miR-206 in linking EPCs and RA pathogenesis has not previously been identified. In this study, we used a miR-206 mimic or inhibitor and a luciferase reporter containing a miR-206 binding site on the VEGF 30 -UTR to verify the direct effects of miR-206 on

REFERENCES 1 Firestein GS. Evolving concepts of rheumatoid arthritis. Nature 2003;423:356–361. 2 Szekanecz Z, Besenyei T, Szentpetery A et al. Angiogenesis and vasculogenesis in rheumatoid arthritis. Curr Opin Rheumatol 2010;22:299–306. 3 Colville-Nash PR, Scott DL. Angiogenesis and rheumatoid arthritis: Pathogenic and therapeutic implications. Ann Rheum Dis 1992;51:919–925. 4 Asahara T, Masuda H, Takahashi T et al. Bone marrow origin of endothelial progenitor cells responsible for postnatal vasculogenesis in physiological and pathological neovascularization. Circ Res 1999;85:221–228. C AlphaMed Press 2015 V

resistin-elevated VEGF regulation. We found that resistin, via the downregulation of miR-206 targeting of VEGF 30 -UTR, functions as a critical angiogenic participant and a regulator of circulating EPCs. In addition, we performed in vivo Matrigel implants and CAM assays to investigate the effects of EPCs on recruitment of cytokines such as VEGF and angiogenesis in articular microenvironments. Our results suggest that resistin when present in high levels in synovial fluid from RA patients promotes EPCs migration and tube formation.

CONCLUSIONS In conclusion, our study is the first to identify the role of EPC homing to the synovium during RA angiogenesis and the signaling pathway involved in resistin-induced VEGF expression in primary EPCs. We found that resistin, which is highly expressed in patients with RA, causes EPC homing to the synovium and VEGF-dependent tube formation through the negative regulation of miR-206 via the PKC-d/AMPK signaling pathway. EPC infiltration into the synovium results in subsequent angiogenesis. Thus, resistin may be a novel therapeutic target in patients with RA. Our findings may open a new aspect on the nature of the signal transduction of progenitor cells and provide a better understanding of the mechanisms underlying RA pathogenesis.

ACKNOWLEDGMENTS This work was supported by grants from the Ministry of Science and Technology of Taiwan (103-2628-B-039-002-MY3, 102-2632-B-715-001-MY3, and 101-2314-B-039-002-MY3) and China Medical University, Taiwan (CMU 103-S-06).

AUTHOR CONTRIBUTIONS C.M.S., C.H.T.: substantial contributions to study conception and design and drafted the article or revised it critically for important intellectual content; C.J.H., C.H.T., C.Y.H., and S.W.W.: substantial contributions to acquisition of human data.

DISCLOSURE

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POTENTIAL CONFLICTS

OF INTEREST

The authors indicate no potential conflicts of interest.

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Resistin Promotes Angiogenesis in Endothelial Progenitor Cells Through Inhibition of MicroRNA206: Potential Implications for Rheumatoid Arthritis.

Endothelial progenitor cells (EPCs) promote angiogenesis and are therefore key contributors to a wide variety of angiogenesis-related autoimmune disea...
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