Mechanism of Fibroblast-Like Synoviocyte Apoptosis Induced by Recombinant Human Endostatin in Rats with Adjuvant Arthritis.код для вставкиСкачать
THE ANATOMICAL RECORD 291:1029–1037 (2008) Mechanism of Fibroblast-Like Synoviocyte Apoptosis Induced by Recombinant Human Endostatin in Rats with Adjuvant Arthritis XUE-YING HUANG,1,2 FEI-HU CHEN,1* JUN LI,1 LI-JUAN XIA,1 YONG-JING LIU,3 XIAO-MING ZHANG,2 AND FENG-LAI YUAN1 1 School of Pharmacy, Anhui Medical University, Hefei, China 2 Department of Anatomy, Anhui Medical University, Hefei, China 3 Department of Cardiothoracic Surgery, PLA 105 Hospital, Hefei, China ABSTRACT Rheumatoid arthritis (RA) is a chronic inﬂammatory disease characterized by pronounced synovial hyperplasia, in which there may be an imbalance between the growth and death of ﬁbroblast-like synoviocytes (FLS). The present study was undertaken to examine the effect of recombinant human endostatin (rhEndostatin) on FLS apoptosis in experimental RA. Adjuvant arthritis (AA) was induced in male Sprague Dawley (SD) rats. Using cultured AA FLS obtained from these rats, the apoptosis process was measured by terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) as well as Annexin V-ﬂuorescein isothiocyanate (FITC) and propidium iodide (PI) labeling methods. In addition, the expression levels of the Fas, c-jun, NFjB, and caspase-3 gene products in synovial tissues were quantiﬁed by quantitative real-time polymerase chain reaction (qPCR) and/or Western blotting assays. Our data revealed that rhEndostatin induced apoptosis in AA FLS. The number and signal density of TUNEL-positive cells were signiﬁcantly increased in rats treated with rhEndostatin (2.5 mg/kg). The percentage of Annexin V-FITC-positive cells was 6.67% after treatment with rhEndostatin at 25 mg/mL for 48 hr, compared with only 3.32% among untreated control cells. There were signiﬁcant increases in Fas mRNA, c-jun mRNA, c-Jun protein, and caspase-3 (p20) protein in AA synovial tissues treated with rhEndostatin (2.5 mg/kg), whereas no signiﬁcant difference in NFjB expression was detected between treated and untreated tissues. These ﬁndings indicate that rhEndostatin has a therapeutic effect on RA by inducing FLS apoptosis, which is strongly associated with increased expression of Fas, c-jun, and caspase-3, but not NFjB. Anat Rec, 291:1029–1037, 2008. Ó 2008 Wiley-Liss, Inc. Key words: rheumatoid arthritis; recombinant human endostatin; apoptosis; Fas; c-Jun; caspase-3; NFjB Rheumatoid arthritis (RA) is characterized by pronounced synovial hyperplasia composed of extensive proliferation of ﬁbroblast-like synoviocytes (FLS) and inﬂammatory cell inﬁltration with neovascularization. Pannus tissue comprising the hyperplastic synovium invades the surface of the articular cartilage and subchondral bone adjacent to the synovial cartilage junction, leading to deterioration of the joint function. In addition, the pannus tissue secretes various kinds of growth factors and inﬂammatory cytokines, which stimulate the growth of the pannus tissue itself. Thus, the synovial hyperplasia has a critical role in the propagation of rheumatoid synovitis (Padula et al., 1986). It is Ó 2008 WILEY-LISS, INC. Abbreviations used: SL 5 synovial lining; BV 5 blood vessel. Grant sponsor: National Science Foundation of China; Grant number: 30572196; Grant sponsor: Natural Science Foundation of the Department of Education, Anhui Province; Grant number; KJ2007B034; Grant sponsor: Postdoctoral Science Foundation of Anhui Province; Grant number: 07-08. *Correspondence to: Fei-hu Chen, School of Pharmacy, Anhui Medical University, No. 81 Meishan Road, Hefei 230032, China. Fax: 86-551-516-7735. E-mail: email@example.com Received 25 October 2007; Accepted 21 February 2008 DOI 10.1002/ar.20722 Published online 28 May 2008 in Wiley InterScience (www. interscience.wiley.com). 1030 HUANG ET AL. generally accepted that there is a close relationship between cell proliferation and cell death, and that there may be an imbalance between the growth and death of FLS in RA, which leads to synovial hyperplasia (Nishioka et al., 1998). Therefore, induction of apoptosis in RA FLS has been proposed as one of the promising strategies for treating RA by way of reducing synovial hyperplasia in situ. Adjuvant arthritis (AA) is a model of experimental arthritis that is induced by injection of complete Freund’s adjuvant (CFA). The similarities between the joint pathologies as well as the cellular and humoral immunities in AA and RA suggest that AA is a relevant animal model that acts as a useful test system for evaluating apoptosis-inducing therapies (Bendele et al., 1999). Endostatin, a 22-kDa fragment of collagen XVIII, is a member of a group of endogenous antiangiogenic proteins (O’Reilly et al., 1997) that are activated by proteolytic processing (Ferreras et al., 2000). In fact, the antiangiogenic activity of endostatin is speculated to be speciﬁcally mediated by the inhibition of endothelial cell adhesion, migration and proliferation, and induction of apoptosis (Dhanabal et al., 1999). Recently, endostatin has been studied not only for its inhibitory effects on vascular endothelial cell function but also its direct antitumor effects on cancer cell migration and proliferation, and induction of apoptosis (Wilson et al., 2003; Cui et al., 2007). Fibroblast-like synoviocytes are the ultimate target cells of the pathologic changes in arthritis (Hui et al., 1997), and endostatin was reported to induce regression of synovial proliferation in SCID mice grafted with human RA cells (Matsuno et al., 2002). Previous studies have shown that systemic administration of recombinant human endostatin (rhEndostatin) attenuates arthritis severity and blocks synovial thickening in AA rats by means of inhibition of angiogenesis and proinﬂammatory factors (Yue et al., 2004, 2007). These ﬁndings suggest that endostatin may be a potential therapeutic agent for RA. However, it remains unclear whether endostatin directly inﬂuences FLS functions such as apoptosis. The aim of the current study was to determine whether endostatin could induce FLS apoptosis in AA rats. The potential mechanisms are also discussed. MATERIALS AND METHODS Animals Male Sprague Dawley (SD) rats weighing 160–180 g were obtained from the Animal Center of Anhui Medical University (Hefei, China). The animals were housed under standard conditions at 228C with a 12-hr light/ 12-hr dark cycle, and given free access to food and water. The animals were acclimatized to the holding room for at least 7 days before the initiation of experiments. All animal care and experimental procedures were carried out in accordance with the Ethical Regulations for the Care and Use of Laboratory Animals of Anhui Medical University, which conform to the Guidelines for Laboratory Animals of the National Research Council of USA (1996). Reagents and Drugs The rhEndostatin used in the present study was kindly provided by the Anhui Sunning Institute of Bio- technology (Hefei, China). Bacille Calmette-Guerin (BCG) was purchased from the Biochemical Factory (Shanghai, China). RPMI 1640 medium was purchased from Gibco Company (Carlsbad, CA). Fetal calf serum (FCS) was purchased from Gibco BRL (Carlsbad, CA). Trypsin was purchased from Sigma Chemical Company (St. Louis, MO). An In Situ Cell Death Detection Kit was obtained from Roche Applied Science (Mannheim, Germany). Phycoerythrin (PE) Mouse Anti-Rat VCAM-1 Kit containing PE-conjugated mouse IgG (negative control) and Annexin V-FITC Apoptosis Detection Kit containing propidium iodide (PI) were purchased from Becton Dickinson Biosciences (PharMingen, San Diego, CA). TRIzol reagent was purchased from Gibco BRL. A RevertAidTM First Strand cDNA Synthesis Kit was obtained from Fermentas Life Sciences (Vilnius, Lithuania). A ShineSybr1 Real Time qPCR MasterMix Kit and a ShineProbe1 Real Time qPCR MasterMix Kit were obtained from ShineGene Molecular Bio-tech Co. Ltd. (Shanghai City, China). Sodium dodecylsulfate (SDS) and polyacrylamide gel electrophoresis (PAGE) reagents were obtained from Sigma Chemical Company. Mouse anti-NFjB p65, goat anti-caspase-3 p20, and rabbit antib-actin primary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). A rabbit anti-cJun primary antibody was purchased from Cell Signaling Technology (Beverly, MA). Horseradish peroxidase (HRP)-conjugated donkey anti-rabbit, anti-mouse and anti-goat secondary antibodies were obtained from Sigma Chemical Company. Induction of AA and Treatment Protocol Rats were immunized on day 0 by intradermal injection of a 0.1-mL aliquot of CFA containing 10 mg of heat-inactivated BCG/mL of parafﬁn oil into the left hind paw. Arthritis, as determined by the ﬁrst signs of redness or swelling of the ankle joints, was observed approximately 10 days after the immunization. Subcutaneous administrations of rhEndostatin (2.5 mg/kg/day) and methotrexate (MTX; 1 mg/kg twice weekly), a disease modifying anti-rheumatic drug (DMARD), to the abdomen were started on day 10 after immunization and continued until day 16 after immunization. These doses were given in the morning. A control group received the same volumes of phosphate-buffered saline (PBS). On day 26, the animals were killed under anesthesia with sodium pentobarbital (45 mg/kg intraperitoneally) and the knee joints were promptly removed for subsequent experiments. Histological Examination After killing on day 26, the knee joints were removed, trimmed, ﬁxed in 4% buffered paraformaldehyde, decalciﬁed, embedded in parafﬁn, sectioned at 5 mm and stained with hematoxylin and eosin (HE) for histopathological analysis. Preparation of AA FLS Fresh synovial tissues were obtained from AA rats. The synovium was minced, incubated in a plastic ﬂask and maintained in RPMI 1640 supplemented with 10 mmol/L HEPES (pH 7.2), 20% FCS, 2 mmol/L glutamine, 50 mmol/L mercaptoethanol, 100 kU/L penicillin ENDOSTATIN INDUCES AA FLS APOPTOSIS sodium and 100 mg/L streptomycin in a humidiﬁed 5% CO2-containing atmosphere at 378C for 7 days. After removal of the synovial pieces, the adherent cells were cultured in the same medium. At 70–80% conﬂuence, nonadherent cells were removed, and adherent cells were trypsinized, split at a 1:2 ratio and recultured in the same medium. The synoviocytes were used in experiments from passages 3. After three passages, most of the cultured synoviocytes comprised a homogeneous population of FLS. The cells were identiﬁed by their morphology and expression of vascular cell adhesion molecule-1 (VCAM-1). Terminal Deoxyribonucleotidyl Transferasemediated dUTP Nick-End Labeling (TUNEL) Analysis for Apoptosis Fibroblast-like synoviocytes obtained from AA rats treated with rhEndostatin (2.5 mg/kg) were seeded in six-well plates containing coverslips at 2 3 105 cells/well and incubated for 48 hr. Apoptotic cells were detected with an In Situ Cell Death Detection Kit according to the manufacturer’s instructions. Brieﬂy, the cells were ﬁxed with 4% paraformaldehyde for 1 hr at room temperature and permeabilized with 0.1% Triton X-100. After addition of the TUNEL reaction mixture, the cells were incubated in a humidiﬁed atmosphere for 1 hr at 378C in the dark. Negative and positive control reactions were performed for each experiment. For positive controls, the cells were incubated with DNase I (grade I; 3– 3,000 U/mL in 50 mM Tris-HCl pH 7.5, 1 mg/mL BSA) for 10 min at 15–258C to induce DNA strand breaks before labeling procedures. For negative controls, terminal transferase was omitted from the reaction mixture. All samples were directly analyzed under a ﬂuorescence microscope. Annexin V-FITC/PI Analysis for Apoptosis To distinguish early apoptosis from late apoptosis (secondary necrosis), the cells were simultaneously stained with FITC-conjugated annexin V and PI. The former reagent binds to phosphatidylserine on the surface of both early and late apoptotic cells, while the latter reagent stains cells that have lost their plasma membrane integrity, as is the case for late apoptotic cells. Fibroblast-like synoviocytes obtained from AA rats were incubated in RPMI 1640 containing rhEndostatin (25 mg/mL) for 48 hr, while control FLS were incubated in RPMI 1640 alone. Next, the cells were trypsinized and collected for detection of apoptosis with an Annexin V-FITC Apoptosis Detection Kit according to the manufacturer’s protocol. Brieﬂy, the cells were washed twice with cold PBS and resuspended in 500 mL of binding buffer (10 mM HEPES-NaOH pH 7.4, 140 mM NaCl, 2.5 mM CaCl2) at a concentration of 1 3 106 cells/mL. After addition of 5 ml of Annexin V-FITC solution and PI (1 mg/mL), the cells were incubated for 15 min at room temperature. The cells were analyzed with a ﬂow cytometer (Beckman Coulter, Fullerton, CA). Total RNA Extraction Total RNA was extracted from each frozen synovial tissue sample using the TRIzol reagent. Brieﬂy, the rats 1031 were randomly divided into normal, AA control and rhEndostatin (2.5 mg/kg)-treated groups. Five rats were taken from each group. The synovial tissues were dissected and rapidly frozen in liquid nitrogen. Aliquots (100 mg) of the tissues were homogenized and treated with 1 mL of TRIzol reagent. After addition of chloroform, the mixture was centrifuged to separate the RNA phase from the DNA phase. The RNA phase was subjected to RNA precipitation using isopropyl alcohol. The obtained RNA samples were rinsed with ethanol and dissolved in RNase-free water. Finally, the RNA samples were treated with RNase-free DNase I to remove any contaminating genomic DNA before reverse transcription. cDNA Synthesis from Total RNA cDNAs were synthesized with a RevertAidTM First Strand cDNA Synthesis Kit according to the manufacturer’s instructions. Brieﬂy, 6 mL of puriﬁed RNA was reverse-transcribed using 1 mL of oligo(dT)18 primer (500 mg/mL), 2 mL of 10 mM dNTP mix, 4 mL of 53 ﬁrststrand buffer, 1 mL of RiboLockTM Ribonuclease Inhibitor (20 kU/mL) and 1 mL of RevertAidTM M-MuLV Reverse Transcriptase (200 kU/mL) in a ﬁnal volume of 20 mL. The contents were incubated at 428C for 60 min before being heat-denatured at 708C for 10 min. Finally, the cDNAs were diluted 1:5 with DEPC-treated H2O and stored in 12-mL aliquots at 2708C. Quantitative Real-time Polymerase Chain Reaction (qPCR) The Fas and c-jun mRNA levels were quantiﬁed by real-time PCR in an FTC-2000 instrument (Funglyn Biotech Inc., Toronto, Canada) using SYBR Green I dye and TaqMan probe detection of ampliﬁcation products, respectively. The primers and probes were designed using the Primer Express software (PE Applied Biosystems, Foster City, CA) and are shown in Table 1. The threshold cycle (Ct) was determined for each sample using the GeneAmp 5700 Sequence Detection System (Applied Biosystems). Standard curves were generated by linear regression using Ct versus log10(copy number). The copy equivalent numbers for samples were calculated using these standard curves. Data were expressed as the ratio between the gene of interest copy equivalent and an endogenous housekeeping gene (b-actin or GAPDH) copy equivalent, yielding the relative expression. Ampliﬁcation of the target Fas cDNA was performed by SYBR Green I real-time PCR in a ﬁnal volume of 50 mL in thin-walled 0.2-mL tubes. The reaction mixture in each tube consisted of 25 mL of 23 PCR buffer, 0.6 mL of each forward and reverse primer (25 pmol/mL), 1 mL of template cDNA and 22.8 mL of DEPC-treated H2O. All tubes were sealed and centrifuged at 1,000 3 g for 30 s, before the mixtures were ampliﬁed under the following cycling conditions: initial activation of PowerQ1 Taq DNA polymerase for 4 min at 948C, followed by 40 cycles of denaturation for 15 s at 948C, annealing for 25 s at 608C and extension for 25 s at 728C. Negative controls consisted of the master mix plus water instead of the cDNA template (nontemplate control). Rat b-actin, an endogenous housekeeping gene, was used as an internal control for this method. The PCR products generated by 1032 HUANG ET AL. TABLE 1. Primers and probes used in real-time reverse transcriptase polymerase chain reaction Forward primer 50 –30 Genes Fas b-actin c-jun GAPDH c-jun probe GAPDH probe Reverse primer 50 –30 Product size TGGCTGTCCTGCCTCTGGT CGAACGCTCCTCTTCAACTCC CCCATCTATGAGGGTTACGC TTTAATGTCACGCACGATTTC GCAATGGGCACATCACCACTAC GTGACACTGGGCAGCGTATTCT TGGAGTCTACTGGCGTCTT TGTCATATTTCTCGTGGTTCA fam1 CGTGACCGACGAGCAGGAGGG 1tamra fam1 CTGAAGGGTGGGGCCAAAAG 1tamra qPCR were conﬁrmed by performing a melting curve analysis at the end of the ampliﬁcation by increasing the temperature from 728C at 0.28C/s and continuously measuring the ﬂuorescence. TaqMan real-time PCR was used to determine the relative expression levels of c-jun mRNA. The ﬁnal volume of 50 mL of PCR mix included 25 mL of 23 Hotstart FluoPCR mix, 0.8 mL of each forward and reverse primer (25 pmol/mL), 0.3 mL of TaqMan probe (25 pmol/mL), 1 mL of template cDNA and 22.1 mL of DEPC-treated H2O. The thermal cycling conditions were 4 min at 948C to allow activation of PowerQ1 Taq DNA polymerase, followed by 40 cycles of 30 s at 948C and 30 s at 608C. Negative controls consisted of the master mix plus water instead of the cDNA template (nontemplate control). Relative c-jun mRNA expression levels were calculated according to the mRNA expression of the housekeeping gene GAPDH. 100bp 150bp 142bp 138bp Differences between groups were deemed signiﬁcant at P < 0.05. RESULTS Induction of the Rat AA Model Adjuvant-induced arthritis developed in rats immunized with CFA, and clinical signs (periarticular redness and swelling) of arthritis appeared at 100% incidence around day 10 after the immunization. The inﬂammatory response in the ankles reached its peak by day 17, and there was signiﬁcant joint pathology by day 24 (Fig. 1b) in comparison with normal control ankles (Fig. 1a). Among the variety of pathological features, the most distinctive characteristics of the AA model were hyperplasia/hypertrophy of FLS accompanied by massive inﬁltration of inﬂammatory cells and neovascularization (Fig. 1d) relative to normal synovial tissues (Fig. 1c). Western Blot Analysis Detection of NFjB, c-Jun, and caspase-3 proteins in synovial tissues was performed by Western blot analysis. The rats were randomly divided into normal, AA control, rhEndostatin (2.5 mg/kg)- treated and MTX (1 mg/kg) -treated groups. Five rats were taken from each group. The tissues were dissected and rapidly frozen in liquid nitrogen. Aliquots (100 mg) of the tissues were homogenized and treated with 400 mL of lysis buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.02% NaN3, 0.1% SDS, 1% NP-40, 0.5% sodium deoxycholate, 1 mg/mL aprotinin, 100 mg/mL PMSF). The protein concentrations of the tissues were determined by absorbance readings at 280 nm on a UV spectrophotometer (Bio-Rad, Hercules, CA). Aliquots of the synovial tissue samples containing 30 mg of protein were resolved by SDS-PAGE using a 10% gel and transferred to a nitrocellulose membrane (Whatman, Kent, England) by electroblotting. The membranes were blocked with Tris-buffered saline containing 0.1% Tween 20 (TBST) and 5% nonfat milk for 1 hr at room temperature, followed by incubation at 48C overnight with one of the following Abs: mouse anti-NFjB (1:200); rabbit antic-Jun (1:200); goat anti-caspase-3 (1:200); and rabbit anti-b-actin (1:200). After three washes with TBST, the membranes were incubated for 1 hr with HRP-conjugated donkey anti-rabbit, anti-mouse or anti-goat secondary antibodies (1:1,000). Antigen-antibody interactions were visualized with chemiluminescence (Pierce, Rockford, IL) using Kodak X-AR ﬁlm (Eastman Kodak, Rochester, NY). Statistical Analysis Data were expressed as means 6 standard deviation (SD) and analyzed by one-way analysis of variance. Identiﬁcation of FLS Fibroblast-like synoviocytes were isolated from knee joints of the AA rats and cultured under permissive conditions. The cells were morphologically homogeneous and exhibited the typical appearance of FLS with a bipolar conﬁguration under inverse microscopy (after more than three passages; Fig. 2a). The isolated FLS most likely corresponded to the intimal subpopulation of FLS, because they all expressed VCAM-1 as evaluated by ﬂow cytometry (FCM) analysis (94.2% VCAM-1-positive cells; Fig. 2b). In contrast, phycoerythrin (PE) -conjugated mouse IgG was positive only in 2.63% of the cultured synoviocytes (Fig. 2c). Effect of rhEndostatin on FLS Apoptosis The effect of rhEndostatin on FLS apoptosis was examined using the TUNEL assay method. The number and signal density of TUNEL-positive cells were signiﬁcantly increased in rats treated with rhEndostatin (2.5 mg/kg; Fig. 3c). There were no TUNEL-positive cells in negative control rats (Fig. 3a), while all the positive control rats exhibited TUNEL-positive cells (Fig. 3b). To further conﬁrm that rhEndostatin induced apoptosis in AA FLS, the cells were treated with rhEndostatin and detected by FCM. As shown in Figure 3e, rhEndostatin did indeed induce apoptosis in AA FLS. Speciﬁcally, the percentage of Annexin-positive cells was 6.67% after treatment with rhEndostatin (25 mg/mL) for 48 hr, compared with only 3.32% among untreated control cells (Fig. 3d). 1033 ENDOSTATIN INDUCES AA FLS APOPTOSIS Fig. 1. Induction of a rat adjuvant-induced arthritis model as evaluated by the appearance and histological features of joints from normal rats and rats immunized with CFA. a: Foot joints of a normal rat. b: Foot joints of an adjuvant-induced arthritis model rat. The disease in this model is a migratory polyarthritis primarily affecting the tarsal, metatarsal and interphalangeal joints. After immunization with CFA, the ankle joints of AA rats swell markedly over a period of 17 days. c,d: Synovial histology of rat knee joints. Synovial tissue sections were stained with hematoxylin and eosin (HE). The AA lining (d) shows redundant folds of the synovial lining, intense inﬁltration of inﬂammatory cells and enhanced angiogenesis. Furthermore, the AA intimal lining layer (d) is hyperplastic, with ten or more layers of cells, compared with a normal lining (c), which is one or two cell layers deep. Scale bar 5 50 mm. Fig. 2. Identiﬁcation of FLS by their morphology and expression of VCAM-1. a: After three passages, most of the cultured synoviocytes are in a ﬁbrocyte-like form with a long fusiform shape and grow in the same direction. b: VCAM-1 is expressed by more than 94% of iso- lated AA FLS, as detected by FCM analysis. c: Phycoerythrin (PE) conjugated mouse IgG is positive only in 2.63% of the cultured synoviocytes. PE-IgG, phycoerythrin (PE) -conjugated mouse IgG; SS, side scatter. Scale bar 5 100 mm. Effects of rhEndostatin on Expression of Apoptotic Molecules in FLS also analyzed by Western blotting. Recombinant human endostatin treatment induced partial caspase-3 activation, as indicated by the signiﬁcantly higher expression of the 20-kDa cleavage product (p20) of caspase-3 compared with normal, AA and MTX-treated samples (P < 0.01; Fig. 5b). However, in three independent experiments, NFjB protein bands were observed in both AA and rhEndostatin-treated groups (Fig. 5a). The difference between these bands was not signiﬁcant (Fig. 5b). To determine whether expression of Fas and c-jun in FLS is due to pretranslational events, we performed real-time PCR to quantify their mRNA levels in synovial tissues. As shown in Figure 4c,d, the Fas and c-jun mRNA expression levels were signiﬁcantly increased by rhEndostatin, compared with the levels in AA and normal control tissues (P < 0.01). The 48-kDa (p48) and 43-kDa (p43) protein bands, representing the subunits of c-Jun, were detected by Western blot analysis (Fig. 5a). Densitometric analysis indicated the intensities of both the p48 and p43 protein bands in synovial tissues treated with rhEndostatin were signiﬁcantly higher than those in normal, and AA and MTX-treated tissues, respectively (P < 0.01; Fig. 5b). The expression of levels of NFjB and caspase-3 proteins in synovial tissues were DISCUSSION Recent investigations have indicated that RA should no longer be considered a benign disease. A considerable amount of data suggests that this disease is associated with diminished long-term survival (Finesilver, 2003). Trials of different treatment strategies in animal models of RA have shown that endostatin has therapeutic 1034 HUANG ET AL. Fig. 3. Effect of rhEndostatin on FLS apoptosis. a–c: TUNEL analysis of rhEndostatin-induced apoptosis. a: Negative control. Terminal transferase was omitted from the reaction mixture. None of the negative control cells show positive signals. b: Positive control. Cells were permeabilized and incubated with DNase I (grade I) for 10 min at 15– 258C to induce DNA strand breaks. All the positive controls exhibit positive signals. c: rhEndostatin (2.5 mg/kg)-induced apoptosis of AA FLS. d,e: Annexin V-FITC/PI analysis for apoptosis. The percentage of Annexin V-FITC-positive cells is 6.67% after treatment with rhEndostatin (25 mg/mL) for 48 hr (e), compared with only 3.32% among untreated control cells (d). Scale bar 5 15 mm. effects on RA by means of inhibition of angiogenesis and proinﬂammatory factors (Kurosaka et al., 2003; Yue et al., 2007). However, FLS are the ultimate target cells of the pathologic changes of arthritis (Hui et al., 1997), and there may be an imbalance between the growth and death of FLS in RA, which leads to synovial hyperplasia (Nishioka et al., 1998). Although abnormal proliferation and/or persistence of FLS has long been described in inﬂammatory arthritic conditions, substantial attention has only relatively recently been drawn to the relevance of abnormal apoptotic processes in disease pathogenesis and treatment. A study by Pope (2002) revealed that insufﬁcient apoptosis represents at least one fundamental underlying process in RA. However, the issue of whether endostatin can directly induce FLS apoptosis in RA remains unclear. In the present study, AA was induced in SD rats to imitate the clinical scenario of RA and the effects of rhEndostatin on FLS apoptosis were assessed in this experimental model. Although animal models of arthritis only approximate RA, AA in rats is among the most commonly used animal models for RA. These rats provide a useful test system for evaluating apoptosis-inducing therapies (Klareskog et al., 1995; Oliver and Brahn, 1996). Our ﬁndings are the ﬁrst to reveal that rhEndostatin can induce apoptosis of FLS in AA. These results suggest that rhEndostatin may play a key regulatory role in the inhibition of rheumatoid synovial hyperplasia in vivo by means of induction of FLS apoptosis. Apoptosis can be initiated through death receptor- or mitochondria-dependent pathways. However, the detailed intracellular mechanism of apoptosis in FLS is ENDOSTATIN INDUCES AA FLS APOPTOSIS 1035 Fig. 4. Quantiﬁcation of Fas and c-jun mRNA levels using an FTC2000 PCR instrument. a,b: Standard curves. a: Standard curve for quantiﬁcation of target Fas cDNA detected by qPCR using SYBR Green I dye. r 5 0.99973; slope 5 23.3872; intercept 5 46.5234. b: Standard curve for quantiﬁcation of target c-jun cDNA detected by qPCR using the TaqMan probe. r 5 0.99938; slope 5 23.3672; intercept 5 46.4281. c,d: Relative expression levels of Fas and c-jun mRNAs. The target Fas and c-jun cDNA copy numbers were quantiﬁed by qPCR using the standard curves shown in panels (a) and (b). The results are expressed as the fold increases of Fas and c-jun mRNAs relative to b-actin and GAPDH mRNAs, respectively. Data are presented as the mean 6 SD of three independent experiments. **P < 0.01 vs. the normal group. ##P < 0.01 vs. the AA control group. still unknown. The death receptor-dependent pathway has received the most attention in inﬂammatory arthritis, particularly RA (Peng, 2006). The major death receptors include Fas and TNFR. Our novel observation that rhEndostatin increases Fas mRNA expression in AA synovial tissues suggests that Fas is up-regulated at the transcriptional level in AA. In a previous study on RA synovial tissues, expression of Fas protein was detected in sub-lining layers and the majority of Fas-expressing cells were FLS (Chou et al., 2001). Thus, the enhanced expression of Fas in AA synovial tissues treated with rhEndostatin in vivo suggests that rhEndostatin may function to induce Fas-mediated apoptosis in FLS. Early studies on Fas-mediated apoptosis suggested that caspase-3 is only required for the execution of death signals triggered by caspase-8 at the site of initiation (Liu and Pope, 2003; Itoh et al., 2004). Consistent with the previous study (Liu and Pope, 2003; Itoh et al., 2004), our present results conﬁrmed a signiﬁcantly higher expression level of the 20-kDa cleavage product (p20) of caspase-3 in rhEndostatin-treated rats compared with AA control rats. These results indicate that rhEndostatininduced apoptosis in AA FLS is strongly associated with the expression of Fas and activation of caspase-3 (p20). In addition, caspase-3 plays key regulatory roles in TNFR-mediated apoptosis (Liu and Pope, 2003). Therefore, in the present study, we cannot conclude that rhEndostatin only speciﬁcally induces Fas expression in AA FLS, since no data for TNFR were obtained. c-Jun, a prominent member of the AP-1 transcriptional factor family, has been implicated in the regulation of a wide range of biological processes including apoptosis, which it can promote or counteract depending on the tissue, the developmental stage and the nature of the death stimulus (Leppa and Bohmann, 1999; Shaulian and Karin, 2002). Its transcriptional activities are regulated by changes in the level of c-jun expression as well as posttranslational modiﬁcations of the c-Jun protein. An early report revealed that increased c-Jun activity is sufﬁcient to trigger apoptotic cell death in NIH 3T3 ﬁbroblasts (Bossy-Wetzel et al., 1997). Another study by Kolbus et al. (2000) revealed that ﬁbroblasts with a targeted null mutation in c-jun exhibit a defect in methyl methanesulfonate-induced apoptosis. c-Jun is highly activated in RA FLS and synovium and regulated at both the transcriptional and posttranslational levels (Boyle et al., 1997; Han et al., 2001). Here, we found that c-jun mRNA and c-Jun protein were overexpressed in AA synovium treated with rhEndostatin compared with AA control synovium. These data, together with the previous ﬁnding described above, strongly suggest that overexpression of c-Jun contributes to rhEndostatin-induced apoptosis in AA FLS. However, direct links between c-Jun activity and the induction of speciﬁc initiators or executors of apoptosis have not yet been identiﬁed by functional means. Early reports revealed that NF-jB is abundant in rheumatoid synovium and plays diverse roles in the 1036 HUANG ET AL. in contrast to c-jun mRNA and c-Jun protein. Taken together, these observations suggest that the p65 subunit of NF-jB is not responsible for rhEndostatin-induced apoptosis in AA FLS. In summary, increased proliferation and/or insufﬁcient apoptosis may contribute to the increased numbers of FLS in RA joints. The invasive front of the synovium forms a pannus that invades the cartilage of affected joints. Therefore, a new therapeutic viewpoint for RA is to stress the importance of further research regarding the intracellular mechanism of FLS apoptosis. In the present study, we have demonstrated that rhEndostatin is sufﬁcient to induce apoptosis in AA FLS. Although we cannot exclude the involvement of molecules other than Fas, caspase-3, c-Jun, and NF-jB, our results reveal that Fas, caspase-3, and c-Jun play important roles in rhEndostatin- induced cell death among AA FLS. These results indicate that rhEndostatin may play a key regulatory role in the inhibition of rheumatoid synovial hyperplasia in vivo by means of induction of FLS apoptosis, thereby suggesting that rhEndostatin should be considered as a therapeutic agent for RA. In addition, our results reveal that therapeutic strategies targeting Fas, caspase-3 and c-Jun may be effective in diseases such as RA. ACKNOWLEDGMENTS The authors thank Dr. Hao Li (The First Afﬁliated Hospital, Anhui Medical University, Hefei, China) for assistance with the histopathology analysis. The authors also thank Dr. Lei Zhang and Cheng-mu Hu (School of Pharmacy, Anhui Medical University, Hefei, China) for help with the induction of the experimental model of rheumatoid arthritis. Fig. 5. Expression levels of c-Jun, caspase-3 and NFjB proteins. Total protein was extracted from synovial tissue lysates of the indicated groups and subjected to Western blot analysis. a: Western blot analysis of the levels of c-Jun (p48), c-Jun (p43), caspase-3 (p20), and NFjB proteins in synovial tissues. Expression of b-actin is included as a control for protein loading. This image is representative of three experiments. b: Histogram representing the relative expression levels of the indicated proteins analyzed in panel (a). The results are expressed as the fold increases in c-Jun, caspase-3 and NFjB proteins relative to b-actin protein. Data are presented as the mean 6 SD of three independent experiments. **P < 0.01 versus the rhEndostatintreated group. initiation and perpetuation of RA (Han et al., 1998; Sioud et al., 1998; Chen et al., 1999; Campbell et al., 2000). The main activated form of NF-jB is a heterodimer of the p65 subunit and either a p50 or p52 subunit, and immunohistochemical analyses demonstrated that p50 and p65 NF-jB proteins are present in the nuclei of cells in the synovial intimal lining. Activated NF-jB is a common feature in human RA synovium and various animal models of RA such as AA in rats. Previous studies have shown that NF-jB activation increases the expression levels of inﬂammatory molecules in FLS and protects cells against TNF-a and Fas ligand-induced apoptosis. In vivo suppression of NF-jB enhanced apoptosis in the synovium of rats with experimentallyinduced arthritis (Chen et al., 1999; Campbell et al., 2000). In the present study, we found that the p65 subunit of NF-jB was only weakly induced by rhEndostatin LITERATURE CITED Bendele A, McComb J, Gould T, McAbee T, Sennello G, Chlipala E, Guy M. 1999. Animal models of arthritis: relevance to human disease. Toxicol Pathol 27:134–142. 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