Cell Tissue Res DOI 10.1007/s00441-015-2194-8
REGULAR ARTICLE
Adenovirus-mediated osteoprotegerin ameliorates cartilage destruction by inhibiting proteoglycan loss and chondrocyte apoptosis in rats with collagen-induced arthritis Zhi-yun Feng 1 & Zhen-nian He 2 & Bin Zhang 2 & Yi-qiao Li 3 & Jian Guo 2 & Yuan-lin Xu 2 & Ming-yuan Han 2 & Zhong Chen 1
Received: 4 October 2014 / Accepted: 9 April 2015 # Springer-Verlag Berlin Heidelberg 2015
Abstract Our aim is to eluc id ate the effects of osteoproteogerin (OPG) on cartilage destruction in rats as a model of collagen-induced arthritis (CIA). To establish the CIA model, Sprague Dawley rats were injected with bovine type II collagen solution subcutaneously via the tails. Adenovirus-mediated OPG (Ad-OPG) was then injected intra-articularly either at the beginning of CIA (early OPG treatment) or one week after CIA establishment (late OPG treatment); vehicle or Ad-green fluorescent protein were injected as controls. The rats were killed 4 weeks after treatment. Ankle-joint sections were obtained for histology. Serum samples were collected for enzyme-linked immunosorbent assay. Safranin O staining showed that proteoglycan loss was inhibited in the early and late Ad-OPG groups. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining revealed that both early and late Ad-OPG
treatments significantly prevented chondrocyte apoptosis in CIA rats. Furthermore, disintegrin and metalloproteinase with thrombospondin motif−5 expression decreased remarkably in the early and late OPG treatment groups. However, the cartilage destruction score, cartilage oligomeric matrix protein level and caspase-3 expression were only decreased in the early Ad-OPG treatment group. Additionally, ankle-joint swelling and the interleukin-1β expression level in CIA rats were not notably altered by Ad-OPG treatment. Taken together, our results suggest that early Ad-OPG treatment has potent protective effects against cartilage destruction during rheumatoid arthritis progression, mainly by reducing proteoglycan loss and chondrocyte apoptosis.
Zhi-yun Feng and Zhen-nian He contributed equally to this work.
Introduction
Electronic supplementary material The online version of this article (doi:10.1007/s00441-015-2194-8) contains supplementary material, which is available to authorized users. * Zhong Chen [email protected] 1
Spine Lab, Department of Orthopedic Surgery, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, People’s Republic of China 310003
2
Department of Orthopedics, Beilun People’s Hospital, No. 1288, Lu shan Road, Ningbo, Zhejiang Province, People’s Republic of China 315806
3
Department of Laboratory Center, Beilun People’s Hospital, No. 1288, Lu shan Road, Ningbo, Zhejiang Province, People’s Republic of China 315806
Keywords Osteoprotegerin . Cartilage destruction . Proteoglycan loss . Chondrocyte apoptosis . Collagen-induced arthritis . Rat (Sprague Dawley)
Rheumatoid arthritis (RA) is a chronic autoimmune disease mainly affecting the synovial joints. It is characterized by synovial inflammation and progressive erosion of bone and cartilage (Gough et al. 1994; Schett et al. 2003; Weissmann 2006). Although synovial hyperplasia leads to joint swelling, it is the bone and cartilage destruction that ultimately leads to work disability (Wolfe et al. 2000) and even life-long physical disability (Welsing et al. 2001). Therefore, we need to explore effective approaches to inhibit joint destruction. Since most therapeutic strategies have been designed to inhibit the inflammation of the synovium of joints, less effort has been made to prevent the destruction of cartilage itself. Generally, cartilage erosion in RA has been considered secondary to inflammatory pannus, which is attached to cartilage (Scott et al. 2003).
Cell Tissue Res
However, no effective therapy exists to suppress both synovium inflammation and joint destruction. Moreover, only a weak correlation has been determined between advancing synovitis and erosive progression in the hand joints of RA patients (Kirwan 1997). Furthermore, the pathophysiological causes of synovitis and cartilage erosions are not always the same (Kim and Song 1999; Kirwan 1997). Thus, other treatments are called for, based on innovative mechanisms of action. As an alternative, we should focus on the inherent mechanisms of cartilage destruction in RA, such as proteoglycan loss and cell apoptosis. Kim and Song (1999) found that the apoptosis of chondrocytes in RA joints is more prominent than those without RA. In addition, they reported that the expression of caspase-3 increases in cartilage of RA patients (Kim and Song 1999), as is the case in animal models (Campo et al. 2008). Proteoglycan is a main component in cartilage and the loss of proteoglycan has been considered as the initiation point of cartilage degradation. Previous reports have shown the remarkable loss of proteoglycan in a collagen-induced arthritis (CIA) model, as assessed by Safranin O staining (Bendele 2001; Xie et al. 2013). During proteoglycan loss, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) -4 and −5 are the primary degrading enzymes in cartilage (Plaas et al. 2007). The corresponding inhibitor targeting ADAMTS-5 has been proposed, with hopes that it can be entered into clinical trials (Gilbert et al. 2008). Osteoprotegerin (OPG) was initially found to prevent bone resorption by blocking osteoclast differentiation and activation (Boyce and Xing 2007). Subsequently, several studies have confirmed that OPG can protect bone against inflammation in various RA model animals (Kong et al. 1999; Romas et al. 2002; Zwerina et al. 2004; Saidenberg-Kermanac'h et al. 2004). To date, research regarding the effects of OPG on cartilage protection in an adjuvant-induced arthritis (AIA) model is still limited (Kong et al. 1999). However, this model has disproportionally mild cartilage destruction relative to evident cartilage damage in the CIA model (Bendele 2001). Recent reports have shown that OPG is able to prevent cartilage destruction in an osteoarthritis (OA) model in mice (Shimizu et al. 2007; Kadri et al. 2008), thus leading to hopes that this protein has potential to attenuate cartilage destruction in RA. Multiple and repeated injections, high cost and a short half life are the main problems concerning the use of biological agents for arthritis therapy. However, gene therapy employs a target gene in a virus vector and thereby endows tissue with the long-term expression of a target gene, thus avoiding the aforementioned disadvantages. Adenovirus has been tested and demonstrated to be a safe vector for gene therapy in animal models (Bolon et al. 2001). Another problem is that pathological cartilage damage might occur before visualization by radiology (Quinn et al. 2001; Fuchs et al. 1989). In order to reduce such damage, previous studies have revealed that
earlier disease modifying anti-rheumatic drug therapy has better efficacies (Anderson et al. 2000; Keystone et al. 2003; Finckh et al. 2006). In this study, we examine the effects of OPG on cartilage destruction in CIA rats and explore the potential mechanisms by over-expressing OPG in the affected joints by using an adenoviral-mediated OPG (Ad-OPG) gene therapy system. We also injected Ad-OPG injections into a group of animals 1 week later to examine whether the treatment given during the middle-late stage of CIA might also be protective.
Materials and methods Generation of recombinant adenoviral vector expressing human OPG Recombinant adenovirus carrying cDNA of human OPG was generated by using Admaxsystem (Micro-bix Bio-systems, Ontario, Canada). All experiments were performed following standard instructions. Briefly, the human OPG gene was subcloned into adenoviral type V, BAGlox△E1, 3 Cre vector (Micro-bix Bio-systems) in 293 cells (ATCC, Va., USA) by homologous recombination. An Ad-GFP (green fluorescent protein) expression cassette was used as control. Amplification of the recombinant adenovirus was performed in HEK 293 cells (ATCC). We obtained high titer viral stocks by caesium-chloride density-gradient centrifugation. Tran-EZ (Sun-bio Medical Bio-technology, Shanghai, China) was used to determine the titer of recombinant adenovirus: 1.2×1010 plaque forming units (pfu)/ml in this study. In addition, the expression of the OPG gene delivered by the adenovirus vector was identified by Western blot. The OPG expression in Ad-OPG infected chondrocytes increased significantly when compared with those infected by Ad-GFP (Fig. S1).
Experimental animals Eight-week-old, male, Sprague Dawley rats (The Center of Experimental Animals, Zhejiang University, Hangzhou, China) were kept under standardized conditions with a 12hour light–dark cycle and appropriate temperature and humidity. These rats were maintained under special pathogen-free conditions and had free access to sterilized water and pellet food. All experiments were performed following the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and were pre-approved by the Review Board of the First Affiliated Hospital of Medical College of Zhejiang University.
Cell Tissue Res
Induction of CIA model CIA was induced by immunization with bovine type II collagen (CII; Chondrex, Redmond, Wash., USA). Dissolved CII (4 mg/ml, solubilized in 0.05 M acetic acid) was emulsified with an equal volume of incomplete Freund’s adjuvant by using an electric homogenizer (IKA, Works Guangzhou, China). The rats were then immunized with 0.1 ml emulsion subcutaneously into the tail base at day 0 and day 7. Seven rats were injected with a vehicle at the same dose and frequency into the same site as normal controls for the CIA rats. We recorded the body weight, diameter of ankle and arthritis score every 3–4 days to monitor arthritic induction. Treatment Arthritis was induced successfully in 37 rats, which showed swelling and redness in both hind legs. The rats were randomly separated into four treatment groups: early OPG treatment group (Ad-OPG-injected at the onset of arthritis, n=10), late OPG treatment group (Ad-OPG-injected 1 week after onset of arthritis, n=7), GFP group (Ad-GFP-injected at the onset of arthritis, n=13) and CIA control group (vehicle-injected at the onset of arthritis, n=7). Each side of the ankle joints was single-injected with 30μl Ad-OPG (containing 3.6×108 pfu adenovirus), Ad-GFP, or a vehicle into the joint cavity. We recorded body weight, diameter of ankle and arthritis score every 3–4 days during treatment. All the animals were killed 4 weeks later. During the experiment, some rats in the GFP group were killed at 2, 7, 14 and 21 days after the GFP injection to examine the location and expression of GFP in the joints by fluorescence microscopy. Two rats in the early OPG group were killed at 2 and 14 days after Ad-OPG injection to show the OPG expression pattern in cartilage.
joints were harvested for histological examination. After removal of the surrounding soft tissue, the joints were fixed in 4 % paraformaldehyde and decalcified in 10 % EDTA for about 2 weeks. Following dehydration and paraffin embedding, serial 5-μm-thick sagittal sections were obtained. First, hematoxylin and eosin (HE) staining was carried out to assess the infiltration of inflammatory cells and bone damage. Second, sections were stained with 0.02 % Fast Green for 10 min and then 0.1 % safranin O for 7 min. A semiquantitative scoring system for proteoglycan loss was employed (Dudler et al. 2000) in which the scoring was as follows: 0, no loss; 1, mild loss; 2, severe loss; 3, complete loss of staining for proteoglycan. Third, the severity of cartilage destruction within the ankle joints was evaluated according to a cartilage destruction score as described by Romas et al. (2002), with minor modification: 0, normal; 1, minimal (loss of safranin O staining only); 2, mild (loss of safranin O staining and mild cartilage thinning and fissuring); 3, moderate (moderate cartilage destruction with 1–3 sites of minor-focal depth reaching the middle zone); 4, marked (marked cartilage destruction with more than three sites of minor-focal depth reaching the deep zone); and 5, severe (severe cartilage destruction with macro-focal cartilage destruction reaching the tidemark). ELISA analysis of COMP and IL-1β in serum The serum concentration of cartilage oligomeric matrix protein (COMP) and interleukin-1β (IL-1β) were determined by ELISA kits (Biorbyt, Montgomery Street, Calif., USA). All samples were performed in triplicate. Immunohistochemical analysis
Assessment of inflammation and cartilage destruction in arthritis Joint swelling was determined by measurement of the ankle diameter with a precision caliper (Stolina et al. 2005). Briefly, we first located the ankle joint and marked it with picric acid to ensure that we measured the same site every time. Second, we put the ankle joint into the slot of the caliper and adjusted the outside jaws of the caliper in order to bring the joint into the slot between the two outside jaws. The values presented in the build-in electronic screen were recorded. Both ankle joints were measured and the mean was calculated. Kappa statistics demonstrated that the agreement and the reproducibility were good (0.84). The arthritis scores were measured by the inflammation of the paws, as described previously (Jin et al. 2010). Rats were killed by the injection of pentobarbital intra-peritoneal. Blood samples from the inferior vena cava were collected for enzyme-linked immunosorbent assay (ELISA). Ankle
Dewaxed and dehydrated sections were first washed in phosphate buffer solution (PBS) and then incubated with 3 % peroxyl in methanol for 15 min for endogenous peroxidases blocking. After being washed in PBS, sections were then immersed in boiled citrate buffer solution for 10 min for antigen retrieval. Following a blocking step with 5 % bovine serum albumin for 20 min, sections were then covered with anti-rat caspase-3 antibody (1:200, Santa Cruz biotechnology, Texas, USA), anti-rat ADAMTS-5 antibody (1:150, Santa Cruz biotechnology), anti-rat OPG antibody (1:100, GeneTex, USA), or anti-rat TRAIL antibody (1:300, Biorbyt) overnight at 4 °C in a humidified chamber. On the following day, sections were incubated with a bio-tinylated anti-rabbit antibody (Boster Biotechnology, Wuhan, China) for 30 min at room temperature and then coupled with diaminobenzidine to visualize the positive expression of the relevant targeted proteins. After being counterstained with hematoxylin, all sections were
Cell Tissue Res
dehydrated in an ascending ethanol series and then mounted in neutral resins. An Olympus microscope, with a computer-aided imagecollection system (NIS-Elements F2.30, Nikon, Japan) was utilized to examine the sections. Positive cells of the targeted protein were counted at magnification ×400 in three fields of each slide. The positive cells number was normalized to the cell number per 100 total cells. TUNEL analysis TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling) assay was performed by using the in situ cell death detection POD kit (Roche, Germany) according to the manufacturer’s protocol. Briefly, after being permeabilized with 20 μg/ml proteinase K (Meck & Millpore, USA) for 1 h at 37 °C, the sections were washed with PBS and then incubated with TUNEL reaction mixture for 60 min at 37 °C in a dark and humid atmosphere. Signals in the sections were then transferred by incubating with POD reactions. As a positive control, we treated sections with DNase I at room temperature for 10 min prior to the labeling procedures. The terminal transferase was not added to the mixture in the negative controls. All sections were counterstained with hematoxylin. These sections were assessed by the same methods as for the immunohistochemical sections. Statistical analysis Data are presented as the means±standard errors and were analyzed with the statistical package from GraphPad Prism (version 5.0, GraphPad Software, San Diego, Calif., USA). Intergroup comparisons were subjected to one-way analysis of variance (ANOVA). The unpaired t-test was used for comparisons of difference in positive cell numbers between the treatment group and control group. P-values less than 0.05 were considered as showing statistical significance.
Results Establishment of CIA model and effects of Ad-OPG on inflammatory conditions Thirty-seven rats developed arthritis by 12–14 days after the first immunization. Redness and swelling were first seen in the small joints of the hind extremities, followed by the bilateral ankle joints. Compared with the normal control (Fig. 1a), the swelling of the ankle joints was evident in CIA rats (Fig.1b). Any rats not developing bilateral ankle-joint swelling were excluded. During the course of treatment, the arthritis had a parallel progression among rats treated with a vehicle (Fig. 1b), Ad-
GFP (Fig. 1c) and Ad-OPG at the early phase (Fig. 1d) or late phase (Fig. 1e), as the increases of diameter of ankle joints were comparable among all treatment groups at days 14, 21, 28, 35 and 42 (Fig. 1f, P>0.05). In addition, as a key factor in triggering CIA, the IL-1β serum level was not decreased significantly by Ad-OPG treatment delivered at either the early or later phase of disease (Fig. 1g, P>0.05). Moreover, HE staining revealed that the inflammatory cell infiltration was remarkable around the affected joints in the vehicle group (Fig. 1i, n) and GFP group (Fig. 1j, o), whereas it was not attenuated in rats treated with Ad-OPG at either the early phase (Fig. 1k, p) or the later phase (Fig. 1l, q). Efficacy of adenovirus infection of cartilage tissue We randomly killed one rat in the GFP group at 2, 7, 14, 21 and 28 days after Ad-GFP treatment. Frozen sections were obtained and immediately observed by fluorescence microscopy. As shown, the expression of GFP could be observed after 2 days (Fig. 2a), became stronger after 7 days (Fig. 2b) and was prominent after 14 days (Fig. 2c). GFP expression could also be seen during the following few days (Fig. 2d, e). These data revealed that the adenovirus-mediated gene exhibited remarkable and sustained expression in cartilage following injection into the articular cavity. Immunohistochemical analysis showed that OPG had a similar expression trend as GFP (Fig. 2f–h). Effect of Ad-OPG on proteoglycan loss and cartilage destruction In comparison with the excellent safrarin O staining in the normal controls (Fig. 3a), CIA rats treated with vehicle (Fig. 3b) or Ad-GFP (Fig. 3c) showed significant proteoglycan loss, as revealed by weakened and uneven safrarin O staining. However, proteoglycan loss was greatly inhibited by both early (Fig. 3d) and late OPG (Fig. 3e) treatment. The proteoglycan loss score was significantly lower in the early OPG and late OPG groups (Fig. 3f, P 0.05, f). The IL-1β (interleukin-1β) serum level was not significantly changed by Ad-OPG treatment (P>0.05, g). HE staining showed remarkable inflammation,
such as inflammatory cell infiltration and synovial hyperplasia in the vehicle group (i, n) and GFP group (j, o). However, the inflammation status was not attenuated by the treatment of OPG at either the early (l, q) or the later (k, p) phase. Boxed areas in h–l (magnification: ×100) are shown at higher magnification in m–q (magnification: ×400), respectively. Values are means ± standard errors. *P < 0.05, **P < 0.001, ***P
REGULAR ARTICLE
Adenovirus-mediated osteoprotegerin ameliorates cartilage destruction by inhibiting proteoglycan loss and chondrocyte apoptosis in rats with collagen-induced arthritis Zhi-yun Feng 1 & Zhen-nian He 2 & Bin Zhang 2 & Yi-qiao Li 3 & Jian Guo 2 & Yuan-lin Xu 2 & Ming-yuan Han 2 & Zhong Chen 1
Received: 4 October 2014 / Accepted: 9 April 2015 # Springer-Verlag Berlin Heidelberg 2015
Abstract Our aim is to eluc id ate the effects of osteoproteogerin (OPG) on cartilage destruction in rats as a model of collagen-induced arthritis (CIA). To establish the CIA model, Sprague Dawley rats were injected with bovine type II collagen solution subcutaneously via the tails. Adenovirus-mediated OPG (Ad-OPG) was then injected intra-articularly either at the beginning of CIA (early OPG treatment) or one week after CIA establishment (late OPG treatment); vehicle or Ad-green fluorescent protein were injected as controls. The rats were killed 4 weeks after treatment. Ankle-joint sections were obtained for histology. Serum samples were collected for enzyme-linked immunosorbent assay. Safranin O staining showed that proteoglycan loss was inhibited in the early and late Ad-OPG groups. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining revealed that both early and late Ad-OPG
treatments significantly prevented chondrocyte apoptosis in CIA rats. Furthermore, disintegrin and metalloproteinase with thrombospondin motif−5 expression decreased remarkably in the early and late OPG treatment groups. However, the cartilage destruction score, cartilage oligomeric matrix protein level and caspase-3 expression were only decreased in the early Ad-OPG treatment group. Additionally, ankle-joint swelling and the interleukin-1β expression level in CIA rats were not notably altered by Ad-OPG treatment. Taken together, our results suggest that early Ad-OPG treatment has potent protective effects against cartilage destruction during rheumatoid arthritis progression, mainly by reducing proteoglycan loss and chondrocyte apoptosis.
Zhi-yun Feng and Zhen-nian He contributed equally to this work.
Introduction
Electronic supplementary material The online version of this article (doi:10.1007/s00441-015-2194-8) contains supplementary material, which is available to authorized users. * Zhong Chen [email protected] 1
Spine Lab, Department of Orthopedic Surgery, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, People’s Republic of China 310003
2
Department of Orthopedics, Beilun People’s Hospital, No. 1288, Lu shan Road, Ningbo, Zhejiang Province, People’s Republic of China 315806
3
Department of Laboratory Center, Beilun People’s Hospital, No. 1288, Lu shan Road, Ningbo, Zhejiang Province, People’s Republic of China 315806
Keywords Osteoprotegerin . Cartilage destruction . Proteoglycan loss . Chondrocyte apoptosis . Collagen-induced arthritis . Rat (Sprague Dawley)
Rheumatoid arthritis (RA) is a chronic autoimmune disease mainly affecting the synovial joints. It is characterized by synovial inflammation and progressive erosion of bone and cartilage (Gough et al. 1994; Schett et al. 2003; Weissmann 2006). Although synovial hyperplasia leads to joint swelling, it is the bone and cartilage destruction that ultimately leads to work disability (Wolfe et al. 2000) and even life-long physical disability (Welsing et al. 2001). Therefore, we need to explore effective approaches to inhibit joint destruction. Since most therapeutic strategies have been designed to inhibit the inflammation of the synovium of joints, less effort has been made to prevent the destruction of cartilage itself. Generally, cartilage erosion in RA has been considered secondary to inflammatory pannus, which is attached to cartilage (Scott et al. 2003).
Cell Tissue Res
However, no effective therapy exists to suppress both synovium inflammation and joint destruction. Moreover, only a weak correlation has been determined between advancing synovitis and erosive progression in the hand joints of RA patients (Kirwan 1997). Furthermore, the pathophysiological causes of synovitis and cartilage erosions are not always the same (Kim and Song 1999; Kirwan 1997). Thus, other treatments are called for, based on innovative mechanisms of action. As an alternative, we should focus on the inherent mechanisms of cartilage destruction in RA, such as proteoglycan loss and cell apoptosis. Kim and Song (1999) found that the apoptosis of chondrocytes in RA joints is more prominent than those without RA. In addition, they reported that the expression of caspase-3 increases in cartilage of RA patients (Kim and Song 1999), as is the case in animal models (Campo et al. 2008). Proteoglycan is a main component in cartilage and the loss of proteoglycan has been considered as the initiation point of cartilage degradation. Previous reports have shown the remarkable loss of proteoglycan in a collagen-induced arthritis (CIA) model, as assessed by Safranin O staining (Bendele 2001; Xie et al. 2013). During proteoglycan loss, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) -4 and −5 are the primary degrading enzymes in cartilage (Plaas et al. 2007). The corresponding inhibitor targeting ADAMTS-5 has been proposed, with hopes that it can be entered into clinical trials (Gilbert et al. 2008). Osteoprotegerin (OPG) was initially found to prevent bone resorption by blocking osteoclast differentiation and activation (Boyce and Xing 2007). Subsequently, several studies have confirmed that OPG can protect bone against inflammation in various RA model animals (Kong et al. 1999; Romas et al. 2002; Zwerina et al. 2004; Saidenberg-Kermanac'h et al. 2004). To date, research regarding the effects of OPG on cartilage protection in an adjuvant-induced arthritis (AIA) model is still limited (Kong et al. 1999). However, this model has disproportionally mild cartilage destruction relative to evident cartilage damage in the CIA model (Bendele 2001). Recent reports have shown that OPG is able to prevent cartilage destruction in an osteoarthritis (OA) model in mice (Shimizu et al. 2007; Kadri et al. 2008), thus leading to hopes that this protein has potential to attenuate cartilage destruction in RA. Multiple and repeated injections, high cost and a short half life are the main problems concerning the use of biological agents for arthritis therapy. However, gene therapy employs a target gene in a virus vector and thereby endows tissue with the long-term expression of a target gene, thus avoiding the aforementioned disadvantages. Adenovirus has been tested and demonstrated to be a safe vector for gene therapy in animal models (Bolon et al. 2001). Another problem is that pathological cartilage damage might occur before visualization by radiology (Quinn et al. 2001; Fuchs et al. 1989). In order to reduce such damage, previous studies have revealed that
earlier disease modifying anti-rheumatic drug therapy has better efficacies (Anderson et al. 2000; Keystone et al. 2003; Finckh et al. 2006). In this study, we examine the effects of OPG on cartilage destruction in CIA rats and explore the potential mechanisms by over-expressing OPG in the affected joints by using an adenoviral-mediated OPG (Ad-OPG) gene therapy system. We also injected Ad-OPG injections into a group of animals 1 week later to examine whether the treatment given during the middle-late stage of CIA might also be protective.
Materials and methods Generation of recombinant adenoviral vector expressing human OPG Recombinant adenovirus carrying cDNA of human OPG was generated by using Admaxsystem (Micro-bix Bio-systems, Ontario, Canada). All experiments were performed following standard instructions. Briefly, the human OPG gene was subcloned into adenoviral type V, BAGlox△E1, 3 Cre vector (Micro-bix Bio-systems) in 293 cells (ATCC, Va., USA) by homologous recombination. An Ad-GFP (green fluorescent protein) expression cassette was used as control. Amplification of the recombinant adenovirus was performed in HEK 293 cells (ATCC). We obtained high titer viral stocks by caesium-chloride density-gradient centrifugation. Tran-EZ (Sun-bio Medical Bio-technology, Shanghai, China) was used to determine the titer of recombinant adenovirus: 1.2×1010 plaque forming units (pfu)/ml in this study. In addition, the expression of the OPG gene delivered by the adenovirus vector was identified by Western blot. The OPG expression in Ad-OPG infected chondrocytes increased significantly when compared with those infected by Ad-GFP (Fig. S1).
Experimental animals Eight-week-old, male, Sprague Dawley rats (The Center of Experimental Animals, Zhejiang University, Hangzhou, China) were kept under standardized conditions with a 12hour light–dark cycle and appropriate temperature and humidity. These rats were maintained under special pathogen-free conditions and had free access to sterilized water and pellet food. All experiments were performed following the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and were pre-approved by the Review Board of the First Affiliated Hospital of Medical College of Zhejiang University.
Cell Tissue Res
Induction of CIA model CIA was induced by immunization with bovine type II collagen (CII; Chondrex, Redmond, Wash., USA). Dissolved CII (4 mg/ml, solubilized in 0.05 M acetic acid) was emulsified with an equal volume of incomplete Freund’s adjuvant by using an electric homogenizer (IKA, Works Guangzhou, China). The rats were then immunized with 0.1 ml emulsion subcutaneously into the tail base at day 0 and day 7. Seven rats were injected with a vehicle at the same dose and frequency into the same site as normal controls for the CIA rats. We recorded the body weight, diameter of ankle and arthritis score every 3–4 days to monitor arthritic induction. Treatment Arthritis was induced successfully in 37 rats, which showed swelling and redness in both hind legs. The rats were randomly separated into four treatment groups: early OPG treatment group (Ad-OPG-injected at the onset of arthritis, n=10), late OPG treatment group (Ad-OPG-injected 1 week after onset of arthritis, n=7), GFP group (Ad-GFP-injected at the onset of arthritis, n=13) and CIA control group (vehicle-injected at the onset of arthritis, n=7). Each side of the ankle joints was single-injected with 30μl Ad-OPG (containing 3.6×108 pfu adenovirus), Ad-GFP, or a vehicle into the joint cavity. We recorded body weight, diameter of ankle and arthritis score every 3–4 days during treatment. All the animals were killed 4 weeks later. During the experiment, some rats in the GFP group were killed at 2, 7, 14 and 21 days after the GFP injection to examine the location and expression of GFP in the joints by fluorescence microscopy. Two rats in the early OPG group were killed at 2 and 14 days after Ad-OPG injection to show the OPG expression pattern in cartilage.
joints were harvested for histological examination. After removal of the surrounding soft tissue, the joints were fixed in 4 % paraformaldehyde and decalcified in 10 % EDTA for about 2 weeks. Following dehydration and paraffin embedding, serial 5-μm-thick sagittal sections were obtained. First, hematoxylin and eosin (HE) staining was carried out to assess the infiltration of inflammatory cells and bone damage. Second, sections were stained with 0.02 % Fast Green for 10 min and then 0.1 % safranin O for 7 min. A semiquantitative scoring system for proteoglycan loss was employed (Dudler et al. 2000) in which the scoring was as follows: 0, no loss; 1, mild loss; 2, severe loss; 3, complete loss of staining for proteoglycan. Third, the severity of cartilage destruction within the ankle joints was evaluated according to a cartilage destruction score as described by Romas et al. (2002), with minor modification: 0, normal; 1, minimal (loss of safranin O staining only); 2, mild (loss of safranin O staining and mild cartilage thinning and fissuring); 3, moderate (moderate cartilage destruction with 1–3 sites of minor-focal depth reaching the middle zone); 4, marked (marked cartilage destruction with more than three sites of minor-focal depth reaching the deep zone); and 5, severe (severe cartilage destruction with macro-focal cartilage destruction reaching the tidemark). ELISA analysis of COMP and IL-1β in serum The serum concentration of cartilage oligomeric matrix protein (COMP) and interleukin-1β (IL-1β) were determined by ELISA kits (Biorbyt, Montgomery Street, Calif., USA). All samples were performed in triplicate. Immunohistochemical analysis
Assessment of inflammation and cartilage destruction in arthritis Joint swelling was determined by measurement of the ankle diameter with a precision caliper (Stolina et al. 2005). Briefly, we first located the ankle joint and marked it with picric acid to ensure that we measured the same site every time. Second, we put the ankle joint into the slot of the caliper and adjusted the outside jaws of the caliper in order to bring the joint into the slot between the two outside jaws. The values presented in the build-in electronic screen were recorded. Both ankle joints were measured and the mean was calculated. Kappa statistics demonstrated that the agreement and the reproducibility were good (0.84). The arthritis scores were measured by the inflammation of the paws, as described previously (Jin et al. 2010). Rats were killed by the injection of pentobarbital intra-peritoneal. Blood samples from the inferior vena cava were collected for enzyme-linked immunosorbent assay (ELISA). Ankle
Dewaxed and dehydrated sections were first washed in phosphate buffer solution (PBS) and then incubated with 3 % peroxyl in methanol for 15 min for endogenous peroxidases blocking. After being washed in PBS, sections were then immersed in boiled citrate buffer solution for 10 min for antigen retrieval. Following a blocking step with 5 % bovine serum albumin for 20 min, sections were then covered with anti-rat caspase-3 antibody (1:200, Santa Cruz biotechnology, Texas, USA), anti-rat ADAMTS-5 antibody (1:150, Santa Cruz biotechnology), anti-rat OPG antibody (1:100, GeneTex, USA), or anti-rat TRAIL antibody (1:300, Biorbyt) overnight at 4 °C in a humidified chamber. On the following day, sections were incubated with a bio-tinylated anti-rabbit antibody (Boster Biotechnology, Wuhan, China) for 30 min at room temperature and then coupled with diaminobenzidine to visualize the positive expression of the relevant targeted proteins. After being counterstained with hematoxylin, all sections were
Cell Tissue Res
dehydrated in an ascending ethanol series and then mounted in neutral resins. An Olympus microscope, with a computer-aided imagecollection system (NIS-Elements F2.30, Nikon, Japan) was utilized to examine the sections. Positive cells of the targeted protein were counted at magnification ×400 in three fields of each slide. The positive cells number was normalized to the cell number per 100 total cells. TUNEL analysis TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling) assay was performed by using the in situ cell death detection POD kit (Roche, Germany) according to the manufacturer’s protocol. Briefly, after being permeabilized with 20 μg/ml proteinase K (Meck & Millpore, USA) for 1 h at 37 °C, the sections were washed with PBS and then incubated with TUNEL reaction mixture for 60 min at 37 °C in a dark and humid atmosphere. Signals in the sections were then transferred by incubating with POD reactions. As a positive control, we treated sections with DNase I at room temperature for 10 min prior to the labeling procedures. The terminal transferase was not added to the mixture in the negative controls. All sections were counterstained with hematoxylin. These sections were assessed by the same methods as for the immunohistochemical sections. Statistical analysis Data are presented as the means±standard errors and were analyzed with the statistical package from GraphPad Prism (version 5.0, GraphPad Software, San Diego, Calif., USA). Intergroup comparisons were subjected to one-way analysis of variance (ANOVA). The unpaired t-test was used for comparisons of difference in positive cell numbers between the treatment group and control group. P-values less than 0.05 were considered as showing statistical significance.
Results Establishment of CIA model and effects of Ad-OPG on inflammatory conditions Thirty-seven rats developed arthritis by 12–14 days after the first immunization. Redness and swelling were first seen in the small joints of the hind extremities, followed by the bilateral ankle joints. Compared with the normal control (Fig. 1a), the swelling of the ankle joints was evident in CIA rats (Fig.1b). Any rats not developing bilateral ankle-joint swelling were excluded. During the course of treatment, the arthritis had a parallel progression among rats treated with a vehicle (Fig. 1b), Ad-
GFP (Fig. 1c) and Ad-OPG at the early phase (Fig. 1d) or late phase (Fig. 1e), as the increases of diameter of ankle joints were comparable among all treatment groups at days 14, 21, 28, 35 and 42 (Fig. 1f, P>0.05). In addition, as a key factor in triggering CIA, the IL-1β serum level was not decreased significantly by Ad-OPG treatment delivered at either the early or later phase of disease (Fig. 1g, P>0.05). Moreover, HE staining revealed that the inflammatory cell infiltration was remarkable around the affected joints in the vehicle group (Fig. 1i, n) and GFP group (Fig. 1j, o), whereas it was not attenuated in rats treated with Ad-OPG at either the early phase (Fig. 1k, p) or the later phase (Fig. 1l, q). Efficacy of adenovirus infection of cartilage tissue We randomly killed one rat in the GFP group at 2, 7, 14, 21 and 28 days after Ad-GFP treatment. Frozen sections were obtained and immediately observed by fluorescence microscopy. As shown, the expression of GFP could be observed after 2 days (Fig. 2a), became stronger after 7 days (Fig. 2b) and was prominent after 14 days (Fig. 2c). GFP expression could also be seen during the following few days (Fig. 2d, e). These data revealed that the adenovirus-mediated gene exhibited remarkable and sustained expression in cartilage following injection into the articular cavity. Immunohistochemical analysis showed that OPG had a similar expression trend as GFP (Fig. 2f–h). Effect of Ad-OPG on proteoglycan loss and cartilage destruction In comparison with the excellent safrarin O staining in the normal controls (Fig. 3a), CIA rats treated with vehicle (Fig. 3b) or Ad-GFP (Fig. 3c) showed significant proteoglycan loss, as revealed by weakened and uneven safrarin O staining. However, proteoglycan loss was greatly inhibited by both early (Fig. 3d) and late OPG (Fig. 3e) treatment. The proteoglycan loss score was significantly lower in the early OPG and late OPG groups (Fig. 3f, P 0.05, f). The IL-1β (interleukin-1β) serum level was not significantly changed by Ad-OPG treatment (P>0.05, g). HE staining showed remarkable inflammation,
such as inflammatory cell infiltration and synovial hyperplasia in the vehicle group (i, n) and GFP group (j, o). However, the inflammation status was not attenuated by the treatment of OPG at either the early (l, q) or the later (k, p) phase. Boxed areas in h–l (magnification: ×100) are shown at higher magnification in m–q (magnification: ×400), respectively. Values are means ± standard errors. *P < 0.05, **P < 0.001, ***P
