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RESEARCH Rosiglitazone decreases angiogenesis in the MCL after ACL rupture - A pilot study Christopher J. DeSutter, BSc, 1 Daniel Miller, MD PhD, 2 Catherine Leonard, MSc, 2 Robert C. Bray, MD, MSc 2 1Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Canada 2McCaig Centre for Joint Injury and Arthritis Research, University of Calgary, Calgary, Canada Correspondence and reprint requests to Dr. R. Bray, Department of Surgery, University of Calgary, 3330 Hospital Dr. NW, Calgary, Alberta, Canada T2N 4N1, Ph: (403) 220-4244, Fax: (403) 270-0617, Email: rcbray@ucalgary.ca ABSTRACT In the anterior cruciate ligament (ACL) transected knee, the medial collateral ligament (MCL) incurs numerous physiological changes that include inflammation and increased angiogenic activity resulting in functional deficiency. Peroxisome proliferator activated receptor - γ(PPAR-γ) agonists show promising results for their possible use in osteoarthritis therapies, but there are limited studies looking at their effects in this area. The purpose of this study was to examine the effect the PPAR-γ agonist rosiglitazone has on the angiogenic response in the osteoarthritic rabbit model. Six rabbits were assigned to one of three groups: control (n=2); 4-week right leg ACL transected (ACL-X) (n=2); 4-week right leg ACL-X treated with 5 mg/kg per day of rosiglitazone (n=2). The two contralateral MCLs in the 4-week ACL-X rabbits treated with rosiglitazone were also used as the drug treated non-ACL transected leg control. In total 8 MCLs were analyzed. To measure the blood vessel volume and angiogenic response in the MCLs, the vascular endothelium (CD-31) and vascular smooth muscle (SMA) volumes of them were determined. In the ACL-X rosiglitazone treated MCL, CD-31 volume decreased 3-fold down to control levels, in comparison to non-treated ACL-X MCLs. Rosiglitazone had a significant effect on SMA, causing decreased volume in comparison to non – treated MCLs. In summary, rosiglitazone has a significant effect on the angiogenic response in the ACL ruptured animal model. INTRODUCTION Joint injury and arthritis are major causes of morbidity in the United States. One of the most clinically important ligament injuries that occur is to the anterior cruciate ligament (ACL). Each year in the USA, there are 80,000 surgical ACL reconstructions performed. 1, 2 Patients possessing ACL ruptures often complain of recurring loss of joint stability that often leads to the 4 premature onset of osteoarthritis, the most common type of degenerative joint 3, 4 disease. In the knee, ACL rupture results in anterior translation of the tibia in relationship to the femur resulting in joint laxity (Figure 1). This abnormal biomechanical environment in an ACL ruptured knee is not only detrimental to cartilage health, but induces a series of adaptive structural and physiological changes in secondary joint stabilizing structures. There is angiogenesis, hyperaemia, inflammation and increased cellularity in the ligaments, meniscus, capsule and synovium of the knee joint. 5-7 In the medial collateral ligament (MCL) there is increased blood flow, increased DNA and RNA synthesis and cellularity7-10 Due to the properties of the MCL being degraded, this leads to further cartilage degeneration and altered joint mechanics in the ACL ruptured knee. 11 There is limited information available on how this physiological adaptation occurs. The modification of adaptive changes in osteoarthritic tissues has the potential to provide new information on underlying mechanisms, leading to new therapies for osteoarthritis. The peroxisome proliferator –activated receptors (PPAR) agonist drugs show great promise in this area as a potential therapeutic treatment. PPAR agonists are ligand activated transcription factors that are part of the nuclear hormone super family. 12 Three different isotypes have been identified: PPAR-α, PPAR-β and PPAR-γ. These PPAR endogenous ligands form a diverse group of fatty acids with their derivatives generated by lipid metabolism. 12 Recently, Figure 1. Anterior view of the knee detailing the major ligaments, bones, menisci and tendons. UpToDate (2011). Anterior knee anatomy adult. http://www.uptodate.com/contents/ image?imageKey=RHEUM%2F26531 (accessed August 7, 2011). University of Alberta Health Sciences Journal • April 2012 • Volume 7 • Issue 1
it has been found that PPAR-γ agonists are involved in vascular biology, inflammatory responses, tissue repair, cell differentiation and proliferation. 12-15 In arthritic synoviocytes, PPAR-γ agonists greatly inhibit inflammatory cytokine expression. 16 PPAR-γ agonists have also been expressed in human chondrocytes acting as a protective 17, 18 mechanism for cartilage. PPAR-γ agonists have therapeutic potential in a variety of clinical conditions. There is literature showing PPAR-γ agonists role in fat metabolism and their anti-inflammatory actions shown through their effect on inhibiting cytokine expression. 19, 20 There are limited published studies looking at the effect of PPAR-γ agonists in osteoarthritis models. One study by Kobayashi et al., using a partial medial menisectomy guinea pig model, found that the PPAR-γ agonist pioglitazone reduced cartilage lesion depth and area. 21 By studying the effect of the PPAR-γ agonist rosiglitazone on the adaptive physiological response in ACL ruptured knees (specifically the angiogenic activity), its potential as a new therapy to treat osteoarthritic patients can be determined. The purpose of this study is to examine the effect of the PPAR-γ agonist rosiglitazone on the physiological and angiogenic responses in the rabbit model of joint laxity and osteoarthritis. The rabbit model was chosen as we can compare the results obtained to our established database on adaptive physiology in rabbit ACL – ruptured joints. It is hypothesized that PPAR-γ agonists will decrease physiological degeneration of the MCL in the ACL ruptured knee. The blood vessel volume and degree of angiogenesis occurring in the MCL of the ACL ruptured knees will be measured using immunohistochemistry for specific markers for vascular endothelium (CD-31) and vascular smooth muscle (SMA) in the MCL. MATERIALS AND METHODS Subjects Six young skeletally mature 1-year-old New Zealand white rabbits (4.5 - 6.5kg; Riemans Fur Ranch, St. Agatha, Ontario) were assigned to one of three groups: control (n=2); 4-week right leg ACL transected (ACL-X) (n=2); 4-week right leg ACL-X treated with a low dose of 5 mg/kg /day of rosiglitazone (n=2). The two contralateral MCLs in the 4-week ACL-X rabbits treated with rosiglitazone were used as the drug treated non-ACL ruptured leg controls. This results in a total of 8 MCLs analyzed in this study. Rabbits were kept on a 12 – hour light/dark cycle and fed standard laboratory chow and tap water ad libitum. All animals were treated and maintained according to the Canadian Council on Animal Care guidelines and this study received approval by the University of Calgary Faculty of Medicine Animal Care Committee. aCL transection and Rosiglitazone injections Rabbits were given 0.18 mL of acepromazine maleate (Atravet ®) intravenously and anesthetized with halothane (2-5%, 1.0 L/ min O ). All ACL transection surgeries were 2 completed on the right leg of the rabbits. An anterior tibial draw test was performed on the right leg to ensure no prior ACL injury existed. The anterior tibial draw test was done by grasping the tibia with both hands below the joint line, thumbs placed on either side of the patella, with the tibia pulled anteriorly. An antero-lateral surgical approach was used. The ligament was exposed by lateral subluxation of the patella and reflection of the intra-articular fat pad. The ACL was isolated using a hooked probe and the ligament was transected at the middle with a #12 hooked blade. A second anterior tibial draw was performed to ensure the transection was complete. Following the unilateral surgery, rabbits were treated with standard antibiotics and allowed to resume normal cage activity for four weeks. Rosiglitazone treated animals were injected subcutaneously with a low dose of 5 mg/ kg of body weight per day for a total of four weeks. In the ACL-X rabbits, injections began on the day following the ACL transection surgery. Since the contralateral MCL was used as the non-operated leg rosiglitazone treated control, the 4-week drug treatment was simultaneous. At the beginning and end of the 4-week dosing period, anterior tibial draw tests were completed on the left leg to ensure no ACL injury existed. Immunohistochemistry MCLs were sectioned and labeled for CD-31 and SMA according to the following doublelabel protocol. Rabbits were euthanized then the MCLs were harvested from control, ACL ruptured and rosiglitazone treated knees then cleaned of any extra tissue. Tissues were cryopreserved in serial sucrose solutions of 10%, 20% and 30% concentrations. Following cryoprotection, ligaments were frozen in isopentane at -80°C, embedded in OCT media then stored at -30°C for one month until processing. MCLs were cut into 100 μm thick longitudinal serial sections and placed individually in 24 well plastic plates. Sections were washed in phosphate buffered saline (PBS) (3 x 10 minutes) then immersed in 10% normal donkey serum (Jackson Immunoresearch, West Grove, PA, USA)/ PBS 1% Triton X100 for 1 hour at room temperature. MCLs were then incubated with mouse anti-human CD-31 antibody (1:50 dilution; Dako, Carpinteria, CA, USA) for 24 hours at 4°C in a humidified chamber. Sections were washed in PBS (3 x 10 minutes) and incubated with donkey antimouse Cy5 conjugated secondary antibody (1:300 dilution; Jackson Immunoresearch, West Grove, PA, USA) for 2 hours at room temperature. Sections were washed in PBS (3 x 10 minutes) then incubated with goat anti-human smooth muscle actin (SMA) antibody (1:400 dilution, Novus Biologicals, Littleton, CO, USA) for 24 hours at 4°C, followed by further PBS washes and incubation with donkey anti-goat Cy2 conjugated antibody (1:400 dilution; Jackson Immunoresearch, West Grove, PA, USA) for 2 hours at room temperature. Sections were then washed for a final time, placed on slides with Fluorsave TM (Calbiochem, Mississauga, Ontario) and coverslipped. Slides were stored in a cardboard slide holder to protect fluorescence loss due to light. Confocal Microscopy MCL sections were analyzed using an Olympus Fluoview FV-1000 confocal microscope. They were visualized under a 10x objective and imaged using 4-micron thick optical z-stack sections. Simultaneous dual channel scanning laser confocal analysis was performed using preconfigured Cy2 and Cy5 channel settings. Images were saved in the OIF file format. Statistical analysis The CD-31 and SMA volumes found in the MCL were determined using Image J software. The volumes of CD-31 and SMA in each image were put into an excel spreadsheet and then summed together to get a total volume converted into milliliters for each ligament. The volumes of CD-31 and SMA for each MCL type had their averages calculated. Since there are only two subjects per group, the mean and the range of data was the chosen method of statistical analysis. If the subjects in each group had increased numbers, inferential analysis using a two – way ANOVA would have been preferred for comparative purposes. By using this method of analysis, one could then interpret if rosiglitazone has an effect on the angiogenic response in this pilot study. University of Alberta Health Sciences Journal • April 2012 • Volume 7 • Issue 1 5 RESEARCH
Page 1 and 2: UAHSJ www.uahsj.ualberta.ca ISSN 17
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