Gene therapy for follistatin decreases systemic inflammation and pain sensitivity following knee injury in high-fat diet induced obese mice

Abstract

Purpose: Both obesity and joint trauma are risk factors for osteoarthritis (OA) and OA-related pain. In addition, muscle may have a role in knee OA development as changes in muscle strength could affect kinematics of joint loading. Furthermore, because of its key function in glucose storage and utilization, muscle strength and physiology are particularly vulnerable to obesity-associated inflammation. However, the mechanical, inflammatory, and metabolic links among obesity, muscle, and OA remain unclear. Follistatin (FST), an activin-binding protein, has been used as an effective treatment for muscle degenerative diseases by increasing muscle growth due to its ability to inhibit myostatin, a negative regulator of muscle mass. Moreover, FST has been reported to reduce the infiltration of inflammatory cells in the synovial membrane of the knee joint. Therefore, we hypothesized that overexpression of FST using a gene therapy approach will mitigate obesity-associated inflammation and pain sensitization in a high-fat diet (HFD) induced obesity model of mouse OA. Methods: Six-week old male C57BL/6 mice were fed either a chow control-diet or HFD (60% fat by kcal, n = 16/dietary group). At 7-weeks of age, either an AAV9-mediated FST 314 gene vector or GFP-labeled control vector (n = 10 for each dietary groups) was delivered via tail vein at a final dose of 6 × 1011 vg/ mouse. At 16-weeks of age, mice in each group (n = 10) underwent surgery to destabilize the medial meniscus (DMM) on the left knee. The remaining mice served as non-surgical controls. At time of surgery, Dual Energy X-ray Absorptiometry was conducted to measure body composition and bone mineral density (BMD). Six-weeks post-surgery (22-weeks of age), blood was collected and analyzed using a 31-plex Luminex® assay. Peripheral thermal sensitivity was determined using a hot/cold plate, while mechanical pain algesia at the knee was evaluated by a pressure-pain test. A two-way ANOVA between groups or a paired t-test within each group was performed to determine statistical significance, as appropriate (α = 0.05). Results: Prior to surgery, HFD resulted in increased body weight for both GFP- and FST-treated animals compared to chow (p < 0.05). However, overexpression of FST significantly decreased percentage of body fat in both chow and HFD treated mice as compared to their own corresponding GFP-controls. There was a main effect of both diet (p = 0.028) and FST-treatment (p = 0.007) on bone mineral density, such that HFD decreased BMD (HFD-GFP: 0.46 ± 0.23g/cm2 vs. chow-GFP: 0.51 ± 0.15g/cm2) while FST increased BMD (HFD-FST: 0.52g/cm2 ± 0.17, chow-FST: 0.56g/cm2 ± 0.17). Six weeks post-surgery, serum inflammatory profiles for HFD-GFP animals demonstrated increased systemic inflammation compared to chow diet, which was attenuated in the HFD-FST animals across fourteen markers (Table 1). HFD increased hot-plate paw-withdrawal latency time in GFP-treated animals compared to all other groups (p < 0.05), and the phenotype was rescued by FST-treatment in the HFD animals. Similarly, reduced cold plate responsiveness was also observed in HFD animals, which was rescued by FST-treatment. No differences in thermal peripheral pain were found with surgery. Additionally, FST treatment protected HFD animals from mechanical algesia at the knee post-DMM surgery (Fig. 1). Conclusions: Overexpression of FST decreased body fat, pain sensitivity, and obesity-associated systemic inflammation, while increasing whole body BMD in mice with HFD-induced obesity. These findings further support the important role of muscle in obesity and inflammation, which are well-known risk factors associated with OA onset and progression. We are currently evaluating the relationship among these factors in these mice, and whether FST treatment will mitigate OA development due to obesity and joint injury.Tabled 1Serum Markers Decreased with FST Treatment in HFD Animals compared to HFD animals with GFP.MarkerHFD-GFP Mean ± SEM (ng/mL)HFD-FST Mean ± SEM (ng/mL)p-valueCXCL1/KC0.25 ± 0.030.16 ± 0.020.050CXCL10/IP-100.09 ± 0.010.06 ± 0.040.005, #IL-1α1.03 ± 0.170.53 ± 0.080.022*IL-1β0.06 ± 0.020.03 ± 0.010.028*Leptin137.93 ± 17.4317.69 ± 4.640.001*Insulin25.22 ± 4.884.30 ± 0.650.001** indicates p < 0.05; # indicates p < 0.05 between DMM animals and control surgery animals. Open table in a new tab * indicates p < 0.05; # indicates p < 0.05 between DMM animals and control surgery animals.