Research ArticlePHYSIOLOGY

Gene therapy for follistatin mitigates systemic metabolic inflammation and post-traumatic arthritis in high-fat diet–induced obesity

See allHide authors and affiliations

Science Advances  08 May 2020:
Vol. 6, no. 19, eaaz7492
DOI: 10.1126/sciadv.aaz7492
  • Fig. 1 Effects of AAV-mediated FST delivery on body composition and systemic inflammatory markers.

    (A) DXA images of mice at 26 weeks of age. (B) DXA measurements of body fat percentage and bone mineral density (BMD; 26 weeks) and body weight measurements over time. (C) Serum levels for adipokines (insulin, leptin, resistin, and C-peptide) at 28 weeks. (D) Metabolite levels for glucose, triglycerides, cholesterol, and FFAs at 28 weeks. (E) Serum levels for cytokines (IL-1α, IL-1β, MCP-1, and VEGF) at 28 weeks. (F) Fluorescence microscopy images of visceral adipose tissue with CD11b:Alexa Fluor 488 (green), CD11c:phycoerythrin (PE) (red), and 4′,6-diamidino-2-phenylindole (DAPI; blue). Scale bars, 100 μm. Data are presented as mean ± SEM; n = 8 to 10; two-way analysis of variance (ANOVA), P < 0.05. Groups not sharing the same letter are significantly different with Tukey post hoc analysis. For IL-1α and VEGF, P < 0.05 for diet effect and AAV effect. For MCP-1, P < 0.05 for diet effect.

  • Fig. 2 Effects of AAV-FST delivery on OA severity, synovitis, inflammatory cytokines, and pain.

    (A) Histologic analysis of OA severity via Safranin O (glycosaminoglycans) and fast green (bone and tendon) staining of DMM-operated joints. (B) Histology [hematoxylin and eosin (H&E) staining] of the medial femoral condyle of DMM-operated joints. Thickened synovium (S) from HFD mice with a high density of infiltrated cells was observed (arrows). (C) Modified Mankin scores compared within the diet. (D) Synovitis scores compared within the diet. (E) Levels of proinflammatory cytokines in the SF compared within the diet. (F) Hot plate latency time and sensitivity to cold plate exposure, as measured using the number of jumps in 30 s, both for non-operated algometry measurements of pain sensitivity compared within the diet. Data are presented as mean ± SEM; n = 5 to 10 mice per group; two-way ANOVA, P < 0.05. Groups not sharing the same letter are significantly different with Tukey post hoc analysis.

  • Fig. 3 Effects of AAV-FST gene therapy on muscle structure and function.

    (A) Photographic images and (B) measured mass of tibialis anterior (TA), gastrocnemius (GAS), and quadriceps (QUAD) muscles; n = 8, diet and AAV effects both P < 0.05. (C) Western blot showing positive bands of FST protein only in FST-treated muscles, with β-actin as a loading control. Immunolabeling of (D) GAS muscle and (E) adipose tissue showing increased expression of FST, particularly in skeletal muscle. (F) H&E-stained sections of GAS muscles were measured for (G) mean myofiber diameter; n = 100 from four mice per group, diet, and AAV effects; both P < 0.05. (H) Oil Red O staining was analyzed for (I) optical density values of FAs; n = 6. (J) Second-harmonic generation imaging of collagen in TA sections was quantified for intensity; n = 6. (K) Western blotting showing the level of phosphorylation markers of protein synthesis in GAS muscle. (L) Functional analysis of grip strength and treadmill time to exhaustion; n = 10. Data are presented as mean ± SEM; two-way ANOVA, P < 0.05. Groups not sharing the same letter are significantly different with Tukey post hoc analysis. Photo credit: Ruhang Tang, Washington University.

  • Fig. 4 Effects of AAV-FST gene delivery on bone structure.

    (A) Three-dimensional (3D) reconstruction of microCT images of non-operated and DMM-operated knees. (B) Tibial plateau (TP) and femoral condyle (FC) regional analyses of trabecular bone fraction bone volume (BV/TV), BMD, and trabecular number (Tb.N). Data are presented as mean ± SEM; n = 8 to 19 mice per group; two-way ANOVA. (C) 3D microCT reconstruction of metaphysis region of DMM-operated joints. (D) Analysis of metaphysis BV/TV, Tb.N, and BMD. (E) 3D microCT reconstruction of cortical region of DMM-operated joints. (F) Analysis of cortical cross-sectional thickness (Ct.Cs.Th), polar moment of inertia (MMI), and tissue mineral density (TMD). (D and F) Data are presented as mean ± SEM; n = 8 to 19 mice per group; Mann-Whitney U test, *P < 0.05.

  • Fig. 5 Effects of AAV-FST gene delivery on browning of WAT and mitochondrial OXPHOS.

    (A) Immunohistochemistry of UCP-1 expression in SAT. Scale bar, 50 μm. (B) Western blotting of SAT for key proteins expressed in BAT, with β-actin as a loading control. (C) Western blot analysis of mitochondria lysates from SAT for OXPHOS proteins using antibodies against subunits of complexes I, II, III, and IV and adenosine triphosphate (ATP) synthase. (D) Change of densitometry quantification normalized to the average FST level of each OXPHOS subunit. Data are presented as mean ± SEM; n = 3. *P < 0.05, t test comparison within each pair.

  • Table 1 Multivariate regression analyses for factors predicting OA severity.

    β, standardized coefficient. ***P < 0.001.

    Predictor variablesModel 1
    β
    Model 2
    β
    SF IL-1α0.130.003
    Serum IL-1β0.0060.073
    Serum TNF-α0.4710.537
    Serum amylin0.0230.026
    Body fat percentage0.142
    Serum leptin0.00003
    Adjusted r20.613***0.379***

Supplementary Materials

  • Supplementary Materials

    Gene therapy for follistatin mitigates systemic metabolic inflammation and post-traumatic arthritis in high-fat diet–induced obesity

    Ruhang Tang, Natalia S. Harasymowicz, Chia-Lung Wu, Kelsey H. Collins, Yun-Rak Choi, Sara J. Oswald, Farshid Guilak

    Download Supplement

    This PDF file includes:

    • Table S1
    • Figs. S1 to S4

    Files in this Data Supplement:

Stay Connected to Science Advances

Navigate This Article