Transforming growth factor beta variation with physical activity in knee osteoarthritis

Abstract

Purpose: Transforming growth factor beta (TGF-β) is a multifunctional cytokine with three different mammalian isoforms. All three isoforms of TGF-β are secreted as inactive latent complexes. Bound to latency-associated peptide, TGF-β is unable to bind to membrane receptors. Therefore, even though TGF-β is constitutively expressed throughout the body, only the free ligand is biologically active and able to modulate the metabolic activity of cartilage. An existing theory likens the latent TGF-β complex to a molecular sensor that monitors the extracellular matrix and responds to perturbations by releasing free TGF-β. In the cartilage, TGF-β1 plays an essential role in regulating cartilage matrix metabolism. The goal of this study was to assess the effects of physical activity on free and total TGF-β isoforms in plasma from individuals with knee osteoarthritis (OA). Methods: To assess the impact of physical activity on biomarker variation, participants (n=20; 65% female; 80% white; mean age 70 years; mean BMI 31.72 kg/m2) were recruited on the basis of symptomatic OA in at least one knee. Knee OA severity was assessed from a postero-anterior semi-flexed radiograph with a SynaFlexer positioning device and quantified by Kellgren Lawrence (K-L) score for each knee. Blood was obtained at 4 different time points (Figure 1). The interval from T0 to T1A was devoted to physical activity including activities of daily living and non-brisk walking for 1 hour, with no more than 10 minutes rest at any one time. All procedures were approved by the Institutional Review Board. Using commercially available ELISA assays, we quantified free and total TGF-β1 (BioLegend) in plasma samples (n=80) and available matched serum (n=79) samples. Total TGF-β2 and TGF-β3 (MSD U-PLEX ELISA) were also quantified in plasma (n=80). The data for TGF-β3 should be interpreted with caution, as 47.5% of samples had concentrations below the LLOD so were imputed as 1/2 LLOD for analyses. To assess variations in TGF-β over this time period, biomarker concentrations for the four time points were normalized to the mean across all four time points for each individual. Significance was assessed by non-parametric Friedman test and Dunn’s post-hoc test. Spearman correlation was used to evaluate associations between non-normalized: free and total plasma TGF-β1; serum and plasma free and total TGF-β1; all total TGF-β isoforms; and free and total TGF-β1 (T1A and change from T0 to T1A) with sum K-L grade. Results: Median plasma concentrations (range pg/ml) were as follows: free TGF-β1: 8.53 (0-40.71); total TGF-β1: 27343.00 (6760.00-81815.00); total TGF-β2: 24.99 (0-170.30); and total TGF-β3: 1.03 (0-3.10). Free plasma TGF-β1 represented 0.032% of total plasma TGF-β1. Free and total TGF-β1, and total TGF-β2, increased significantly after one hour of activity (Figure 2; time point T0 to T1A). The mean fold-change increase with activity was 1.7 for free TGF-β1, 1.4 for total TGF-β1, and 1.5 for total TGF-β2. After food consumption (T1B), TGF-β3 concentrations decreased an average of 62% from T1A, but no significant changes occurred in the other TGF-β isoforms at this time point. In plasma, total TGF-β1 strongly correlated with free TGF-β1 (rs=0.36, p=0.008), total TGF-β2 (rs=0.69, p<0.0001), and total TGF-β3 (rs=0.36, p=0.001). Total TGF-β2 also correlated with Total TGF-β3 (rs=0.29, p=0.01). Serum TGF-β1 concentrations were consistently higher than plasma concentrations (a mean 17.5-fold for free TGF-β1 and a mean 1.6-fold for total TGF-β1). Plasma and matched sera did not correlate for free TGF-β1 (rs=0.09, p=0.43) or total TGF-β1 (rs=0.19, p=0.09). Neither free nor total TGF-β1 were associated with sum K-L grade. Conclusions: This study demonstrates that plasma concentrations of both free and total TGF-β1, and total TGF-β2, all significantly increased with physical activity, suggesting release from a mechanically sensitive reservoir. These results are consistent with known release of TGF-β1 from extracellular matrix of myofibroblasts upon mechanical contraction in vitro. We observed an effective 1.7-fold increase of circulating free (active) TGF-β1 during an hour of activity (from a mean 8.45 to a mean 13.08 pg/ml). Based upon previous published studies, these concentrations may represent a physiologically relevant modulation. These results suggest the need for standardization of activity prior to biofluid sampling intended for TGF-β analyses to assess its impact on musculoskeletal health and disease.View Large Image Figure ViewerDownload Hi-res image Download (PPT)