Simvastatin and atorvastatin reduce the mechanical properties of tendon constructs in vitro and introduce catabolic changes in the gene expression pattern

Abstract

Treatment with lipid-lowering drugs, statins, is common all over the world. Lately, the occurrence of spontaneous tendon ruptures or tendinosis have suggested a negative influence of statins upon tendon tissue. But how statins might influence tendons is not clear. In the present study, we investigated the effect of statin treatment on mechanical strength, cell proliferation, collagen content and gene expression pattern in a tendon-like tissue made from human tenocytes in vitro. Human tendon fibroblasts were grown in a 3D tissue culture model (tendon constructs), and treated with either simvastatin or atorvastatin, low or high dose, respectively, for up to seven days. After seven days of treatment, mechanical testing of the constructs was performed. Collagen content and cell proliferation were also determined. mRNA levels of several target genes were measured after one or seven days. The maximum force and stiffness were reduced by both statins after 7 days (p<0.05), while the cross sectional area was unaffected. Further, the collagen content was reduced by atorvastatin (p = 0.01) and the cell proliferation rate was decreased by both types of statins (p<0.05). Statin treatment also introduced increased mRNA levels of MMP-1, MMP-3, MMP-13, TIMP-1 and decreased levels of collagen type 1 and 3. In conclusion, statin treatment appears to have a negative effect on tendon matrix quality as seen by a reduced strength of the tendon constructs. Further, activated catabolic changes in the gene expression pattern and a reduced collagen content indicated a disturbed balance in matrix production of tendon due to statin administration.

Fig 1. Stress-strain curves during mechanical testing.

Representative curves from each treatment group (all from the same cell line). Grey is untreated-controls, black is DMSO-controls, blue is low dose simvastatin treatment (0.05μM), green is high dose simvastatin treatment (0.5μM), purple is low dose atorvastatin treatment (0.05μM) and red is high dose atorvastatin treatment (0.5μM). The samples treated with high dose simvastatin had a significantly lower maximum stress compared to the DMSO-control.

Fig 2. Construct mechanical data.

Maximum force, maximum stiffness, cross sectional area, maximum stress and maximum modulus of tendon constructs with or without statin treatment for 7 days. Low dose (L) statin treatment corresponds to 0.05μM (simvastatin or atorvastatin) and high dose (H) corresponds to 0.5μM. Significant changes from DMSO-control are indicated by * (p<0.05), ** (p<0.01) or *** (p<0.001). The line represents median value. n = 6 different donor-specific cell lines, where each symbol represent the mean value from three replicates from one cell line. The force, stiffness, stress and modulus were all reduced after 7 days of high dose simvastatin treatment. High dose treatment with atorvastatin also reduced the force and stiffness and tended to reduce the maximum modulus (p = 0.07).

Fig 3. Collagen quantification.

Total collagen content in tendon constructs with or without statin treatment for 7 days. Low dose (L) statin treatment corresponds to 0.05μM (simvastatin or atorvastatin) and high dose (H) corresponds to 0.5μM. The line represents median value. Individual donor-specific cell lines are represented by the six different symbols. The collagen content was approximately 10% of the construct dry weight. High dose treatment with atorvastatin reduced the collagen content compared to DMSO-controls, this was not seen after simvastatin treatment. Significant changes from DMSO-control are indicated by ** (p = 0.01).

Fig 4. Cell proliferation.

Cell proliferation in tendon constructs, with or without statin treatment for 7 days, measured by a MTT assay. The data is presented as optical density (OD) value. Low dose (L) statin treatment corresponds to 0.05μM (simvastatin or atorvastatin) and high dose (H) corresponds to 0.5μM. The line represents median value. Individual donor-specific cell lines are represented by the six different symbols. The cell proliferation was reduced in constructs treated with high dose simvastatin or high dose atorvastatin. Significant changes from DMSO-control are indicated by * (p<0.05).

Fig 5. Gene expression analysis: Effect of treatment on GAPDH, heat shock proteins and tendon related genes.

Gene expression after 1 or 7 days of statin treatment. The samples were analyzed for GAPDH, heat shock proteins and tendon related genes. Low dose (L) statin treatment corresponds to 0.05μM of either simvastatin or atorvastatin and high dose (H) corresponds to 0.5μM. The DMSO-control is set as baseline, and mRNA expression in constructs treated with statins are shown relative to DMSO-control for each cell line and day. Data is presented on a logarithmic y scale as geometric means ± 95% confidence interval (CI). Individual donor-specific cell lines are represented by dots. Significant interaction in the repeated measures 2-way ANOVA (time*treatment) are below each graph.

Fig 6. Gene expression analysis: Effect of treatment on collagen, MMPs and TIMP-1.

Gene expression after 1 or 7 days of statin treatment. The samples were analyzed for collagens, MMPs and TIMP-1. Low dose (L) statin treatment corresponds to 0.05μM of either simvastatin or atorvastatin and high dose (H) corresponds to 0.5μM. The DMSO-control is set as baseline, and mRNA expression in constructs treated with statins are shown relative to DMSO-control for each cell line and day. Significant changes from DMSO-control are indicated by * (p<0.05), ** (p<0.01), and *** (p<0.001). Data is presented on a logarithmic y scale as geometric means ± 95% confidence interval (CI). Individual donor-specific cell lines are represented by dots. Significant interaction in the repeated measures 2-way ANOVA (time*treatment) are indicated below each graph.