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Abstract
Background:
Statins can regulate the production of pro-inflammatory cytokines and inhibit MMP production or activation in a variety of types of cells. This study evaluated whether statins would inhibit MMP release from human lung fibroblasts, which play a major role in remodeling processes.
Methods:
This study, using an in-vitro model (three-dimensional collagen gel contraction system), evaluated the effect of cytokines (tumor necrosis factor-α, TNF-a and interleukin-1β, IL-1b) on the MMP release and MMP activation from human lung fibroblasts. Collagen degradation induced by cytokines and neutrophil elastase (NE) was evaluated by quantifying hydroxyproline.
Results:
In three-dimensional collagen gel cultures (3D cultures) where cytokines (TNF-a and IL-1b) can induce the production of MMPs by fibroblasts, it was found that simvastatin inhibited MMP release. In 3D cultures, cytokines together with NE induced collagen degradation and can lead to activation of the MMP, which was inhibited by simvastatin.
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Figure 1.
Effect of Simvastatin on Three-dimensional Collagen Gel (3D-gel) Contraction in the Presence or Absence of Cytokines and Neutrophil Elastase. Gels were released into tissue culture dishes containing 5 mL of serum-free DMEM with or without simvastatin (5 mM). After 1 hour incubation, cytokines (TNF-a 5 ng/mL, IL-1b 5 ng/mL) and NE (20 nM) were added in to the gel floating medium. Vertical axis presents gel size as % of initial area, and horizontal axis presents time after release (days). The data presented are mean±SEM from three separate experiments, each of which included triplicate gels for each condition. When simvastatin added together with the combination of the neutrophil elastase and cytokines, simvastatin (5μM) significantly inhibit collagen gel contraction induced by the combination of the neutrophil elastase and cytokines. ∗p＜0.01 compared with combination of neutrophil elastase and cytokines. CK: cytokines (TNF-α＋IL-1b); NE: neutrophil elastase. Statin: simvastatin; SEM: standard error of the mean.
Figure 2.
Effect of Simvastatin on Collagen Gel Degradation Induced by Cytokines and NE. Gels were prepared and cultured as shown above in figure 1. On day 5, gels were harvested and subjected to hydroxyproline assay. Vertical axis presents hydroxyproline (% of control), and horizontal axis presents culture conditions. The data presented are mean±SEM from three separate experiments, each per formed in duplicate. Cytokines (TNF-a 5 ng/mL, IL-1b 5 ng/mL) alone or NE (20 nM) alone slightly decreased the hydroxyproline content. The combination of cytokines and NE, however, resulted in a significant decrease of hydroxyproline content in collagen gels. Simvastatin (5μM) significantly blocked collagen gel degradation induced by cytokines plus neutrophil elastase. ∗p ＜0.01 compared with combination of neutrophil elastase and cytokines, †p＜0.01 compared with Cytokines alone or NE, ‡p＜0.01 compared with control cultures. CK: cytokines (TNF-a＋IL-1b); NE: neutrophil elastase; SEM: standard error of the mean.
Figure 3.
Effect of simvastatin on MMP-2 and -9 production and activation by the combination of cytokines and NE. Gels were prepared and cultured as shown above in figure 1. On day 2 (Figure 3A) and day 5 (Figure 3B), media (500μL on day 2 and the rest on day 5) were harvested and subjected to gelatin zymography. Supernatant from HT1080 cell monolayer culture was used as a positive control. Under control conditions, HFL-1 fibroblasts cultured in three-dimensional collagen gels primarily released MMP-2 (gelatinase A) into surrounding media, as identified by its characteristic molecular weights of 72 kD (latent form) and 66 kD (active form) (Figure 3A and B). MMP-9 (gelatinase B) was not produced by HFL-1 cells under control conditions. In the presence of cytokines (TNF-a and IL-1b), however, latent form of MMP-9 (92 kD) was produced and this latent MMP-9 was converted to the active form (84 kD) by NE (10 nM, Figure 3). Simvastatin (5μM) not only significantly blocked the MMP-9 conversion from latent to active form (Figure 3A), but also inhibited MMP-9 and MMP-2 production (Figure 3B). CK: cytokines (TNF-a＋IL-1b); NE: neutrophil elastase.
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