Thrombin Suppresses Matrix Metalloproteinase 2 Activity and Increases Tissue Inhibitor of Metalloproteinase 1 Synthesis in Cultured Human Peritoneal Mesothelial Cells

Abstract

Objective

Recently, high levels of intraperitoneally generated thrombin were found in the effluent of patients treated with continuous ambulatory peritoneal dialysis (CAPD). The aim of the present study was to investigate in human peritoneal mesothelial cells (HMCs) the effect of thrombin on the activity and synthesis of matrix metalloproteinases (MMPs), which regulate the degradation of basement membrane collagen.

Methods

Cultured HMCs were isolated from omental tissue and used at confluence for the experiments. Conditioned media were obtained by incubating cells with serum-free M199 containing the relevant doses of thrombin. Activity of MMP-2 and MMP-9 were determined by an activity assay system. The antigen levels of MMPs and of the specific tissue inhibitor of metalloproteinases 1 (TIMP-1) were measured by ELISA. Northern blot analysis was applied to analyze mRNA expression of MMP-2 and TIMP-1.

Results

Incubation of HMCs with increasing doses of thrombin resulted in a concentration- and time-dependent suppression of MMP-2 activity. No changes in MMP-9 activity were seen. After a 48-hour stimulation period with thrombin (5 U/mL), MMP-2 activity decreased to 53% of that seen in control conditions. Antigen measurements revealed that this decrease was paralleled by a slight reduction in MMP-2 levels, which became significant at a thrombin dose of 5 U/mL [50.65 ± 7.5 ng/10⁵ cells (48 hours, 5 U/mL) vs 64.6 ± 10.1 ng/10⁵ cells (control)]. Under the same conditions, TIMP-1 levels were considerably increased [3.9 ± 0.46 μg/10⁵ cells (48 hours, 5 U/mL) vs 1.2 ± 0.14 μg/10⁵ cells (control)]. Hirudin (10 U/mL) completely inhibited the thrombin-induced effects on MMP-2 and TIMP-1 synthesis. These results were also reflected by Northern blot hybridization, where a slight decrease in MMP-2 and an increase in TIMP-1 mRNA expression were observed in response to thrombin.

Conclusions

Our results suggest that high thrombin levels suppress MMP-2 activity through decreased MMP-2 and increased TIMP-1 synthesis. Thus, thrombin may promote the accumulation of basement membrane collagen. In addition to fibrin formation, this mechanism may represent a further contribution by thrombin to peritoneal thickening during CAPD.