Mechanisms of the Inhibitory Effect of Epigallocatechin-3-Gallate on Cultured Human Vascular Smooth Muscle Cell Invasion

Abstract

Although we recently showed that the administration of catechins reduced the neointimal formation in a rat balloon-injury model, the precise molecular mechanisms are largely unknown. In the present study, we tried to determine these mechanisms using an in vitro SMC invasion system. Boyden chamber assay was used to examine the effect of catechins on the invasive behavior of SMCs. The invasive activity of SMCs through collagen gel was restrained by EGCG in a concentration-dependent manner. The data from gelatin and collagen zymography and Western blot revealed that EGCG blocks the activation of pro-matrix metalloproteinase (MMP)-2 during an invasion assay and in the conditioned medium of cultured SMCs as well as the activities of MMP-2 and membrane type 1-MMP (MT1-MMP) even at 0.1 to 0.3 micromol/L of EGCG. EGCG was found to restrain MT1-MMPcat-dependent pro-MMP-2 activation. EGCG upregulated the expression of tissue inhibitor of MMP-2 (TIMP-2) protein. Reverse zymography showed that the increased TIMP-2 to expression was validated by an increased activity. The data from decreased TIMP-2 activity using its siRNA suggested that upregulation of TIMP-2 expression may be one of the major mechanisms for inhibition of SMC invasion by EGCG. These results indicate that EGCG targets multiple MMP-mediated SMC cellular events and provides a new major mechanism for the SMC invasion through upregulation of TIMP-2 expression to modulate MMP activity.