Role of matrix metalloproteinases 1, 2, and 9 and tissue inhibitor of matrix metalloproteinase-1 in chronic venous insufficiency

Abstract

Purpose: Increased transforming growth factor-beta(1) (TGF-beta(1)) activity is associated with chronic venous insufficiency (CVI) disease progression and dermal skin pathology. Because TGF-beta(1) stimulates collagen synthesis and alters the levels of matrix metalloproteinases (MMPs) and their inhibitors (TIMPs), we investigated the hypothesis that increased TGF-beta(1) activity is associated with differences in messenger RNA and protein levels of MMPs and TIMP-1 in patients with CVI.

Methods: One hundred ten biopsies of the lower calf and lower thigh in 73 patients were snap frozen in liquid nitrogen and stratified into six groups according to the clinical etiologic anatomic distribution pathophysiology disease classification. One set of lower-calf and lower-thigh biopsies were analyzed for MMP-1 and TIMP-1 gene expression with quantitative reverse transcription and competitive polymerase chain reaction. A second set of biopsies was analyzed for the active and latent forms of MMP-1, MMP-2, and MMP-9 as well as for TIMP-1 by western blotting, gelatin zymography, and tissue localization by immunohistochemistry (IHC).

Results: Compared with the control, MMP-1 messenger RNA was increased in class-4 and class-6 patients (P < or =.01), whereas TIMP-1 was increased in class-6 patients only (P < or =.05). However, there were no differences in total protein between MMP-1 and TIMP-1. Active MMP-2 protein increased in class-4 and class-5 patients compared with active MMP-1 and TIMP-1 (P < or =.01). Western blotting did not identify the active component of MMP-9. Similarly, only the latent form of MMP-9 was observed by gelatin zymography, whereas both the latent and active forms of MMP-2 were observed. IHC demonstrated MMP-1 and MMP-2 in dermal fibroblasts and in perivascular leukocytes. TIMP-1 was observed in basal-layer keratinocytes of the epidermis only. MMP-9 was not detected by IHC.

Conclusion: MMP synthesis is regulated at both the transcriptional and post-transcriptional levels in CVI. Our data suggest that post-translational modifications are key to functional regulation. Dermal fibroblasts and migrating leukocytes are probable cellular sources of MMPs. Increased active MMP-2 levels in class-4 and class-5 patients indicate tissue remodeling caused by pre-ulcer and postulcer environmental stimuli. These data suggest that alterations in MMP-2 activity, in conjunction with TGF-beta(1)-mediated events, cause an imbalance in tissue remodeling leading to a pro-ulcer-forming environment.