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The matrix metalloproteinases (MMP) are a large group of enzymes responsible for matrix degradation. They contribute to joint destruction in rheumatoid arthritis (RA) by directly degrading the cartilage and bone and indirectly promoting angiogenesis (formation of new blood vessels). Inhibition of MMPs is a primary therapeutic target in RA. However, the results of limited clinical trials performed to date are disappointing. Improvements in therapeutic benefit may be achieved by targetting specific MMPs. A subclass of the MMPs, the gelatinases, contribute directly to joint destruction as well as being vital during angiogenesis. Gelatinase A is released as a latent enzyme and must be activated to degrade the matrix. It has a unique mechanism of activation on the cell surface involving membrane-type MMP (MT-MMP). Recently, the serine protease, activated protein C (APC), has been shown to directly activate gelatinase A, without requiring MT-MMP Inhibition of APC represents a selective approach to prevent gelatinase A activation and may prove to be of therapeutic benefit in RA.

Human gelatinase B is involved in tissue remodeling and angiogenesis. It is thought to be synthesized and rapidly secreted as an inactive precursor. In this report, we have shown that human endothelial cells accumulate active forms of gelatinase B in the cytosol. Microvascular but not macrovascular endothelial cells dramatically increased the expression of cytosolic gelatinase B in response to phorbol myristate acetate. Western blotting showed that tissue inhibitor of metalloproteinase-1 (TIMP1) was also present in the cytosol. Whereas gelatinase B was complexed with TIMP1 in the conditioned medium, it existed as a free enzyme in the cytosol, suggesting that the formation of gelatinase B and TIMP1 complex occurs after their secretion. Immunogold electron microscopy revealed that gelatinase B was localized in secretory vesicles which were especially prominent in invading pseudopodia. In contrast, TIMP1 was found throughout the cytoplasm but was not present in the gelatinase vesicles. The accumulation of intracellular activated gelatinase B, ready for rapid release, may facilitate the migration of microvascular endothelial cells during angiogenesis.

Objectives-Angiogenesis (the formation of new blood vessels) is a major component of the inflammatory pannus in rheumatoid arthritis (RA). Matrix metalloproteinase (MMP) secretion by microvascular endothelial cells is an essential step in angiogenesis. The secretion of MMP1, MMP2, MMP9, and TIMP1 by human microvascular endothelial cells derived from RA synovium (RASE) to normal synovium (NSE) and neonatal foreskin (FSE) was compared. Methods-Confluent monolayers of endothelial cells in basal medium were preincubated for 24 hours in the presence or absence of phorbol myristate acetate (PMA, 100 ng/ml). MMP1 activity was measured using a spectrophotometric assay and western blotting. MMP2 and MMP9 were measured using zymography. TIMP1 was measured by enzyme linked immunosorbent assay and western blotting. Results-There was little diVerence between the amounts of MMP2 secreted by any of the cell lines. In response to PMA both synovial cell types showed a significantly higher MMP1 and MMP9 activity compared with FSE, although there was no diVerence between RASE and NSE. Tumour necrosis factor had minimal eVect on MMP activity. There was a striking decrease in the amount of TIMP1 secreted by RASE compared with normal synovium. Conclusions-As overall MMP activity is a balance between the amount of MMP and TIMP1 present, the low levels of TIMP1 produced by RASE would shift the balance in favour of increased MMP activity by these cells. This is likely to contribute to the angiogenic potential of RASE.

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