Tissue inhibitor of metalfoproteinase-2 (TIMP-2), a protease inhibitor that binds to the latent and active forms of 72 kDa type IV collagenase (gelatinase A), was found to inhibit the in vitro proliferation of human microvascular endothelial (HME) cells stimulated with bFGF and 5% serum. The maximal inhibitory effect of TIMP-2 on incorporation of 3H-thymidine was evident 24 hours after bFGF stimulation of these cells and ranged between 45 and 60%. The half-maximal effective concentration of TIMP-2 was 107 * 12 n M (S.D.). In contrast, TIMP-1 was not found to slow the growth of HME cells. The inhibition of cell proliferation observed with TIMP-2 was not mimicked by addition to the culture medium of 8894, a general matrix metalloproteinase inhibitor, nor antibodies to the 72 kDa type IV collagenase. In addition to growth, two other cell functions associated with the angiogenic process were tested for sensitivity to TIMP-2. Cell adhesion to tissue culture plastic was slightly stimulated by TIMP-2, and cell migration was inhibited with short-term exposure to TIMP-2, but neither process was affected by longer-term exposure. The ability of TIMP-2 to inhibit cultured endothelial cell proliferation independent of protease inhibitory activity suggests that TIMP-2 may have additional actions which may limit neovascularization associated with solid tumor growth and metastasis in vivo.
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Normal physiological capillary growth and morphogenesis occur only during embryonic growth, menses, and wound healing. However, in a number of pathological conditions, such as in solid tumor growth, diabetic retinopathy, and rheumatoid arthritis, the process of angiogenesis actually contributes to the disease state. In particular, significant data have accumulated which suggest that the growth of solid tumors is dependent on angiogenesis (for review, see Folkman and Klagsbrun, 1987). This process may be initiated by tumor cells which are capable of elaborating a number of factors that either directly or indirectly stimulate neovascularization, such as heparin binding growth factors, angiogenin, and transforming growth factor beta (Folkman and Klagsbrun, 1987). Inhibitors of angiogenesis may be clinically useful in limiting the contributions of this process to pathological progression of the underlying disease states.
Angiogenesis and tumor cell invasion and metastasis share common mechanistic steps comprising interactions between the cells and their extracellular matrix including adhesion, proteolysis, cell migration, and proliferation (Liotta et al., 1991;Van Roy and Mareel, 1992). In vitro assays of both processes reveal that endothelial cells and invasive tumor cells secrete proteases that mediate degradation of components of the extracellular matrix. Specifically, collagenases and other matrix metalloproteinases appear to play a key role in this proteolysis (Liotta et al., 1991;Mignatti et al., 1986). Potential therapeutic approaches to the inhi-