Examining the relationship between the gelatinolytic balance and the invasive capacity of endothelial cells

Alain Puyraimond 1 Jonathan B. Weitzman 0 Emeline Babiole 1 Suzanne Menashi ) 1 0 Unite des Virus Oncogenes, URA1644 CNRS, Institut Pasteur , 75724 Paris , France 1 U353 INSERM, Hopital Saint Louis , 75010 Paris , France - capacity of endothelial cells SUMMARY Angiogenesis and the formation of new blood vessels requires coordinated regulation of matrix proteolysis and endothelial cell migration. Cellular proteolytic capacity is the balance between secreted matrix metalloproteinases (MMP) and their inhibitors (TIMPs). We have examined the regulation of the gelatinase/TIMP balance by transforming growth factor-b 1 (TGF-b 1) and phorbol myristate acetate (PMA) in bovine endothelial cells. The low constitutive expression of gelatinase A/MMP-2 was upregulated by TGF-b 1 in a dose-dependent manner. Gelatinase B/MMP-9 was only detected upon treatment with either PMA or TGF-b 1. However, addition of both factors together revealed a striking synergistic effect causing upregulation of MMP-9 and downregulation of TIMPs, thereby increasing the net MMP-9/TIMP balance and the gelatinolytic capacity. These effects were observed Angiogenesis plays a fundamental role in many physiological and pathological processes including wound healing and tumor growth. The formation of new vessels, which involves the migration of stimulated endothelial cells and subsequent tube formation, depends on a tightly controlled proteolysis of the components of the extracellular matrix. The secretion of matrix metalloproteinase (MMP) activity was suggested to constitute an essential step in the angiogenic process since the MMP inhibitors TIMP-1 and TIMP-2 have been shown to inhibit angiogenesis in both in vitro and in vivo model systems (Johnson et al., 1994; Murphy et al., 1993), as have synthetic inhibitors of metalloproteinases activity (Galardy et al., 1994; Taraboletti et al., 1995). However, since these inhibitors are not specific for any particular MMP and can inhibit all members of the MMP family, these studies could not determine which MMP may be involved. The two mammalian gelatinases (gelatinase A/MMP-2 and gelatinase B/MMP-9) are members of the MMP family that specifically degrade gelatin, type-IV, type-V and type-XI collagen (Matrisian, 1990; Murphy et al., 1991). It is assumed that the secretion of gelatinases having specificity for type IV at both the protein and mRNA levels. We demonstrate that changes in different members of the Jun oncogene family with distinct transactivation properties may account for this synergistic effect. We investigated the contribution of these changes in gelatinolytic balance to endothelial cell migration and invasion. The endothelial cells showed increased cell motility in response to PMA, but the addition of TGF-b 1 had an inhibitory effect. Hence, regulation of the MMP-9/TIMP balance failed to correlate with the migratory or invasive capacity. These results question a direct role for MMP-9 in endothelial cell motility and suggest that gelatinases may contribute in alternative ways to the angiogenic process. collagen would endow endothelial cells with an advantage for degradation of the extracellular matrix (ECM) and subsequent migration across the basement membrane. The gelatinases can also cleave a variety of non-ECM molecules such as the basic fibroblast growth factor (bFGF) receptor, galectin-3, IL-1b , substance P, myelin basic protein and amyloid b peptide (Levi et al., 1996; Ochieng et al., 1994; Vu and Werb, 1998). Although MMP-9 can be induced in most cells in culture by specific cytokines and by tumor promoters such as PMA, its expression in vivo is mainly restricted to inflammatory cells and to pathological states such as inflammatory arthritis, tumor invasion, corneal ulcers and Alzheimers disease (Vu and Werb, 1998). MMP-9 is implicated in the invasive behaviour of metastatic tumor cells and trophoblasts (Alexander et al., 1996; Bernhard et al., 1994; Bischof et al., 1995; Himelstein et al., 1994; Stetler-Stevenson, 1990). Furthermore, a role for the gelatinases in angiogenesis has recently been suggested by studies of genetically modified knockout mice (Itoh et al., 1998; Vu et al., 1998). The delayed skeletal growth plate vascularization phenotype of mutant animals implicated MMP9 in the release of angiogenic activators (Vu et al., 1998) and host MMP-2 in tumor progression (Itoh et al., 1998). Tumor progression and the angiogenic switch are regulated by a large range of tumor-secreted factors. Many of these molecules have been shown to have diverse effects on cell proliferation, migration and proteinase secretion. Transforming growth factor-b 1 (TGF-b 1) is a multi-functional cytokine which has both positive and negative effects on endothelial cell functions (Madri et al., 1988; Pepper et al., 1990, 1993; Yang and Moses, 1990). TGF-b 1 can promote angiogenesis in vivo, but is a potent inhibitor of endothelial cell proliferation and migration in vitro. Furthermore, TGF-b 1 is known to regulate ECM by stimulating the synthesis of its components (collagens, fibronectin, tenascin and proteoglycans), by inhibiting matrix degradation through the down-regulation of proteinases such as plasminogen activators, collagenase-1 and stromelysin-1, and by up-regulating proteinase inhibitors such as PAI-1 and TIMP-1 (Kerr et al., 1990; Matrisian et al., 1992; Mauviel et al., 1993). However, TGF-b 1 has been shown to upregulate both the 72 kDa and 92 kDa gelatinases (MMP-2 and MMP-9) in cultured fibroblasts (Overall et al., 1991), keratinocytes (Salo et al., 1991), and in several tumor cell lines (Shimizu et al., 1996; Welch et al., 1990). The tumor promoter phorbol myristate acetate (PMA) has been shown to induce MMP secretion by a large number of mesenchymal cell and invasive tumor cell lines (Crawford and Matrisian, 1996). PMA is also capable of inducing cell invasion and tube formation of both microvascular and large vessel endothelial cells in vitro (Montesano and Orci, 1985; Montesano and Orci, 1987). Although the association between MMPs and tumor progression is well established, few studies have addressed their involvement in the migration of endothelial cells which is essential for neo-vascularization. Furthermore, the effects of several angiogenic regulators on MMP induction has been studied in tumor cell lines, but the mechanisms of MMP regulation in endothelial cells and their role in angiogenesis are still unclear. To address these issues, we have studied the regulation of MMP-2, MMP-9, TIMP-1 and TIMP-2 secretion in bovine endothelial cells treated with TGF-b 1 and PMA, alone or in combination. The consequences of the regulation of the gelatinolytic potential on the migratory and invasive capacity of endothelial cells were also investigated. MATERIALS AND METHODS Calf pulmonary artery endothelial cells (CPAE) were kindly provided by Dr J. Badet (Laboratoire de Biotechnologie des Cellules Eucaryotes, University de Crteil, France) and were gr (...truncated)