ERK1-2 and p38 MAPK regulate MMP/TIMP balance and function in response to thrombospondin-1 fragments in the microvascular endothelium

Abstract

We found that thrombospondin-1 (TSP-1) has opposite functions on angiogenesis depending on the nature of the proteolytic fragment released in vivo by the action of proteases. We studied the effect of the 25 and 140 kDa fragments of TSP-1 generated by its proteolytic cleavage on the cascade of mitogen activated protein kinase (MAPK) activation and matrix-metalloproteinase (MMP)/tissue inhibitor of metalloproteinase (TIMP) function and expression in microvascular endothelium. Post-capillary endothelial cells (CVEC) isolated from bovine heart were used. The 25 kDa fragment enhanced the upregulation of MMP-2 and -9 and reduced TIMP-2 expression leading to CVEC chemoinvasion. Conversely, the 140 kDa fragment blocked MMP-2 and -9 stimulation and doubled TIMP-2 expression, leading to inhibition of endothelial chemoinvasion induced by fibroblast growth factor-2 (FGF-2). MAPK activity (ERK1-2) was induced by TSP-1 and by the 25 kDa fragment, but not by the 140 kDa fragment which, however, promoted MAPK p38 activation. This evidence indicates that fragments originating from TSP-1 switch the pro- or anti-angiogenic phenotype in endothelium by targeting MAPK cascades with opposite functions on MMP/TIMP balance.

Introduction

Angiogenesis is becoming an attractive therapeutic approach for cardiovascular diseases because the recently discovered families of regulators, both stimulators and inhibitors, have facilitated its practical application (Folkman, 1995). It is now thought that a shift in the balance between stimulators and inhibitors in favor of the former triggers the angiogenic switch in endothelial cells, producing a distinct survival advantage (Bouck, 1996). During angiogenesis, endothelial cells release matrix metalloproteinases (MMPs) and endogenous inhibitors, tissue inhibitor of metalloproteinases (TIMPs), which are essential to extracellular matrix (ECM) turnover. Remodeling of ECM is an important regulator of neovascularization, vascular morphogenesis and vascular invasion. The initiating step in angiogenesis is believed to be the proteolytic degradation of basement membrane by MMPs and other proteases. MMP-2 and -9, in particular, have important roles in angiogenesis Hiraoka et al., 1998, Werb et al., 2001, Stetler-Stevenson, 2001. Mice deficient in MMP-2 and -9 show reduced angiogenesis in vivo (Itoh et al., 1998). By inhibiting MMP activity, TIMPs are important in regulating matrix composition and affect a wide range of physiological processes including cell growth and invasion, angiogenesis and apoptosis.

Thrombospondin-1 (TSP-1), a trimeric 450 kDa glycoprotein, is a matricellular protein that interacts with several matrix components, cell receptors, soluble growth factors and proteolytic enzymes. TSP-1 expression is associated with atherosclerotic lesions, acute vascular injury, hypercholesterolemia and hypertension Reed et al., 1995, Roth et al., 1998. In experimental models and in clinical studies, the loss of TSP-1 production has been associated with the acquisition of an angiogenic phenotype and with tumor progression Bertin et al., 1997, Clezardin et al., 1993, Grossfeld et al., 1997, while restoration of TSP-1 production prevents angiogenesis and reduces tumor growth (Sheibani and Frazier, 1995). Administration of anti-TSP-1 antibodies facilitates re-endothelization and inhibits neointimal thickening in baloon-injured rat carotid artery (Chen et al., 1999). However TSP-1 can also stimulate microvessel formation in rat aorta explants and favours proangiogenic activities in endothelial cells such as invasion and production of MMPs (Qian et al., 1997). TSP-1 activates the ERK1-2 mitogen activated protein kinase (MAPK), influencing important cell functions during hemostasis, wound repair and other inflammatory responses (Wilson et al., 1999). TSP-1 also appears to promote pathologic angiogenesis in tumors, in animal models and patients (Tuszynski and Nicosia, 1994). Recently a positive correlation has been reported between TSP-1 expression and tumor angiogenesis in pancreatic adenocarcinoma (Qian et al., 2001), ductal breast carcinoma (Motegi et al., 2002), and gastric carcinoma (Zhang et al., 2003). We have shown that TSP-1 exerts opposite effects on angiogenesis depending on which domain/fragment is functional in a given biological setting (Taraboletti et al., 2000).

Several distinct MAPKs have been identified that act in independent signalling pathways to affect the pleiotropic functions of this kinase family. These include p42/p44 extracellular signal-related kinase (ERK1-2), c-Jun N-terminal protein kinase (JNK)/stress-activated protein kinase (SAPK), and p38 MAPK Boulton et al., 1991, Kyriakis et al., 1994, Han et al., 1994. These kinases are the terminal targets of signalling cascades, triggered by the binding of growth factors/survival factors or death receptors. Activation of JNK/SAPK and p38 MAPK is generally associated with promotion of apoptosis, while ERK1-2 activity inhibits apoptosis. TSP-1 directly activates the intracellular kinase ERK1-2 in inflammatory and stromal cells Wilson et al., 1999, Gahtan et al., 1999, but nothing is known about how its fragments affect MAPK activity or the implications of MAPK activity in the opposing effects of TSP-1 peptides on endothelial cell functions related to angiogenesis.

The aim of this study was to assess whether in coronary endothelium TSP-1 and the two fragments (25 and 140 kDa) generated by its cleavage affected endothelial cell angiogenic activity (invasion and balance of MMPs/TIMPs) targeting the MAPKs.