Protein targets of inflammatory serine proteases and cardiovascular disease

Serine proteases are a key component of the inflammatory response as they are discharged from activated leukocytes and mast cells or generated through the coagulation cascade. Their enzymatic activity plays a major role in the body's defense mechanisms but it has also an impact on vascular homeostasis and tissue remodeling. Here we focus on the biological role of serine proteases in the context of cardiovascular disease and their mechanism(s) of action in determining specific vascular and tissue phenotypes. Protease-activated receptors (PARs) mediate serine protease effects; however, these proteases also exert a number of biological activities independent of PARs as they target specific protein substrates implicated in vascular remodeling and the development of cardiovascular disease thus controlling their activities. In this review both PAR-dependent and -independent mechanisms of action of serine proteases are discussed for their relevance to vascular homeostasis and structural/functional alterations of the cardiovascular system. The elucidation of these mechanisms will lead to a better understanding of the molecular forces that control vascular and tissue homeostasis and to effective preventative and therapeutic approaches

Urokinase Plasminogen Activator and Gelatinases Are Associated with Membrane Vesicles Shed by Human HT1080 Fibrosarcoma Cells

Membrane vesicles are shed by tumor cells both in vivo and in vitro. Although their functions are not well understood, it has been proposed that they may play multiple roles in tumor progression. We characterized membrane vesicles from human HT1080 fibrosarcoma cell cultures for the presence of proteinases involved in tumor invasion. By gelatin zymography and Western blotting, these vesicles showed major bands corresponding to the zymogen and active forms of gelatinase B (MMP-9) and gelatinase A (MMP-2) and to the MMP-9. tissue inhibitor of metalloproteinase 1 complex. Both gelatinases appeared to be associated with the vesicle membrane. HT1080 cell vesicles also showed a strong, plasminogen-dependent fibrinolytic activity in 125I fibrin assays; this activity was associated with urokinase plasminogen activator, as shown by casein zymography and Western blotting. Urokinase was bound to its high affinity receptor on the vesicle membrane. Addition of plasminogen resulted in activation of the progelatinases associated with the vesicles, indicating a role of the urokinase-plasmin system in MMP-2 and MMP-9 activation. We propose that vesicles shed by tumor cells may provide a large membrane surface for the activation of membrane-associated proteinases involved in extracellular matrix degradation and tissue invasion

The cardiovascular effects of proteolysis of high molecular weight basic fibroblast growth factor by inflammatory serine proteases

Abstract Cardiovascular disease is characterized by structural and functional changes of blood vessel's wall that lead to reduced blood flow and eventually occlusion. The integrity of the vascular wall is maintained by homeostatic mechanisms controlled by the endothelium. Stress caused by age, oxidants, mechanical injury, and inflammation can result in endothelial dysfunction leading to remodeling of the vessel wall. We found that thrombin, a key protease of the coagulation cascade and inflammatory response, cleaves the high molecular weight (HMW) forms of basic fibroblast growth factor (FGF-2), a ubiquitous protein with trophic effects on vascular cells. The C-terminal fragment of FGF-2 generated by thrombin is similar to low molecular weight (LMW; 18 kDa) FGF-2, and induces vascular cell activation of the mitogen-activated protein kinases ERK-1/2, migration and proliferation. The N-terminal fragment generated by thrombin cleavage of HMW FGF-2 contains a sequence rich in asymmetric-dimethyl-arginine (ADMA) residues. In free form ADMA inhibits key reactions for blood vessel homeostasis such as nitric oxide synthesis, and its serum levels, elevated in diabetes, renal failure, hypertension, and hypercholesterolemia, correlate with a poor prognosis in cardiovascular patients. We found that thrombin cleavage of HMW FGF-2 dramatically upregulates intracellular ADMA levels in cultured cells. Thus, the C-terminal cleavage product of HMW FGF-2 can activate intracellular signaling and control vascular cell functions, while the N-terminal fragment of FGF-2 generates ADMA, a powerful inhibitor of nitric oxide synthesis. We hypothesize that upon vascular injury HMW FGF-2 is processed by inflammatory serine proteases such as thrombin generating molecules that accelerate the development of intimal hyperplasia. This novel mechanism implicates human HMW FGF-2 in the pathogenetic mechanisms of vascular injury occurring in hypertension, diabetes, and dyslipidemia, conditions that are all characterized by elevated serum levels of ADMA. The elucidation of these mechanisms will foster the development of new pharmacological tools for the treatment of the cardiovascular disorders associated with these diseases

Vascular endothelial growth factor increases urokinase receptor expression in vascular endothelial cells

Vascular endothelial growth factor (VEGF) is a potent angiogenic factor and endothelial cell-specific mitogen that stimulates urokinase-type plasminogen activator (uPA) activity in vascular endothelial cells. Here, we report that VEGF increases the high affinity binding of uPA to the same cells and that this binding is prevented by a peptide corresponding to the uPA receptor (uPAR) binding growth factor-like domain of uPA. Ligand cross-linking, ligand blotting, and uPA-Sepharose affinity chromatography revealed an increase in a cell surface uPA binding protein that corresponds to the uPAR on the basis of its affinity for uPA, M(r) of 50,000-55,000, and phosphatidylinositol-specific phospholipase C sensitivity. By Scatchard analysis, VEGF increased the number of uPAR molecules by 2.8-3.5-fold and concomitantly decreased their affinity for uPA. By northern blotting uPAR mRNA was increased in a dose- and time-dependent manner in response to VEGF. Taken together, these findings demonstrate that VEGF-induced angiogenesis is accompanied by increased uPAR expression and uPA activity on the endothelial cell surface. These observations are consistent with the notion that the uPA-uPAR interaction facilitates cellular invasion

Principi e norme pratiche di radioprotezione:breve guida ad uso degli operatori del Dipartimento di Genetica e Microbiologia

ed. Università degli Studi di Pavi

TIMP-2 interaction with MT1-MMP activates the AKT pathway and protects tumor cells from Apoptosis

Membrane-type 1 matrix metalloproteinase (MT1-MMP), a transmembrane proteinase with an extracellular catalytic domain and a short cytoplasmic tail, degrades a variety of extracellular matrix (ECM) components. In addition, MT1-MMP activates intracellular signaling through proteolysis-dependent and independent mechanisms. We have previously shown that binding of tissue inhibitor of metalloproteinases-2 (TIMP-2) to MT1-MMP controls cell proliferation and migration, as well as tumor growth in vivo by activating the Ras-extracellular signal regulated kinase-1 and -2 (ERK1/2) pathway through a mechanism that requires the cytoplasmic but not the proteolytic domain of MT1-MMP. Here we show that in MT1-MMP expressing cells TIMP-2 also induces rapid and sustained activation of AKT in a dose- and time-dependent manner and by a mechanism independent of the proteolytic activity of MT1-MMP. Fibroblast growth factor receptor-1 mediates TIMP-2 induction of ERK1/2 but not of AKT activation; however, Ras activation is necessary to transduce the TIMP-2-activated signal to both the ERK1/2 and AKT pathways. ERK1/2 and AKT activation by TIMP-2 binding to MT1-MMP protects tumor cells from apoptosis induced by serum starvation. Conversely, TIMP-2 upregulates apoptosis induced by three-dimensional type I collagen in epithelial cancer cells. Thus, TIMP-2 interaction with MT1-MMP provides tumor cells with either pro- or anti-apoptotic signaling depending on the extracellular environment and apoptotic stimulus

Membrane-Type 1 matrix metalloproteinase downregulates fibroblast growth factor-2 binding to the cell surface and intracellular signaling

Membrane-type 1 matrix metalloproteinase (MT1-MMP, MMP-14), a transmembrane proteinase with an extracellular catalytic domain and a short cytoplasmic tail, degrades extracellular matrix components and controls diverse cell functions through proteolytic and non-proteolytic interactions with extracellular, intracellular, and transmembrane proteins. Here we show that in tumor cells MT1-MMP downregulates fibroblast growth factor-2 (FGF-2) signaling by reducing the amount of FGF-2 bound to the cell surface with high and low affinity. FGF-2 induces weaker activation of ERK1/2 MAP kinase in MT1-MMP expressing cells than in cells devoid of MT1-MMP. This effect is abolished in cells that express proteolytically inactive MT1-MMP but persists in cells expressing MT1-MMP mutants devoid of hemopexin-like or cytoplasmic domain, showing that FGF-2 signaling is downregulated by MT1-MMP proteolytic activity. MT1-MMP expression results in downregulation of FGFR-1 and -4, and in decreased amount of cell surface-associated FGF-2. In addition, MT1-MMP strongly reduces the amount of FGF-2 bound to the cell surface with low affinity. Because FGF-2 association with low-affinity binding sites is a prerequisite for binding to its high-affinity receptors, downregulation of low-affinity binding to the cell surface results in decreased FGF-2 signaling. Consistent with this conclusion, FGF-2 induction of tumor cell migration and invasion in vitro is stronger in cells devoid of MT1- MMP than in MT1-MMP expressing cells. Thus, MT1-MMP controls FGF-2 signaling by a proteolytic mechanism that decreases the cell's biological response to FGF-2

Expression of the urokinase receptor in vascular endothelial cells is stimulated by basic fibroblast growth factor

Abstract. Basic fibroblast growth factor, a potent angiogenesis inducer, stimulates urokinase (uPA) production by vascular endothelial cells. In both basic fibroblast growth factor-stimulated and-nonstimulated bovine capillary endothelial and human umbilical vein endothelial cells single-chain uPA binding is mediated by a membrane protein with a Mr of 42,000. Exposure of bovine capillary or endothelial human umbilical vein endothelial cells to pmolar concentrations of basic fibroblast growth factor results in a dose-dependent, protein synthesis-dependent increase in the number of membrane receptors for uPA (19,500-187,000) and in a parallel decrease in their affinity (KD = 0.144-0.790 nM). With both cells, single-chain uPA binding is competed by synthetic peptides whose sequence correspond