Giuseppe Pintucci

TRANSFORMING GROWTH FACTOR-BETA 1 (TGF-β1) INDUCES ANGIOGENESIS THROUGH VASCULAR ENDOTHELIAL GROWTH FACTOR (VEGF)-MEDIATED APOPTOSIS

VEGF and TGF‐β1 induce angiogenesis but have opposing effects on endothelial cells. VEGF protects endothelial cells from apoptosis; TGF‐β1 induces apoptosis. We have previously shown that VEGF/VEGF receptor‐2 (VEGFR2) signaling mediates TGF‐β1 induction of apoptosis. This finding raised an important question: Does this mechanism stimulate or inhibit angiogenesis? Here we report that VEGF‐mediated apoptosis is required for TGF‐β1 induction of angiogenesis. In vitro the apoptotic effect of TGF‐β1 on endothelial cells is rapid and followed by a long period in which the cells are refractory to apoptosis induction by TGF‐β1. Inhibition of VEGF/VEGFR2 signaling abrogates formation of cord‐like structures by TGF‐β1 with an effect comparable to that of z‐VAD, an apoptosis inhibitor. Similarly, genetic deficiency of VEGF abolishes TGF‐β1 upregulation of endothelial cell differentiation and formation of vascular structures in embryoid bodies. In vivo TGF‐β1 induces endothelial cell apoptosis as rapidly as in vitro. Inhibition of VEGF blocks TGF‐β1 induction of both apoptosis and angiogenesis, an effect similar to that of z‐VAD. Thus, TGF‐β1 induction of angiogenesis requires a rapid and transient apoptotic effect mediated by VEGF/VEGFR2. This novel, unexpected role of VEGF and VEGFR2 indicates VEGF‐mediated apoptosis as a potential target to control angiogenesis. J. Cell. Physiol. 219: 449–458, 2009.

VEGF, a prosurvival factor, acts in concert with TGF-β1 to induce endothelial cell apoptosis

VEGF and TGF-β1 are potent angiogenesis inducers with opposing effects on endothelial cells. TGF-β1 induces apoptosis; VEGF protects endothelial cells from apoptosis. We found that TGF-β1 promotes endothelial cell expression of FGF-2, which up-regulates VEGF synthesis. Inhibition of VEGF signaling through VEGF receptor 2 (flk-1) abrogates TGF-β1-induced apoptosis and p38MAPK activation. Inhibition of p38MAPK blocks TGF-β1-induced apoptosis, showing that VEGF/flk-1-mediated activation of p38MAPK is required for TGF-β1 induction of apoptosis. In the absence of TGF-β1, VEGF activates p38MAPK and promotes endothelial cell survival. However, in context with TGF-β1, VEGF/flk-1-mediated activation of p38MAPK results in apoptosis. Thus, cross-talk between TGF-β1 and VEGF signaling converts VEGF/flk-1-activated p38MAPK into a proapoptotic signal. This finding illustrates an unexpected role of VEGF and indicates that VEGF can be pharmacologically converted into an apoptotic factor, a novel approach to antiangiogenesis therapy.

Lack of ERK activation and cell migration in FGF-2-deficient endothelial cells

The formation of blood capillaries from preexisting vessels (angiogenesis) and vascular remodeling secondary to atherosclerosis or vessel injury are characterized by endothelial cell migration and proliferation. Numerous growth factors control these cell functions. Basic fibroblast growth factor (FGF‐2), a potent angiogenesis inducer, stimulates endothelial cell proliferation, migration, and proteinase production in vitro and in vivo. However, mice genetically deficient in FGF‐2 have no apparent vascular defects. We have observed that endothelial cell migration in response to mechanical damage in vitro is accompanied by activation of the extracellular signal‐regulated kinase (ERK) pathway, which can be blocked by neutralizing anti‐FGF‐2 antibodies. Endothelial cells from mice that are genetically deficient in FGF‐2 neither migrate nor activate ERK in response to mechanical wounding. Addition of exogenous FGF‐2 restores a normal cell response, which shows that impaired migration results from the genetic deficiency of this growth factor. Injury‐induced ERK activation in endothelial cells occurs only at the edge of the wound. In addition, FGF‐2‐induced ERK activation mediates endothelial cell migration in response to wounding without a significant effect on proliferation. These data show that FGF‐2 is a key regulator of endothelial cell migration during wound repair.

MAPK-dependent expression of p21WAF and p27kip1 in PMA-induced differentiation of HL60 cells

Treatment of HL60 cells with phorbol 12‐myristate 13‐acetate (PMA) results in growth arrest and differentiation towards the macrophage lineage. PMA‐induced changes are easily monitored by morphological changes while cells in suspension start adhering onto substrate. PMA induces rapid activation of the extracellular signal‐regulated kinases (ERKs). Activation of the ERK pathway is essential to PMA‐induced differentiation of HL60 cells. PMA also induces the expression of the cyclin‐dependent kinase inhibitors p21WAF and p27kip1, which is modulated by the use of an inhibitor of the ERK cascade. This implies that a link exists between ERK activation and p21WAF and p27kip1 induction in the process of terminal differentiation.

Tissue Inhibitor of Metalloproteinases-2 Binding to Membrane-type 1 Matrix Metalloproteinase Induces MAPK Activation and Cell Growth by a Non-proteolytic Mechanism

Membrane-type 1 matrix metalloproteinase (MT1-MMP), a transmembrane proteinase with a short cytoplasmic domain and an extracellular catalytic domain, controls a variety of physiological and pathological processes through the proteolytic degradation of extracellular or transmembrane proteins. MT1-MMP forms a complex on the cell membrane with its physiological protein inhibitor, tissue inhibitor of metalloproteinases-2 (TIMP-2). Here we show that, in addition to extracellular proteolysis, MT1-MMP and TIMP-2 control cell proliferation and migration through a non-proteolytic mechanism. TIMP-2 binding to MT1-MMP induces activation of ERK1/2 by a mechanism that does not require the proteolytic activity and is mediated by the cytoplasmic tail of MT1-MMP. MT1-MMP-mediated activation of ERK1/2 up-regulates cell migration and proliferation in vitro independently of extracellular matrix proteolysis. Proteolytically inactive MT1-MMP promotes tumor growth in vivo, whereas proteolytically active MT1-MMP devoid of cytoplasmic tail does not have this effect. These findings illustrate a novel role for MT1-MMP-TIMP-2 interaction, which controls cell functions by a mechanism independent of extracellular matrix degradation.

Basic fibroblast growth factor (FGF‐2): The high molecular weight forms come of age

After over thirty years from its discovery, research on basic fibroblast growth factor (FGF‐2) keeps revealing new aspects of the complexity of its gene expression as it evolved in the eukaryotic organisms. The discovery of multiple forms of FGF‐2 generated by alternative translation from AUG and non‐canonical CUG codons on the same mRNA transcript has led to the characterization of a low molecular weight (LMW) FGF‐2 form and various high molecular weight (HMW) forms (four in humans). In this review, we discuss the biochemical features and biological activities of the different FGF‐2 forms. In particular, we focus on the properties that are unique to the HMW forms and its biological functions. J. Cell. Biochem. 100: 1100–1108, 2007.

Correlation between plasma osteopontin levels and aortic valve calcification: Potential insights into the pathogenesis of aortic valve calcification and stenosis

OBJECTIVE The inflammatory process of aortic stenosis involves the differentiation of aortic valve myofibroblasts into osteoblasts. Osteopontin, a proinflammatory glycoprotein, both stimulates differentiation of myofibroblasts and regulates the deposition of calcium by osteoblasts. Osteopontin levels are increased in patients with such conditions as end-stage renal disease, ectopic calcification, and autoimmune disease. We hypothesized that increased plasma osteopontin levels might be associated with the presence of aortic valve calcification and stenosis. METHODS Venous blood from volunteers older than 65 years undergoing routine echocardiographic analysis or aortic valve surgery for aortic stenosis was collected. Plasma osteopontin levels were measured by means of enzyme-linked immunosorbent assay. The presence of aortic stenosis was defined as an aortic valve area of less than 2.0 cm(2). Aortic valve calcification was assessed by using a validated echocardiographic grading system (1, none; 2, mild; 3, moderate; 4, severe). Comparisons were performed with nonpaired t tests. RESULTS Aortic stenosis was present in 23 patients (mean age, 78 years) and was absent in 7 patients (mean age, 72 years). Aortic valve calcification scores were 3.5 +/- 0.6 and 1.3 +/- 0.5 in patients with and without aortic stenosis, respectively (P < .001). Patients with no or mild aortic valve calcification had lower osteopontin levels compared with patients with moderate or severe aortic valve calcification (406.1 +/- 165.8 vs 629.5 +/- 227.5 ng/mL, P = .01). Similarly, patients with aortic stenosis had higher osteopontin levels compared with patients without aortic stenosis (652.2 +/- 218.7 vs 379.7 +/- 159.9 ng/mL, P < .01). CONCLUSION Increased levels of plasma osteopontin are associated with the presence of aortic valve calcification and stenosis. These findings suggest that osteopontin might play a functional role in the pathogenesis of calcific aortic stenosis.

Induction of stromelysin‐1 (MMP‐3) by fibroblast growth factor‐2 (FGF‐2) in FGF‐2−/− microvascular endothelial cells requires prolonged activation of extracellular signal‐regulated kinases‐1 and ‐2 (ERK‐1/2)

Basic fibroblast growth factor (FGF‐2) and matrix metalloproteinases (MMPs) play key roles in vascular remodeling. Because FGF‐2 controls a number of proteolytic activities in various cell types, we tested its effect on vascular endothelial cell expression of MMP‐3 (stromelysin‐1), a broad‐spectrum proteinase implicated in coronary atherosclerosis. Endothelial cells (EC) from FGF‐2−/− mice are highly responsive to exogenous FGF‐2 and were therefore used for this study. The results showed that treatment of microvascular EC with human recombinant FGF‐2 results in strong induction of MMP‐3 mRNA and protein expression. Upregulation of MMP‐3 mRNA by FGF‐2 requires de novo protein synthesis and activation of the ERK‐1/2 pathway. FGF‐2 concentrations (5–10 ng/ml) that induce rapid and prolonged (24 h) activation of ERK‐1/2 upregulate MMP‐3 expression. In contrast, lower concentrations (1–2 ng/ml) that induce robust but transient (<8 h) ERK‐1/2 activation are ineffective. Inhibition of ERK‐1/2 activation at different times (−0.5 h to +8 h) of EC treatment with effective FGF‐2 concentrations blocks MMP‐3 upregulation. Thus, FGF‐2 induces EC expression of MMP‐3 with a threshold dose effect that requires sustained activation of the ERK‐1/2 pathway. Because FGF‐2 controls other EC functions with a linear dose effect, these features indicate a unique role of MMP‐3 in vascular remodeling.

Mechanical endothelial damage results in basic fibroblast growth factor–mediated activation of extracellular signal-regulated kinases

BACKGROUND Endothelial damage, such as that associated with balloon angioplasty or preparation of veins for bypass grafts, results in intimal hyperplasia. Growth factors and cytokines that modulate endothelial cell functions through various intracellular signaling pathways mediate rapid endothelial repair, which may prevent or reduce restenosis. Here we investigated the effect of mechanical injury of endothelial cells on the mitogen-activated kinase signaling pathways, extracellular-signal-regulated kinases (ERKs), C-Jun N-terminal kinase (JNK/SAPK), and p38. METHODS Confluent human umbilical vein endothelial cells or bovine aortic endothelial cells were wounded with a razor blade; mitogen-activated kinase activation was monitored by immunoblotting with antibodies to active ERK, JNK/SAPK, or p38. RESULTS Wounding of human umbilical vein endothelial cell or bovine aortic endothelial cell monolayers resulted in rapid (5-minute) activation of ERK-1 and -2, which was abolished by monoclonal antibody to basic fibroblast growth factor (FGF-2). This antibody or an inhibitor of ERK activation, PD98059, also blocked endothelial cell migration after the wounding. Thus FGF-2-induced ERK activation mediates the endothelial response to wounding. CONCLUSIONS ERK-1 and -2 are activated by FGF-2 released from endothelial cells in response to injury. Therapeutic strategies aimed at reducing FGF-2-induced intimal hyperplasia should preserve ERK activation in endothelial cells while abolishing it in smooth muscle cells.

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 bodys 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.