Portal de Periódicos da CAPES

The role of mast cells in tumour angiogenesis

2001; Wiley; Volume: 115; Issue: 3 Linguagem: Inglês

10.1046/j.1365-2141.2001.03202.x

1365-2141

Doménico Ribatti, Angelo Vacca, Beatrice Nico, Enrico Crivellato, Luisa Roncali, Franco Dammacco,

Cell Adhesion Molecules Research

Mast cells (MCs) were first described by Paul Ehrlich in his doctoral thesis (Ehrlich, 1878). He discovered these cells in various organs of several animal species and 'recognized their tendency to concentrate around blood vessels, nerves and glandular ducts as well as in inflammatory and neoplastic foci' (Selye, 1965). MCs are derived from precursors of the haematopoietic lineage and complete their differentiation in peripheral tissues (Galli, 1990). All mammalian MCs express common characteristics, including plasma membrane receptors binding IgE antibodies and cytoplasmic granules storing biogenic amines, proteoglycans, cytokines and neutral proteases 1, 2. However, MC populations show marked differences in their phenotypic expression in distinct anatomical sites, a phenomenon called 'mast cell heterogeneity' (Galli, 1990). Semi-thin section of three rat peritoneal mast cells stained with toluidine blue. Numerous cytoplasmic metachromatic granules are recognizable. Original magnification ×1000. Ultrastructural finding of a resting human mast cell showing a monolobed nucleus, narrow surface folds and numerous electron-dense cytoplasmic granules. Original magnification ×15 000. Human MCs are conventionally divided into two types depending on the expression of different proteases in their granules. MCT cells (also regarded as immune cell associated) contain tryptase and are predominantly located in the respiratory and intestinal mucosa, where they co-localize around T lymphocytes. MCTC cells contain both tryptase and chymase and are predominantly found in connective tissue areas, such as skin, conjunctiva and synovium. Typically, MCs stain metachromatically, because of the presence of the sulphated glycosaminoglycan heparin. In many organs and under physiological conditions, MCs are numerous close to capillaries (Eady et al, 1979; Rhodin & Fujita, 1989; Rakusan et al, 1990). Maintenance of MCs by endothelial cells is possible, as human dermal endothelial cells express the MC growth and chemotactic factor stem cell factor (SCF) (Nilsson et al, 1994; Meininger & Zetter, 1995). Eady et al (1979) found that the number of MCs in the dermis correlates with blood vessel density. Rakusan et al (1990) demonstrated that a rapid increase in MC density takes place in the myocardium in the same post-natal period as rapid capillary formation. MCs are the recognized key cells of type I hypersensitivity reactions. However, their ubiquitous distribution throughout both serosal and mucosal tissues and their close proximity to blood vessels have suggested their involvement in several other diseases. Their numbers, in fact, rise in pathological conditions accompanied by an increase in angiogenic activity, such as psoriasis, atherosclerosis, rheumatoid arthritis, haemangioma and other neoplasms (Schubert & Christophers, 1985; Mican & Metcalfe, 1990; Meininger et al, 1992; Norrby & Woolley, 1993; Atkinson et al, 1994; Dimitriadou & Koutsilieris, 1997). Angiogenesis refers to the formation of capillaries from pre-existing vessels, i.e. capillaries and post-capillary venules (Risau, 1997), and is the result of endothelial sprouting or intussusceptive (non-sprouting) microvascular growth (Ausprunk & Folkman, 1977; Burri & Tarek, 1990). Under physiological conditions, angiogenesis is dependent on the balance between positive and negative angiogenic modulators within the vascular microenvironment (Pepper, 1997) and requires the activities of a number of molecules, including angiogenic factors, extracellular matrix proteins, adhesion receptors and proteolytic enzymes. The distinct gene expression pattern of angiogenic endothelial cells is therefore characterized by a switch of the cell proteolytic balance towards an invasive phenotype as well as by the expression of specific adhesion molecules (Korff & Augustin, 1999). Angiogenic factors are potent growth factors that promote proliferation and differentiation of vascular endothelial cells. Several have been identified, including vascular endothelial growth factor (VEGF), fibroblast growth factor-2 (FGF-2), transforming growth factor β (TGF-β), hepatocyte growth factor (HGF), tumour necrosis factor α (TNF-α), angiogenin, interleukin-8 (IL-8) and the angiopoietins-1 and -2 (Ang-1 and Ang-2). Ang-1 is associated with developing vessels, and its absence leads to a defect in vascular remodelling, whereas Ang-2, which antagonizes Ang-1, plays a role in the destabilization of existing vessels and is found in tissues such as the ovary, uterus and placenta that undergo transient or periodic growth and vascularization, followed by regression (Holash et al, 1999). The major natural negative regulators of angiogenesis include thrombospondin-1 (TSP-1), interferon-α (IFN-α), platelet factor 4 (PF-4), tissue inhibitors of metalloproteinases (TIMPs), angiostatin and endostatin. MCs are a rich source of preformed and newly synthesized cytokines and growth factors that induce or modulate angiogenesis. The most important of these mediators are TNF-α, IL-8, FGF-2 and VEGF (Gordon & Galli, 1990; Moller et al, 1993; Grutzkau et al, 1998). Qu et al (1998) have demonstrated FGF-2 immunolocalization in secretory granules of both resting and activated MCs. The ability of MCs to store angiogenic growth factors and their cell-specific release of preformed factors into the surrounding tissue by piecemeal degranulation (Dvorak & Kissel, 1991) indicate that their granules are a depot for endothelial survival factors. Piecemeal degranulation is also of clinical significance in diseases characterized by fibroproliferation and neovascularization (Dvorak, 1992). MCs are also a rich source of proteases, specifically tryptase (Hopsu & Glenner, 1963; Schwartz et al, 1981) and chymase (Schechter et al, 1983; Wintroub et al, 1986; Sayama et al, 1987). Both enzymes are involved in angiogenesis after their release from the granules of activated MCs. Their proteolytic activities directly degrade extracellular matrix components or release matrix-associated growth factors (Taipale et al, 1995), and they also act more indirectly by activating latent matrix-degrading metalloproteases (Gruber et al, 1989). Blair et al (1997) have investigated in vitro the angiogenic potential of tryptase released by MCs and demonstrated its important role in neovascularization. Tryptase added to microvascular endothelial cells cultured on Matrigel caused a pronounced increase in capillary growth, and this was suppressed by specific tryptase inhibitors. Moreover, tryptase directly induced endothelial cell proliferation in a dose-dependent fashion. Other MC-specific mediators with angiogenic properties include histamine and heparin (Norrby, 1985; Ribatti et al, 1987). Heparin stimulates endothelial cell proliferation and migration in vitro (Thorton et al, 1983; Alessandri et al, 1984). In vivo, however, it has been found to stimulate (Ribatti et al, 1987; Norrby & Sorbo, 1992; Norrby, 1993), inhibit (Jakobson & Hahnenberger, 1991; Wilks et al, 1991; Norrby, 1993) or have no effect (Castellot et al, 1982; Taylor & Folkman, 1982), although these differences seem to be related to its molecular size and degree of sulphation. The 22-kDa and 2·4-kDa heparin fractions display stimulatory and inhibitory properties respectively (Norrby, 1993). N-sulphate, but not O-sulphate, groups are necessary for the release of the extracellular matrix (heparan sulphate)-bound FGF-2, in that their total replacement by acetyl or hexanoyl groups, despite the normal O-sulphate content, abolishes the FGF-2-releasing activity (Ishai-Michaeli et al, 1992). Heparin acts as a soluble form of the low-affinity FGF-2 receptor (Folkman & Shing, 1992), which displaces FGF-2 in the biologically active form, allowing its rapid interaction with endothelial cells (Yayou et al, 1991). Protamine sulphate, a heparin inhibitor, blocks angiogenesis (Taylor & Folkman, 1982). Heparanase is closely involved in angiogenesis, both directly by promoting invasion of endothelial cells (vascular sprouting) and indirectly by releasing heparan sulphate-bound FGF-2 and generating heparan sulphate degradation fragments that promote FGF-2 activity (Vlodavski et al, 2000). Histamine might also contribute to the hyperpermeable nature of newly formed microvessels during pathological angiogenesis. The increased vascular permeability induced by histamine may increase leakage of plasma proteins and hence deposition of fibrin. Degradation products of fibrin are angiogenic in the chick embryo chorioallantoic membrane (CAM) (Thompson et al, 1995). Norrby et al (1986) demonstrated that active MC secretion induced by repeated intraperitoneal injection of compound 48/80, a highly selective MC secretagogue, resulted in marked mesenteric neovascularization in rats and mice, as determined by the vascularized area and the vascular density. Further indirect support for a functional link between MCs and neovascularization was obtained by Roche (1985) in cell culture experiments showing that isolated rat and mouse MC granules are mitogenic to human venous endothelial cells. Moreover, rat MC granules added to cultured human microvascular endothelial cells exerted a marked proliferative effect (Marks et al, 1986). The chorioallantoic membrane (CAM) is an extraembryonic membrane that serves as a gas exchange surface (Romanoff, 1960), and its function is supported by an extensive capillary network (Fig 3). It is currently used as a target for the study of angiogenic and anti-angiogenic compounds (Ribatti et al, 1996a; 2000a). Macroscopic figure of the chick embryo chorioallantoic membrane (asterisk), showing in ovo distribution pattern of the allantoic vessels. Original magnification ×25. Histamine and heparin stimulate the proliferation of endothelial cells and induce the formation of new blood vessels in the CAM assay (Ribatti et al, 1987; Thompson & Brown, 1987). Activation of connective tissue MCs by compound 48/80 promoted angiogenesis in the CAM (Clinton et al, 1988). In addition, Kessler et al (1976) reported that a 40-fold increase in the number of MCs preceded angiogenesis induced by tumour implants in the CAM. We have compared the angiogenic potential of cell suspensions of MCs isolated from rats, degranulated MCs and their secretory granules adsorbed on gelatin sponges and implanted on top of the developing CAM (Ribatti et al, 2001). The results showed that isolated MCs and their secretory granules, but not degranulated MCs, induced an angiogenic response. The addition of anti-FGF-2 or anti-VEGF antibodies reduced these responses by 50% and 30% respectively. Angiogenesis and the production of angiogenic factors are fundamental for tumour progression in the form of growth, invasion and metastasis (Folkman, 1995a). New vessels promote growth by conveying oxygen and nutrients and removing catabolities, whereas endothelial cells secrete growth factors for tumour cells (Hamada et al, 1992; Folkman, 1995a) and a variety of matrix-degrading proteinases that facilitate invasion (Mignatti & Rifkin, 1993). An expanding endothelial surface also gives tumour cells more opportunities to enter the circulation and metastasize (Aznavoorian et al, 1993), whereas the release of anti-angiogenic factors by tumour cells explains the control exerted by primary tumours over metastasis (Chen et al, 1995; Folkman, 1995b). These observations suggest that tumour angiogenesis is linked to a switch in the equilibrium between positive and negative regulators (Hanahan & Folkman, 1996). In normal tissues, vascular quiescence is maintained by the dominant influence of endogenous angiogenesis inhibitors over angiogenic stimuli. Tumour angiogenesis, on the other hand, is induced by increased secretion of angiogenic factors and/or downregulation of angiogenesis inhibitors. Solid tumour growth consists of an avascular and a subsequent vascular phase. Assuming that it is dependent on angiogenesis and that this depends on the release of angiogenic factors, the acquisition of angiogenic capability can be seen as an expression of progression from neoplastic transformation to tumour growth and metastasis (Folkman, 1990). Practically all solid tumours, including tumours of the colon, lung, breast, cervix, bladder, prostate and pancreas, progress through these phases. Tumour cells may not be the only source of angiogenic factors within a tumour. Host inflammatory cells, including lymphocytes, neutrophils, fibroblasts, macrophages and MCs, which are recruited and activated by tumour cells via paracrine mechanisms, act synergically with these cells by secreting the same or other factors (Polverini, 1996). Ehrlich (1879) found many MCs in tumours, especially carcinomas. It was left to his pupil, Westphal (1891), to recognize that they tended to congregate at the periphery of carcinomatous nodules, rather than in the core of a tumour. Experimentally induced tumours display MC accumulation close to the tumour cells before the onset of angiogenesis (Kessler et al, 1976). Likewise, an increase in MC number has been observed in tumour invasion around rat adenocarcinoma (Dabbous et al, 1986) and in hamster epidermal carcinoma (Dabbous et al, 1986). Tumours induced in MC-deficient mice display both reduced angiogenesis and the ability to metastasize (Starkey et al, 1988; Dethlefsen et al, 1994). Angiogenesis is restored after local reconstitution of MCs (Starkey et al, 1988). In tumours, MCs are recruited and activated via several factors secreted by tumour cells: the c-kit receptor or stem cell factor (Poole & Zetter, 1983; Norrby & Wooley, 1993), as well as FGF-2, VEGF and platelet-derived endothelial cell growth factor (PD-ECGF), which are operative at picomolar concentrations (Gruber et al, 1995). MC accumulation has been associated with enhanced growth and invasion of human mammary carcinoma (Hartveit, 1981; Hartveit et al, 1984), cervical carcinoma (Dunn & Montogomery, 1957), gastric cancer (Yano et al, 1999), rectal cancer (Fisher et al, 1989), haemangioma (Glowacki & Milltan, 1982; Qu et al, 1995), Kaposi's sarcoma (Hagiwara et al, 1999), lung cancer (Takanami et al, 2000; Tomita et al, 2000), laryngeal squamous cell carcinoma (Sawatsubashi et al, 2000) and a variety of skin tumours (Cawley & Hoch-Ligeti, 1961). Glowacki & Milltan (1982) found that the density of MCs in rapidly forming haemangiomas was at least five times higher than in normal skin and that the MC number fell to normal when they regressed. The functional significance of tumour-infiltrating MCs is not entirely clear. They are thought to act as a host response to neoplasia (Nakanishi et al, 1994) and display tumoricidal activity in some in vitro assays (Farram & Nelson, 1980; Henderson et al, 1981). However, their accumulation has also been observed in experimentally induced tumours before the onset of tumour-associated angiogenesis (Kessler et al, 1976). On the other hand, tumours in MC-deficient mice have reduced vascularity and produce fewer metastases (Starkey et al, 1988; Dethlefsen et al, 1994). These findings point to a direct involvement of MCs in tumour angiogenesis by releasing several angiogenic factors through the release of specific angiogenic cytokines or modulators of angiogenesis from their secretory granules. The role of angiogenesis in the growth, progression and metastatic spread of solid tumours is well established. The progression of several cancers of haematopoietic lineage, including non-Hodgkin's lymphomas (NHL), lymphoblastic leukaemia, B-cell chronic lymphocytic leukaemia, acute myeloid leukaemia and multiple myeloma (MM), is also clearly related to the degree of angiogenesis (Bertolini et al, 2000). We have demonstrated previously that angiogenesis is correlated with tumour growth (S-phase fraction) in monoclonal gammopathies of undetermined significance (MGUS) and MM grouped according to a pathway of progression (Vacca et al, 1994, 1999), and with progression stages in both B-cell NHL (B-NHL) (Ribatti et al, 1996b) and mycosis fungoides (Vacca et al, 1997). Patients with active MM have elevated levels of angiogenic cytokines, such as FGF-2 and VEGF, with a role in both tumour cell growth and survival and in bone marrow angiogenesis (Vacca et al, 1999). Furthermore, neovascularization, plasma cell angiogenic potential and matrix metalloproteinase-2 secretion parallel disease progression (Vacca et al, 1999). Our investigation of the role of MCs in the angiogenic response in these malignancies has shown that angiogenesis in benign lymphadenopathies and B-NHL, measured as microvessel counts, is correlated with the total metachromatic and MC tryptase-positive counts, and that both counts increase in step with the increase in Working Formulation malignancy grades (Ribatti et al, 1998; 2000b). Moreover, we have shown that bone marrow angiogenesis, evaluated as microvessel area, and MC counts are highly correlated in patients with inactive and active MM and in those with MGUS, and that both parameters increase simultaneously in active MM (Ribatti et al, 1999). In B-NHL and MM, MCs rest near or around blood or lymphatic capillaries. Their ultrastructural picture includes a morphological semi-lunar feature (Fig 4), or piecemeal partial degranulation of their secretory granules, unlike the IgE-mediated massive degranulation that occurs during immediate hypersensitivity reactions. This morphology is typical of the slow degranulation that takes place in delayed hypersensitivity reactions and chronic inflammation (Kops et al, 1984; Ribatti et al, 1988) and has been observed in gastric cancer (Caruso et al, 1997). In B-NHL and MM, the semi-lunar appearance may reflect slow but progressive release of angiogenic factors, favouring chronic and progressive stimulation of MC degranulation. Ultrastructural finding of bone marrow biopsy from patient with active multiple myeloma. A cytoplasmic granule with a semi-lunar aspect (arrow) is recognizable. Original magnification ×30 000. MCs are critical for a number of physiological and pathological events, including acute inflammation such as type 1 hypersensitivity, chronic inflammation and tumours, where their roles are thought to be mainly dependent on the selective expression and secretion of specific MC mediators. The challenge for the future is to determine the mechanisms used by MCs to stimulate angiogenesis and how they can be controlled in a clinical situation. MC-stabilizing agents may help to prevent neovascularization in tumour progression. This study was supported in part by grants from the Associazione Italiana per la Ricerca sul Cancro, Milan, to D.R. and A.V., the Associazione Italiana per la Lotta al Neuroblastoma, Genoa, to D.R., and Ministero dell'Università e della Ricerca Scientifica e Tecnologica (local funds) to D.R.