Chymase mediates mast cell-induced angiogenesis in hamster sponge granulomas

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Abstract

We investigated the contribution of mast cell chymase in mast cell-dependent angiogenesis using the hamster sponge-implant model, where angiogenesis in the granulation tissue surrounding the subcutaneously implanted sponge was evaluated by measuring the hemoglobin content. Daily local injection of compound 48/80 (3–100 μg/site/day), a potent mast cell activator, induced formation of granulomas and angiogenesis in time- and dose-dependent manners. This angiogenic response was inhibited by chymase inhibitors including chymostatin (≥1 nmol/site/day), soybean trypsin inhibitor (SBTI; ≥1.4 nmol/site/day) and lima bean trypsin inhibitor (LBTI; ≥3.3 nmol/site/day), but not by a tryptase inhibitor like leupeptin (≥700 nmol/site/day). Although pyrilamine (≥2,580 nmol/site/day), a histamine H1 receptor antagonist, and protamine (300 μg/site/day) also inhibited angiogenesis, these effects were much less pronounced than those by chymase inhibitors. Furthermore, antigen-induced angiogenesis in hamsters pre-sensitized with ovalbumin was also inhibited by the chymase inhibitors by 60–70%. Our results suggest that chymase is a major mediator in mast cell-mediated angiogenesis.

Introduction

Angiogenesis is an important physiological phenomenon implicated in integrated biological processes such as wound healing, embryonic development and formation of collateral circulation in ischaemic tissues. It is also involved in the pathogenesis of several diseases, including chronic inflammatory diseases, diabetic retinopathy and tumour growth. Angiogenesis is a multistep event and each step is regulated by different mechanisms. Many mediators and cell types are involved in this complex process, and the progression of angiogenesis may be positively or negatively regulated by numerous factors Fan and Brem, 1992, Folkman and Shing, 1992.

Mast cells are topographically associated with microvessels Rakusan et al., 1990, Rhodin and Fujita, 1989 and accumulate in a number of angiogenesis-dependent events such as ovulation, wound healing, synovial proliferation in rheumatoid arthritis and tumour growth (reviewed in Meininger and Zetter, 1992, Norrby and Woolley, 1993). These histological studies implicate mast cells, especially connective tissue type mast cells, in angiogenesis. The role of mast cells in angiogenesis has also been suggested by the results of experiments showing that activation of connective tissue type mast cells in situ by compound 48/80 promoted angiogenesis in several models, such as rat mesenteric microvessels (Norrby et al., 1986) or chick chorioallantoic membrane (Clinton et al., 1988). However, the significance of mast cells in angiogenesis under physiological and/or pathophysiological conditions is still unclear.

Mast cell granules contain numerous substances including histamine, proteoglycans, such as heparin (Metcalfe et al., 1979), and proteases such as tryptase Schwartz et al., 1981, Hopsu and Glenner, 1963, chymase Schechter et al., 1983, Wintroub et al., 1986, Sayama et al., 1987 and carboxypeptidase A (Goldstein et al., 1987). These mediators are released from the granules upon activation of mast cells. Lipid mediators are also produced by activated mast cells Hogaboam et al., 1992, Lewis et al., 1982, Razin et al., 1982. Although some of these mast cell-derived mediators, such as histamine Zauberman et al., 1969, Sorbo and Norrby, 1992 and heparin Jakobsson et al., 1990, Unger et al., 1991, Norrby and Sorbo, 1995, exhibit angiogenic properties both in vitro and in vivo, the precise mechanism and the factors responsible for mast cell-dependent angiogenesis remain to be elucidated.

A major focus of our research is related to the physiological/pathophysiological roles of chymase. Chymase is a serine protease with chymotrypsin-like substrate specificity and is one of the major components of mast cell granules (Lagunoff and Pritzl, 1976). Marked species differences in chymase substrate specificity have been reported. For example, in humans, dogs and hamsters, chymase generates angiotensin II from its inactive precursor angiotensin I, whereas chymase cleaves angiotensin I into inactive fragments in rats, mice or rabbits Le Trong et al., 1987, Okunishi et al., 1993, Chandrasekharan et al., 1996, Balcells et al., 1997. In this regard, to investigate the physiological and/or pathophysiological roles of mast cell chymase in angiogenesis in humans, hamsters should be used as the experimental animal. Recently, we established a hamster sponge granuloma-angiogenesis model (Muramatsu et al., 2000), which was originally developed in rats by Fan et al. Andrade et al., 1987, Hu et al., 1996 who measured 133Xe clearance rate to quantify angiogenesis. In our modified model, the hemoglobin content in the sponge granulomas is used for the quantification of angiogenesis. Using this model, we have demonstrated that chymase acts as a pro-angiogenic factor and that endogenous chymase is involved in basic fibroblast growth factor (FGF)-induced angiogenesis probably via the local generation of angiotensin II (Muramatsu et al., 2000). However, we have not confirmed a source of chymase responsible for angiogenesis under physiological and/or pathophysiological conditions.

The aims of the present study were to elucidate the role of mast cell-derived chymase in angiogenesis using the hamster sponge granuloma model. Our results showed that direct injections of compound 48/80 into sponge implants induced angiogenesis, suggesting that activation of connective tissue type mast cells is sufficient for the induction of angiogenesis in this model. Furthermore, antigen-dependent activation of mast cells also resulted in angiogenesis in sensitized hamsters. These angiogenic responses elicited by activated mast cells were inhibited by treatment with chymase inhibitors including chymostatin and soybean trypsin inhibitor (SBTI). Our results suggest that chymase is one of the factors that mediate mast cell-dependent angiogenesis under certain physiological and/or pathophysiological conditions.

Section snippets

Hamster sponge-implant mode

Circular sponge discs, 5-mm thick×13-mm diameter, weighing 14.2±0.1 mg, were prepared from a polyurethane foam sheet. The discs were soaked in 70% ethanol overnight and then sterilized with an autoclave. Male Syrian hamsters (70–90 g, purchased from Japan SLC) were used in all experiments. The dorsal hair of ether-anaesthetized hamsters was shaved and the skin was sterilized with 70% ethanol. A midline incision was made and sponge discs were implanted in the subcutaneous space. Before suturing,

Compound 48/80-induced angiogenesis

In this hamster sponge-implant model, spontaneous angiogenesis (PBS-injected controls in Fig. 1A) occurred after day 7, as evident by a gradual increase in hemoglobin contents. Daily injection of compound 48/80 significantly induced angiogenesis in the sponge implants. As shown in Fig. 1A, the hemoglobin contents in compound 48/80 (100 μg/site/day)-injected sponge granulomas increased progressively from day 2 (2.2±0.17 mg/g of wet tissue) to day 16 (7.5±0.42 mg/g of wet tissue). The hemoglobin

Discussion

In the present study, we showed that chymase, derived from activated mast cells, was involved in mast cell-mediated angiogenesis. This finding complements our recent study (Muramatsu et al., 2000), in which we confirmed that hamster chymase is angiogenic when injected directly into the subcutaneously implanted sponges. While our previous study suggested that basic FGF-induced angiogenesis might be partly mediated through the endogenous chymase, the exact origin of chymase in sponge granulomas

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