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Eur J Cardiothorac Surg 2005;28:617-621
© 2005 Elsevier Science NL

Original articles

The relationship of tryptase- and chymase-positive mast cells to angiogenesis in stage I non-small cell lung cancer

^a Department of Pharmacology, Osaka Medical College, Takatsuki City, Osaka 569-8686, Japan
^b Department of Thoracic and Cardiovascular Surgery, Osaka Medical College, Takatsuki City, Osaka 569-8686, Japan
^c Department of Pathology, Hoshigaoka Koseinenkin Hospital, 4-8-1, Hoshigaoka, Hirakata City, Osaka 573-8511, Japan
^d Department of Thoracic Surgery, Hoshigaoka Koseinenkin Hospital, 4-8-1, Hoshigaoka, Hirakata City, Osaka 573-8511, Japan

Received 25 March 2005; received in revised form 6 June 2005; accepted 9 June 2005.

^* Corresponding author. Tel.: +81 726 84 7292; fax: +81 726 84 6518. (Email: pha010{at}art.osaka-med.ac.jp).

Abstract

Objective: There are two types of human mast cells, tryptase-positive mast cells (MC_T) and tryptase- and chymase-positive mast cells (MC_TC). Although MC_T have been reported to be related to the generation of angiogenesis, little is known about the involvement of MC_TC in tumor angiogenesis. In this study, to clarify the relationship between MC_TC and lung cancer angiogenesis, we evaluated MC_TC, MC_T, and microvessel counts in normal, border, and central lung cancer regions. Methods: Tumor sections from 32 cases of adenocarcinoma and 13 cases of squamous cell carcinoma were immunostained for chymase to evaluate MC_TC, tryptase to evaluate MC_T, and CD34 to evaluate microvessel counts. Results: Both MC_TC and MC_T counts in the border lung cancer region were significantly higher than in the central region, and the MC_TC and MC_T counts in the central region were significantly higher than those in the normal regions. The microvessel counts in the border region were higher than those in the central region. The ratio of MC_TC to MC_T in the border region, but not in the central region, was significantly higher than that in the normal region. In the border region, significant correlations not only between MC_T and microvessel count, but also between MC_TC and microvessel count were observed. In the central region, a significant correlation between MC_TC and the microvessel count was observed, but there was no significant correlation between MC_T and the microvessel count. Conclusions: These findings suggest that MC_TC may be involved in the pathogenesis of angiogenesis in lung cancer.

Key Words: Angiogenesis • Lung cancer • Chymase • Mast cells • Human

1. Introduction

Human mast cells contain two types of serine protease, tryptase and chymase. Tryptase is a trypsin-like enzyme, and chymase is a chymotrypsin-like enzyme. Human mast cells are divided into the tryptase-positive mast cells (MC_T), which contain tryptase but not chymase, and the tryptase- and chymase-positive mast cells (MC_TC), which contain both tryptase and chymase. Mast cells are thought to be closely related to the generation of angiogenesis in various types of lung cancers [1,2]. We previously reported that transfection of human pro-chymase cDNA and an injection of purified chymase increased the generation of angiogenesis in a hamster sponge angiogenesis model [3]. Human chymase in vascular tissues converts angiotensin I to angiotensin II, which is a potent angiogenic factor via the induction of vascular endothelial growth factor (VEGF) [4,5]. Therefore, chymase-generating angiotensin II induces VEGF expression and results in the generation of angiogenesis. In fact, chymase-induced generation of angiogenesis has been shown to be partly, though not completely, prevented by an angiotensin II receptor blocker [3]. Chymase also activates promatrix metalloproteinase-9 to matrix metalloproteinase (MMP)-9, which is well known to be associated with tumor cell invasion and tumor-induced angiogenesis [6]. However, little is known about the correlation between MC_TC and angiogenesis in lung cancer.

Previous reports of significant correlations between tryptase and angiogenesis [7,8] suggest that tryptase may be an important enzyme in the generation of angiogenesis. Recently, Nagata et al. [9] demonstrated for the first time that, in small sized adenocarcinoma of the lung, the generation of angiogenesis was significantly correlated not only to MC_T but also to MC_TC. However, this correlation has not yet been studied in other adenocarcinomas or squamous cell carcinoma of the lung. Furthermore, though angiogenesis in lung cancer is particularly expressed in the border region of the cancer, the precise localization of MC_T and MC_TC activity within a lung cancer has not yet been established.

Thus, in the present study, in order to clarify the involvement of MC_TC in lung cancer angiogenesis, we evaluated patients with stage I non-small cell lung cancer for MC_T, MC_TC, and microvessel counts in the normal, border, and central lung cancer regions in each patient. We excluded patients treated with chemotherapy and/or radiotherapy, since both of these therapies affect mast cell expression [10,11].

2. Materials and methods

2.1 Patient profiles and surgical specimens
The primary stage I lung cancers of 45 patients (36 men and 9 women) who were treated by surgery only at Hoshigaoka Koseinenkin Hospital (Hirakata, Japan) between 1996 and 1999 were evaluated in this study. There were 32 cases of adenocarcinoma and 13 cases of squamous cell carcinoma. The patients' average age was 64±2 years old. Tissues from patients with atopic diseases, including atopic dermatitis, asthma, and allergic rhinitis, were excluded, since these conditions may increase mast cell number. For each patient we identified three areas: the non-invasion area was the normal lung tissue; the invasion front was the border area; and the complete tumor area included the central lung cancer regions.

2.2 Immunohistochemistry
For the immunohistochemical analysis we used mouse monoclonal antibodies 2D11G10D [12] to human mast cell chymase (a gift from Dr Suzuki, Katakura Industries Co., Saitama, Japan) to evaluate mast cell chymase, we used AA1 to human mast cell tryptase (Dako Japan, Kyoto, Japan) to evaluate tryptase, and we used QBEnd10 to human CD34 (Dako Japan) to evaluate CD34. Surgical specimens were fixed with 10% buffered formalin and embedded in paraffin.

Five-micrometer-thick sections were mounted on silanized slides (Matsunami Glass, Osaka, Japan) and deparaffinized with xylene and ethanol. To retrieve the antigen, sections were pretreated in 10mM citrate buffer and autoclaved for 5min at 120°C before immunostaining for chymase, tryptase, and CD34 as has been previously described [8]. The sections were then washed three times with phosphate-buffered saline (PBS) for 5min. Next, the sections were soaked in absolute methanol containing 1% hydrogen peroxide for 10min at room temperature to remove endogenous peroxidase activity. To suppress nonspecific binding, the sections were incubated with non-immune goat serum for 30min. The sections were then incubated with mouse monoclonal antibody, 2D11G10D to human mast cell chymase (1µg/ml), AA1 to human mast cell tryptase, and QBEnd10 to human CD34, for 60min at room temperature. After being washed with PBS, the sections were incubated with biotin-conjugated goat antimouse immunoglobulin G antibody for 30min, and this was followed by incubation with avidin–biotin-peroxidase complex (Vectastain Elite ABC kit; Vector Laboratories, Berlingame, CA, USA) for 30min. The sections were subsequently washed three times with PBS and incubated with 0.03% hydrogen peroxide and 0.05% 3,3'-diaminobenzidine for 3min. This was followed by counterstaining of the nuclei with hematoxylin solution.

To assess MC_TC, MC_T, and microvessel counts in the sections, we evaluated the immunohistochemical reactivities for chymase, tryptase, and CD34, and classified chymase-positive mast cells as MC_TC, tryptase-positive mast cells as MC_T, and CD-34 positive cells as representing a microvessel. Furthermore, to clarify the difference in the distribution of MC_TC, MC_T, and microvessel counts at the central, border, and normal regions of the lung cancer, we separately evaluated these locations. Using a x200 microscopic field, we counted chymase-positive and tryptase-positive mast cells at the sites where they had accumulated most intensely [8]. We excluded immunochemical-positive cells in the perivascular and peribronchial regions, since mast cells are known to accumulate in these areas. In the areas that were considered to have the most active neovascularization, vessels stained as CD34-positive cells were also counted using a x200 microscopic field. The MC_TC, MC_T, and microvessel counts were determined by two investigators who were blinded to the patients' clinical data. The averages of their counts in three fields were calculated as has been previously described [8]. Correlations between the two observers were excellent for MC_TC (r=0.925, P<0.001), MC_T (r=0.938, P<0.001), and microvessel counts (r=0.878, P<0.001).

2.3 Statistical analysis
All numerical data shown in the text are expressed as the mean±SD. Significant differences among the mean values of multiple groups were evaluated first by one-way ANOVA and then Fisher's test. Using linear regression analysis [Pearson's correlation coefficient], we assessed the statistical correlations between the MC_TC and microvessel counts and between the MC_T and microvessel counts. Differences were considered statistically significant at P<0.05.

3. Results

3.1 MC_TC and MC_T counts
Typical photographs of chymase-, tryptase-, and CD34-positive cells are shown in Fig. 1 . The average MC_TC count was 12.4±6.73 in the border region, 6.2±6.30 in the central region, and 1.33±1.90 in the normal region. The MC_TC counts in the border and central regions were significantly higher than those in the normal region. Furthermore, the count in the border region was significantly higher than the count in the central region (Fig. 2 , left panel).

View larger version (104K): [in this window] [in a new window]   Fig. 1. Typical photographs of immunostaining with anti-chymase (Chymase), anti-tryptase (Tryptase) and anti-CD34 antibodies (CD34) in the normal lung region, the border region, and the central lung tumor region.

View larger version (17K): [in this window] [in a new window]   Fig. 2. MCTC and MCT counts in the normal (N), border (B) and central (C) lung tumor regions. **P<0.01.

3.2 Ratio of MC_TC to MC_T counts
The ratio of the MC_TC count to MC_T count was 0.50±0.41 in the border region, 0.38±0.83 in the central region, and 0.23±0.42 in the normal region. The border region's ratio was significantly higher than the normal region's ratio. There was no significant difference between the central region's ratio and the normal region's ratio (Fig. 3 ).

View larger version (15K): [in this window] [in a new window]   Fig. 3. The ratio of the MCTC count to the MCT count in the normal, border and central lung tumor regions. *P<0.05.

View larger version (9K): [in this window] [in a new window]   Fig. 4. Microvessel counts in the border and the central lung tumor regions. *P<0.05.

View larger version (36K): [in this window] [in a new window]   Fig. 5. The correlations between the MCTC and microvessel counts and between the MCT and microvessel counts in the border and central lung tumor regions.

In this study, we found that both MC_TC and MC_T counts in the border lung cancer region were significantly higher than those in the normal region. This result is in agreement with a recent report by Nagata et al. [9]. However, in addition to this finding, we analyzed three lung cancer regions (normal, border, and central) and demonstrated for the first time that both MC_TC and MC_T counts in the lung cancer border region were significantly higher than those in the central region. The MC_T count in the border region was 125% greater than the in central region, and the MC_TC count in the border region was 200% greater than the central region count. The microvessel analysis showed that the count in the border region was significantly increased by 238% in comparison with the count in the central region. Furthermore, there was a significant correlation between the MC_TC count and the microvessel count in the border and the central regions. However, while there was a significant correlation between the MC_T count and the microvessel count in the border region, no significant difference was noted in the central region. The fact that no significant correlation between the MC_T count and the microvessel count was found is likely due to the high MC_T count and the low microvessel count in the central region. On the other hand, the MC_TC count in the central region was low. These findings suggest that microvessel expression may be more correlated with MC_TC accumulation than with MC_T accumulation.

Mast cells can produce angiogenic factors such as VEGF, basic fibroblast growth factor (bFGF) and transforming growth factor-ß (TGF-ß) [13–16]. In patients with lung cancer, a significant correlation between mast cell number and angiogenesis has been previously reported, and patients in the high-count mast cell group showed a significantly worse outcome than those in the low-count group [1]. In mast cell-deficient mice, it was found that there was a decreased rate of tumor angiogenesis, and that hematogenous metastasis was also reduced [17]. In mouse Lewis lung carcinoma, a mast cell-stabilizer, tranilast, significantly reduced tumor size and microvessel density [18]. Thus, numerous reports have provided strong evidence for the role of mast cells in tumor invasion and metastasis. However, angiogenic factors are associated with both MC_TC and MC_T, and it has been unclear as to which of the two is more correlated with angiogenesis. The present study demonstrated for the first time that MC_TC is significantly associated with lung cancer angiogenesis.

Chymase is a chymotrypsin-like enzyme that is stored in the secretory granules of mast cells. The enzymatic character of chymase recognizes aromatic amino acids such as phenylalanine (Phe), tyrosine (Tyr), and tryptophan (Trp) and cleaves the carboxy-terminal side of the protein that is found at the end of their amino acids [19]. In several animal models, chymase has been found to activate angiotensin I to angiotensin II, which enhances neovascularization via the induction of vascular endothelial growth factor and increases blood flow in ischemia-induced angiogenesis [20–23]. In a hamster sponge implant model, exogenous injection directly into the sponges of both angiotensin II and angiotensin I enhanced angiogenesis, whereas the presence of chymase inhibitors significantly prevented the angiogenesis induced by angiotensin I, but not by angiotensin II [3,24]. This finding suggests the importance of chymase-dependent angiotensin II formation in angiogenesis. However, although chymase acts as a proangiogenic factor, since injections of the chymase gene or purified chymase into the implanted sponges strongly facilitated angiogenesis in this model, chymase-induced angiogenesis could only be partially prevented by an angiotensin II receptor blocker [3]. Thus, this finding suggests that chymase-induced angiogenesis is involved in the activation of other angiogenic factors. It is known, for example, that chymase activates MMP-9 which is associated with tumor cell invasion and tumor-induced angiogenesis [6]. MMP activity is generally balanced by the tissue inhibitor of metalloproteinase (TIMP), and tissue degradation is dependent on an imbalance between MMP and TIMP. However, chymase also cleaves TIMP into inactive fragments, as well as cleaving MMP and TIMP complexes, which have no MMP activity, to form active MMP [25]. Therefore, chymase-induced angiogenesis may depend not only on angiotensin II formation but also on the activation of other factors such as MMP-9.

In the present study, in order to analyze MC_T, MC_TC, and microvessel counts, we divided the lung cancer lesions of each patient into three regions (normal, border, and central regions). We demonstrated that significant correlations existed not only between microvessel and MC_T counts but also between microvessel and MC_TC counts in the border region, though in the central region, a significant correlation between microvessel and MC_TC counts, but not between microvessel and MC_T counts, was observed. Therefore, MC_TC may be involved in the pathogenesis of lung cancer angiogenesis, and the inhibition of MC_TC may be useful as an anti-tumor therapy by reducing tumor-induced angiogenesis.

Acknowledgments

This study was supported in part by Grant-in-Aid for Exploratory Research 16659068 from the Ministry of Education, Science, Sports and Culture, Japan.

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to Personal Folders Download to citation manager Author home page(s): Takahiro Katsumata Permission Requests Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Ibaraki, T. Articles by Miyazaki, M. Search for Related Content PubMed PubMed Citation Articles by Ibaraki, T. Articles by Miyazaki, M. Related Collections Lung - cancer Lung - basic science