Re-Evaluation of Microcirculatory Endothelial Cell as an Endocrine System of Leptin: Its Significance During Ulcer Healing and Helicobacter pylori Infection

Re-Evaluation of Microcirculatory Endothelial Cell as an Endocrine System of Leptin: Its Significance

During Ulcer Healing and Helicobacter pylori Infection

Masahiko Nakamura

, Yasutada Akiba

, Hidenori Matsui

, Naoki Kumagai

, Hiroshi Serizawa

, Noriaki Watanabe

, Satoshi Tsunematsu

, Fumihiko Kaneko

, Kouichiro Atsuta

, Hiromasa Ishii

, and Kanji Tsuchimoto

Key words.

Leptin, Helicobacter pylori, Acetic acid-induced ulcer, Endothelial cell, Myofibroblast

Introduction

The myofibroblast, i.e., activated or modified fibroblast, has been shown to have growth-promoting effects on endothelial cells and be closely associated to angiogenesis [1]. Our recent histochemical studies have revealed a marked increase in the number and size of myofibroblasts in the Helicobacter pylori (Hp)-infected human and Mongolian gerbil fundic mucosa [2,3]. On the other hand, the mediators which stimulate the conversion of undifferentiated mesenchymal cells or fibroblasts to the myofibroblasts remain to be clar ified.

Recently, leptin, a protein product of obese gene expressed primarily by adipocytes, has been shown to be related to angiogenesis [4], and the relation of leptin and myofibroblast is an interesting point, because myofibroblast derives from fat-storing or Ito cells. In this chapter, we discuss the immuno- histochemical changes of leptin and its Ob receptors during the healing of

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1Department of Clinical Pharmacy and Clinical Sciences, School of Pharmaceutical Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan

2Department of Internal Medicine, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan

3Kitasato Institute for Life Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan

4Department of Internal Medicine, Kitasato Institute Hospital, 5-9-1 Shirokane, Minato- ku, Tokyo 108-8642, Japan

acetic acid-induced ulcer in rats and Hp-induced gastric mucosal damage in Mongolian gerbils.

Alteration of Leptin and Ob Receptors During Healing of Acetic Acid-Induced Ulcer

Stomach tissues were treated with Zamboni’s fixative, and the indirect immunofluorescence method using monoclonal antibodies against leptin (YC-040, Yanaihara Institute, Shizuoka, Japan) was performed, counterstained with Alexa Fluor 594 phalloidin (Molecular Probes, Leiden, the Netherlands), and observed by confocal laser microscopy (Leica TCS NT). In the control group, leptin immunoreactivity was recognized in the mesenchymal cells in the lamina propria mucosa, i.e., endothelial cells of the microcirculatory network and fibroblasts showing weak alexa phalloidin reactivity. Some of the surface epithelial cells showed very weak leptin immunoreactivity (Fig. 1).

Ob-R immunoreactivity was recognized in the mesenchymal cells surround- ing the microvascular network as well as in the endothelial cells.

In the acetic acid-treated group, 100% acetic acid was attached to the serosal surface of the stomach for 30 s 7 days before the experiments. One week after the acetic acid-induced gastric ulcer formation, leptin receptor immunoreactivity significantly increased in the endothelial cells and sur- rounding fibroblasts. In addition, Ob-R immunoreactivity was shown to accu- mulate in the tip portion of the regenerated fundic glandular mesenchymal cells.

As to the leptin localization in the microcirculation, a relationship to angio- genesis has recently been pointed out, because leptin administration has been reported to bring about new fenestrated blood vessels [4]. Leptin has also been shown to synergistically stimulate angiogenesis with basic fibroblast growth factor and vascular endothelial growth factor, the two most potent and commonly expressed angiogenic factors. As a whole, leptin has been sug- gested to be closely linked to the regeneration of the gastric mucosa after acetic acid-induced gastric mucosal injury.

Alteration of Leptin and Ob Receptors in Hp-Induced Gastric Mucosal Damage

Helicobacter pylori infection was evoked in Mongolian gerbils by the oral

administration of CagA- and VacA-positive Hp strain (ATCC 43504) 12

months before the experiments. In this group leptin was markedly recognized

in the mesenchymal cells including myofibroblasts which showed strong

Fig. 1a–f. Confocal laser microscopic observation of leptin and Ob-R immunoreactivities in control and acetic acid-treated rat fundic mucosa. a In the base of the fundic mucosa, many endothelial cells (arrows) show a positive reaction to leptin. b In the tip of the fundic mucosa, many mesenchymal cells (arrows) surrounding the microvascular network are immunoreactive to Ob-R. c,d In the acetic acid-treated gastric mucosa, erosive lesion is observed (c). In the regenerating mucosa, many enlarged endothelial cells are reactive to leptin (arrows). e,f In the regenerating mucosa, strong Ob-R immunoreactivity is recog- nized in the mesenchymal cells (arrows)

110 M. Nakamura

alexa-phalloidin reactivity and endothelial cells (Fig. 2) [5]. Ob-R immunore- activity accumulated markedly in the myofibroblasts, which were identified by myofibroblast-specific PR 2D3 immunoreactivity [6]. We have reported a markedly increased number of myofibroblasts during acetic acid-induced ulcer healing in rats [2] and Hp infection in gerbils [3]. Our result sug- gests the interaction of leptin with this transformation of fibroblast to myofibroblast.

Alteration of Leptin and Ob Receptors and Transformation of Fibroblast in Two-Layered Culture Model

In the in vitro experiment, GSM06 cells, i.e., gastric surface mucous cell line from a primary culture of gastric fundic mucosal cells of transgenic mice, were incubated in several media with an air–liquid surface on the collagen gel layer containing transformed mouse fibroblast cell line L929 for 2 weeks before experiments. The medium containing Hp bacilli (ATCC 43504) at mul- tiplicity of infection of 10–100 were added from the luminal side and incu- bated with 5% CO

for 1–24 h. Helicobacter pylori was found to attach to the GSM06 cells with pedestal formation and the number of the microvilli was increased on the surface plasma membrane of these mucous cells (Fig. 3). L929 cells in the collagen gel showed a spindle shape with poor cytoplasm. In the Hp-treated group, some of the L929 cells under GSM06 cells showed immunoreactivity of leptin and Ob-RL as well as PR 2D3. On electron micro- scopic observation, these cells had indented nuclei, well-developed r-ER, and actin bundles with dense patches just under plasma membrane, characteris- tic of the myofibroblast. The L929 cells incubated with Hp alone could not show differentiation to myofibroblasts. Pretreatment with leptin antibody pre- vented the transformation of the fibroblast. These data support the idea that leptin may stimulate fibroblasts to differentiate into myofibroblasts. Many cytokines play a role in the regulation of fibroblast activities, including growth factors, interleukins, and retinoic acid. The inter-relationship between leptin and these factors requires clarification.

Conclusion

Fibroblasts can be transformed to myofibroblasts during the healing process

and Hp infection at least, in part, through the effect of leptin from the

endothelial cells.

Fig. 2a–f. Confocal laser microscopic observation of leptin and Ob-R immunoreactivities in control and Hp-infected Mongolian gerbil fundic mucosa. a,b In the control Mongolian gerbil fundic mucosa, leptin immunoreactivity is seen in the endothelial cells of the col- lecting venules in the tip portion and the metarterioles in the base of the mucosal layer (arrows). c,d In the Hp-infected Mongolian gerbil fundic mucosa, the cytoplasm of the endothelial cells has enlarged and is positive to leptin (arrows). e,f Ob-R immunoreactiv- ity is found in the endothelial cells and mesenchymal cells in the tip portion of the fundic mucosa of the Hp-infected Mongolian gerbils (arrowheads)

112 M. Nakamura

Fig. 3a–f. Confocal laser and electron microscopic observation of the GSM06 cells and L929 cells. a,b The culture cells are composed of the GSM06 cells (G) and L929 cells (L).

Attached Hp bacilli are seen in the luminal surface of the GSM06 cells (arrows). c Electron microscopic observation of Hp (H) almost attached to the GSM06 cells. d Many micro- filaments are recognized in the L929 cells after Hp attachment to the GSM06 cells. e In the control group, leptin immunoreactivity is very weak in GSM06 cell layers (arrowhead).

f In the Hp-treated group, leptin immunoreactivity in the GSM06 cells (arrowhead) and Ob-R reactivity in the L929 cells (arrows) are clearly recognized

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