Recruitment of a Prostaglandin E Receptor Subtype, EP3-Expressing Bone Marrow Cells Is Crucial in Wound-Induced Angiogenesis
2006; Elsevier BV; Volume: 169; Issue: 4 Linguagem: Inglês
10.2353/ajpath.2006.051358
ISSN1525-2191
AutoresEmi Kamoshita, Yasuhiro Ikeda, Mamoru Fujita, Hideki Amano, Atsuhiko Oikawa, Tastunori Suzuki, Y. Ogawa, Shohei Yamashina, Sadahiro Azuma, Shuh Narumiya, Nobuya Unno, Masataka Majima,
Tópico(s)NF-κB Signaling Pathways
ResumoE-type prostaglandins have been reported to be proangiogenic in vivo. Thus, we examined prostaglandin receptor signaling relevant to wound-induced angiogenesis. Full-thickness skin wounds were created on the backs of mice, and angiogenesis in wound granulation tissues was estimated. Wound closure and re-epithelization in EP3 receptor knockout mice (EP3−/−) were significantly delayed compared with their wild-type (WT) mice, whereas those in EP1−/−, EP2−/−, and EP4−/− were not delayed. Wound-induced angiogenesis estimated with CD31 immunohistochemistry in EP3−/− mice was significantly inhibited compared with that in WT mice. Immunoreactive vascular endothelial growth factor (VEGF) in wound granulation tissues in EP3−/− mice was markedly less than that in WT mice. Wound closure in WT mice was delayed significantly by VEGF neutralizing antibody compared with control IgG. Wound-induced angiogenesis and wound closure were significantly suppressed in EP3−/− bone marrow transplantation mice compared with those in WT bone marrow transplantation mice. These were accompanied with the reductions in accumulation of VEGF-expressing cells in wound granulation tissues and in mobilization of VEGF receptor 1-expressing leukocytes in peripheral circulation. These results indicate that the recruitment of EP3-expressing cells to wound granulation tissues is critical for surgical wound healing and angiogenesis via up-regulation of VEGF. E-type prostaglandins have been reported to be proangiogenic in vivo. Thus, we examined prostaglandin receptor signaling relevant to wound-induced angiogenesis. Full-thickness skin wounds were created on the backs of mice, and angiogenesis in wound granulation tissues was estimated. Wound closure and re-epithelization in EP3 receptor knockout mice (EP3−/−) were significantly delayed compared with their wild-type (WT) mice, whereas those in EP1−/−, EP2−/−, and EP4−/− were not delayed. Wound-induced angiogenesis estimated with CD31 immunohistochemistry in EP3−/− mice was significantly inhibited compared with that in WT mice. Immunoreactive vascular endothelial growth factor (VEGF) in wound granulation tissues in EP3−/− mice was markedly less than that in WT mice. Wound closure in WT mice was delayed significantly by VEGF neutralizing antibody compared with control IgG. Wound-induced angiogenesis and wound closure were significantly suppressed in EP3−/− bone marrow transplantation mice compared with those in WT bone marrow transplantation mice. These were accompanied with the reductions in accumulation of VEGF-expressing cells in wound granulation tissues and in mobilization of VEGF receptor 1-expressing leukocytes in peripheral circulation. These results indicate that the recruitment of EP3-expressing cells to wound granulation tissues is critical for surgical wound healing and angiogenesis via up-regulation of VEGF. Nonsteroidal anti-inflammatory drugs that inhibit the enzyme cyclooxygenase (COX) and suppress prostaglandin (PG) synthesis have been widely used as anti-inflammatory, antipyretic, and analgesic agents. It was frequently reported that nonsteroidal anti-inflammatory drugs delay healing of experimental gastric ulcers by arresting epithelial proliferation in the margins of ulcers, interfering with re-epithelialization, and inhibiting angiogenesis in the granulation tissue.1Levi S Goodlad RA Lee CY Stamp G Walport MJ Wright NA Hodgson HJ Inhibitory effect of non-steroidal anti-inflammatory drugs on mucosal cell proliferation associated with gastric ulcer healing.Lancet. 1990; 336: 840-843Abstract PubMed Scopus (198) Google Scholar, 2Wang JY Yamasaki S Takeuchi K Okabe S Delayed healing of acetic acid-induced gastric ulcers in rats by indomethacin.Gastroenterology. 1989; 96: 393-402PubMed Scopus (171) Google Scholar, 3Tarnawski A Stachura J Douglass TG Krause WJ Gergely H Sarfeh IJ Indomethacin impairs quality of experimental gastric ulcer healing: a quality histologic and ultrastructural analysis.in: Garner A O'Brien PE Mechanisms of Injury, Protection and Repair of the Upper Gastrointestinal Tract. 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Two COX isoforms have been identified: COX-1 is constitutively expressed in various tissues, whereas COX-2 is induced by mitogens, cytokines, and tumor promoters. Disruption of the COX-2 gene in mice reduced the size of intestinal polyps generated by a mutation in the adenomatous polyposis (APC) gene. This reduction in the size of intestinal polyps was accompanied with reduced angiogenesis.22Ohshima M Dinchuk JE Kargman SL Oshima H Hancock B Kwong E Trzaskos JM Evans JF Taketo MM Suppression of intestinal polyposis in ApcΔ716 knockout mice by inhibition of cyclooxygenase 2 (COX-2).Cell. 1996; 87: 803-809Abstract Full Text Full Text PDF PubMed Scopus (2291) Google Scholar In this regard, endogenous PGs could be one of the contributors to angiogenesis. For mechanistic analysis of angiogenesis in vivo, we have developed a sponge implantation model in which a polyurethane sponge disk implanted subcutaneously induces extensive angiogenesis in the surrounding proliferative granulation tissues.23Majima M Isono M Ikeda Y Hayashi I Hatanaka K Harada Y Katsumata O Yamashina S Katori M Yamamoto S Significant roles of inducible cyclooxygenase (COX)-2 in angiogenesis in rat sponge implants.Jpn J Pharmacol. 1997; 75: 105-114Crossref PubMed Scopus (98) Google Scholar, 24Muramatsu M Katada J Hayashi I Majima M Chymase as a proangiogenic factor. 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Furthermore, we have transplanted EP receptor-lacking bone marrow (BM) cells into irradiated mice and have clarified the crucial roles of recruitment of EP receptor-expressing hematopoietic cells from BM on the wound-induced angiogenesis and the wound-healing processes. Our present results indicate that PGE2-EP3 signaling on the BM-derived cells appears to be crucial for the wound-induced angiogenesis and the wound-healing processes and that the EP3 signaling pathway linked to the induction of a potent proangiogenic growth factor, VEGF, certainly has a proangiogenic action and facilitates wound closure. Female 8-week-old C57BL/6 wild-type mice (WT) and EP3 receptor knockout mice (EP3−/−) were used for wound angiogenesis studies.36Amano H Hayashi I Endo H Kitasato H Yamashina S Maruyama T Kobayashi M Satoh K Narita M Sugimoto Y Murata T Yoshimura H Narumiya S Majima M Host prostaglandin E(2)-EP3 signaling regulates tumor-associated angiogenesis and tumor growth.J Exp Med. 2003; 197: 221-232Crossref PubMed Scopus (286) Google Scholar In some experiments, other types of EP receptor knockout mice lacking EP1, EP2, and EP4, respectively, were used for comparison.27Narumiya S Sugimoto Y Ushikubi F Prostanoid receptors: structures, properties, and function.Physiol Rev. 1999; 79: 1193-1226Crossref PubMed Scopus (0) Google Scholar, 36Amano H Hayashi I Endo H Kitasato H Yamashina S Maruyama T Kobayashi M Satoh K Narita M Sugimoto Y Murata T Yoshimura H Narumiya S Majima M Host prostaglandin E(2)-EP3 signaling regulates tumor-associated angiogenesis and tumor growth.J Exp Med. 2003; 197: 221-232Crossref PubMed Scopus (286) Google Scholar Surgical wounds were made on the back of mice as reported previously.41Straino S Germani A Di Carlo A Porcelli D De Mori R Mangoni A Napolitano M Martelli F Biglioli P Capogrossi MC Enhanced arteriogenesis and wound repair in dystrophin-deficient mdx mice.Circulation. 2004; 110: 3341-3348Crossref PubMed Scopus (52) Google Scholar, 42Mori R Kondo T Nishie T Ohshima T Asano M Impairment of skin wound healing in beta-1,4-galactosyltransferase-deficient mice with reduced leukocyte recruitment.Am J Pathol. 2004; 164: 1303-1314Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar After shaving the dorsal hair and cleaning the exposed skin with 70% ethanol, full-thickness excisional skin wounds of 8-mm diameter were made aseptically on either side of the dorsal midline using a 8-mm biopsy punch (Kai Industries, Tokyo, Japan). Four wounds were made on the same animal. Twelve hours after the above-mentioned surgery, after it was confirmed that the bleeding was completely stopped, the size of the wound was measured to give the day 1 result. Each wound region was digitally photographed at the fixed time, and the areas of the wounds were calculated by PhotoShop software, version 7.0 (Adobe Systems, San Jose, CA). Changes in the wound areas were expressed as the percentage of the wound areas at day 1. In some series of experiments, wounds and their surrounding area, including the scab and epithelial margins, were harvested with a fine pair of scissors in a genotype-blind manner after the mice were killed with an overdose of diethyl ether. All mice had free access to tap water and rodent chow and were housed individually with a 12-hour light/dark cycle and at a constant temperature (25 ± 1°C) and humidity (60 ± 5%). All experiments were performed in accordance with the guidelines for animal experiments of Kitasato University School of Medicine. Microvessel density in the wound granulation tissues was assessed as a parameter of wound-induced angiogenesis in a blind manner according to the established methods described previously.16Ikeda Y Hayashi I Kamoshita E Yamazaki A Endo H Ishihara K Yamashina S Tsutsumi Y Matsubara H Majima M Host stromal bradykinin B2 receptor signaling facilitates tumor-associated angiogenesis and tumor growth.Cancer Res. 2004; 64: 5178-5185Crossref PubMed Scopus (79) Google Scholar, 43Borre M Offersen BV Nerstrom B Overgaard J Microvessel density predicts survival in prostate cancer patients subjected to watchful waiting.Br J Cancer. 1998; 78: 940-944Crossref PubMed Scopus (216) Google Scholar, 44Weidner N Intratumor microvessel density as a prognostic factor in cancer.Am J Pathol. 1995; 147: 9-19PubMed Google Scholar, 45Fujita M Hayashi I Yamashina S Itoman M Majima M Blockade of angiotensin AT1a receptor signaling reduces tumor growth, angiogenesis, and metastasis.Biochem Biophys Res Commun. 2002; 294: 441-447Crossref PubMed Scopus (210) Google Scholar The individual microvessels were counted in 10 100 μm × 100 μm fields of a wound granulation tissue, and the numbers were averaged. CD31-immunoreactive endothelial cells were stained with CD31 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) and were differentiated from other connective tissue elements. Microvessel density was expressed in terms of microvessel number per observed area (vessel number/mm2).41Straino S Germani A Di Carlo A Porcelli D De Mori R Mangoni A Napolitano M Martelli F Biglioli P Capogrossi MC Enhanced arteriogenesis and wound repair in dystrophin-deficient mdx mice.Circulation. 2004; 110: 3341-3348Crossref PubMed Scopus (52) Google Scholar A COX-2 selective inhibitor, NS-39846Futaki N Yoshikawa K Hamasaka Y Arai I Higuchi S Iizuka H Otomo S NS-398, a novel non-steroidal anti-inflammatory drug with potent analgesic and antipyretic effects, which causes minimal stomach lesions.Gen Pharmacol. 1993; 24: 105-110Crossref PubMed Scopus (361) Google Scholar or JTE522,47Wakitani K Nanayama T Masaki M Matsushita M Profile of JTE-522 as a human cyclooxygenase-2 inhibitor.Jpn J Pharmacol. 1998; 78: 365-371Crossref PubMed Scopus (57) Google Scholar was suspended in 5% gum Arabic at a concentration of 6 mg/ml and was orally administered (0.05 ml/10 g body weight, 30 mg/kg, twice a day) to the WT mice. For vehicle control mice, distilled water with 5% gum Arabic was administered orally (0.05 ml/10 g body weight, twice a day). Aspirin was also administered to WT mice orally (30 mg/kg, twice a day). An anti-angiogenic agent, FR118487,48Mu J Abe Y Tsutsui T Yamamoto N Tai XG Niwa O Tsujimura T Sato B Terano H Fujiwara H Hamaoka T Inhibition of growth and metastasis of ovarian carcinoma by administering a drug capable of interfering with vascular endothelial growth factor activity.Jpn J Cancer Res. 1996; 87: 963-971Crossref PubMed Scopus (48) Google Scholar, 49Matsuo Y Li Y Taniguchi H Motoda M Amemiya T Inhibition of experimental choroidal neovascularization by an anti-growth agent inhibiting vascular endothelial development.Jpn J Ophthalmol. 2003; 47: 454-458Crossref PubMed Scopus (2) Google Scholar was administered to WT mice orally in the same manner as that of COX-2 inhibitors. The dose of FR118487 was 6 mg/kg, twice a day. The administration of above-mentioned compounds was started from the day of wounding throughout the experimental periods. Neutralizing antibody against mouse VEGF (IgG; Genzyme, Cambridge, MA),16Ikeda Y Hayashi I Kamoshita E Yamazaki A Endo H Ishihara K Yamashina S Tsutsumi Y Matsubara H Majima M Host stromal bradykinin B2 receptor signaling facilitates tumor-associated angiogenesis and tumor growth.Cancer Res. 2004; 64: 5178-5185Crossref PubMed Scopus (79) Google Scholar which can block the biological activity of recombinant mouse VEGF164 and VEGF120 with the neutralizing dose50 (100 ng/ml determined in the presence of 10 ng/ml of mouse VEGF164), was dissolved in physiological saline (14 μg/0.1 ml) and subcutaneously infused to WT mice with an Alzat miniosmotic pump (0.1 ml/7 days). For control mice, a nonimmune IgG fraction (14 μg/0.1 ml/7 days; Genzyme) was infused subcutaneously with the same osmotic pump. One day after pump implantation, full-thickness skin wounds were made. An orally active, low-molecular weight inhibitor of VEGF receptor (KDR/VEGFR 2) tyrosine kinase (ZD6474)50Wedge SR Ogilvie DJ Dukes M Kendrew J Chester R Jackson JA Boffey SJ Valentine PJ Curwen JO Musgrove HL Graham GA Hughes GD Thomas AP Stokes ES Curry B Richmond GH Wadsworth PF Bigley AL Hennequin LF ZD6474 inhibits vascular endothelial growth factor signaling, angiogenesis, and tumor growth following oral administration.Cancer Res. 2002; 62: 4645-4655PubMed Google Scholar was administered to WT mice orally, in the same manner as that used for COX-2 inhibitors. The dose of ZD6474 was 50 mg/kg (twice a day). For EP3−/− mice, human VEGF (Genzyme) dissolved in physiological saline (14 μg/0.1 ml) was subcutaneously infused with an Alzat miniosmotic pump (infusion rate was 0.1 ml/14 days). For vehicle-treated EP3−/− mice, physiological saline was infused subcutaneously with the same osmotic pump (infusion rate was 0.1 ml/14 days). One day after pump implantation, full-thickness skin wounds were made. Bone marrow (BM) cells were obtained by flushing the cavities of freshly dissected femurs, tibias, and pelves of donor male EP3 receptor knockout mice (EP3−/−) with phosphate-buffered saline (PBS). Donor BM cells of their WT counterparts (male C57BL/6 mice, 8 weeks old) were also harvested by the same method. The flushed BM cells of each donor were dispersed by pipetting and resuspended in PBS at a density of 1 × 107 cells/ml. WT mice (male C57BL/6 mice, 8 weeks old) were lethally irradiated with 9.0 Gy using an MBR-1505R X-ray irradiator (Hitachi Medico Co., Tokyo, Japan) with a filter (copper, 0.5 mm; aluminum, 2 mm) monitoring the cumulative radiation dose. Each donor's BM mononuclear cells (2 × 106 cells) in 200 μl of PBS were transplanted via the tail vein of irradiated WT mice. After 12 weeks, peripheral blood was collected, and the smear preparation was stained with β-galactosidase to confirm the chimerism. The mice in which more than 95% of the peripheral leukocytes exhibited a positive reaction to β-galactosidase were used in the present experiment. GFP transgenic mice (a gift from Dr. M. Okabe, Genome Information Research Center, Osaka University, Osaka, Japan) with C57BL/6 background were used as WT mice to confirm the chimerism. The mice in which more than 95% of the peripheral leukocytes exhibited GFP-positive in fluorescence-activated cell sorting analysis were used in the present experiment as a control group. Sponge disks (thickness, 5 mm; diameter, 1.3 cm)23Majima M Isono M Ikeda Y Hayashi I Hatanaka K Harada Y Katsumata O Yamashina S Katori M Yamamoto S Significant roles of inducible cyclooxygenase (COX)-2 in angiogenesis in rat sponge implants.Jpn J Pharmacol. 1997; 75: 105-114Crossref PubMed Scopus (98) Google Scholar, 24Muramatsu M Katada J Hayashi I Majima M Chymase as a proangiogenic factor. A possible involvement of chymase-angiotensin-dependent pathway in the hamster sponge angiogenesis model.J Biol Chem. 2000; 275: 5545-5552Crossref PubMed Scopus (107) Google Scholar, 25Majima M Hayashi I Muramatsu M Katada J Yamashina S Katori M Cyclooxygenase-2 enhances basic fibroblast growth factor-induced angiogenesis through the induction of vascular endothelial growth factor in rat sponge implants.Br J Pharmacol. 2000; 130: 641-649Crossref PubMed Scopus (186) Google Scholar were implanted under light ether anesthesia into the subcutaneous tissues of the backs of WT mice. After a fixed number of days, the granulation tissues that had formed around the sponge were taken immediately after death induced by the intraperitoneal administration of excessive doses of sodium pentobarbital. Neovascularization was assessed by measuring the concentration of hemoglobin in the granulation tissue
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