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• W2051239865 abstract "Angiotensin II is involved in tumor growth; however, the precise mechanism is not known. Platelets also contribute to tumor growth, and angiotensin II type 1 receptor (AT1) is expressed on the platelet surface. We hypothesized that interaction of platelets with tumor cells through AT1 receptor signaling promotes tumor metastasis. B16F1 melanoma cells were intravenously injected into Agtr1a knockout mice (AT1a−/−) and wild-type littermates (WT); the AT1a−/− mice exhibited a reduction in lung colonies. Angiotensin II induced expression of P-selectin on platelets in WT but not in AT1a−/− mice. A selective P-selectin neutralizing antibody decreased lung colony numbers in WT but not in AT1a−/− mice. Levels of vascular endothelial growth factor (VEGF) and stromal cell-derived factor 1 (SDF-1) receptor in platelets at metastatic locus were lower in AT1a−/− mice. Treatment of neutralizing antibodies against VEGF and CXCR4 decreased lung colony numbers in WT but not in AT1a−/− mice. In AT1a−/− mice, and both mobilization of progenitor cells expressing CXCR4+VEGFR1+ cells from bone marrow and their recruitment to lung tissues were suppressed. These results suggest that AT1A signaling plays a critical role in tumor metastasis through P-selectin–mediated interactions of platelets with tumor and endothelial cells and through the AT1A signaling-dependent production of VEGF and SDF-1, which may be involved in mobilization of CXCR4+VEGFR1+ cells. Angiotensin II is involved in tumor growth; however, the precise mechanism is not known. Platelets also contribute to tumor growth, and angiotensin II type 1 receptor (AT1) is expressed on the platelet surface. We hypothesized that interaction of platelets with tumor cells through AT1 receptor signaling promotes tumor metastasis. B16F1 melanoma cells were intravenously injected into Agtr1a knockout mice (AT1a−/−) and wild-type littermates (WT); the AT1a−/− mice exhibited a reduction in lung colonies. Angiotensin II induced expression of P-selectin on platelets in WT but not in AT1a−/− mice. A selective P-selectin neutralizing antibody decreased lung colony numbers in WT but not in AT1a−/− mice. Levels of vascular endothelial growth factor (VEGF) and stromal cell-derived factor 1 (SDF-1) receptor in platelets at metastatic locus were lower in AT1a−/− mice. Treatment of neutralizing antibodies against VEGF and CXCR4 decreased lung colony numbers in WT but not in AT1a−/− mice. In AT1a−/− mice, and both mobilization of progenitor cells expressing CXCR4+VEGFR1+ cells from bone marrow and their recruitment to lung tissues were suppressed. These results suggest that AT1A signaling plays a critical role in tumor metastasis through P-selectin–mediated interactions of platelets with tumor and endothelial cells and through the AT1A signaling-dependent production of VEGF and SDF-1, which may be involved in mobilization of CXCR4+VEGFR1+ cells. Tumor metastasis is a complex multistep process, requiring the acquisition of many distinct properties if it is to progress. Loss of cellular adhesion and increased invasiveness, intravasation, and survival and proliferation in a new site are all prerequisites for establishment of distant macrometastases.1Fidler I.J. The pathogenesis of cancer metastasis: the seed and soil hypothesis revisited.Nat Rev Cancer. 2003; 3: 453-458Crossref PubMed Scopus (3532) Google Scholar, 2Joyce J.A. Pollard J.W. Microenvironmental regulation of metastasis.Nat Rev Cancer. 2009; 9: 239-252Crossref PubMed Scopus (2749) Google Scholar, 3Chaffer C.L. Weinberg R.A. A perspective on cancer cell metastasis.Science. 2011; 331: 1559-1564Crossref PubMed Scopus (3331) Google Scholar Despite progress in other areas of cancer therapeutics, the complexities of this process remain poorly understood. Much evidence supports the concept that cancer metastasis is significantly facilitated by interaction between disseminating tumor cells and blood platelets.4Mehta P. Potential role of platelets in the pathogenesis of tumor metastasis.Blood. 1984; 63: 55-63Crossref PubMed Google Scholar Interference by antiplatelet agents or anticoagulants in tumor cell–platelet interaction potently inhibit both spontaneous and experimental metastases.5Trikha M. Nakada M.T. Platelets and cancer: implications for antiangiogenic therapy.Semin Thromb Hemost. 2002; 28: 39-44Crossref PubMed Scopus (80) Google Scholar In addition, measures that lower circulating-platelet counts have resulted in a decrease in distant metastases. Platelets contain vascular endothelial growth factor (VEGF), stromal cell-derived factor-1 (SDF-1), fibroblast growth factor-4 (FGF-4), and transforming growth factor (TGF), proteins that may be released into the peritumor space after platelet activation and influence tumor metastasis.6English D. Garcia J.G. Brindley D.N. Platelet-released phospholipids link haemostasis and angiogenesis.Cardiovasc Res. 2001; 49: 588-599Crossref PubMed Scopus (131) Google Scholar, 7Sierko E. Wojtukiewicz M.Z. Platelets and angiogenesis in malignancy.Semin Thromb Hemost. 2004; 30: 95-108Crossref PubMed Scopus (254) Google Scholar, 8Gunsilius E. Petzer A. Stockhammer G. Nussbaumer W. Schumacher P. Clausen J. Gastl G. Thrombocytes are the major source for soluble vascular endothelial growth factor in peripheral blood.Oncology. 2000; 58: 169-174Crossref PubMed Scopus (143) Google Scholar, 9Jin D.K. Shido K. Kopp H.G. Petit I. Shmelkov S.V. Young L.M. Hooper A.T. Amano H. Avecilla S.T. Heissig B. Hattori K. Zhang F. Hicklin D.J. Wu Y. Zhu Z. Dunn A. Salari H. Werb Z. Hackett N.R. Crystal R.G. Lyden D. Rafii S. Cytokine-mediated development of SDF-1 induces revascularization through recruitment of CXCR4+ hemangiocytes.Nat Med. 2006; 12 ([Erratum appeared in Nat Med 2006, 12:978]): 557-567Crossref PubMed Scopus (559) Google Scholar, 10van den Dolder J. Mooren R. Vloon A.P. Stoelinga P.J. Jansen J.A. Platelet-rich plasma: quantification of growth factor levels and the effect on growth and differentiation of rat bone marrow cells.Tissue Eng. 2006; 12: 3067-3073Crossref PubMed Scopus (204) Google Scholar These findings suggest that the interaction of activated platelets with tumor cells plays a critical role in the process of tumor metastasis. The renin-angiotensin system plays important roles in the regulation of cardiovascular homeostasis and blood pressure.11Ibrahim J. Hughes A.D. Sever P.S. Action of angiotensin II on DNA synthesis by human saphenous vein in organ culture.Hypertension. 2000; 36: 917-921Crossref PubMed Scopus (13) Google Scholar There is increasing evidence that angiotensin II (Ang II), which is generated by chymase or angiotensin-converting enzyme (ACE), in addition to its effects on blood pressure, may play a role in various pathological situations involving regulation of cell proliferation, inflammation, angiogenesis, and tissue repair and development.12George A.J. Thomas W.G. Hannan R.D. The renin-angiotensin system and cancer: old dog, new tricks.Nat Rev Cancer. 2010; 10: 745-759Crossref PubMed Scopus (362) Google Scholar Many reports have suggested that Ang II has a significant role as a growth factor.13Tamarat R. Silvestre T. Durie M. Levy B.I. Angiotensin II angiogenic effect in vivo involves vascular endothelial growth factor- and inflammation-related pathways.Lab Invest. 2002; 82: 747-756Crossref PubMed Scopus (210) Google Scholar, 14Bell L. Madri J.A. Influence of the angiotensin system on endothelial and smooth muscle cell migration.Am J Pathol. 1990; 137: 7-12PubMed Google Scholar, 15Sasaki K. Murohara T. Ikeda H. Sugaya T. Shimada T. Shintani S. Imaizumi T. Evidence for the importance of angiotensin II type 1a receptor in ischemia-induced angiogenesis.J Clin Invest. 2002; 109: 603-611Crossref PubMed Scopus (146) Google Scholar Other studies, including some from our research group, have also investigated the angiogenic effects of exogenous Ang II in several in vivo angiogenesis models.16Tufan H. Zaki B.M. Tecder-Unal M. Erdem S.R. Take G. Angiotensin II captopril cotreatment augments angiogenesis in abdominal skin flap in rats.Ann Plast Surg. 2007; 58: 441-448Crossref PubMed Scopus (6) Google Scholar, 17Muramatsu 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, 18Katada J. Muramatsu M. Hayashi I. Tsutsumi M. Konishi Y. Majima M. Significance of vascular endothelial cell growth factor up-regulation mediated via a chymase-angiotensin-dependent pathway during angiogenesis in hamster sponge granulomas.J Pharmacol Exp Ther. 2002; 302: 949-956Crossref PubMed Scopus (34) Google Scholar The pathophysiological activities of Ang II are known to be mediated by seven transmembrane receptors, and two major subtypes of Ang II receptors have been identified: AT1 and AT2, the former having subtypes A and B.19Kim S. Iwao H. Molecular and cellular mechanisms of angiotensin II-mediated cardiovascular and renal diseases.Pharmacol Rev. 2000; 52: 11-34PubMed Google Scholar Some recent studies using AT1 receptor antagonist and AT1A receptor-deficient mice [knockout of the Agtr1a gene (alias AT1a)] have shown that the blockade of AT1A receptor signaling inhibits growth of the following tumor cells: melanoma, lung, prostate, renal cell carcinoma, and pancreas.20Egami K. Murohara T. Shimada T. Sasaki K. Shintani S. Sugaya T. Ishii M. Akagi T. Ikeda H. Matsuishi T. Imaizumi T. Role of host angiotensin II type I receptor in tumor angiogenesis and growth.J Clin Invest. 2003; 112: 67-75Crossref PubMed Scopus (304) Google Scholar, 21Fujita M. Hayashi I. Yamashina S. Fukamizu A. Itoman M. Majima M. Angiotensin type 1a receptor signaling-dependent induction of vascular endothelial growth factor in stroma is relevant to tumor-associated angiogenesis and tumor growth.Carcinogenesis. 2005; 26: 271-279Crossref PubMed Scopus (132) Google Scholar, 22Uemura H. Ishiguro H. Nagashima Y. Sasaki T. Nakaigawa N. Hasumi H. Kato S. Kubota Y. Antiproliferative activity of angiotensin II receptor blocker through cross talk between stromal and epithelial prostate cancer cells.Mol Cancer Ther. 2005; 4: 1699-1709Crossref PubMed Scopus (70) Google Scholar, 23Miyajima A. Koasaka T. Asano T. Asano T. Seta K. Kawai T. Hayakawa M. Angiotensin II type I antagonist prevent pulmonary metastasis of murine renal cancer by inhibiting tumor angiogenesis.Cancer Res. 2002; 62: 4176-4179PubMed Google Scholar, 24Fujimito Y. Sasaki T. Tsuchida A. Chayama K. Angiotensin II type 1 receptor expression in human pancreatic cancer and growth inhibition by angiotensin II type 1 receptor antagonist.FEBS Lett. 2001; 495: 197-200Crossref PubMed Scopus (142) Google Scholar We have also found that the AT1 receptor antagonist TCV-116, or a deficient gene for the AT1A receptor, inhibits tumor growth, tumor-associated angiogenesis, and metastasis in a murine model.17Muramatsu 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, 21Fujita M. Hayashi I. Yamashina S. Fukamizu A. Itoman M. Majima M. Angiotensin type 1a receptor signaling-dependent induction of vascular endothelial growth factor in stroma is relevant to tumor-associated angiogenesis and tumor growth.Carcinogenesis. 2005; 26: 271-279Crossref PubMed Scopus (132) Google Scholar, 25Fujita 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 (208) Google Scholar However, the precise mechanisms regarding the contribution of AT1 receptor signaling to tumor metastasis remain to be determined. Ang II has the effect of a potent angiogenesis stimulator, both directly and by supplying angiogenic factors by platelet aggregation.26Senchenkova E.Y. Russell J. Almeida-Paula L.D. Harding J.W. Granger D.N. Angiotensin II-mediated microvascular thrombosis.Hypertension. 2010; 56: 1089-1095Crossref PubMed Scopus (69) Google Scholar Platelets express AT1 receptors on their surface, and AT1 receptor antagonists reduce P-selectin expression in platelets27Alexandru N. Popov D. Dragan E. Andrei E. Georgescu A. Platelet activation in hypertension associated with hypercholesterolemia: effects of irbesartan.J Thromb Haemost. 2011; 9: 173-184Crossref PubMed Scopus (28) Google Scholar and inhibit P-selectin–mediated platelet adhesion to the microvessels.28Ishikawa M. Sekizuka E. Yamaguchi N. Nakadate H. Terao S. Granger D.N. Minamitani H. Angiotensin II type 1 receptor signaling contributes to platelet-leukocyte-endothelial cell interactions in the cerebral microvasculature.Am J Physiol Heart Circ Physiol. 2007; 292: H2306-H2315Crossref PubMed Scopus (46) Google Scholar These findings led us to hypothesize that tumor metastasis is facilitated by interactions between platelets and metastatic tumor cells through AT1 receptor signaling. The objective of the present study was to investigate the role of AT1A signaling in colony formation and to explore the underlying mechanisms of tumor metastasis regulated by the AT1A signaling pathway. Male C57BL/6 mice, 6 to 8 weeks old and weighing 25 to 30 g, were obtained from the CLEA Japan Shizuoka Laboratory Animal Center (Fuji, Japan). AT1A receptor knockout mice (AT1a−/−) on the C57BL/6 hybrid background were generated by our research group.13Tamarat R. Silvestre T. Durie M. Levy B.I. Angiotensin II angiogenic effect in vivo involves vascular endothelial growth factor- and inflammation-related pathways.Lab Invest. 2002; 82: 747-756Crossref PubMed Scopus (210) Google Scholar The mice (males, 8 weeks old) were maintained at a constant humidity (60 ± 5%) and temperature (20 ± 1°C) and were kept continuously on a 12-hour light/dark cycle. All animals were provided food and water ad libitum. All experiments were performed in accordance with the guidelines for animal experiments of Kitasato University School of Medicine. B16F1 melanoma cells originally isolated from C57BL/6 mice were cultured at 37°C in RPMI 1640 medium suspended in 10% fetal bovine serum in a humidified atmosphere containing 5% CO2. B16F1 cells were harvested and washed three times with PBS. The cells were suspended in PBS at a density of 3.0 × 106 cells/mL, and 100 μL of the resulting suspension was injected into the tail vein.29Matsui Y. Amano H. Ito Y. Eshima K. Suzuki T. Ogawa F. Iyoda A. Satoh Y. Kato S. Nakamura M. Kitasato H. Narumiya S. Majima M. Thromboxane A2 receptor signaling facilitates tumor colonization through P-selectin-mediated interaction of tumor cells with platelets and endothelial cells.Cancer Sci. 2012; 103: 700-707Crossref PubMed Scopus (31) Google Scholar An AT1R antagonist, TCV-116 (100 mg/kg per day; Takeda Chemical Industries, Osaka, Japan), or an angiotensin-converting enzyme (ACE) agonist or Ang II was orally administrated every day (3.6 μg/day; Sigma-Aldrich, Tokyo, Japan). VEGF neutralized antibody (10 μg per mouse; Genzyme Japan, Tokyo, Japan), CXCR4 neutralized antibody (10 μg per mouse; clone 2B11; BD Biosciences, San Jose, CA), P-selectin neutralized antibody (30 μg per mouse; RB40.34; BD Biosciences), and isotype control IgG (rat IgG) were intraperitoneally injected daily. On day 21 after injection of B16F1 cells, AT1a−/− and WT mice were sacrificed with ether, and the lungs were resected. The isolated lungs were fixed with Bouin’s fluid solution,30Amano H. Ito Y. Suzuki T. Kato S. Matsui Y. Ogawa F. Murata T. Sugimoto Y. Senior R. Kitasato H. Hayashi I. Satoh Y. Narumiya S. Majima M. Roles of a prostaglandin E-type receptor, EP3, in upregulation of matrix metalloproteinase-9 and vascular endothelial growth factor during enhancement of tumor metastasis.Cancer Sci. 2009; 100: 2318-2324Crossref PubMed Scopus (51) Google Scholar and metastatic colonies were counted under a light microscope. The numbers of white blood cells and platelet numbers were measured using a Celltacα automatic cell counter (MEK-6450; Nihon Kohden, Tokyo, Japan). Anesthetized WT C57BL/6 mice were bled via heart puncture into heparinized microtubes, and the blood was collected into acid citrate dextrose. Platelet-rich plasma (PRP) was obtained by centrifugation (20 × g for 20 minutes). The PRP was transferred to a fresh tube, platelets were centrifuged, and the pellet was resuspended in RPMI 1640 medium. B16F1 cells were maintained in RPMI 1640 medium containing 10% fetal bovine serum. B16F1 cells (six-well tissue culture plates at 3 × 105 cells per well) were incubated with platelets (1.0 × 105 per well). Tumor cells and platelets were stimulated with Ang II (10 μg/mL per well) and P-selectin antibody (10 μg/mL per well) for 6 hours. To determine plasma levels of VEGF, SDF-1, and stem cell factor (SCF) and bone marrow (BM) levels of pro-matrix metallopeptidase 9 (pro-MMP-9), plasma and BM via the femur were collected and stored at −20°C until assay. Plasma levels of VEGF, SDF-1, and SCF and BM levels of pro-MMP-9 were assessed with specific enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems, Minneapolis, MN). We also measured VEGF and SDF-1 in PRP and cell culture supernatants with ELISA kits. These experiments were performed in duplicate. After lung tissues were extracted, tissues were rinsed in ice-cold PBS (0.02 mol/L, pH 7.0 to 7.2) to remove excess blood thoroughly and were weighed before homogenization. Tissues were minced into small pieces and then were homogenized in 1 mL of PBS with homogenizer on ice. The resulting suspension was sonicated with an ultrasonic cell disrupter, and the homogenates were centrifuged for 5 minutes at 5000 × g. Supernatants were used to measure VEGF, SDF-1, and PSGL-1 (mouse PSGL-1 ELISA kit; USCN Life Science, Houston, TX). All levels were normalized to total protein concentrations as determined by BCA assay (Pierce; Thermo Fisher Scientific, Rockford, IL). Blood was drawn via the tail vein on day 1 after surgery. The white blood cell fraction, including platelets, was obtained by Ficoll separation, and flow cytometric analysis was performed as described previously.31Hattori K. Hessig B. Wu Y. Dias S. Tejada R. Ferris B. Hicklin D.J. Zhu Z. Bohlen P. Witte L. Hendrikx J. Hackett N.R. Crystal R.G. Moore M.A. Werb Z. Lyden D. Rafii S. Placental growth factor reconstitutes hematopoiesis by recruiting VEGFR1(+) stem cells from bone-marrow microenvironment.Nat Med. 2002; 8: 841-849Crossref PubMed Scopus (556) Google Scholar, 32Eshima K. Suzuki H. Shinohara N. Cross-positive selection of thymocytes expressing a single TCR by multiple major histocompatibility complex molecules of both classes: implications for CD4+ versus CD8+ lineage commitment.J Immunol. 2006; 176: 1628-1636Crossref PubMed Scopus (17) Google Scholar In another set of experiments, platelets (1.0 × 105 per well) were incubated in RPMI 1640 medium for 24 hours and then were treated with Ang II (10 μg/mL per well) for 30 minutes. Cells were labeled with fluorescein isothiocyanate anti-mouse CD41 antibody (eBioscience, San Diego, CA) and phycoerythrin-labeled anti-mouse P-selectin antibody or fluorescein isothiocyanate–labeled anti-VEGFR1 and phycoerythrin-labeled anti-CXCR4 isotype control antibody (BD Pharmingen, San Diego, CA), in the presence of an anti-FcR monoclonal antibody (2.4G2; BD Biosciences). After a washing, the cells were analyzed with a FACSCalibur flow cytometer (BD Biosciences), and small cells (with low forward scatter) were gated for peripheral blood analysis. For immunofluorescent cytochemistry, lung tissues were immediately fixed with 4% paraformaldehyde in a 0.1 mol/L phosphate buffer solution (pH 7.4). After fixation, the tissues were dehydrated with a graded ethanol series and then embedded in paraffin. Sections of the paraffin-embedded tissues (4 μm thick) were mounted on glass slides, deparaffinized with xylene, and placed in 4°C acetone. The sections were blocked with 1% bovine serum albumin-PBS and incubated with anti-mouse CXCR4 antibody (1:200; LifeSpan Biosciences, Seattle, WA), anti-mouse VEGFR-1 antibody (1:200; Santa Cruz Biotechnology, Santa Cruz, CA), mouse anti-human integrin αIIb monoclonal antibody (1:50; Santa Cruz Biotechnology), rabbit anti-rat VEGF antibody (1:500; Santa Cruz Biotechnology), goat anti-human SDF antibody (1:100; Santa Cruz Biotechnology), and anti-mouse P-selectin antibody (1:50; BD Pharmingen). After a washing in PBS, the sections were incubated with Alexa Fluor 488 goat anti-rabbit IgG (1:1000; Invitrogen; Life Technologies, Carlsbad, CA) and Alexa Fluor 568 goat anti-rat IgG (1:1000; Invitrogen; Life Technologies). Negative control staining was performed by replacing the primary antibodies with 1% bovine serum albumin-PBS. Images were captured with a confocal scanning laser microscope (LSM710; Carl Zeiss, Jena, Germany), as described previously.33Katoh H. Hosono K. Ito Y. Suzuki T. Ogawa Y. Kubo H. Kamata H. Mishima T. Tamaki H. Sakagami H. Sugimoto Y. Narumiya S. Watanabe M. Majima M. COX-2 and prostaglandin EP3/EP4 signaling regulate the tumor stromal proangiogenic microenvironment via CXCL12-CXCR4 chemokine systems.Am J Pathol. 2010; 176: 1469-1483Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 34Hosono K. Suzuki T. Tamaki H. Sakagami H. Hayashi I. Narumiya S. Alitalo K. Majima M. Roles of prostaglandin E2-EP3/EP4 receptor signaling in the enhancement of lymphangiogenesis during fibroblast growth factor-2-induced granulation formation.Arterioscler Thromb Vasc Biol. 2011; 31: 1049-1058Crossref PubMed Scopus (47) Google Scholar B16F1 and human umbilical vein endothelial cells (HUVECs) were grown from 50% to 60% confluent cultures on Lab-Tek II chamber slides (Nalge Nunc International; Thermo Scientific, Rochester, NY). The monolayers were fixed with 3.7% paraformaldehyde for 10 minutes, permeabilized in 0.2% Triton X-100 for 10 minutes, and then incubated with anti–PSGL-1 goat polyclonal antibody (1:100; Santa Cruz Biotechnology) overnight. The secondary antibody was Alexa Fluor 568-conjugated donkey anti-goat IgG (Invitrogen; Life Technologies). Sections were then observed under a confocal scanning laser microscope. Transcripts encoding PSGL-1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were quantified by real-time RT-PCR analysis. After 2 hours of treatment with Ang II, HUVECs were collected and homogenized with TRIzol reagent. The real-time RT-PCR primers were designed using Primer 3 software (http://primer3.sourceforge.net) on the basis of data from GenBank. The following primers were used for real-time RT-PCR: PSGL-1, 5′-GAGAAGATTGCCACCACTGAC-3′ (sense) and 5′-GTTGGACGGTCTCTACTGAGG-3′ (antisense); GAPDH, 5′-ACATCAAGAAGGTGGTGAAGC-3′ (sense) and 5′-AAGGTGGAAGAGTGGGAGTTG-3′ (antisense); human PSGL-1, 5′-CAATTTGTCCGTCAACTACCC-3′ (sense) and 5′-TGCACACGAAGAAGATAGTGG-3′ (antisense); human P-selectin, 5′-GCCAGAATGAATACGTGAG-3′ (sense) and 5′-AGCTGCACTGCGAGTTAAAG-3′ (antisense); and human GAPDH, 5′-GAAGGTGAAGGACGGACTC-3′ (sense) and 5′-GAAGATGGTGATGGGATTTC-3′ (antisense) (Sigma-Aldrich). Total cellular proteins were isolated using radioimmunoprecipitation assay buffer [50 mmol/L Tris-HCl (pH 7.2), 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate]. Nuclear and cytoplasmic fractions were also prepared using NE-PER nuclear and cytoplasmic extraction reagents (Pierce; Thermo Fisher Scientific). Aliquots of 1 to 10 μg of protein were resolved by SDS-PAGE and transferred to membranes. Immunoblotting was performed with mouse polyclonal anti–PSGL-1 antibody (1:400; Abnova, Taipei, Taiwan) and monoclonal anti-mouse β-actin (1:10000; Sigma-Aldrich), followed by incubation with anti-rabbit horseradish peroxidase conjugated secondary antibody (1:2000; GE Healthcare, Little Chalfont, UK), and anti-mouse horseradish peroxidase conjugated secondary antibody (1:2000; GE Healthcare). The protein was visualized by a commercial chemiluminescent method with an Amersham ECL detection system (GE Healthcare). BM transplantation was performed as reported previously.35Ogawa Y. Suzuki T. Oikawa A. Hosono K. Kubo H. Amano H. Ito Y. Kitasato H. Hayashi I. Kato T. Sugimoto Y. Narumiya S. Watanabe M. Majima M. Bone marrow-derived EP3-expressing stromal cells enhance tumor-associated angiogenesis and tumor growth.Biochem Biophys Res Commun. 2009; 382: 720-725Crossref PubMed Scopus (23) Google Scholar Donor BM cells from AT1a−/− mice and their WT counterparts were harvested using the same method. The BM mononuclear cells of each donor (2.0 × 106 cells in 200 mL PBS) were transplanted via the tail veins of irradiated WT. Data are expressed as means ± SD. Comparisons among multiple groups were performed by analysis of variance. Comparisons between the two groups were made using Student’s t-test. Survival experiments were analyzed using the log-rank test and are presented as Kaplan-Meier survival curves. P <0.05 was considered statistically significant. We examined the effect of the AT1 antagonist TCV-116 on lung metastasis after i.v. injection of B16F1 cells into mice. The number of lung colonies in mice treated with the AT1 antagonist was significantly lower than in control mice: TCV-116, 45.8 ± 18.9 colonies; PBS, 98.7 ± 17.0 colonies (P < 0.01) (Figure 1A). In contrast, mice treated with Ang II exhibited significantly induced colony formation, compared with PBS-treated mice: Ang II, 128.6 ± 13.3 colonies; PBS, 98.7 ± 17.0 colonies (P < 0.05). These results suggest that AT1 plays an important role for lung metastasis in this model. We have previously reported that AT1A receptor signaling is a key regulator of tumor associated angiogenesis and tumor growth.21Fujita M. Hayashi I. Yamashina S. Fukamizu A. Itoman M. Majima M. Angiotensin type 1a receptor signaling-dependent induction of vascular endothelial growth factor in stroma is relevant to tumor-associated angiogenesis and tumor growth.Carcinogenesis. 2005; 26: 271-279Crossref PubMed Scopus (132) Google Scholar To investigate the role of AT1A signaling in tumor metastasis, we compared the numbers of lung colonies in AT1a−/− mice with those in WT mice (Figure 1B). There were significantly fewer colonies formed in AT1a−/− than in WT mice: WT, 116.2 ± 25.9 colonies; AT1a−/−, 45.8 ± 18.9 colonies (P < 0.05) (Figure 1C). In addition, on day 21 after injection of tumor cells, almost all of the WT mice had died, whereas all of the AT1a−/− mice survived (Figure 1D). These results suggest that AT1A signaling facilitates B16F1 lung colony formation and is also a prognostic factor for metastasis. Because platelets may play a role in tumor metastasis formation, we determined platelet numbers during the formation of lung metastasis. Baseline numbers did not differ between WT and AT1a−/− mice (P > 0.172). On days 7 and 14, however, platelet numbers were significantly decreased in AT1a−/− mice, compared with WT mice: 75.8 × 104 ± 1.54/μL for AT1a−/− mice versus 107.6 × 104 ± 1.45/μL for WT mice on day 7, and 71.0 × 104 ± 1.23/μL for AT1a−/− mice versus 99.8 × 104 ± 1.46/μL for WT mice on day 14 (P < 0.05) (Figure 2A). These results suggest that increased platelet numbers are associated with enhanced colony formation through AT1A signaling. To examine whether Ang II induces platelet activation, we isolated platelets from WT and AT1a−/− mice. The platelets were then incubated for 30 minutes under Ang II stimulation, and the expression of P-selectin (CD62P) +CD41+ platelets was determined. The percentage of CD62P+CD41+ platelets in Ang II-treated WT mice was significantly enhanced, compared with PBS-treated WT mice (P < 0.05) (Figure 2B). In contrast, for the AT1a−/− mice platelets did not differ significantly between the Ang II and PBS treatments (Figure 2C). These results suggest that AT1A receptor signaling on platelets is required for Ang II-induced P-selectin expression. Platelets activated by Ang II would interact with tumor cells as well as endothelial cells. To further examine whether AT1A signaling affects platelet activity during tumor metastasis formation, the population of CD62P +CD41+ platelets was determined by flow cytometry analysis. The percentage of CD62P+CD41+ platelets in AT1a−/− mice on day 1 was significantly reduced, compared with WT mice: AT1a−/−, 0.58 ± 0.29%; WT, 1.27 ± 0.18% (P < 0.05) (Figure 2D). This result suggests that AT1A signaling plays a role in platelet activation during the initial step of lung metastasis. To investigate the interaction of platelets with tumor cells at the metastatic sites, double-immunostaining analysis of P-selectin with integrin αIIb, a specific marker for platelets, was performed. The expression of P-selectin was colocalized with cells positive for αIIb around the metastatic area, and coexpression of P-selectin and αIIb was reduced in AT1a−/− mice, compared with WT mice (Figure 3A). These results indicate that AT1A signaling induces metastasis formation via interaction of activated platelets with tumor cells. To clarify the role of P-selectin in tumor metastasis formation via AT1A signaling, P-selectin neutralizing antibody was injected. In WT mice, treatment with P-selectin neutralizing antibody markedly reduced the number of lung colonies, compared with IgG treatment: P-selectin neutralizing antibody, 53.6 ± 18.5 colonies; IgG, 118.2 ± 23.1 colonies (P < 0.05). In the AT1a−/− mice, however, there was no difference in the number of lung colonies formed: P-selectin neutralizing antibody, 46.6 ± 19.2 colonies; IgG, 35.4 ± 10.1 colonies (P > 0.4) (Figure 3B). These results suggest that P-selectin–mediated tumor metastasis formation is AT1A signaling dependent. We further examined whether the interact" @default.
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• W2051239865 title "Angiotensin II Type 1A Receptor Signaling Facilitates Tumor Metastasis Formation through P-Selectin–Mediated Interaction of Tumor Cells with Platelets and Endothelial Cells" @default.
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