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B Lymphocyte-Specific c-Myc Expression Stimulates Early and Functional Expansion of the Vasculature and Lymphatics during Lymphomagenesis

2003; Elsevier BV; Volume: 163; Issue: 6 Linguagem: Inglês

10.1016/s0002-9440(10)63581-x

1525-2191

Alanna Ruddell, Pau Mezquita, Kimberly A Brandvold, Andrew G. Farr, Brian M. Iritani,

Angiogenesis and VEGF in Cancer

Expression of the c-myc proto-oncogene is deregulated in many human cancers. We examined the role of c-Myc in stimulating angiogenesis and lymphangiogenesis in a highly metastatic murine model of Burkitt's lymphoma (Eμ-c-myc), where c-Myc is expressed exclusively in B lymphocytes. Immunohistochemical analysis of bone marrow and lymph nodes from young (preneoplastic) Eμ-c-myc transgenic mice revealed increased growth of blood vessels, which are functional by dye flow assay. Lymphatic sinuses also increased in size and number within the lymph nodes, as demonstrated by immunostaining for with a lymphatic endothelial marker 10.1.1. The 10.1.1 antibody recognizes VEGFR-2- and VEGFR-3-positive lymphatic sinuses and vessels within lymph nodes, and also recognizes lymphatic vessels in other tissues. Subcutaneously injected dye traveled more efficiently through draining lymph nodes in Eμ-c-myc mice, indicating that these hypertrophic lymphatic sinuses increase lymph flow. Purified B lymphocytes and lymphoid tissues from Eμ-c-myc mice expressed increased levels of vascular endothelial growth factor (VEGF) by immunohistochemical or immunoblot assays, which could promote blood and lymphatic vessel growth through interaction with VEGFR-2, which is expressed on the endothelium of both vessel types. These results indicate that constitutive c-Myc expression stimulates angiogenesis and lymphangiogenesis, which may promote the rapid growth and metastasis of c-Myc-expressing cancer cells, respectively. Expression of the c-myc proto-oncogene is deregulated in many human cancers. We examined the role of c-Myc in stimulating angiogenesis and lymphangiogenesis in a highly metastatic murine model of Burkitt's lymphoma (Eμ-c-myc), where c-Myc is expressed exclusively in B lymphocytes. Immunohistochemical analysis of bone marrow and lymph nodes from young (preneoplastic) Eμ-c-myc transgenic mice revealed increased growth of blood vessels, which are functional by dye flow assay. Lymphatic sinuses also increased in size and number within the lymph nodes, as demonstrated by immunostaining for with a lymphatic endothelial marker 10.1.1. The 10.1.1 antibody recognizes VEGFR-2- and VEGFR-3-positive lymphatic sinuses and vessels within lymph nodes, and also recognizes lymphatic vessels in other tissues. Subcutaneously injected dye traveled more efficiently through draining lymph nodes in Eμ-c-myc mice, indicating that these hypertrophic lymphatic sinuses increase lymph flow. Purified B lymphocytes and lymphoid tissues from Eμ-c-myc mice expressed increased levels of vascular endothelial growth factor (VEGF) by immunohistochemical or immunoblot assays, which could promote blood and lymphatic vessel growth through interaction with VEGFR-2, which is expressed on the endothelium of both vessel types. These results indicate that constitutive c-Myc expression stimulates angiogenesis and lymphangiogenesis, which may promote the rapid growth and metastasis of c-Myc-expressing cancer cells, respectively. The c-myc proto-oncogene has a central role in regulation of cell growth and differentiation.1Schmidt EV The role of c-myc in cellular growth control.Oncogene. 1999; 18: 2988-2996Crossref PubMed Scopus (306) Google Scholar Overexpression of c-Myc via translocations or other mechanisms is a common feature of many cancers including colon, breast, prostate, and gastrointestinal cancers, as well as leukemias and lymphomas.2Nesbit CE Tersak JM Prochownik EV MYC oncogenes and human neoplastic disease.Oncogene. 1999; 18: 3004-3016Crossref PubMed Scopus (957) Google Scholar Experimental c-Myc overexpression in chickens and mice indicates that c-Myc rapidly induces neoplastic transformation of many cell types, including B lymphocytes. In all of these models, increased c-Myc expression impairs cell cycle exit, slows differentiation,3Brandvold KA Ewert DL Kent SC Neiman P Ruddell A Blocked B cell differentiation and emigration support the early growth of Myc-induced lymphomas.Oncogene. 2001; 20: 3226-3234Crossref PubMed Scopus (14) Google Scholar, 4Iritani BM Eisenman RN c-Myc enhances protein synthesis and cell size during B lymphocyte development.Proc Natl Acad Sci USA. 1999; 96: 13180-13185Crossref PubMed Scopus (295) Google Scholar and induces blood vessel growth.5Brandvold KA Neiman P Ruddell A Angiogenesis is an early event in the generation of myc-induced lymphomas.Oncogene. 2000; 19: 2780-2785Crossref PubMed Scopus (87) Google Scholar, 6Ngo CV Gee M Akhtar N Yu D Volpert O Auerbach R Thomas-Tikhonenko A An in vivo function for the transforming Myc protein: elicitation of the angiogenic phenotype.Cell Growth Differ. 2000; 11: 201-210PubMed Google Scholar, 7Pelengaris S Littlewood T Khan M Elia G Evan G Reversible activation of c-Myc in skin: induction of a complex neoplastic phenotype by a single oncogenic lesion.Mol Cell. 1999; 3: 565-577Abstract Full Text Full Text PDF PubMed Scopus (398) Google Scholar Studies using conditional c-Myc expression indicate that c-Myc is continuously required to support c-Myc-induced tumors; loss of c-Myc expression in these tumors results in differentiation, apoptosis, and blood vessel degeneration, followed by tumor resorption.7Pelengaris S Littlewood T Khan M Elia G Evan G Reversible activation of c-Myc in skin: induction of a complex neoplastic phenotype by a single oncogenic lesion.Mol Cell. 1999; 3: 565-577Abstract Full Text Full Text PDF PubMed Scopus (398) Google Scholar, 8Felsher DW Bishop JM Transient excess of MYC activity can elicit genomic instability and tumorigenesis.Proc Natl Acad Sci USA. 1999; 96: 3940-3944Crossref PubMed Scopus (360) Google Scholar These findings indicate that c-Myc is a potent oncogene able to mediate many features of tumor induction, including angiogenesis.While these studies make plain that c-Myc is important for cell growth and the maintenance of tumors, it is still unclear whether c-Myc may also contribute to tumor metastasis, which is a critical feature in determining the prognosis of cancer patients. Clinical studies indicate that c-Myc expression generally correlates with a less favorable prognosis,2Nesbit CE Tersak JM Prochownik EV MYC oncogenes and human neoplastic disease.Oncogene. 1999; 18: 3004-3016Crossref PubMed Scopus (957) Google Scholar and is associated with increased metastasis of breast,9Watson PH Safneck JR Le K Dubik D Shiu RP Relationship of c-myc amplification to progression of breast cancer from in situ to invasive tumor and lymph node metastasis.J Natl Cancer Inst. 1993; 85: 902-907Crossref PubMed Scopus (80) Google Scholar colorectal,10Sato K Miyahara M Saito T Kobayashi M c-myc mRNA overexpression is associated with lymph node metastasis in colorectal cancer.Eur J Cancer. 1994; 30A: 1113-1117Abstract Full Text PDF PubMed Scopus (26) Google Scholar and cervical cancers.11Riou G Le MG Favre M Jeannel D Bourhis J Orth G Human papillomavirus-negative status and c-myc gene overexpression: independent prognostic indicators of distant metastasis for early-stage invasive cervical cancers.J Natl Cancer Inst. 1992; 84: 1525-1526Crossref PubMed Scopus (30) Google Scholar In addition, amplification of L-myc has been closely associated with metastasis of lung cancer to lymph nodes and other organs.12Kawashima K Shikama H Imoto K Izawa M Naruke T Okabayashi K Nishimura S Close correlation between restriction fragment length polymorphism of the L-MYC gene and metastasis of human lung cancer to the lymph nodes and other organs.Proc Natl Acad Sci USA. 1988; 85: 2353-2356Crossref PubMed Scopus (83) Google Scholar Among the potential mechanisms for increasing the metastasis potential of tumor cells, the sprouting of new blood vessels that help feed tumor cells (angiogenesis), and the growth of lymphatic vessels that drain extravasated fluid, proteins, and tumor cells (lymphangiogenesis),13Alitalo K Carmeliet P Molecular mechanisms of lymphangiogenesis in health and disease.Cancer Cell. 2002; 1: 219-227Abstract Full Text Full Text PDF PubMed Scopus (574) Google Scholar are leading candidates for modulation by c-Myc.Angiogenesis is essential for growth and metastasis of solid tumors, and is involved in hematopoietic malignancies.14Saaristo A Karpanen T Alitalo K Mechanisms of angiogenesis and their use in the inhibition of tumor growth and metastasis.Oncogene. 2000; 19: 6122-6129Crossref PubMed Scopus (251) Google Scholar For example, bone marrow from leukemia and multiple myeloma patients show increased vascularization,15Aguayo A Kantarjian H Manshouri T Gidel C Estey E Thomas D Koller C Estrov Z O'Brien S Keating M Freireich E Albitar M Angiogenesis in acute and chronic leukemias and myelodysplastic syndromes.Blood. 2000; 96: 2240-2245PubMed Google Scholar, 16Perez-Atayde AR Sallan SE Tedrow U Connors S Allred E Folkman J Spectrum of tumor angiogenesis in the bone marrow of children with acute lymphoblastic leukemia.Am J Pathol. 1997; 150: 815-821PubMed Google Scholar, 17Vacca A Ribatti D Presta M Minischetti M Iurlaro M Ria R Albini A Bussolino F Dammacco F Bone marrow neovascularization, plasma cell angiogenic potential, and matrix metalloproteinase-2 secretion parallel progression of human multiple myeloma.Blood. 1999; 93: 3064-3073PubMed Google Scholar and non-Hodgkin's lymphomas also feature increased vessels.18Vacca A Ribatti D Ruco L Giacchetta F Nico B Quondamatteo F Ria R Iurlaro M Dammacco F Angiogenesis extent and macrophage density increase simultaneously with pathological progression in B-cell non-Hodgkin's lymphomas.Br J Cancer. 1999; 79: 965-970Crossref PubMed Scopus (128) Google Scholar Hematopoietic tumors can produce pro-angiogenic factors including vascular endothelial growth factor (VEGF), a potent growth factor for vascular endothelium, and metalloproteinases involved in matrix remodeling.19Kossakowska AE Hinek A Edwards DR Lim MS Zhang C-L Breitman DR Proteolytic activity of human non-Hodgkins lymphomas.Am J Pathol. 1998; 152: 565-576PubMed Google Scholar, 20Vacca A Ribatti D Iurlaro M Albini A Minischetti M Bussolino F Pellegrino A Ria R Rusnati M Presta M Vincenti V Persico MG Dammacco F Human lymphoblastoid cells produce extracellular matrix-degrading enzymes and induce endothelial cell proliferation, migration, morphogenesis, and angiogenesis.Int J Clin Lab Res. 1998; 28: 55-68Crossref PubMed Scopus (88) Google Scholar Tumor lymphangiogenesis has recently gained attention for its potential involvement in lymphatic dissemination and metastasis of tumor cells.21Stacker SA Baldwin ME Achen MG The role of tumor lymphangiogenesis in metastatic spread.EMBO J. 2002; 16: 922-934Google Scholar Lymph vessel growth is promoted by VEGF-C or VEGF-D activation of VEGFR-3, whose expression is generally restricted to lymphatic endothelium. For example, VEGF-C- or VEGF-D-expressing tumor cell line implants in immunodeficient mice induced growth of lymph vessels, which promoted metastasis.22Mandriota SJ Jussila L Jeltsch M Compagni A Baetens D Prevo R Banerji S Huarte J Montesano R Jackson DG Orci L Alitalo K Christofori G Pepper MS Vascular endothelial growth factor-C-mediated lymphangiogenesis promotes tumour metastasis.EMBO J. 2001; 20: 672-682Crossref PubMed Scopus (831) Google Scholar, 23Skobe M Hawighorst T Jackson DG Prevo R Janes L Velasco P Riccardi L Alitalo K Claffey K Detmar M Induction of tumor lymphangiogenesis by VEGF-C promotes breast cancer metastasis.Nat Med. 2001; 7: 192-198Crossref PubMed Scopus (1479) Google Scholar, 24Stacker SA Caesar C Baldwin ME Thornton GE Williams RA Prevo R Jackson DG Nishikawa S Kubo H Achen MG VEGF-D promotes the metastatic spread of tumor cells via the lymphatics.Nat Med. 2001; 7: 186-191Crossref PubMed Scopus (1051) Google Scholar Moreover, inhibition of VEGFR-3 signaling blocked lymphatic vessel growth and tumor metastasis,25He Y Kozaki K Karpanen T Koshikawa K Yla-Herttuala S Takahashi T Alitalo K Suppression of tumor lymphangiogenesis and lymph node metastasis by blocking vascular endothelial growth factor receptor 3 signaling.J Natl Cancer Inst. 2002; 94: 819-825Crossref PubMed Scopus (448) Google Scholar and it also inhibited corneal lymphangiogenesis.26Kubo H Cao R Brakenhielm E Makinen T Cao Y Alitalo K Blockade of vascular endothelial growth factor receptor-3 signaling inhibits fibroblast growth factor-2-induced lymphangiogenesis in mouse cornea.Proc Natl Acad Sci USA. 2002; 99: 8868-8873Crossref PubMed Scopus (260) Google Scholar Thus far, few experimental models have been developed to examine the natural role of angiogenesis and lymphangiogenesis in tumor dissemination.To more fully investigate the ability and mechanism of c-Myc-induced angiogenesis and lymphangiogenesis during hematopoietic cell transformation, we used the well-characterized Eμ-c-myc mouse model of Burkitt's lymphoma,27Adams JM Harris AW Pinkert CA Corcoran LM Alexander WS Cory S Palmiter RD Brinster RL The c-myc oncogene driven by immunoglobulin enhancers induces lymphoid malignancy in transgenic mice.Nature. 1985; 318: 533-538Crossref PubMed Scopus (1314) Google Scholar where the immunoglobulin heavy chain gene enhancer drives c-Myc expression in B-cell progenitors. Overexpression of c-Myc results in accumulation of pre-B cells within the bone marrow, lymph nodes, and spleen beginning before birth.28Langdon WY Harris AW Cory S Adams JM The c-myc oncogene perturbs B lymphocyte development in Eu-myc transgenic mice.Cell. 1986; 47: 11-18Abstract Full Text PDF PubMed Scopus (318) Google Scholar This polyclonal B-cell expansion is followed by the appearance of oligoclonal or monoclonal tumors after 12 to 16 weeks of age, which metastasize to many organs, including the lung, liver, and bone.29Harris AW Pinkert CA Crawford M Langdon WY Brinster RL Adams JM The E μ-c-myc transgenic mouse: a model for high-incidence spontaneous lymphoma and leukemia of early B cells.J Exp Med. 1988; 167: 353-371Crossref PubMed Scopus (316) Google Scholar While Eμ-c-myc mice have been extensively studied to examine the effects of c-Myc on proliferation, differentiation, and apoptosis, the ability of c-Myc to induce angiogenesis and lymphangiogenesis has not yet been examined in this model. We find that young preneoplastic and older tumor-bearing Eμ-c-myc mice show increased growth of functional blood and lymphatic vessels in hematolymphoid tissues relative to otherwise genetically identical normal littermate control mice, which may be promoted at least in part by VEGF production from B lymphocytes.Materials and MethodsAnimalsEμ-c-myc (C57Bl/6J) transgenic mice27Adams JM Harris AW Pinkert CA Corcoran LM Alexander WS Cory S Palmiter RD Brinster RL The c-myc oncogene driven by immunoglobulin enhancers induces lymphoid malignancy in transgenic mice.Nature. 1985; 318: 533-538Crossref PubMed Scopus (1314) Google Scholar obtained from Jackson Laboratories (West Grove, PA) were maintained under specific pathogen-free conditions, and were genotyped by polymerase chain reaction (PCR) according to the supplier's instructions. Mice were euthanized with CO2, and dissected tissues were snap-frozen in OCT (Sakura Finetek, Torrance, CA) before cryosectioning. Femur marrow was extruded by cutting off the ends of the bone and applying air pressure from a 10-mlsyringe with a 21-gauge needle, before freezing in ornithine carbamyl transferase (OCT). For paraffin sectioning, whole femur bones were formalin- or zinc-fixed30Beckstead J A simple technique for preservation of fixation-sensitive antigens in paraffin-embedded tissues.J Histochem Cytochem. 1994; 42: 8-13Crossref Scopus (226) Google Scholar and decalcified by 10% nitric acid treatment before embedding. Experimental methods involving animals were approved by the University of Washington and Fred Hutchinson Cancer Research Center Animal Care and Use Committees.ImmunostainingSerially sectioned organs from 4 to 10 pairs of Eμ-c-myc and wild-type C57Bl/6J littermates were examined in each study. For MECA-32 immunohistochemistry of zinc-fixed (BD Biosciences, San Jose, CA) and decalcified paraffin sections of bone marrow, 2-μm sections were incubated sequentially with MECA-32 antibody (BD Biosciences), biotinylated rabbit anti-rat antibody (Dako, Carpinteria, CA), and Vectastain Universal Elite ABC kit (Vector Laboratories, Burlingame, CA), followed by DAB/NiCl2 detection and Acridine Orange/Safronin O counterstaining. For laminin immunohistochemistry (Sigma, St. Louis, MO), formalin-fixed sections were treated with proteinase K (ProK; Dako) for 5 minutes before immunostaining for laminin.Eight micrometer cryosections were immunostained with the following antibodies and fixation conditions: for c-Myc (Oncogene Research Products, La Jolla, CA) or Prox-1 antibodies (generously provided by G. Oliver31Wigle JT Harvey N Detmar M Lagutina I Grosveld G Gunn MD Jackson DG Oliver G An essential role for Prox1 in the induction of the lymphatic endothelial cell phenotype.EMBO J. 2002; 21: 1505-1513Crossref PubMed Scopus (713) Google Scholar) sections were fixed in 4% paraformaldehyde for 10 minutes, and permeabilized with 0.2% Triton X-100 in saline for 2 minutes; for MECA-32, laminin, digoxygenin-labeled-10.1.1,32Farr A Nelson A Hosier S Kim A A novel cytokine-responsive cell surface glycoprotein defines a subset of medullary thymic epithelium in situ.J Immunol. 1993; 150: 116-1171Google Scholar and VEGF (sc-507; Santa Cruz Biotechnology, Santa Cruz, CA) sections were fixed in acetone for 10 minutes, dried for 15 minutes, and fixed in 10% formalin for 10 minutes; and for affinity-purified anti-VEGFR-2 (generously provided by R. Brekken33Brekken RA Huang X King SW Thorpe PE Vascular endothelial growth factor as a marker of tumor endothelium.Cancer Res. 1998; 58: 1952-1959PubMed Google Scholar) or VEGFR-3 (R and D Systems Inc., Minneapolis, MN) sections were fixed in acetone for 5 minutes. Endogenous peroxidase or alkaline phosphatase was inhibited by 30 minutes of treatment with 3% hydrogen peroxide, or with levamisole (Vector Laboratories), respectively. Immunohistochemical staining was detected with Vector VIP or Vector Black, followed by Methyl Green counterstaining (Vector Laboratories). For immunofluorescent detection, sections were mounted in Vectashield (Vector Laboratories), and imaged by confocal microscopy, using a Leica SP1 confocal microscope. Stacks of optical sections were collected by optical z sectioning (z step = 0.73 μm). For electron microscopy, BALB/C mice were briefly perfused with 2% paraformaldehyde, 1 mmol/L CaCl2, and 0.1 mol/L cacodylate pH 7.4, before mesenteric lymph nodes were dissected and embedded, and 10.1.1 immunostaining detected with horseradish peroxidase and 3,3′-diaminobenzidine (DAB), as previously described.32Farr A Nelson A Hosier S Kim A A novel cytokine-responsive cell surface glycoprotein defines a subset of medullary thymic epithelium in situ.J Immunol. 1993; 150: 116-1171Google ScholarDye Injection Analysis of Blood and Lymph CirculationEight- to 10-week-old mice were anesthetized by intraperitoneal injection of 87 mg/kg ketamine and 13 mg/kg xylazine. Fluorescein isothiocyanate (FITC)-labeled Lycopersicon esculentum lectin (0.1 ml; Vector Laboratories) was injected into the tail vein to label functional blood vessels.34Hashizume H Baluk P Morikawa S McLean JW Thurston G Roberge S Jain RK McDonald DM Openings between defective endothelial cells explain tumor vessel leakiness.Am J Pathol. 2000; 156: 1363-1380Abstract Full Text Full Text PDF PubMed Scopus (1297) Google Scholar Two minutes later, tissues were rapidly dissected and snap-frozen in OCT. Bone marrow was extruded from the bone before freezing in OCT. In some experiments mice were perfused before harvesting tissues, using an 18-gauge blunt needle placed in the left ventricle to deliver 4% paraformaldehyde at 120 mmHg. The right atrium was incised to allow flow of fixative. Serial sections through each tissue were analyzed by fluorescence or confocal microscopy.Functional blood vessel density was assessed by fluorescence microscope examination of representative 50-μm cryosections of bone marrow or lymph nodes from FITC-lectin-injected mice. The number of discrete FITC-labeled vessels in three representative and non-overlapping ×400 fields from each section (covering most of the tissue) were counted in a double-blind manner and were averaged together. Vessels in sections from five pairs of mice were counted, and Student's t-tests of significance were performed.For lymph flow measurement, the rear footpads of anesthetized mice were injected with 20 μl TRITC-dextran (Sigma), and popliteal and iliac lymph nodes were harvested 5 to 30 minutes later. Cryosections of lymph nodes from TRITC-dextran-injected mice were dried for 1 hour at room temperature, fixed with 4% paraformaldehyde for 20 minutes, and mounted in Vectashield. Representative sections were imaged by confocal microscopy at ×200 magnification. The MetaMorph computer program (Universal Imaging Corp., Downington, PA) was used to measure the spread of dye through each lymph node. The area filled with dye in confocal z stack images covering most of each node was measured in Eμ-c-myc and wild-type lymph nodes. Images from five pairs of mice were detected under the same imaging conditions through each section, and pixel intensity was thresholded relative to uninjected lymph node sections.B-Cell Purification and Immunoblotting AnalysisTissues from 4-week-old Eμ-c-myc mice and wild-type littermates were homogenized, and protein was measured by Bradford assay. Splenic B cells were purified by mashing spleens between the frosted ends of microscope slides, followed by erythrocyte lysis, as previously described.35Iritani BM Forbush KA Farrar MA Perlmutter RM Control of B cell development by Ras-mediated activation of Raf.EMBO J. 1997; 16: 7019-7031Crossref PubMed Scopus (132) Google Scholar T cells were depleted by incubating samples with anti-Thy2 monoclonal antibody, followed by treatment with guinea pig complement (Invitrogen, Carlsbad, CA). The resulting B cells are typically 90% pure, as determined by flow cytometry. B cells were lysed by incubation in 50 mmol/L Tris pH 8.0, 150 mmol/L NaCl, 1% Nonidet P40, 1 mmol/L dithiothreitol, with complete protease inhibitor cocktail (Roche, Indianapolis, IN) on ice for 30 minutes, with occasional mixing, and centrifuged 10 minutes at 14,000 × g to remove debris. Lysate protein was bound to 50 μl heparin-agarose (Sigma) for 1 hour at 4°C, eluted by 0.5% sodium dodecyl sulfate treatment at 95°C for 5 minutes, and protein was measured by Bradford assay. Twenty micrograms of protein from each preparation were immunoblotted and probed with VEGF antibody, and then reprobed with actin antibody (Sigma).ResultsAngiogenesis and Increased Blood Circulation in Bone Marrow of Young Eμ-c-myc Micec-Myc-expressing B-cell progenitors proliferate and accumulate within the bone marrow of young Eμ-c-myc mice,28Langdon WY Harris AW Cory S Adams JM The c-myc oncogene perturbs B lymphocyte development in Eu-myc transgenic mice.Cell. 1986; 47: 11-18Abstract Full Text PDF PubMed Scopus (318) Google Scholar suggesting that this model could be useful for analysis of bone marrow angiogenesis. Immunohistochemical staining confirmed that c-Myc-expressing B cells predominate within the marrow of 4-week-old Eμ-c-myc mice (Figure 1A), while other hematopoeitic progenitors are reduced, in agreement with previous studies.28Langdon WY Harris AW Cory S Adams JM The c-myc oncogene perturbs B lymphocyte development in Eu-myc transgenic mice.Cell. 1986; 47: 11-18Abstract Full Text PDF PubMed Scopus (318) Google Scholar In contrast, bone marrow from C57Bl6/J wild-type littermates contained few c-Myc-expressing cells. To determine whether c-Myc-expressing B cells induce angiogenesis within the bone marrow, the vasculature of marrow from Eμ-c-myc and wild-type littermates was assessed by immunostaining decalcified and paraffin-embedded femur sections with the MECA-32 antibody, which recognizes most vascular endothelium.36Leppink DM Bishop DK Sedmak DD Henry ML Ferguson RM Streeter PR Butcher EC Orosz CG Inducible expression of an endothelial cell antigen on murine myocardial vasculature in association with interstitial cellular infiltration.Transplantation. 1989; 48: 874-877Crossref PubMed Scopus (48) Google Scholar Bone marrow from 4- or 8-week-old Eμ-c-myc mice showed numerous small vessels dispersed throughout the marrow, while vessels were sparse in wild-type marrow (Figure 1B, arrows). Venous sinuses, which are a major feature of bone marrow vasculature, can be detected by their discontinuous laminin coat.37Nilsson SK Debatis ME Dooner MS Madri JA Quesenberry PJ Becker PS Immunofluorescence characterization of key extracellular matrix proteins in murine bone marrow in situ.J Histochem Cytochem. 1998; 46: 371-377Crossref PubMed Scopus (139) Google Scholar Immunostaining for laminin revealed these larger erythrocyte-filled sinuses, which are markedly increased in size and number in Eμ-c-myc bone marrow relative to littermate control bone marrow (Figure 1C, arrows). These findings indicate that accumulation of c-Myc-expressing progenitors promotes growth of blood vessels and venous sinuses within the marrow. The laminin coating of these sinuses and vessels is much thicker in Eμ-c-myc bone marrow, suggesting that c-Myc also increases laminin deposition as the vessels grow.While tumor cells can induce a strong angiogenic response, the newly formed vasculature can be defective.34Hashizume H Baluk P Morikawa S McLean JW Thurston G Roberge S Jain RK McDonald DM Openings between defective endothelial cells explain tumor vessel leakiness.Am J Pathol. 2000; 156: 1363-1380Abstract Full Text Full Text PDF PubMed Scopus (1297) Google Scholar, 38Holash J Maisonpierre PC Compton D Boland P Alexander CR Zagzag D Yancopoulos GD Wiegand SJ Vessel cooption, regression, and growth in tumors mediated by angiopoeitins and VEGF.Science. 1999; 284: 1994-1997Crossref PubMed Scopus (1889) Google Scholar We therefore examined whether c-Myc-induced angiogenesis produces functional vessels, by intravenously injecting 8-week-old mice with FITC-labeled L. esculentum lectin, which specifically binds to vascular endothelium.34Hashizume H Baluk P Morikawa S McLean JW Thurston G Roberge S Jain RK McDonald DM Openings between defective endothelial cells explain tumor vessel leakiness.Am J Pathol. 2000; 156: 1363-1380Abstract Full Text Full Text PDF PubMed Scopus (1297) Google Scholar Functional vessels labeled with circulating FITC-labeled lectin were compared with those labeled by Texas Red-laminin immunostaining. Confocal imaging demonstrates that intravenously injected FITC-labeled lectin fills nearly all of the laminin-coated venous sinuses and smaller blood vessels of Eμ-c-myc mice (Figure 1D). In wild-type bone marrow, blood vessels are small and sparse, and show faint laminin staining (Figure 1D). The co-localization of laminin-coated vessels and injected lectin indicates that the venous sinuses and blood vessels identified by laminin immunostaining in Eμ-c-myc mice are functional. Moreover, these studies indicate that laminin may be a useful marker to reveal the venous sinuses of the bone marrow, which are not detected using standard vascular markers such as MECA-32 (Figure 1B).The lectin injection assay was used to analyze the effect of c-Myc-induced angiogenesis on the bone marrow blood supply, using 50 μm thick cryosections to survey the entire marrow from wild-type and Eμ-c-myc mice. Confocal imaging of FITC-labeled lectin demonstrated that blood flow is greatly increased in marrow from Eμ-c-myc mice, while functional vessels are rare in control marrow (Figure 1E). This increase in circulation is specific to Eμ-c-myc bone marrow, as the kidney glomeruli (Figure 1F), liver, intestine, and skin (data not shown) from wild-type and Eμ-c-myc mice showed equivalent blood flow. The increased density of functional vessels in Eμ-c-myc marrow was confirmed by counting discrete FITC-lectin-labeled vessels in 50-μm cryosections from five pairs of mice. Functional vessel density increases more than threefold in bone marrow from Eμ-c-myc mice (Figure 2). This increase is significant by t-test (P < 0.01%). These results indicate that c-Myc-induced angiogenesis increases the blood supply to the bone marrow, which could support the sustained production of B-cell progenitors observed in bone marrow of Eμ-c-myc mice.Figure 2.Functional blood vessel density is increased in bone marrow from Eμ-c-myc mice. The number of discrete FITC-lectin-labeled vessels were counted per 400-μm field, in 50-μm bone marrow cryosections from five pairs of wild-type and Eμ-c-myc FITC lectin-injected mice. Mean functional vessel density and standard errors are shown.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Angiogenesis and Lymphangiogenesis in Eμ-c-myc Lymph NodesLymph nodes of Eμ-c-myc mice grow 2 to 4 times larger than wild-type nodes by 4 weeks of age, due to accumulation of B lymphocytes.29Harris AW Pinkert CA Crawford M Langdon WY Brinster RL Adams JM The E μ-c-myc transgenic mouse: a model for high-incidence spontaneous lymphoma and leukemia of early B cells.J Exp Med. 1988; 167: 353-371Crossref PubMed Scopus (316) Google Scholar We sought to determine whether c-Myc produced by mature B lineage cells in the periphery also induces angiogenesis within lymph nodes. Cryosections of lymph nodes from 4-week-old Eμ-c-myc or control mice were first immunostained with c-Myc antisera, to evaluate the representation of c-Myc-positive cells. c-Myc-expressing cells are distributed throughout the cortex and medulla of lymph nodes from Eμ-c-myc mice (Figure 3A) and they show