The regulation of angiogenesis by tissue cell-macrophage interactions

Angiogenesis is the physiological process where new blood vessels grow from existing ones, in order to replenish tissues suffering from inadequate blood supply. Perhaps the most studied angiogenic process occurs in solid tumors whose growing mass and expanding cells create a constant demand for additional supply of oxygen and nutrients for survival. However, other physiological and clinical conditions, such as wound healing, ischemic events, autoimmune and age-related diseases also involve angiogenesis. Angiogenesis is a well-structured process that begins when oxygen and nutrients are depleted, leading to the release of chemokines and growth factors that attract immune cells, particularly macrophages and endothelial cells to the site. Macrophages that are recruited to the site, as well as tissue cells and endothelial cells, secrete pro-angiogenic mediators that affect endothelial cells and promote angiogenesis. These mediators include growth factors such as vascular endothelial cell growth factor (VEGF), matrix metalloproteinases (MMPs), as well as low levels of mediators that are usually seen as pro-inflammatory but are pro-angiogenic when secreted in low levels (e.g. nitric oxide (NO) and TNFa). Thus, macrophages play a major role in angiogenesis. Macrophages exhibit high plasticity and are capable of shifting between different activation modes and functions according to their changing microenvironment. Small differences in the composition of activating factors (e.g. TLR ligands such as LPS, anti-inflammatory cytokines, ECM molecules) in the microenvironment may differently activate macrophages to yield classically activated macrophages (or M1 macrophages) that can kill pathogen and tumor cells, alternatively activated macrophages (or M2 macrophages) that secrete antiinflammatory cytokines, resolution macrophages (rM?) that are involved in the resolution of inflammation, or regulatory macrophages (e.g. Myeloid-Derived Suppressor Cells - MDSCs) that control the function of other immune cells. In fact, macrophages may be activated in a spectrum of subsets that may differently contribute to angiogenesis, and in particular non-classically activated macrophages such as tumor-associated macrophages (TAMs) and Tie2-expressing monocytes (TEMs) can secrete high amounts of pro-angiogenic factors (e.g. VEGF, MMPs) or low levels of pro-inflammatory mediators (e.g. NO or TNFa) resulting in pro-angiogenic effects. Although the importance of macrophages as major contributors and regulators of the angiogenic process is well documented, less is known about the interactions between macrophages and other cell types (e.g. tumor cells, normal epithelial cells, endothelial cells) that regulate angiogenesis. We still have only limited understanding which proteins or complexes mediate these interactions and whether they require cell-cell contact (e.g. through integrins) or soluble factors (e.g. the EGF-CSF-1 loop), which signaling pathways are triggered in each of the two corresponding cell types, and how this leads to secretion of pro- or antiangiogenic factors in the microenvironment. The regulation of such interactions and through them of angiogenesis, whether through post-translational modifications of proteins or via the involvement of microRNA, is still unclear. The goal of this Research Topic is to highlight these interactions and their regulation in the context of both physiological and pathological conditions

Molecular events involved in the increased expression of matrix metalloproteinase-9 by T lymphocytes of mammary tumor-bearing mice

Matrix metalloproteinases (MMPs) are a family of extracellular proteinases whose contributions to cancer progression have been studied because of their matrix-degrading abilities and elevated expression in advanced stage tumors. Recent findings suggest a role for MMPs during the multiple stages of tumor progression including establishment and growth, migration, invasion, metastasis, and angiogenesis. MMP-9 regulation at the molecular level can be studied by measuring the effect(s) of a variety of physiological and pharmacological agents on cells. Multiple signaling molecules such as protein kinase C, pertussis toxin-sensitive guanine nucleotide-binding protein G, and protein tyrosine kinases are known to mediate the secretion of MMPs in cell lines. We previously reported an upregulation of MMP-9 in T cells of mammary tumor-bearing mice. In this study, pharmacologic inhibitors were used to dissect the signaling pathways involved in the upregulation of MMP-9 in the splenic T cells of normal and mammary tumor-bearing mice. Staurosporine, a protein kinase inhibitor, stimulated MMP-9 secretion by normal T lymphocytes, while the constitutively high levels of MMP-9 produced by tumor bearers' T cells were decreased by Genistein, a specific tyrosine kinase inhibitor, and Rottlerin, a PKC inhibitor. Using a NF-kappaB specific probe to the murine MMP-9 promoter, electromobility shift assays of nuclear proteins from normal and tumor bearers' splenic T cells revealed a pattern of higher intensity bands from the tumor bearers' nuclear extracts, indicating a greater amount of these transcription factors bound to the recognition motif. When mammary tumor bearers' T cells were cultured with the NF-kappaB inhibitors, N-p-Tosyl-L-lysine chloromethyl ketone hydrochloride and Bay 11-7082, there was a subsequent decreased production of MMP-9. These results suggest that the tumor burden may be activating various signaling pathways within splenic T lymphocytes to upregulate MMP-9 expression

GM-CSF up-regulates the expression of CCL2 by T lymphocytes in mammary tumor-bearing mice

CP-1/CCL2 (monocyte chemoattractant protein-1/CC chemokine ligand 2) is a β or CC chemokine that is expressed by a variety of cell types, including fibroblasts, endothelial, smooth muscle, and glial cells. In addition, cells involved in immunity, such as monocytes/macrophages, neutrophils, and eosinophils have also been shown to express this chemoattractant. Using a murine model of the D1-DMBA-3 mammary adenocarcinoma, we demonstrated the unique production of CCL2 by splenic T lymphocytes from tumor-bearing animals. Because this tumor produces GM-CSF, and this factor is also up-regulated in the B lymphocytes of tumor-bearing mice, we looked at the ability of GM-CSF to induce CCL2 production by T cells. Treatment of normal and tumor bearers' T cells with GM-CSF resulted in an increased secretion of this chemokine. This up-regulation was seen with or without stimulation by Concanavalin A, although these treatments were additive in their effects. The induction of CCL2 was studied at the molecular level by analyzing the effect(s) of a variety of physiological and pharmacological agents on cultured T cells. These results suggest that the tumor-derived factor GM-CSF activates various signaling pathways within splenic T cells to up-regulate CCL2 expression

Abstract 792: Tumor-induced thymic atrophy: Alteration in interferons and Jak/Stats signaling pathways

Abstract The thymus is the major site of T cell differentiation and a key organ of the immune system. Most important, it provides a specialized environment for the selection of rearranged clones that will function appropriately in the adaptive immune response. Thymic involution has been observed in several model systems; including graft-vs-host disease, aging, and tumor development, however, the mechanisms involved in this phenomenon remain to be elucidated. Previous results from our laboratory have reported that the severe thymic atrophy and impaired T cell development seen in mammary tumor bearers are associated with an arrest in at least two steps of T cell differentiation, changes in the levels of crucial cytokines expressed in the thymus microenvironment, and a progressive increase in apoptosis during the tumor development mainly due to downregulation of important molecules that control programmed cell death. Cytokines regulate numerous aspects of hematopoiesis via activation of the Jak/Stat pathways. In the present study we have used our mammary tumor model to investigate whether changes in the levels of cytokines in the thymus could affect the normal expression of the aforementioned pathways. RNA and protein analysis revealed an overexpression of the different interferons, a downregulation of most of the Jak/Stat pathways, and an increased expression of several suppressors of cytokine signaling (SOCS) in the thymuses of tumor bearers. Collectively, our data suggest a mechanism by which the impaired Jak/Stat signaling pathways observed in the whole thymus of tumor-bearing mice could be contributing to the abnormal T cell development and apoptosis observed during the tumor-induced thymic atrophy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 792. doi:10.1158/1538-7445.AM2011-792</jats:p

Exploring the role of CHI3L1 in “pre-metastatic” lungs of mammary tumor-bearing mice

Elevated levels of chitinase-3-like-1 (CHI3L1) are associated with poor prognosis, shorter recurrence-free intervals and low survival in breast cancer patients. Breast cancer often metastasizes to the lung. We hypothesized that molecules expressed in the “pre-metastatic” lung microenvironment could support the newly immigrant tumor cells by providing growth and angiogenic factors. Macrophages are known to play an important role in tumor growth by releasing pro-angiogenic molecules. Using mouse mammary tumor models, we have previously shown that during neoplastic progression both the mammary tumor cells and splenic macrophages from tumor-bearing mice express higher levels of CHI3L1 compared to normal control mice. However, the role of CHI3L1 in inducing angiogenesis by macrophages at the pulmonary microenvironment to support newly arriving breast cancer cells is not yet known. In this study, we determined the expression of CHI3L1 in bronchoalveolar lavage macrophages and interstitial macrophages in regulating angiogenesis that could support the growth of newly immigrant mammary tumor cells into the lung. Here we show that in vitro treatment of pulmonary macrophages with recombinant murine CHI3L1 resulted in enhanced expression of pro-angiogenic molecules including CCL2, CXCL2, and MMP-9. We and others have previously shown that inhibition of CHI3L1 decreases the production of angiogenic molecules. In this study, we explored if in vivo administration of chitin microparticles has an effect on the expression of CHI3L1 and pro-angiogenic molecules in the lungs of mammary tumor-bearing mice. We show that treatment with chitin microparticles decreases the expression of CHI3L1 and pro-angiogenic molecules in the “metastatic” lung. These studies suggest that targeting CHI3L1 may serve as a potential therapeutic agent to inhibit angiogenesis and thus possibly tumor growth and metastasis

Induction of proinflammatory mediators by CHI3L1 is reduced by chitin treatment: decreased tumor metastasis in a breast cancer model

Disseminated metastasis accounts for over 90% of breast cancer deaths. Recently, elevated serum levels of a glycoprotein known as chitinase-3 like-protein-1 (CHI3L1) has been correlated with poor prognosis and shorter survival of patients with metastatic breast cancer. In this study, we show that there are increased levels of CHI3L1 in plasma of tumor-bearing mice and that both tumor cells and immune cells express and secrete CHI3L1. However, the biological and physiological functions of CHI3L1 are still unclear. We demonstrate that while CHI3L1 has an inhibitory role in the expression of interferon-gamma (IFN-γ), CHI3L1 up-regulates pro-inflammatory mediators, C-chemokine ligand 2 (CCL2), Chemokine CX motif ligand 2 (CXCL2) and matrix metalloproteinase-9 (MMP-9) all of which contribute to tumor growth and metastasis. We found that in vitro inhibition of CHI3L1 by siRNA suppressed the production of CCL2, CXCL2 and MMP-9 by macrophages. In vivo treatment of mammary tumor-bearing mice with chitin (β-(1–4)-poly-N-acetyl D-glucosamine), a TH 1 adjuvant and a ligand for CHI3L1, promoted immune effector functions with increased production of IFN-γ and decreased CCL2, CXCL2 and MMP-9 expression. In vivo administration of chitin to mammary tumor-bearing mice significantly decreased lung metastasis. These studies show that CHI3L1 plays a role in tumor progression and that chitin can inhibit the pleiotropic effects of CHI3L1 giving support to the idea that CHI3L1 is a useful therapeutic target for treatment of breast cancer

Decreased levels of both Stat1 and Stat3 in T lymphocytes from mice bearing mammary tumors

Lymphocytes from tumor-bearing animals have been shown to lack antitumor function. The objective of this study was to investigate the status of the signal transducers, Stat1 and Stat3, in T lymphocytes of animals bearing D1-DMBA-3 mammary tumors and to elucidate if any alterations in these signal transducers can be explained by the presence of tumor-derived factors and correlated with the lack of antitumor function in these cells. T Lymphocytes from spleens of normal and tumor-bearing mice were purified and assayed for the presence of Stat1 and Stat3 by Western blot analysis. It was found that levels of both Stat1 and Stat3 were reduced in T lymphocytes of tumor-bearers not only in their active, phosphorylated form but in total protein levels. These findings indicate that during mammary tumor progression, alteration of various transcription factors may contribute to the down-regulation of immune function

Abstract 448: Mechanisms involved in the tumor-induced thymic involution

Abstract The thymus is a central lymphoid organ critical for the development and maintenance of an effective peripheral T cell repertoire. Most important, it provides a specialized environment for the selection of rearranged clones that will function appropriately in the adaptive immune response. T cell maturation progresses through well-defined stages in which proliferation, differentiation, and apoptosis are central to the selection of a functional TCR repertoire. During mammary tumorigenesis, there is a profound thymus involution associated with a severe depletion of the most abundant subset of thymocytes, CD4+CD8+ double positive (DP) immature cells. Thymic involution has been observed in several model systems; including graft-vs-host disease, aging, and tumor development, however, the precise mechanisms involved in this phenomenon remain poorly defined. Previous results from our laboratory have reported that the severe thymic atrophy and impaired T cell development seen in mammary tumor bearers are associated with a profound decrease in thymus size accompanied by a disrupture of the normal thymic architecture, an arrest in at least two steps of T cell differentiation, and an increase in thymic apoptosis in tumor-bearers. In our studies we have used the murine D1-DMBA-3 mammary tumor model to elucidate the exact mechanisms associated to the tumor-induced impaired T cell development and thymic involution. We have found that the disruption in the thymic architecture of tumor bearers appears to be related to a downregulated expression of hepatocyte growth factor in the medullar zone of the thymus. Moreover, the progressive increase of apoptosis in thymocytes during the tumor development is associated to a downregulation of Bcl-xL and A1, two important molecules that control programmed cell death. Additionally, the impaired T cell development observed during the tumor-induced thymic involution is associated to a profound decrease of IL-7 and IL-15, two crucial cytokines involved in T cell development; a downregulation in the expression of most of the Jak/Stat signaling proteins; and an increased expression of several suppressor of cytokine signaling (SOCS) members. Our data provides the bases for understanding the mechanisms associated to the impaired T cell development and thymic involution present in the thymuses of tumor bearers. Citation Format: Roberto Carrio-Jardines, Vijaya Iragavarapu-Charyulu, Stephania Libreros, Ramón A. García-Areas, Marta Torroella-Kouri, Diana M. Lopez. Mechanisms involved in the tumor-induced thymic involution. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 448. doi:10.1158/1538-7445.AM2013-448</jats:p