ABSTRACT
The future ability to manipulate the growth of new blood vessels (angiogenesis) holds great promise for treating ischemic disease and cancer. Several models of human in vivo angiogenesis have been described, but they seem to depend on transgenic support and have not been validated in a therapeutic context. Here, we describe an in vivo assay that mimics human angiogenesis in which native human umbilical vein-derived endothelial cells are suspended in a liquid laminin/collagen gel (Matrigel), injected into immunodeficient mice, and develop into mature, functional vessels that vascularize the Matrigel plug in the course of 30 d. Moreover, we demonstrate how to target this process therapeutically by sustained delivery of the angiogenesis inhibitor endostatin from subcutaneously implanted microosmotic pumps.
Subject(s)
Collagen , Drug Evaluation, Preclinical/methods , Endothelium, Vascular/cytology , Laminin , Neovascularization, Physiologic/drug effects , Proteoglycans , Angiogenesis Inducing Agents/pharmacology , Animals , Cells, Cultured , DNA-Binding Proteins/genetics , Drug Combinations , Endostatins/pharmacology , Endothelium, Vascular/drug effects , Humans , Immunologic Deficiency Syndromes/genetics , Injections, Subcutaneous , Mice , Mice, Knockout , Platelet Endothelial Cell Adhesion Molecule-1/analysis , Umbilical Veins/cytologyABSTRACT
Gene-targeted mice have recently revealed a role for lymphocytes and interferon-gamma (IFNgamma) in conferring protection against cancer, but the mechanisms remain unclear. Here, we have characterized a successful primary antitumor immune response initiated by naive CD4+ T cells. Major histocompatibility complex class II (MHC-II)-negative myeloma cells injected subcutaneously into syngeneic mice were surrounded within 3 days by macrophages that captured tumor antigens. Within 6 days, naive myeloma-specific CD4+ T cells became activated in draining lymph nodes and subsequently migrated to the incipient tumor site. Upon recognition of tumor-derived antigenic peptides presented on MHC-II by macrophages, the myeloma-specific CD4+ T cells were reactivated and started to secrete cytokines. T cell-derived IFNgamma activated macrophages in close proximity to the tumor cells. Tumor cell growth was completely inhibited by such locally activated macrophages. These data indicate a mechanism for immunosurveillance of MHC-II-negative cancer cells by tumor-specific CD4+ T cells through collaboration with macrophages.
Subject(s)
CD4-Positive T-Lymphocytes/immunology , Immunologic Surveillance , Lymphocyte Activation/immunology , Multiple Myeloma/immunology , Animals , Cell Line, Tumor , Collagen , Drug Combinations , Drug Therapy , Enzyme-Linked Immunosorbent Assay , Flow Cytometry , Histocompatibility Antigens Class II/immunology , Immunohistochemistry , Interferon-gamma/immunology , Interferon-gamma/metabolism , Laminin , Macrophages/immunology , Mice , Mice, SCID , Mice, Transgenic , Neoplasm Transplantation , Proteoglycans , Receptors, Antigen, T-Cell/geneticsABSTRACT
Endostatin is a proteolytic fragment of collagen XVIII that inhibits endothelial cell migration in vitro and experimental tumor growth in vivo. To determine how endostatin affects the in vivo behavior of endothelial cells, we took advantage of a surrogate model of human angiogenesis, in which human endothelial cells are transferred to immunodeficient mice and develop into complex vessels in the course of 30 days. Systemic delivery of human yeast-derived endostatin (serum levels of 30-35 ng/mL) inhibited the number of human vessels dramatically (95% at day 20), as most endothelial cells remained suspended as single cells. The fraction of cells with a migratory phenotype (F-actin-positive, extending pseudopods) was strongly reduced (from 50% to 13% at day 10), while the number of apoptotic and mitotic cells remained unchanged. Endostatin also hampered the recruitment of alpha-smooth muscle actin-expressing perivascular cells and thus reduced the number of mature vessels (from 64.3% to 28.6% at day 30). Moreover, transcripts of pericyte-recruiting platelet-derived growth factor-B (PDGFB) were strongly reduced in endothelial cells of endostatin-treated mice. Our results are strong evidence that endostatin inhibits angiogenesis at several levels in vivo, including perivascular cell recruitment.
Subject(s)
Angiogenesis Inhibitors/pharmacology , Angiostatins/pharmacology , Cell Movement/physiology , Endothelium, Vascular/physiology , Proto-Oncogene Proteins c-sis/physiology , Animals , Capillaries/drug effects , Capillaries/physiology , Cell Movement/drug effects , Endothelium, Vascular/drug effects , Humans , Mice , Mice, Inbred BALB C , Mice, SCID , Morphogenesis/drug effects , Morphogenesis/physiologyABSTRACT
To establish a model for adoptive transfer of endothelial cells, we transferred human umbilical vein endothelial cells (HUVECs) to immunodeficient mice (Rag 2(-/-)). HUVECs were suspended as single cells in Matrigel and injected subcutaneously in the abdominal midline. Within 10 days after injection, HUVECs expressed pseudopod-like extensions and began to accumulate in arrays. By day 20, we observed human vessels that contained erythrocytes, and on day 30 we observed perivascular cells that expressed smooth muscle actin, thus resembling mature vessels. Throughout the experimental period, HUVECs bound Ulex europaeus lectin and expressed CD31, VE-cadherin, von Willebrand factor, as well as ICAM-2. A fraction of the cells also expressed the proliferation marker Ki67. Moreover, the sialomucin CD34, which is rapidly down-regulated in cultured HUVECs, was reinduced in vivo. However, we found no reinduction of CD34 in cells cultured inside or on top of Matrigel in vitro. We also injected cells suspended in Matrigel around the catheter tip of implanted osmotic pumps. Delivery of recombinant human interferon-gamma by this route led to strong induction of MHC class II and ICAM-1 on the human vessels. In conclusion, isolated human endothelial cells can integrate with the murine vascular system to form functional capillaries and regain in vivo properties.
