Transferrin receptor on endothelium of brain capillaries

The blood/brain barrier prevents the passive diffusion of proteins and metabolites from cerebral blood vessels into tissue spaces around neuronal and glial cells. To provide nutrients for these cells, transport mechanisms must exist and indeed have been demonstrated for metabolites. We now show that monoclonal antibodies against rat and human transferrin receptors label blood capillaries in the brain but not in other tissues. In the rat this labelling occurs after injection of antibody into the blood, thus the receptors seem to be accessible at the endothelial surface. It is possible that transferrin receptors are expressed on these cells to allow transport of transferrin (and thus iron) into brain tissues.     

Therapy with monoclonal antibodies by elimination of T-cell subsets in vivo

A major aim in immunology has been to understand how the immune system evokes characteristic responses to infection, foreign tissue grafts and tumours. The current view of immunoregulation is based mainly on studies of lymphocyte subsets, either in vitro or by adoptive transfer to irradiated recipients. Many reagents are available for defining T-cell subsets, but only recently have there been helper T-cell-specific antibodies against the mouse equivalent of the Leu3/T4 (man) and W3/25 (rat) antigens. It is clear that monoclonal antibodies will eventually replace antilymphocyte globulin for immunosuppression in organ grafting, but although there has been some clinical success, most monoclonal reagents cause only transient reductions in their target cells in vivo. This uncertainty in the potency of monoclonal antibodies has led some workers to consider them as targeting agents for such highly cytotoxic drugs as ricin A (ref. 21). We show here that unmodified monoclonal antibodies can be extremely effective at depleting cells in vivo and can be used for the selective manipulation of different aspects of the immune response.     

Application of phorbol ester to mouse skin causes a rapid and sustained loss of protein kinase C

It is now widely accepted that tumour-promoting phorbol esters activate a Ca2+- and phospholipid-dependent protein kinase (protein kinase C) both in vitro and in intact cells, and that the kinase represents a major cellular phorbol ester-binding protein. The phorbol esters act as analogues of diacylglycerol, a natural regulator of protein kinase C, and stabilize the membrane-association of the kinase. Although other molecular targets may exist, protein kinase C activation is probably important in mediating the diverse responses of cultured cells to phorbol esters and in promoting in vivo tumours. The enzyme comprises a family of closely related proteins and has been detected in extracts from mouse epidermal cells, the likely targets for two-stage carcinogenesis in mouse skin. In this report we show that application of a single dose of TPA (12-O-tetradecanoyl phorbol-13-acetate) to mouse skin results in a rapid and complete loss of protein kinase C activity which is maintained for 3-4 days. This is associated with a loss of immunologically detectable protein kinase C and the accumulation of a smaller protein detectable by antibodies recognizing the regulatory domain of protein kinase C.     

Capillary endothelial cells express basic fibroblast growth factor, a mitogen that promotes their own growth

Angiogenesis, the formation of new capillaries, which is observed in embryonic and injured tissue and is particularly prominent in the vicinity of solid tumours, involves the migration and proliferation of capillary endothelial cells. It is probably triggered by agents, such as basic fibroblast growth factor (bFGF), thought to be released from tissues adjacent to proliferating capillaries. As well as being a potent inducer of cell division in capillary endothelial cells in vitro, bFGF can act as an angiogenic agent in vivo. It is present in a wide variety of richly vascularized tissues including brain, pituitary, retina, adrenal gland, kidney, corpus luteum, placenta and various tumours. So far, however, the normal bFGF-producing cell species in these tissues have not been identified. We report here that capillary endothelial cells express the bFGF gene, that they produce and release bFGF and that bFGF derived from them can stimulate the proliferation of capillary endothelial cells. We conclude that bFGF can act as a self-stimulating growth factor for capillary endothelial cells, and that it is possible that the formation of new capillaries is induced by capillary endothelial cells themselves.     

Tumour prevention and rejection with recombinant vaccinia

Tumour-specific antigens (TSA; ref. 1) have been exploited in the diagnosis and imaging of human cancer and anti-TSA antibodies have therapeutic potential. Vaccination with TSA or anti-idiotypic (TSA) antibodies has also been used to control tumour growth in model systems. An effective immune response nevertheless demands copresentation of antigen with host histocompatibility determinants. We therefore examined whether live vaccinia virus recombinants expressing TSA in cells of the vaccinated host might better elicit tumour immunity. Polyoma virus (PY) is tumorigenic in rodents; because killed PY-transformed cells can elicit tumour immunity, a PY-specific TSA has been postulated. Tumorigenesis involves expression of three early PY proteins, large-T (LT), middle-T (MT) and small-T (ST), but their role as TSAs is unclear. We therefore expressed the three T proteins in separate vaccinia recombinants. Rejection of PY tumours was observed in rats immunized with recombinants expressing either LT or MT. Further, tumour-bearing animals could be induced to reject their tumours by inoculation of recombinants.     

Antibodies against metal chelates

Because monoclonal antibodies can recognize and bind to specific groups of atoms such as tumour antigens, they have promise for use in vivo as carriers of radionuclides, drugs or other appended molecules for diagnosis and treatment of disease. Attachment of metal ions to antibodies by means of bifunctional chelating agents can add the diverse nuclear, physical and chemical properties of the metallic elements to these specific binding proteins (ref. 4 and refs therein). With the ultimate aim of engineering probe-binding properties into the antibodies themselves, we have now prepared monoclonal antibodies against the EDTA chelate of indium. These antibodies show a remarkable preference for indium chelates; changing to another metal such as scandium or gallium can decrease the antibody-binding constant by more than three orders of magnitude. These antibodies also introduce a new degree of control over the biological distributions of chelated radionuclides, markedly altering their uptake in tumours and normal organs.     

Inhibition of Dll4 signalling inhibits tumour growth by deregulating angiogenesis

Haploinsufficiency of Dll4, a vascular-specific Notch ligand, has shown that it is essential for embryonic vascular development and arteriogenesis. Mechanistically, it is unclear how the Dll4-mediated Notch pathway contributes to complex vascular processes that demand meticulous coordination of multiple signalling pathways. Here we show that Dll4-mediated Notch signalling has a unique role in regulating endothelial cell proliferation and differentiation. Neutralizing Dll4 with a Dll4-selective antibody rendered endothelial cells hyperproliferative, and caused defective cell fate specification or differentiation both in vitro and in vivo. In addition, blocking Dll4 inhibited tumour growth in several tumour models. Remarkably, antibodies against Dll4 and antibodies against vascular endothelial growth factor (VEGF) had paradoxically distinct effects on tumour vasculature. Our data also indicate that Dll4-mediated Notch signalling is crucial during active vascularization, but less important for normal vessel maintenance. Furthermore, unlike blocking Notch signalling globally, neutralizing Dll4 had no discernable impact on intestinal goblet cell differentiation, supporting the idea that Dll4-mediated Notch signalling is largely restricted to the vascular compartment. Therefore, targeting Dll4 might represent a broadly efficacious and well-tolerated approach for the treatment of solid tumours.     

Endothelial tubes assemble from intracellular vacuoles in vivo

The formation of epithelial tubes is crucial for the proper development of many different tissues and organs, and occurs by means of a variety of different mechanisms. Morphogenesis of seamless, properly patterned endothelial tubes is essential for the development of a functional vertebrate circulatory system, but the mechanism of vascular lumenization in vivo remains unclear. Evidence dating back more than 100 years has hinted at an important function for endothelial vacuoles in lumen formation. More than 25 years ago, in some of the first endothelial cell culture experiments in vitro, Folkman and Haudenschild described "longitudinal vacuoles" that "appeared to be extruded and connected from one cell to the next", observations confirmed and extended by later studies in vitro showing that intracellular vacuoles arise from integrin-dependent and cdc42/Rac1-dependent pinocytic events downstream of integrin-extracellular-matrix signalling interactions. Despite compelling data supporting a model for the assembly of endothelial tubes in vitro through the formation and fusion of vacuoles, conclusive evidence in vivo has been lacking, primarily because of difficulties associated with imaging the dynamics of subcellular endothelial vacuoles deep within living animals. Here we use high-resolution time-lapse two-photon imaging of transgenic zebrafish to examine how endothelial tubes assemble in vivo, comparing our results with time-lapse imaging of human endothelial-cell tube formation in three-dimensional collagen matrices in vitro. Our results provide strong support for a model in which the formation and intracellular and intercellular fusion of endothelial vacuoles drives vascular lumen formation.     

Imaging in the era of molecular oncology

New technologies for imaging molecules, particularly optical technologies, are increasingly being used to understand the complexity, diversity and in vivo behaviour of cancers. 'Omic' approaches are providing comprehensive 'snapshots' of biological indicators, or biomarkers, of cancer, but imaging can take this information a step further, showing the activity of these markers in vivo and how their location changes over time. Advances in experimental and clinical imaging are likely to improve how cancer is understood at a systems level and, ultimately, should enable doctors not only to locate tumours but also to assess the activity of the biological processes within these tumours and to provide 'on the spot' treatment.     

Inhibition of antigen-induced T-cell clone proliferation by antigen-specific antibodies

Attempts to inhibit the recognition of soluble antigens by T lymphocytes using antibodies specific for the antigen in question have been uniformally unsuccessful, in contrast to the observed specific inhibition of antibody generation by B cells. One exception is the unique situation whereby anti-hapten antisera inhibit the T-cell proliferative responses observed when hapten-specific T lymphocytes or clones are cultured with hapten-derivatized cells or proteins. The inability to inhibit T-cell functions by antigen-specific antibodies has been interpreted in several ways: (1) T cells possess a different repertoire from B cells; (2) the antibodies tested recognize epitopes present on the native antigen, whereas T cells recognize non-native (processed) structures; (3) the antigenic determinant(s) recognized by T cells on the surface of antigen presenting cells are either not accessible to antibodies, or are present in low amounts. The development of antigen-specific T-cell clones and monoclonal antibodies both specific for the same antigenic determinants now allows this question to be investigated definitively. Here, we report for the first time the specific inhibition of antigen-induced T-cell clone proliferation by a monoclonal antibody directed against the relevant soluble protein antigen.     

Bv8 regulates myeloid-cell-dependent tumour angiogenesis

Bone-marrow-derived cells facilitate tumour angiogenesis, but the molecular mechanisms of this facilitation are incompletely understood. We have previously shown that the related EG-VEGF and Bv8 proteins, also known as prokineticin 1 (Prok1) and prokineticin 2 (Prok2), promote both tissue-specific angiogenesis and haematopoietic cell mobilization. Unlike EG-VEGF, Bv8 is expressed in the bone marrow. Here we show that implantation of tumour cells in mice resulted in upregulation of Bv8 in CD11b+Gr1+ myeloid cells. We identified granulocyte colony-stimulating factor as a major positive regulator of Bv8 expression. Anti-Bv8 antibodies reduced CD11b+Gr1+ cell mobilization elicited by granulocyte colony-stimulating factor. Adenoviral delivery of Bv8 into tumours was shown to promote angiogenesis. Anti-Bv8 antibodies inhibited growth of several tumours in mice and suppressed angiogenesis. Anti-Bv8 treatment also reduced CD11b+Gr1+ cells, both in peripheral blood and in tumours. The effects of anti-Bv8 antibodies were additive to those of anti-Vegf antibodies or cytotoxic chemotherapy. Thus, Bv8 modulates mobilization of CD11b+Gr1+ cells from the bone marrow during tumour development and also promotes angiogenesis locally.     

Proteomic analysis of active multiple sclerosis lesions reveals therapeutic targets

Understanding the neuropathology of multiple sclerosis (MS) is essential for improved therapies. Therefore, identification of targets specific to pathological types of MS may have therapeutic benefits. Here we identify, by laser-capture microdissection and proteomics, proteins unique to three major types of MS lesions: acute plaque, chronic active plaque and chronic plaque. Comparative proteomic profiles identified tissue factor and protein C inhibitor within chronic active plaque samples, suggesting dysregulation of molecules associated with coagulation. In vivo administration of hirudin or recombinant activated protein C reduced disease severity in experimental autoimmune encephalomyelitis and suppressed Th1 and Th17 cytokines in astrocytes and immune cells. Administration of mutant forms of recombinant activated protein C showed that both its anticoagulant and its signalling functions were essential for optimal amelioration of experimental autoimmune encephalomyelitis. A proteomic approach illuminated potential therapeutic targets selective for specific pathological stages of MS and implicated participation of the coagulation cascade.     

Notch-dependent VEGFR3 upregulation allows angiogenesis without VEGF-VEGFR2 signalling

Developing tissues and growing tumours produce vascular endothelial growth factors (VEGFs), leading to the activation of the corresponding receptors in endothelial cells. The resultant angiogenic expansion of the local vasculature can promote physiological and pathological growth processes. Previous work has uncovered that the VEGF and Notch pathways are tightly linked. Signalling triggered by VEGF-A (also known as VEGF) has been shown to induce expression of the Notch ligand DLL4 in angiogenic vessels and, most prominently, in the tip of endothelial sprouts. DLL4 activates Notch in adjacent cells, which suppresses the expression of VEGF receptors and thereby restrains endothelial sprouting and proliferation. Here we show, by using inducible loss-of-function genetics in combination with inhibitors in vivo, that DLL4 protein expression in retinal tip cells is only weakly modulated by VEGFR2 signalling. Surprisingly, Notch inhibition also had no significant impact on VEGFR2 expression and induced deregulated endothelial sprouting and proliferation even in the absence of VEGFR2, which is the most important VEGF-A receptor and is considered to be indispensable for these processes. By contrast, VEGFR3, the main receptor for VEGF-C, was strongly modulated by Notch. VEGFR3 kinase-activity inhibitors but not ligand-blocking antibodies suppressed the sprouting of endothelial cells that had low Notch signalling activity. Our results establish that VEGFR2 and VEGFR3 are regulated in a highly differential manner by Notch. We propose that successful anti-angiogenic targeting of these receptors and their ligands will strongly depend on the status of endothelial Notch signalling.     

Synthetic analogues of fumagillin that inhibit angiogenesis and suppress tumour growth.

Neovascularization is critical for the growth of tumours and is a dominant feature in a variety of angiogenic diseases such as diabetic retinopathy, haemangiomas, arthritis and psoriasis. Recognition of the potential therapeutic benefit of controlling unabated capillary growth has led to a search for safe and effective angiogenesis inhibitors. We report here the synthesis of a family of novel inhibitors that are analogues of fumagillin, a naturally secreted antibiotic of Aspergillus fumigatus fresenius. We first isolated this fungus from a contaminated culture of capillary endothelial cells. Purified fumagillin inhibited endothelial cell proliferation in vitro and tumour-induced angiogenesis in vivo; it also inhibited tumour growth in mice, but prolonged administration was limited because it caused severe weight loss. Synthesis of fumagillin analogues yielded potent angiogenesis inhibitors ('angioinhibins') which suppress the growth of a wide variety of tumours with relatively few side-effects.     

CCL2 recruits inflammatory monocytes to facilitate breast-tumour metastasis

Macrophages, which are abundant in the tumour microenvironment, enhance malignancy. At metastatic sites, a distinct population of metastasis-associated macrophages promotes the extravasation, seeding and persistent growth of tumour cells. Here we define the origin of these macrophages by showing that Gr1-positive inflammatory monocytes are preferentially recruited to pulmonary metastases but not to primary mammary tumours in mice. This process also occurs for human inflammatory monocytes in pulmonary metastases of human breast cancer cells. The recruitment of these inflammatory monocytes, which express CCR2 (the receptor for chemokine CCL2), as well as the subsequent recruitment of metastasis-associated macrophages and their interaction with metastasizing tumour cells, is dependent on CCL2 synthesized by both the tumour and the stroma. Inhibition of CCL2-CCR2 signalling blocks the recruitment of inflammatory monocytes, inhibits metastasis in vivo and prolongs the survival of tumour-bearing mice. Depletion of tumour-cell-derived CCL2 also inhibits metastatic seeding. Inflammatory monocytes promote the extravasation of tumour cells in a process that requires monocyte-derived vascular endothelial growth factor. CCL2 expression and macrophage infiltration are correlated with poor prognosis and metastatic disease in human breast cancer. Our data provide the mechanistic link between these two clinical associations and indicate new therapeutic targets for treating metastatic breast cancer.     

Blocking VEGFR-3 suppresses angiogenic sprouting and vascular network formation

Angiogenesis, the growth of new blood vessels from pre-existing vasculature, is a key process in several pathological conditions, including tumour growth and age-related macular degeneration. Vascular endothelial growth factors (VEGFs) stimulate angiogenesis and lymphangiogenesis by activating VEGF receptor (VEGFR) tyrosine kinases in endothelial cells. VEGFR-3 (also known as FLT-4) is present in all endothelia during development, and in the adult it becomes restricted to the lymphatic endothelium. However, VEGFR-3 is upregulated in the microvasculature of tumours and wounds. Here we demonstrate that VEGFR-3 is highly expressed in angiogenic sprouts, and genetic targeting of VEGFR-3 or blocking of VEGFR-3 signalling with monoclonal antibodies results in decreased sprouting, vascular density, vessel branching and endothelial cell proliferation in mouse angiogenesis models. Stimulation of VEGFR-3 augmented VEGF-induced angiogenesis and sustained angiogenesis even in the presence of VEGFR-2 (also known as KDR or FLK-1) inhibitors, whereas antibodies against VEGFR-3 and VEGFR-2 in combination resulted in additive inhibition of angiogenesis and tumour growth. Furthermore, genetic or pharmacological disruption of the Notch signalling pathway led to widespread endothelial VEGFR-3 expression and excessive sprouting, which was inhibited by blocking VEGFR-3 signals. Our results implicate VEGFR-3 as a regulator of vascular network formation. Targeting VEGFR-3 may provide additional efficacy for anti-angiogenic therapies, especially towards vessels that are resistant to VEGF or VEGFR-2 inhibitors.     

The accessible surface area and stability of oligomeric proteins

Protein structures are stabilized by hydrophobic and van der Waals forces, and by hydrogen bonds. The relation between these thermodynamic quantities and the actual three-dimensional structure of proteins can not be calculated precisely. However, certain empirical relations have been discovered. Hydrophobic energy is gained by the reduction of surface in contact with water. For monomeric proteins, the area of the surface accessible to solvent, and of that buried in the interior, is a simple function of molecular weight. Proteins with different shapes and secondary structures, but of the same molecular weight, have the same accessible surface area. It has been argued that there is no similar relationship for large oligomeric proteins. In this paper we show that the surface areas of oligomeric proteins, and the areas of the surface buried within them, are directly related to relative molecular mass. Although oligomers of the same molecular weight bury the same amounts of surface, the proportions buried within and between subunits vary. This has important implications for the role of subunit interfaces in the stability and activity of oligomeric proteins.     

Tumour necrosis factor alpha stimulates resorption and inhibits synthesis of proteoglycan in cartilage

During inflammatory reactions, activated leukocytes are thought to produce a variety of small proteins (cytokines) that influence the behaviour of other cells (including other leukocytes). Of these substances, which include the interleukins, interferons and tumour necrosis factors (TNFs), interleukin-1 (IL-1) has been considered potentially a most important inflammatory mediator because of its wide range of effects. In vivo it is pyrogenic and promotes the acute phase response; in vitro it activates lymphocytes and stimulates resorption of cartilage and bone. Cartilage resorption is a major feature of inflammatory diseases such as rheumatoid arthritis, and IL-1 is the only cytokine hitherto known to promote it. TNFs are characterized by their effects on tumours and cytotoxicity to transformed cells, but share some actions with IL-1. I report here that recombinant human TNF alpha stimulates resorption and inhibits synthesis of proteoglycan in explants of cartilage. Its action is similar to and additive with IL-1, and it is a second macrophage-derived cytokine whose production in rheumatoid arthritis, or inflammation generally, could contribute to tissue destruction.     

Diverse behavioural defects caused by mutations in Caenorhabditis elegans unc-43 CaM kinase II

Calcium/calmodulin-dependent serine/threonine kinase type II (CaMKII) is one of the most abundant proteins in the mammalian brain, where it is thought to regulate synaptic plasticity and other processes. Activation of the multisubunit kinase by calcium is effectively cooperative and can persist long after transient calcium rises. Despite extensive biochemical characterization of CaMKII and identification of numerous in vitro kinase targets, little is known about its function in vivo. Here we report that unc-43 encodes the only Caenorhabditis elegans CaMKII. A gain-of-function unc-43 mutation reduces locomotory activity, alters excitation of three muscle types and lengthens the period of the motor output of a behavioural clock. Null unc-43 mutations cause phenotypes generally opposite to those of the gain-of-function mutation. Mutations in the unc-103 potassium channel gene suppress a gain-of-function phenotype of unc-43 in one tissue without affecting other tissues; thus, UNC-103 may be a tissue-specific target of CaMKII in vivo.