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1.
Proc Natl Acad Sci U S A ; 103(42): 15491-6, 2006 Oct 17.
Article in English | MEDLINE | ID: mdl-17030814

ABSTRACT

Angiopoietin (Ang)-2, a context-dependent agonist/antagonist for the vascular-specific Tie2 receptor, is highly expressed by endothelial cells at sites of normal and pathologic angiogenesis. One prevailing model suggests that in these settings, Ang-2 acts as an autocrine Tie2 blocker, inhibiting the stabilizing influence of the Tie2 activator Ang-1, thereby promoting vascular remodeling. However, the effects of endogenous Ang-2 on cells that are actively producing it have not been studied in detail. Here, we demonstrate that Ang-2 expression is rapidly induced in endothelial cells by the transcription factor FOXO1 after inhibition of the phosphatidylinositol 3-kinase/Akt pathway. We employ RNAi and blocking antibodies to show that in this setting, Ang-2 unexpectedly functions as a Tie2 agonist, bolstering Akt activity so as to provide negative feedback on FOXO1-regulated transcription and apoptosis. In addition, we show that Ang-2, like Ang-1, activates Tie2/Akt signaling in vivo, thereby inhibiting the expression of FOXO1 target genes. Consistent with a role for Ang-2 as a Tie2 activator, we demonstrate that Ang-2 inhibits vascular leak. Our data suggests a model in which Ang-2 expression is induced in stressed endothelial cells, where it acts as an autocrine Tie2 agonist and protective factor.


Subject(s)
Angiopoietin-2/metabolism , Autocrine Communication , Endothelial Cells/physiology , Oxidative Stress , Androstadienes/metabolism , Angiopoietin-2/genetics , Animals , Apoptosis/physiology , Cells, Cultured , Endothelial Cells/cytology , Forkhead Box Protein O1 , Forkhead Transcription Factors/genetics , Forkhead Transcription Factors/metabolism , Humans , Mice , Phosphatidylinositol 3-Kinases/metabolism , Phosphorylation , Protein Kinase Inhibitors/metabolism , Proto-Oncogene Proteins c-akt/metabolism , RNA Interference , Receptor, TIE-2/genetics , Receptor, TIE-2/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Signal Transduction/physiology , Transcriptional Activation , Wortmannin
2.
J Bone Joint Surg Am ; 85(12): 2332-42, 2003 Dec.
Article in English | MEDLINE | ID: mdl-14668502

ABSTRACT

BACKGROUND: The formation of the skeleton requires inductive signals that are balanced with their antagonists in a highly regulated negative feedback system. Inappropriate or excessive expression of BMPs (bone morphogenetic proteins) or their antagonists results in genetic disorders affecting the skeleton, such as fibrodysplasia ossificans progressiva. BMP signaling mediated through binding to its receptors is a critical step in the induction of abnormal ossification. Therefore, we hypothesized that engineering more effective inhibitors of this BMP-signaling process may lead to the development of therapies for such conditions. METHODS: BMP4-induced heterotopic ossification was used as a model for testing the ability of the BMP antagonist Noggin to block de novo bone formation, either by local or systemic delivery. Since Noggin naturally acts locally, a Noggin mutein, hNOGDeltaB2, was engineered and was shown to circulate systemically, and its ability to block heterotopic ossification was tested in a mouse model with use of adenovirus-mediated somatic cell gene transfer. RESULTS: A mouse model of BMP4-induced heterotopic ossification was developed. Local delivery of wild-type NOG inhibited heterotopic ossification, but systemic administration was ineffective. In contrast, systemic delivery of the adenovirus encoding hNOGDeltaB2 resulted in systemic levels that persisted for more than two weeks and were sufficient to block BMP4-induced heterotopic ossification. CONCLUSIONS: BMP4-induced heterotopic ossification can be prevented in vivo either by local delivery of wild-type Noggin or after somatic cell gene transfer of a Noggin mutein, hNOGDeltaB2. Furthermore, the data in the present study provide proof of concept that a naturally occurring factor can be engineered for systemic delivery toward a desirable pharmacological outcome. CLINICAL RELEVANCE: Blocking bone formation is clinically relevant to disorders of heterotopic ossification in humans, such as fibrodysplasia ossificans progressiva. Furthermore, development of BMP antagonists as therapeutic agents may provide modalities for the treatment of other pathologic conditions that arise from aberrant expression of BMPs and/or from a lack of their antagonists.


Subject(s)
Myositis Ossificans/genetics , Ossification, Heterotopic/prevention & control , Proteins/pharmacology , Adenoviridae , Animals , Biopsy, Needle , Bone Morphogenetic Protein 4 , Bone Morphogenetic Proteins , Carrier Proteins , Cells, Cultured , Disease Models, Animal , Enzyme-Linked Immunosorbent Assay , Gene Transfer Techniques , Genetic Engineering , Immunohistochemistry , Injections, Intralesional , Injections, Intraperitoneal , Mice , Mice, Inbred C57BL , Myositis Ossificans/pathology , Osteogenesis/genetics , Osteogenesis/physiology , Photomicrography , Sensitivity and Specificity , Time Factors
3.
Nat Med ; 9(1): 47-52, 2003 Jan.
Article in English | MEDLINE | ID: mdl-12483208

ABSTRACT

Cytokines can initiate and perpetuate human diseases, and are among the best-validated of therapeutic targets. Cytokines can be blocked by the use of soluble receptors; however, the use of this approach for cytokines such as interleukin (IL)-1, IL-4, IL-6 and IL-13 that use multi-component receptor systems is limited because monomeric soluble receptors generally exhibit low affinity or function as agonists. We describe here a generally applicable method to create very high-affinity blockers called 'cytokine traps' consisting of fusions between the constant region of IgG and the extracellular domains of two distinct cytokine receptor components involved in binding the cytokine. Traps potently block cytokines in vitro and in vivo and represent a substantial advance in creating novel therapeutic candidates for cytokine-driven diseases.


Subject(s)
Antigens, CD/metabolism , Cytokines/antagonists & inhibitors , Cytokines/metabolism , Immunoglobulin Fc Fragments/metabolism , Membrane Glycoproteins/metabolism , Receptors, Interleukin-6/metabolism , Animals , Antigens, CD/genetics , Antigens, CD/immunology , Cell Division/physiology , Cell Line , Cytokine Receptor gp130 , Cytokines/immunology , Dimerization , Humans , Immunoglobulin Fc Fragments/genetics , Immunoglobulin Fc Fragments/immunology , Immunoglobulin G/genetics , Immunoglobulin G/immunology , Macaca fascicularis , Male , Membrane Glycoproteins/genetics , Membrane Glycoproteins/immunology , Mice , Mice, Inbred Strains , Protein Binding , Random Allocation , Receptors, Interleukin-6/genetics , Receptors, Interleukin-6/immunology , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , Recombinant Fusion Proteins/metabolism , Time Factors
5.
Proc Natl Acad Sci U S A ; 99(17): 11393-8, 2002 Aug 20.
Article in English | MEDLINE | ID: mdl-12177445

ABSTRACT

Vascular endothelial growth factor (VEGF) plays a critical role during normal embryonic angiogenesis and also in the pathological angiogenesis that occurs in a number of diseases, including cancer. Initial attempts to block VEGF by using a humanized monoclonal antibody are beginning to show promise in human cancer patients, underscoring the importance of optimizing VEGF blockade. Previous studies have found that one of the most effective ways to block the VEGF-signaling pathway is to prevent VEGF from binding to its normal receptors by administering decoy-soluble receptors. The highest-affinity VEGF blocker described to date is a soluble decoy receptor created by fusing the first three Ig domains of VEGF receptor 1 to an Ig constant region; however, this fusion protein has very poor in vivo pharmacokinetic properties. By determining the requirements to maintain high affinity while extending in vivo half life, we were able to engineer a very potent high-affinity VEGF blocker that has markedly enhanced pharmacokinetic properties. This VEGF-Trap effectively suppresses tumor growth and vascularization in vivo, resulting in stunted and almost completely avascular tumors. VEGF-Trap-mediated blockade may be superior to that achieved by other agents, such as monoclonal antibodies targeted against the VEGF receptor.


Subject(s)
Antineoplastic Agents/pharmacology , Endothelial Growth Factors/antagonists & inhibitors , Endothelial Growth Factors/immunology , Endothelium, Vascular/physiology , Lymphokines/antagonists & inhibitors , Lymphokines/immunology , Melanoma, Experimental/drug therapy , Proto-Oncogene Proteins/antagonists & inhibitors , Receptor Protein-Tyrosine Kinases/antagonists & inhibitors , 3T3 Cells , Animals , Antineoplastic Agents/therapeutic use , Bone Neoplasms/blood supply , Bone Neoplasms/drug therapy , Cell Division , Drug Design , Endothelial Growth Factors/pharmacology , Extracellular Matrix/physiology , Humans , Immunoglobulin Constant Regions/genetics , Immunoglobulin G/genetics , Lymphokines/pharmacology , Melanoma, Experimental/blood supply , Mice , Mice, Inbred BALB C , Phosphorylation , Protein Engineering , Rhabdomyosarcoma/blood supply , Rhabdomyosarcoma/drug therapy , Umbilical Veins , Vascular Endothelial Growth Factor A , Vascular Endothelial Growth Factor Receptor-1 , Vascular Endothelial Growth Factors
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