ABSTRACT
OBJECTIVE: We evaluated the stages of VEGF-A(164) driven angiogenesis that are inhibited by therapeutic doses of rapamycin and the potential role of S6K1 in that response. METHODS AND RESULTS: We assessed the effects of rapamycin on the several stages of angiogensis and lymphangiogenesis induced with an adenovirus expressing VEGF-A(164) (Ad-VEGF-A(164)) in the ears of adult nude mice. Rapamycin (0.5 mg/kg/d) effectively inhibited mTOR and downstream S6K1 signaling and partially inhibited Akt signaling, likely through effects on TORC2. The earliest stages of angiogenesis, including mother vessel formation and increased vascular permeability, were strikingly inhibited by rapamycin, as was subsequent formation of daughter glomeruloid microvasular proliferations. However, later stage formation of vascular malformations and lymphangiogenesis were unaffected. Retrovirally delivered isoforms and shRNAs demonstrated that S6K1 signaling plays an important role in early VEGF-A(164)-angiogenesis. CONCLUSIONS: Rapamycin potently inhibited early and mid stages of VEGF-A(164)-driven angiogenesis, but not late-stage angiogenesis or lymphangiogenesis. Rapamycin decreased phosphorylation of both Akt and S6, suggesting that both the TORC1 and TORC2 pathways are impacted. Inhibition of S6K1 signaling downstream of mTOR is a major component of the antiangiogenesis action of rapamycin.
Subject(s)
Carrier Proteins/antagonists & inhibitors , Neovascularization, Pathologic/drug therapy , Phosphotransferases (Alcohol Group Acceptor)/antagonists & inhibitors , Ribosomal Protein S6 Kinases/antagonists & inhibitors , Signal Transduction/drug effects , Sirolimus/pharmacology , Skin/blood supply , Animals , Carrier Proteins/metabolism , Cells, Cultured , Endothelial Cells/metabolism , Endothelial Cells/pathology , Female , Immunohistochemistry , Immunosuppressive Agents/pharmacology , Mice , Mice, Nude , Neovascularization, Pathologic/metabolism , Neovascularization, Pathologic/pathology , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Ribosomal Protein S6 Kinases/metabolism , TOR Serine-Threonine Kinases , Umbilical Veins/metabolism , Umbilical Veins/pathologyABSTRACT
Vascular endothelial growth factor (VEGF)-A has essential roles in vasculogenesis and angiogenesis, but the downstream steps and mechanisms by which human VEGF-A acts are incompletely understood. We report here that human VEGF-A exerts much of its angiogenic activity by up-regulating the expression of TR3 (mouse homologue Nur77), an immediate-early response gene and orphan nuclear receptor transcription factor previously implicated in tumor cell, lymphocyte, and neuronal growth and apoptosis. Overexpression of TR3 in human umbilical vein endothelial cells (HUVECs) resulted in VEGF-A-independent proliferation, survival, and induction of several cell cycle genes, whereas expression of antisense TR3 abrogated the response to VEGF-A in these assays and also inhibited tube formation. Nur77 was highly expressed in several types of VEGF-A-dependent pathological angiogenesis in vivo. Also, using a novel endothelial cell-selective retroviral targeting system, overexpression of Nur77 DNA potently induced angiogenesis in the absence of exogenous VEGF-A, whereas Nur77 antisense strongly inhibited VEGF-A-induced angiogenesis. B16F1 melanoma growth and angiogenesis were greatly inhibited in Nur77-/- mice. Mechanistic studies with TR3/Nur77 mutants revealed that TR3/Nur77 exerted most of its effects on cultured HUVECs and its pro-angiogenic effects in vivo, through its transactivation and DNA binding domains (i.e., through transcriptional activity).
Subject(s)
DNA-Binding Proteins/metabolism , Gene Expression Regulation/physiology , Neovascularization, Physiologic/physiology , Receptors, Cytoplasmic and Nuclear/metabolism , Receptors, Steroid/metabolism , Transcription Factors/metabolism , Animals , Apoptosis/drug effects , Apoptosis/immunology , Cell Cycle/drug effects , Cell Cycle/physiology , Cell Line, Tumor , DNA-Binding Proteins/genetics , Female , Fetal Blood/cytology , Fetal Blood/metabolism , Gene Expression Regulation/drug effects , Humans , Lymphocytes/metabolism , Mice , Mice, Knockout , Mice, Nude , Neovascularization, Physiologic/drug effects , Nuclear Receptor Subfamily 4, Group A, Member 1 , Protein Structure, Tertiary/genetics , Receptors, Cytoplasmic and Nuclear/genetics , Receptors, Steroid/genetics , Retroviridae , Transcription Factors/genetics , Transduction, Genetic/methods , Vascular Endothelial Growth Factor A/administration & dosageABSTRACT
Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF, VEGF-A) is a multifunctional cytokine with important roles in pathological angiogenesis. Using an adenoviral vector engineered to express murine VEGF-A(164), we previously investigated the steps and mechanisms by which this cytokine induced the formation of new blood vessels in adult immunodeficient mice and demonstrated that the newly formed blood vessels closely resembled those found in VEGF-A-expressing tumors. We now report that, in addition to inducing angiogenesis, VEGF-A(164) also induces a strong lymphangiogenic response. This finding was unanticipated because lymphangiogenesis has been thought to be mediated by other members of the VPF/VEGF family, namely, VEGF-C and VEGF-D. The new "giant" lymphatics generated by VEGF-A(164) were structurally and functionally abnormal: greatly enlarged with incompetent valves, sluggish flow, and delayed lymph clearance. They closely resembled the large lymphatics found in lymphangiomas/lymphatic malformations, perhaps implicating VEGF-A in the pathogenesis of these lesions. Whereas the angiogenic response was maintained only as long as VEGF-A was expressed, giant lymphatics, once formed, became VEGF-A independent and persisted indefinitely, long after VEGF-A expression ceased. These findings raise the possibility that similar, abnormal lymphatics develop in other pathologies in which VEGF-A is overexpressed, e.g., malignant tumors and chronic inflammation.
