BackgroundDll4/Notch and Ephrin-B2/EphB4 pathways play critical roles in tumor vessel development and maturation. This study evaluates the efficacy of the inhibition of both signaling pathways, alone and in combination, in reducing the growth of an autochthonous mouse tumor and assesses potential adverse effects.MethodsWe used the transgenic RIP1-Tag2 tumor model to study the effects of 1) inhibition of Dll4/Notch by either Dll4 allelic deletion or use of a soluble extracellular Dll4 (sDll4), 2) inhibition of Ephrin-B2/EphB4 signaling by a soluble extracellular EphB4 fused to albumin (sEphB4-Alb), and 3) inhibition of both pathways by sEphB4-Alb combined with either Dll4 allelic deletion or sDll4. To investigate adverse effects, we used inducible endothelial-specific Dll4 knock-out mice, treated with sEphB4-Alb, and carried out histopathological analysis.ResultsDll4 allele deletion or soluble Dll4 treatment resulted in increased tumor vessel density, reduced mural cell recruitment and vessel perfusion which resulted in reduced tumor size. The soluble EphB4 instead reduced vessel density and vessel perfusion, leading to reduction of tumor size. Greater efficacy was observed when sEphB4-Alb was combined with either Dll4 allele deletion or sDll4 in regards to tumor size, vessel perfusion and mural cell recruitment. Induced endothelial specific Dll4 loss-of-function caused hepatic vascular alterations, which were prevented by concomitant sEphB4-Alb treatment.ConclusionCombination targeting of Dll4/Notch and Ephrin-B2/EphB4 has potential for clinical investigation, providing cumulative efficacy and increased safety over Dll4/Notch inhibition alone.

Studies in vitro have established that free tryptophan induces tna operon expression by binding to the ribosome that has just completed synthesis of TnaC-tRNA Pro , the peptidyl-tRNA precursor of the leader peptide of this operon. Tryptophan acts by inhibiting Release Factor 2-mediated cleavage of this peptidyl-tRNA at the tnaC stop codon. Here we analyze the ribosomal location of free tryptophan, the changes it produces in the ribosome, and the role of the nascent TnaC-tRNA Pro peptide in facilitating tryptophan binding and induction. The positional changes of 23S rRNA nucleotides that occur during induction were detected by using methylation protection and binding͞competition assays. The ribosomeTnaC-tRNA Pro complexes analyzed were formed in vitro; they contained either wild-type TnaC-tRNA Pro or its nonfunctional substitute, TnaC(W12R)-tRNA Pro . Upon comparing these two peptidyltRNA-ribosome complexes, free tryptophan was found to block methylation of nucleotide A2572 of wild-type ribosome-TnaCtRNA Pro complexes but not of ribosome-TnaC(W12R)-tRNA Pro complexes. Nucleotide A2572 is in the ribosomal peptidyl transferase center. Tryptophanol, a noninducing competitor of tryptophan, was ineffective in blocking A2572 methylation; however, it did reverse the protective effect of tryptophan. Free tryptophan inhibited puromycin cleavage of TnaC-tRNA Pro ; it also inhibited binding of the antibiotic sparsomycin. These effects were not observed with TnaC(W12R)-tRNA Pro mutant complexes. These findings establish that Trp-12 of TnaC-tRNA Pro is required for introducing specific changes in the peptidyl transferase center of the ribosome that activate free tryptophan binding, resulting in peptidyl transferase inhibition. Free tryptophan appears to act at or near the binding sites of several antibiotics in the peptidyl transferase center.transcription termination ͉ tryptophan induction ͉ tryptophanase operon ͉ peptidyl transferase inhibition ͉ antibiotic inhibitors of translation T he Escherichia coli tna operon consists of a promoter-leader regulatory region followed by the structural genes tnaA and tnaB, encoding, respectively, the enzyme tryptophanase and a tryptophan-specific permease (1). Tryptophanase catalyzes the reversible hydrolysis of tryptophan to indole, pyruvate, and ammonia. Transcription of the structural genes of the tna operon is regulated by both catabolite-repression and tryptophaninduced inhibition of Rho factor-dependent transcription termination in the leader region of the operon (2, 3). The transcript of the tna operon leader region contains a coding region for a 24-residue leader peptide, tnaC, followed by a Rho factorbinding site. In the presence of free

Axl is an oncogenic receptor tyrosine kinase that plays multiple roles in tumorigenesis and metastasis of many cancers. This study is the first to demonstrate that Axl is induced in Kaposi sarcoma and Kaposi sarcoma herpesvirus (KSHV) transformed endothelial cells. Conditionally, expression of one KSHV latency protein vFLIP induces Axl expression in endothelial cells. This induction can be blocked by nuclear factor-B inhibitor, consistent with the known vFLIP mechanism of action. KS cell lines lacking KSHV also have elevated Axl expression, which probably resulted from hypomethylation of AXL promoter. Axl activation activates downstream phosphoinositol-3 kinase signaling, and Axl knockdown by siRNA impairs phosphoinositol-3 kinase signaling. Furthermore, Axl knockdown inhibits KS cell growth and invasion. To explore the potential for translation of these findings, we generated monoclonal antibodies to block the biologic functions of Axl. MAb173, which induces receptor degradation, showed activity in vitro to inhibit KS cell invasion. Moreover, in vivo xenograft studies with KS cells with or without KSHV infection showed that MAb173 reduced tumor growth, increased tumor cell apoptosis, and markedly decreased Axl protein level in tumors. Axl thus has a potential role in KS pathogenesis and is a candidate for prognostic and therapeutic investigations. (Blood. 2010;116(2): 297-305) IntroductionKaposi sarcoma (KS) is a common cancer in HIV-infected persons. It originates from endothelial cells transformed by KS-associated herpesvirus (KSHV). 1 KS tumor cells have spindle cell morphology and characteristically produce excessive and abnormal vascular structures, which contain red blood cells and pigment from their lysis. 1 Nearly all KS cells carry KSHV with viral gene expression profile of latency. 2 It is thus assumed that latency proteins play an important role in KS pathogenesis.Gene expression analysis in KS cells versus endothelial cells by subtractive hybridization showed induction of Axl receptor kinase in KS cells (supplemental Figure 1, available on the Blood Web site; see the Supplemental Materials link at the top of the online article). Axl is a member of TAM receptor tyrosine kinases family that also includes Tyro3 and Mer. 3,4 Axl is composed of 2 immunoglobin-like domains and dual fibronectin type III repeats in the extracellular region, a single transmembrane and a cytoplasmic domain with kinase activity. 4 Protein S and growth arrest-specific 6 (Gas6) are the ligands for Axl, whereas only the latter has high affinity to Axl. 5,6 Axl activation and signaling have been implicated in multiple cellular responses, including cell survival, proliferation, migration, and adhesion. 7 In vascular biology, Axl receptor signaling has been shown to regulate vascular smooth muscle homeostasis, endothelial cell migration, and vascular network formation. [8][9][10] The primary downstream Axl signaling pathway appears to be phosphatidylinositol 3-kinase (PI3K) pathway. 9,11 However, the Janus kinase-signal transducers ...

The results of this prospective analysis showed that acute aortic dissection itself activated the hemostatic system even before surgery. After hemostatic therapy, fibrin formation was more impaired than platelet function. In this setting, we proposed that hemostatic therapy should focus on rapid and sufficient supplementation of fibrinogen. Thus, we recommend further increases in fibrinogen concentration to improve coagulopathy in patients with acute aortic dissection.