Multiple Pro-angiogenic Pathways Research Articles

Adenosine-to-Inosine Editing of MicroRNA-487b Alters Target Gene Selection After Ischemia and Promotes Neovascularization.

Adenosine-to-inosine editing of microRNAs has the potential to cause a shift in target site selection. 2'-O-ribose-methylation of adenosine residues, however, has been shown to inhibit adenosine-to-inosine editing. To investigate whether angiomiR miR487b is subject to adenosine-to-inosine editing or 2'-O-ribose-methylation during neovascularization. Complementary DNA was prepared from C57BL/6-mice subjected to hindlimb ischemia. Using Sanger sequencing and endonuclease digestion, we identified and validated adenosine-to-inosine editing of the miR487b seed sequence. In the gastrocnemius muscle, pri-miR487b editing increased from 6.7±0.4% before to 11.7±1.6% (P=0.02) 1 day after ischemia. Edited pri-miR487b is processed into a novel microRNA, edited miR487b, which is also upregulated after ischemia. We confirmed editing of miR487b in multiple human primary vascular cell types. Short interfering RNA-mediated knockdown demonstrated that editing is adenosine deaminase acting on RNA 1 and 2 dependent. Using reverse-transcription at low dNTP concentrations followed by quantitative-PCR, we found that the same adenosine residue is methylated in mice and human primary cells. In the murine gastrocnemius, the estimated methylation fraction increased from 32.8±14% before to 53.6±12% 1 day after ischemia. Short interfering RNA knockdown confirmed that methylation is fibrillarin dependent. Although we could not confirm that methylation directly inhibits editing, we do show that adenosine deaminase acting on RNA 1 and 2 and fibrillarin negatively influence each other's expression. Using multiple luciferase reporter gene assays, we could demonstrate that editing results in a complete switch of target site selection. In human primary cells, we confirmed the shift in miR487b targeting after editing, resulting in a edited miR487b targetome that is enriched for multiple proangiogenic pathways. Furthermore, overexpression of edited miR487b, but not wild-type miR487b, stimulates angiogenesis in both in vitro and ex vivo assays. MiR487b is edited in the seed sequence in mice and humans, resulting in a novel, proangiogenic microRNA with a unique targetome. The rate of miR487b editing, as well as 2'-O-ribose-methylation, is increased in murine muscle tissue during postischemic neovascularization. Our findings suggest miR487b editing plays an intricate role in postischemic neovascularization.

Abstract 1812: In vitro and in vivo antiangiogenic activity of desacetylvinblastine monohydrazide through inhibition of VEGFR2 and Axl pathways

Abstract Background and Objective: Tumor angiogenesis process is regulated by multiple proangiogenic pathways, such as VEGFR2 and Axl. Inhibition of VEGF/VEGFR2 signaling alone fails to block tumor neovascularization, and anti-VEGF resistance is often associated with Axl. Hence, discovery of novel agents that target multiple angiogenesis pathways is in demand. Here, we describe desacetylvinblastine monohydrazide (DAVLBH), a derivative of vinblastine (VLB) that exerts a more potent antiangiogenic effect than VLB in vitro and in vivo by inhibiting VEGFR2 and Axl pathways. Methods: The antiangiogenic effects of DAVLBH were studied in vitro (proliferation, migration, and tube formation assays) and ex vivo (aortic ring assay). In vitro pericyte migration to endothelial tubes was assessed using a three-dimensional co-culture assay. In vivo assay was performed in HeLa xenograft model. Western blotting, immunohistochemical and immunofluorescence assays were conducted to evaluate the key proteins of the VEGF/VEGFR2 and Gas6/Axl pathways. Results: DAVLBH (1 nM) inhibited VEGF- and Gas6-induced angiogenesis in vitro. At 0.75 μmol/kg, DAVLBH significantly delayed tumor growth and reduced vascular density in vivo, which was associated with the inactivation of VEGF/VEGFR2 and Gas6/Axl signalling pathways. DAVLBH blocked the compensatory upregulation of Axl in response to bevacizumab treatment in HUVECs. DAVLBH also suppressed the recruitment of pericytes to well-established endothelial tubes and reduced pericyte coverage in vivo, which was accompanied by inhibition of Axl. Conclusions: DAVLBH potently inhibited angiogenesis-mediated tumor growth by blocking the activation of VEGF/VEGFR2 and Gas6/Axl pathways. DAVLBH might serve as a promising antiangiogenic agent for cancer therapy. Note: This abstract was not presented at the meeting. Citation Format: Minfeng Chen, Xueping Lei, Qiulin Nie, Jianyang Hu, Zhenjian Zhuo, Anita Yiu, Heru Chen, Nanhui Xu, Maohua Huang, Kaihe Ye, Liangliang Bai, Wencai Ye, Dong-Mei Zhang. In vitro and in vivo antiangiogenic activity of desacetylvinblastine monohydrazide through inhibition of VEGFR2 and Axl pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1812. doi:10.1158/1538-7445.AM2017-1812

Therapeutic options targeting angiogenesis in nonsmall cell lung cancer

There is a major unmet medical need for effective and well-tolerated treatment options for patients with advanced nonsmall cell lung cancer (NSCLC), in both first-line and relapsed/refractory settings. Experimental evidence has validated signalling pathways that regulate tumour angiogenesis, including the vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF) pathways, as valid anti-cancer drug targets. However, to date, bevacizumab (an anti-VEGF monoclonal antibody) is the only antiangiogenic agent to be approved for the treatment of NSCLC. Many other agents, including antibodies, small-molecule tyrosine kinase inhibitors and vascular disrupting agents, have been assessed in phase III trials but have generally failed to demonstrate clinically meaningful benefits. This lack of success probably reflects the redundancy of proangiogenic pathways and the molecular and clinical heterogeneity of NSCLC. In this review we summarise recently completed and ongoing randomised clinical trials of emerging antiangiogenic agents in patients with NSCLC. We highlight recent promising data with agents that simultaneously inhibit multiple proangiogenic pathways, including the PDGF and FGF pathways, as well as the VEGF pathway. Finally, we discuss the outlook for antiangiogenic agents in NSCLC, emphasising the need for clinically validated prognostic and predictive biomarkers to identify patients most likely to respond to therapy.

Novel VEGF-independent Strategies Targeting Tumor Vasculature: Clinical Aspects

In the last decades, the active research in the field of tumor angiogenesis has led to the development of a class of agents providing an effective inhibition of neo-vessel formation through the blockade of VEGF related pathways. More recently, the identification of other factors involved in tumor angiogenesis, such as platelet-derived growth factor, fibroblast growth factor and Angiopoietins has emphasized the need to develop agents targeting multiple pro-angiogenic pathways. Although contrasting data are currently available regarding the clinical efficacy of multikinase inhibitors, Sunitinib, Sorafenib and Pazopanib have displayed encouraging results, and have fuelled further evaluations. Moreover, definitive data are also eagerly awaited regarding the clinical role of angiopoietins inhibitors. On the other hand, the existence of morphological, functional and architectural differences between normal and tumor vasculature has provided solid basis for the development of a novel class of compounds, known as Vascular Disrupting Agents (VDAs) able to selectively disrupt existing tumor vessels. After initial concerns related to the potential development of severe cardiovascular toxicities, further clinical investigations have shown a safe toxicity profile for these agents. Moreover, despite the discouraging data on dolostatin-10 and ASA404, several VDAs, including CAP4, Ombrabulin and Pinabulin have already shown promising activity in phase I-II clinical trials warranting more advanced evaluations. In this review we aimed at summarizing the most relevant VEGF-independent strategies targeting tumor vasculature, focusing on the clinical development of novel antiangiogenic agents including multikinase and angiopoietins inhibitors as well as VDAs.

Hypoxia-Induced Angiogenesis: Good and Evil

The vascular network delivers oxygen (O(2)) and nutrients to all cells within the body. It is therefore not surprising that O(2) availability serves as a primary regulator of this complex organ. Most transcriptional responses to low O(2) are mediated by hypoxia-inducible factors (HIFs), highly conserved transcription factors that control the expression of numerous angiogenic, metabolic, and cell cycle genes. Accordingly, the HIF pathway is currently viewed as a master regulator of angiogenesis. HIF modulation could provide therapeutic benefit for a wide array of pathologies, including cancer, ischemic heart disease, peripheral artery disease, wound healing, and neovascular eye diseases. Hypoxia promotes vessel growth by upregulating multiple pro-angiogenic pathways that mediate key aspects of endothelial, stromal, and vascular support cell biology. Interestingly, recent studies show that hypoxia influences additional aspects of angiogenesis, including vessel patterning, maturation, and function. Through extensive research, the integral role of hypoxia and HIF signaling in human disease is becoming increasingly clear. Consequently, a thorough understanding of how hypoxia regulates angiogenesis through an ever-expanding number of pathways in multiple cell types will be essential for the identification of new therapeutic targets and modalities.