Vinblastine inhibits the angiogenic response induced by adrenomedullin in vitro and in vivo

Ribatti, Doménico; Guidolin, Diego; Conconi, Maria Teresa; Baiguera, Silvia; Parnigotto, Pier Paolo; Vacca, Angelo; Nussdorfer, Gastone G.

doi:10.1038/sj.onc.1206789

Cited by 55 publications

(28 citation statements)

References 17 publications

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“…Hence, the inhibitory effect on network formation is likely due to impaired elongation and migration characteristic of pathfinding EC during angiogenesis. Our findings confirm the reported anti-angiogenic effects of vinblastine and paclitaxel observed in other in vitro and in vivo angiogenesis assays (22,34).…”

Section: Dose-response Experimentssupporting

confidence: 92%

“…Upon binding tubulin, vinblastine inhibits microtubule formation, thereby arresting cells in the M phase of the cell cycle and inhibiting cell division. Vinblastine blocks formation of the endothelial tubes at picomolar concentrations and is a potent anti-angiogenic agent in the in vivo chick embryo chorioallantoic membrane (CAM) assay (22). Congruent with this, we observe a dose-dependent inhibition of capillary-like network formation (IC50 5 0.26 nM) in the EC-vSMC co-culture assay (Fig.…”

Section: Dose-response Experiments With Screening Hitsmentioning

confidence: 62%

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A novel imaging‐based high‐throughput screening approach to anti‐angiogenic drug discovery

et al. 2009

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The successful progression to the clinic of angiogenesis inhibitors for cancer treatment has spurred interest in developing new classes of anti-angiogenic compounds. The resulting surge in available candidate therapeutics highlights the need for robust, highthroughput angiogenesis screening systems that adequately capture the complexity of new vessel formation while providing quantitative evaluation of the potency of these agents. Available in vitro angiogenesis assays are either cumbersome, impeding adaptation to high-throughput screening formats, or inadequately model the complex multistep process of new vessel formation. We therefore developed an organotypic endothelial-mural cell co-culture assay system that reflects several facets of angiogenesis while remaining compatible with high-throughput/high-content image screening. Co-culture of primary human endothelial cells (EC) and vascular smooth muscle cells (vSMC) results in assembly of a network of tubular endothelial structures enveloped with vascular basement membrane proteins, thus, comprising the three main components of blood vessels. Initially, EC are dependent on vSMC-derived VEGF and sensitive to clinical anti-angiogenic therapeutics. A subsequent phenotypic VEGFswitch renders EC networks resistant to anti-VEGF therapeutics, demarcating a mature vascular phenotype. Conversely, mature EC networks remain sensitive to vascular disrupting agents. Therefore, candidate anti-angiogenic compounds can be interrogated for their relative potency on immature and mature networks and classified as either vascular normalizing or vascular disrupting agents. Here, we demonstrate that the EC-vSMC co-culture assay represents a robust high-content imaging high-throughput screening system for identification of novel anti-angiogenic agents. A pilot highthroughput screening campaign was used to define informative imaging parameters and develop a follow-up dose-response scheme for hit characterization. High-throughput screening using the EC-vSMC co-culture assay establishes a new platform to screen for novel anti-angiogenic compounds for cancer therapy. ' 2009 International Society for Advancement of Cytometry Key termshigh-throughput screening; endothelial/mural cell co-culture; angiogenesis INHIBITING angiogenesis is a validated therapeutic modality for cancer (1). Hence, new agents that potently inhibit pathological angiogenesis are a critical component of future combination cancer therapies (2). The identification of candidate therapeutics relies on current in vitro angiogenesis assays that measure effects on endothelial cell migration, proliferation, apoptosis and tube formation, and in vivo models such as the chick chorioallantoic membrane assay, corneal neovascularization assay, and Matrigel plug assays (3-5). Current in vitro angiogenesis assays are generally focused on specific behaviors of monocultured endothelial cells (EC) (e.g., migration, proliferation) and fail to model heterotypic perivascular interactions, whereas in vivo systems are not compatible ...

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Section: Dose-response Experimentssupporting

confidence: 92%

Section: Dose-response Experiments With Screening Hitsmentioning

confidence: 62%

A novel imaging‐based high‐throughput screening approach to anti‐angiogenic drug discovery

et al. 2009

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“…Endothelial cells, seeded on specific substrate, such as Matrigel, spread and align with each other to form branching anastomosing tubes with multicentric junctions that give rise to a meshwork of capillary-like structures. This morphogenesis is strongly inhibited by low concentrations of MTDs as effectively in vitro (13,14,(16)(17)(18) as in vivo (15,19). Moreover, noncytotoxic concentrations of MTDs can also inhibit endothelial cell migration and chemotaxis (12,14,19,20).…”

Section: Introductionmentioning

confidence: 98%

“…Human endothelial cells have been shown to be very sensitive to MTDs compared with epithelial cells or other anticancer drugs (12,13). In fact, angiogenesis can be inhibited by very low concentrations of MTDs (13)(14)(15)(16) without any mitotic cell block or apoptosis induction (17,18). Endothelial cells, seeded on specific substrate, such as Matrigel, spread and align with each other to form branching anastomosing tubes with multicentric junctions that give rise to a meshwork of capillary-like structures.…”

Section: Introductionmentioning

confidence: 99%

Antiangiogenic Concentrations of Vinflunine Increase the Interphase Microtubule Dynamics and Decrease the Motility of Endothelial Cells

et al. 2006

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Angiogenesis is a key event in tumor progression and metastasis. This complex process, which constitutes a potent target for cancer therapy, is inhibited by very low concentrations of microtubule-targeting drugs (MTD). However, the intimate mechanisms of the antiangiogenic activity of MTDs remain unclear. Recently, we have shown that low antiangiogenic and noncytotoxic concentrations of paclitaxel induced an unexpected increase in microtubule dynamics in endothelial cells. In this study, we showed that vinflunine, the newest Vinca alkaloid, increased microtubule dynamic instability in human endothelial cells after 4-hour incubation at low concentrations (29% and 54% at 0.1 and 2 nmol/L). The growth and shortening rates were increased, and the percentage of time spent in pause and the mean duration of pauses were decreased, as previously observed with paclitaxel. As opposed to paclitaxel, the transition frequencies were not significantly disturbed by vinflunine. Moreover, low concentrations of vinflunine did not affect mitotic index and anaphase/metaphase ratio. Interestingly, these low vinflunine concentrations that increased microtubule dynamics exhibited an antiangiogenic effect through the inhibition of both morphogenesis and random motility. Capillary tube formation on Matrigel was decreased up to 44%. The cell speed and the random motility coefficient were decreased (13% and 19% and 13% and 33% at 0.1 and 2 nmol/L, respectively) and the persistent time was statistically increased. Altogether, our results confirm that the increase in microtubule dynamics is involved in MTD antiangiogenic activity and highlight the crucial role of interphase microtubule dynamics in angiogenesis. (Cancer Res 2006; 66(6): 3256-63)

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“…The AM receptor-mediated proangiogenic action of AM is well known and the sensitivity of in vitro Matrigel assay to test this effect of AM is well recognized (35)(36). The good efficiency of our protocol for RAMP2 gene silencing and the ensuing impairment of AM1 receptor activity has been confirmed by the demonstration that control but not transfected HUVECs display a normal in vitro angiogenic response to AM.…”

Section: Discussionmentioning

confidence: 65%

Gene silencing of human RAMP2 mediated by short-interfering RNA

Albertin

Ruggero²,

Guidolin³

et al. 2006

Int J Mol Med

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Abstract. Adrenomedullin (AM) is a regulatory peptide widely expressed, along its receptors, in cells and tissues, of which it controls many basic and specific functions acting in an autocrine-paracrine manner. However, the unequivocal demonstration of the physiological relevance of the regulatory role of AM would require the study of cells where the endogenous AM system has been suppressed. For this task we developed a technique to silence the AM gene in human umbilical vein endothelial cells (HUVECs) and the human embryonal kidney cell line (HEK-293). AM acts via two subtypes of receptor, named AM1 and AM2, which derive from the interaction of the calcitonin receptor-like receptors with two chaperones, called receptor activity modifying proteins (RAMP2 and RAMP3). Hence, we developed a protocol to suppress the human AM1 receptor by silencing the RAMP2 gene by transfection with short interfering RNAs (siRNAs). HUVECs were transfected using a new Ambion transfection reagent. RAMP2 gene silencing was determined in HUVECs by measuring RAMP2 mRNA levels in transfected and control cells by realtime polymerase chain reaction. The RAMP2 gene silencing was ~60% and was observed 48 h after transfection. Matrigel assay in vitro was carried out to evaluate the effects of siRNA sequences. HUVECs cells were plated on matrigel and the analysis of capillary-like tubule formation showed that the cells were viable. The knockdown of the RAMP2 gene decreased the formation of tubes in response to 10 -8 M AM. The conclusion is drawn that siRNA technology can be successfully used in the investigations on AM and AM receptor functions.

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Vinblastine inhibits the angiogenic response induced by adrenomedullin in vitro and in vivo

Cited by 55 publications

References 17 publications

A novel imaging‐based high‐throughput screening approach to anti‐angiogenic drug discovery

A novel imaging‐based high‐throughput screening approach to anti‐angiogenic drug discovery

Antiangiogenic Concentrations of Vinflunine Increase the Interphase Microtubule Dynamics and Decrease the Motility of Endothelial Cells

Gene silencing of human RAMP2 mediated by short-interfering RNA

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