Blood vessel maturation in a 3‐dimensional spheroidal coculture model: direct contact with smooth muscle cells regulates endothelial cell quiescence and abrogates VEGF responsiveness

Korff, Thomas; Kimmina, Sarah; Martiny-Baron, Georg; Augustin, Hellmut G.

doi:10.1096/fj.00-0139com

Cited by 335 publications

(352 citation statements)

References 34 publications

(57 reference statements)

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“…Previous findings demonstrate that EC in co-culture with vSMC do not proliferate (8,31). It is therefore somewhat unexpected that vinblastine and paclitaxel have such potent inhibitory effects as they are primarily used as antimitotic drugs in cancer treatment.…”

Section: Dose-response Experimentsmentioning

confidence: 98%

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 Experimentsmentioning

confidence: 98%

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

et al. 2009

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“…3 of 15,34,35 ). Similarly, using PAE/VEGFR2, PAE/VEGFR2/NRP1 and PAE/VEGFR2/NRP2 cell lines expressing similar VEGFR2 levels, the authors showed that both VEGF-A165a and VEGF-A121a substantially enhanced VEGFR2 activation (Y951 and Y1175 phosphorylation) when NRP1 is present (Figure 5 of 15 ).…”

Section: Evidence Supporting Neuropilins Modulating Vegf-a121a-inducementioning

confidence: 99%

VEGF-A121a binding to Neuropilins – A concept revisited

Sarabipour

Gabhann

2017

Cell Adhesion & Migration

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All known splice isoforms of vascular endothelial growth factor A (VEGF-A) can bind to the receptor tyrosine kinases VEGFR-1 and VEGFR-2. We focus here on VEGF-A121a and VEGF-A165a, two of the most abundant VEGF-A splice isoforms in human tissue1, and their ability to bind the Neuropilin co-receptors NRP1 and NRP2. The Neuropilins are key vascular, immune, and nervous system receptors on endothelial cells, neuronal axons, and regulatory T cells respectively. They serve as co-receptors for the Plexins in Semaphorin binding on neuronal and vascular endothelial cells, and for the VEGFRs in VEGF binding on vascular and lymphatic endothelial cells, and thus regulate the initiation and coordination of cell signaling by Semaphorins and VEGFs.2 There is conflicting evidence in the literature as to whether only heparin-binding VEGF-A isoforms – that is, isoforms with domains encoded by exons 6 and/or 7 plus 8a – bind to Neuropilins on endothelial cells. While it is clear that VEGF-A165a binds to both NRP1 and NRP2, published studies do not all agree on the ability of VEGF-A121a to bind NRPs. Here, we review and attempt to reconcile evidence for and against VEGF-A121a binding to Neuropilins. This evidence suggests that, in vitro, VEGF-A121a can bind to both NRP1 and NRP2 via domains encoded by exons 5 and 8a; in the case of NRP1, VEGF-A121a binds with lower affinity than VEGF-A165a. In in vitro cell culture experiments, both NRP1 and NRP2 can enhance VEGF-A121a-induced phosphorylation of VEGFR2 and downstream signaling including proliferation. However, unlike VEGFA-165a, experiments have shown that VEGF-A121a does not ‘bridge’ VEGFR2 and NRP1, i.e. it does not bind both receptors simultaneously at their extracellular domain. Thus, the mechanism by which Neuropilins potentiate VEGF-A121a-mediated VEGFR2 signaling may be different from that for VEGF-A165a. We suggest such an alternate mechanism: interactions between NRP1 and VEGFR2 transmembrane (TM) and intracellular (IC) domains.

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“…EC monolayer therefore mimics immature microvessels. 47 Thus, these characteristics make the EC/mpc cocultures a model for interplays at work during muscle regeneration. Therefore, what happens once the steady state is reached and the homeostasis of the muscle recovered?…”

Section: The Ménage à Trois In the Skeletal Muscle Vascular Bedmentioning

confidence: 99%

Regulation of myogenic stem cell behaviour by vessel cells: The "ménage à trois" of satellite cells, periendothelial cells and endothelial cells

Abou-Khalil¹,

Mounier²,

Chazaud³

2010

Cell Cycle

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Blood vessel maturation in a 3‐dimensional spheroidal coculture model: direct contact with smooth muscle cells regulates endothelial cell quiescence and abrogates VEGF responsiveness

Cited by 335 publications

References 34 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

VEGF-A121a binding to Neuropilins – A concept revisited

Regulation of myogenic stem cell behaviour by vessel cells: The "ménage à trois" of satellite cells, periendothelial cells and endothelial cells

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