The vascular endothelial growth factor (VEGF) family and its receptors have roles in the development, maintenance and remodeling of the vasculature. This ligand-receptor system is very complex (): five ligand genes give rise to at least 17 homodimeric proteins and an unknown number of heterodimeric proteins that each have distinct binding characteristics for the products of the five receptor genes (). The receptors themselves heterodimerize [96
], resulting in multiple parallel nonexclusive downstream signaling pathways per ligand (). Extracellular processing of ligands and receptors results in a complex picture of VEGF transport in tissue ().
Complexity of expression regulation and control for VEGF family ligands and receptors
VEGF ligands have different binding profiles to cell surface VEGF receptors on the cell surface and to proteoglycans in the matrix
Three heterodimerizing receptors lead to nine distinct signaling pathway-initiating receptor states
Tissue-level view of the trafficking of VEGF and its receptors
For a complex system such as this, a systems biology approach can be very useful. With so many interacting components, experiments focusing on any one or small number of molecules at the expense of the others risk making incomplete or even flawed interpretations of results. Computational models based on current biological knowledge allow us to design and make predictions for future experiments that can fill gaps in our knowledge. The results of computational models can either confirm or conflict with our mechanistic understanding, and in both cases we learn more about the system, generating new testable hypotheses. Additional, possibly counterintuitive results can give us further unique insights. Here we review the computational and experimental systems biology work that has been done in the field of VEGF research, and identify areas in which more work would be vital to the advancement of designed VEGF-based or VEGF-targeting therapeutics.
The computational models described in the text vary widely in scope, and none include all of the processes described in . Instead, each model focuses on a subset of the VEGF system, e.g. the regulation of a transcription factor HIF1α that regulates VEGF, or the interstitial transport of VEGF, or the activation of matrix metalloproteinases that degrade the matrix, releasing stored VEGF. In this way, each component of the overall VEGF system is modeled and validated independently. To truly describe the overall systems these modules will be coupled together to create a meta-model that allows simultaneous simulation at multiple scales: inside the nucleus, in the cytoplasm, at the cell surface, throughout the tissue and throughout the body.
Most of the models presented here are molecularly-detailed, meaning that each molecular species in the network being studied is explicitly represented along with their interactions and transport signaling pathways. This approach allows for the computational testing of therapeutic approaches, if the interaction of the drug with the components of the model is known. That is, molecular therapeutics can be tested with a molecularly-detailed model, without resorting to lumped or effective parameters.
The first molecularly-detailed models of VEGF transport were developed to simulate the interactions of exogenous VEGF with receptors expressed on cultured cells in vitro [93
]. These models were useful for hypothesis testing, for example, the shifting of ligands from VEGFR1 to VEGFR2 was predicted not to be central to the observed synergy between placental growth factor and VEGF-A [93
]; and the mechanism of action of an antibody to VEGF co-receptor Neuropilin-1 was elucidated from an application of the computational models to experimental data on VEGF receptor activation [94
]. The validated model of ligand-receptor interactions was then used to build compartmental models of VEGF transport in vivo in multiple tissues, including human breast cancer [95
] and human vastus lateralis muscle [97
]. Three-dimensional anatomically-detailed models of VEGF transport in rat extensor digitorum longus muscle, including predictions of VEGF gradients, have also been generated [66
Molecularly-detailed models of other ligand-receptor systems have been made, including epidermal growth factor (EGF) [78
] and platelet-derived growth factor (PDGF) [114
]. Transport of a hypothetical generic tumor angiogenic factor (TAF), that reflects some of the VEGF transport processes, is included in models of neovascularization [23
]. Elements of VEGF transport are also considered in concert with models that simulate many cells undergoing cellular-level responses (e.g., migration and proliferation) during angiogenesis [11
]. The role of the Notch-Dll4 system combined with VEGF transport has been simulated as part of a three-dimensional model of cellular behavior during angiogenic sprouting [120
]. The role of Notch-Dll4 and VEGF interactions in filopodial extension and tip cell selection have also been modeled [14
]. Stochastic models of VEGF (i.e. that take into account the effects of low concentrations by including each individual molecule in the simulation) have been developed to study both in vitro and in vivo behavior [3
In this review we focus on the kinetics of the biochemical reactions between VEGF and its receptors (), and the transport processes that involve these species (), and will not include the signaling downstream of the VEGF receptors. Several excellent reviews on the multiple signaling pathways induced by VEGF through its receptors are available [113
]. We also focus on the transport of VEGF and its receptors in the adult animal rather than in development. There is extensive literature on the behavior of the VEGF system during development, including intriguing results such as the haploinsufficiency of the VEGF-A ligand gene [20
] but not of the receptor genes [44
]. We will not explore that literature here. Finally, the VEGF system is also critical for the lymphatic network, but we will focus on the vascular system here.
Box 1. Emerging questions in VEGF systems biology
A sampling of the opportunities for further exploration of a complex system.
- Overall multiplicative complexity of the system. From the ligand and receptor genes, to multiple alternatively spliced mRNA, to homo- and hetero-dimerized ligands and receptors, and extracellular proteolytic cleavage into alternative, active forms: how should this progressive complexity in experimental and computational studies be managed?
- Alternative splicing of pro- and anti-angiogenic isoforms. The same-size isoforms VEGF165 and VEGF165b have opposite effects, but may have for years been observed with a single antibody. How do we reinterpret previous results and design experiments going forward?
- Generation and maintenance of VEGF gradients. VEGF gradients are believed to be necessary for vascular sprout guidance; however, current experimental techniques have not yielded measurements of VEGF gradients at the microscale. Can the gradients be estimated by molecularly-detailed computational modeling?
- Matrix-bound VEGF may be available to cell surface receptors. Sequestered VEGF may be able to ligate cell-surface receptors, presenting a different signaling context and a mechanical cell-matrix connection. How do we differentiate ligand presentation modes?
- Proteolysis and cleaved VEGF isoforms. Matrix-sequestered VEGF outside of the reach of VEGF receptors accounts for a significant fraction of VEGF in adult tissue. Is this stored VEGF a source of chemotactic gradients or an isotropic source of VEGF for potentiation of endothelial activation?
- Differential signaling by heterodimerized VEGF receptors. Three receptor tyrosine kinases, homo-and hetero-dimerized by the same ligand, can form nine different membrane-based signal initiating units. How do we model and measure the multiple cross-talking pathways downstream of these activation patterns?
- Context-dependent signaling. VEGF receptors cluster with, and may form multimolecular complexes with, integrins, cadherins and other localized microenvironmental sensors. Do VEGF receptors in cell-cell junctions generate different signals from those in focal adhesions on the ablumenal side of the endothelial cell and from those located on the lumenal side?
- Signaling by internalized VEGF receptors. Activated cell surface receptors are internalized, but this is not the end of the signaling; perinuclear and nuclear translocation occurs. How can we differentiate between, or understand the integration of, cell-surface-derived and nuclear-derived VEGFR signals?
- VEGF receptor expression on non-endothelial cells. Parenchymal receptors may transduce signals, but they may also interfere with drug targeting of endothelial receptors. How do we study molecular targeting in a multicellular milieu?
- Global VEGF communication. Plasma VEGF concentration may be an effective diagnostic and prognostic marker. If this is so, then VEGF exchange between the blood and tissues may extend beyond the diseased tissue and affect other, ‘normal’ tissues. What secondary effects would pro- or anti-VEGF treatment have on non-target tissues?
- Formed element VEGF sequestration and release. Both platelets and leukocytes effectively scavenge and sequester VEGF in the blood. The VEGF may be released at a later time. The function of this is not clear: sensor, effector or both?
- Soluble VEGF receptors. sVEGFR1 (sFlt-1) has been identified as a major factor in several disease conditions. Evidence exists that other receptors, including Neuropilin, also exist in soluble form. What is their role under physiological and pathological conditions?