Systems biology of pro-angiogenic therapies targeting the VEGF system

Abstract

Vascular endothelial growth factor (VEGF) is a family of cytokines for which the dysregulation of expression is involved in many diseases; for some, excess VEGF causes pathological hypervascularization, while for others VEGF-induced vascular remodeling may alleviate ischemia and/or hypoxia. Anti-angiogenic therapies attacking the VEGF pathway have begun to live up to their promise for treatment of certain cancers and of age-related macular degeneration. However, the corollary is not yet true: in coronary artery disease and peripheral artery disease, clinical trials of pro-angiogenic VEGF delivery have not, so far, proven successful. The VEGF and VEGF-receptor system is complex, with at least five ligand genes, some encoding multiple protein isoforms and five receptor genes. A systems biology approach for designing pro-angiogenic therapies, using a combination of quantitative experimental approaches and detailed computational models, is essential to deal with this complexity and to understand the effects of drugs targeting the system. This approach allows us to learn from unsuccessful clinical trials and to design and test novel single therapeutics or combinations of therapeutics. Among the parameters that can be varied in order to determine optimal strategy are dosage, timing of multiple doses, route of administration, and the molecular target.

Figure 1. Overview of the HIF-VEGF-VEGFR angiogenic axis

A, The angiogenic balance is maintained by pro- and anti-angiogenic proteins. To push the system towards angiogenesis, either an increase in the pro-angiogenic factors or a decrease in (or inhibition of) anti-angiogenic factors can be employed. Exercise does both by increasing expression of both the ligands and the pro-angiogenic receptor expression, thus decreasing binding of VEGF to anti-angiogenic or modulatory receptors. B, Stimuli such as hypoxia, mechanical stress and other cytokines can cause an increase in expression or stability of transcription factors (TFs), notably HIF-1 and HIF-2, but also PGC-1 and others. Upon binding to the hypoxia response element (HRE) or other upstream regulatory element on the ligand genes, these TFs increase production and secretion of one or more of the many VEGF ligands, including isoforms of VEGF-A and PlGF. Following diffusion through the extracellular matrix, and possibly communication between tissues via the blood, the ligands bind to receptors on endothelial cells, initiating pro-angiogenic signals. Contextual stimuli such as cell-communication and cell-matrix interactions can further modify the response to VEGF signals.

Figure 2. Transcriptional regulation of VEGF

While HIF binding to the HRE is the best-studied part of the vegfa promoter system, several other transcriptional activators and co-activators are known, both hypoxia-dependent and – independent. The hypoxia-independent pathways are often dysregulated in angiogenic tumors. Note that vegfa was the first gene shown to display haploinsufficiency, and thus its expression appears to normally be tightly regulated, particularly during development.

Figure 3. VEGF and VEGF receptor proteins and their interactions

The mRNA products of vegf and plgf gene expression are spliced into multiple isoforms. The protein products have different profiles of binding to each of the five cell-surface receptors in the vegfr and nrp families. In addition, the receptors themselves have alternate splice forms that are truncated and non-cell-surface associated. Typically, these soluble forms of the receptors bind the same ligands as the full-length receptor.

Figure 4. Sample results from computational models of VEGF gene therapy

As noted in the text, clinical trials involving gene delivery of VEGF ligands, or of ligand-upregulating transcription factors, have not been successful in treating ischemic diseases. Using a computational model of VEGF transport in skeletal muscle, we predict that VEGF ligand upregulation results in simultaneous upregulation of both pro-angiogenic VEGFR2 activation (VEGFR2*) and modulatory VEGFR1 activation (VEGFR1*). By calculating the change in the ratio of the competing VEGFR2 and VEGFR1 signals following treatment, we note that there is actually a slight decline in this anti-angiogenic metric (yellow bar). Interestingly, this result is independent of the relative strengths of the VEGFR kinases. In contrast, by increasing VEGFR2 or NRP1 expression, or by decreasing VEGFR1 expression, the model predicts lower increases in VEGFR2 activation, but decreases in VEGFR1 activation, resulting in a substantial improvement in the signaling ratio pro-angiogenic metric. Data for this graph was taken from unpublished results, based on models developed in [57].