Highly tunable delivery of matrix-bound growth factors for therapeutic angiogenesis

Abstract

Therapeutic angiogenesis is a promising strategy to treat pathologies characterized by insufficient blood supply to tissue, such as coronary and peripheral artery diseases, but also to promote the engraftment and survival of tissue-engineered constructs after transplantation into the host. Vascular endothelial growth factor (VEGF) is the master regulator of both physiological and pathological vascular growth. However, initial clinical trials of VEGF gene delivery failed to establish clinical benefit. Retrospective analyses identified several issues that compromised the efficacy of those trials, particularly the difficulty to deliver a sufficient VEGF dose into the target tissue at safe vector doses. VEGF is absolutely required to induce new blood vessel formation: however growth of normal blood vessels is a complex multi-step process that requires the temporal and spatial co-ordination of different cell types. In fact, blood vessel maturation and subsequent stabilization depends on the coordinated cross-talk of multiple signaling pathways between the endothelium and pericytes recruited by the platelet-derived-growth-factor-BB (PDGF-BB). By cell-based gene delivery, we previously found that, to effectively exploitOs VEGF therapeutic potential, it is key to achieve sustained expression for at least 4 weeks at homogenous microenvironmental concentration, as VEGF binds tightly to the extracellular matrix (ECM). On the other hand, coordinated co-expression of PDGF-BB and VEGF by a single bicistronic construct completely normalized aberrant angiogenesis induced by heterogeneous VEGF levels. The delivery of recombinant growth factors (GFs) presents several desirable features for clinical translation of these biological concepts compared to gene-therapy, such as defined duration of treatment, homogenous dose distribution and the absence of genetic modifications. The controlled release of factors from biopolymers is an attractive approach to protect them from rapid clearance while ensuring homogenous and sustained release. Pro-angiogenic factors such as VEGF and PDGF-BB have heparin-binding domains that localize them in the extracellular matrix after secretion and their restricted spatial organization within the microenvironment is crucial to induce physiological angiogenesis. Therefore, we previously developed a protein engineering approach that enzymatically links recombinant GFs to fibrin, a biopolymer physiologically degraded by cell-associated proteases. Engineered factors are produced as fusion proteins with the Factor XIIIa substrate sequence ?2-PI1-8 at the N-terminal, to allow covalent binding by factor XIIIa during the fibrin cross-linking reaction and subsequent release by enzymatic cleavage. Thus, in this work we aimed to establish an optimized platform to induce stable and functional angiogenesis in relevant target tissues using recombinant proteins engineered to covalently bind the fibrin gel. We first optimized a fibrin platform to ensure controlled and dose-dependent delivery of ?2-PI1-8-VEGF over at least 4 weeks, capable of inducing normal and stable angiogenesis into the healthy skeletal mouse muscle tissue over a 500-fold range of ?2-PI1-8-VEGF concentrations. Remarkably, newly induced vessels did not regress for at least 3 months, whereas the injected gels were almost completely degraded by 4 weeks, demonstrating that they achieved independence from exogenous VEGF signaling for their survival and suggesting that they could likely persist indefinitely. This is particularly relevant for the therapeutic potential of this approach, since until now one of the main limits has been the insufficient duration of GF release to achieve stable and persistent effects. The treatment with ?2-PI1-8-VEGF was able to promote functional improvement into a wound-healing ischemia model. Further studies should take advantage of the exquisite dose control afforded by this system to identify the therapeutic window of fibrin-bound ?2-PI1-8-VEGF for the treatment of limb and cardiac ischemia. In the second part, we determined that balanced co-delivery of ?2-PI1-8-PDGF-BB and ?2-PI1-8-VEGF from fibrin hydrogels had the potential to normalize aberrant angiogenesis, providing a tool to expand the range of ?2-PI1-8-VEGF concentrations within which normal and stable angiogenesis can be reliably induced. Further, taking advantage of the robust flexibility of this platform, we could determine, with two different ?2-PI1-8-VEGF concentrations, that complete normalization of induced angiogenesis was ensured by a ratio ?2-PI1-8-PDGF-BB: ?2-PI1-8-VEGF as low as 1:20. Further investigations will be needed in order to rigorously determine the minimum duration of growth factor co-delivery necessary to achieve vessel stabilization and indefinite persistence, to define a complete set of parameters (dose, ratio and duration) for therapeutic applications.