Long-lasting fibrin matrices ensure stable and functional angiogenesis by highly tunable, sustained delivery of recombinant VEGF(164)

Clinical trials of therapeutic angiogenesis by vascular endothelial growth factor (VEGF) gene delivery failed to show efficacy. Major challenges include the need to precisely control in vivo distribution of growth factor dose and duration of expression. Recombinant VEGF protein delivery could overcome these issues, but rapid in vivo clearance prevents the stabilization of induced angiogenesis. Here, we developed an optimized fibrin platform for controlled delivery of recombinant VEGF, to robustly induce normal, stable, and functional angiogenesis. Murine VEGF(164) was fused to a sequence derived from alpha 2-plasmin inhibitor (alpha(2)-PI1-8) that is a substrate for the coagulation factor fXIIIa, to allow its covalent cross-linking into fibrin hydrogels and release only by enzymatic cleavage. An alpha(2)-PI1-8-fused variant of the fibrinolysis inhibitor aprotinin was used to control the hydrogel degradation rate, which determines both the duration and effective dose of factor release. An optimized aprotinin-alpha(2)-PI1-8 concentration ensured ideal degradation over 4 wk. Under these conditions, fibrin-alpha(2)-PI1-8-VEGF(164) allowed exquisitely dose-dependent angiogenesis: concentrations >= 25 mu g/mL caused widespread aberrant vascular structures, but a 500-fold concentration range (0.01-5.0 mu g/mL) induced exclusively normal, mature, nonleaky, and perfused capillaries, which were stable after 3 mo. Optimized delivery of fibrin-alpha(2)-PI1-8-VEGF(164) was therapeutically effective both in ischemic hind limb and wound-healing models, significantly improving angiogenesis, tissue perfusion, and healing rate. In conclusion, this optimized platform ensured (i) controlled and highly tunable delivery of VEGF protein in ischemic tissue and (ii) stable and functional angiogenesis without introducing genetic material and with a limited and controllable duration of treatment. These findings suggest a strategy to improve safety and efficacy of therapeutic angiogenesis.

Engineering of Signaling Microenvironments with Fibronectin Domains to Enhance Tissue Regeneration

Mikaël Martino

Conducting tissue healing and regeneration through biomaterials and morphogens is still an unrealized goal. Understand the multiple roles of the extracellular matrix (ECM) is indeed essential for the design of successful regenerative medicine strategies. During tissue repair and healing, cells receive numerous signals from their immediate ECM microenvironment and adhere by receptor-mediated interactions with ECM components by specialized adhesion receptors, such as integrins. As such, design and modulation of ECM analogs to ligate specific integrins is a promising approach to control cellular processes. Through production of variants of the 9th to 10th type III repeat of fibronectin (FN, FN III9-10) with variable stabilities, we engineered ligands that present different specificities for the integrin α5β1. Furthermore, the FN fragments have been engineered in order to be covalently incorporated into fibrin, a clinical relevant matrix for regenerative medicine. We demonstrated the capacity of α5β1 integrin-specific engagement to influence human mesenchymal stem cell behavior, showing that α5β1 integrin has an important role in the control of their osteogenic differentiation. Specifically, compared to FN, FN fragments with increased specificity for α5β1 versus αvβ3 integrins (FN III9*-10) results in significantly enhanced osteogenic differentiation in 2D and in a clinically relevant fibrin matrix system, while cell attachment/spreading and proliferation were comparable. On the other hand, growth factors (GFs) are key molecules for tissue morphogenesis and healing. However, while they are really promising molecules for a use in regenerative medicine applications, they often fail to prove cost-effective or even clinically efficacious during clinical trials. One of the reasons for this poor translation may lie in the rapid clearance of GFs from tissue sites in vivo, leading to the development of strategies controlling their release. Since FN has been shown to bind GFs from very different families, we first explored the possibility of FN to bind GFs much more broadly, and secondly the possibility of using FN fragments as an anchor for GF retention into fibrin matrix. We found that the 12th to 14th type III repeats of FN (FN III12-14) promiscuously bind GFs from the platelet-derived GF, fibroblast GF, transforming GF-β and neurotrophin families. Overall, 25 new binding interactions were demonstrated, supporting that GF binding may be one of FN's main physiological functions. However, the reasons for such promiscuous binding capacity were still unclear, while evidences from the literature suggested that the close proximity of the major integrin-binding domain, FN III9-10, allows joint integrin/GF-receptor signaling triggered by a complex FN/GFs. Accordingly, we found that FN fragments containing both the integrin- and GF-binding domains (FN III9()-10/12-14) could drastically enhance GF activities in vitro. In addition, testing which integrins were involved within these synergistic effects, we found that α5β1 integrin was mainly involved. By the use of FN III9()-10/12-14 and fibrin, we could engineer a specific microenvironment allowing sequestration of multiple wild-type GFs, while triggering synergistic signaling between GF-receptors and integrins. In a delayed wound healing model in mouse and in a calvarial bone defect model in rat, GFs delivered with the FN fragment microenvironment were drastically improved in their ability to induce tissue healing, even though a single low dose of GFs was used. Specifically, we established integrin/GF-receptor synergistic activities as a key parameter for GF translation into regenerative medicine treatments and demonstrate a method to exploit this phenomenon. This thesis highlights the absolutely critical role of the microenvironment in modulating signaling of GFs and in driving these molecules forward toward more widespread clinical use.

EPFL2011

An in vivo neovascularization assay for screening regulators of angiogenesis and assessing their effects on pre-existing vessels

Witold Waldemar Kilarski, Marcin Stefan Zielinski

Therapeutic regulation of tissue vascularization has appeared as an attractive approach to treat a number of human diseases. In vivo neovascularization assays that reflect physiological and pathological formation of neovessels are important in this effort. In this report we present an assay where the effects of activators and inhibitors of angiogenesis can be quantitatively and qualitatively measured. A provisional matrix composed of collagen I and fibrin was formed in a plastic cylinder and implanted onto the chick chorioallantoic membrane. A nylon mesh separated the implanted matrix from the underlying tissue to distinguish new from pre-existing vessels. Vascularization of the matrix in response to fibroblast growth factor-2 or platelet-derived growth factor-BB was scored in a double-blinded manner, or vessel density was measured using a semi-automated image analysis procedure. Thalidomide, fumagillin, U0126 and TGF beta inhibited neovessel growth while hydrocortisone exerted a negative and wortmannin a toxic effect on the pre-existing vasculature. This quantitative, inexpensive and rapid in vivo angiogenesis assay might be a valuable tool in screening and characterizing factors that influence wound or tumor induced vascularization and in assessing their effects on the normal vasculature.

Springer Netherlands2012

Local induction of lymphangiogenesis with engineered fibrin-binding VEGF-C promotes wound healing by increasing immune cell trafficking and matrix remodeling

Priscilla Suhasna Maithili Briquez, Manuel Andreas Fankhauser, Esra Güç, Jeffrey Alan Hubbell, Witold Waldemar Kilarski, Melody Swartz

Lymphangiogenesis occurs in inflammation and wound healing, yet its functional roles in these processes are not fully understood. Consequently, clinically relevant strategies for therapeutic lymphangiogenesis remain underdeveloped, particularly using growth factors. To achieve controlled, local capillary lymphangiogenesis with protein engineering and determine its effects on fluid clearance, leukocyte trafficking, and wound healing, we developed a fibrin-binding variant of vascular endothelial growth factor C (FB-VEGF-C) that is slowly released upon demand from infiltrating cells. Using a novel wound healing model, we show that implanted fibrin containing FB-VEGF-C, but not free VEGF-C, could stimulate local lymphangiogenesis in a dose-dependent manner. Importantly, the effects of FB-VEGF-C were restricted to lymphatic capillaries, with no apparent changes to blood vessels and downstream collecting vessels. Leukocyte intravasation and trafficking to lymph nodes were increased in hyperplastic lymphatics, while fluid clearance was maintained at physiological levels. In diabetic wounds, FB-VEGF-C-induced lymphangiogenesis increased extracellular matrix deposition and granulation tissue thickening, indicators of improved wound healing. Together, these results indicate that FB-VEGF-C is a promising strategy for inducing lymphangiogenesis locally, and that such lymphangiogenesis can promote wound healing by enhancing leukocyte trafficking without affecting downstream lymphatic collecting vessels. (C) 2017 The Authors. Published by Elsevier Ltd.

2017

Engineering of Signaling Microenvironments with Fibronectin Domains to Enhance Tissue Regeneration

Mikaël Martino

EPFL2011