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

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.

The heparin binding domain of von Willebrand factor binds to growth factors and promotes angiogenesis in wound healing

Kazuto Fukunaga, Jeffrey Alan Hubbell, Jun Ishihara, Matthias Lütolf, Yoji Tabata

During wound healing, the distribution, availability, and signaling of growth factors (GFs) are orchestrated by their binding to extracellular matrix components in the wound microenvironment. Extracellular matrix proteins have been shown to modulate angiogenesis and promote wound healing through GF binding. The hemostatic protein von Willebrand factor (VWF) released by endothelial cells (ECs) in plasma and in the subendothelial matrix has been shown to regulate angiogenesis; this function is relevant to patients in whom VWF deficiency or dysfunction is associated with vascular malformations. Here, we show that VWF deficiency in mice causes delayed wound healing accompanied by decreased angiogenesis and decreased amounts of angiogenic GFs in the wound. We show that in vitro VWF binds to several GFs, including vascular endothelial growth factor-A (VEGF-A) isoforms and platelet-derived growth factor-BB (PDGF-BB), mainly through the heparin-binding domain (HBD) within the VWF A1 domain. VWF also binds to VEGF-A and fibroblast growth factor-2 (FGF-2) in human plasma and colocalizes with VEGF-A in ECs. Incorporation of the VWF A1 HBD into fibrin matrices enables sequestration and slow release of incorporated GFs. In vivo, VWF A1 HBD-functionalized fibrin matrices increased angiogenesis and GF retention in VWF-deficient mice. Treatment of chronic skin wounds in diabetic mice with VEGF-A165 and PDGF-BB incorporated within VWF A1 HBD-functionalized fibrin matrices accelerated wound healing, with increased angiogenesis and smooth muscle cell proliferation. Therefore, the VWF A1 HBD can function as a GF reservoir, leading to effective angiogenesis and tissue regeneration.

AMER SOC HEMATOLOGY2019

Engineering the growth factor microenvironment with fibronectin domains to promote wound and bone tissue healing

Jeffrey Alan Hubbell, Mikaël Martino, Mayumi Mochizuki Martino, Federico Tortelli

Although growth factors naturally exert their morphogenetic influences within the context of the extracellular matrix microenvironment, the interactions among growth factors, their receptors, and other extracellular matrix components are typically ignored in clinical delivery of growth factors. We present an approach for engineering the cellular microenvironment to greatly accentuate the effects of vascular endothelial growth factor-A (VEGF-A) and platelet-derived growth factor-BB (PDGF-BB) for skin repair, and of bone morphogenetic protein-2 (BMP-2) and PDGF-BB for bone repair. A multifunctional recombinant fragment of fibronectin (FN) was engineered to comprise (i) a factor XIIIa substrate fibrin-binding sequence, (ii) the 9th to 10th type III FN repeat (FN III9-10) containing the major integrin-binding domain, and (iii) the 12th to 14th type III FN repeat (FN III12-14), which binds growth factors promiscuously, including VEGF-A165, PDGF-BB, and BMP-2. We show potent synergistic signaling and morphogenesis between α(5)β(1) integrin and the growth factor receptors, but only when FN III9-10 and FN III12-14 are proximally presented in the same polypeptide chain (FN III9-10/12-14). The multifunctional FN III9-10/12-14 greatly enhanced the regenerative effects of the growth factors in vivo in a diabetic mouse model of chronic wounds (primarily through an angiogenic mechanism) and in a rat model of critical-size bone defects (through a mesenchymal stem cell recruitment mechanism) at doses where the growth factors delivered within fibrin only had no significant effects.

2011

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

Martin Ehrbar, Elena Groppa, Jeffrey Alan Hubbell, Kristen Lorentz, Mikaël Martino

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.

Natl Acad Sciences2014

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

Martin Ehrbar, Elena Groppa, Jeffrey Alan Hubbell, Kristen Lorentz, Mikaël Martino

Natl Acad Sciences2014