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

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.

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

Growth Factors Engineered for Super-Affinity to the Extracellular Matrix Enhance Tissue Healing

Priscilla Suhasna Maithili Briquez, Esra Güç, Jeffrey Alan Hubbell, Witold Waldemar Kilarski, Mikaël Martino, Stéphanie Elisabeth Metzger, Jeffrey James Rice, Melody Swartz, Federico Tortelli

Growth factors (GFs) are critical in tissue repair, but their translation to clinical use has been modest. Physiologically, GF interactions with extracellular matrix (ECM) components facilitate localized and spatially regulated signaling; therefore, we reasoned that the lack of ECM binding in their clinically used forms could underlie the limited translation. We discovered that a domain in placenta growth factor-2 (PlGF-2(123-144)) binds exceptionally strongly and promiscuously to ECM proteins. By fusing this domain to the GFs vascular endothelial growth factor-A, platelet-derived growth factor-BB, and bone morphogenetic protein-2, we generated engineered GF variants with super-affinity to the ECM. These ECM super-affinity GFs induced repair in rodent models of chronic wounds and bone defects that was greatly enhanced as compared to treatment with the wild-type GFs, demonstrating that this approach may be useful in several regenerative medicine applications.

Amer Assoc Advancement Science2014

A multichamber fluidic device for 3D cultures under interstitial flow with live imaging: Development, characterization, and applications

Carmen Bonvin, James Brandon Dixon, Jan Overney, Adrian Chung-Kai Shieh, Melody Swartz

Interstitial flow is an important biophysical cue that can affect capillary morphogenesis, tumor cell migration, and fibroblast remodeling of the extracellular matrix, among others. Current models that incorporate interstitial flow and that are suitable for live imaging lack the ability to perform multiple simultaneous experiments, for example, to compare effects of growth factors, extracellular matrix composition, etc. We present a nine-chamber radial flow device that allows simultaneous 3D fluidic experiments for relatively long-term culture with live imaging capabilities. Flow velocity profiles were characterized by fluorescence recovery after photobleaching (FRAP) for flow uniformity and estimating the hydraulic conductivity. We demonstrate lymphatic and blood capillary morphogenesis in fibrin gels over 10 days, comparing flow with static conditions as well as the effects of an engineered variant of VEGF that binds fibrin via Factor XIII. We also demonstrate the culture of contractile fibroblasts and co-cultures with tumor cells for modeling the tumor microenvironment. Therefore, this device is useful for studies of capillary morphogenesis, cell migration, contractile cells like fibroblasts, and multicellular cultures, all under interstitial flow. © 2009 Wiley Periodicals, Inc.

Wiley-Blackwell2010