Interplay between vascular endothelial growth factor-A and extracellular matrix in angiogenesis: molecular and cellular mechanisms

The induction of angiogenesis by stimulation of physiological vessel growth using pro- angiogenic growth factors is currently under intense investigation in medical research. It is well accepted, that angiogenesis is a rate-limiting step in skin regeneration, as it ensures supply of novel tissue with nutrients and oxygen. Chronic wounds are characterized by a lack of angiogenesis and thus represent a major target for the induction of angiogenesis by therapeutic means. For the delivery of VEGF-A165 to chronic wounds and the induction of effective angiogenesis, in general it is proposed that first, the stabilization of the growth factor against protease activities, and second, the tight control of its release are beneficial. The aim of this study was to optimize wound angiogenesis in non-healing wounds in response to recombinant VEGF-A165 by improving bioavailability and potency of the growth factor. To stabilize the growth factor in the chronic wound environment, recently a mutated form of VEGF-A165 (VEGFmut) resistant to plasmin cleavage was generated in our group. This mutant was used in this study for the analysis of protein delivery. For this purpose, a hybrid protein composed of VEGFmut and a factor XIIIa transglutaminase substrate sequence (TG) was generated. This sequence allows covalent incorporation of the recombinant protein a into fibrin matrix. Furthermore, a second strategy to increase the angiogenic response was investigated which aimed at the stimulation of synergistic signaling downstream of VEGFR-2 and integrin αvβ3. To this end, bi-functional proteins consisting of the fibronectin type III domain 10 (FNIII10) and VEGFmut denoted as FLV were generated for the concomitant activation of both receptors. The recombinant proteins were expressed in E. coli. FNIII10 and TG-FNIII10 were purified as GST-fusion proteins, whereas VEGF-containing constructs were produced in bacterial inclusion bodies, refolded, dimerized, and purified by heparin affinity chromatography. However, purification of the bi-functional proteins was challenging, as they tended to precipitate and dimerization was ineffective. To overcome these problems, in a subsequent approach, FNIII10, TG-FNIII10, VEGFmut, TG-VEGFmut, FLV and TG-FLV were expressed in eucaryotic cells (HEK293-EBNA) and purified by a C-terminal poly-histidine tag. The biological activity of these proteins was confirmed in various in vitro assays. First, HUVECs were shown to attach to TG-FNIII10 in a concentration dependent manner, and this attachment was reduced by integrin αvβ3 function-blocking antibodies. Second, recombinant proteins fused to a TG sequence were covalently incorporated into fibrin gels by the activity of factor XIIIa, and were retained by up to 90 % after two days of washing, whereas their soluble counterparts were released in a burst release during the first 8 hours. Third, the biological activity of the VEGF-variants was shown in vitro by their ability to induce VEGFR-2 phosphorylation in HUVECs by western blot analysis and ELISA. Intriguingly, the bi-functional protein FLV failed to induce detectable synergistic signaling on the receptor level when it was added to HUVECs. In contrast, when HUVECs were seeded on microscopy slides coated with the recombinant proteins, FLV promoted attachment and spreading to a higher degree than FNIII10 or VEGFmut alone. Together, these findings indicate that the immobilized proteins show different potential in the induction of cellular responses. The potency of these proteins to induce angiogenesis was also assessed in vivo using wound healing as a model. Fibrin gels containing 0.468 µM (corresponding to 20 µg/mL effective VEGF-concentration) of either VEGFmut, TG-VEGFmut, TG-FLV or no recombinant protein were applied to full-thickness punch biopsy wounds created on the back of db/db mice, which are used as a model of impaired wound healing. When compared to fibrin treatment only, wound closure was accelerated upon treatment with various VEGF-proteins. More important, both TG-VEGF and TG-FLV proved to be significantly more potent in inducing blood vessel growth into the wound area, when compared to soluble VEGFmut. Differences between the two TG- isoforms were observed in the maturity of neovessels as indicated by the recruitment of pericytes: pericyte recruitment was more efficient in fibrin/TG-VEGF treated wounds than in fibrin/TG-FLV treated wounds at day 10. Collectively, the findings of this study support a critical role for the interplay between VEGF-A and extracellular matrix during wound angiogenesis, and suggest that protein engineering provides a novel molecular approach to use these interactions for therapeutic angiogenesis.