Roser Sabater i Serra

Engineered microenvironments for synergistic VEGF – Integrin signalling during vascularization

We have engineered polymer-based microenvironments that promote vasculogenesis both in vitro and in vivo through synergistic integrin-growth factor receptor signalling. Poly(ethyl acrylate) (PEA) triggers spontaneous organization of fibronectin (FN) into nanonetworks which provide availability of critical binding domains. Importantly, the growth factor binding (FNIII12-14) and integrin binding (FNIII9-10) regions are simultaneously available on FN fibrils assembled on PEA. This material platform promotes synergistic integrin/VEGF signalling which is highly effective for vascularization events in vitro with low concentrations of VEGF. VEGF specifically binds to FN fibrils on PEA compared to control polymers (poly(methyl acrylate), PMA) where FN remains in a globular conformation and integrin/GF binding domains are not simultaneously available. The vasculogenic response of human endothelial cells seeded on these synergistic interfaces (VEGF bound to FN assembled on PEA) was significantly improved compared to soluble administration of VEGF at higher doses. Early onset of VEGF signalling (PLCγ1 phosphorylation) and both integrin and VEGF signalling (ERK1/2 phosphorylation) were increased only when VEGF was bound to FN nanonetworks on PEA, while soluble VEGF did not influence early signalling. Experiments with mutant FN molecules with impaired integrin binding site (FN-RGE) confirmed the role of the integrin binding site of FN on the vasculogenic response via combined integrin/VEGF signalling. In vivo experiments using 3D scaffolds coated with FN and VEGF implanted in the murine fat pad demonstrated pro-vascularization signalling by enhanced formation of new tissue inside scaffold pores. PEA-driven organization of FN promotes efficient presentation of VEGF to promote vascularization in regenerative medicine applications.

Controlled Assembly of Fibronectin Nanofibrils Triggered by Random Copolymer Chemistry

Fibronectin fibrillogenesis is the physiological process by which cells elaborate a fibrous FN matrix. Poly(ethyl acrylate), PEA, has been described to induce a similar process upon simple adsorption of fibronectin (FN) from a protein solution-in the absence of cells-leading to the so-called material-driven fibronectin fibrillogenesis. Poly(methyl acrylate), PMA, is a polymer with very similar chemistry to PEA, on which FN is adsorbed, keeping the globular conformation of the protein in solution. We have used radical polymerization to synthesize copolymers with controlled EA/MA ratio, seeking to modulate the degree of FN fibrillogenesis. The physicochemical properties of the system were studied using dynamic-mechanical analysis, differential scanning calorimetry, and water contact angle. Both the degree of FN fibrillogenesis and the availability of the integrin binding region of FN directly depend on the percentage of EA in the copolymer, whereas the same total amount of FN was adsorbed regardless the EA/MA ratio. Cell morphology adhesion and differentiation of murine C2C12 were shown to depend on the degree of FN fibrillogenesis previously attained on the material surface. Myogenic differentiation was enhanced on the copolymers with higher EA content, i.e. more interconnected FN fibrils.

Water and protein dynamics in protein-water mixtures over wide range of composition

Water and protein dynamics in two globular protein-water systems, water-lysozyme and water-BSA (bovine serum albumine), were studied by differential scanning calorimetry (DSC), dielectric relaxation spectroscopy (DRS) and thermally stimulated depolarization currents (TSDC) techniques. Water equilibrium sorption isotherms (ESI) measurements were also recorded at room temperature. The samples covered a wide range of composition, from practically dry solid pellets (2wt% of water) to dilute solutions (82wt% of water). Crystallization and melting events of water were studied by DSC and the amount of uncrystallized water was calculated. The evolution of dynamics with hydration level was followed for various dielectric relaxation processes, the emphasis being given to relaxation processes of polar groups on the surface of the proteins and of uncrystallized water molecules. A relationship between the formation of a conductive percolating water cluster and the saturation of the water v process was found.

Role of chemical crosslinking in material-driven assembly of fibronectin (nano)networks: 2D surfaces and 3D scaffolds

Graphical abstract

Water and protein dynamics in protein - Water mixtures over wide ranges of composition

Glass transition and water and protein dynamics were studied in water mixtures of two globular proteins, lysozyme and BSA, by DSC, DRS and TSDC measurements. DSC measurements were used for the study of crystallization and melting phenomena of absorbed water. The noncrystallized water has been determined to be about 0.20 and 0.24 for Lysozyme and BSA, respectively, independent on hw. Dielectric measurements reveal the α relaxation process associated with the glass transition of the hydrated proteins for both systems, in agreement with DSC results. For water fractions where no crystallization of water occurs during cooling our results shown a strong plasticization of Tg. Splitting of the the α relaxation process, observed for hw > 0.20, is maybe due to microphase separation occurred on both hydrated proteins at that hydration levels.

Zinc uptake promotes myoblast differentiation via Zip7 transporter and activation of Akt signalling transduction pathway

Myogenic regeneration occurs through a chain of events beginning with the output of satellite cells from quiescent state, formation of competent myoblasts and later fusion and differentiation into myofibres. Traditionally, growth factors are used to stimulate muscle regeneration but this involves serious off-target effects, including alterations in cell homeostasis and cancer. In this work, we have studied the use of zinc to trigger myogenic differentiation. We show that zinc promotes myoblast proliferation, differentiation and maturation of myofibres. We demonstrate that this process occurs through the PI3K/Akt pathway, via zinc stimulation of transporter Zip7. Depletion of zinc transporter Zip7 by RNA interference shows reduction of both PI3K/Akt signalling and a significant reduction of multinucleated myofibres and myotubes development. Moreover, we show that mature myofibres, obtained through stimulation with high concentrations of zinc, accumulate zinc and so we hypothesise their function as zinc reservoirs into the cell.