Journal: Biomaterials

Article Title: Integrin-Directed Modulation of Macrophage Responses to Biomaterials

doi: 10.1016/j.biomaterials.2014.01.007

Figure Lengend Snippet: The presence of surface-adsorbed lipopolysaccharide (LPS) endotoxin on polystyrene microparticle (MP) does not influence uptake by C57BL6/J bone marrow-derived macrophages, but is required for cytokine production. A.) Microparticles opsonized with various proteins (BSA: bovine serum albumin; Fg: fibrinogen; FN: fibronectin; Serum: fetal bovine serum; VN: vitronectin) were either tested and found to be endotoxin-free (white bars), or were coated with LPS to a level of 5 EU/million MPs/mL (grey bars). After incubating 7.5 h at the MP:cell ratio of 20:1, MP uptake was quantified by pooling data from at least 9 samples from 3 separate runs, and the mean and standard error were plotted. No statistical differences were found when comparing MPs with and without LPS coating for each protein coating. B.) Secretion of the inflammatory cytokine, TNF-α, was quantified after 24 h exposure to MPs with LPS coating (grey bars) or without LPS coating (white bars). Cytokine concentrations were quantified by pooling data from 6 samples from 2 separate runs. Plotted are mean and standard error. Pair-wise significant difference between MPs coated with or without LPS for each protein coating (by ANOVA and Tukey significance test) is denoted by the * symbol (p value < 0.05).

Article Snippet: The LPS coated MPs were then protein coated by incubating with various extracellular matrix proteins such as human plasma-derived fibronectin (FN) (BD Bioscience), human plasma-derived vitronectin (VN) (BD Bioscience) and bovine plasma fibrinogen (FG) (MP Biomedicals) as well as with fetal bovine serum (Serum) and bovine serum albumin (BSA) (Fisher Bioreagents).

Techniques: Derivative Assay

Journal: Biomaterials

Article Title: Engineered microenvironments for synergistic VEGF – Integrin signalling during vascularization

doi: 10.1016/j.biomaterials.2017.02.024

Figure Lengend Snippet: Integrin-VEGF synergistic signalling triggered by FN organized into nanonetworks on PEA. a) Fibronectin molecule with domains depicted; b) fibronectin assembly on two different polymer substrates; on PMA it remains in globular conformation, whereas on PEA, FN assembly is triggered and networks are assembled; c) scheme of synergistic effect of VEGF bound to FN on cell signalling: the presentation of VEGF bound to FN in close vicinity of integrin binding site effectively enhances outside-in signalling and allows to VEGFR and integrins to work in synergy.

Article Snippet: Spin-coated polymer samples were sterilized under UV for 20 min. Polymers were coated with a human plasma fibronectin (FN) solution (20 μg/ml, recombinant human VEGF 165, R&D Systems) for 1 h, washed with PBS, and incubated in VEGF (25 ng/ml, R&D) for 1 h. Control samples without either FN or VEGF were incubated in PBS for the same time as coated samples.

Techniques: Polymer, Binding Assay

Journal: Biomaterials

Article Title: Engineered microenvironments for synergistic VEGF – Integrin signalling during vascularization

doi: 10.1016/j.biomaterials.2017.02.024

Figure Lengend Snippet: Effect of fibronectin RGD → RGE mutation on HUVEC behavior on PEA. a) Representative fluorescence images of HUVECs cultures after 6 days of incubation showed lower cell attachment and spreading on mFN-RGE coated surfaces when compared to mFN-WT (RGD). Scale bar represents 200 μm b) Image analysis of parameters characterizing cell attachment and formation of aligned structures revealed that the mutated mFN-RGE significantly decreased HUVEC numbers and spreading on PEA surfaces when compared to mFN-WT; this was valid also in presence of VEGF for both VEGF in coating (VEGFc) and VEGF in media (VEGFm) (blue, green and grey bar graphs). mFN-RGE also impaired the network formation in comparison to mFN-WT, in VEGFm samples (yellow, red and cream bar graphs), and partially in VEGFc samples (red bar graph). For statistical evaluation, one way ANOVA with Tukey's multiple comparison post-test was performed; *P < 0.05; **P < 0.01; ***P < 0.001; ns = non-significant. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Spin-coated polymer samples were sterilized under UV for 20 min. Polymers were coated with a human plasma fibronectin (FN) solution (20 μg/ml, recombinant human VEGF 165, R&D Systems) for 1 h, washed with PBS, and incubated in VEGF (25 ng/ml, R&D) for 1 h. Control samples without either FN or VEGF were incubated in PBS for the same time as coated samples.

Techniques: Mutagenesis, Fluorescence, Incubation, Cell Attachment Assay, Comparison, Cream

Journal: Biomaterials

Article Title: Engineered microenvironments for synergistic VEGF – Integrin signalling during vascularization

doi: 10.1016/j.biomaterials.2017.02.024

Figure Lengend Snippet: ERK1/2, PLCγ1 and FAK phosphorylation in HUVEC on PEA and PMA coated with fibronectin: a) ELISA quantification of pERK1/2 after 30 min incubation showed significantly higher phosphorylation in cells on PEA (green bars) than on PMA (orange bars). On PEA+FN substrates, VEGF-coated sample (VEGFc) showed higher level of ERK1/2 phosphorylation when compared to PEA+FN without VEGF (FN). PEA+FN sample with VEGF present in both coating and media (VEGFcm) showed no difference from PEA+FN+VEGFc, and PEA+FN with VEGF in media only (VEGFm) did not vary from PEA+FN control. b) Representative images of western blot membranes with detected phospho-PLCγ1 and phospho-FAK proteins in HUVEC lysates after 30 min and 2 h incubation, respectively; bands were normalized against α-tubulin; c) Quantification of phosphorylated proteins from phospho-PLCγ1 and phospho-FAK western blot bands. Enhanced VEGF signalling is observed on PEA+FN+VEGFc d) Scheme of individual VEGFR-2 and integrin signalling pathways towards ERK1/2 stimulation depicting the role of PLCγ1 and FAK as early effectors of VEGFR-2 and integrin signal transduction; phosphorylated mitogen-activated kinase 1 and 2 (ERK1/2) leads to activation of the c-Fos transcription factor. Its formation of heterodimers with c-Jun and binding to DNA. e) Scheme of synergistic VEGFR-2 and integrin signalling towards pERK1/2 can lead to enhanced ERK1/2 stimulation. For statistical evaluation, one way ANOVA with Tukey's multiple comparison post-test was performed; *P < 0.05; **P < 0.01; ***P < 0.001; ns = non-significant. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Spin-coated polymer samples were sterilized under UV for 20 min. Polymers were coated with a human plasma fibronectin (FN) solution (20 μg/ml, recombinant human VEGF 165, R&D Systems) for 1 h, washed with PBS, and incubated in VEGF (25 ng/ml, R&D) for 1 h. Control samples without either FN or VEGF were incubated in PBS for the same time as coated samples.

Techniques: Phospho-proteomics, Enzyme-linked Immunosorbent Assay, Incubation, Control, Western Blot, Transduction, Activation Assay, Binding Assay, Comparison