Journal: bioRxiv

Article Title: Heparin flexibility within the extracellular matrix determines the bioactivity of bound vascular endothelial growth factor

doi: 10.1101/2025.03.11.642311

Figure Lengend Snippet: a Schematic representation of a DexHepMA hydrogel. A mixture of DexMA (functionalized with the cell-adhesive ligand CGRGDS) and HepMA polymer chains are crosslinked with MMP-cleavable peptides. VEGF binds to the hydrogel surface through electrostatic interactions with the negatively charged HepMA. b Sandwich assay set-up. VEGF-functionalized hydrogels are brought in contact with a HUVEC monolayer for the required assay time. Then, the hydrogel is removed and cells are lysed for further analysis. c Images of dimethylmethylene blue (DMMB)-stained hydrogels (outlined by dotted lines) with (right) and without (left) HepMA. Scale bar, 3 mm. d Activation of VEGFR-2 in HUVECs treated with VEGF-tethered hydrogels. HUVEC monolayers were exposed to VEGF-functionalized (330 ng/mL) hydrogels with (+) and without (-) HepMA containing 26 mM crosslinker for 2 min, followed by immunoprecipitation of VEGFR-2 and Western blot analysis for pan-phosphorylation of tyrosine residues (using 4G10 antibody) (top panel). As loading control, the blots were reprobed with an antibody against total human VEGFR-2 (bottom panel). e Quantification of fold change of pVEGFR-2 signal intensities, adjusted to the level of precipitated VEGFR-2 and normalized to HUVEC sample treated with VEGF-functionalized (330 ng/mL) hydrogels without HepMA. All data are reported as mean ± SD, p < 0.05 is considered to be statistically significant (two-tailed unpaired t-test with Welch’s correction) (n = 3 independent experiments).

Article Snippet: HUVECs pre-screened for VEGF response were obtained from PromoCell ® (#C-12205).

Techniques: Adhesive, Polymer, Staining, Activation Assay, Immunoprecipitation, Western Blot, Control, Two Tailed Test

Journal: bioRxiv

Article Title: Heparin flexibility within the extracellular matrix determines the bioactivity of bound vascular endothelial growth factor

doi: 10.1101/2025.03.11.642311

Figure Lengend Snippet: a Design of DexHepMA hydrogels with tunable stiffness (determined by concentration of MMP-cleavable crosslinker). b Young’s moduli of DexHepMA hydrogels as a function of crosslinker concentration (n = 3 independent samples). c HUVEC monolayers were exposed to VEGF-functionalized (330 ng/mL) DexHepMA hydrogels with varying crosslinker concentrations for 2 min, followed by immunoprecipitation of VEGFR-2 and Western blot analysis for pan-phosphorylation of tyrosine residues (using 4G10 antibody) (top panel). As loading control, the blots were reprobed with an antibody against total human VEGFR-2 (bottom panel). d Quantification of fold change of pVEGFR-2 signal intensities, adjusted to the level of precipitated VEGFR-2 and normalized to HUVEC sample treated with VEGF-functionalized (330 ng/mL) DexHepMA hydrogel containing 26 mM crosslinker (n = 14 independent experiments). e HUVEC monolayers were exposed to VEGF-functionalized (330 ng/mL) DexHepMA hydrogels with varying concentrations of crosslinker for 15 min, followed by Western blot analysis of cell lysate using an antibody against human phospho-ERK 1/2. As loading control, the blots were reprobed with an antibody against total human ERK 1/2 (bottom panel). f Quantification of fold change of phospho-ERK 1/2 signal intensities, adjusted to the level of ERK 1/2 and normalized to HUVEC sample treated with VEGF-functionalized (330 ng/mL) DexHepMA hydrogel containing 26 mM crosslinker (n = 3 independent experiments). All data are reported as mean ± SD, p < 0.05 is considered to be statistically significant (two-tailed unpaired t-test with Welch’s correction).

Article Snippet: HUVECs pre-screened for VEGF response were obtained from PromoCell ® (#C-12205).

Techniques: Concentration Assay, Immunoprecipitation, Western Blot, Control, Two Tailed Test

Journal: bioRxiv

Article Title: Heparin flexibility within the extracellular matrix determines the bioactivity of bound vascular endothelial growth factor

doi: 10.1101/2025.03.11.642311

Figure Lengend Snippet: a Design of DexHepMA hydrogels with control over matrix positive charge density and crosslinking. b Schematic representation of a stiff DexHepMA hydrogel with embedded negatively charged PGA shielding peptide crosslinker positive charges. c VEGF-functionalized (330 ng/mL) DexHepMA hydrogels with (+) or without (-) PGA (85.3 mM) and crosslinked with 43.4 mM peptide crosslinker were presented to HUVEC monolayers for 2 min, followed by immunoprecipitation of VEGFR-2 and Western blot analysis for pan-phosphorylation of tyrosine residues (using 4G10 antibody) (top panel). As loading control, the blots were reprobed with an antibody against total human VEGFR-2 (bottom panel). d Quantification of fold change of pVEGFR-2 signal intensities, adjusted to the level of precipitated VEGFR-2 and normalized to HUVEC sample treated with VEGF-functionalized (330 ng/mL) DexHepMA hydrogel containing 43.4 mM crosslinker without PGA (n = 4 independent experiments). e Schematic representation of a soft DexHepMA hydrogel functionalized with mono-Cys peptide to match the positive charge density of a stiff hydrogel. f VEGF-functionalized (330 ng/mL) DexHepMA hydrogels of varying positive charges densities (value reported relative to the positive charge density of DexHepMA hydrogel containing 26 mM crosslinker without mono-Cys peptide) were presented to HUVEC monolayers for 2 min, and analysed as described in c . g Quantification of fold change of pVEGFR-2 signal intensities, adjusted to the level of precipitated VEGFR-2 and normalized to HUVEC sample treated with VEGF-functionalized (330 ng/mL) DexHepMA hydrogel containing 26 mM crosslinker without mono-Cys peptide (n = 3 independent experiments). All data are reported as mean ± SD, p < 0.05 is considered to be statistically significant. (One-way ANOVA with Tukeýs post-hoc test performed in g , two-tailed unpaired t-test with Welch’s correction in d ).

Article Snippet: HUVECs pre-screened for VEGF response were obtained from PromoCell ® (#C-12205).

Techniques: Control, Immunoprecipitation, Western Blot, Two Tailed Test

Journal: bioRxiv

Article Title: Heparin flexibility within the extracellular matrix determines the bioactivity of bound vascular endothelial growth factor

doi: 10.1101/2025.03.11.642311

Figure Lengend Snippet: a , b Representative QCM-D binding assays of different VEGF isoforms (a, VEGF-A 165 ; b, VEGF-A 189 ) (5 µg/mL) interacting with heparin-functionalized SLBs. After protein interaction (black arrow) and buffer washing (PBS), soluble heparin (0.5 mg/mL) (green arrow) was flushed into the QCM-D chamber to compete with SLB-bound heparin chains. c Δ D as a function of -Δ F for the VEGF isoforms (red, VEGF-A 165 ; blue, VEGF-A 189 ) derived from the protein interaction portion of the QCM-D plots (black arrow) reported in ( a , b ). d VEGF-A 165 and VEGF-A 189 -functionalized (330 ng/mL) DexHepMA hydrogels with varying concentrations of positively charged peptide crosslinker were presented to HUVEC monolayers for 2 min, followed by immunoprecipitation of VEGFR-2 and Western blot analysis for pan-phosphorylation of tyrosine residues (using 4G10 antibody) (top panel). As loading control, the blots were reprobed with an antibody against total human VEGFR-2 (bottom panel). e Quantification of fold change of pVEGFR-2 signal intensities, adjusted to the level of precipitated VEGFR-2 and normalized to HUVEC sample treated with VEGF-A 165 -functionalized (330 ng/mL) DexHepMA hydrogel containing 26 mM of peptide crosslinker (n = 3 independent experiments). All data are reported as mean ± SD, p < 0.05 is considered to be statistically significant (one-way ANOVA with Tukeýs post-hoc test).

Article Snippet: HUVECs pre-screened for VEGF response were obtained from PromoCell ® (#C-12205).

Techniques: QCM-D, Binding Assay, Derivative Assay, Immunoprecipitation, Western Blot, Control

Journal: bioRxiv

Article Title: Heparin flexibility within the extracellular matrix determines the bioactivity of bound vascular endothelial growth factor

doi: 10.1101/2025.03.11.642311

Figure Lengend Snippet: a Representative cell outlines of HUVECs cultured atop VEGF-functionalized (330 ng/mL) DexHepMA hydrogels, with (right) or without (left) 25.9 mM mono-Cys peptide, crosslinked with 17.4 mM crosslinker peptide. b Quantification of cell aspect ratio for conditions shown in ( a ) (n > 200 cells, pooled from 3 independent experiments). c Representative high magnification confocal images of HUVECs on hydrogels described in ( a ). Composite fluorescence images show nuclei (magenta) and F-actin (cyan). d Quantification of F-actin fluorescence intensity per cell after overnight culture (> 16 h) on hydrogels described in ( a ) (n > 50 cells, pooled from 3 independent experiments). e Representative confocal images of HUVECs after overnight culture (> 16 h) on hydrogels described in ( a ) and treated for 4 h with EdU. Composite fluorescence images show nuclei (blue) and EdU (red). f Quantification of EdU + cells for conditions shown in ( e ). The number of EdU + cells were normalized to the number of EdU + HUVECs cultured on VEGF-functionalized (330 ng/mL) DexHepMA hydrogels crosslinked with 17.4 mM crosslinker peptide and containing 0 mM mono-Cys peptide (n = 3 independent experiments). Data in ( b ) and ( d ) are reported as box-and-whiskers plots (box, 25–75 percentile; bar-in-box, median; whiskers, the minimum and maximum values); data in ( f ) are represented as mean ± SD, p < 0.05 is considered to be statistically significant (two-tailed unpaired t-test with Welch’s correction). Scale bar, 100 µm.

Article Snippet: HUVECs pre-screened for VEGF response were obtained from PromoCell ® (#C-12205).

Techniques: Cell Culture, Fluorescence, Two Tailed Test