Differential effects of cell adhesion, modulus and VEGFR-2 inhibition on capillary network formation in synthetic hydrogel arrays

Abstract

Efficient biomaterial screening platforms can test a wide range of extracellular environments that modulate vascular growth. Here, we used synthetic hydrogel arrays to probe the combined effects of Cys-Arg-Gly-Asp-Ser (CRGDS) cell adhesion peptide concentration, shear modulus and vascular endothelial growth factor receptor 2 (VEGFR2) inhibition on human umbilical vein endothelial cell (HUVEC) viability, proliferation and tubulogenesis. HUVECs were encapsulated in degradable poly(ethylene glycol) (PEG) hydrogels with defined CRGDS concentration and shear modulus. VEGFR2 activity was modulated using the VEGFR2 inhibitor SU5416. We demonstrate that synergy exists between VEGFR2 activity and CRGDS ligand presentation in the context of maintaining HUVEC viability. However, excessive CRGDS disrupts this synergy. HUVEC proliferation significantly decreased with VEGFR2 inhibition and increased modulus, but did not vary monotonically with CRGDS concentration. Capillary-like structure (CLS) formation was highly modulated by CRGDS concentration and modulus, but was largely unaffected by VEGFR2 inhibition. We conclude that the characteristics of the ECM surrounding encapsulated HUVECs significantly influence cell viability, proliferation and CLS formation. Additionally, the ECM modulates the effects of VEGFR2 signaling, ranging from changing the effectiveness of synergistic interactions between integrins and VEGFR2 to determining whether VEGFR2 upregulates, downregulates or has no effect on proliferation and CLS formation.

Keywords: Angiogenesis; Cell encapsulation; ECM; Growth factors; Hydrogel.

Figure 1

Molecules included in PEG hydrogels. A) The hydrogels are composed of (i) 8-arm PEG molecules, with each arm functionalized with a norbornene molecule; (ii) Di-thiolated PEG crosslinking molecules bridge multiple 8-arm PEG molecules together into an ordered polymer network. A di-thiolated PEG molecule acts as an inert crosslinking molecule that is not cell-degradable; (iii) In bioactive hydrogels, PEG molecules are decorated with CRGDS adhesion peptide or CRDGS scrambled peptide to modulate cell adhesion to the hydrogel; (iv) Di-thiolated matrix metalloproteinase (MMP) labile crosslinking peptides enable cell-driven hydrogel degradation. B) “Background” hydrogels are void of cell adhesion molecules and are not subject to cell-driven degradation. C) “Hydrogel spots” modulate cell behavior through covalently attached adhesion molecules and are biodegradable via MMP activity.

Figure 2

Schematic representation of hydrogel array fabrication. 1) Separate hydrogel spot solutions containing various ratios of CRGDS adhesion peptide (Red circles) and a scrambled CRDGS non-functional peptide (Blue circles) are pipetted into wells of a PDMS stencil. Total pendant peptide concentration is fixed at 2 mM in all solutions. 2) The hydrogel spots are crosslinked in the stencil using UV light. 3) A crosslinked 1-mm thick “background” hydrogel slab is laid on top of the crosslinked bioactive hydrogel spots. A thin layer of background hydrogel solution is added to the slab to anchor the cured spots to the background. 4) The hydrogel spots are anchored to the background after treatment with UV light. 5) The completed hydrogel array is removed from the stencil. Red boxes highlight the raised spots in the schematic and side view images of the arrays.

Figure 3

Characterizing mechanical properties and pendant peptide incorporation into the hydrogel array. A) Equilibrium swelling ratios of degradable (left) and background (right) and hydrogels used in low, medium and high hydrogel modulus conditions. B) Complex shear modulus of degradable (left) and background (right) hydrogels using in low, medium and high hydrogel modulus conditions. Error bars indicate standard deviation. C) Reduction in norbornene alkene protons due to covalent coupling of CRGDS and CRDGS as measured using NMR. D) N-terminal amines of CRGDS were labeled with Alexa Fluor® 488 (Green). Green fluorescence intensity was quantified from the left to right columns (Black lines: PEG polymer and crosslinker. Red circles: CRGDS).

Figure 4

Viability of HUVECs encapsulated inside the hydrogel array spots. A) Cell viability as determined by counting live cell and dead cell nuclei 48 hours after encapsulation. B) Cell viability measured when VEGFR2 was inhibited by 10 μM SU5416 supplementation. *, p < 0.05. &, p < 0.05 compared to all equivalent CRGDS concentration in other modulus conditions C) Viability of SU5416-treated HUVECs normalized to HUVEC viability in growth medium. *, p < 0.05; **, p < 0.01; ***, p < 0.001 compared to growth medium control.

Figure 5

Proliferation of HUVECs encapsulated inside the hydrogel array spots. A) Cell proliferation as determined by Click-it EdU staining 24 hours after encapsulation B) Cell proliferation measured when VEGFR2 was inhibited by 10 μM SU5416 supplementation. *, p < 0.05. &, p < 0.05 compared to all equivalent CRGDS concentration in other modulus conditions C) Cell proliferation during SU5416 treatment normalized to proliferation in growth medium. *, p < 0.05; **, p < 0.01; ***, p < 0.001 compared to growth medium control. D) Proliferating cells (arrowheads) were localized to multicellular structures. Green: Cell Tracker Green. Blue: Hoescht nuclear stain. Red: Alexa Fluor® 594 labeling nuclei of cells in S-phase.

Figure 6

Tubulogenesis of HUVECs encapsulated inside the hydrogel array spots. A) Total tubule length was determined by manually measuring tubule lengths throughout the spots from epifluorescence Z-stack images. The cells were stained using Cell Tracker Green and Hoescht nuclear stain 24 hours after encapsulation. B) Tubulogenesis when VEGFR2 was inhibited by 10 μM SU5416 supplementation. *, p < 0.05. &, p < 0.05 compared to all equivalent CRGDS concentration in other modulus conditions C) Tubulogenesis during SU5416 treatment normalized to tubulogenesis in growth medium. *, p < 0.05; **, p < 0.01; ***, p < 0.001 compared to growth medium. D) Confocal microscopy images of low tubulogenesis in low modulus, 2 mM RGDS spots and increased tubulogenesis levels with SU5416 treatment. Bottom: Enlarged examples of capillary-like structures seen in the VEGFR2-inhibited condition. Scale bars: 100 μm. Green: Cell Tracker Green. Blue: Hoescht nuclear stain.

Figure 7

Effects of VEGFR2 inhibition in standard model systems. A) HUVEC proliferation with and without SU5416 supplementation on tissue culture-treated polystyrene (TCPS). *, p < 0.05. B) HUVEC tubulogenesis with and without SU5416 supplementation in growth factor-reduced Matrigel. C) HUVEC CLS formation in 0.4 μL Matrigel spots. In each pair of pictures, the tubules in the right hand copy were highlighted. Green: Cell Tracker Green. *, p < 0.05 between EGM2 and SU5416-treated conditions.