Enhanced trophic factor secretion by mesenchymal stem/stromal cells with Glycine-Histidine-Lysine (GHK)-modified alginate hydrogels

Abstract

Recombinant proteins and cytokines are under broad preclinical and clinical investigation to promote angiogenesis, but their success is limited by ineffective delivery, lack of long-term stability and excessive cost. Mesenchymal stem/stromal cells (MSC) secrete bioactive trophic factors, and thus, may provide an effective alternative to address these challenges. Glycine-Histidine-Lysine (GHK) is a peptide fragment of osteonectin, a matricellular protein with reported proangiogenic potential. We examined the capacity of GHK to up-regulate secretion of proangiogenic factors from human MSC in culture and when covalently coupled to alginate hydrogels. GHK had no apparent cytotoxic effects on MSC in culture over a wide range of concentrations. We detected a dose-dependent increase in vascular endothelial growth factor (VEGF) concentration in media conditioned by GHK-treated MSC, which increased endothelial cell proliferation, migration and tubule formation. We covalently coupled GHK to alginate using carbodiimide chemistry, and human MSC were entrapped in alginate hydrogels to assess VEGF secretion. Similar to monolayer culture, MSC responded to GHK-modified gels by secreting increased concentrations of VEGF and basic fibroblast growth factor compared to unmodified gels. The pre-treatment of MSC with antibodies to α6 and β1 integrins prior to entrapment in GHK-modified gels abrogated VEGF secretion, suggesting that the proangiogenic response of MSC was integrin-mediated. These data demonstrate that the proangiogenic potential of MSC can be significantly increased by the presentation of GHK with a biodegradable carrier, therefore increasing their clinical potential when used for tissue repair.

Keywords: Alginate; Angiogenesis; GHK; Hydrogel; Vascular endothelial growth factor.

Figure 1

Cell viability of MSC treated with increasing concentration of GHK for 3 days in monolayer culture: (A) metabolic activity assayed by percent reduction of alamarBlue; (B) protein concentration; (C) caspase 3/7 activity for n=4.

Figure 2

VEGF secretion in conditioned media from GHK-treated or untreated MSC at 3 days. *p<0.05 (n=3).

Figure 3

Mitogenic activity of conditioned medium from GHK-treated MSC on ECFC. (A) ECFC proliferation exhibits a dose-dependent response to increasing GHK dosage on MSC. (B) ECFC proliferation is inhibited upon the addition of a pan-VEGF antibody. *p<0.05 (n=3).

Figure 4

ECFC migration and tubule formation in response to conditioned media from GHK-treated MSC. (A) Fluorescence microscopy images of ECFC that migrated through transwell. Images are taken at 40x magnification and are representative of three independent experiments. Scale bar represents 100 μm. (B) Quantification of fluorescence on underside of transwell following calcein staining of migrated ECFC. ***p<0.001 vs. media; *p<0.05 vs all other groups (n=3). (C) Phase contrast images of ECFC morphology on Matrigel reveal tubulogenesis as a function of GHK dose on MSC. Images are taken at 40x; scale bar represents 100 μm.

Figure 5

Covalent linkage of GHK to alginate was confirmed. (A) Attenuated total reflection–Fourier transform infrared (ATR–FTIR) spectra of alginate and GHK modified alginate reveals unique peaks in GHK-modified alginate. (B) ¹H NMR spectra of alginate, GHK peptide and GHK-modified alginate recorded at 800 MHz using D₂O as a solvent.

Figure 6

MSC adhesion on peptide-modified alginate gels. (A) Fluorescence microscopy reveals that MSC did not attach on unmodified or GHK-modified alginate gels, but cells are visible and spread on RGD-modified gels after 1 and 3 days. Magnification is 100x; scale bar represents 200 μm. (B) Quantification of fluorescence from GFP-expressing MSC on gel surface after 3 days. ***p<0.001 vs. ALG or GHK (n=3).

Figure 7

Cells survive and exhibit increased angiogenic factor secretion when entrapped in GHK-modified alginate gels. (A) Fluorescence microscopy reveals MSC distribution within gels after 7 days. Magnification is 100x; scale bar represents 100 μm. (B) GHK presentation from alginate gels increases VEGF secretion by entrapped MSC into conditioned media, as determined by ELISA. ***p<0.001 (n=3). (C) Stacked column chart of fluorescence (×1000) of predominant angiogenic cytokines, as determined from MSC conditioned media using an angiogenic protein array. Data are presented as median subtracted background fluorescence intensity and normalized to positive controls and excluding inherent serum levels for pooled conditioned media for n = 4.

Figure 8

VEGF secretion stimulated by GHK is integrin-mediated. Blocking of integrin dimers suppressed VEGF secretion by MSC, with maximal suppression occurring for cells treated with α₆ and β₁ antibodies. VEGF secretion measured by ELISA using conditioned media from MSC entrapped in alginate hydrogels. ***p<0.001 vs. ALG. #p<0.001 vs. GHK. †p<0.05 vs. α₂β₁ (n=3).