Heparan sulfates facilitate harmless amyloidogenic fibril formation interacting with elastin-like peptides

Abstract

Heparan sulfates (HSs) modulate tissue elasticity in physiopathological conditions by interacting with various matrix constituents as tropoelastin and elastin-derived peptides. HSs bind also to protein moieties accelerating amyloid formation and influencing cytotoxic properties of insoluble fibrils. Interestingly, amyloidogenic polypeptides, despite their supposed pathogenic role, have been recently explored as promising bio-nanomaterials due to their unique and interesting properties. Therefore, we investigated the interactions of HSs, obtained from different sources and exhibiting various degree of sulfation, with synthetic amyloidogenic elastin-like peptides (ELPs), also looking at the effects of these interactions on cell viability and cell behavior using in vitro cultured fibroblasts, as a prototype of mesenchymal cells known to modulate the soft connective tissue environment. Results demonstrate, for the first time, that HSs, with differences depending on their sulfation pattern and chain length, interact with ELPs accelerating aggregation kinetics and amyloid-like fibril formation as well as self-association. Furthermore, these fibrils do not negatively affect fibroblasts' cell growth and parameters of redox balance, and influence cellular adhesion properties. Data provide information for a better understanding of the interactions altering the elastic component in aging and in pathologic conditions and may pave the way for the development of composite matrix-based biomaterials.

Figure 1

Thioflavin T (ThT) fluorescence for real-time monitoring of fibrillation kinetics. (a) (VGGVG), (b) (VGGVG)₃, (c) (VGGVG)_n and (d) (LGGVG)_n were incubated in aqueous solution for 24 h at 37 °C in the absence or in the presence of natural (n-HS) or of semisynthetic (ss-HS) heparan sulfates. In each panel, data of ThT assay for n-HS and ss-HS alone (50 μg/mL) are also shown. Data are expressed as the mean ± SD of three experiments.

Figure 2

Transmission electron microscopy. Images show (VGGVG), (VGGVG)₃, (VGGVG)_n and (LGGVG)_n incubated in water for 24 h at 37 °C in the absence or in the presence of natural (n-HS) or of semisynthetic (ss-HS) heparan sulfates. Samples are negatively stained on formvar coated copper grids. Bars: 1 μm.

Figure 3

Spectroscopic assay by Congo red (CR). Natural (n-HS) or semisynthetic (ss-HS) heparan sulfate alone or elastin-like peptide (P) alone or their mixture were mantained for 24 h at 37 °C in acqueous solution. Data are normalized to CR background set at 1 (dotted line) and are expressed as the mean ± SD of three experiments. *p < 0.05; **p < 0.01; ****p < 0.0001 peptide vs other conditions; ^#p < 0.05 peptide + n-HS vs same peptide + ss-HS.

Figure 4

Intracellular content of reactive oxygen species (ROS) evaluated by flow cytometry. The fluorescent probes H₂DCF-DA and DH₂ were used to detect hydrogen peroxide (H₂O₂) and superoxide anion (O₂^•−), respectively. Cells were incubated with natural (n-HS) or semisynthetic (ss-HS) heparan sulfate alone, or with peptides (P) i.e. (VGGVG), (VGGVG)₃, (VGGVG)_n and (LGGVG)_n alone or in combination with HSs. Values are expressed as percentage of H₂O₂ + O₂^•− normalized to ROS content obtained in cells cultured in standard medium (dotted line set at 100). Data are represented as mean values ± SD of three experiments.

Figure 5

A schematic hypothetical model of HS-mediated enhancement of ELPs self-assembly into amyloid-like fibrils in aqueous environment at 37 °C. Initially, free monomers in solution self-assemble into small and/or large intermediate oligomers and amorphous aggregates acting as nucleation seeds and leading to the formation of protofibrils with different morphology, which merge and intertwine yielding to elongated mature amyloid fibrils. In parallel, higher molecular weight n-HS interact with amyloidogenic intermediates/species more effectively than the lower molecular weight ss-HS. This event can generate a high local concentration of peptides on the HS scaffold that drives the association of amyloidogenic ELPs. Moreover, the hydrophilic nature of HSs may help the β-sheets to associate with one another by sequestering the water molecules through interactions with the disaccharide moieties. The final result of these events combined with the accelerated release of water previously trapped in protofibrils may explain the different acceleration of the overall aggregation pathway leading to amyloid fibril formation, the marked enhancement of their lateral aggregation in more packed fibrillar assemblies as well as the differences in cell adhesion on fibrillar coated surfaces.