Supramolecular nanostructures that mimic VEGF as a strategy for ischemic tissue repair

Abstract

There is great demand for the development of novel therapies for ischemic cardiovascular disease, a leading cause of morbidity and mortality worldwide. We report here on the development of a completely synthetic cell-free therapy based on peptide amphiphile nanostructures designed to mimic the activity of VEGF, one of the most potent angiogenic signaling proteins. Following self-assembly of peptide amphiphiles, nanoscale filaments form that display on their surfaces a VEGF-mimetic peptide at high density. The VEGF-mimetic filaments were found to induce phosphorylation of VEGF receptors and promote proangiogenic behavior in endothelial cells, indicated by an enhancement in proliferation, survival, and migration in vitro. In a chicken embryo assay, these nanostructures elicited an angiogenic response in the host vasculature. When evaluated in a mouse hind-limb ischemia model, the nanofibers increased tissue perfusion, functional recovery, limb salvage, and treadmill endurance compared to controls, which included the VEGF-mimetic peptide alone. Immunohistological evidence also demonstrated an increase in the density of microcirculation in the ischemic hind limb, suggesting the mechanism of efficacy of this promising potential therapy is linked to the enhanced microcirculatory angiogenesis that results from treatment with these polyvalent VEGF-mimetic nanofibers.

Fig. 1.

The chemical structure of the VEGF-mimetic peptide amphiphile (A), designed to assemble into cylindrical nanostructures (G). The VEGF PA forms nanofibers, visualized by cryogenic TEM (B), and entangled nanofiber gel networks, imaged by SEM (C). Circular dichroism for the VEGF PA demonstrating α-helical secondary structure (D), and melting analysis performed about the 220-nm α-helical signature for VEGF PA (E) and the peptide epitope control (F).

Fig. 2.

Results from an ELISA assay for the receptor phosphorylation of VEGFR1 (A) and VEGFR2 (B) as well as a time course of phosphorylation for both VEGFR1 and VEGFR2 (C). Significance is shown relative to VEGF PA treatment. VEGF protein is shown as an assay control for verification of phosphorylation and was not included in statistical analysis as a comparative group.

Fig. 3.

Quantified results from the CAM assay beginning on embryonic day 10 (t = day 0) and extending for 4 d along with representative images from day 3 for treatments of VEGF PA, VEGF peptide, mutant PA, and an untreated control. Significance shown is for VEGF PA treatment compared to all other treatment groups. The scale bar shown corresponds to 2 mm in all micrographs.

Fig. 4.

Results from in vivo hind-limb ischemia study examining the tissue salvage score (A) and the motor function score (B) of the various treatment groups over time, as well as the endpoint analysis at day 28 of failure time for a Rota Rod motor functional performance test (C). Laser Doppler perfusion imaging (D) for mouse hind-limb ischemia studies quantified as the perfusion ratio of treated to untreated limb along with representative LDPI images from the same animal at day 0 and day 28 for treatments of VEGF PA, VEGF peptide, mutant PA, and an untreated control. Significance is shown for the VEGF PA relative to other treatments (A, B, and D) and for other treatments compared to the VEGF PA treatment (C).

Fig. 5.

Results from quantification of immunohistological staining for CD31⁺ capillary forms in the ischemic tissue at day 28 of the hind-limb ischemia study, as well as representative immunohistological images staining for CD31 (green), smooth muscle α-actin (red), and nuclei (blue) for treatments of VEGF PA, VEGF peptide, mutant PA, and an untreated control. Significance is shown relative to VEGF PA treatment, and the scale bar shown corresponds to 100 μm in all micrographs.