Coupling growth-factor engineering with nanotechnology for therapeutic angiogenesis

Abstract

Therapeutic angiogenesis is an emerging paradigm for the management of ischemic pathologies. Proangiogenic Therapy is limited, however, by the current inability to deliver angiogenic factors in a sustained manner at the site of pathology. In this study, we investigated a unique nonglycosylated active fragment of hepatocyte growth factor/scatter factor, 1K1, which acts as a potent angiogenic agent in vitro and in a zebrafish embryo and a murine matrigel implant model. Furthermore, we demonstrate that nanoformulating 1K1 for sustained release temporally alters downstream signaling through the mitogen activated protein kinase pathway, and amplifies the angiogenic outcome. Merging protein engineering and nanotechnology offers exciting possibilities for the treatment of ischemic disease, and furthermore allows the selective targeting of downstream signaling pathways, which translates into discrete phenotypes.

Fig. 1.

Structural features of the 1K1-heparin complex and interaction with the MET receptor (A) Domain structure of full length multidomain HGF/SF. The α-chain consists of N-terminal domain (amino acids 32–121) and four kringle domains(K1, K2, K3, and K4) and the β-chain contains serine protinease domain (spdh domain). (B) Crystal structure of NK1-heparin complex (16) (PDB accession 1GMO). Two NK1 dimers are shown bridged by heparin. N and K domains are shown in blue and yellow, heparin is shown in red (C) Crystal structure of 1K1-heparin complex (PDB accession 3MPK). Two 1K1 dimers are shown bridged by heparin. N and K domains are shown in blue and gray, heparin is shown in red. The figures in B and C have been drawn with Pymol. Reverse mutations K132E and R134E were introduced into K1 domain of 1K1 to inactivate the low-affinity heparin-binding sites. (D) and (E). (D) Amino acid sequence of 1K1 demonstrating the mutated sites. Binding of NK1 (E) and 1K1 (F) to MET567 in the presence of 12mer heparin using surface plasmon resonance. Twofold dilutions of each protein were used from a concentration of top concentration of 200 nM. The concentration of 12mer heparin in the sample and in the reaction buffer was 10 μM. (G) and (H). Velocity sedimentation analysis of ternary complexes of NK1-heparin-MET567 (G) and 1K1-heparin-MET567 (H). Data show plots of c(s) against s*20,w. In the presence of 10mer heparin, the amount of ternary complex is significantly higher for 1K1 than for NK1.

Fig. 2.

(A) Effect of pharmacological inhibitors on the 1K1-induced on HUVECs tube formation on growth-factor-reduced matrigel. Graphs (B) and (C) show the quantification of the images using three morphometric analyses of average length of tubes and average number of nodes respectively. The data represent mean ± SEM pixels from n = 3. ** P < 0.01 vs. vehicle-treated control; # P < 0.05 vs. 1K1(10^-7 M]). (D) Representative Western blot showing phospho Erk and total Erk and also phospho Akt and total Akt expression in HUVECs treated with Met inhibitor PHA 665752 (10^-6 M), LY294002 (50 μM) and PD98059 (50 μM) for 2 h, followed by 1K1 for 10 mins. The numbers indicate 1: vehicle, 2: HGF/SF (10^-8 M), 3: 1K1 (10^-7 M), 4: 1K1 + PHA 665752 (10^-6 M) 5: 1K1 + LY294002 (50 μM), 6: 1K1 + PD98059 (50 μM). For sample no. 4, 5, and 6, 1K1 (10^-7 M) were used along with the inhibitors.

Fig. 3.

(A) Transmission electron microscopy (TEM) image of 1K1-encapsulated nanoparticles (1K1-NP) (Bar = 500 nm). (B) Size distribution (in nm) of the 1K1-encapsulated nanoparticles by dynamic light scattering experiment. (C) Graph shows release kinetics of 1K1 from the nanoparticle when incubated in PBS at room temperature. The values on the Y-axis represent the amount of 1K1 released from the 1K1-NP in ug unit. (D) Effect of Met inhibitor, PHA 665752, on 1K1-NP induced HUVECs proliferation at 48 h. (E) Effect of PI3 kinase inhibitor, LY294002, on 1K1-NP induced HUVECs proliferation at 48 h. (F) Effect of MAP kinase inhibitor, PD98059, on 1K1-NP induced HUVECs proliferation at 48 h. The data represent mean ± SEM from n = 3. *** P < 0.001 vs. vehicle-treated control; ** P < 0.01 vs. vehicle-treated control; ### P < 0.001 vs. 1K1(10^-7 M); ## P < 0.01 vs. 1K1(10^-7 M); # P < 0.05 vs. 1K1(10^-7 M).

Fig. 4.

(A) Effect of pharmacological inhibitors on the 1K1-NP induced on HUVECs tube formation on matrigel. (B) Graphs show the quantification of the images using average length of tubes. The data represent mean ± SEM pixels from n = 3. * P < 0.05 vs. vehicle-treated control; ## P < 0.01 vs. 1K1(10^-7 M); # P < 0.05 vs. 1K1(10^-7 M). (C) Representative Western blot showing phospho Erk and total Erk and also phospho Akt and total Akt in HUVECs treated with Met inhibitor PHA 665752 (10^-6 M), LY294002 (50 μM) and PD98059 (50 μM) for 2 h, followed by 10 min 1K1-NP and 1K1. The numbers indicate 1: vehicle (PBS), 2: vehicle (empty PLGA), 3: freshly prepared 1K1 (0.5 × 10^-7 M), 4: 1K1 incubated at 37 °C for 24 h (0.5 × 10^-7 M), 5: 1K1-NP incubated at 37 °C for 24 h (0.5 × 10^-7 M), 6: 1K1-NP + PHA665752 (10^-6 M)7: 1K1-NP + LY294002 (50 μM), 8: 1K1-NP + PD98059 (50 μM). For sample no. 6, 7, and 8, (0.5 × 10^-7 M) 1K1-NP were used along with the inhibitors. (D) Representative Western blot showing phospho Erk and total Erk in HUVECs treated with (0.5 × 10^-7 M) 1K1 and (0.5 × 10^-7 M) 1K1-NP for 8.5 h.

Fig. 5.

Effect of 1K1 and 1K1-NP mediated angiogenesis in vivo using zebrafish model. 1K1 or 1K1-NP were injected with growth-factor-reduced matrigel (Mgel) near the subintestinal vessel. SIVs were then stained with alkaline phosphate and visualized in bright field. (A): vehicle (Mgel); (B) empty PLGA in Mgel (C) 1K1 in Mgel, (D) 1K1-NP in Mgel and (E) 1K1-NP. (F) Graph shows morphometric quantification of the effect of treatment on angiogenesis. Data shown mean SEM ± from n = 3.

Fig. 6.

Effect of 1K1 (200 ng/plug) and 1K1-NP (200 ng/plug) induced angiogenesis in growth-factor-reduced matrigel implants in vivo. Upper (A–D) shows gross morphology of the implants after everting the murine skin, and Bottom (E–H) shows cross sections where blood vessels are delineated with von Willebrand factor immunolabeling and appear red. Nuclei were counterstained with DAPI and appear blue. Images were captured at 512 × 512 pixels resolution. A and E: vehicle, B and F: empty NP, C and G: 1K1, D and H: 1K1 NP.