Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2016 Nov;24(11):1974-1986.
doi: 10.1038/mt.2016.158. Epub 2016 Aug 10.

Dual Therapeutic Action of a Neutralizing Anti-FGF2 Aptamer in Bone Disease and Bone Cancer Pain

Ling Jin  1 , Yosuke Nonaka  1 , Shin Miyakawa  1 , Masatoshi Fujiwara  1 , Yoshikazu Nakamura  2
Affiliations

Dual Therapeutic Action of a Neutralizing Anti-FGF2 Aptamer in Bone Disease and Bone Cancer Pain

Ling Jin et al. Mol Ther. 2016 Nov.

Abstract

Fibroblast growth factor 2 (FGF2) plays a crucial role in bone remodeling and disease progression. However, the potential of FGF2 antagonists for treatment of patients with bone diseases has not yet been explored. Therefore, we generated a novel RNA aptamer, APT-F2, specific for human FGF2 and characterized its properties in vitro and in vivo. APT-F2 blocked binding of FGF2 to each of its four cellular receptors, inhibited FGF2-induced downstream signaling and cells proliferation, and restored osteoblast differentiation blocked by FGF2. APT-F2P, a PEGylated form of APT-F2, effectively blocked the bone disruption in mouse and rat models of arthritis and osteoporosis. Treatment with APT-F2P also exerted a strong analgesic effect, equivalent to morphine, in a mouse model of bone cancer pain. These findings demonstrated dual therapeutic action of APT-F2P in bone diseases and pain, providing a promising approach to the treatment of bone diseases.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Binding profiles of anti-FGF2 aptamer. (a) SPR sensorgrams monitoring the affinity of APT-F2 to human FGF2 and other FGF family proteins or heparin-binding growth factors. The 3′-biotine labeled APT-F2 RNA was immobilized to a streptavidin sensor chip and the test proteins (100 nmol/l) were injected at the indicated time periods. Experimental conditions and procedures are described in Methods. (b) SPR sensorgrams showing the affinity of APT-F2 to the indicated different FGF2 species. (c) SPR sensorgrams showing the ability of APT-F2 to block the FGF2•FGFR1 interaction. Human FGFR1α(IIIc)/Fc fusion protein (100 nmol/l) was immobilized to a protein A sensor chip, and human FGF2 (100 nmol/l) was injected in the presence of heparin (100 nmol/l) with APT-F2 (150 nmol/l). FGF2, Fibroblast growth factor 2; SPR, surface plasmon resonance; FGFR, FGF receptor.
Figure 2
Figure 2
Inhibition of FGF2-induced signaling pathways in NIH3T3 cells by APT-F2P. NIH3T3 cells were treated with FGF2 (2 nmol/l) or PDGF (2 nmol/l) with or without APT-F2P (2, 10, and 100 nmol/l) for 0.5 hour, and proteins were analyzed by Western blotting using the indicated antibodies to detect pErk phosphorylation levels and protein loading control (actin and Erk). FGF2, Fibroblast growth factor 2; PDGF, platelet-derived growth factor.
Figure 3
Figure 3
Attenuation of FGF2-induced proliferation of HUVEC cells by APT-F2P. (a) HUVEC cells growth was examined in the presence of FGF2 (0.5 nmol/l) and the neutralizing antibody 3H3 or APT-F2P at the indicated doses for 72 hours. Scramble denotes a negative control RNA in which the original sequence of APT-F2 was scrambled. (b) Inhibition profiles of 3H3 and APT-F2P. Experimental conditions and procedures are described in Methods. Values are the mean and SD of three or four independent experiments. FGF2, Fibroblast growth factor 2; HUVEC, human umbilical vein endothelial cells.
Figure 4
Figure 4
Attenuation of FGF2-induced proliferation of normal HFLS cells by APT-F2P. (a) HFLS cells growth was examined in the presence of FGF2 (0.5 nmol/l) and the neutralizing anti-FGF2 antibody or APT-F2 or APT-F2P at the indicated doses for 72 hours. (b) Inhibition profiles of APT-F2 and APT-F2P. Values are the mean and SD of three or four independent experiments. **P < 0.01 versus HFLS cells growth in the presence of FGF2 (Dunnett's test). FGF2, Fibroblast growth factor 2; HFLS, human fibroblast-like synovial.
Figure 5
Figure 5
Effects of APT-F2P on premature osteoblasts differentiation and OPG production in HFLS cells. (a) Attenuation of FGF2-induced differentiation arrest of premature osteoblasts by APT-F2P. MC3T3-E1 cells were grown in minimum essential medium alpha (α-MEM) supplemented 10% FBS, and transferred to the differentiation medium supplemented with the indicated compounds, followed by the ALP activity measurement. Experimental conditions and procedures are described in Methods. (b) Inhibition profiles of APT-F2P in FGF2-induced differentiation arrest. (c) FGF2 blocks OPG production and APT-F2P liberates the production in HFLS cells. HFLS cells were grown in the presence of FGF2 (1 nmol/l) and the neutralizing anti-FGF2 antibody or APT-F2P at the indicated doses for 72 hours. The secreted OPG level was monitored with ELISA kits (Pierce) using anti-OPG antibody according to the manufacturer's instructions. ALP, alkaline phosphatase; ELISA, enzyme-linked immunosorbent assay; FBS, fetal bovine serum; FGF2, Fibroblast growth factor 2; HFLS, human fibroblast-like synovial; MEM-α, minimum essential medium alpha; OPG, osteoprotegerin.
Figure 6
Figure 6
Attenuation of CIA development in mice by APT-F2P. (a) Procedures of mouse CIA experiments. Experimental details are described in Methods. (b) CIA clinical scores for vehicle and APT-F2P administered mice. Values are the mean and SEM of 5–6 mice per group. *P < 0.05 and **P < 0.01 versus vehicle (Mann-Whitney's U-test or Dunnett's test). (c) Histopathological analysis of the synovitis and bone and cartilage damage. Synovitis is represented by “inflammation in synovium”, “formation of granulation tissue/vascular/synovial cavity”, and “inflammation in dermis/muscle/tendon”, while bone and cartilage damage is represented by “decreased cartilage”, “bone resorption”, “formation of cartilaginous tissue”, and “formation of callus”. CIA, collagen-induced arthritis; SEM, standard error of mean.
Figure 7
Figure 7
Blockade of bone density loss in OVX rats by APT-F2P. (a,b) Femur (a) and lumbar (b) bone mineral density in three months post-OVX measure by DEXA. Values are the mean and SEM of eight mice per group. ##P < 0.01 versus Sham group (Student's t- test). Experimental conditions and procedures are described in Methods. (c) Femur diaphysis bone density in 3 months post-OVX measured by pQCT. DEXA, dual-energy X-ray absorptiometry; OVX, ovariectomized; pQCT, peripheral quantitative computed tomography; SEM, standard error of mean.
Figure 8
Figure 8
Analgesic impact on mouse FBC pain. (a) Procedures of mouse FBC pain experiments. Experimental details are described in Methods. (b,c) Weight-bearing tests in Experiments 1 and 2, respectively. (d) von Frey filament test in Experiment 2. Values are the mean and SEM of 7–13 mice per group. S, V1, V2, and M were represented sham, vehicle for APT-F2P, vehicle for morphine and morphine administration groups, respectively. ##P < 0.01 versus S (Aspin Welch's t-test), **P < 0.01 versus V1 (Dunnett's test), ††P < 0.01 versus V2 (Aspin Welch's t-test). FBC, femur bone cancer; SEM, standard error of mean.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Krejci, P, Prochazkova, J, Bryja, V, Kozubik, A and Wilcox, WR (2009). Molecular pathology of the fibroblast growth factor family. Hum Mutat 30: 1245–1255. - PMC - PubMed
    1. Marie, PJ, Miraoui, H and Sévère, N (2012). FGF/FGFR signaling in bone formation: progress and perspectives. Growth Factors 30: 117–123. - PubMed
    1. Okada-Ban, M, Thiery, JP and Jouanneau, J (2000). Fibroblast growth factor-2. Int J Biochem Cell Biol 32: 263–267. - PubMed
    1. Bolander, ME (1992). Regulation of fracture repair by growth factors. Proc Soc Exp Biol Med 200: 165–170. - PubMed
    1. Mignatti, P, Morimoto, T and Rifkin, DB (1992). Basic fibroblast growth factor, a protein devoid of secretory signal sequence, is released by cells via a pathway independent of the endoplasmic reticulum-Golgi complex. J Cell Physiol 151: 81–93. - PubMed

MeSH terms

Substances