Tat PTD–endostatin: A novel anti-angiogenesis protein with ocular barrier permeability via eye-drops

doi:10.1016/j.bbagen.2015.01.019

Biochimica et Biophysica Acta (BBA) - General Subjects

Volume 1850, Issue 6, June 2015, Pages 1140-1149

https://doi.org/10.1016/j.bbagen.2015.01.019 Get rights and content

Highlights

•
Tat PTD–Es was obtained by bioengineering technology.
•
Tat PTD–Es performed excellent anti-angiogenesis activity.
•
Tat PTD–Es could cross ocular barriers after eye-drop administration.
•
Tat PTD is a promising ocular delivery tool.
•
Tat PTD–Es is a potential drug for curing fundus oculi angiogenesis diseases.

Abstract

Background

Endostatin, a specific inhibitor of endothelial cell proliferation and angiogenesis, has been proved to have effects on ocular neovascular diseases by intraocular injection. In order to increase its permeability to ocular barriers and make it effective on fundus oculi angiogenesis diseases via non-invasive administration (eye drops), endostatin was fused to Tat PTD via a genetic engineering method.

Methods

Most of the Tat PTD– endostatin was expressed as inclusion bodies in Escherichia coli, so pure and active Tat PTD–endostatin was prepared by a series of operations, including inclusion body denaturation, refolding and chromatography. The anti-angiogenesis activity of Tat PTD–endostatin was investigated by cell proliferation experiments and chick embryo chorioallantoic membrane assay. In addition, its translocating ability and concrete entry mechanism into cells were also investigated by fluorescence microscope and flow cytometry. The penetrating ability to ocular barriers was also studied by immunohistochemistry. A mouse choroidal neovascularization model was established to investigate the pharmacodynamics of Tat PTD–endostatin.

Results

The obtained Tat PTD–endostatin had excellent anti-angiogenesis activity and was superior to Es in cellular translocating. Macropinocytosis may be the dominant route of entry of Tat PTD–endostatin into cells. Tat PTD–endostatin could cross ocular barriers and arrive at the retina after eye-drop administration. In addition, it displayed inhibitory effects on choroidal neovascularization via eye drops.

Conclusions

Tat PTD–endostatin possessed excellent ocular penetrating ability and anti-angiogenesis effects.

General significance

Tat PTD is a promising ocular delivery tool, and Tat PTD–endostatin is a potential drug for curing fundus oculi angiogenesis diseases.

Introduction

Endostatin (Es), a 20 kDa C-terminal fragment of collagen XVIII, is a specific inhibitor of endothelial cell proliferation and angiogenesis [1], [2], [3], [4]. Its analogue, Endostar, has been approved by the China Food and Drug Administration (CFDA) for the treatment of patients with non-small-cell lung cancer [5]. In addition, researchers have made some achievements with Es on the prevention and treatment of ocular neovascular diseases [6]. For example, researchers have identified that Es and the Es gene could inhibit ocular neovascularization by bulbar conjunctival injection or intravitreal injection [7], [8]. However, due to ocular barriers, Es has to be administered by intraocular injection to cure fundus oculi diseases. This mode of operation is difficult and can result in irreversible damage to the eyeball [9], and so it is critical to develop a simple, safe and effective route for the administration of Es to treat these ocular diseases. Although eye-drops seem to be an ideal administration route, ocular barriers prevent penetration of Es into fundus oculi sites.

Tat PTD, a protein transduction domain of the Tat protein of HIV-1, has been studied extensively for its ability to pass through biological membranes with different cargoes, including peptides, proteins, and oligonucleotides [10], [11], [12], [13]. Many in vitro and in vivo studies have shown that Tat PTD and its cargos [14], [15] were able to pass through most cell line membranes [16]. After being fused with Tat PTD, some proteins with poor cell membrane permeability could cross the blood–brain barrier (BBB) and even the eye barriers [12], [17], [18], [19]. It is, therefore, a promising tool for non-invasive cellular import of cargos and for making therapeutic agents more efficient for the treatment of many diseases. We hypothesized that Tat PTD might assist Es in penetrating the ocular barriers and playing its anti-angiogenesis role on the fundus oculi via eye-drops.

Thus, in this study, Tat PTD was conjugated to the N-terminus of Es through a bioengineering method and expressed in Escherichia coli. After a series of operations, including inclusion body denaturation, refolding and chromatography, pure Tat PTD–Es, which was expected to have both ocular barrier-penetrating ability and anti-angiogenesis effects, was obtained. In order to verify whether the purified Tat PTD–Es possesses ocular penetrating ability and anti-angiogenesis effects, its in vitro and in vivo activities were investigated. The anti-angiogenesis activity of Tat PTD–Es in vitro was determined using cell proliferation experiments and the chorioallantoic membrane (CAM) assay. The ocular barrier-penetrating activity and inhibitory activity on choroidal neovascularization (CNV) of Tat PTD–Es via eye-drops were evaluated in vivo. Furthermore, its concrete entry mechanism into cells was also investigated, since understanding its mechanism will aid in its application in clinical treatment.

Section snippets

Strains, vectors, cells and reagents

E. coli DH5α cells were used for plasmid propagation and E. coli BL21 (DE3) cells were used for expression of the fusion protein. A His-trap™ HP column, was purchased from GE Healthcare (Sweden). EAHY926 endothelial cells were obtained from Shanghai Cell Bank, the Institute of Cell Biology, China Academy of Sciences (Shanghai, China). Avastin was produced by Genentech/Roche (US). Mouse anti-His polyclonal antibody was purchased from Zhongshan Golden Bridge Biotechnology Co. Ltd (Beijing,

Cloning, expression, renaturation and purification of Tat PTD–Es and Es

In this study, the Tat PTD sequence (YGRKKRRQRRR) was fused to the N-terminus of Es protein and the fusion protein contained an N-terminal polyhistidine purification tag (His-tag). The recombinant fusion protein was expressed in E. coli at a level up to 20% of the total cell proteins, and most of the fusion protein was detected as inclusion bodies (Fig. 1). The molecular weight of Tat PTD–Es was 22 kDa, which was consistent with the size deduced from its coding sequence. The purity of the fusion

Discussion

In our study, though His-tag and Tat PTD were fused to the N-terminus of Es protein, no activity decrease was detected. Tat PTD–Es even displayed an enhanced inhibitory effect on EAHY926 cells compared with Es (p < 0.05) at low concentrations (0.2 and 0.8 μM), which implies that the fusion of Tat PTD to Es enhanced the inhibitory activity of Es on endothelial cells.

In the activity assay, both Tat PTD–Es and Es displayed excellent ability to inhibit endothelial cell proliferation. Tat PTD–Es even

Conflict of interest

We declare that we have no conflict of interest.

Acknowledgements

This work was supported by the National Natural Science Foundation of China under grant No. 81273417 and No. 81302686.

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Tat PTD–endostatin: A novel anti-angiogenesis protein with ocular barrier permeability via eye-drops

Highlights

Abstract

Background

Methods

Results

Conclusions

General significance

Introduction

Section snippets

Strains, vectors, cells and reagents

Cloning, expression, renaturation and purification of Tat PTD–Es and Es

Discussion

Conflict of interest

Acknowledgements

Cell

Exp. Cell Res.

Biochem. Biophys. Res. Commun.

Cell

J. Biol. Chem.

Adv. Drug Deliv. Rev.

J. Control. Release

J. Control. Release

Am. J. Pathol.

J. Control. Release

J. Control. Release

Biomaterials

J. Biol. Chem.

Biochim. Biophys. Acta

Mol. Ther.

J. Biol. Chem.

Research advances of endostatin and its short internal fragments

Curr. Protein Pept. Sci.

Characterization and secondary structure analysis of endostatin covalently modified by polyethylene glycol and low molecular weight heparin

J. Biochem.