Biochimica et Biophysica Acta (BBA) - General Subjects
Tat PTD–endostatin: A novel anti-angiogenesis protein with ocular barrier permeability via eye-drops
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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