| The abnormal angiogenesis in retina and choroid is the main cause of blindness in many diseases, such as diabetic retinopathy (DR), age-related macular degeneration (AMD), retinophathy of prematurity (RP), central and branch retinal vein occlusion and so on. Currently, there are three main methods for retinal neovascularization treatment, laser photocoagulation, vitreous surgery and pharmaceutical therapy. The pharmaceutical method has great potential in clinical application. However, because of the existence of membrance barriers in ocular, it is hard for macromolecular drugs to reach the fundus oculi to play efficacy via eye drops, which makes it can only be used by vitreous injection. All these methods may cause irreversible damage to patients and bring much inconvenience. Hence, a safe, effective and convenient way to treat retinal neovascularization is in need. Endostatin (Es), a 20 kDa C-terminal fragment of collagen XVIII, is produced by hemangioendothelioma as an angiogenesis inhibitor which has good prospects in clinical application. It not only can be used in tumor treatment but it can also be used in the treatment of retinal neovascularization. But, served as a macromolecule, Es can not pass through ocular barriers to reach fundus oculi by itself. The vitreous injection may bring much pain to the patients. In order to increase the penetration ability of Es, the protein transduction domain of the transacting activator of transcription (Tat PTD) was genetically fused to Es to generate the recombinant protein Tat PTD-Es in our previous work. It was proved that Tat PTD-Es could reach fundus oculi to play efficacy. Even though Tat PTD-Es shows promising anti-angiogenesis capability and ocular barrier permeability both in vitro and in vivo, its biological functions are not cell type specific. In order to increase the endothelial cell targeting ability of Tat PTD-Es, a tripeptide, arginine-glycine-aspartic (RGD) that is well known to bind preferentially to the αvβ3 integrin expressed on endothelial cells in new blood vessels, was introduced to the structure of Tat PTD-Es and a novel recombinant protein, Tat PTD-Es-RGD, was constructed. The goal of our study is to get a new recombinant protein Tat PTD-Es-RGD that not only has high penetration ability through ocular barriers and but also targets the abnormal angiogenesis in retina.The main contents and results are as follows:(1) Expression and purification of Tat PTD-Es-RGD in E.coliE. coli expression vector pET28a(+) was linearized by NcoI and XhoI for constructing Tat PTD-Es and Es containing no additional affinity tags, while pET28a(+) was linearized by BamHI and XhoI for constructing Tat PTD-Es-RGD and Es-RGD with an additional 6×His-tag. The recombinant plasmids were transformed into E. coli Origami 2 (DE3) to express the recombinant proteins. The influence of different temperatures on the soluble expression of recombinant proteins was examined. All target proteins were purified using affinity chromatography. Ni2+-Sepharose was used for purifying Es-RGD and Tat PTD-Es-RGD while Heparin-Sepharpse was used for purifying Es and Tat PTD-Es. Finally, all proteins were desalted using a Sephadex G-50 column. In order to increase the production of the recombinant proteins, the proteins were also prepared from inclusion bodies. Different ratios of GSSG/GSH were investigated to select the best renaturation condition. The renaturation protein was also purified by the methods above.The results showed that the optimal expression condition for soluble protein was induction with 0.25 mmol/L IPTG at 25 ℃ for 16 h for all recombinant proteins except Tat PTD-Es, which is optimally expressed at 37 ℃. High yield of recombinant proteins in inclusion bodies was obtained when the expression was induced with 0.25 mmol/L IPTG at 37 ℃ for 4 h. About 0.5 mg of Es,4.2 mg of Tat PTD-Es,3.4 mg of Es-RGD and 3.2 mg of Tat PTD-Es-RGD were obtained from soluble expression, wherease about 5.8 mg of Es,9.6 mg of Tat PTD-Es,18.9 mg of Es-RGD and 15.1 mg of Tat PTD-Es-RGD were obtained from inclusion bodies.(2) Study on the activity and ocular barriers penetration ability of Tat PTD-Es-RGD in vitroCell Counting Kit-8 (CCK-8) was used to detect the inhibition ratio of recombinant proteins to the endothelial cells EAHY926 proliferation. The anti-angiogenesis effect of the recombinant proteins was tested using a chick embryo CAM assay. A wound healing assay was performed to measure the inhibitory effect of Tat PTD-Es-RGD on endothelial cell EAHY926 migration. A tube formation assay of EAHY926 was used to evaluate the anti-angiogenesis effect of Tat PTD-Es-RGD. The results showed that each recombinant protein inhibited the EAHY926 endothelial cell proliferation in a dose-dependent manner. At the concentration of 8 μmol/L, the inhibition ratio of each recombinant protein could reach approximately 80%. The recombinant proteins significantly inhibited the development of new embryonic blood vessels induced by bFGF. Compared with the bFGF group, the ratio of vessel area was reduced from (38.4 ± 3.2)% to (17.9 ± 2.5)% in the Tat PTD-Es-RGD group. Tat PTD-Es-RGD could significantly restrict the migration of EAHY926 by (54.92 ± 3.36)% for 24 h incubation and by (42.30 ± 7.92)% for 48 h incubation compared with the control group. The inhibitory effect of Tat PTD-Es-RGD on endothelial cell migration was similar to those of Es, Tat PTD-Es and Es-RGD. Es, Es-RGD, Tat PTD-Es and Tat PTD-Es-RGD could significantly reduce the tubular formation of endothelial cells by (60.77 ± 8.42)%, (64.89 ± 6.38)%, (60.30 ± 8.41)% and (61.70 ± 6.38)%, respectively, when compared with the control. The results above indicated that after fused with Tat PTD and RGD, the activity of anti-angiogenesis of Es was not changed any more.Air-lifting multilayer cultures of rat corneal epithelial cell (RCEC) was selected to simulate corneal structure and rat retinal microvascular endothelial cell-rat retina microvascular pericytes (RMEC-RRPC) cocultures were selected to establish the BRB model in vitro. Trans-epithelial electrical resistance (TEER) and the permeability of sodium flourescein (Na-F) through the barrier were used to evaluate the integrity of the barriers. The penetration abilities of recombinant proteins of Es, Es-RGD, Tat PTD-Es and Tat PTD-Es-RGD labeled with FITC on ocular barriers were determined by a fluorescent multiwell plate reader. The penetration ability Tat PTD-Es and Tat PTD-Es-RGD on BRB which stimulated by a NO donor of S-nitroso-N-acetylpenicillamine (SNAP) was determined to reflect binding affinity of proteins to integrin αvβ3 on RMEC indirectly. The results showed that for corneal barriers, air-lifting multilayer cultures of RCEC showed high values of TEER and very low permeability to Na-F. Similar results were seen in the BRB model. For corneal barrier, Tat PTD-Es and Tat PTD-Es-RGD showed a higher penetration abilty than Es, but Es-RGD did not. The results expressed in BRB models were same to corneal barriers. Compared with penetration ability on BRB without SNAP treatment, it was decreased significantly in the Tat PTD-Es-RGD group but was not in the Tat PTD-Es group. These results indicated that proteins fused with Tat PTD could take higher barriers penetration ability and fused with RGD could increase the binding affnity of recombinant protein to integrin αvβ3 on the surface of RMEC.(3) Retinal pharmacokinetics and pharmacodynamics of Tat PTD-Es-RGDThe retinal pharmacokinetics was used to deternine the metabolism rate of recombinant proteins in retina after eye drops. The results indicated that the Cmax values of Tat PTD-Es and Tat PTD-Es-RGD were (8.43 ± 0.85) ng/mg and (9.42 ± 0.77) ng/mg, respectively, which were much higher than that of Es of (0.68±0.10) ng/mg. The half-life times of Tat PTD-Es and Tat PTD-Es-RGD were (9.64 ± 1.31) h and (11.03 ± 1.95) h, respectively.Oxygen-induced retinopathy (OIR) model was used to evaluate the inhibition ability of the recombinant proteins on retinal neovascularization. For the groups treated with Tat PTD-Es and Tat PTD-Es-RGD via eye drops, the avascular areas and vessel tufts were reduced significantly compared with those of the negative control group. Compared with the Tat PTD-Es eye drops group, the Tat PTD-Es-RGD eye drops group had significantly lower percentage of tuft area and avascular area. Another evaluation index for hyperoxia-induced neovascularization was the number of endothelial cells that broke through inner limiting membrane (ILM) of retinas. Compared with the negative control group, the number of nuclei in Tat PTD-Es and Tat PTD-Es-RGD eye drops groups and all intravitreal injection groups were significantly decreased. The number of nuclei on the vitreal side of the ILM in Tat PTD-Es eye drops groups was more than that in Tat PTD-Es-RGD eye drops group. Immunohistochemistry was used to investigate the expression level of VEGF that induces angiogenesis. The VEGF expression level in the Tat PTD-Es and Tat PTD-Es-RGD eye drops groups and all intravitreal injection groups was much lower than that of the negative control group, and the VEGF expression level of the Tat PTD-Es-RGD eye drops group was much lower than that of the other eye drops groups. The distribution of the recombinant proteins was examined with collagen XVIII antibody by immunohistochemistry. The results showed that Tat PTD-Es and Tat PTD-Es-RGD eye drop groups had significantly higher level of Es distribution than the negative control group. The quantity of Es in the Tat PTD-Es-RGD eye drops group was higher than that in the Tat PTD-Es eye drops groups. All the results indicated that the recombinant proteins with Tat PTD could penetrate ocular barriers to reach the fundus oculi to play efficacy and the RGD could increase the efficacy of recombinant proteins in retina.(4) The anti-tumor metastatic ability of Tat PTD-Es-RGDThe anti-tumor metastatic effect of the recombinant proteins against B16-F10 melanoma cells was investigated in vivo. All recombinant proteins could reduce the number of nodules compared to negative control group. The number of nodules in Tat PTD-Es-RGD groups was decreased significantly compared with those in Es, Tat PTD-Es and Es-RGD groups. The H&E staining results indicated that, compared with negative control group, the areas of noduls were reduced in the recombinant protein treatment groups, and the areas in the Tat PTD-Es-RGD group was the smallest. The CD34-positive blood vessels were analyzed by immunohistochemical staining. Compared with the negative control group, the area of CD34-positive blood vessels per tumor area in recombinant protein treatment groups were decreased significantly, and the area of CD34-positive blood vessels per tumor area in Tat PTD-Es-RGD group were the smallest among all groups.The main conclusions are as follows:(1) The expression strain of pET28a(+)-Tat PTD-Es-RGD was firstly constructed. The recombinant protein of Tat PTD-Es-RGD was successfully expressed in E.coli Origami 2 (DE3) and pure recombinant protein was obtained after reanturation and purification.(2) The recombinant proteins of Es, Tat PTD-Es, Es-RGD and Tat PTD-Es-RGD could inhibit the proliferation, migration and tube formation of endothelial cells EAHY926. They could also inhibit the development of new embryonic blood vessels induced by bFGF.(3) Tat PTD could carry Es to penetrate corneal barriers and BRB in vitro. The recombinant protein of Tat PTD-Es-RGD had high binding affinity to integrin αvβ3 in the surface of RMEC.(4) Tat PTD-Es and Tat PTD-Es-RGD could reach retina and play efficacy by eye drops, and the efficacy of Tat PTD-Es-RGD was better than Tat PTD-Es.(5) The recombinant proteins of Es, Tat PTD-Es, Es-RGD and Tat PTD-Es-RGD could inhibit tumor metastasis, and the activity of Tat PTD-Es-RGD was better than those of the other recombinant proteins. |
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