Study On The Penetration Of Eye Barrier And Inhibition Of Ocular Angiogenesis Of Endostatin With The Mediation Of Tat Ptd

Many diseases (such as diabetic retinopathy and age-related macular degeneration) can induce vascular proliferation of eyes which makes serious damage to visual function or even results in blindness. Until now, intraocular injection or laser photocoagulation is commonly adopted to treat retinal angiogenesis in clinic. But these methods often result in serious irreversible damage on retina. Therefore, it has major social and economic significance to search some novel drugs which can prevent angiogenesis on eyes. Endostatin (Es) is an endogenous potent inhibitor of vascular endothelial cell proliferation. Its analogue, Endostar, has been approved by the State Food and Drug Administration (SFDA) in China for the treatment of patients with non-small-cell lung cancer. In addition, researchers have made some achievements of Es on prevention and treatment of ocular neovascular diseases. For example, researchers have identified that Es and Es gene can inhibit the formation of ocular neovascularization by bulbar conjunctiva injection or intravitreal injection. However, due to the presence of eye barrier, Es has to be administrated only by intraocular injection. This mode of operation is difficult and might damage the eyeball organizations, even can result in irreversible damage to patients. So it is important to look for a simple, safe, effective route of administration to treat these ocular diseases. Protein transduction domain of the transacting activator of transcription (Tat PTD) can transverse the cell membrane with different cargoes and it has the potential to cross eye barriers. Thus, in this study, Tat PTD and Es was fused together by biotechnology. And this fusion protein (Tat PTD-Es) was expected to cross ocular barriers via the mediation of Tat PTD and perform its inhibition effect on vascular proliferation of retina. The research contents and the main results are as follows.1Fusion expression and purification of Tat PTD-endostatin (Tat PTD-Es) in Pichia pastorisTat PTD-Es gene was prepared and recombinant plasmid pGAPAaA/Tat PTD-Es (pG/TE) and pGAPZaA/Es (pG/E) was constructed. Plamids was transformed into Pichia pastoris by electroporation and positive colonies were screened. The expressed product was analyzed by SDS-PAGE. The target protein was preliminarily purified with nickel affinity chromatography column.The results showed the Pichia pastoris system was constructed successfully but expressed protein was low.2Fusion expression, renaturation, purification of Tat PTD-Es in E.coliTat PTD-Es and Es were expressed in E.coli expression system. Corresponding expression vectors pET28a/Tat PTD-Es(pET28a/TE) and pET28a/Es(pET28a/E) were constructed. E. coli Rosetta (DE3) cells containing recombinant plasmid were grown at37℃. After4h induction, the cells was harvested and analyzed by SDS-PAGE. The inclusion body was renatured by direct dilution method. Subsequently, His-trapTM HP column was used to purify the protein. After purification, high purity of the target protein was obtained. The molecular weight of Tat PTD-Es was22kDa by SDS-PAGE, which was consistent with the deduced size from its coding sequence. Western blot analysis showed that Tat PTD-Es and Es reacted with6×His antibody, which showed that the fused protein has6×His tag.3Study on the activity in vitro and in vivo and endocytosis mechanism of Tat PTD-EsCCK-8assay was performed to assay the anti-proliferative effects of Tat PTD-Es and Es on endothelial cell EAHY926. Both of the two proteins significantly inhibited EAHY926proliferation in a dose dependent manner. The activity of Tat PTD-Es was higher than Es at low concentrations. Both Tat PTD-Es and Es showed more than80%inhibition on EAHY926proliferation at high concentrations(>4μmol/L).The activity of Tat PTD-Es and Es was also tested by a chick embryo chorioallantoic membrane (CAM) assay. For the control groups, the CAM were treated with saline (blank control) and bFGF (negative control) respectively. The inhibition of Tat PTD-Es and Es on angiogenesis of CAM induced by bFGF was observed. Compared with bFGF group(30.74±6.58), the number of blood vessels of CAM treated with Tat PTD-Es and Es were reduced from to (12.18±4.92) and (14.56±6.21) respectively(p<0.05). The results showed that Tat PTD-Es and Es could significantly inhibit angiogenesis of CAM induced by bFGF.The abilities of cellular internalization of Tat PTD-Es and Es were compared by fluorescence microscope and flow cytometry, respectively. Intracellular accumulation of FITC labeled proteins which incubated with EAHY926cells for4h was observed and the fluorescence intensity in cells exposed to Tat PTD-Es increased notably compared with Es. The percentage of positive cells after exposure to Tat PTD-Es and Es were99.28%and33.61%respectively. The results showed that Tat PTD significantly (p<0.05) enhanced Es delivery into EAHY926cells. The impacts of Tat PTD-Es concentration and incubation time on its uptake were studied. The uptake of Tat PTD-Es by EAHY926cells was clearly dependent on the tested concentration range of1μmol/L to10μmol/L (fluorescence intensity from20.72to305.23). In terms of the effect of the treatment time on the internalization process of Tat PTD-Es, with the incubation time increase, the fluorescence intensity inside EAHY926cells increased respectively. Rapid entrance into cells of Tat PTD-Es was observed, and the fluorescence intensity increased sharply from0min-2h (fluorescence intensity from17.16to221.37). Then it increased to249.18in a more gentle way until4h.To understand the concrete uptake pathway of Tat PTD-Es, the effects of some inhibitors on the transduction of Tat PTD-Es were investigated. Compared with the control group, the fluorescence intensity of cells that were treated with4℃and NaN3(ATP inhibitor) was greatly reduced and it decreased by92.49%and94.91%. The result indicated that ATP played an important role in the uptake pathway of Tat PTD-Es in EAHY926cells and Tat PTD-Es appeared to be taken up inside the cells via an energy-dependent active process. The uptake of Tat PTD-Es by EAHY926cells which were treated with sucrose (the inhibitor of clathrin pathway), chlorpromazine (the inhibitor of clathrin pathway), genistein (the inhibitor of caveolin pathway), cytochalasin D (the inhibitor of macropinocytosis) respectively were all decreased, but the decreases were different. When treated with sucrose and chlorpromazine, uptakes of Tat PTD-Es by EAHY926cells were all reduced by about60%. The result suggested that Tat PTD-Es entered EAHY926cell by the clathrin-mediated endocytosis. When cells were pre-incubated with genistein, the cell uptake of Tat PTD-Es was inhibited significantly by33%, indicating that a portion of Tat PTD-Es was internalized into the cells through caveolae-mediated endocytosis. The cell uptake of Tat PTD-Es was significantly reduced by72%after treating cells with cytochalasin D. The result implied that macropinocytosis might be a major uptake mechanism for Tat PTD-Es by EAHY926cells.By these results, we speculated that the cellular entrance of Tat PTD-Es into EAHY926cells may not only follow a single way, but several pathways.4Study on the ability to penetrate eye barriers and pharmacodynamics in vivo of Tat PTD-EsAfter mice were grouped, Tat PTD-Es or Es was eye-dropped or intravitreally injected. Physiological saline was eye-dropped as control. Mouse eyes were harvested and the fusion protein was examined with6×His antibody by immunohistochemistry. The results showed that both Tat PTD-Es and Es appeared in retina after intravitreal injection. It indicated that drugs can quickly reach the retinal after intravitreal injection. But Es did not appear in retina after eye-dropping. It was implicated that Es can not arrive in retina through eye-dropping. Tat PTD-Es appeared in retinal after eye-dropping. It was verified Tat PTD can carry protein to penetrate eye barriers through eye-dropping. These results achieved the intend purpose.Mouse choroidal neovascularization (CNV) models were established by laser-burning. Then Tat PTD-Es or Es was eye-dropped or intravitreally injected. Physiological saline was eye-dropped as negative control, and Avastin was intravitreally injected as positive control. The results indicated that the CNV areas of mouse intravitreally injected with Tat PTD-Es and Es were respectively (961.2±86.9)μm2and (944.6±88.3)μm2, which obviously decreased comparing to negative control, of which the CNV area was(2623.6±240.9)μm2(p<0.01), and was not significantly different from positive control with an CNV area of (863.5±50.1)μm2. When Es was eye-dropped, the CNV area was (2514.7±260.)μm2. It was not significantly different from the negative control, showing no inhibition of CNV angiogenesis. The CNV area of Tat PTD-Es eye-dropping mice was (1378.4±154.3)μm2, which significantly decreased compared to negative control (p<0.01). It had significant differences from those of intravitreal injection of positive control Avastin and Tat PTD-Es. Tat PTD-Es via eye drops was able to inhibit CNV angiogenesis, and these results further demonstrated that Tat PTD-Es had the capacity to penetrate the eye barrier, reach the retina choroid and play its role via eye-dropping.Results and conclusions:(1) The expression strain of Tat PTD Es in Pichia was constructed.(2) The expression strain of Tat PTD-Es in E.coli was firstly constructed. The protein was expressed successfully and high purity of target proteins was obtained by renaturation and purification from inclusion bodies.(3) Tat PTD-Es and Es had excellent activity in vitro and they can significantly inhibit EAHY926endothelial cell proliferation and CAM angiogenesis.(4) Tat PTD-Es has better cell penetrating ability than Es and it followed several cellular mechanisms entering into EAHY926.(5) Tat PTD-Es could penetrate the eye barrier and reached the retina choroid via eye drops. It also showed a better inhibition choroidal angiogenesis in vivo.