¡¡¡¡MODIFICATION OF ES

¡¡¡¡There are a lot of obstacles on ES¬ðs clinical application, such as need of high dose to maintain its efficacy, poor stability, etc. In order to overcome these shortcomings, structural modification has received particular attention. Many scholars reformed ES to enhance the stability and improve targeted therapy of ES, these measures indeed improve the antiangioª²genic ability of ES.

¡¡¡¡Chemical Modification  Polyethylene glycol (PEG) is a highly investigated polymer for the covalent modification of peptides and proteins. PEG possesses superiorities in shielding antigenic and immunogenic epitopes in compared with other modifiers[18].PEG shows better amphipathic properties and biocompatibility. Li et al[19] first applied polyethylene glycol ES(PEGª²ES) to inhibit corneal NV induced by alkali burn in experimental model, the result demonstrated that PEGª²ES possesses more antiª²angiogenic activities than ES, and there were no toxicity and adverse reactions when local administrated.

¡¡¡¡The chemical modification of ES with low molecular weight heparin(LMWH) is mainly based on the sequence of LMWH sugar molecules exist twoª²oª²hydroxy structure, after the periodic acid oxidation a highly active aldehyde can be formed, which combined with the free amino of ES protein to form covalent modification of ES. Tan et al[20] modified ES by LMWH(LMWHª²ES), the changes of the secondary structure of the modified products were studied by Fourier transform infrared spectroscopy and Circular dichroism spectra. Their study demonstrated that the modified products have a better heat tolerance and higher activity than ES towards. Zhu et al[21] first administrated LMWHª²ES by subconjuctival injection in rabbit corneal NV model, the result showed that LMWHª²ES was superior to ES in the inhibition of NV.

¡¡¡¡Genetic Modification

¡¡¡¡P125A  Recent studies have shown that a point mutation in human ES at position 125 can obtain a mutant ES, called P125Aª²ES(P125Aª²ES)[22].This genetically engineered ES showed  improved endothelial cell binding and antiangiogenic biological activity when compared to the native protein. P125Aª²ES can be acquired through genetically replaced the amino acid proline at position 125 with alanine .

¡¡¡¡RGD sequence  Neovascular tissue express high levels of ¦Áv¦Â3/av¦Â5 and a5¦Â1 integrins. Consequently, peptides containing the RGD (Argª²Glyª²Asp) sequence, which is present in ligands of integrins, is effective in targeting therapeutic reagents to neovascular endothelium[23]. Yokoyama et al[24] added RGD sequence to either the amino or carboxyl terminus of P125Aª²ES to get further modification of P125Aª²ES with the RGD motif. RGDª²modified P125Aª²ES showed increased binding to endothelial cells and improved antiangiogenic properties. Ren et al[25] changed GRIRGAD sequence of ES into RGDRGD by  the method of siteª²directed mutagenesis to raise its antiª²angiogenic activity.

¡¡¡¡NGR motif  Human ES has an internal asparagineª²glycineª²arginine (NGR) motif at position 126ª²128. Peptides that contain NGR sequence have been shown to target tumor vasculature and inhibit aminopeptidase N activity[26]. Yokoyama et al added NGR sequence to the amino terminus of the human ES through genetical modification, NGRª²ES showed improved inhibition of tumor growth, endothelial cell homing and biologic activity.

¡¡¡¡Endostar  Endostar, a novel recombinant human ES, was purified in Escherichia coli with an additional nineª²amino acid sequence (MGGSHHHHH)[27].The protein can be folded into a soluble one, the antiangiogenic effects of endostar were correlated with the VEGFª²triggered signaling[28]. Endostar suppressed the VEGFª²stimulated proliferation, migration, and tube formation.

¡¡¡¡CONCLUSION

¡¡¡¡Recent studies demonstrated that modification of a vascular targeting sequence to enhance the biology characteristics and therapeutic value of human ES is encouraging. However, its action mechanisms have not been fully elucidated. The relationship between the structure and function of ES warrant further investigation, so as to explore new substitution with native ES. Most of the modified ES have not been used in ocular diseases yet, we are longing for more and more administration in ophthalmology fields.

¡¡¡¡Gene transfer provides a means to treat ocular NV without the development of toxicity and tolerance. Modified ES in combined with gene therapy may be harnessed to provide us broader prospects in the treatment of ocular NV.

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  ¡¡¡¡1 Zhang P, Yue T, Zhu ZY, Zheng JL, Lin JX, Zhang WX, Feng GG. The preparation of endostatin protein and the measurement of its biologic activity. Int J Ophthalmol(Guoji Yanke Zazhi) 2005;5(5):841ª²846

¡¡¡¡2 O¬ðReilly MS, Boehm T, Shing Y, Fukai N, Vasios G, Lane WS, Flynn E, Birkhead JR, Olsen BR, Folkman J.Endostatin:an endogenous inhibitor of angiogensis and tumor growth. Cell 1997;88(2):277ª²285

¡¡¡¡3 Sasaki T, Hohenester E, Timpl R.Structure and function of collagenª²derived endostatin inhibitors of angiogenesis. IUBMB Life 2002;53(2):77ª²84

¡¡¡¡4 Ricardª²Blum S, F¨¦raud O, Lortatª²Jacob H, Rencurosi A, Fukai N, Dkhissi F, Vittet D, Imberty A, Olsen BR, van der Rest M. Characª²terization of endostatin binding to heparin and heparan sulfate by surface plasmon resonance and molecular modeling: role of divalent cations. J Biol Chem 2004;279(4):2927ª²2936

¡¡¡¡5 Boehm T, O¬ðReilly MS, Keough K, Shiloach J, Shapiro R, Folkman J.Zincª²binding of endostatin is essential for its antiangigenic activity.Biochem. Biophys Res Commun 1998;252(1):190ª²194

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