【摘要】 内皮抑素是一种强有力的内源性血管生成抑制因子。它抑制血管内皮细胞的增生，迁移，且无毒性、无耐药性。内皮抑素在体外不稳定，需长期大剂量用药，如何提高内皮抑素生物活性是当今一个热门话题。内皮抑素可以通过化学共价修饰或基因突变改变其结构，提高其稳定性与抑制新生血管活性。我们旨在探讨改良内皮抑素与眼部新生血管的研究进展，并讨论改良内皮抑素的优越性。研究改良内皮抑素也有助于我们了解内皮抑素作用的分子机制.改良内皮抑素有望作为一种新的血管生成抑制剂应用于临床，从而取代内皮抑素。
【关键词】 改良内皮抑素 眼部新生血管 抑制新生血管活性
INTRODUCTION
Most of diseases that cause catastrophic loss of vision can be blamed to ocular neovascularization(NV),such as corneal NV, diabetic retinopathy (DR) and agerelated macular degeneration (AMD). The exact pathogenicity of ocular NV is not yet well understood, and there is no satisfactory therapy for ocular NV. Vascular endothelial cells migrate and proliferate to form new blood vessels. Endostatin(ES) has been characterized and was identified by its ability to inhibit endothelial cell proliferation, migration and cord formation and to suppress angiogenesis[1].It is believed to be promising in the treatment of ocular NV in the near future. We reviews recent progress in studies on the mechanisms and therapeutic potential of modified ES in ocular NV.
THE STRUCTURE OF ES
ES was first identified in the conditioned medium of hemangioendothelioma cells by OReilly et al. ES is derived from the nontriplehelical Cterminal NC1 domains of collagens XVIII, which is released proteolytically in trimeric form and further converted to monomeric ESs of about 20kDa. The fragment has been characterized with antiangiogenic properties[2]. Xray diffraction demonstrated ES possesses a compact globular folding and a core structure related to the carbohydrate recognition domain of Ctype lectins[3]. Analogous to many other angiogenesis inhibitors, ES has a strong affinity for heparin. Two heparinbinding domains have been identified in ES involving two clusters of arginine residues[4], and a zinc binding site is located in the Nterminal part of the molecule[5]. The possibility that its antiangiogenic effect might be related to displacement of angiogenic factors from the surface of endothelial cells through binding of heparan sulfate has prompted several investigations of its interaction with heparin and heparan sulfate (HS)[6]. The role of zinc in the biological activity of ES remains controversial. Zincbinding has been reported to be essential for the antiangiogenic activity of ES[7]. Later studies have failed to confirm the relationship between zincbinding and inhibition of endothelial cell migration or angiogenesis[8].
THE MECHANISMS OF ES
Receptor Pathway Evidence suggests that ES binds to cell surface receptors, such as vascular endothelial growth factor(VEGF), integrins, HS, nucleolin receptor. Kim et al[9] demonstrated that ES binds directly to VEGF receptors but not to VEGF, and that binding of ES to VEGF receptor blocks VEGFinduced tyrosyl phosphorylation of VEGF receptors (KDR/flk1), MAP kinases, and FAK in human umbilical vein endothelial cells. Rehn et al[10] demonstrated that soluble ES binds to integrin α5 and αv to inhibit human vascular endothelial cell migration. Javaherian et al [11] demonstrated that oligomeric ES binds to HS on the cell surface to regulate migration and morphogenesis of vascular endothelial cells. Shi et al[12]found that ES is internalized and transported into cell nuclei of endothelial cell via nucleolin. The phosphorylation of nucleolin, which is critical for cell proliferation, can be inhibited by ES in the nucleus.
Multiple Mechanisms The rest of multiple mechanisms for ES functioning are characterized as follows: 1) ES inhibits vascular endothelial tube formation by inhibiting nitric oxide synthase;2) ES induces endothelial cell apoptosis by activating caspase3 enzymatic activity, reducing antiapoptotic protein BcL2, and reducing MAP kinases activities; 3) ES regulates the Wnt signaling pathway by promoting β catenin degradation; 4) ES causes G1 arrest of endothelial cells and downregulating cmyc mRNA expression and decreasing the mRNA and protein of cyclin D1 [13].
ES AND OCULAR NV
ES was found universal expression in ocular structure, namely the basement membranes (BMs) of the corneal and conjunctival epithelia, the BMs of the pigment epithelium of the retina, and the internal limiting membrane and so on. The ubiquitous distribution of ES in human ocular tissues may be related to the avascularity of the eye[14].
ES exerts powerful antiangiogenic effect and without the development of resistance and toxicity, therefore it is drawing more and more ophthalmologists attention. ES can be administrated by topical instillation, subconjunctival injection, intravitreous injection and gene therapy. Gene transfer provides a strategy to achieve sustained release of ES and can circumvent difficulties arising from handling the protein. The effect of intraocular delivery of recombinant viruses carrying genes encoding angiostatic proteins has been demonstrated in experimental models of ocular NV[15].Lai et al[16] used a recombinant adenoassociated viral (rAAV) vector carrying ES gene to examine the inhibition of corneal NV induced by silver nitrate cauterization in mice. They concluded the rAAV was capable of directly delivering genes to the ocular surface epithelium by way of subconjunctival injection and was able to deliver sustained high levels of gene expression in vivo to inhibit angiogenesis. Zhang et al[17] subconjunctively injected pBlasthES to investigate gene therapy of rat corneal NV induced by acid cauterization.
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