内皮抑素与眼部新生血管

眼部新生血管导致眼结构和功能的破坏,造成严重的视功能损害,是当前最难解决的眼科难题之一。内皮抑制素(endostatin,ES)是目前公认的最强的血管生成抑制因子之一,已经用于抑制眼部新生血管的实验研究,表明内皮抑制素具有强大的抗眼部新生血管作用。本文就内皮抑素的结构、功能及与眼部新生血管的相关研究进行综述。     

内皮抑素在抗新生血管性眼病治疗中的研究进展

眼部新生血管性疾病严重危害视力,目前的动物实验已证明内皮抑素(endostatin,ES)有望成为一种新型、有效的抑制眼部新生血管的药物。我们就ES在抗新生血管性眼病治疗中的研究进展作一综述。     

抗血管内皮生长因子Bevacizumab在眼部新生血管中的应用新进展

眼部新生血管性病变是致盲的主要原因之一,大量的研究证据表明血管内皮生长因子(vascular endothelial growth factor,VEGF)是新生血管形成的关键调控因子。Bevaci-zumab是世界上首个批准上市的血管内皮因子抑制剂由于其强大的抗新生血管作用,在眼科新生血管性疾病治疗中有广泛的应用前景。本文对VEGF的特性、VEGF在眼部新生血管形成中的作用和Bevacizumab作用机制、应用范围进行综述。     

贝伐单抗治疗眼部新生血管性疾病的研究进展

从分子生物学水平阐述血管内皮生长因子(VEGF)与眼部新生血管性疾病发病的关系,总结近5年来国内外应用抗VEGF药物贝伐单抗(Bevacizumab,Avastin)治疗眼部新生血管性疾病的基础和临床研究,评价其疗效及安全性.     

血管内皮生长因子抑制剂治疗视网膜新生血管性疾病

视网膜新生血管性疾病是致盲的主要病因,早期预防、控制视网膜新生血管的发生发展显得尤为重要。目前,关于视网膜新生血管性疾病的确切发病机制尚不清楚,但对血管内皮生长因子(vascular endothelial growth factor,VEGF)在其形成过程中起关键作用已达成共识,现在已有多种VEGF抑制剂被用于治疗眼部新生血管性疾病。现将治疗视网膜新生血管性疾病的最新几种VEGF抑制剂作一综述。     

血管内皮生长因子-B与眼部新生血管性疾病

异常的新生血管会导致多种眼部疾病,而血管内皮生长因子（VEGF）在新生血管的发生及发展中起重要作用.VEGF-B作为VEGF家族的一员,对血管生成和血管通透性无显著影响,但可通过血管生存作用和细胞凋亡作用对新生血管的生长进行调控,从而抑制新生血管的发生和发展.同时,VEGF-B对心脏和神经元等具有保护作用.抗VEGF治疗作为目前明确有效的治疗新生血管的方法,一直受到广泛关注,而因VEGF-B的两面性,对于抗VEGF-B的药物在治疗眼科新生血管性疾病方面的作用尚需进一步研究.就VEGF-B对机体血管、细胞、神经元及心脏所起的作用及其在眼部新生血管性疾病治疗中的应用进行综述.     

改良内皮抑素与眼部新生血管的研究进展(英文)

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。     

重组人内皮抑素滴眼液抑制小鼠角膜新生血管形成的研究

目的　检验重组人内皮抑素对角膜新生血管形成的抑制效果,探索其临床应用价值。 方法　用MTT方法检验重组内皮抑素对血管内皮细胞的增殖抑制活性;用重组人内皮抑素滴眼液,治疗小鼠角膜碱烧伤导致的角膜新生血管形成。 结果　重组人内皮抑素 10μg/mL时可抑制bFGF刺激下HUVEC的增殖,抑制率达到 42 1% (P<0 05); 100μg/mL的内皮抑素可抑制碱烧伤诱导的小鼠角膜新生血管形成,治疗 7d时新生血管长度较未治疗组减少 30 9%(P<0 01),新生血管化面积减少了 13 7% (P<0 01)。 结论　内皮抑素特异地抑制血管内皮细胞增殖,抑制碱烧伤诱导的小鼠角膜新生血管形成,可能成为临床上预防、治疗相关眼科疾患的药物。     

贝伐单抗在角膜新生血管性疾病与翼状胬肉中的应用

贝伐单抗是近年来出现的新型血管生成靶向治疗药物,具有抑制血管内皮生长因子亚型,阻止血管渗漏和新生血管形成,抗纤维增生的作用,在治疗多种眼部新生血管相关性疾病中取得较好的疗效,本文就贝伐单抗在角膜新生血管性疾病与翼状胬肉中的应用作一综述.     

眼部新生血管与抗血管内皮生长因子治疗

眼部新生血管性病变是致盲的主要原因之一,可见于多种眼底疾病,其确切的发病机制尚不完全清楚.大量的研究证据表明血管内皮生长因子(VEGF)是新生血管形成的关键调控因子.本文对VEGF的特性、VEGF在眼部新生血管形成中的作用和抗VEGF治疗进行综述.     

Outcome of vitreous surgery and the balance between vascular endothelial growth factor and endostatin

PURPOSE: To predict the results of vitreous surgery in patients with proliferative diabetic retinopathy (PDR), the correlation between vitreous fluid levels of vascular endothelial growth factor (VEGF) or endostatin and the postoperative outcome was investigated. METHODS: VEGF and endostatin levels in vitreous fluid specimens obtained during vitreous surgery were measured by enzyme-linked immunosorbent assay. Expression of VEGF and endostatin in epiretinal membranes was assessed immunohistochemically. Patients were prospectively followed up for 6 months. RESULTS: No improvement and/or progression of PDR was seen in 11 (25%) of 44 eyes (progression group). The vitreous fluid level of VEGF was significantly higher in the progression group than in the regression group (P = 0.0023). Conversely, the vitreous fluid level of endostatin was significantly higher in the regression group than in the progression group (P = 0.0299). Eyes with a high vitreous fluid level of VEGF and a low endostatin level had a significantly greater risk of progression of PDR after vitreous surgery than did eyes with low VEGF and high endostatin levels (odds ratio = 10.00, P = 0.047). VEGF and endostatin were detected immunohistochemically in the fibrovascular epiretinal membranes resected from the subjects. CONCLUSIONS: In this study both VEGF and endostatin were expressed in eyes with PDR. VEGF and endostatin levels in the vitreous fluid correlated with the outcome of vitreous surgery for PDR. Therefore, the outcome of PDR surgery can probably be predicted by measuring cytokines and/or growth factors in the vitreous fluid, with VEGF and endostatin being good candidates.     

Effect of verteporfin photodynamic therapy on endostatin and angiogenesis in human choroidal neovascular membranes

AIM: To evaluate the effect of verteporfin photodynamic therapy (PDT) on endostatin with regard to expression of vascular endothelial growth factor (VEGF) in human choroidal neovascular membranes (CNVs) secondary to age-related macular degeneration. METHODS: A retrospective review of an interventional case series of 68 patients who underwent removal of CNV. 29 patients were treated with PDT 3-655 days before surgery. 39 CNVs without previous treatment were used as controls. CNVs were stained for CD34, CD105, Ki-67, cytokeratin 18, endostatin, E-selectin and VEGF. "Predominance score of VEGF over endostatin" (mean) was defined as the difference between VEGF and endostatin staining scores. RESULTS: In four CNVs treated by PDT 3 days previously, PS was significantly higher in the retinal pigment epithelium (mean = 2.5, p = 0.006) and stroma (mean = 2, p = 0.015) than in the control group (mean = 0). At longer post-PDT intervals, PS was significantly decreased in the retinal pigment epithelium (mean = 0, p = 0.019) and stroma (mean = 0, p = 0.015). Proliferative activity was high (p = 0.023), but mostly related to inflammatory cells. PDT did not influence E-selectin expression significantly. CONCLUSIONS: VEGF predominance over endostatin early after PDT might contribute to enhanced angiogenic activity associated with recurrences. Strategies upregulating or replacing endostatin early after PDT might increase the effectiveness of PDT.     

Endostatin modulates VEGF-mediated barrier dysfunction in the retinal microvascular endothelium

Recent evidence indicates that the anti-angiogenic peptide endostatin may modulate some of the vasomodulatory effects of vascular endothelial growth factor (VEGF) in the retina, including reduction of blood retinal barrier function although it remains uncertain how endostatin promotes endothelial barrier properties. The current study has sought to examine how physiological levels of endostatin alters VEGF-induced inner BRB function using an in vitro model system and evaluation of occludin and ZO-1 regulatory responses. In addition, the ability of exogenous endostatin to regulate VEGF-mediated retinal vascular permeability in vivo was investigated. Retinal microvascular endothelial cells (RMEC's) were exposed to various concentrations of endostatin. In parallel studies, RMEC monolayers were treated with vascular endothelial growth factor (VEGF165). Vasopermeability of RMEC monolayers and occludin expression were determined. Blood retinal barrier integrity was quantified in mouse retina using Evans Blue assay following intravitreal delivery of VEGF165, endostatin or a VEGF/endostatin combination. Endostatin increased the levels of expression of occludin whilst causing no significant change in FITC-dextran flux across the RMEC monolayer. Endostatin reversed the effects of VEGF165-enhanced permeability between microvascular endothelial cells and induced phosphorylation of occludin. Evans Blue leakage from retinas treated with VEGF was 2.0 fold higher than that of contra-lateral untreated eyes (P<0.05) while leakage of eyes from endostatin treated animals was unchanged. When eyes were injected with a combination of VEGF165 and endostatin there was a significant reduction in retinal vasopermeability when compared to VEGF-injected eyes (P<0.05). We conclude that endostatin can promote integrity of the retinal endothelial barrier, possibly by preventing VEGF-mediated alteration of tight junction integrity. This suggests that endostatin may be of clinical benefit in ocular disorders where significant retinal vasopermeability changes are present.     

Expression of endostatin in human choroidal neovascular membranes secondary to age-related macular degeneration

Endostatin is an endogenous angiogenesis inhibitor which requires E-selectin for its antiangiogenic activity. The aim of this study was to investigate the expression of endostatin in human choroidal neovascular membranes (CNV) secondary to age-related macular degeneration (AMD) with regard to vascularization and proliferative activity. An interventional case series of 36 patients who underwent removal of CNV were retrospectively investigated. Thirty-six CNV were analyzed by light microscopic immunohistochemistry for the expression of CD34 (endothelial cells, EC), CD105 (activated EC), Ki-67 (cell proliferation), Cytokeratin 18 (epithelial cells), VEGF (vascular endothelial growth factor), E-selectin and endostatin. Donor eyes (n=7) including one with AMD were used as controls. Endostatin immunoreactivity was present in choroidal vessels of five as well as in the retinal pigment epithelium (RPE)-Bruch's membrane complex of two donor eyes without AMD. In one eye with AMD, endostatin was detected in RPE, Bruch's membrane and choroidal vessels. Ninety-two percent (33/36) of CNV disclosed endostatin staining. RPE-Bruch's membrane complex, choroidal vessels and stroma were positive in 50% (18/36), 72% (26/36), and 78% (28/36) of the membranes, respectively. Both control eyes and CNV expressed all the investigated markers except E-selectin being positive only in membranes. Endostatin, an endogenous angiogenesis inhibitor, is expressed in CNV and its therapeutic up-regulation may be a new strategy in the treatment of neovascular AMD.     

Matrix metalloproteinases in human choroidal neovascular membranes excised following verteporfin photodynamic therapy

AIM: To evaluate expression of proangiogenic matrix metalloproteinases (MMP) 2 and 9 at distinct intervals after verteporfin photodynamic therapy (PDT) in human choroidal neovascular membranes (CNV) secondary to age-related macular degeneration (AMD). METHODS: Retrospective review of an interventional case series of 49 patients who underwent removal of CNV. Twenty-six patients were treated with PDT 3 to 383 days prior to surgery. Twenty-three CNV without previous treatment were used as controls. CNV were stained for CD34, cytokeratin 18, endostatin, MMP-2 and MMP-9 by immunohistochemistry. RESULTS: CNV without previous therapy disclosed MMP-2, MMP-9 in RPE-Bruch's membrane, vessels and stroma in different intensities. Three days after PDT, MMP-9 expression was significantly weaker in stroma (p = 0.0019). Endostatin was significantly reduced in vessels (p<0.001). At longer post-PDT intervals, a significant increase of MMP-9 in stroma (p = 0.037) and of endostatin in RPE-Bruch's membrane (p = 0.02), vessels (p = 0.005) and stroma (p<0.001) were disclosed. No significant changes in MMP-2 expression were detected. CONCLUSIONS: PDT induced an early, temporary decrease in MMP-9 and endostatin expression. At longer intervals, MMP-9 increase is possibly associated with the angiogenic process responsible for recurrence after PDT. MMP-9, however, acts as a double-edged sword by concomitant induction of endostatin, an endogenous inhibitor of angiogenesis.     

Generation of endostatin by matrix metalloproteinase and cathepsin from human limbocorneal epithelial cells cultivated on amniotic membrane

PURPOSE: Cultured human limbocorneal epithelial (HLE) cells secrete endostatin-related molecules that are augmented when the cells are cultivated on denuded amniotic membrane (DAM). This study is to identify mechanisms for enhanced endostatin production by HLE cells cultivated on AM. METHODS: HLE cells were cultured on dish, on intact AM (IAM) or on DAM. Collagen XVIII alpha1 mRNA was analyzed by real-time quantitative PCR. In HLE/DAM cultures, inhibitors of MMPs (GM-6001; 1,10-phenanthroline), cathepsins (E64; cathepsin B inhibitor II), elastase (elastatinal), and serine proteases (AEBSF; aprotinin) were added. Endostatin in the conditioned medium (CM) was detected by Western blot. MMP-7; MMP-9; and cathepsins B, K, L, and V in the CM were quantitated by ELISA. Exogenous cathepsin B or V was added to the concentrated HLE/DAM CM to see the effect on endostatin production. RESULTS: The expression of collagen XVIII alpha1 mRNA in the three groups was similar. Elastatinal, AEBSF, and aprotinin had no effect on endostatin generation. MMP inhibitors inhibited the generation of all the 20- and 28- to 30-kDa endostatin-related fragments, while cathepsin inhibitors inhibited only the 20-kDa endostatin. The level of MMP-7 and cathepsin B but not cathepsin V increased as the culture time increased, and paralleled with endostatin production. However, cathepsins K and L were absent in the CM. Exogenous cathepsins B and V further augmented the generation of endostatin. CONCLUSIONS: MMP-7 and cathepsins B and V are involved in the generation of endostatin by HLE cells. Facilitating endostatin generation may be a novel physiological function of the cornea-specific cathepsin V.     

Collagen XVIII/endostatin shows a ubiquitous distribution in human ocular tissues and endostatin-containing fragments accumulate in ocular fluid samples

Background The endostatin domain of type XVIII collagen (ColXVIII) inhibits neovascularization and regulates cell migration and matrix turnover. This study was designed to demonstrate the protein and gene expression patterns of ColXVIII/endostatin in the human eye and to ascertain whether endostatin is detectable in ocular fluid samples.Methods Twenty human eyes enucleated on account of choroidal melanoma were used for immunohistochemical stainings with antibodies against ColXVIII and endostatin. In situ hybridization was used to localize cells responsible for the production of mRNA for ColXVIII. Tear fluid, aqueous humor, and vitreous gel samples were used for Western immunoblotting to detect endostatin fragments in these samples.Results ColXVIII was immunolocalized to almost all ocular structures, namely the basement membranes (BMs) of the corneal and conjunctival epithelia, Descement’s membrane, the anterior border layer and posterior pigmented epithelium of the iris, the BMs of the pigmented and non-pigmented ciliary epithelia, the internal wall of Schlemm’s canal and trabeculae, the ciliary and iris muscle cells, the BMs of the pigment epithelium of the retina, and the internal limiting membrane. Universal expression was seen in the BMs of vascular endothelial cells, and in fibroblasts located in the conjunctiva, the iris, and the ciliary body. Endostatin showed a corresponding pattern, but additional immunostaining was present in the corneal and conjunctival epithelial cells. Most epithelial and mesenchymal cells expressed the mRNA for ColXVIII. Endostatin-containing fragments varying in size were detected in tear fluid, aqueous humor and vitreous gel samples.Conclusions Practically all structures of the human eye contain ColXVIII/endostatin, emphasizing its possible important structural and functional role in the human eye. Furthermore, ocular fluid samples contain endostatin fragments, which may contribute to the antiangiogenic properties of the eye.     

Stimulation and inhibition of angiogenesis in diabetic retinopathy.

PURPOSE: To investigate the role of stimulators and inhibitors of angiogenesis in the pathogenesis of diabetic retinopathy. METHODS: Undiluted vitreous samples and simultaneous paired plasma samples were obtained from 30 diabetic patients (35 eyes) undergoing vitreous surgery. The levels of vascular endothelial growth factor (VEGF), endostatin, and platelet factor-4 (PF-4) were measured simultaneously in each specimen by enzyme-linked immunosorbent assay. The severity of diabetic retinopathy was evaluated according to the modified Early Treatment Diabetic Retinopathy Study retinopathy severity scale. RESULTS: Vitreous levels of VEGF and endostatin were significantly correlated with the severity of diabetic retinopathy (rho = 0.52, rho = 0.48, respectively), but the vitreous level of PF-4 was not (rho = 0.12). Vitreous levels of VEGF, endostatin, and PF-4 were not significantly correlated with their plasma levels. The vitreous level of VEGF was significantly correlated with that of endostatin (rho = 0.42). The VEGF concentration was significantly higher in the vitreous than in the plasma, while the endostatin concentration was not. CONCLUSIONS: The present study showed that VEGF and endostatin were expressed in the vitreous of patients with diabetic retinopathy and may be involved in the pathogenesis of this condition.     

Localization of collagen XVIII and endostatin in the human eye

PURPOSE: To investigate the localization of endostatin, a potent angiogenesis inhibitor, and its progenitor collagen XVIII in the human eye. METHODS: Twelve normal human eyes were investigated. Immunohistochemistry of the anterior and posterior eye segment was performed using a polyclonal endostatin and collagen XVIII antibody and a monoclonal collagen XVIII antibody. Specificity of the antibodies was confirmed by Western blot analysis. RESULTS: The antibody against collagen XVIII stained Bowman's membrane, the lens capsule, the trabecular meshwork, and all epithelial and endothelial basal membranes in the anterior and posterior eye segment. In contrast, the antibody against endostatin showed a more distinct staining pattern. Intense intracellular staining for endostatin was present in the lens epithelium and in the non-pigmented epithelium of the ciliary body. Extracellular presence of endostatin could be detected in the lens capsule and all border membranes lining the aqueous humor including the anterior surface of the iris. The choroid was unstained. In the retina, staining was restricted to the inner limiting membrane and to endothelial cells of larger vessels. CONCLUSIONS: Our results show that there is a ring of specifically endostatin expressing structures forming a "barrier" around the anterior chamber and the vitreous. This might physiologically prevent vessels from sprouting into these avascular compartments.