角膜淋巴管新生在眼部疾病中的研究进展

角膜淋巴管新生在眼部疾病中发挥重要作用。通常情况下,角膜是一种无血管、无淋巴管的组织,这种无血管、无淋巴管的状态对角膜的透明性和功能性至关重要。然而,一些疾病或创伤可能引起角膜血管和淋巴管的新生,从而破坏角膜的结构和功能。尽管已有多种针对角膜血管新生的药物在临床应用,但尚无特异性针对角膜淋巴管新生的药物。因此,本综述将介绍与角膜淋巴管新生相关的因素,探讨与之相关的眼部疾病,同时对目前的治疗现状进行分析,旨在为淋巴管新生相关的眼部疾病治疗提供更多的选择和可能性,为基于角膜淋巴管新生的研究和药物开发提供参考。     

单纯疱疹性角膜炎发病机制的研究进展

单纯疱疹性角膜炎(HSK)是一种全球高发的严重感染性致盲眼病,视力的慢性损害常与感染的复发、角膜瘢痕、角膜白斑、新生血管以及新生淋巴管等相关.根据目前的研究,角膜的损伤是免疫系统对单纯疱疹病毒(HSV)抗原反应的结果.由于HSK发病机制尚不明确,治疗效果不满意,因此全面了解其初次感染、潜伏、复发的机制是有效治疗的前提.本文分别阐述自噬、免疫系统、细胞因子和微小RNA等几个方面在HSK感染与发病中的作用机制,以了解HSK的发生和发展过程,在一定程度上为HSK的诊断提供参考,并为HSK的治疗及药物研制等研究带来新的启发.     

角膜移植后角膜新生淋巴管与新生血管间的关联

目的:研究角膜移植后角膜新生淋巴管与新生血管间关联。方法:人角膜取自行二次角膜移植的19名患者。5核苷酸酶-碱性磷酸酶(5-nase-Alkaline phosphatase,5-NA-ALP)双重酶组织化学染色及淋巴内皮细胞受体(lymphatic vessel endothelial receptor,LYVE-1)、内皮细胞黏附因子-1(platelet endothelial cell adhesion modecule-1,PECAM-1)双重免疫组化法标记角膜中的新生血管和淋巴管,并进行淋巴管计数(lymphatic vessels counting,LVC)和血管计数(blood vessels counting,BVC),比较BVC与LVC之间的关联。结果:角膜中存在角膜新生血管12例(63%),存在角膜新生淋巴管5例(26%)。角膜新生淋巴管仅出现在血管化角膜中。角膜移植后BVC与LVC间呈显著性正相关(r=0.725;P<0.01)。结论:人角膜移植后角膜新生淋巴管与新生血管之间存在密切关联。     

角膜新生淋巴管的研究进展

近年来随着淋巴管内皮细胞特异性标记物VEGFR-3、LYVE-1、Podoplanin和Prox 1等的发现,对于角膜新生淋巴管的研究有了较大进展,成为近几年的研究热点。本文就角膜新生淋巴管是如何生成,其与新生血管之间的关系,新生淋巴管在角膜移植免疫排斥反应发生过程中的作用作一综述。     

角膜新生淋巴管的研究进展

近年来随着淋巴管内皮细胞特异性标记物VEGFR-3、LYVE-1、Podoplanin和Prox 1等的发现，对于角膜新生淋巴管的研究有了较大进展，成为近几年的研究热点。本文就角膜新生淋巴管是如何生成，其与新生血管之间的关系，新生淋巴管在角膜移植免疫排斥反应发生过程中的作用作一综述。     

角膜新生淋巴管的作用研究进展

角膜新生淋巴管作为角膜免疫反应的传入弧,与许多疾病的发生密切相关,角膜新生淋巴管与角膜新生血管的关系一直是研究的热点,他们共同破坏了角膜的免疫赦免机制.因此,深入研究角膜新生淋巴管的作用机制,探讨相关眼科疾病的治疗方法,是目前亟待解决的问题.就角膜新生淋巴管与感染、角膜移植术后的并发症、干眼、翼状胬肉等疾病的关系展开综述,以期为临床治疗提供理论依据.     

单纯疱疹病毒性角膜炎中细胞凋亡的作用机制研究进展

单纯疱疹病毒性角膜炎（HSK）是一种严重的世界性致盲性角膜疾病,发病机制目前尚不明确。近年来的研究表明,该病的发病机制与细胞凋亡密切相关。病毒感染角膜后,可诱导角膜上皮细胞发生凋亡,随后抑制凋亡过程。同时亦可引起浅层基质发生细胞凋亡,以防止病毒从感染的上皮细胞传染至角膜深部。而当病变累及角膜深层时,常可发生角膜基质混浊、坏死、瘢痕形成、新生血管化以及角膜内皮皱褶等。就HSK发病过程中角膜上皮、基质、内皮发生细胞凋亡的机制进行综述。     

荧光标记大鼠全角膜新生血管和淋巴管的方法研究

目的探讨标记大鼠全角膜新生血管和淋巴管的方法。方法应用心脏灌注荧光联合全角膜免疫荧光法．共焦显微镜下区分并标记全角膜的新生血管和淋巴管。结果适度的抗体浓度和灌注时间下可标记出全角膜新生血管和淋巴管。血管呈黄绿色，淋巴管呈红色。结论心脏灌注荧光联合全角膜免疫荧光法可用于标记大鼠全角膜新生血管和淋巴管。     

角膜新生淋巴管的分子免疫学研究进展

角膜新生淋巴管构成了角膜免疫反应的传入弧,它与角膜新生血管的协同作用是破坏角膜免疫赦免机制的关键因素.近年来,随着淋巴内皮细胞特异性标志物的相继发现,有关角膜淋巴管生成因子的生物学特性、调节机制及临床意义等方面的研究有很大进展.角膜新生淋巴管的免疫调节作用及与移植排斥反应的关系成为研究热点.为此,本文结合角膜免疫赦免特点对角膜新生淋巴管生成的调控机制及其与角膜移植排斥反应之间的关系作一综述.     

Blockade of the VEGF isoforms in inflammatory corneal hemangiogenesis and lymphangiogenesis

Background

The VEGF-A family plays a crucial role in the induction of pathological corneal neovascularization. The role of the different VEGF-A isoforms during lymphangiogenesis is only little-known. Current anti-angiogenic therapies in the eye and other organs inhibit all VEGF-A isoforms, and have effects on both blood and lymphatic vessels. Here we investigate whether selective targeting of the isoform VEGF 165 is able to inhibit corneal lymphangiogenesis under inflammatory conditions.

Methods

The mouse model of suture-induced corneal neovascularization was used to assess the antihem- and antilymphangiogenic effect of topically applied pegaptanib. Corneal blood and lymph vascularized areas were analyzed morphometrically. Furthermore, we analyzed the proliferative effects of VEGF A 121, 165, and 189 on blood and lymphatic endothelial cells (BEC/LEC) via a cell-proliferation assay.

Results

Pegaptanib significantly inhibited inflammatory corneal hemangiogenesis (p?<?0.01), but not lymphangiogenesis in vivo (p?>?0.05), both topically as well as systemically, in the inflamed cornea. In vitro, BECs were more susceptible to pegaptanib than LECs.

Conclusions

Targeting VEGF-A 165 significantly inhibits hem- but not lymphangiogenesis, suggesting VEGF-A 165 to be critical for hem-, but dispensable for lymphangiogenesis, at least in the inflamed cornea.     

角膜新生淋巴管与血管内皮生长因子C

角膜新生淋巴管（CL）存在于血管化角膜上，常引起并加重角膜混浊和角膜移植排斥反应，是重要的致盲原因之一。血管内皮生长因子C（VEGF—C）是血管内皮生长因子（VEGF）家族成员之一，是VEGFR-2和VEGFR-3受体的配体。VEGF-C通过与VEGFR-3结合促进淋巴管增生，是一个特异性的淋巴管生长因子，在CL形成中起着重要的作用。就近年来CL研究进展及与VEGF—C的关系进行综述。     

Time course of angiogenesis and lymphangiogenesis after brief corneal inflammation

PURPOSE: To study the time course of angiogenesis and lymphangiogenesis in the cornea after a short inflammatory insult. This might be helpful for the timing of corneal transplantation in high-risk eyes. METHODS: The mouse model of suture-induced inflammatory corneal neovascularization was used. After placement of 3 interrupted 11-0 sutures into the corneal stroma of BALB/c mice (left in place for 14 days), corneas were excised 2, 3, 5, 7, 14, and 21 days as well as 1, 2, 3, 6, and 8 months after surgery. Hem- and lymphangiogenesis were evaluated using double immunohistochemistry of corneas with CD31/PECAM1 as panendothelial and LYVE-1 as lymphatic endothelial marker. RESULTS: Both blood and lymphatic vessels grew into the cornea as early as day 2 after suture placement. The outgrowth was initially parallel. Hem- and lymphangiogenesis peaked around day 14. Thereafter, both vessel types started to regress. Regression of lymphatic vessels started earlier and was more pronounced than that of blood vessels. Whereas at 6 and 8 months (partly) perfused CD31+++/LYVE-1(-) blood vessels and (nonperfused) ghost vessels could still be observed, there were no CD31+/LYVE-1+++ lymphatic vessels detectable beyond 6 months after this short inflammation. CONCLUSIONS: After a temporary inflammatory insult to the cornea, there is initially parallel outgrowth of both blood and lymphatic vessels. But thereafter, lymphatic vessels regress earlier than blood vessels and are completely regressed by 6 months. Earlier regression of pathologic corneal lymph versus blood vessels suggests that corneal graft survival in high-risk eyes might best be delayed for a prolonged interval following an inflammatory insult.     

Antiangiogenic therapy at the ocular surface: when, what and why?

There are numerous indications for local antiangiogenic therapy at the ocular surface. These include vision-limiting corneal neovascularization, corneal blood and lymphatic vessels endangering corneal graft survival and tumor-associated lymphangiogenesis. A literature review in PubMed and own clinical and experimental data form the basis for a discussion of the indications, current therapeutic options and potential side-effects of local antiangiogenic therapy at the ocular surface.     

Lymphangiogenesis concurrent with haemangiogenesis in the human cornea

BACKGROUND: Corneal transplant rejection can occur with and without neovascularization; therefore, it is necessary to elucidate what other factors allow for rejection. It has been suggested that the lymphatic system may play a role in graft failure, but it has also been held that the cornea is devoid of lymphatics. Use of a new monoclonal antibody against a lymphatic endothelial marker, D2-40, has been used to detect lymphatics in other tissues. The purpose of this study was to use this new tool to determine if the human cornea can undergo lymphangiogenesis. METHODS: Twelve corneal buttons submitted for routine pathology were subjected to immunohistochemical staining with a monoclonal antibody against D2-40 to detect the presence/absence of lymphatics by light microscopy. RESULTS: By the criteria defined, lymphatic vessels were identified in seven out of 12 corneal buttons. In these cases, there was also evidence of neovascularization. Lymphatic positive buttons included four cases where there were histological markers of inflammation. There were no identifiable lymphatics in the remaining five cases and no sign of vascularization. CONCLUSIONS: Corneal lymphatics were identified in association with corneal neovascularization, via the use of a monoclonal antibody against D2-40. In non-vascularized corneas, lymphatics were absent.     

Age-related changes in murine limbal lymphatic vessels and corneal lymphangiogenesis

Important risk factors for graft rejection after corneal transplantation are pathologic corneal lymphangiogenesis and young recipient age. Purpose of this study was to investigate whether there are age-related differences in normal murine limbal and pathologic corneal lymphatic vessels, which could partly explain the unequal outcome of corneal transplantation in young versus old recipients. Furthermore, we investigated whether these observed differences correlate with changes in allograft survival in the murine model of corneal transplantation. Corneal whole mounts from untreated young (aged 6-8 weeks), untreated old (aged 9-15 months) and young and old mice after suture-induced, inflammatory corneal neovascularization were prepared and stained with LYVE-1 as a lymphendothelial marker. Angles of corneal parts with and without a main circumferential limbal lymphatic vessel were measured and then related to the total 360° of corneal circumference. Centrally directed vascular extensions from the main limbal lymphatic vessel (“sprouts”) of previously untreated old mice were counted. Concerning the outgrowth of pathologic lymphatic vessels after inflammatory corneal neovascularization, the area covered with pathologic lymphatic vessels was detected by an algorithm on digitized whole mounts using cell^F^® software. Low-risk allogeneic (C57Bl/6 to BALB/c) corneal transplantations were performed with one recipient group being young, the other group being old mice. In young, untreated mice, 70.5% of the total corneal circumference was covered by a main circumferential limbal lymphatic vessel versus 60.8% in old, untreated mice. Comparing the number of centripedal vascular extensions from the main limbal lymphatic vessel (“sprouts”), untreated old mice had significantly less extensions than young, untreated mice (p < 0.001). After an inflammatory stimulus, old mice had significantly less pathologic corneal lymphatic vessels than young mice (42% less, p < 0.001). Comparing the survival proportions after corneal transplantation, old recipient mice showed a significantly better graft survival 6 weeks after transplantation (65% versus 33%, p < 0.05). Thus, limbal lymphatic vascular sprouts and inflammation-induced pathologic corneal lymphangiogenesis decrease with age. The lower lymphangiogenic potency of older mice may explain the better outcome of corneal transplantations in old recipients, supporting the concept that lymphangiogenesis is an important risk-factor for corneal transplant rejection.     

Corneal lymphangiogenesis: evidence,mechanisms, and implications for corneal transplant immunology

PURPOSE: The normal cornea is devoid of blood and lymphatic vessels but can become vascularized secondary to a variety of corneal diseases and surgical manipulations. Whereas corneal (hem)angiogenesis, i.e., the outgrowth of new blood vessels from preexisting limbal vessels, is obvious both clinically and histologically, proof of associated corneal lymphangiogenesis has long been hampered by invisibility and lack of specific markers. This has changed with the recent discovery of the lymphatic endothelial markers vascular endothelial growth factor receptor 3, LYVE-1 (a lymphatic endothelium-specific hyaluronan receptor), Prox 1, and Podoplanin. METHODS: We herein summarize the current evidence for lymphangiogenesis in the cornea and describe its molecular markers and mediators. Furthermore, the pathophysiologic implications of corneal lymphangiogenesis for corneal transplant immunology are discussed. RESULTS: Whereas corneal angiogenesis in vascularized high-risk beds provides a route of entry for immune effector cells to the graft, lymphangiogenesis enables the exit of antigen-presenting cells and antigenic material from the graft to regional lymph nodes, thus inducing alloimmunization and subsequent graft rejection. CONCLUSIONS: Antilymphangiogenic strategies may improve transplant survival both in the high- and low-risk setting of corneal transplantation.     

高危角膜移植大鼠排斥反应及VEGF-C/D表达的研究

目的：探讨鼠角膜碱烧伤后VEGF-C/D的表达和意义,以及新生淋巴管在高危角膜移植后排斥反应中的作用。
　　方法：制作角膜碱烧伤模型,取不同时间段角膜进行电镜观察,观察角膜血管化情况;采用免疫组织化学方法检测l、3、5、7、14、28 d角膜组织VEGF-C/D及VEGFR-3的表达;并在角膜中仅有血管(A组),同时存在新生血管及新生淋巴管(B组),新生淋巴管消退期(C组),角膜新生血管消退期(D组)以及正常组(N 组)进行穿透性角膜移植,比较不同角膜植片的排斥反应指数( rejection index, RI)值及存活时间。
　　结果：电镜观察发现,在碱烧伤后第7d时鼠角膜出现新生血管,未出现新生淋巴管,在碱烧伤2 wk时出现新生血管的同时出现淋巴管,5wk时无明显的新生淋巴管,8wk时新生血管逐渐消退;大鼠角膜组织中 VEGF-C/D 及VEGFR-3的表达从第3 d开始明显上升,并于第5 d达到最高峰。角膜移植后N、A、B、C、D组的植片平均存活时间分别为14.25±0.62、9.35±1.02、5.06±1.13、8.71±0.83、9.44±1.05d。组间比较发现,B组植片平均存活时间显著性缩短(P<0.05),A、C、D的存活时间均显著性延长(P<0.05)。
　　结论：角膜碱烧伤后存在VEGF-C/D及VEGFR-3的高表达,而且新生淋巴管能加速高危角膜移植后的免疫排斥反应。     

Bevacizumab as a potent inhibitor of inflammatory corneal angiogenesis and lymphangiogenesis

PURPOSE: To analyze whether bevacizumab can inhibit inflammatory angiogenesis and lymphangiogenesis in the cornea. Bevacizumab (Avastin; Roche, Welwyn Garden City, UK) is a recombinant, humanized, monoclonal antibody against VEGF-A that has been approved by the U.S. Food and Drug Administration for the treatment of colon carcinomas. METHODS: The mouse model of suture-induced corneal neovascularization was used to assess the antihemangiogenic and antilymphangiogenic effect of bevacizumab by systemic and topical application. Corneal flatmounts were stained with LYVE-1 as a specific lymphatic vascular endothelial marker and CD31 as a pan-endothelial marker, and blood and lymph vascularized areas were analyzed morphometrically. The inhibitory effect of bevacizumab on lymphatic endothelial cells (LECs) was analyzed with a colorimetric (BrdU) proliferation ELISA. The binding ability of bevacizumab to murine VEGF-A was analyzed by Western blot, ELISA, and surface plasmon resonance. RESULTS: The systemic and topical applications of bevacizumab significantly inhibited the outgrowth of blood (P < 0.006 and P < 0.0001, respectively) and lymphatic (P < 0.002 and P < 0.0001, respectively) vessels. Inhibition of the proliferation of LECs was also significant (P < 0.0001). Western blot analysis, ELISA, and the surface plasmon resonance assay showed that bevacizumab binds murine VEGF-A. CONCLUSIONS: Topical or systemic application of bevacizumab inhibits both inflammation-induced angiogenesis and lymphangiogenesis in the cornea. This finding suggests an important role of VEGF-A in corneal lymphangiogenesis. Bevacizumab may be useful in preventing immune rejections after penetrating keratoplasty or tumor metastasis via lymphatic vessels.