角膜移植后患者角膜新生淋巴管与新生血管和炎症的关联

目的:研究角膜移植后角膜新生淋巴管与新生血管和炎症的关联。方法:人角膜取自行二次角膜移植的患者19例。淋巴内皮细胞受体(lymphatic vessel endothelial hyaluronan receptor,LYVE-1)和内皮细胞黏附因子-1(platelet endothelial celladhesion 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,进行淋巴管计数(lymphatic ves-sels counting,LVC)和血管计数(blood vessels counting,BVC),比较BVC、炎症指数(inflammation index,IF)、移植历史(transplantation history,TH)与LVC之间的关联。结果:角膜移植后BVC,IF与LVC间均呈显著性正相关,而TH与LVC间呈显著性负相关。角膜移植后新生淋巴管、血管、眼表炎症间大致成平行发展,新生淋巴管最先退化,其次是眼表炎症,新生血管最后消退。结论:人角膜移植后角膜新生淋巴管与新生血管、眼表炎症之间存在着极为密切的关联。     

碱烧伤后角膜新生淋巴管的实验研究

目的动态检测大鼠角膜碱烧伤后的角膜新生淋巴管和血管的变化,并阐明二者之间的关联。方法制备大鼠角膜碱烧伤模型,于碱烧伤后1周、2周行角膜组织电镜检查,检测角膜新生淋巴管与新生血管。5’核苷酸酶-碱性磷酸酶(5’-nase-alkaline phos-phatase,5’-NA-ALP)双重酶组织化学染色及全角膜免疫荧光染色分别检测碱烧伤后6h、1d、3d、1周、2周、3周、4周、5周、6周、7周、8周的角膜新生淋巴管和新生血管的动态变化,并进行淋巴管计数(lymphatic vessels counting,LVC)和血管计数(blood vessels counting,BVC)比较。结果电镜观察结果:角膜碱烧伤后1周,角膜基质层出现新生血管,未见淋巴管;碱烧伤后2周,新生血管和新生淋巴管均出现。酶组织化学染色结果显示:碱烧伤后6h有新生血管,1周时角膜基质层存在新生淋巴管,2周时LVC和BVC均达到高峰,分别为(16.41±1.00)个和(50.40±1.56)个;以后逐渐下降,5周时LVC为(0.33±0.50)个,BVC为(12.52±2.51)个;8周时均为0。碱烧伤后,新生淋巴管和新生血管呈显著性正...     

碱烧伤后角膜新生淋巴管与炎症反应指数间的关联

目的 探讨角膜碱烧伤后的角膜新生淋巴管与炎症反应指数间的关联.方法 实验研究.制备大鼠角膜碱烧伤模型.采用5'核苷酸酶-碱性磷酸酶(5'-NA-ALP)双重酶组织化学染色及全角膜免疫荧光法分别检测碱烧伤后1、3 d,1、2、3、4、5、6、7及8周的角膜新生淋巴管和血管的动态变化,并进行淋巴管计数(LVC)和血管计数(BVC).同时,于裂隙灯显微镜下观察角膜炎症反应的变化,记录炎症反应指数(IF),并比较LVC和IF之间的关联.11例人角膜取自碱烧伤后行角膜移植的11例患者.淋巴管内皮细胞受体(LYVE-1)免疫组织化学染色法标记人角膜中的新生淋巴管,LVC和IF之间的关联运用Pearson's相关分析,采用配对t检验比较角膜中存在淋巴管和不存在淋巴管的患者之间IF、炎性细胞计数、碱烧伤病史、年龄的差异.结果 碱烧伤后,角膜基质层存在着新生淋巴管.碱烧伤后3 d时出现角膜新生淋巴管,2周末达到高峰,5周末消退.新生淋巴管的出现滞后于炎症反应,但先于炎症反应和新生血管而消退.LVC与IF之间呈正相关(r=0.572,P＜0.01).11例患者中3例存在着角膜新生淋巴管.与另8例角膜中无新生淋巴管的患者相比,前者IF显著性升高(t=3.28,P＜0.05)、炎性细胞计数显著性增加(t=2.42,P＜0.05),年龄显著性下降(t=2.62,P＜0.05),而碱烧伤病史无显著性差异(t=1.28,P＞0.05).结论 角膜碱烧伤后有淋巴管生成,角膜新生淋巴管和炎症反应指数之间存在着密切的关联.     

碱烧伤人角膜组织新生淋巴管的检测

目的 探讨人角膜碱烧伤后新生淋巴管的生长情况及其影响因素.方法 回顾性系列病例研究.分析2005年1至12月期间在中山大学中山眼科中心因角膜碱烧伤住院手术治疗的22例(22只眼)患者.记录患眼的烧伤时间(IT)和损伤等级(ID),并测量炎性反应指数(II)和角膜新生血管的相对面积(BVA);应用双重酶组织化学、免疫组织化学染色及透射电镜检测角膜标本的新生淋巴管(LVC)和血管(BVC);采用苏木素-伊红染色检测多形核白细胞(PMN)浸润情况.结果 采用配对Student t检验、Pearson相关检验及Stepwise回归进行统计学分析.结果 22例患者观测结果 IT为(57.62±31.72)个月、ID为(12.00±2.76)分、II为(2.32±2.63)分、BVA为29.79%±18.61%、BVC为(14.45±9.29)个、LVC为(2.73±4.57)个、PMN为(13.45±13.09)个,其中7例(占32%,IT<64个月)同时存在角膜新生淋巴管(8.6±3.8)个和血管(22.3±11.1)个,LVC总数为60个,占发生新生淋巴管的角膜中总管腔数(BVC和LVC,378个)的16%.LVC与IT、ID、BVA、PMN、II的相关系数分别为-0.673、0.604、0.755、0.806、0.873,均P<0.05;进一步回顾分析得出LVC近似等于II和BVA分别和特定的常数相乘后取和所得的淋巴管指数(LI).透射电镜从微观上证实了碱烧伤人角膜组织中存在具有典型结构特征的新生淋巴管和炎性细胞浸润.结论 碱烧伤后部分角膜组织存在新生淋巴管,通过II和BVA可间接估计其发生情况.LI是一项评估碱烧伤角膜新生淋巴管的有用临床参数.(中华眼科杂志,2009,45:115-121)     

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.     

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。     

角膜新生淋巴管的研究进展

角膜新生淋巴管多见于感染、炎症、外伤或角膜移植术后.临床上,如何有效的防治角膜移植术后排斥反应是角膜移植手术成功的关键.随着近年来角膜新生淋巴管内皮特异性标记物及重要淋巴管生长因子的相继发现,人们对角膜新生淋巴管的生成机制及与角膜移植排斥反应的关系有了深入的研究,此文就新生淋巴管的生成及其在角膜移植排斥中的作用作一综述.     

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.     

Corneal lymphangiogenesis correlates closely with hemangiogenesis after keratoplasty

AIM: To examine the relationship between corneal lymphangiogenesis and hemangiogenesis after keratoplasty. · METHODS: Nineteen human corneas were obtained from 19 patients undergoing a second corneal transplantation in Zhongshan Ophthalmic Center in 2005. Blood and lymphatic vessels in human transplanted corneas were identified by lymphatic vessel endothelial receptor (LYVE-1) and platelet endothelial cell adhesion modecule-1 (PECAM-1) immunohi- stochemistry, and double enzyme-histochemistry; then the association of corneal blood vessel counting (BVC) with lymphatic vessel counting (LVC) was examined. · RESULTS: Corneal hemangiogenesis was present in 12 cases (63%), and lymphangiogenesis occurred in 5 cases (26%) human transplanted corneas. In addition, corneal lymphangiogenesis was only present in vascularized corneas. LVC was strongly and positively correlated with BVC(r=0.725, P <0.01). · CONCLUSION: Corneal lymphangiogenesis develops after keratoplasty and strongly associates with hemangiogenesis.     

Clinical and experimental research of corneal lymphangiogenesis after keratoplasty

The objective of this study is to provide further evidence that corneal lymphangiogenesis occurs after keratoplasty, and to explore the association of corneal hemangiogenesis, corneal inflammation and transplantation history with corneal lymphangiogenesis. Rat corneal lymphangiogenesis was examined by electron microscopy, lymphatic vessel endothelial receptor (LYVE-1) immunohistochemistry, and whole-mount immunofluorescence at 1, 3, 7, 10 and 14 days after corneal transplantation. Blood and lymphatic vessels in human transplanted corneas were identified by LYVE-1 and CD(31) immunohistochemistry, then the association between corneal blood vessel counting, inflammatory index and transplantation history with the lymphatic vessel counting was examined. The results showed that corneal lymphangiogenesis was present in all rat corneas and 26% of human transplanted corneas. Lymphatic vessel counting was significantly associated with blood vessel counting, inflammatory index and transplantation history (all p values <0.0001). We conclude that corneal lymphangiogenesis develops after keratoplasty, and is strongly associated with hemangiogenesis, inflammation and the history of transplantation.     

Lymphatic vessels in vascularized human corneas: immunohistochemical investigation using LYVE-1 and podoplanin

PURPOSE: To determine whether lymphatic vessels exist in vascularized human corneas, by using immunohistochemistry with novel markers for lymphatic endothelium. METHODS: Human corneas exhibiting neovascularization secondary to keratitis, transplant rejection, trauma, and limbal insufficiency (n = 21) were assessed for lymphatic vessel content by conventional transmission electron microscopy and by immunostaining and immunoelectron microscopy with antibodies specific for the lymphatic endothelial markers, lymphatic vessel endothelial hyaluronan receptor (LYVE-1) and the 38-kDa integral membrane glycoprotein podoplanin. In addition, corneas were stained for the lymphangiogenic growth factor VEGF-C, and its receptor VEGFR3 by immunohistochemistry and in situ RNA hybridization, respectively. RESULTS: Thin-walled, erythrocyte-free vessels staining with lymphatic markers (LYVE-1 and podoplanin) were found to constitute 8% of all vessels, to be more common in the early course of neovascularization, to be always associated with blood vessels and stromal inflammatory cells, and to correlate significantly with the degree of corneal hemangiogenesis (r = 0.6; P = 0.005). VEGF-C, VEGFR3, podoplanin, and LYVE-1 colocalized on the endothelial lining of lymphatic vessels. With immunogold labeling, LYVE-1 and podoplanin antigen were found on endothelial cells lining vessels with ultrastructural features of lymph vessels. CONCLUSIONS: Immunohistochemistry with novel lymph-endothelium markers and ultrastructural analyses indicate the existence of lymphatic vessels in vascularized human corneas. Human corneal lymphangiogenesis appears to be correlated with the degree of corneal hemangiogenesis and may at least partially be mediated by VEGF-C and its receptor VEGFR3.     

Topical Ranibizumab inhibits inflammatory corneal hem‐ and lymphangiogenesis

Purpose: Ranibizumab (Lucentis^®) is a Fab‐Fragment of a recombinant, humanized, monoclonal VEGF (anti‐vascular endothelial growth factor) antibody. This study analyzed the ability of topical Ranibizumab to inhibit lymphangiogenesis in addition to hemangiogenesis after acute corneal inflammation in vivo. In addition, the effect of Ranibizumab on the proliferation of human lymphatic endothelial cells (LECs) and blood endothelial cells (BECs) in vitro was studied. Methods: The inhibitory effect of Ranibizumab on LECs and BECs was studied in vitro using a proliferation enzyme‐linked immunosorbent assay (ELISA) assay. To study the in vivo effects of Ranibizumab, the mouse model of suture induced inflammatory corneal neovascularization was used. Study mice received topical Ranibizumab as eye drops. After 1 week excised corneas were stained with LYVE‐1 and CD31. Hemangiogenesis and lymphangiogenesis were analyzed morphometrically by using a semiautomatic method based on the image analyzing program Cell^F. Results: An antiproliferative effect of Ranibizumab was seen in vitro on both human BECs and LECs with a significance of p < 0.0001 and p < 0.0004, respectively. In vivo experiments showed that topical application of Ranibizumab significantly inhibits both hemangiogenesis (p = 0.0026) and lymphangiogenesis (p = 0.0026) in the cornea. Conclusion: Ranibizumab is a potent inhibitor of inflammatory corneal hemangiogenesis and lymphangiogenesis in vivo with a direct inhibitory effect on both endothelial cell types in vitro. This study for the first time demonstrates an inhibitory effect of Ranibizumab on lymphatic vessels which could have a wider range of clinical applications.     

Development of new lymphatic vessels in alkali‐burned corneas

Purpose: Corneal lymphangiogenesis provides an exit route for antigen‐presenting cells to regional lymph nodes, inducing immune response. The purpose of this study was to examine the development of corneal lymphatic vessels in alkali‐burned corneas. Methods: Corneal lymphatic vessels were examined by electron microscopy, 5′‐nase‐alkaline phosphatase (5′‐NA‐ALP) double enzyme‐histochemistry and whole mount immunofluorescence at 6 hr, 1 day, 3 days, and 1, 2, 3, 4, 5, 6, 7 and 8 weeks after rat corneal alkali injury. The expression of vascular endothelial growth factor‐C (VEGF‐C) protein and mRNA was examined 1, 3, 5, 7, 9, 11 and 14 days after the injury. Results: Corneal lymphangiogenesis developed 3 days after alkaline burns, reached its peak 2 weeks after the injury, decreased gradually and disappeared at the end of the fifth week. The expression of VEGF‐C in burned corneas increased dramatically on the third day but disappeared the 14th day after the injury. Conclusion: Corneal lymphatic vessels develop after alkaline burns and VEGF‐C may play an important role in corneal lymphangiogensis.     

Combined blockade of VEGFR-3 and VLA-1 markedly promotes high-risk corneal transplant survival

PURPOSE. High-risk corneal transplantation refers to grafting performed on inflamed and highly vascularized host beds. It represents a clinical dilemma because the rejection rate can be as high as 90%, irrespective of current treatment modalities. This study was conducted to investigate whether combined blockade of VEGFR-3 (vascular endothelial growth factor receptor-3) and VLA-1 (very late antigen-1) promotes high-risk transplant survival and how it correlates with corneal lymphangiogenesis and hemangiogenesis before and after transplantation. METHODS. High-risk corneal transplantation was performed between normal C57BL/6 (donor) and inflamed BALB/c (recipient) mice. The recipients were randomized to receive intraperitoneal injections of VEGFR-3 and VLA-1-neutralizing antibodies or their controls twice a week for up to 8 weeks after transplantation. Corneal grafts were evaluated by ophthalmic slit-lamp biomicroscopy and analyzed by Kaplan-Meier survival curve. Additionally, whole-mount corneas before and after transplantation were examined by immunofluorescent microscopic assays, and the correlation between lymphatic or blood vessel distribution and transplant outcome was analyzed. RESULTS. The combined blockade markedly promotes 90% survival of high-risk transplants. This strategy specifically modified host beds by selective inhibition of lymphangiogenesis but not hemangiogenesis. A strong correlation was also identified between high-risk transplant rejection and severe lymphatic invasion reaching the donor-graft border. CONCLUSIONS. These novel findings not only provide a new and potentially powerful strategy to promote high-risk transplant survival, they also confirm a critical role of high-degree lymphangiogenesis in mediating high-risk transplant rejection. Results from this study may also shed new light on our understanding and management of other lymphatic- and immune-related diseases in general.