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.     

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.     

Short- and long-term corneal vascular effects of tafluprost eye drops

Background

Prostaglandin analogs are first line therapy in the treatment of glaucoma, but also display side effects during ocular inflammation. In this context, the potential side effects of prostaglandin analogs on the normally avascular cornea, the main application route for eye drops, are so far not fully defined. Therefore, the aim of this study was to evaluate the vascular effects of the prostaglandin analog tafluprost on the healthy and inflamed cornea.

Methods

For in vitro studies, blood and lymphatic endothelial cells were treated with tafluprost; cell proliferation was assessed after 48 h. For long-term in vivo studies under healthy conditions, naïve corneas of BALB/c mice were treated with tafluprost eye drops for 4 weeks. For short-term in vivo studies under inflammatory conditions, corneal inflammation was induced by suture placement; mice then received tafluprost eye drops for 1 week. Afterwards, corneas were stained with CD31 as panendothelial and LYVE-1 as lymphendothelial (and macrophage) marker.

Results

In vitro, tafluprost did not alter blood or lymphatic endothelial cell proliferation. In vivo, there was no change in limbal blood or lymphatic vessel anatomy after long-term treatment with tafluprost. Short-term treatment with tafluprost under inflammatory conditions did not influence the recruitment of LYVE-1 positive macrophages into the cornea. Moreover, treatment of inflamed corneas with tafluprost did not significantly influence corneal hem- and lymphangiogenesis.

Conclusions

Tafluprost does not affect blood and lymphatic vessel growth, neither under resting nor under inflammatory conditions. These findings suggest a safe vascular profile of tafluprost eye drops at the inflammatory neovascularized cornea.     

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.     

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。     

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.     

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.     

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。     

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.     

Inhibition of hemangiogenesis and lymphangiogenesis after normal-risk corneal transplantation by neutralizing VEGF promotes graft survival

PURPOSE: To evaluate the occurrence and time course of hem- and lymphangiogenesis after normal-risk corneal transplantation in the mouse model and to test whether pharmacologic strategies inhibiting both processes improve long-term graft survival. METHODS: Normal-risk allogeneic (C57BL/6 to BALB/c) and syngeneic (BALB/c to BALB/c) corneal transplantations were performed and occurrence and time course of hem- and lymphangiogenesis after keratoplasty was observed, by using double immunofluorescence of corneal flatmounts (with CD31 as a panendothelial and LYVE-1 as a lymphatic vascular endothelium-specific marker). A molecular trap designed to eliminate VEGF-A (VEGF Trap(R1R2); 12.5 mg/kg) was tested for its ability to inhibit both processes after keratoplasty and to promote long-term graft survival (intraperitoneal injections on the day of surgery and 3, 7, and 14 days later). RESULTS: No blood or lymph vessels were detectable immediately after normal-risk transplantation in either donor or host cornea, but hem- and lymphangiogenesis were clearly visible at day 3 after transplantation. Both vessel types reached donor tissue at 1 week after allografting and similarly after syngeneic grafting. Early postoperative trapping of VEGF-A significantly reduced both hem- and lymphangiogenesis and significantly improved long-term graft survival (78% vs. 40%; P < 0.05). CONCLUSIONS: There is concurrent, VEGF-A-dependent hem- and lymphangiogenesis after normal-risk keratoplasty within the preoperatively avascular recipient bed. Inhibition of hem- and lymphangiogenesis (afferent and efferent arm of an immune response) after normal-risk corneal transplantation improves long-term graft survival, establishing early postoperative hem- and lymphangiogenesis as novel risk factors for graft rejection even in low-risk eyes.     

Absence of blood and lymphatic vessels in the developing human cornea

PURPOSE: The normal human cornea is devoid of both blood and lymphatic vessels and actively maintains this avascularity (corneal angiogenic privilege). Whether and when corneal angiogenic privilege is achieved during development is unknown. METHODS: This study analyzed whether the cornea is primarily devoid of both blood and lymphatic vessels during intrauterine development or whether secondary regression of pre-existing vessels occurs before delivery. Indirect double immunohistochemistry was performed on 4-microm serial pupil-optic disc sections of paraffin-embedded human eyes stillborn at gestational ages of 17 to 41 weeks with antibodies against von Willebrand factor (vWF; factor VIII-associated antigen) as a panendothelial marker and with antibodies against lymphatic vessel endothelial hyaluronate receptor 1 (LYVE1) as a marker specific for lymphatic vascular endothelium. RESULTS: Human corneas were devoid of both vWF+++/LYVE-1(-) blood vessels and vWF+/LYVE-1+++ lymphatic vessels at all time-points analyzed. In contrast, there were numerous blood and lymphatic vessels detectable in the adjacent conjunctiva. CONCLUSION: The normal human cornea is primarily avascular and devoid of both blood and lymphatic vessels. Corneal angiogenic privilege is already achieved very early during fetal intrauterine development. This suggests early and strong expression of both antiangiogenic and antilymphangiogenic factors in the human cornea during development.     

Novel expression and characterization of lymphatic vessel endothelial hyaluronate receptor 1 (LYVE-1) by conjunctival cells

PURPOSE: Lymphatic vessel endothelial hyaluronic acid receptor (LYVE-1) is a newly discovered lymphatic-specific marker. To date, there is no report of its expression on conjunctival cells. The purpose of this study was to investigate the expression of LYVE-1 in normal conjunctiva, to phenotype LYVE-1+ cells, and to study changes in their expression levels during corneal inflammation. METHODS: Flat-mounted conjunctivae or cross sections of eyeballs were harvested from BALB/c mice (6-8 weeks of age) for immunofluorescent confocal microscopic studies. RESULTS: The data demonstrate, for the first time, that in addition to its expression on lymphatic vessels, LYVE-1 was expressed on CD45+, CD11b+, and CD31- conjunctival cells, indicating a bone-marrow-derived monocytic lineage. Surprisingly, the number of cells that expressed LYVE-1 decreased during corneal inflammation, in conjunction with ingrowth of lymphatics into the cornea. CONCLUSIONS: A new population of monocytic cells has been found to express LYVE-1 in normal conjunctiva. These cells that normally express LYVE-1 may act as a reservoir for lymphangiogenesis and cell recruitment when the immune system is challenged.     

鼠角膜碱烧伤后新生淋巴管上血管内皮生长因子受体-3的表达和意义

目的探讨鼠角膜碱烧伤后血管内皮生长因子受体-3(VEGFR-3)在新生淋巴管上的表达和意义,进一步证实角膜新生淋巴管的存在。方法在大鼠角膜上制作碱烧伤模型,应用免疫组化法检测第3、5、7天角膜中VEGFR-3蛋白的表达。电镜观测伤后第5、7、10、14天角膜新生淋巴管的情况。结果碱烧伤后,大鼠角膜组织中VEGFR-3表达从第3天开始明显上升,并于第5天达到最高峰,伤后第14天出现新生淋巴管。结论角膜碱烧伤后有新生淋巴管长入,VEGFR-3可能成为抑制角膜新生淋巴管的一个新靶点。     

Blockade of VEGFR3-signalling specifically inhibits lymphangiogenesis in inflammatory corneal neovascularisation

Purpose Inflammatory corneal hem- and lymphangiogenesis occurring both prior to as well as after penetrating keratoplasty significantly increase the risk for subsequent immune rejections. The purpose of this study was to analyze whether the blocking anti-VEGFR3 antibody mF4-31C1 is able to inhibit the outgrowth of pathologic new lymphatic vessels in a mouse model of suture-induced, inflammatory corneal neovascularisation, and whether this antibody specifically inhibits lymphangiogenesis without affecting hemangiogenesis. Methods Three interrupted 11-0 nylon sutures were placed into the corneal stroma of BALB/c mice (6 weeks old) and left in place for 7 days to induce neovascularisation. The treatment group (n = 9) received the anti-VEGFR3 antibody mF4-31C1 intraperitoneally on the day of surgery and 3 days later (0.5 mg/mouse). Control mice received an equal amount of control IgG solution. For immunohistochemistry, corneal flat mounts were stained with LYVE-1 as a specific lymphatic vascular endothelial marker and with CD31 as panendothelial marker. Morphometry was performed with the image analysis software analySIS^B (Soft Imaging Systems GmbH, Münster, Germany). To improve the objectivity and precision of the morphometrical analysis, we established a modified method using grey filter sampling on monochromatic pictures. Results The mF4-31C1 antibody-treated mice displayed nearly complete inhibition of lymphangiogenesis compared with IgG controls (p < 0.006). In contrast, there was no significant inhibitory effect observed with respect to blood vessel growth (p > 0.05). Conclusions Inflammatory corneal lymphangiogenesis seems to depend on VEGFR3-signalling. By blocking this receptor the ingrowths of lymphatic vessels can be inhibited almost completely, and specifically without affecting hemangiogenesis. This may open new treatment options to promote (corneal) graft survival without affecting hemangiogenesis. Interdisciplinary Centre for Clinical Research (IZKF) Erlangen (A9), ELAN Funds Erlangen, DFG (Cu 47/2-1).     

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。     

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.     

Improved semiautomatic method for morphometry of angiogenesis and lymphangiogenesis in corneal flatmounts

Purpose of the study was to describe a novel semiautomatic, quantitative image analysis method based on threshold analysis for morphometry of corneal (lymph)angiogenesis and to test its validity, reliability and objectivity. Murine corneas were vascularized by using a suture-induced neovascularization assay. For immunohistochemistry, flatmounts of the vascularized corneas were stained with LYVE-1 as a specific lymphatic vascular endothelial marker and with CD31 as panendothelial marker. Morphometry of corneal hem and lymphangiogenesis was performed semi-automatically on digital images using image analysis software. Data were analyzed by a paired t-test, intraclass-correlation and systemic difference analysis compared to a manual method. The semiautomatic method based on threshold analysis was more valid in measuring the area covered by blood or lymphatic vessels. Both methods had a good reproducibility with respect to both vessel types (blood vessels: manual: 0.969, semiautomatic: 0.982; lymphatic vessels: manual: 0.951, semiautomatic: 0.966), whereas the systemic difference was significant for both groups measuring lymphatic vessels (manual: p < 0.003; semiautomatic: p < 0.035) and for the manual method measuring blood vessels (manual: p < 0.0001; semiautomatic: p < 0.419). The new semiautomatic morphometry method based on threshold analysis provides higher accuracy, is more valid than and at least as reproducible and objective as the manual outlining method. Therefore the semiautomatic method can be used to detect even small effects on hem and lymphangiogenesis in murine corneal flatmounts with greater precision.     

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.     

Relationship between angiogenesis and lymphangiogenesis in recurrent pterygium

AIM: To examine the relationship between angiogenesis and lymphangigenesis in recurrent pterygia. METHODS: Tissues from 34 excised recurrent pterygia (including 12 Grade 1, 10 Grade 2, and 12 Grade 3) were involved in the study and tissues from 7 nasal epibulbar conjunctivae segments were used as controls. Sections from each pterygium were immunostained with CD₃₁ and LYVE-1 monoclonal antibodies to evaluate lymphatic microvessel density (LMVD) and blood microvessel density (BMVD), and the relationship between LMVD and BMVD in the pterygium was examined. RESULTS: There was a large number of CD₃₁⁽⁺⁾LYVE-1^(-) blood vessels but only a few CD₃₁⁽⁺⁾LYVE-1⁽⁺⁾ lymphatic vessels in grades 1 and 2 pterygium. However, lymphatic vessels were dramatically increased in grade 3 pterygium. LMVD correlated closely with BMVD in all pterygia, including grades 1, 2 and 3 peterygium patients (all P values <0.01). Although both the density of blood and lymphatic vessels increased in recurrent pterygia, lymphatic vessels developed much faster than blood vessels, especially in grade 3 pterygia. CONCLUSION: There is a significant but not parallel relationship between angiogenesis and lymphangiogenesis in recurrent pterygium. The outgrowth of blood and lymphatic vessels provide evidence that immunological mechanism may play a role in the development and recurrence of pterygium.