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).     

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.     

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.     

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.     

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.     

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.     

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.     

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。     

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.     

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.     

Age alters ADPase positive dendritic (Langerhans) cell response to P. aeruginosa ocular challenge

The morphology, distribution and quantitation of dendritic (Langerhans) cells (LC) was determined by analysis of ADPase stained epithelial flat mounts from 6-8 week young adult (resistant) and 24 month old (susceptible) aged mice before and after experimental infection with P. aeruginosa topically applied to the scarified cornea. The contralateral eye (controls) was also scarified and phosphate buffered saline applied similarly. This study has examined the changes in ADPase positive cell populations of the conjunctival limbal epithelium and corneal epithelium of naturally resistant mice (Swiss-Webster and CD2F1) following corneal infection with Pseudomonas aeruginosa at two different ages, young adult (8 week old) and aged (24 month old). The young adult mice recover from their infection and restore corneal clarity while the aged mice have extensive ocular destruction and corneal scarring. Conjunctival limbal dendritic cell numbers in young adult mice were found to be significantly increased at day seven post infection and then returned to baseline levels. In contrast, conjunctival limbal dendritic cell numbers in aged mice were found to increase slowly and to peak at fourteen days after infection. Other differences between the two ages (young adult and aged) included an initial increase in dendritic cells five hours post infection in the young adult groups and an initial decrease at five hours in the aged groups of mice.     

Corneal lymphangiogenesis in dry eye disease is regulated by substance P/neurokinin-1 receptor system through controlling expression of vascular endothelial growth factor receptor 3

PurposeTo evaluate the role of substance P (SP)/neurokinin-1 receptor (NK1R) system in the regulation of pathologic corneal lymphangiogenesis in dry eye disease (DED).MethodsImmunocytochemistry, angiogenesis assay, and Western blot analysis of human dermal lymphatic endothelial cells (HDLECs) were conducted to assess the involvement of SP/NK1R system in lymphangiogenesis. DED was induced in wild-type C57BL/6 J mice using controlled-environment chamber without scopolamine. Immunohistochemistry, corneal fluorescein staining, and phenol red thread test were used to evaluate the effect of SP signaling blockade in the corneal lymphangiogenesis. The expression of lymphangiogenic factors in the corneal and conjunctival tissues of DED mouse model was quantified by real-time polymerase chain reaction.ResultsNK1R expression and pro-lymphangiogenic property of SP/NK1R system in HDLECs were confirmed by Western blot analysis and angiogenesis assay. Blockade of SP signaling with L733,060, an antagonist of NK1R, or NK1R-targeted siRNA significantly inhibited lymphangiogenesis and expression of vascular endothelial growth factor (VEGF) receptor 3 stimulated by SP in HDLECs. NK1R antagonist also suppressed pathological corneal lymphangiogenesis and ameliorated the clinical signs of dry eye in vivo. Furthermore, NK1R antagonist effectively suppressed the lymphangiogenic factors, including VEGF-C, VEGF-D, and VEGF receptor 3 in the corneal and conjunctival tissues of DED.ConclusionsSP/NK1R system promotes lymphangiogenesis in vitro and NK1R antagonism suppresses pathologic corneal lymphangiogenesis in DED in vivo.     

新生淋巴管与角膜移植排斥的研究进展

角膜新生淋巴管构成了角膜免疫反应的传入弧,在免疫反应中发挥了重要作用。由于新生淋巴管的出现破坏了免疫机制使角膜移植术后排斥反应发生率升高。随着淋巴管内皮标记物的相继出现和对淋巴管生成因子的研究深入,众多学者对角膜淋巴管在角膜免疫排斥反应机制、治疗和预防等方面进行了大量研究,通过抑制新生淋巴管提高植片存活率。     

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.     

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。     

Inflammatory corneal (lymph)angiogenesis is blocked by VEGFR-tyrosine kinase inhibitor ZK 261991, resulting in improved graft survival after corneal transplantation

PURPOSE: To analyze whether tyrosine kinase inhibitors blocking VEGF receptors (PTK787/ZK222584 [PTK/ZK] and ZK261991 [ZK991]) can inhibit not only hemangiogenesis but also lymphangiogenesis and whether treatment with tyrosine kinase inhibitors after corneal transplantation can improve graft survival. METHODS: Inflammatory corneal neovascularization was induced by corneal suture placement. One treatment group received PTK/ZK, and the other treatment group received ZK991. Corneas were analyzed histomorphometrically for pathologic corneal hemangiogenesis and lymphangiogenesis. The inhibitory effect of tyrosine kinase inhibitors on lymphatic endothelial cells (LECs) in vitro was analyzed with a colorimetric (BrdU) proliferation ELISA. Low-risk allogeneic (C57Bl/6 to BALB/c) corneal transplantations were performed; the treatment group received ZK991, and grafts were graded for rejection (for 8 weeks). RESULTS: Treatment with tyrosine kinase inhibitors resulted in a significant reduction of hemangiogenesis (PTK/ZK by 30%, P < 0.001; ZK991 by 53%, P < 0.001) and lymphangiogenesis (PTK/ZK by 70%, P < 0.001; ZK991 by 71%, P < 0.001) in vivo. Inhibition of proliferation of LECs in vitro was also significant and dose dependent (PTK/ZK, P < 0.001; ZK991, P < 0.001). Comparing the survival proportions after corneal transplantation, treatment with ZK991 significantly improved graft survival (68% vs. 33%; P < 0.02). CONCLUSIONS: Tyrosine kinase inhibitors blocking VEGF receptors are potent inhibitors not only of inflammatory corneal hemangiogenesis but also lymphangiogenesis in vivo. Tyrosine kinase inhibitors seem to have the ability to restrain the formation of the afferent and efferent arm of the immune reflex arc and are therefore able to promote graft survival after corneal transplantation.     

Change with age in limbal conjunctival epithelial Langerhans cells

The distribution of Langerhans cells (LC) in corneal and conjunctival epithelial flat mounts obtained from outbred albino Swiss-Webster mice of various ages was examined by histochemical methods. The LC stain positively with ATPase and were negative to DOPA-oxidase. Mouse pup limbal conjunctival epithelium showed one or two LC/mm2. LC began to significantly increase in number by day 31 and ranged from 150-205/mm2 in 7-48 week old mice. A slight decrease in LC was seen in 12 and 24 month old mice.     

培养角膜缘上皮细胞深低温保存后异体移植实验研究

目的深低温保存培养的角膜缘上皮细胞,复苏后进行异体移植实验,为临床应用提供实验依据。方法将浅层角膜基质载体上培养的角膜缘上皮细胞深低温保存,同时制作兔角膜缘干细胞损伤模型,分别用新鲜培养的角膜缘上皮细胞和冷冻复苏后培养的细胞及角膜基质进行异体移植实验。结果角膜缘上皮细胞能成功地培养于载体上,将培养细胞保存于-196℃的液氮罐中二月,可见深低温保存前后细胞的形态结构及生物学特性无显著差异。异体移植实验显示：新鲜培养的细胞及深低温保存后的细胞异体移植后,模型兔角膜表面逐渐正常上皮化,而角膜基质组异体移植后,角膜表面仍结膜化并可见大量新生血管生长,各项检测结果与培养细胞组相比差异显著（P〈0．05）。结论以浅层角膜基质为载体培养的角膜缘上皮细胞深低温保存后仍具有上皮细胞特性并具有高增殖能力,可用以进行异体移植治疗角膜缘干细胞缺陷的角膜病。