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.     

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。     

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。     

Endothelin-1 and ETA/ETB receptor protein and mRNA: expression in normal and vascularized human corneas

PURPOSE: Neovascularization of the cornea causes blindness and increases the risk of immune rejections after keratoplasty. The purpose of this study was to investigate involvement of the potent angiogenic growth factor endothelin (ET)-1 and its receptors, ETA and ETB, in corneal neovascularization. METHODS: ET-1, ETA, and ETB receptor protein expression was evaluated in nonvascularized and vascularized human corneas by immunohistochemistry. Epithelial ET-1 protein expression of both groups was compared using a semiquantitative scoring system. Double immunofluorescence was used to colocalize ETA and ETB receptor with CD31. In situ hybridization and immunoelectron microscopy analyzed ET-1 and its receptors in normal and vascularized corneas. RESULTS: Nonvascularized corneas displayed ET-1 and ETA/ETB receptor protein and mRNA in epithelial and some corneal endothelial cells. ETA more than ETB receptors were expressed on some keratocytes. In vascularized corneas, ET-1 and ETA/ETB receptor expression was found in the endothelial lining of new blood vessels (as shown by CD31-colocalization). ET-1 protein expression was significantly increased in the epithelium of vascularized corneas (P < 0.001). Immunogold localized ET-1 and its receptors to the nuclear/perinuclear space and to the luminal side of endothelial cells of new blood vessels. CONCLUSIONS: In corneal neovascularization, ET-1 protein and mRNA expression is upregulated in epithelial cells. Together with ET-1, ETA, and ETB receptor expression on endothelial cells of ingrown new blood vessels, this points to an involvement of ET-1 and its receptors in corneal angiogenesis. As potent ETA and ETB receptors are available, the endothelin system may represent an additional target for corneal antiangiogenic therapy.     

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.     

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.     

Orbital lymphangioma with positive immunohistochemistry of lymphatic endothelial markers (vascular endothelial growth factor receptor 3 and podoplanin)

BACKGROUND: Existence of true orbital lymphangiomas has been questioned in recent years. Therefore an orbital lymphangioma was analyzed with two new specific markers of lymphatic endothelium. METHODS: Case-report with clinicopathological, immunohistochemical, and ultrastructural findings. A 25-year-old man presented with recurrent lower lid "hematomas" and a pea-sized tumor palpable in the left lower lid. Magnetic resonance imaging showed an inferonasally located orbital tumor which extended to the posterior pole of the eye. The highly vascularized tumor was excised by medial orbitotomy. RESULTS: Histopathologically, the mass consisted of large, erythrocyte-filled cavernous vessels without evidence of smooth muscle cells or pericytes surrounding them. Numerous lymph follicles and small arterioles were scattered between them. Immunohistochemically, endothelial cells lining the lumina of the cavernous vessels were partly positive for podoplanin and vascular endothelial growth factor receptor 3 (flt-4), two markers of lymphatic endothelium. These markers did not react with endothelial cells lining the arterioles. Ultrastructurally, cavernous vessels displayed features characteristic of lymphatic vessels, and the smaller vessels demonstrated signs of arterioles. CONCLUSION: Ultrastructural analysis and immunohistochemistry using two new markers of lymphatic endothelium suggest a lymphatic nature of large vessels in an orbital lymphangioma. A greater series of vascular orbital tumors must be studied with these new lymph endothelial markers to confirm the existence of true orbital lymphangiomas and to analyze different profiles of lymph endothelial marker expression.     

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.     

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.     

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.     

LYVE-1-positive macrophages are present in normal murine eyes

PURPOSE: A functioning lymphatic system is necessary not only to permit the organism to mount a rapid and effective immune response but, even more so, to maintain tissue fluid homeostasis. However, no clear evidence of lymphatic vessels draining intraocular and orbital tissues--retina, choroid, sclera, and extraocular muscles--exists. METHODS: Ocular tissue flatmounts from normal or enhanced green fluorescence protein (EGFP) chimeric mice were immunostained for lymphatic endothelium hyaluronan receptor (LYVE-1, a routinely used lymphatic endothelial marker), podoplanin, Flt4/VEGFR3, Sca-1, CD11b, or F4/80 and were observed by confocal microscopy. Single-cell suspensions from ocular tissues were also prepared and were analyzed by flow cytometry. RESULTS: Lymphatic vessels were detected in the posterior regions of the extraocular muscles and the connective tissues of the extraocular muscle cones in the normal mouse. No typical lymphatic vessels were found within the eye. A large population of LYVE-1(+) nonendothelial cells, distributed as single cells, was detected in all ocular tissues except the central cornea. These cells also express another lymphatic endothelial cell marker, Flt4/VEGFR3, but not podoplanin, and they have hyaluronan-binding ability. Bone marrow chimerism studies indicated that the LYVE-1(+) cell populations are bone marrow derived and have a slow turnover in ocular tissues (3-6 months). Phenotype analysis revealed that nonendothelial LYVE-1(+)cells in the sclera, choroid, and iris included CD11b(+)F4/80(+) macrophages, CD11b(+)F4/80(-) macrophages, and CD11b(-)F4/80(-) bone marrow-derived cells. All LYVE-1(+) cells in the retina were CD11b(+)F4/80(+) macrophages. Cells in the limbus and the iris root also express Sca-1, suggesting that they are hematopoietic lymphatic vessel progenitor cells. CONCLUSIONS: These observations suggest that a lymphatic system exists for the transport of immune cells and fluids from the posterior segment of the eye, that ocular tissues are rich in bone marrow-derived LYVE-1(+) macrophages under normal physiological conditions, and that a subpopulation of these cells may represent resident precursor cells necessary for the de novo formation of ocular/orbital lymphatic vessels in pathologic conditions.     

Expression of vascular endothelial growth factor and its receptors in inflamed and vascularized human corneas

PURPOSE: To help further define the possible role of vascular endothelial growth factor (VEGF) in the pathogenesis of corneal neovascularization, the expression of VEGF and of its receptors Flt-1 and Flk-1 was investigated in various inflammatory corneal diseases. METHODS: Polyclonal antibodies to VEGF and its receptors were used for immunohistochemical staining of frozen sections of 38 human corneas with various degrees of neovascularization and inflammation. In addition, a panel of monoclonal antibodies was used to characterize the composition of the inflammatory infiltrates and to confirm the presence of neovascularization. Furthermore, VEGF concentrations were determined in vascularized corneas using a sensitive enzyme-linked immunosorbent assay. RESULTS: VEGF was expressed by epithelial cells, by corneal endothelial cells, by vascular endothelial cells of limbal vessels and of newly formed vessels in the stroma, and weakly by keratocytes. Furthermore, VEGF expression was often markedly increased in inflamed corneas on epithelial cells and on vascular endothelial cells, particularly in the vicinity of macrophage infiltrates, and on fibroblasts in scar tissue. Correspondingly, VEGF concentrations were significantly higher in vascularized corneas compared with normal control corneas (P < 0.001). Expression of both VEGF receptors, Flt-1 and Flk-1, was increased on endothelial cells of newly formed vessels in the stroma of inflamed corneas compared with limbal vessels of normal control corneas. In addition, Flt-1 was also expressed by corneal endothelial cells and by macrophages, whereas Flk-1 expression was lacking. CONCLUSIONS: These results demonstrate that VEGF, Flt-1, and Flk-1 are strongly expressed in inflamed and vascularized human corneas and, thus, may play an important role in corneal neovascularization.     

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).     

Expression of tissue inhibitor of metalloproteinase-4 in normal human corneal cells and experimental corneal neovascularization

PURPOSE: To study the expression of TIMP-4 in cultured corneal cells and in corneal neovascularization. METHODS: Human limbo-corneal epithelial cells, fibroblasts, and endothelial cells were cultured in serum-free, PMA- or basic fibroblast growth factor (bFGF)-treated condition. Neovascularization in rat cornea was induced by suturing. The expression of TIMP-4 was examined by immunohistochemistry, Western blot and RT-PCR. RESULTS: TIMP-4 was constitutively expressed in cultured human corneal cells. The expression was only mildly enhanced after mitogen treatment. TIMP-4 immunoreactivity was predominantly expressed in normal rat corneal epithelium, and also in ingrowing blood vessels following suturing, which persisted up to day 28. Increased staining in corneal epithelium and blood vessels were also noted in vascularized human corneas. CONCLUSIONS: TIMP-4 is expressed in the cornea, which may play a role in modulating extracellular matrix remodeling associated with corneal wound healing and angiogenesis.     

A Case of Abnormal Lymphatic-Like Differentiation and Endothelial Progenitor Cell Activation in Neovascularization Associated with Hemi-Retinal Vein Occlusion

PurposePathological vascular differentiation in retinal vein occlusion (RVO)-related neovessel formation remains poorly characterized. The role of intraocular lymphatic-like differentiation or endothelial progenitor cell activity has not been studied in this disease.MethodsVitrectomy was performed in an eye with hemi-RVO; the neovessel membrane located at the optic nerve head was removed and subjected to immunohistochemistry. Characterization of the neovascular tissue was performed using hematoxylin and eosin, α-smooth muscle actin, and the pan-endothelial cell (EC) adhesion molecule CD31. The expression of lymphatic EC markers was studied by lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1), podoplanin (PDPN), and prospero-related homeobox protein 1 (Prox-1). Potential vascular stem/progenitor cells were identified by active cellular proliferation (Ki67) and expression of the stem cell marker CD117.ResultsThe specimen contained blood vessels lined by ECs and surrounded by pericytes. Immunoreactivity for LYVE-1 and Prox-1 was detected, with Prox-1 being more widely expressed in the active Ki67-positive lumen-lining cells. PDPN expression was instead found in the cells residing in the extravascular tissue. Expression of the stem cell markers CD117 and Ki67 suggested vascular endothelial progenitor cell activity.ConclusionsIntraocular lymphatic-like differentiation coupled with progenitor cell activation may be involved in the pathology of neovessel formation in ischemia-induced human hemi-RVO.Key Words: Hemi-retinal vein occlusion, Endothelial progenitor cell, Platelet endothelial cell adhesion molecule 1, Podoplanin, Prospero-related homeobox gene 1, Lymphatic endothelial cell, Lymphatic vessel endothelial hyaluronan receptor 1, Immunohistochemistry, Proliferative diabetic retinopathy, Vascular endothelial stem cell     

A quantitative electron microscopic study of growing corneal lymphatic vessels

Following silver nitrate injury to rabbit corneas, injection of carbon into the corneas showed the presence of new corneal lymphatic vessels in addition to the new corneal blood vessels.Quantitative measurements of several vessel parameters including endothelial thickness, area of vessel and lumen and circumference of vessel and lumen were made using electron micrographs of cross-sections of corneal lymphatic vessels of various ages. The thickness of the endothelium was found to decrease as the vessel matures, whereas both the area and the circumference of both vessel and lumen increase with age. This is in agreement with qualitative observations.Procedures for estimating the accurate age of vessels from their physical dimensions and for assessing the degree of tortuosity of the endothelial surfaces are presented and discussed.     

Activation of ocular surface mast cells promotes corneal neovascularization

PurposeMast cells, historically known for their effector function in the induction of allergic diseases, reside in all vascularized tissues of the body in particular proximity to blood and lymphatic vessels. As neighboring sentinel cells to blood vessels, mast cells have been associated with angiogenesis. Here we assess the direct contribution of mast cells to neovascularization at the ocular surface.MethodsCorneal neovascularization was induced by placing a single figure-of-eight intrastromal suture 1 mm from the limbus in mast cell-deficient (cKit^W-sh), C57BL/6, and Balb/c mice. Corneas were harvested at 6 h post-suture to quantify cKit⁺FcεR1⁺ mast cells using flow cytometry and tear wash was collected within 6 h to measure β-hexosaminidase and tryptase. Neovascularization was assessed using slit-lamp biomicroscope and immunohistochemistry analysis of corneas harvested on day 4 post-suture. To investigate the effects of mast cells on blood vessel growth, mast cells were co-cultured with vascular endothelial cells (VECs), and tube formation and proliferation of VECs were measured. 2% cromolyn was administered locally to inhibit mast cell activation in vivo.ResultsPlacement of corneal suture activates ocular surface mast cells, which infiltrate into the cornea adjacent to new vessels. Mast cell-deficient mice develop significantly fewer new vessels following suture placement. Mast cells directly promote VEC proliferation and tube formation, partly through secreting high levels of VEGF-A. Pharmacological inhibition of mast cell activation results in significantly less corneal neovascularization.ConclusionOur data demonstrate that ocular surface mast cells are critical to corneal neovascularization, suggesting mast cells as a potential therapeutic target in the treatment of corneal neovascularization.