实验性角膜新生血管形成的形态学研究

采用氢氧化钠灼伤角膜方法，诱导２０只兔眼角膜新生血管形成，并对其发生、发展过程进行裂隙灯显微镜、光镜和透射电镜观察。结果发现，伤后８小时，角膜缘组织已有中性粒细胞浸润等明显急性炎症变化；渗出于血管外的中性粒细胞胞质内吞噬溶酶体异常丰富。在伤后２天，角膜缘才出现新生血管芽，角膜新生血管周围可见中性粒细胞浸润及其崩解产物。提示白细胞，特别是中性粒细胞，参与角膜新生血管形成过程，并可能起介导作用。     

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.     

血管内皮钙黏蛋白对实验性角膜新生血管的作用及机制

目的 探讨血管内皮钙黏蛋白（VE-cadherin）在实验性角膜新生血管发生过程中的作用及机制。方法 动物实验研究。以碱烧伤诱导构建小鼠角膜新生血管模型;RT-PCR法检测VE-cadherin在碱烧伤角膜组织中的表达;碱烧伤1周后角膜局部应用阻断性抗小鼠VE-cadherin抗体进行干预,碱烧伤3周后大体观察角膜新生血管的发生情况以及全角膜铺片CD31荧光组织化学法检测角膜组织新生血管的面积;RT-PCR检测烧伤角膜组织内caspase-3 mRNA的表达;流式细胞术检测角膜组织血管内皮细胞凋亡数目;体外实验进一步验证VE-cadherin对血管内皮细胞（HREC）血管网形成的影响。采用成组t检验方法处理数据。结果 碱烧伤3周后,VE-cadherin抗体干预组角膜新生血管数目较对照组明显减少。RT-PCR结果显示VE-cadherin抗体干预组caspase-3基因水平表达增高;进一步流式细胞术检测结果显示VE-cadherin抗体干预后,角膜组织内凋亡的血管内皮细胞明显增多。体外实验证实VE-cadherin抗体明显抑制HREC细胞系血管网的形成。结论 VE-cadherin钙黏蛋白能通过维护细胞间黏附以及抑制细胞的凋亡等作用保护实验性角膜新生血管内皮细胞的生长,从而维持新生血管的发生发展。     

角膜干细胞与角膜表层新生血管

从干细胞角度探讨角膜碱烧伤后表层新生血管的成因。对大鼠角膜缘部进行碱性烧灼造成不同程度的损害，连续观察5周，结果，缘部基底细胞完全破坏者，角膜以新生血管化愈合，而保留缘部基底细胞者，角膜无新生血管化。本实验进一步证实了角膜干细胞缘部基底层定位的观点，提示干细胞对碱性烧伤后阻止角膜的新生血管化，并重建角膜表面的完整性具有重要意义。     

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.     

大鼠角膜缘上皮细胞培养与同体移植的实验研究

目的：观察以明胶为载体培养的角膜缘上皮细胞移植治疗角膜缘干细胞缺乏症的疗效。 方法：大鼠角膜缘上皮细胞在铺有明胶载体的细胞培养板上进行培养5d后,角膜上皮细胞移植术前24h用3H胸腺嘧啶核苷标记培养的角膜缘上皮细胞,培养标记的角膜缘上皮细胞对角膜缘干细胞缺乏的大鼠动物模型行角膜缘上皮细胞同体移植术,对移植术后角膜进行观察、病理学检查及同位素检测。 结果：大鼠角膜缘上皮细胞可以在明胶载体上培养、增殖、分化为密集角膜上皮细胞层;角膜缘上皮细胞移植术后角膜上皮完整、基质细胞浸润减轻、新生血管减少。病理学检查角膜缘及周边部上皮细胞为多层结构,角膜新生血管消失及基质中炎性细胞浸润减轻。角膜缘上皮细胞移植术后4wk受眼角膜仍可测到3H胸腺嘧啶核苷。 结论：角膜缘上皮细胞移植治疗角膜缘干细胞缺乏症可恢复角膜缘干细胞缺乏病变角膜上皮结构的完整性,减少角膜新生血管的形成,维持角膜缘干细胞的屏障功能,为角膜移值提供更好的条件。     

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.     

The role of PDGF receptor inhibitors and PI3-kinase signaling in the pathogenesis of corneal neovascularization

PURPOSE: Corneal neovascularization remains an unsolved therapeutic problem. Platelet-derived growth factor (PDGF) is directly linked to vessel formation and stabilization. This study was undertaken to elucidate the mechanisms by which PDGF exerts its effects on corneal angiogenesis. METHODS: Corneal neovascularization was induced in C57 mice by removal of the limbal epithelium. When mature vessels appeared after 7 days, mice were treated with the PDGF receptor-beta inhibitor AG 1296 or the phosphatidylinositol 3-kinase (PI3-K)-inhibitors wortmannin and LY294002, respectively, using an intraperitoneally implanted miniosmotic pump. At day 14 after scraping, corneas of treated and untreated (control) mice were dissected and immunostained with FITC-CD31 antibody for endothelial cells and with Cy3-SMA (smooth muscle actin) for pericytes. VEGF (vascular endothelial growth factor), ang1/2 (angiopoietin 1 and 2), and PDGF mRNA levels of treated and untreated corneas were determined by real-time RT-PCR. RESULTS: Mice treated with the PDGF inhibitor AG 1296 showed an inhibition of corneal neovascularization of 21.1% and a reduction of pericytes of 52% in the newly formed vessels compared with untreated animals. VEGF, ang1, ang2, and PDGF mRNA expression was reduced in the corneas of AG 1296-treated mice compared with the respective control. Treatment with the PI3-K inhibitors wortmannin and LY29002 had similar effects, inducing a decrease in corneal neovascularization and a reduction of VEGF, ang1, ang2, and PDGF mRNA levels. CONCLUSIONS: Inhibition of the PDGF signal pathway results in loss of pericytes and a reduction in vessel density in the neovascularized cornea that correlates with reduced expression of PDGF, ang1/2, and VEGF mRNA. Furthermore, PI3-K was shown to be involved in the regulation of VEGF, ang1, and PDGF, as the PI3-K inhibitors wortmannin or LY294002 had similar effects. Because PDGF is a known stimulus for PI3-K activation, it can be postulated that the observed decrease in VEGF, ang1/2, and PDGF mRNA levels on administration of the PDGF inhibitor is caused by the decreased activation of the PI3-K signaling cascade.     

Advances in corneal stem-cell transplantation in rabbits with severe ocular alkali burns

PURPOSE: To evaluate the efficacy of autologous corneal epithelial sheet implantation in restoring transparency of rabbit corneas severely injured by alkaline and the effect of photocoagulation in arresting corneal neovessel ingrowth. SETTING: Ophthalmology Department, School of Biomedical Sciences, Universidad Austral, Buenos Aires, Argentina. METHODS: Limbal stem-cell deficiency (LSCD) was induced in 14 rabbits by alkali burns. A limbal cell biopsy was done in the contralateral eye, and the cells were cultured on a fibroblast feeder layer grown on autologous clotted platelet-poor plasma or commercial fibrin for 21 days. Anterior keratectomy was followed by suturing corneal cell sheets over the stroma. If regrowth of vessels occurred, argon laser photocoagulation was applied to them. Rabbits were killed at 30, 60, 90, 180, and 360 days and the corneas processed for histopathology and inmunohistochemistry. RESULTS: A small (2.5 mm(2)) limbal biopsy achieved stem-cell replication in vitro. Corneal clarity and epithelial defects evolved with a trend toward improvement. There was a significant reduction in corneal neovascularization. Histology showed a multilayered stratified epithelium including several epithelial-like cells with clear cytoplasm in the deepest part. There were no signs of intraepithelial mucin cells on the implanted corneas. Immunohistochemical results showed expression of cytokeratins 3 and 12 in the central corneal epithelium and an absence of cytokeratin 19. CONCLUSIONS: Autologous limbal epithelial cell transplantation improved the corneal surface in eyes with LSCD. Photocoagulation of neovessel ingrowth was effective over the 1-year follow-up. Results may facilitate the application of this technique in patients.     

Morphological study of experimental corneal neovascularization after anterior uveal ischemia

We morphologically investigated what kinds of inflammatory cells infiltrate the corneoscleral limbus by light and electron microscopy and what manner of keratocytes and vascular endothelial cells appear at the corneal limbus, when the corneal edema was aggravated severely and clinical corneal new vessels were found initially, i.e. until three days after anterior segmental ischemia in rabbit eyes. The majority of infiltrating inflammatory cells were polymorphonuclear leukocytes and the others were lymphocytes and histiocytes etc. Peripheral keratocytes around limbal vessels were stimulated and transformed to fibroblastic cells as a result of anterior segment ischemia. Proliferating endothelial cells of comparatively minor limbal vessels invaded between corneal lamellar layers toward the corneal center. Polymorphonuclear leukocytes were often found near activated keratocytes proliferating vascular endothelial cells, hear and there. Consequently, it is considered that the stimulated polymorphonuclear leukocytes play an important role when corneal neovascularization occurs after anterior segmental ischemia.     

Macroscopic observations on corneal epithelial wound healing in the rabbit

A newly-developed macroscope was applied to observe the healing process of corneal epithelial wound in vivo. After removing epithelium of the central cornea, the changes of the corneal surface were observed with the macroscope and the findings were compared with histological examinations. At 12 hours after abrasion, areas unstained with Richardson's staining (R staining) appeared. In the histological section, a single layer of regenerating epithelial cells covered the same area. At 24 and 36 hours after abrasion, the epithelial defects became smaller but surrounding epithelium was rough and showed dot-like staining with R solution. By 2 days, the epithelial defects disappeared. On macroscopic observation, the central corneal surface showed a pavement-like appearance. Histology revealed that the regenerating epithelium still consisted of one or two layers. At 3 days, dot-like stainings were present only in the center and the corneal surface appeared considerably smooth. Histology also showed that regenerating epithelium became columnar and multilayered, thereby suggesting stratification. By 7 days, the abraded corneal surface had recovered its smooth appearance. Histologic sections also demonstrated that the epithelium had regained its normal structure. Thus, using this macroscope, findings suggesting the process of epithelial migration and proliferation could be observed.     

小鼠角膜碱烧伤后前炎性因子、趋化因子及其受体的表达

目的检测前炎性因子、趋化因子及其受体在小鼠角膜碱烧伤后表达的动态变化。方法建立小鼠碱烧伤模型，用RT—PCR法检测角膜碱烧伤后不同时间点前炎性因子、趋化因子及其受体mRNA的表达。结果RT-PCR检测显示，角膜碱烧伤后角膜组织中前炎性因子IL-1d、IL-1B及IL-6表达增强；调控单核细胞迁移的CCR1-CCL2、CCL3、CCIA、CCR2-CCL2以及调控中性粒细胞迁移的CXCR2-CXCL2表达增强。结论角膜碱烧伤后前炎性因子以及参与调控白细胞迁移的趋化因子及其受体明显上调，提示其参与角膜碱烧伤后白细胞向局部的迁移。     

Delayed inflammation-associated corneal neovascularization in MMP-2-deficient mice

Corneal neovascularization is a significant, sight-threatening, complication of many ocular surface disorders, but its underlying molecular background is still not fully understood. In the present study, we analysed the expression and role of matrix metalloproteinase-2 (MMP-2), a proteolytic enzyme suggested to regulate angiogenesis, in a mouse model of inflammation-related corneal neovascularization. A silk suture was placed centrally in pigmented mice corneas causing limbal vasculature to sprout, forming new vessels. Neovascularization progressed centrally involving the entire cornea after about 12 days. Histological analysis revealed vascularization of the corneal stroma accompanied by a marked inflammatory response. The neovascularization correlated with an increased expression of MMP-2 mRNA and protein that was mainly found in cells that stained positively for S100A4, a marker for activated keratocytes. MMP-2-deficient mice and wild-type mice were compared in a kinetic study, showing a statistically significant delay of neovascularization in MMP-2-deficient mice. These results implicate a role for MMP-2 in experimental inflammation-associated corneal neovascularization.     

Effects of salicylate and steroid on neutrophil migration and corneal blood vessel growth

Cauterization of the cornea results in emigration of neutrophils from the limbal blood vessels into the corneal tissue. Blood vessel proliferation follows, the stimulus for which is unknown. In this study, 0.1 M sodium salicylate drops administered topically to cauterized rat corneas over a 48-h period had an inhibitory effect on the migration of neutrophils from the limbal vessels 6 h after injury, but this was not maintained at 48 h. After 6 days of treatment, the salicylate had no effect on vessel growth into the cauterized rat cornea. Application of prednisolone disodium phosphate ointment to cauterized corneas also inhibited neutrophil migration at 6 h, but increased the extravascular neutrophils at 48 h. After 6 days of treatment, corneal blood vessel growth was significantly reduced. It was concluded that there is no consistent relation between the number of extravascular neutrophils at the corneal limbus and the extent of corneal blood vessel growth.     

Leukocytes in the early events of corneal neovascularization

PURPOSE: To study the early events in corneal neovascularization after alkali injury and their relationship to the presence and absence of leukocytes. METHODS: A standardized 5.5-mm diameter penetrating central corneal alkali wound was induced in one eye in each of ten New Zealand white rabbits (2.5 kg). In five of the ten rabbits, 1.5 mL 5% fucoidin was given intravenously every 2 hours to prevent leukocytes from leaving the blood stream. Presence of hyaluronan (HA) and proliferating cell nuclear antigen (PCNA) in the corneas were analyzed using immunohistochemical staining 36 hours after injury. RESULTS: In the alkali wounded corneas, HA was expressed intensively in the limbal area where a massive infiltration of leukocytes was seen. PCNA was expressed in the vascular endothelium as well as in the corneal cells. In the leukocyte-free corneas, HA staining intensity and distribution were the same as in uninjured corneas. No positive PCNA staining was seen in the vascular endothelial cells in these corneas. CONCLUSIONS: Extravasated leukocytes in the alkali-burned corneas caused enhanced production of HA and proliferation of vascular endothelial cells.     

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.     

Limbal vascular response prior to corneal vascularization

In spite of claims of a significant latent period of up to 2 days between corneal injury and the beginning of corneal vascularization, there is evidence of a limbal reaction occurring only hours after injury. In this study, the central area of rat corneas was cauterized to give an injury which was remote from the limbal vessels. The resulting changes in vascular permeability were investigated. Significant emigration of neutrophil polymorphonuclear leucocytes began at 36 min and leakage of Trypan blue was discernible at about 1 hr after injury. Eosinophilic leucocytes do not appear to be involved in the response and mast cells, while not showing signs of increased degranulation, may be decreased in number over several days. The significance of these results in relation to the stimulus factors involved in these changes, and the importance of separating inflammatory and proliferative changes, are diseussed.     

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.