EphrinB2-EphB4 signals regulate formation and maintenance of funnel-shaped valves in corneal lymphatic capillaries.

PURPOSE: To elucidate the role of signals mediated by EphB4 receptor tyrosine kinase and its transmembrane ephrinB2 ligand in corneal lymphatic capillaries. METHODS: To detect expression of ephrinB2 and EphB4 in mouse corneas, immunohistochemistry of flat-mount corneas from 6- to 10-week-old wild-type, Efnb2-lacZ, and Ephb4-lacZ mice on a C57BL/6 background was performed. To induce formation of new blood vessels and lymphatic vessels, mouse corneal epithelia were swabbed with 0.1 M sodium hydroxide. To antagonize endogenous receptor-ligand interactions in corneal lymphatic vessels, recombinant EphB4/Fc proteins were injected into the subconjunctival spaces. To visualize the corneal lymphatic flow, FITC-dextran was injected subconjunctivally. RESULTS: In lymphatic capillaries of adult mouse corneas, EphB4 was intensively expressed in lymphatic endothelial cells (LECs) of funnel-shaped valves, which were segregated from ephrinB2-expressing LECs. The number of corneal lymphatic valves was significantly decreased by Efnb2 haploinsufficiency, and subconjunctival EphB4/Fc injections resulted in the deformation of preexisting valves of corneal lymphatic capillaries. In alkali-burn corneas, ephrinB2 and EphB4 were highly expressed in LECs of valve-forming areas. Subconjunctival EphB4/Fc injections perturbed the morphologic maturation of new lymphatic valves, leading to reflux of FITC-dextran to peripheral lymphatic branches. CONCLUSIONS: The results demonstrate a pivotal role of ephrinB2-EphB4 signals in the formation and maintenance of funnel-shaped valves in corneal lymphatic capillaries, and further suggest the potential of ephrinB2-EphB4 signals as a target to therapeutically manipulate corneal lymphangiogenesis.

Angiogenesis in the mouse retina: a model system for experimental manipulation.

The vascular system of the mouse retina provides a useful model for analyzing the molecular and cellular mechanisms regulating angiogenesis because (1) hierarchical vascular networks are newly formed only after birth, (2) the cellular components involved in angiogenesis are well characterized, and (3) all the processes are accessible for monitoring and manipulation. In this article, we present an overview of our current understanding of the process of retinal angiogenesis and describe a number of methodologies applicable to experimental manipulation of the retinal vascular system.

Thrombin inhibitor reduces leukocyte-endothelial cell interactions and vascular leakage after scatter laser photocoagulation.

PURPOSE: Macular edema is one of the most serious adverse effects after retinal scatter laser photocoagulation. It has been suggested that the inflammatory reaction after photocoagulation may be involved in the pathogenesis of macular edema. This study was designed to evaluate quantitatively the inhibitory effects of argatroban, a direct thrombin inhibitor, on leukocyte-endothelial cell interactions and vascular permeability after scatter laser photocoagulation. METHODS: Argon laser photocoagulation was performed in one half of the retina in pigmented male rats (n = 114). Argatroban was administered just before scatter laser photocoagulation. In the other half of the retina, AO leukocyte fluorography was used to evaluate in vivo leukocyte rolling along the retinal vein and accumulation in the retinal capillary bed. The expressions of P-selectin and intercellular adhesion molecule (ICAM)-1 were evaluated by reverse transcription-polymerase chain reaction. Retinal vessel permeability was quantified by using fluorescein isothiocyanate (FITC)-conjugated dextran. RESULTS: Scatter laser photocoagulation caused significant inflammatory leukocyte-endothelial cell interactions in the nonphotocoagulated half of the retina. Treatment with argatroban suppressed leukocyte-endothelial cell interactions. The maximum number of rolling and accumulating leukocytes was reduced by 46.6% (P < 0.01) and 51.4% (P < 0.01), respectively. The expression of P-selectin and ICAM-1 mRNA was suppressed significantly in the argatroban-treated retinas (P < 0.05). Retinal vascular permeability was also suppressed significantly (P < 0.05). CONCLUSIONS: Argatroban suppressed leukocyte-endothelial cell interactions and blood-retinal barrier breakdown after scatter laser photocoagulation, suggesting that argatroban prevents postlaser macular edema.

Intravitreal injection of corticosteroid attenuates leukostasis and vascular leakage in experimental diabetic retina.

PURPOSE: Recently, intravitreal injection of corticosteroids has been in wide use as a treatment for diabetic macular edema, and the outcomes have been favorable. However, the exact mechanism remains unclear. The hypothesis for the current study was that intravitreal corticosteroids may improve diabetic retinal edema by amelioration of blood-retinal barrier (BRB) breakdown, by inhibiting leukocyte stasis (leukostasis). METHODS: Diabetes was induced in 6-week-old male Long-Evans rats by intraperitoneal injection of streptozotocin (75 mg/kg). Three weeks after induction of diabetes, intravitreal injection of dexamethasone (40 microg/10 microL) was performed. At 2 days after intravitreal injection, accumulated leukocytes were counted in vivo by acridine orange leukocyte fluorography, and BRB breakdown was evaluated by measurement of retinal vascular permeability. The mRNA expression and protein levels of intercellular adhesion molecule (ICAM)-1 in the retina were also studied. RESULTS: The number of leukocytes accumulated in the retina, once increased in the diabetic group, was decreased by 31.6% (P = 0.0001) after dexamethasone injection. The level of BRB breakdown, also elevated in the diabetic group, was suppressed by 61.1% (P = 0.0046) after dexamethasone injection. The level of ICAM-1 mRNA expression and its protein, upregulated in the diabetic group, were downregulated by dexamethasone treatment by 70.0% (P < 0.0001) and 56.4% (P = 0.0003). CONCLUSIONS: Intravitreal injection of corticosteroids improves diabetic retinal edema through inhibiting leukocyte recruitment in the diabetic retina.

High level of endothelial cell-specific gene expression by a combination of the 5' flanking region and the 5' half of the first intron of the VE-cadherin gene.

To develop a tool to obtain a high level of gene expression specifically in endothelial cells (ECs), we assessed enhancer activity of fragments in the first intron of the VE-cadherin gene using 3 different experimental systems: luciferase assay in the F2 EC line, green fluorescent protein (GFP) expression in ECs generated in embryonic stem (ES) cell differentiation culture, and GFP expression in transgenic mice. Although the 2.5-kbp (kilobase pair) 5' flanking sequence of the VE-cadherin gene is EC specific, adding 4 kbp of the 5' half of the first intron affected an enhancement of the gene expression level in all 3 assay systems. No other fragments tested in this study could confer such effects. Compared with other gene expression units, the unit described in this study would be the most optimum one available to date for EC-specific gene expression. Because this unit can express genes in VE-cadherin(+) progenitors of hematopoietic cells but not in fully committed hematopoietic cells, it will be useful to manipulate specifically the uncommitted progenitor stage during hematopoietic cell differentiation.

In vivo three-dimensional evaluation of leukocyte behavior in retinal microcirculation of mice.

PURPOSE: To evaluate new physiologic and three-dimensional methods for monitoring leukocyte behavior in mouse retina. METHODS: Endotoxin-induced uveitis (EIU) was produced in mice by footpad injection of lipopolysaccharide (LPS). Leukocytes were labeled with acridine orange (AO). Leukocyte rolling in the retinal microcirculation was evaluated in vivo with AO digital fluorography. The number of migrated leukocytes was counted in flatmounted retina. The behavior of leukocyte migration was observed three-dimensionally at the time of peak migration. After leukocytes were labeled with AO, the mice were perfused with rhodamine-labeled concanavalin A lectin to stain the vascular endothelium. Leukocyte migration into the retina was then monitored three-dimensionally with confocal microscopy, and the velocity of the migration was measured. RESULTS: Both leukocyte rolling and migration peaked at 48 hours after LPS injection. Leukocytes were seen to extravasate from the deeper capillary layers and to migrate toward the outer layer of the retina. The traveling velocity of extravasated leukocytes in retinal tissue was 2.0 +/- 0.1 microm/h. CONCLUSIONS: New methods have been demonstrated for the three-dimensional and quantitative evaluation of leukocyte behavior in mouse retina.

Simvastatin inhibits leukocyte accumulation and vascular permeability in the retinas of rats with streptozotocin-induced diabetes.

Leukocytes play important roles in the pathogenesis of diabetic retinopathy. Recently, 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors have been reported to exert various effects in addition to their lipid-lowering ability. We investigated the effects of simvastatin, a 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor, on leukocyte-induced diabetic changes in retinas. Diabetes was induced in Long-Evans rats with streptozotocin, and simvastatin administration was begun immediately after the induction of diabetes. Two weeks of treatment with simvastatin suppressed significantly the number of leukocytes adhering to retinal vessel endothelium and the number of leukocytes accumulated in the retinal tissue by 72.9% and 41.0%, respectively (P < 0.01). The expression of intercellular adhesion molecule-1 (ICAM-1) and the CD18 (the common beta-chain of ICAM-1 ligands) were both suppressed with simvastatin. The amount of vascular endothelial growth factor in the retina was attenuated in the simvastatin-treated group. To evaluate the effects of simvastatin on leukocyte-induced endothelial cell damage, vascular permeability in the retina was measured with fluorescein-labeled dextran. Treatment with simvastatin markedly reduced retinal permeability (P = 0.014). This suggests that simvastatin attenuates leukocyte-endothelial cell interactions and subsequent blood-retinal barrier breakdown via suppression of vascular endothelial growth factor-induced ICAM-1 expression in the diabetic retina. Simvastatin may thus be useful in the prevention of diabetic retinopathy.

Laser photocoagulation of indocyanine green angiographically identified feeder vessels to idiopathic polypoidal choroidal vasculopathy.

PURPOSE: To evaluate the efficacy of laser photocoagulation of indocyanine green angiographically identified feeder vessels to idiopathic polypoidal choroidal vasculopathic lesions. DESIGN: Interventional case series. METHODS: Fifteen eyes of 14 consecutive patients with idiopathic polypoidal choroidal vasculopathy lesions treated by laser photocoagulation of indocyanine green angiographically identified feeder vessels were investigated. RESULTS: In 10 of the 15 eyes, serous retinal detachment of sensory retina in the macula disappeared after photocoagulation of the feeder vessels. The best-corrected visual acuity improved by 2 or more lines in eight of the 15 eyes and worsened in two eyes during the mean follow-up period of 13.6 months. CONCLUSIONS: Laser photocoagulation targeted exclusively to the feeder vessels supplying the idiopathic polypoidal choroidal vasculopathy lesions is a safe and effective method and can improve vision in eyes in which a serous retinal detachment is present in the macula. Indocyanine green angiography-guided laser photocoagulation should be considered as an optional treatment for idiopathic polypoidal choroidal vasculopathy.

In vivo evaluation of retinal injury after transient ischemia in hypertensive rats.

A number of studies have suggested that hypertension affects the pathogenesis of inflammatory reactions in various organs. The objective of this study was to evaluate the effects of hypertension on leukocyte-endothelial interactions after transient retinal ischemia. Transient retinal ischemia was induced for 60 minutes in spontaneously hypertensive rats (SHR) and in age-matched normotensive Wistar-Kyoto rats (WKY). At 4, 12, 24, 48, and 72 hours after reperfusion, flat-mount retinas were prepared to evaluate the density of leukocytes that had been accumulated in the retina. Intercellular adhesion molecule-1 (ICAM-1) mRNA expression was studied by semiquantitative polymerase chain reaction and ICAM-1 protein levels were studied by enzyme-linked immunosorbent assay. At 14 days after reperfusion, the retinal damage and the effect of superoxide dismutase on the damage were evaluated histologically. In SHR, the number of accumulated leukocytes peaked at 48 hours after reperfusion, and it was upregulated to 5.2-fold, as compared with that of WKY (P<0.001). ICAM-1 mRNA expression and ICAM-1 protein levels were increased significantly in the ischemia-reperfused retina in SHR compared with WKY (P<0.05). Histological examination demonstrated marked increase in the retinal ischemia/reperfusion damage in SHR (P<0.01) and a significant amelioration of the damage by treatment with superoxide dismutase in SHR (P<0.05). Oxidative stress may thus be an important mechanism for the deterioration seen in ischemia/reperfusion injury in the SHR retina.

Platelets adhering to the vascular wall mediate postischemic leukocyte-endothelial cell interactions in retinal microcirculation.

PURPOSE: Recent evidence suggests that platelets play a major role in ischemia-reperfusion injury, not only through thrombus formation but also through participation in inflammatory reactions with leukocytes. This study was designed to investigate the contribution of platelets in leukocyte recruitment to inflamed regions in vivo. METHODS: Thrombocytopenia was produced in male Long-Evans rats by intravenous injection of anti-platelet serum at 4 hours before ischemia-reperfusion. Leukocyte behavior in retinal microcirculation was evaluated with acridine orange digital fluorography. Expression of P-selectin in the postischemia retina was investigated by reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry. After 14 days of reperfusion, ischemia-induced retinal damage was evaluated histologically. RESULTS: Leukocyte rolling along major retinal veins of thrombocytopenic rats was dramatically suppressed, and subsequent leukocyte accumulation in the postischemia retina was also significantly reduced (72.3%; P < 0.001) at 24 hours after reperfusion. Although RT-PCR revealed no significant reduction of P-selectin mRNA in platelet-depleted rat retina after transient ischemia, immunohistologic examination showed suppression of P-selectin expression on the vascular wall. Another immunologic examination using anti-platelet antibody detected adherent platelets, which can also express P-selectin on their surfaces, on postischemic vascular endothelium in vehicle-treated retina. Moreover, blockage of platelet glycoprotein IIb/IIIa resulted in substantial inhibition of leukocyte rolling. In addition, histologic examination showed the participation of platelets in retinal ischemia-reperfusion injury. CONCLUSIONS: This study demonstrated that the expression of P-selectin on platelets may contribute to the recruitment of leukocytes to tissues after ischemia.

Argatroban attenuates leukocyte- and platelet-endothelial cell interactions after transient retinal ischemia.

BACKGROUND AND PURPOSE: Argatroban, a direct thrombin inhibitor, has been shown to reduce neural injury after transient cerebral ischemia. It has also been reported that this neuroprotective effect results from an anticoagulant function. This study was designed to evaluate quantitatively the inhibitory effects of argatroban on leukocyte- and platelet-endothelial cell interactions after transient retinal ischemia. METHODS: Retinal ischemia was induced for 60 minutes in male Long-Evans rats by temporary ligation of the optic sheath (n=342). Argatroban was administered just after induction of ischemia. Leukocyte and platelet behavior in the retinal microcirculation was then evaluated in vivo with scanning laser ophthalmoscopy. The expression of P-selectin and intracellular adhesion molecule-1 (ICAM-1) was evaluated by reverse transcription-polymerase chain reaction. After 10 days of reperfusion, ischemia-induced retinal damage was evaluated histologically. RESULTS: Treatment with argatroban suppressed leukocyte-endothelial cell interactions; the maximum numbers of rolling and accumulated leukocytes were reduced by 90.1% (P<0.05) and 58.7% (P<0.05), respectively, at 12 hours after reperfusion. Treatment with argatroban also suppressed platelet-endothelial cell interactions; the maximum numbers of rolling and adhering platelets were reduced by 91.8% (P<0.01) and 78.9% (P<0.01), respectively, at 12 hours after reperfusion. The expression of P-selectin and ICAM-1 mRNA was suppressed significantly in the argatroban-treated retinas (P<0.01). Histologic examination demonstrated the protective effect of argatroban on ischemia-induced retinal damage (P<0.01). CONCLUSIONS: Argatroban treatment suppressed leukocyte- and platelet-endothelial cell interactions after transient retinal ischemia. This inhibitory effect on postischemic blood cell-endothelial cell interactions might partially contribute to its neuroprotective effects.

Cancer-associated retinopathy associated with invasive thymoma.

PURPOSE: To report a case of cancer-associated retinopathy associated with invasive thymoma. DESIGN: Interventional case report. METHOD: A 41-year-old Japanese woman was observed between February 1998 and May 2001. Ophthalmologic examinations and systemic examinations were performed. The patient received treatment including corticosteroid pulse therapy, plasmapheresis, and thymectomy. RESULTS: The patient developed progressive visual dysfunction including bilateral visual acuity loss, concentric contraction of visual fields, and color vision loss. In both eyes, retinal vessel attenuation and retinal pigment epithelium degeneration were observed with fundus ophthalmoscopy and fluorescein angiography. Response in electroretinogram was reduced, suggesting both rod and cone dysfunction. Autoantibody against 23-kD cancer-associated retinopathy (CAR) antigen (antirecoverin antibody) was detected in the patient's serum. A mediastinal tumor that was histopathologically diagnosed as invasive thymoma was detected and was surgically resected. During more than 3 years of follow-up, no other malignancy was detected despite extensive systemic evaluation. The patient also suffered from subclinical myasthenia gravis. Although temporary improvement of visual function was observed after treatment with steroid pulse therapy and plasmapheresis' light perception of each eye was lost in the end. CONCLUSIONS: The patient was diagnosed as having CAR. Invasive thymoma was considered to be the causative tumor because there had been no evidence that suggested other systemic malignancy during more than 3 years of follow-up.