Hideto Katsuta

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.

In Vivo Evaluation of Retinal Injury After Transient Ischemia in Hypertensive Rats

Abstract—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.

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.

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.

In vivo evaluation of ocular inflammatory responses in experimental diabetes

Aims: Diabetic patients may have abnormal inflammatory reactions to foreign or endogenous stimuli. This study was designed to evaluate inflammatory reactions in the diabetic eye through retinal leucocyte dynamics in the inflamed eyes of diabetic rats. Methods: Three weeks after diabetes induction in Long-Evans rats, endotoxin induced uveitis was produced by footpad injection of lipopolysaccharide (LPS). After LPS injection, leucocyte behaviour was evaluated in vivo by acridine orange digital fluorography. Results: The number of rolling leucocytes increased in a biphasic manner at 12 hours and 48 hours. The number of leucocytes accumulating in the retina reached a peak at 72 hours. The maximal numbers of rolling and accumulating leucocytes in the diabetic retina decreased by 56.3% (p<0.01) and 46.7% (p<0.0001), respectively, compared with the non-diabetic retina. The levels of mRNA expression of adhesion molecules in the retina, which were upregulated after LPS injection, were also lower in diabetic rats than in non-diabetic rats. Conclusion: This study is the first to show that endotoxin induced inflammation is disturbed in the diabetic eye, based on evidence that the leucocyte-endothelial cell interactions stimulated by LPS were suppressed in the diabetic retina. These findings support the theory that ocular inflammatory reactions are impaired in diabetic patients.