F. Tayyari

Rho Kinase Inhibition by Fasudil Ameliorates Diabetes-Induced Microvascular Damage

OBJECTIVE—Leukocyte adhesion in retinal microvasuculature substantially contributes to diabetic retinopathy. Involvement of the Rho/Rho kinase (ROCK) pathway in diabetic microvasculopathy and therapeutic potential of fasudil, a selective ROCK inhibitor, are investigated. RESEARCH DESIGN AND METHODS—Localization of RhoA/ROCK and Rho activity were examined in retinal tissues of rats. Impact of intravitreal fasudil administration on retinal endothelial nitric oxide synthase (eNOS) and myosin phosphatase target protein (MYPT)-1 phosphorylation, intercellular adhesion molecule-1 (ICAM-1) expression, leukocyte adhesion, and endothelial damage in rat eyes were investigated. Adhesion of neutrophils from diabetic retinopathy patients or nondiabetic control subjects to cultured microvascular endothelial cells was quantified. The potential of fasudil for endothelial protection was investigated by measuring the number of adherent neutrophils and terminal transferase-mediated dUTP nick-end labeling–positive endothelial cells. RESULTS—RhoA and ROCK colocalized predominantly in retinal microvessels. Significant Rho activation was observed in retinas of diabetic rats. Intravitreal fasudil significantly increased eNOS phosphorylation, whereas it reduced MYPT-1 phosphorylation, ICAM-1 expression, leukocyte adhesion, and the number of damaged endothelium in retinas of diabetic rats. Neutrophils from diabetic retinopathy patients showed significantly higher adhesion to cultured endothelium and caused endothelial apoptosis, which was significantly reduced by fasudil. Blockade of the Fas-FasL interaction prevented endothelial apoptosis. The protective effect of fasudil on endothelial apoptosis was significantly reversed by Nω-nitro-l-arginine methyl ester, a NOS inhibitor, whereas neutrophil adhesion remained unaffected. CONCLUSIONS—The Rho/ROCK pathway plays a critical role in diabetic retinal microvasculopathy. Fasudil protects the vascular endothelium by inhibiting neutrophil adhesion and reducing neutrophil-induced endothelial injury. ROCK inhibition may become a new strategy in the management of diabetic retinopathy, especially in its early stages.

An animal model of spontaneous metabolic syndrome: Nile grass rat

Metabolic syndrome (MetS) is a prevalent and complex disease, characterized by the variable coexistence of obesity, dyslipidemia, hyperinsulinaemia, and hypertension. The alarming rise in the prevalence of metabolic disorders makes it imperative to innovate preventive or therapeutic measures for MetS and its complications. However, the elucidation of the pathogenesis of MetS has been hampered by the lack of realistic models. For example, the existing animal models of MetS, i.e., genetically engineered rodents, imitate certain aspects of the disease, while lacking other important components. Defining the natural course of MetS in a spontaneous animal model of the disease would be desirable. Here, we introduce the Nile grass rat (NGR), Arvicanthis niloticus, as a novel model of MetS. Studies of over 1100 NGRs in captivity, fed normal chow, revealed that most of these animals spontaneously develop dyslipidemia (P < 0.01), and hyperglycemia (P < 0.01) by 1 yr of age. Further characterization showed that the diabetic rats develop liver steatosis, abdominal fat accumulation, nephropathy, atrophy of pancreatic islets of Langerhans, fatty streaks in the aorta, and hypertension (P < 0.01). Diabetic NGRs in the early phase of the disease develop hyperinsulinemia, and show a strong inverse correlation between plasma adiponectin and HbA1c levels (P < 0.01). These data indicate that the NGR is a valuable, spontaneous model for exploring the etiology and pathophysiology of MetS as well as its various complications.—Noda, K., Melhorn, M. I., Zandi, S., Frimmel, S., Tayyari, F., Hisatomi, T., Almulki, L., Pronczuk, A., Hayes, K. C, Hafezi‐Moghadam, A. An animal model of spontaneous metabolic syndrome: Nile grass rat. FASEBJ. 24, 2443–2453 (2010). www.fasebj.org

Characterization of Azurocidin as a Permeability Factor in the Retina: Involvement in VEGF-Induced and Early Diabetic Blood-Retinal Barrier Breakdown

PURPOSE Azurocidin, released by neutrophils during leukocyte-endothelial interaction, is a main cause of neutrophil-evoked vascular leakage. Its role in the retina, however, is unknown. METHODS Brown Norway rats received intravitreal injections of azurocidin and vehicle control. Blood-retinal barrier (BRB) breakdown was quantified using the Evans blue (EB) dye technique 1, 3, and 24 hours after intravitreal injection. To block azurocidin, aprotinin was injected intravenously before the intravitreal injections. To investigate whether azurocidin plays a role in vascular endothelial growth factor (VEGF)-induced BRB breakdown, rats were treated intravenously with aprotinin, followed by intravitreal injection of VEGF(164). BRB breakdown was quantified 24 hours later. To investigate whether azurocidin may mediate BRB breakdown in early diabetes, aprotinin or vehicle was injected intravenously each day for 2 weeks to streptozotocin-induced diabetic rats, and BRB breakdown was quantified. RESULTS Intravitreal injection of azurocidin (20 microg) induced a 6.8-fold increase in vascular permeability compared with control at 1-3 hours (P < 0.05), a 2.7-fold increase at 3 to 5 hours (P < 0.01), and a 1.7-fold increase at 24 hours (P < 0.05). Aprotinin inhibited azurocidin-induced BRB breakdown by more than 95% (P < 0.05). Furthermore, treatment with aprotinin significantly suppressed VEGF-induced BRB breakdown by 93% (P < 0.05) and BRB breakdown in early experimental diabetes by 40.6% (P < 0.05). CONCLUSIONS Azurocidin increases retinal vascular permeability and is effectively blocked by aprotinin. The inhibition of VEGF-induced and early diabetic BRB breakdown with aprotinin indicates that azurocidin may be an important mediator of leukocyte-dependent BRB breakdown secondary to VEGF. Azurocidin may become a new therapeutic target in the treatment of retinal vascular leakage, such as during diabetic retinopathy.

Noninvasive molecular imaging reveals role of PAF in leukocyte-endothelial interaction in LPS-induced ocular vascular injury

Uveitis is a systemic immune disease and a common cause of blindness. The eye is an ideal organ for light‐based imaging of molecular events underlying vascular and immune diseases. The phospholipid platelet‐activating factor (PAF) is an important mediator of inflammation, the action of which in endothelial and immune cells in vivo is not well understood. The purpose of this study was to investigate the role of PAF in endothelial injury in uveitis. Here, we use our recently introduced in vivo molecular imaging approach in combination with the PAF inhibitors WEB 2086 (WEB) and ginkgolide B (GB). The differential inhibitory effects of WEB and GB in reducing LPS‐induced endothelial injury in the choroid indicate an important role for PAF‐like lipids, which might not require the PAF receptor for their signaling. P‐selectin glycoprotein ligand‐1‐mediated rolling of mouse leukocytes on immobilized P‐selectin in our autoperfused microflow chamber assay revealed a significant reduction in rolling velocity on the cells’ contact with PAF. Rolling cells that came in contact with PAF rapidly assumed morphological signs of cell activation, indicating that activation during rolling does not require integrins. Our results show a key role for PAF in mediating endothelial and leukocyte activation in acute ocular inflammation. Our in vivo molecular imaging provides a detailed view of cellular and molecular events in the complex physiological setting.—Garland, R. C., Sun, D., Zandi, S., Xie, F., Faez, S., Tayyari, F., Frimmel, S. A. F., Schering, A., Nakao, S., Hafezi‐Moghadam, A. Noninvasive molecular imaging reveals role of PAF in leukocyte‐endothelial interaction in LPS‐induced ocular vascular injury. FASEB J. 25, 1284–1294 (2011). www.fasebj.org

In vivo imaging of endothelial injury in choriocapillaris during endotoxin-induced uveitis

Early detection of ocular inflammation may prevent the occurrence of structural damage or vision loss. Here, we introduce a novel noninvasive technique for molecular imaging and quantitative eval uation of endothelial injury in the choriocapillaris of live animals, which detects disease earlier than cur rently possible. Using an established model of ocular inflammation, endotoxin‐induced uveitis (EIU), we vi sualized the rolling and adhesive interaction of fluores cent microspheres conjugated to recombinant P‐selec‐ tin glycoprotein ligand‐Ig (rPSGL‐Ig) in choriocapillaris using a scanning laser ophthalmoscope (SLO). The number of rolling microspheres in the choriocapillaris peaked 4–10 h after LPS injection. The number of the accumulated microspheres peaked 4 h after LPS injection in the temporal choriocapillaris and 4 and 36 h after LPS injection in the central areas around the optic disk. Furthermore, we semiquantified the levels of P‐selectin mRNA expression in the choroidal vessels by reverse transcription‐PCR and found its pattern to match the functional microsphere interactions, with a peak at 4 h after LPS injection. These results indicate that PSGL‐1‐conjugated fluorescent microspheres allow specific detection of endothelial P‐selectin expression in vivo and noninvasive assessment of endothelial in jury. This technique may help to diagnose subclinical signs of ocular inflammatory diseases.— Miyahara, S., Almulki, L., Noda, K., Nakazawa, T., Hisatomi, T., Nakao, S., Thomas, K. L., Schering, A., Zandi, S., Frimmel, S., Tayyari, F., Garland, R. C., Miller, J. W., Gragoudas, E. S., Masli, S., Hafezi‐Moghadam, A. In vivo imaging of endothelial injury in choriocapillaris during endotoxin‐induced uveitis. FASEB J. 22, 1973–1980 (2008)