L. Almulki

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.

HIV protease inhibitors provide neuroprotection through inhibition of mitochondrial apoptosis in mice.

Neuroprotection can be achieved by preventing apoptotic death of postmitotic cells. Apoptotic death can occur by either a caspase-dependent mechanism, involving cytochrome c, apoptosis protease-activating factor-1 (Apaf-1), and caspase-9, or a caspase-independent mechanism, involving apoptosis-inducing factor (AIF). HIV protease inhibitors (PIs) avert apoptosis in part by preventing mitochondrial outer membrane permeabilization (MOMP), but the precise mechanism by which they work is not known. Here, we evaluated the impact of the PIs in a mouse model of retinal detachment (RD) in vivo and in murine primary retinal cell cultures in vitro. Oral administration of the PIs nelfinavir and ritonavir significantly inhibited photoreceptor apoptosis, while preventing the translocation of AIF from mitochondria to the nucleus as well as the activation of caspase-9. RD-induced photoreceptor apoptosis was similarly inhibited in mice carrying hypomorphic mutations of the genes encoding AIF or Apaf-1. Nelfinavir attenuated apoptosis as well as mitochondrial release of AIF and cytochrome c, and subsequent activation of caspase-9 in vitro, in photoreceptor cultures exposed to starvation or monocyte chemoattractant protein-1-stimulated (MCP-1-stimulated) macrophages. Our results suggest that the MOMP inhibition by PIs involved interruption of both caspase-dependent and caspase-independent apoptosis pathways and that PIs may be clinically useful for the treatment of diseases caused by excessive apoptosis.

Vascular adhesion protein-1 blockade suppresses choroidal neovascularization

Vascular adhesion protein‐1 (VAP‐1) is an endothelial cell adhesion molecule involved in leukocyte recruitment. Leukocytes and, in particular, macrophages play an important role in the development of choroidal neovascularization (CNV), an integral component of age‐related macular degeneration (AMD). Previously, we showed a role for VAP‐1 in ocular inflammation. Here, we investigate the expression of VAP‐1 in the choroid and its role in CNV development. VAP‐1 was expressed in the choroid, exclusively in the vessels, and colocalized in the vessels of the CNV lesions. VAP‐1 blockade with a novel and specific inhibitor significantly decreased CNV size, fluorescent angiographic leakage, and the accumulation of macrophages in the CNV lesions. Furthermore, VAP‐1 blockade significantly reduced the expression of inflammation‐associated molecules such as tumor necrosis factor (TNF) ‐α, monocyte chemoattractant protein (MCP) ‐1, and intercellular adhesion molecule (ICAM) ‐1. This work provides evidence for an important role of VAP‐1 in the recruitment of macrophages to CNV lesions, establishing a novel link between VAP‐1 and angiogenesis. Inhibition of VAP‐1 may become a new therapeutic strategy in the treatment of AMD.—Noda, K., She, H., Nakazawa, T., Hisatomi, T., Nakao, S., Almulki, L., Zandi, S., Miyahara, S., Ito, Y., Thomas, K. L., Garland, R. C., Miller, J. W., Gragoudas, E. S., Mashima, Y., Hafezi‐Moghadam, A. Vascular adhesion protein‐1 blockade suppresses choroidal neovascularization. FASEB J. 22, 2928‐2935 (2008)

Inhibition of vascular adhesion protein-1 suppresses endotoxin-induced uveitis

Inflammatory leukocyte accumulation is a common feature of major ocular diseases, such as uveitis, diabetic retinopathy, and age‐related macular degeneration. Vascular adhesion protein‐1 (VAP‐1), a cell surface and soluble molecule that possesses semi‐carbazide‐sensitive amine oxidase (SSAO) activity, is involved in leukocyte recruitment. However, the expression of VAP‐1 in the eye and its contribution to ocular inflammation are unknown. Here, we investigated the role of VAP‐1 in an established model of ocular inflammation, the endotoxin‐induced uveitis (EIU), using a novel and specific inhibitor. Our inhibitor has a half‐maximal inhibitory concentration (IC50) of 0.007 μM against human and 0.008 μM against rat SSAO, while its IC50 against the functionally related monoamine oxidase (MAO) ‐A and MAO‐B is > 10 μM. In the retina, VAP‐1 was exclusively expressed in the vasculature, and its expression level was elevated during EIU. VAP‐1 inhibition in EIU animals significantly suppressed leukocyte recruitment to the anterior chamber, vitreous, and retina, as well as retinal endothelial P‐selectin expression. Our data suggest an important role for VAP‐1 in the recruitment of leukocytes to the immune‐privileged ocular tissues during acute inflammation. VAP‐1 inhibition may become a novel strategy in the treatment of ocular inflammatory diseases. Noda, K., Miyahara, S., Nakazawa, T., Almulki, L., Nakao, S., Hisatomi, T., She, H., Thomas, K. L., Garland, R. C., Miller, J. W., Gragoudas, E. S., Kawai, Y., Mashima, Y., Hafezi‐Moghadam, A. Inhibition of vascular adhesion protein‐1 suppresses endotoxin‐in‐duced uveitis. FASEB J. 22, 1094–1103 (2008)

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

Endogenous PMN sialidase activity exposes activation epitope on CD11b/CD18 which enhances its binding interaction with ICAM-1.

Diapedesis is a dynamic, highly regulated process by which leukocytes are recruited to inflammatory sites. We reported previously that removal of sialyl residues from PMNs enables these cells to become more adherent to EC monolayers and that sialidase activity within intracellular compartments of resting PMNs translocates to the plasma membrane following activation. We did not identify which surface adhesion molecules were targeted by endogenous sialidase. Upon activation, β2 integrin (CD11b/CD18) on the PMN surface undergoes conformational change, which allows it to bind more tightly to the ICAM‐1 and ICAM‐2 on the EC surface. Removal of sialyl residues from CD18 and CD11b, by exogenous neuraminidase or mobilization of PMN sialidase, unmasked activation epitopes, as detected by flow cytometry and enhanced binding to ICAM‐1. One sialidase isoform, Neu1, colocalized with CD18 on confocal microscopy. Using an autoperfused microflow chamber, desialylation of immobilized ICAM‐1 enhanced leukocyte arrest in vivo. Further, treatment with a sialidase inhibitor in vivo reversed endotoxin‐induced binding of leukocytes to ICAM‐1, thereby suggesting a role for leukocyte sialidase in the cellular arrest. These data suggest that PMN sialidase could be a physiologic source of the enzymatic activity that removes sialyl residues on β2 integrin and ICAM‐1, resulting in their enhanced interaction. Thus, PMN sialidase may be an important regulator of the recruitment of these cells to inflamed sites.

VLA-4 blockade suppresses endotoxin-induced uveitis: in vivo evidence for functional integrin up-regulation

Leukocyte adhesion to the vascular wall is a critical early step in the pathogenesis of inflammatory diseases and is mediated in part by the leukocyte integrin, VLA‐4, which binds to endothelial vascular cell adhesion molecule (VCAM) −1. Here, we investigate VLA‐4s role in endotoxin‐induced uveitis (EIU). At various time points (6–48 h) after EIU induction, the severity of the inflammation was evaluated by quantifying cell and protein content in the aqueous fluid, firm leukocyte adhesion in the retinal vessels, and the number of extravasated leukocytes into the vitreous. Functional activation of VLA‐4 in vivo was investigated in our previously introduced autoperfused micro flow chamber assay. Firm adhesion of EIU leukocytes to immobilized VCAM‐1 under physiological blood flow conditions was significantly increased compared with normal controls (P<0.05), suggesting an important role for VLA‐4 in EIU. VLA‐4 blockade in vivo significantly suppressed all uveitis‐related inflammatory parameters studied, decreasing the clinical score by 45% (P<0.01), protein content in the aqueous fluid by 21% (P<0.01), retinal leukostasis by 68% (P<0.01), and leukocyte accumulation in the vitreous by 75% (P<0.01). Our data provide novel evidence for functional up‐regulation of VLA‐4 during EIU and suggest VLA‐4 blockade as a promising therapeutic strategy for treatment of acute inflammatory eye diseases.—Hafezi‐Moghadam, A., Noda, K., Almulki, L., Iliaki, E. F., Poulaki, V., Thomas, K. L., Nakazawa, T., Hisatomi, T., Miller, J. W., Gragoudas, E. S. VLA‐4 blockade suppresses endotoxin‐induced uveitis: in vivo evidence for functional integrin up‐regulation. FASEB J. 21, 464–474 (2006)

Localization of vascular adhesion protein-1 (VAP-1) in the human eye.

Recently we showed a critical role for Vascular Adhesion Protein-1 (VAP-1) in rodents during acute ocular inflammation, angiogenesis, and diabetic retinal leukostasis. However, the expression of VAP-1 in the human eye is unknown. VAP-1 localization was therefore investigated by immunohistochemistry. Five micrometer thick sections were generated from human ocular tissues embedded in paraffin. Sections were incubated overnight with primary mAbs against VAP-1 (5 microg/ml), smooth muscle actin (1 microg/ml), CD31 or isotype-matched IgG at 4 degrees C. Subsequently, a secondary mAb was used for 30 min at room temperature, followed by Dako Envision + HRP (AEC) System for signal detection. The stained sections were examined using light microscopy and the signal intensity was quantified by two evaluators and graded into 4 discrete categories. In all examined ocular tissues, VAP-1 staining was confined to the vasculature. VAP-1 labeling showed the highest intensity in both arteries and veins of neuronal tissues: retina and optic nerve, and the lowest intensity in the iris vasculature (p < 0.05). Scleral and choroidal vessels showed moderate staining for VAP-1. VAP-1 intensity was significantly higher in the arteries compared to veins (p < 0.05). Furthermore, VAP-1 staining in arteries colocalized with both CD31 and smooth muscle actin (sm-actin) staining, suggesting expression of VAP-1 in endothelial cells, smooth muscle cells or potentially pericytes. In conclusion, immunohistochemistry reveals constitutive expression of VAP-1 in human ocular tissues. VAP-1 expression is nearly exclusive to the vasculature with arteries showing significantly higher expression than veins. Furthermore, VAP-1 expression in the ocular vasculature is heterogeneous, with the vessels of the optic nerve and the retina showing highest expressions. These results characterize VAP-1 expression in human ocular tissues.

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.

The Regulatory Roles of Apoptosis-Inducing Factor in the Formation and Regression Processes of Ocular Neovascularization

The role of apoptosis in the formation and regression of neovascularization is largely hypothesized, although the detailed mechanism remains unclear. Inflammatory cells and endothelial cells both participate and interact during neovascularization. During the early stage, these cells may migrate into an angiogenic site and form a pro-angiogenic microenvironment. Some angiogenic vessels appear to regress, whereas some vessels mature and remain. The control mechanisms of these processes, however, remain unknown. Previously, we reported that the prevention of mitochondrial apoptosis contributed to cellular survival via the prevention of the release of proapoptotic factors, such as apoptosis-inducing factor (AIF) and cytochrome c. In this study, we investigated the regulatory role of cellular apoptosis in angiogenesis using two models of ocular neovascularization: laser injury choroidal neovascularization and VEGF-induced corneal neovascularization in AIF-deficient mice. Averting apoptosis in AIF-deficient mice decreased apoptosis of leukocytes and endothelial cells compared to wild-type mice and resulted in the persistence of these cells at angiogenic sites in vitro and in vivo. Consequently, AIF deficiency expanded neovascularization and diminished vessel regression in these two models. We also observed that peritoneal macrophages from AIF-deficient mice showed anti-apoptotic survival compared to wild-type mice under conditions of starvation. Our data suggest that AIF-related apoptosis plays an important role in neovascularization and that mitochondria-regulated apoptosis could offer a new target for the treatment of pathological angiogenesis.