Souska Zandi

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.

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)

ROCK-Isoform-Specific Polarization of Macrophages Associated with Age-Related Macular Degeneration

Age is a major risk factor in age-related macular degeneration (AMD), but the underlying cause is unknown. We find increased Rho-associated kinase (ROCK) signaling and M2 characteristics in eyes of aged mice, revealing immune changes in aging. ROCK isoforms determine macrophage polarization into M1 and M2 subtypes. M2-like macrophages accumulated in AMD, but not in normal eyes, suggesting that these macrophages may be linked to macular degeneration. M2 macrophages injected into the mouse eye exacerbated choroidal neovascular lesions, while M1 macrophages ameliorated them, supporting a causal role for macrophage subtypes in AMD. Selective ROCK2 inhibition with a small molecule decreased M2-like macrophages and choroidal neovascularization. ROCK2 inhibition upregulated M1 markers without affecting macrophage recruitment, underlining the plasticity of these macrophages. These results reveal age-induced innate immune imbalance as underlying AMD pathogenesis. Targeting macrophage plasticity opens up new possibilities for more effective AMD treatment.

Vascular adhesion protein-1 regulates leukocyte transmigration rate in the retina during diabetes.

Vascular adhesion protein-1 (VAP-1) is an endothelial adhesion molecule that possesses semicarbazide-sensitive amine oxidase (SSAO) activity and is involved in leukocyte recruitment. Leukocyte adhesion to retinal vessels is a predominant feature of experimentally induced diabetic retinopathy (DR). However, the role of VAP-1 in this process is unknown. Diabetes was induced by i.p. injection of Streptozotocin in Long-Evans rats. The specific inhibitor of VAP-1, UV-002, was administered by daily i.p. injections. The expression of VAP-1 mRNA in the retinal extracts of normal and diabetic animals was measured by real-time quantitative polymerase chain reaction (PCR). Firm leukocyte adhesion was quantified in retinal flatmounts after intravascular staining with concanavalin A (ConA). Leukocyte transmigration rate was quantified by in vivo acridine orange leukocyte staining (AOLS). In diabetic rats, the rate of leukocyte transmigration into the retinal tissues of live animals was significantly increased, as determined by AOLS. When diabetic animals were treated with daily injections of the VAP-1 inhibitor (0.3 mg/kg), leukocyte transmigration rate was significantly reduced (P < 0.05). However, firm adhesion of leukocytes in diabetic animals treated with the inhibitor did not differ significantly from vehicle-treated diabetic controls. This work provides evidence for an important role of VAP-1 in the recruitment of leukocyte to the retina in experimental DR. Our results reveal the critical contribution of VAP-1 to leukocyte transmigration, with little impact on firm leukocyte adhesion in the retinas of diabetic animals. VAP-1 inhibition might be beneficial in the treatment of DR.

Lymphangiogenesis and angiogenesis: concurrence and/or dependence? Studies in inbred mouse strains

Genetic background significantly affects angiogenesis in mice. However, lymphangiogenic response to growth factors (GFs) in different strains has not been studied. We report constitutive expression of corneal lymphatics that extends beyond the limits of normal limbal vessels. In untreated corneas, the total number (P=0.006), the number above blood vessels (P=10−8), and the area of preexisting lymphatics (P= 0.007) were significantly higher in C57BL/6 than in BALB/c mice. Normal corneas of three other strains, the nu/nu, 129E, and Black Swiss mice, showed in most parameters intermediate phenotypes. FGF‐2−/− mice showed significantly less preexisting lymphatics than control (P= 0.009), which suggests a role for this GF in lymphatic development. VEGF‐A‐induced corneal lymphangiogenic response was significantly higher in BALB/c mice (P=0.03), but it did not differ significantly in C57BL/6 mice, when compared to PBS‐implanted control. FGFR‐3 expression was higher in C57BL/6 than BALB/c mice, which suggests GF‐receptor heterogeneity as a possible explanation for strain‐dependent differences. The heterogeneity of preexisting lymphatic vessels in the limbal area significantly correlated with the extent of corneal lymphangiogenesis (VEGF‐A r=0.7, P=0.01;FGF‐2: r=0.96, P=10−5) in BALB/c but not in C57BL/6 mice. Removal of conjunctival lymphatics did not affect GF‐induced lymphangiogenesis. This work introduces physiological expression of lymphatics without blood vessels, which indicates that angiogenesis and lymphangiogenesis, even though intricately related, may occur independently. Furthermore, we show strain‐dependence of normal and GF‐induced lymphangiogenesis. These differences may affect disease development in various strains.—Nakao, S., Maruyama, K., Zandi, S., Melhorn, M. I., Taher, M., Noda, K., Nusayr, E., Doetschman, T., Hafezi‐Moghadam, A. Lymphangiogenesis and angiogenesis: concurrence and/or dependence? Studies in inbred mouse strains. FASEB J. 24, 504–513 (2010). www.fasebj.org

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

Blood vessel endothelial VEGFR-2 delays lymphangiogenesis: an endogenous trapping mechanism links lymph- and angiogenesis.

Angio- and lymphangiogenesis are inherently related processes. However, how blood and lymphatic vessels regulate each other is unknown. This work introduces a novel mechanism explaining the temporal and spatial relation of blood and lymphatic vessels. Vascular endothelial growth factor-A (VEGF-A) surprisingly reduced VEGF-C in the supernatant of blood vessel endothelial cells, suggesting growth factor (GF) clearance by the growing endothelium. The orientation of lymphatic sprouting toward angiogenic vessels and away from exogenous GFs was VEGF-C dependent. In vivo molecular imaging revealed higher VEGF receptor (R)-2 in angiogenic tips compared with normal vessels. Consistently, lymphatic growth was impeded in the angiogenic front. VEGF-C/R-2 complex in the cytoplasm of VEGF-A-treated endothelium indicated that receptor-mediated internalization causes GF clearance from the extracellular matrix. GF clearance by receptor-mediated internalization is a new paradigm explaining various characteristics of lymphatics.

VAP-1–Mediated M2 Macrophage Infiltration Underlies IL-1β– but Not VEGF-A–Induced Lymph- and Angiogenesis

Vascular adhesion protein-1 (VAP-1) contributes to inflammatory and angiogenic diseases, including cancer and age-related macular degeneration. It is expressed in blood vessels and contributes to inflammatory leukocyte recruitment. The cytokines IL-1β and vascular endothelial growth factor A (VEGF-A) modulate angiogenesis, lymphangiogenesis, and leukocyte infiltration. The lymphatic endothelium expresses intercellular adhesion molecule-1 and vascular adhesion molecule-1, which facilitate leukocyte transmigration into the lymphatic vessels. However, whether lymphatics express VAP-1 and whether they contribute to cytokine-dependent lymph- and angiogenesis are unknown. We investigated the role of VAP-1 in IL-1β- and VEGF-A-induced lymph- and angiogenesis using the established corneal micropocket assay. IL-1β increased VAP-1 expression in the inflamed cornea. Our in vivo molecular imaging revealed significantly higher VAP-1 expression in neovasculature than in the preexisting vessels. VAP-1 was expressed in blood but not lymphatic vessels in vivo. IL-1β-induced M2 macrophage infiltration and lymph- and angiogenesis were blocked by VAP-1 inhibition. In contrast, VEGF-A-induced lymph- and angiogenesis were unaffected by VAP-1 inhibition. Our results indicate a key role for VAP-1 in lymph- and angiogenesis-related macrophage recruitment. VAP-1 might become a new target for treatment of inflammatory lymph- and angiogenic diseases, including cancer.

Atrial Natriuretic Peptide Reduces Vascular Leakage and Choroidal Neovascularization

Atrial natriuretic peptide (ANP) is a hormone with diuretic, natriuretic, and vasodilatory properties. ANP blocks vascular endothelial growth factor (VEGF) production and signaling in vitro; however, its role in vascular leakage and angiogenesis is unknown. In vitro, retinal barrier permeability (transepithelial electrical resistance (TEER)) was measured in cultured retinal endothelial (HuREC) and retinal epithelial (ARPE-19) cells with VEGF (10 ng/ml), ANP (1 pM to 1 micromol/L), and/or isatin, an ANP receptor antagonist. In vivo, blood-retinal barrier (BRB) leakage was studied using the Evans Blue dye technique in rats treated with intravitreal injections of ANP, VEGF, or vehicle. Choroidal neovascularization was generated by laser injury, and 7 days later, lesion size and leakage was quantitated. ANP significantly reversed VEGF-induced BRB TEER reduction in both HuREC and ARPE-19 cells, modeling the inner and the outer BRB, respectively. Isatin, a specific ANP receptor antagonist, reversed ANPs effect. ANP reduced the response of ARPE-19 cells to VEGF apically but not basolaterally, suggesting polarized expression of the ANP receptors in these cells. ANPs TEER response was concentration but not time dependent. In vivo, ANP significantly reduced VEGF-induced BRB leakage and the size of laser-induced choroidal neovascularization lesions. In sum, ANP is an effective inhibitor of VEGF-induced vascular leakage and angiogenesis in vivo. These results may lead to new treatments for ocular diseases where VEGF plays a central role, such as age-related macular degeneration or diabetic retinopathy.

Superior sensitivity of novel molecular imaging probe: simultaneously targeting two types of endothelial injury markers

The need remains great for early diagnosis of diseases. The special structure of the eye provides a unique opportunity for noninvasive light‐based imaging of fundus vasculature. To detect endothelial injury at the early and reversible stage of adhesion molecule up‐regulation, we generated novel imaging agents that target two distinct types of endothelial molecules, a mediator of rolling, P‐selectin, and one that mediates firm adhesion, ICAM‐1. Interactions of these double‐conjugated fluorescent microspheres (MSs) in retinal or choroidal microvasculature were visualized in live animals by scanning laser ophthalmoscopy. The new imaging agents showed significantly higher sensitivity for detection of endothelial injury than singly conjugated MSs (rPSGL‐1‐ or α‐ICAM‐1‐conjugated), both in terms of rolling (P<0.01) and firm adhesion (P<0.01). The rolling flux of α‐ICAM‐1‐conjugated MSs did not differ in EIU animals, whereas double‐conjugated MSs showed significantly higher rolling flux (P<0.01), revealing that ICAM‐1 in vivo supports rolling, once MS interaction with the endothelium is initiated. Double‐conjugated MSs specifically detected firmly adhering leukocytes (P<0.01), allowing in vivo quantification of immune response. Antiinflammatory treatment with dexamethasone led to reduced leukocyte accumulation (P<0.01) as well as MS interaction (P<0.01), which suggests that treatment success and resolution of inflammation is quantitatively reflected with this molecular imaging approach. This work introduces novel imaging agents for noninvasive detection of endothelial injury in vivo. Our approach may be developed further to diagnose human disease at a much earlier stage than currently possible.—Sun, D., Nakao, S., Xie, F., Zandi, S., Schering, A., Hafezi‐Moghadam, A. Superior sensitivity of novel molecular imaging probe: simultaneously targeting two types of endothelial injury markers. FASEB J. 24, 1532–1540 (2010). www.fasebj.org