Sonja Frimmel

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

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.

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)

Molecular imaging reveals elevated VEGFR-2 expression in retinal capillaries in diabetes: a novel biomarker for early diagnosis

Diabetic retinopathy (DR) is a microvascular complication of diabetes and a leading cause of vision loss. Biomarkers and methods for early diagnosis of DR are urgently needed. Using a new molecular imaging approach, we show up to 94% higher accumulation of custom designed imaging probes against vascular endothelial growth factor receptor 2 (VEGFR‐2) in retinal and choroidal vessels of diabetic animals (P<0.01), compared to normal controls. More than 80% of the VEGFR‐2 in the diabetic retina was in the capillaries, compared to 47% in normal controls (P<0.01). Angiography in rabbit retinas revealed microvascular capillaries to be the location for VEGF‐A‐induced leakage, as expressed by significantly higher rate of fluorophore spreading with VEGF‐A injection when compared to vehicle control (26±2 vs. 3±1 μM/s, P<0.05). Immunohistochemistry showed VEGFR‐2 expression in capillaries of diabetic animals but not in normal controls. Macular vessels from diabetic patients (n=7) showed significantly more VEGFR‐2 compared to nondiabetic controls (n=5) or peripheral retinal regions of the same retinas (P<0.01 in both cases). Here we introduce a new approach for early diagnosis of DR and VEGFR‐2 as a molecular marker. VEGFR‐2 could become a key diagnostic target, one that might help to prevent retinal vascular leakage and proliferation in diabetic patients.—Sun, D., Nakao, S., Xie, F., Zandi, S., Bagheri, A., Kanavi, M. R., Samiei, S., Soheili, Z.‐S., Frimmel, S., Zhang, Z., Ablonczy, Z., Ahmadieh, H., Hafezi‐Moghadam, A. Molecular imaging reveals elevated VEGFR‐2 expression in retinal capillaries in diabetes: a novel biomarker for early diagnosis. FASEB J. 28, 3942‐3951 (2014). www.fasebj.org

Molecular imaging of retinal endothelial injury in diabetic animals

Purpose: Diabetic retinopathy is a leading cause of vision loss. There is a great need for early diagnosis prior to the occurrence of irreversible structural damages. Expression of endothelial adhesion molecules is observed before the onset of diabetic vascular damage; however, to date, these molecules cannot be visualized in vivo. Methods: To quantify the expression of endothelial surface molecules, we generated imaging probes that bind to ICAM-1. The α-ICAM-1 probes were characterized via flow cytometry under microfluidic conditions. Probes were systemically injected into normal and diabetic rats, and their adhesion in the retinal microvessels was visualized via confocal scanning laser ophthalmoscopy. Histology was performed to validate in vivo imaging results. Vascular pathologies were visualized using trypsin-digested retinal preparations. Results: The α-ICAM-1 probes showed significantly higher adhesion to retinal microvessels in diabetic rats than in normal controls (P < 0.01), whereas binding of control probes did not differ between the two groups. Western blotting results showed higher ICAM-1 expression in retinas of T1D animals than in normal controls. Retinal endothelial ICAM-1 expression was observed via molecular imaging before markers of structural damage, such as pericyte ghosts and acellular capillaries. Conclusion: Results indicate that molecular imaging can be used to detect subtle changes in the diabetic retina prior to the occurrence of irreversible pathology. Thus, ICAM-1 could serve as a diagnostic target in patients with diabetes. This study provides a proof of principle for non-invasive subclinical diagnosis in experimental diabetic retinopathy. Further development of this technology could improve management of diabetic complications.