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PURPOSE: Inflammation, angiogenesis and fibrosis are pathological hallmarks of proliferative diabetic retinopathy (PDR). The CD146/sCD146 pathway displays proinflammatory and proangiogenic properties. We investigated the role of this pathway in the pathophysiology of PDR. METHODS: Vitreous samples from 41 PDR and 27 nondiabetic patients, epiretinal fibrovascular membranes from 18 PDR patients, rat retinas, human retinal microvascular endothelial cells (HRMECs) and human retinal Müller glial cells were studied by ELISA, Western blot analysis, immunohistochemistry and immunofluorescence microscopy analysis. Blood-retinal barrier breakdown was assessed with fluorescein isothiocyanate-conjugated dextran. RESULTS: sCD146 and VEGF levels were significantly higher in vitreous samples from PDR patients than in nondiabetic patients. In epiretinal membranes, immunohistochemical analysis revealed CD146 expression in leukocytes, vascular endothelial cells and myofibroblasts. Significant positive correlations were detected between numbers of blood vessels expressing CD31, reflecting angiogenic activity of PDR, and numbers of blood vessels and stromal cells expressing CD146. Western blot analysis showed significant increase of CD146 in diabetic rat retinas. sCD146 induced upregulation of phospho-ERK1/2, NF-κB , VEGF and MMP-9 in Müller cells. The hypoxia mimetic agent cobalt chloride, VEGF and TNF-α induced upregulation of sCD146 in HRMECs. The MMP inhibitor ONO-4817 attenuated TNF-α-induced upregulation of sCD146 in HRMECs. Intravitreal administration of sCD146 in normal rats significantly increased retinal vascular permeability and induced significant upregulation of phospho-ERK1/2, intercellular adhesion molecule-1 and VEGF in the retina. sCD146 induced migration of HRMECs. CONCLUSIONS: These results suggest that the CD146/sCD146 pathway is involved in the initiation and progression of PDR. ispartof: INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE vol:62 issue:9 ispartof: location:United States status: published

PURPOSE: To investigate the expression of IL-11 and its receptor IL-11Rα and to quantify density of CD163+ M2 macrophages in proliferative diabetic retinopathy (PDR). METHODS: Vitreous samples from 29 PDR and 19 nondiabetic patients, epiretinal fibrovascular membranes from 15 patients with PDR and Müller cells were studied by enzyme-linked immunosorbent assay, immunohistochemistry and Western blot analysis. RESULTS: We showed a significant increase in expression of IL-11, soluble(s) IL-11Rα, sCD163 and VEGF in vitreous samples from PDR patients compared to nondiabetic controls. Significant positive correlations were found between levels of VEGF and levels of IL-11 and sCD163. Significant positive correlations were found between microvessel density and number of blood vessels and stromal cells expressing IL-11, IL-11Rα and CD163 in PDR epiretinal membranes. The hypoxia mimetic agent cobalt chloride induced upregulation of IL-11 and IL-11Ra in cultured Müller cells. CONCLUSIONS: IL-11/IL-11Rα signaling and CD163+ M2 macrophages might be involved in PDR angiogenesis. ispartof: OCULAR IMMUNOLOGY AND INFLAMMATION vol:28 issue:4 pages:575-588 ispartof: location:England status: published

PURPOSE Galectin-1 regulates endothelial cell function and promotes angiogenesis. We investigated the hypothesis that galectin-1 may be involved in the pathogenesis of proliferative diabetic retinopathy (PDR). METHODS Vitreous samples from 36 PDR and 20 nondiabetic patients, epiretinal fibrovascular membranes from 13 patients with PDR, rat retinas and human retinal Muller glial cells were studied by enzyme-linked immunosorbent assay (ELISA), immunohistochemistry and Western blot analysis. In vitro angiogenesis assays were performed and the adherence of leukocytes to galectin-1-stimulated human retinal microvascular endothelial cells (HRMECs) was assessed. RESULTS The ELISA analysis revealed that galectin-1 and vascular endothelial growth factor (VEGF) levels were significantly higher in vitreous samples from PDR patients than in those from nondiabetics (p

Background— The fibronectin-splicing variant containing extra domain A (Fn-EDA) is present in negligible amounts in the plasma of healthy humans but markedly elevated in patients with comorbid conditions, including diabetes mellitus and hypercholesterolemia, which are risk factors for stroke. It remains unknown, however, whether Fn-EDA worsens stroke outcomes in such conditions. We determined the role of Fn-EDA in stroke outcome in a model of hypercholesterolemia, the apolipoprotein E–deficient (Apoe −/− ) mouse. Methods and Results— In a transient cerebral ischemia/reperfusion injury model, Apoe −/− mice expressing fibronectin deficient in EDA (Fn-EDA −/− Apoe −/− mice) exhibited smaller infarcts and improved neurological outcomes at days 1 and 8 ( P −/− mice). Concomitantly, intracerebral thrombosis [assessed by fibrin(ogen) deposition] and postischemic inflammation (phospho–nuclear factor-κB p65, phospho–IκB kinase α/β, interleukin 1β, and tumor necrosis factor-α) within lesions of Fn-EDA −/− Apoe −/− mice were markedly decreased ( P −/− mice). In an FeCl 3 injury–induced carotid artery thrombosis model, thrombus growth rate and the time to occlusion were prolonged in Fn-EDA −/− Apoe −/− mice ( P −/− mice). Genetic ablation of TLR4 improved stroke outcome in Apoe −/− mice ( P −/− Apoe −/− mice. Bone marrow transplantation experiments revealed that nonhematopoietic cell–derived Fn-EDA exacerbates stroke through Toll-like receptor-4 expressed on hematopoietic cells. Infusion of a specific inhibitor of Fn-EDA into Apoe −/− mouse 15 minutes after reperfusion significantly improved stroke outcome. Conclusions— Hypercholesterolemic mice deficient in Fn-EDA exhibit reduced cerebral thrombosis and less inflammatory response after ischemia/reperfusion injury. These findings suggest that targeting Fn-EDA could be an effective therapeutic strategy in stroke associated with hypercholesterolemia.

To investigate the effects of blocking Rho kinase pathway on the expression of inflammatory signaling pathways in the retina of diabetic mice and in human retinal Müller glial cells stimulated with high-glucose to replicate hyperglycemia.Retinas from diabetic mice and human retinal Müller glial cells (MIO-M1) were studied. Western blot analysis, immunofluorescence, and enzyme-linked immunosorbent assay were utilized to study the effect of the Rho kinase inhibitor fasudil on the expression of Rho-associated protein kinase-1 (ROCK-1), extracellular signal-regulated kinases12(ERK ½), phosphorylated nuclear factor kappa-light-chain-enhancer of activated B cells (p-NF-κB), inducible nitric oxide synthase (iNOS), vascular endothelial growth factor (VEGF), and monocyte chemoattractant protein-1 (MCP-1/CCL2).Treatment of human retinal Müller cells with high-glucose induced significant upregulation of ROCK-1, VEGF, and MCP-1/CCL2. Fasudil co-treatment normalized the high-glucose-induced upregulation of these mediators. Similarly, fasudil attenuated high-glucose-induced enhanced immunoreactivity for ROCK-1 and VEGF. Diabetes induced upregulation of ROCK-1, p-ERK ½, p-NF-κB and iNOS expression in retinas of mice. Constant fasudil intake from the onset of diabetes did not affect the metabolic status of diabetic mice but it attenuated diabetes-induced upregulation of these inflammatory signaling pathways.Our finding suggests that Rho-associated protein kinase-1 activation mediates regulation of inflammatory signaling pathways in diabetic retina.

Purpose Extracellular matrix metalloproteinase inducer (EMMPRIN) promotes angiogenesis through matrix metalloproteinases (MMPs) and vascular endothelial growth factor (VEGF) production. We investigated the expression levels of EMMPRIN and correlated these levels with VEGF, MMP-1 and MMP-9 in proliferative diabetic retinopathy (PDR). In addition, we examined the expression of EMMPRIN in the retinas of diabetic rats and the effect of EMMPRIN on the induction of angiogenesis regulatory factors in human retinal microvascular endothelial cells (HRMECs). Methods Vitreous samples from 40 PDR and 19 non-diabetic patients, epiretinal membranes from 12 patients with PDR, retinas of rats and HRMECs were studied by enzyme-linked immunosorbent assay (ELISA), immunohistochemistry, Western blot analysis, zymography analysis and RT-PCR. Results We showed a significant increase in the expression of EMMPRIN, VEGF, MMP-1 and MMP-9 in vitreous samples from PDR patients compared with non-diabetic controls (p

Purpose We investigated the link among the proinflammatory cytokine high-mobility group box 1 (HMGB1) and 8-hydroxy-2’-deoxyguanosine (8-OHdG) as a marker of oxidative DNA damage, the endothelial adhesion molecule and oxidase enzyme vascular adhesion protein-1 (VAP-1), and the inducible cytoprotective molecule heme oxygenase-1 (HO-1) in proliferative diabetic retinopathy (PDR). We correlated the levels of these molecules with clinical disease activity and studied the proinflammatory activities of HMGB1 on rat retinas and human retinal microvascular endothelial cells (HRMECs). Methods Vitreous samples from 47 PDR and 19 non-diabetic patients, epiretinal membranes from 11 patients with PDR, human retinas (16 from diabetic patients and 16 from non-diabetic subjects), rat retinas, and HRMECs were studied by enzyme-linked immunosorbent assay, immunohistochemistry, western blot immunofluorescence, and RT-PCR analyses. In addition, we assessed the adherence of leukocytes to HMGB1-stimulated HRMECs. Results HMGB1, 8-OHdG, and soluble VAP-1 (sVAP-1) levels were significantly higher in vitreous samples from PDR patients than in those from non-diabetics (p = 0.001

Objective: Cerebral ischaemia is the third leading cause of death after cancer and myocardial infarction. The protective effect of some homoeopathic drugs has been studied in the Middle Cerebral Artery Occlusion (MCAO) model of rat. Materials and Methods: The rats were pretreated with 200C potency once daily for 5 days orally (1 drop or 21 μl) and post treated after 24 hr of MCAO with 30C potency three times a day for 5 days orally (1 drop or 21 μl) with homoeopathic medicines Crotalus, Phosphorus, Arnica and Crocus. Results: The content of antioxidants, Thiobarbituric Acid Reactive Substances (TBARS) was elevated significantly whereas the level of Glutathione (GSH) was depleted significantly in the MCAO of rats as compared to the sham group. The activities of antioxidant enzymes, Glutathione Peroxidase (GPx), Glutathione Reductase (GR) and Glutathione-S-Transferase were decreased significantly in MCAO group as compared to sham group. The medicines used on rats (pre and post treated in potencies of 200C and 30C respectively) have protected the activities of these enzymes significantly when compared with the animals of MCAO group. Conclusion: The study has shown that the homoeopathic drugs have protected most of the studied parameters significantly but further studies are required to comment on the mechanism and reproducibility of homoeopathic drugs.

Purpose Tissue inhibitors of metalloproteinases (TIMPs) block the catalysis by matrix metalloproteinases (MMPs) and have additional biologic activities, including regulation of cell growth and differentiation, apoptosis, angiogenesis and oncogenesis. We investigated the expression levels of all the four human TIMPs and correlated these levels with those of MMP-9 and vascular endothelial growth factor (VEGF) in proliferative diabetic retinopathy (PDR). Methods Vitreous samples from 38 PDR and 21 nondiabetic control patients and epiretinal membranes from 14 patients with PDR and 10 patients with proliferative vitreoretinopathy (PVR) were studied by enzyme-linked immunosorbent assay, Western blot analysis and immunohistochemistry. Results Tissue inhibitor of metalloproteinases-1, TIMP-4, MMP-9 and VEGF levels were significantly higher in vitreous samples from PDR patients than in nondiabetic controls (p

Background and Hypothesis: Fibronectin (FN) containing the alternatively-spliced extra domain A (EDA + -FN) is absent in the arteries or circulation of healthy humans and mice, but specifically expressed in the endothelium of atherosclerotic arteries and elevated in circulation during pathological settings including stroke and atherosclerosis. We tested the hypothesis that EDA + -FN exacerbates atherosclerosis. Model and Methods: We generated EDA hi/hi /ApoE -/- mice (over express EDA + -FN in plasma) and EDA -/- /ApoE -/- mice (EDA-deleted allele of FN). ApoE -/- mice, which express EDA + -FN in plasma (~3-5 μg/mL) similar to levels found in human pathological settings including stroke and atherosclerosis, were used as control. Male mice (n=14/group) were fed a high-fat Western diet beginning at 6 weeks until they were sacrificed at 5 and 8 months of age. Extent of atherosclerosis was evaluated in whole aortae (Oil Red O stain) and in the cross section area of the aortic sinus (Verhoeff-Van Gieson stain). Markers of inflammation were quantified by immunohistochemistry. Results: We report that atherosclerotic plaque formation in the aortae and aortic sinus of EDA hi/hi /ApoE -/- mice were increased by 2-fold, concomitant with increased plaque inflammatory content (neutrophils, macrophages, and NF-κB p65 expression; P-/- ). Conversely, EDA -/- /ApoE -/- mice had smaller plaques (P-/- mice), concordant with decreased inflammatory content within plaque. Because EDA + -FN activates toll-like receptor 4 (TLR4) in vitro , we investigated the role of TLR4 in the EDA + -FN-mediated accelerated atherosclerosis. Genetic ablation of TLR4 in EDA hi/hi /ApoE -/- mice partially reversed exacerbated atherosclerosis (Phi/hi /ApoE -/- mice), whereas TLR4 deficiency in EDA -/- /ApoE -/- had no effect. Total cholesterol and triglycerides levels were similar among groups. Finally, we report co-localization of EDA + -FN with TLR4 on macrophages in human coronary atherosclerotic plaque samples. Conclusions: Endogenous EDA + -FN exacerbates atherosclerosis via TLR4. Targeting EDA + -FN/TLR4 pathway may have the potential to reduce atherosclerosis and improve outcomes in patients at high risk for coronary artery disease.

Background and Hypothesis: Thrombospondin 1 (TSP1) is a large adhesive glycoprotein that is stored in platelet α-granules. Upon platelet activation, TSP1 is released in large amount (up to 30 μg/mL) in circulation. Previous studies have shown smaller plasma von Willebrand factor (VWF) multimer size in Tsp1 -/- mice compared with wild-type (WT), suggesting that TSP1 protects VWF from excessive proteolysis by ADAMTS13. It remains unclear whether TSP1/VWF axis determines the rate of thrombus growth in injured vessels. We tested the hypothesis that TSP1 modulates arterial thrombosis via VWF. Model and Methods: We generated Vwf -/- / Tsp1 -/- mice and compared with Tsp1 -/- , Vwf -/- and WT mice. Platelet adhesion and susceptibility to thrombosis was assessed in a ferric-chloride injury-induced mesenteric artery thrombosis model. Using high-resolution intravital microscopy, we measured the time to form 1 st thrombus (>20 μm), thrombus growth kinetics and occlusion time in the injured arterioles. Results: We found that the number of fluorescently labeled platelets that adhered transiently to 250μm vessel wall segment within 3-4 min following ferric-chloride injury was similar in Tsp1 -/- mice compared with WT mice. The mean time to form first thrombus was significantly increased in Tsp1 -/- mice compared with WT (9.98±0.52 vs. 7.12±0.55min, PTsp1 -/- mice ( P Tsp1 -/- mice compared with WT mice (15.8±0.59 vs. 13.3±0.63min, PVwf -/- mice, Vwf -/- / Tsp1 -/ - littermates exhibited similar platelet adhesion, time to form first thrombus, and non-occlusive thrombus growth kinetics, suggesting that TSP1 modulates thrombus growth via VWF. Conclusion: We provide evidence for the first time that TSP1 does not mediate initial platelet adhesion to injured vessel wall but modulates arterial thrombus growth via a mechanism that requires VWF. These results suggest that TSP1 may participate in amplifying platelet aggregation following injury by protecting VWF from excessive cleavage by ADAMTS13.

Background and Hypothesis: Fibronectin containing the alternatively-spliced extra domain A (EDA + -FN) is absent in the arteries or circulation of healthy humans and mice; but is specifically expressed in the endothelium of atherosclerotic arteries, and elevated in circulation during pathological settings, including stroke. We tested the hypothesis that deletion of EDA in FN protects hypercholesterolemic apolipoprotein E-deficient (ApoE -/- ) mice from acute stroke injury. Methods: We generated EDA hi/hi /ApoE -/- mice (constitutively overexpress EDA + -FN in plasma) and EDA -/- /ApoE -/- mice (EDA-deleted allele of FN). ApoE -/- mice (express EDA + -FN in plasma (~3-5 μg/mL) similar to levels found in human pathological settings) were used as control. Susceptibility to thrombosis was assessed using FeCl 3 -induced carotid thrombosis model. Stroke outcome was evaluated in male mice following 60 min of ischemia/ 23 h of reperfusion injury by measuring infarct size, neurological deficit and markers of inflammation within the infarct and surrounding regions (immunohistochemistry). Results: Using intravital microscopy, we found that the time to stable occlusion of the carotid artery was accelerated in EDA hi/hi /ApoE -/- mice (P-/- mice), whereas EDA -/- /ApoE -/- mice showed opposite effect. EDA -/- /ApoE -/- mice had smaller infarcts and improved neurological outcome (P-/- mice), concordant with decreased vascular inflammation (neutrophils, macrophages, and NF-κB p65 expression). Conversely, EDA hi/hi /ApoE -/- mice had larger infarcts and more severe neurological deficits, concordant with increased vascular inflammation (P-/- mice). Because EDA + -FN activates toll-like receptor 4 (TLR4) in vitro , we investigated the role of TLR4 in the EDA + -FN-mediated aggravated stroke injury. Genetic ablation of TLR4 in EDA hi/hi /ApoE -/- mice completely reversed aggravated stroke injury (Phi/hi /ApoE -/- mice), whereas TLR4 deficiency in EDA -/- /ApoE -/- had no effect. Conclusions: EDA + -FN is pro-thrombotic and pro-inflammatory, and promotes acute stroke injury via a TLR4 -dependent mechanism. We suggest that monitoring plasma EDA + -FN levels may have prognostic value for patients at high risk for stroke.

Summary Background ADAMTS13 reduces the adhesiveness of hyperactive ultra-large von Willebrand factor (ULVWF) multimers by cleaving them into smaller, less active multimers. Recently, we and others have demonstrated that ADAMTS13 reduces atherosclerosis in hypercholesteremic apolipoprotein E (ApoE−/−) deficient mice. It is not known whether ADAMTS13 modulates atherosclerosis directly or indirectly by cleaving ULVWF multimers. Objective We generated triple knockout Adamts13−/−/Vwf−/−/ApoE−/− mice to determine whether ADAMTS13 modulates atherosclerosis through its proteolytic effects on VWF or other potential mechanisms. Methods Female mice were fed a high-fat Western diet beginning at 6 weeks of age until they were sacrificed at 4 months. We compared the extent of atherosclerosis in the serial cross-sections of the aortic sinus using the Verhoeff-Van Gieson stain. Macrophage and neutrophil infiltration were quantified by immunohistochemistry. Under plain polarized light interstitial collagen content in the serial cross-sections of the aortic sinus was quantified using picrosirius red stain. Results Deficiency of VWF in Adamts13−/−/ApoE−/− mice (Adamts13−/−/Vwf−/−/ApoE−/−) completely reversed exacerbated atherosclerosis (P

Background and Purpose - Minocycline provides neurovascular protection reducing acute cerebral injury. However, it is unclear whether minocycline is effective in females. We tested minocycline in both sexes and aged animals using an embolic stroke model in mice that closely mimics acute thromboembolic stroke in humans. The aim was to determine if there is a sex-specific change in MMP-9 levels. Methods - In the context of the revised preclinical STAIR criteria call for testing of neuroprotective agents in female mice and aged mice, minocycline was tested in five groups of mice subjected to thromboembolic stroke: adult males, aged males, adult females, aged females, and adult ovariectomized females (n=9-23 animals/group). Behavioral outcomes, infarct volumes, and cerebral blood flow as well as expression and activity of MMP-9 were assessed. Statistical ANOVA analyses were performed using SAS® 9.2. Results - The model resulted in reproducible infarct in the experimental groups. As expected, adult females were significantly more resistant to cerebral ischemic injury than males. This advantage was abolished by aging and ovariectomy. Minocycline significantly reduced the infarct volume (P=0.0001) and also improved neurologic score (P Conclusion - In a thromboembolic stroke model minocycline is neuroprotective inhibiting MMP-9 irrespective of mouse sex and age.

Restoration of blood flow to an ischemic brain region is associated with generation of reactive oxygen species (ROS) with consequent reperfusion injury. ROS cause lipid peroxidation, protein oxidation, and DNA damage, all of which are deleterious to cells. So diminishing the production of free radicals and scavenging them may be a successful therapeutic strategy for the protection of brain tissue in cerebral stroke. The present study investigated the neuroprotective effect of sesamin (Sn) to reduce brain injury after middle cerebral artery occlusion (MCAO). The middle cerebral artery (MCA) of adult male Wistar rat was occluded for 2 h and reperfused for 22 h. Sesamin is the most abundant lignan in sesame seed oil is a potent antioxidant. Sesamin (30 mg/kg) was given orally twice, 30 min before the onset of ischemia and 12 h after reperfusion. The initial investigations revealed that sesamin reduced the neurological deficits in terms of behavior and reduced the level of thiobarbituric acid reactive species (TBARS), and protein carbonyl (PC) in the different areas of the brain when compared with the MCAO group. A significantly depleted level of glutathione and its dependent enzymes (glutathione peroxidase [GPx] and glutathione reductase [GR]) in MCAO group were protected significantly in MCAO group treated with sesamin. The present study suggests that sesamin may be able to attenuate the ischemic cell death and plays a crucial role as a neuroprotectant in regulating levels of reactive oxygen species in the rat brain. Thus, sesamin may be a potential compound in stroke therapy.

Background Minocycline provides neurovascular protection reducing acute cerebral injury. However, it is unclear whether minocycline is effective in females. We tested minocycline in both sexes and aged animals using a novel embolic stroke model in mice that closely mimics acute thromboembolic stroke in humans. Methods Five groups of mice were subjected to thromboembolic stroke: adult males, aged males, adult females, aged females, and adult ovariectomized females. They were treated with phosphate saline (vehicle) or minocycline (6 mg/kg) immediately after stroke onset. Behavioral outcomes, infarct volumes and cerebral blood flow were assessed. The effect of minocycline on expression and activity of MMP-9 was analyzed. Results The model resulted in reproducible infarct in the experimental groups. As expected, adult females were significantly more resistant to cerebral ischemic injury than males. This advantage was abolished by aging and ovariectomy. Minocycline significantly reduced the infarct volume (P < 0.0001) and also improved neurologic score (P < 0.0001) in all groups. Moreover, minocycline treatment significantly reduced mortality at 24 hours post stroke (P = 0.037) for aged mice (25% versus 54%). Stroke up-regulated MMP-9 level in the brain, and acute minocycline treatment reduced its expression in both genders (P < 0.0001). Conclusion In a thromboembolic stroke model minocycline is neuroprotective irrespective of mouse sex and age.