High Glucose and Hypoxia-Mediated Damage to Human Brain Microvessel Endothelial Cells Induces an Altered, Pro-Inflammatory Phenotype in BV-2 Microglia In Vitro

Diabetes is strongly linked to the development of Alzheimer’s disease (AD), though the mechanisms for this enhanced risk are unclear. Because vascular inflammation is a consistent feature of both diabetes and AD, the cerebral microcirculation could be a key target for the effects of diabetes in the brain. The goal of this study is to explore whether brain endothelial cells, injured by diabetes-related insults, glucose and hypoxia, can affect inflammatory and activation processes in microglia in vitro. Human brain microvascular endothelial cells (HBMVECs) were either treated with 5 mM glucose (control), 30 mM glucose (high glucose), exposed to hypoxia, or exposed to hypoxia plus high glucose. HBMVEC-conditioned medium was then used to treat BV-2 microglia. Alterations in microglia phenotype were assessed through measurement of nitric oxide (NO), cytokine production, microglial activation state markers, and microglial phagocytosis. HBMVECs were injured by exposure to glucose and/or hypoxia, as assessed by release of LDH, interleukin (IL)-1β, and reactive oxygen species (ROS). HBMVECs injured by glucose and hypoxia induced increases in microglial production of NO, tumor necrosis factor-α (TNFα) and matrix metalloproteinase (MMP)-9. Injured HBMVECs significantly increased microglial expression of CD11c and CLEC7A, and decreased expression of the homeostatic marker P2RY12. Finally, bead uptake by BV-2 cells, an index of phagocytic ability, was elevated by conditioned media from injured HBMVECs. The demonstration that injury to brain endothelial cells by diabetic-associated insults, glucose and hypoxia, promotes microglial inflammation supports the idea that the cerebral microcirculation is a critical locus for the deleterious effects of diabetes in the AD brain

Dabigatran reduces thrombin-induced neuroinflammation and AD markers in vitro: Therapeutic relevance for Alzheimer\u27s disease

Background: Vascular risk factors such as atherosclerosis, diabetes, and elevated homocysteine levels are strongly correlated with onset of Alzheimer\u27s disease (AD). Emerging evidence indicates that blood coagulation protein thrombin is associated with vascular and non-vascular risk factors of AD. Here, we examined the effect of thrombin and its direct inhibitor dabigatran on key mediators of neuro-inflammation and AD pathology in the retinoic acid (RA)-differentiated human neuroblastoma cell line SH-SY5Y. Methods: SH-SY5Y cells exposed to thrombin concentrations (10–100 nM) +/- 250 nM dabigatran for 24 h were analyzed for protein and gene expression. Electrophoretic mobility shift assay (EMSA) was used to determine DNA binding of NFkB. Western blotting, qRT-PCR and ELISA were used to measure the protein, mRNA, and activity levels of known AD hallmarks and signaling molecules. Results: Dabigatran treatment attenuated thrombin-induced increase in DNA binding of NFκB by 175% at 50 nM and by 77% at 100 nM thrombin concentration. Thrombin also augmented accumulation of Aβ protein expression and phosphorylation of p38 MAPK, a downstream molecule in the signaling cascade, expression of pro-apoptotic mediator caspase 3, APP, tTau and pTau. Additionally, thrombin increased BACE1 activity, GSK3β expression, and APP, BACE1, Tau and GSK3β mRNA levels. Co-incubation with dabigatran attenuated thrombin-induced increases in the protein, mRNA, and activities of the aforesaid molecules to various extents (between −31% and −283%). Conclusion: Our data demonstrates that thrombin promotes AD-related pathological changes in neuronal cultures and suggests that use of direct oral anticoagulants may provide a therapeutic benefit against thrombin-driven neuroinflammation and downstream pathology in AD

Dabigatran reduces thrombin-induced neuroinflammation and AD markers in vitro: Therapeutic relevance for Alzheimer's disease

Background: Vascular risk factors such as atherosclerosis, diabetes, and elevated homocysteine levels are strongly correlated with onset of Alzheimer's disease (AD). Emerging evidence indicates that blood coagulation protein thrombin is associated with vascular and non-vascular risk factors of AD. Here, we examined the effect of thrombin and its direct inhibitor dabigatran on key mediators of neuro-inflammation and AD pathology in the retinoic acid (RA)-differentiated human neuroblastoma cell line SH-SY5Y. Methods: SH-SY5Y cells exposed to thrombin concentrations (10–100 nM) +/- 250 nM dabigatran for 24 h were analyzed for protein and gene expression. Electrophoretic mobility shift assay (EMSA) was used to determine DNA binding of NFkB. Western blotting, qRT-PCR and ELISA were used to measure the protein, mRNA, and activity levels of known AD hallmarks and signaling molecules. Results: Dabigatran treatment attenuated thrombin-induced increase in DNA binding of NFκB by 175% at 50 nM and by 77% at 100 nM thrombin concentration. Thrombin also augmented accumulation of Aβ protein expression and phosphorylation of p38 MAPK, a downstream molecule in the signaling cascade, expression of pro-apoptotic mediator caspase 3, APP, tTau and pTau. Additionally, thrombin increased BACE1 activity, GSK3β expression, and APP, BACE1, Tau and GSK3β mRNA levels. Co-incubation with dabigatran attenuated thrombin-induced increases in the protein, mRNA, and activities of the aforesaid molecules to various extents (between −31% and −283%). Conclusion: Our data demonstrates that thrombin promotes AD-related pathological changes in neuronal cultures and suggests that use of direct oral anticoagulants may provide a therapeutic benefit against thrombin-driven neuroinflammation and downstream pathology in AD

The 5HT 1a Receptor Agonist 8-Oh DPAT Induces Protection from Lipofuscin Accumulation and Oxidative Stress in the Retinal Pigment Epithelium

Age-related macular degeneration (AMD), a major cause of blindness in the elderly, is associated with oxidative stress, lipofuscin accumulation and retinal degeneration. The aim of this study was to determine if a 5-HT1A receptor agonist can reduce lipofuscin accumulation, reduce oxidative damage and prevent retinal cell loss both in vitro and in vivo. Autophagyderived and photoreceptor outer segment (POS)-derived lipofuscin formation was assessed using FACS analysis and confocal microscopy in cultured retinal pigment epithelial (RPE) cells in the presence or absence of the 5-HT1A receptor agonist, 8-OH DPAT. 8-OH DPAT treatment resulted in a dose-dependent reduction in both autophagy- and POS-derived lipofuscin compared to control. Reduction in autophagy-induced lipofuscin was sustained for 4 weeks following removal of the drug. The ability of 8-OH DPAT to reduce oxidative damage following exposure to 200 mM H 2O 2 was assessed. 8-OH DPAT reduced superoxide generation and increased mitochondrial superoxide dismutase (MnSOD) levels and the ratio of reduced glutathione to the oxidized form of glutathione in H2O2-treated cells compared to controls and protected against H 2O 2-initiated lipid peroxidation, nitrotyrosine levels and mitochondrial damage. SOD2 knockdown mice, which have an AMD-like phenotype, received daily subcutaneous injections of either saline, 0.5 or 5.0 mg/kg 8-OH DPAT and were evaluated at monthly intervals. Systemic administration of 8-OH DPAT improved the electroretinogram response in SOD2 knockdown eyes of mice compared to knockdown eyes receiving vehicle control. There was a significant increase in th

8-OH DPAT reduces superoxide anion generation and increases antioxidant capacity in cultured RPE cells.

<p>Cells were exposed to H₂O₂ (200 µM) for 1 hour and either pre-or post treated with 8-OH DPAT (10 µM) for 24 hours. In the case of pretreatment all measurements were made 24 hr after H₂O₂ and for post treatment 8-OH DPAT was added immediately following H₂O₂ exposure. <b>A</b> - Superoxide generation was measured using FACS analysis following staining with MitoSOX and results are expressed as the mean fluorescence intensity. <b>B</b> - SOD2 levels were determined by Western blot analysis. <b>C</b> - The ratio of reduced glutathione (GSH) to the oxidized form of glutathione (GSSG) was measured by ELISA. Data represent the mean of three experiments.</p

8-OH DPAT decreases oxidative stress in the RPE of SOD2 knockdown eyes of mice.

<p>Eyes received injection of AAV-<i>SOD2</i> ribozyme or AAV-mCherry and animals received subcutaneous 8-OH DPAT or saline control for up to 4 months. Sections were stained for 8-hydroxydeoxyguanosine (8OHdG) (green). <b>A</b> – Representative sections of 8OHdG expression in the RPE of control and SOD2 eyes receiving different concentrations of 8-OH DPAT. <b>B</b> - Graph shows quantitation of 8OHdG fluorescence in the RPE layer.</p

8-OH DPAT reduces lipofuscin accumulation in cultured RPE cells.

<p><b>A</b> - The effect of different concentrations of 8-OH DPAT on lipofuscin accumulation from autophagy in human ARPE19 cells. Cells were maintained in basal medium and received 10 µM 8-OH DPAT every two days for four weeks. <b>B</b> - Autofluorescent intensity of ARPE19 cells treated with 10 µM 8-OH DPAT every two days for up to 4 weeks. Control cells received vehicle alone. <b>C</b> - Fluorescent micrographs of ARPE19 cells as described in B showing reduced lipofuscin granules in 8-OH DPAT treated cells compared to controls. <b>D</b> - Autofluorescent intensity of ARPE19 cells fed POS treated with 10 µM 8-OH DPAT every two days for up to 14 days. Control cells received POS and saline vehicle alone. <b>E</b> - Fluorescent micrographs of ARPE19 cells described in D showing reduced lipofuscin granule accumulation in 8-OH DPAT treated cells fed POS compared to untreated controls. Fluorescence intensity was determined by flow cytometric analysis. Data represent the mean of three experiments. Bar marker is 50 µM.</p

ERG a-wave and b-wave amplitudes and retinal thickness measured in AAV-ribozyme SOD2 knockdown eyes and control eyes treated with 8-OH DPAT.

<p>Eyes received injection of AAV-<i>SOD2</i> ribozyme or AAV-mCherry and animals received subcutaneous 8-OH DPAT or saline control for up to 4 months. ERGs were obtained at 1 and 4 months following virus injection: <b>A & B</b> - ERG wave-forms from mice treated with AAV-mCherry (<b>A</b>) or AAV-VMD2-<i>SOD2</i> Rz (<b>B</b>). Simultaneous full-field ERG measurements were recorded in dark-adapted mice four months after injection with AAV. The scale units on the ordinate are 100 microvolts. The graphs show wave forms for mice treated with saline, blue lines, low dose (0.5 mg/kg) 8-HO-DPAT, red lines, or high dose (5 mg/kg) 8-HO-DPAT, green lines. <b>C</b> – a-wave; <b>D</b> – b-wave. <b>E</b> - ONL thickness measured by SD-OCT at 1 and 4 months. Untreated wild type animals acted as the baseline control. (10 animals per group, P≤0.01 for all doses and time points).</p