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Angiopoietin 2 induces astrocyte apoptosis via ?v?5-integrin signaling in diabetic retinopathy.

ABSTRACT: The vascular leakage in diabetic retinopathy leads to macular edema and vision loss. Although astrocyte play an important role in regulating blood-brain barrier integrity in the brain, the precise role of astrocyte in blood-retinal barrier was yet to be elucidated. This study aimed to investigate the role of angiopoietin 2 (Ang2) in astrocyte loss and vascular leakage in the early streptozotocin-induced diabetic retinopathy. We demonstrated that vascular leakage occurred with astrocyte loss in early diabetic mice retina as Ang2 increased. The astrocyte loss and vascular leakage were inhibited by intravitreal injection of Ang2-neutralizing antibody. In vitro, Ang2 aggravated high glucose-induced astrocyte apoptosis via GSK-3? activation. Ang2 directly bound to ?v?5 integrin, which was abundant in astrocyte, and the blockade of ?v?5 integrin, in vitro, effectively attenuated Ang2-induced astrocyte apoptosis. In vivo, intravitreal injection of anti-?v?5-integrin antibody inhibited astrocyte loss in early diabetic retinopathy. Taken together, Ang2 induced astrocyte apoptosis under high glucose via ?v?5-integrin/GSK-3?/?-catenin pathway. Therefore, we suggest that Ang2/integrin signaling could be a potential therapeutic target to prevent the vascular leakage by astrocyte loss in early diabetic retinopathy.

SUBMITTER: Yun JH

PROVIDER: S-EPMC5399183 | biostudies-literature | 2016 Feb

REPOSITORIES: biostudies-literature

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Angiopoietin 2 induces astrocyte apoptosis via αvβ5-integrin signaling in diabetic retinopathy.

Yun J-H JH Park S W SW Kim J H JH Park Y-J YJ Cho C-H CH Kim J H JH

Cell death & disease 20160218

The vascular leakage in diabetic retinopathy leads to macular edema and vision loss. Although astrocyte play an important role in regulating blood-brain barrier integrity in the brain, the precise role of astrocyte in blood-retinal barrier was yet to be elucidated. This study aimed to investigate the role of angiopoietin 2 (Ang2) in astrocyte loss and vascular leakage in the early streptozotocin-induced diabetic retinopathy. We demonstrated that vascular leakage occurred with astrocyte loss in e ...[more]

PMID: 26890140

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Project description:HIV-1 glycoprotein 120 (gp120) is known to cause neurotoxicity via several mechanisms including production of proinflammatory cytokines/chemokines and oxidative stress. Likewise, drug abuse is thought to have a direct impact on the pathology of HIV-associated neuroinflammation through the induction of proinflammatory cytokines/chemokines and oxidative stress. In the present study, we demonstrate that gp120 and methamphetamine (MA) causes apoptotic cell death by inducing oxidative stress through the cytochrome P450 (CYP) and NADPH oxidase (NOX) pathways. The results showed that both MA and gp120 induced reactive oxygen species (ROS) production in concentration- and time-dependent manners. The combination of gp120 and MA also induced CYP2E1 expression at both mRNA (1.7±0.2- and 2.8±0.3-fold in SVGA and primary astrocytes, respectively) and protein (1.3±0.1-fold in SVGA and 1.4±0.03-fold in primary astrocytes) levels, suggesting the involvement of CYP2E1 in ROS production. This was further confirmed by using a selective inhibitor of CYP2E1, diallylsulfide (DAS), and CYP2E1 knockdown using siRNA, which significantly reduced ROS production (30-60%). As the CYP pathway is known to be coupled with the NOX pathway, including Fenton-Weiss-Haber (FWH) reaction, we examined whether the NOX pathway is also involved in ROS production induced by either gp120 or MA. Our results showed that selective inhibitors of NOX, diphenyleneiodonium (DPI), and FWH reaction, deferoxamine (DFO), also significantly reduced ROS production. These findings were further confirmed using specific siRNAs against NOX2 and NOX4 (NADPH oxidase family). We then showed that gp120 and MA both induced apoptosis (caspase-3 activity and DNA lesion using TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling) assay) and cell death. Furthermore, we showed that DAS, DPI, and DFO completely abolished apoptosis and cell death, suggesting the involvement of CYP and NOX pathways in ROS-mediated apoptotic cell death. In conclusion, this is the first report on the involvement of CYP and NOX pathways in gp120/MA-induced oxidative stress and apoptotic cell death in astrocytes, which has clinical implications in neurodegenerative diseases, including neuroAIDS.

Project description:BackgroundDiabetic retinopathy (DR) is a main complication of diabetes mellitus (DM). Recent studies have implicated microRNAs in human retinal microvascular endothelial cell (HRMEC) dysfunction. In this study, we aim to investigate the apoptotic promotion of miR-29b-3p by blocking SIRT1 in HRMEC for DR situation.MethodBlood samples were obtained from DR patients and controls. Dual-luciferase reporter assay using HEK-293T cells was performed to show the direct interaction of miR-29b-3p and the 3'UTR of SIRT1. HRMECs were exposed to 5.5 mmol/L of glucose (normal control), 5.5 mmol/L of glucose and 24.5 mmol/L of mannitol (osmotic pressure control), 30 mmol/L of glucose [hyperglycemia (HG)], 150 μmol/L of CoCl2 (hypoxia), and 30 mmol/L of glucose plus 150 μmol/L of CoCl2 (HG-CoCl2). To identify the regulating relationship between miR-29b-3p and SIRT1, HRMECs were transfected with miR-29b-3p mimics/inhibitors or their negative controls. SRT1720 was used as a SIRT1 agonist. Cell viability was assessed with the cell counting kit-8 (CCK-8) assay, and apoptotic cells were stained by one-step terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay kit. Gene and protein expression were assayed by quantitative real-time reverse transcriptase-PCR (RT-qPCR) and western blotting separately.ResultMiR-29b-3p was upregulated to 3.2-fold, and SIRT1 protein was downregulated to 65% in DR patients. Dual-luciferase reporter assay showed the direct interaction of miR-29b-3p and SIRT1. HRMECs were identified as >95% positive for CD31 and von Willebrand factor (vWF). MiR-29b-3p and Bax/Bcl-2 ratio was upregulated, whereas SIRT1 was downregulated in HRMECs in the HG-CoCl2 condition. Decreased cell viability and upregulated apoptosis were also found in HRMECs of the HG-CoCl2 condition. Upregulated miR-29b-3p decreased the expression of SIRT1 and increased the ratio of Bax/Bcl-2, whereas downregulated miR-29b-3p increased the expression of SIRT1 protein and downregulated the ratio of Bax/Bcl-2. SRT1720 rescued miR-29b-3p-induced HRMEC apoptosis via upregulating the expression of SIRT1 protein.ConclusionThe dysregulation of miR-29b-3p/SIRT1 is a potential mechanism of HRMEC apoptosis in DR. MiR-29b-3p/SIRT1 may be a potential therapeutic target for DR.

Dataset Information

Angiopoietin 2 induces astrocyte apoptosis via ?v?5-integrin signaling in diabetic retinopathy.

Publications

Angiopoietin 2 induces astrocyte apoptosis via αvβ5-integrin signaling in diabetic retinopathy.

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