MicroRNA-34a promotes apoptosis of retinal vascular endothelial cells by targeting SIRT1 in rats with diabetic retinopathy.
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ABSTRACT:
SUBMITTER: Ji Q
PROVIDER: S-EPMC7714500 | biostudies-literature |
REPOSITORIES: biostudies-literature
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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.
| S-EPMC7000655 | biostudies-literature
Project description:BackgroundRetinal neovascularization, which is characterized by the increased proliferation, migration, and tube formation of retinal microvascular endothelial cells (RMECs), contributes to the progression of diabetic retinopathy (DR). MiR-409-5p has been reported to be upregulated in peripheral blood of DR patients and in vascular endothelial growth factor (VEGF)-induced RMECs. However, the role of miR-409-5p in retinal neovascularization of DR remains unelucidated.MethodThe expression of miR-409-5p was measured in retinal tissues of streptozocin-induced and db/db diabetic mice, in high glucose-induced mouse RMECs (mRMECs), and in vitreous fluid of proliferative DR patients. Antagomir of miR-409-5p was intravitreally injected into diabetic mice. Proliferation, migration, and tube formation were detected using cell counting kit-8 assay, transwell assay, and microscope observation, respectively. Luciferase reporter assay was used to detect the direct interaction between miR-409-5p and peroxisome proliferator-activated receptor-α (PPARα).ResultMiR-409-5p was upregulated in retinal tissues of diabetic mice, in high glucose-induced mRMECs, and in vitreous fluid of proliferative DR patients. The knockdown of miR-409-5p attenuated retinal neovascularization in vivo. The overexpression of miR-409-5p promotes the proliferation, migration, and tube formation, and increased VEGF expression and secretion, while the knockdown of miR-409-5p suppressed the VEGF-induced retinal neovascularization in vitro. PPARα is a downstream target of miR-409-5p, and PPARα overexpression negated the promotion of miR-409-5p overexpression on the proliferation, migration, and tube formation of mRMECs.ConclusionOur findings demonstrated that miR-409-5p acted as a neovasculogenic factor in DR, and anti-miR-409-5p therapy may provide a novel strategy in treating DR.
| S-EPMC7469530 | biostudies-literature
Project description:OBJECTIVE:Diabetes mellitus causes vascular endothelial dysfunction and alters vascular microRNA expression. We investigated whether endothelial microRNA-34a (miR-34a) leads to diabetic vascular dysfunction by targeting endothelial sirtuin1 (Sirt1) and asked whether the oxidative stress protein p66Shc governs miR-34a expression in the diabetic endothelium. APPROACH AND RESULTS:MiR-34a is upregulated, and Sirt1 downregulated, in aortic endothelium of db/db and streptozotocin-induced diabetic mice. Systemic administration of miR-34a inhibitor, or endothelium-specific knockout of miR-34a, prevents downregulation of aortic Sirt1 and rescues impaired endothelium-dependent aortic vasorelaxation induced by diabetes mellitus. Moreover, overexpression of Sirt1 mitigates impaired endothelium-dependent vasorelaxation caused by miR-34a mimic ex vivo. Systemic infusion of miR-34a inhibitor or genetic ablation of endothelial miR-34a prevents downregulation of endothelial Sirt1 by high glucose. MiR-34a is upregulated, Sirt1 is downregulated, and oxidative stress (hydrogen peroxide) is induced in endothelial cells incubated with high glucose or the free fatty acid palmitate in vitro. Increase of hydrogen peroxide and induction of endothelial miR-34a by high glucose or palmitate in vitro is suppressed by knockdown of p66shc. In addition, overexpression of wild-type but not redox-deficient p66Shc upregulates miR-34a in endothelial cells. P66Shc-stimulated upregulation of endothelial miR-34a is suppressed by cell-permeable antioxidants. Finally, mice with global knockdown of p66Shc are protected from diabetes mellitus-induced upregulation of miR-34a and downregulation of Sirt1 in the endothelium. CONCLUSIONS:These data show that hyperglycemia and elevated free fatty acids in the diabetic milieu recruit p66Shc to upregulate endothelial miR-34a via an oxidant-sensitive mechanism, which leads to endothelial dysfunction by targeting Sirt1.
| S-EPMC5293179 | biostudies-literature
Project description:MicroRNA 34a (miR-34a) is a tumor suppressor gene, but how it regulates cell proliferation is not completely understood. We now show that the microRNA miR-34a regulates silent information regulator 1 (SIRT1) expression. MiR-34a inhibits SIRT1 expression through a miR-34a-binding site within the 3' UTR of SIRT1. MiR-34 inhibition of SIRT1 leads to an increase in acetylated p53 and expression of p21 and PUMA, transcriptional targets of p53 that regulate the cell cycle and apoptosis, respectively. Furthermore, miR-34 suppression of SIRT1 ultimately leads to apoptosis in WT human colon cancer cells but not in human colon cancer cells lacking p53. Finally, miR-34a itself is a transcriptional target of p53, suggesting a positive feedback loop between p53 and miR-34a. Thus, miR-34a functions as a tumor suppressor, in part, through a SIRT1-p53 pathway.
| S-EPMC2533205 | biostudies-literature
Project description:Endothelial progenitor cells (EPCs) are involved in the pathogenesis of microvascular dysfunction in diabetic retinopathy (DR). MicroRNAs (miRNAs) serve as crucial regulators in many biological process and human diseases. Herein, to investigate the expression profile and possible role of miRNAs in EPCs, small RNA sequencing was conducted to identify EPC dysfunction-related miRNAs in DR. A total of 72 miRNAs were differentially expressed in EPCs following high glucose stress. Based on Gene Ontology (GO) analysis, the target genes of differentially expressed miRNAs were targeted to "protein binding," "cell differentiation," and "cytoskeleton." Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that cGMP-PKG signaling pathway was tightly associated with miRNA-mediated EPC function. Furthermore, miR-375-3p was verified to be up-regulated in the clinical samples of DR patients. Inhibition of miR-375-3p protected against hyperglycemic stress- or hypoxic stress-induced EPC injury, which increased the viability, proliferation, migration, and tube formation ability of EPCs and retarded the development of apoptosis. Collectively, this study provides a novel insight into the pathogenesis of EPC dysfunction in DR. miR-375-3p is a potential target for the diagnosis or treatment of DR.
| S-EPMC8717624 | biostudies-literature
Project description:Diabetic retinopathy (DR) is a serious microvascular complication of diabetes and a major cause of blindness in the developing world. Early diabetic retinopathy is characterized by a loss of pericytes and vascular endothelial cells, a breakdown of the blood-retinal barrier, vascular dysfunction and vascular-neuroinflammation. However, optimal treatment options and related mechanisms are still unclear. MicroRNA-126 (miR-126) plays a potential role in the pathogenesis in DR, which may regulate VEGF, Ang-1 and VCAM-1 expressions. This study investigated the therapeutic effects and mechanisms of Niaspan treatment of DR in diabetes (DM) rats. DM rats exhibits significantly decreased miR-126 and tight junction Claudin-5/Occludin/ZO-1 genes expression, and increased Blood retinal-barrier (BRB) breakdown, retinal apoptosis and VEGF/VEGFR, as well as VCAM-1/CD45 expressions in the retina compared to normal control group. Niaspan treatment significantly improved clinical and histopathological outcomes; decreased the expressions of VEGF/VEGFR, VCAM-1/CD45, apoptosis and BRB breakdown, significantly increased tight junction proteins and Ang-1/Tie-2 expressions, as well as increased retinal miR-126 expression compared to non-treatment diabetic rats. These data are the first to show that Niaspan treatment ameliorates DR through its repair vascular and inhibits inflammatory effects, and also suggest that the miR-126 pathway may contribute to Niaspan treatment induced benefit effects.
| S-EPMC4880890 | biostudies-other
Project description:Endothelial cells (EC) play essential roles in retinal vascular homeostasis. This study aimed to characterize retinal EC heterogeneity and functional diversity using single-cell RNA sequencing (scRNA-seq). Systematic analysis of cellular compositions and cell-cell interaction networks identified a unique EC cluster with high inflammatory gene expression in the diabetic retina; sphingolipid metabolism in this cluster is a prominent aspect correlated with changes in retinal function. Among the sphingolipid-related genes, alkaline ceramidase 2 (ACER2) showed the most significant increase. We collected plasma and vitreous samples from patients with non-proliferative diabetic retinopathy (NPDR) and PDR for mass spectrometry analysis. Metabolomic profiling revealed that the ceramide levels were significantly elevated in NPDR and further increased in PDR compared with control patients. In vitro analyses showed that the ACER2 overexpression retarded endothelial barrier breakdown induced by ceramide, while silencing of ACER2 further disrupted the injury. Moreover, intravitreal injection of the recombinant ACER2 adeno-associated virus rescued diabetes-induced vessel leakiness, inflammatory response, and neurovascular disease in diabetic mouse models. Together, this study revealed a new diabetes-specific retinal EC population and a negative feedback regulation pathway that reduces ceramide content and endothelial dysfunction by upregulating ACER2 expression. These findings provide insights into cell-type targeted interventions for diabetic retinopathy.
2024-01-01 | GSE243100 | GEO
Project description:ADAM17, a disintegrin and metalloproteinase 17, is a transmembrane metalloproteinase that regulates bioavailability of multiple membrane-bound proteins via ectodomain shedding. ADAM17 activity was shown to contribute to a number of vascular pathologies, but its role in the context of diabetic retinopathy (DR) is not determined. We found that expression and enzymatic activity of ADAM17 are upregulated in human diabetic postmortem retinas and a mouse model of streptozotocin-induced diabetes. To further investigate the contribution of ADAM17 to vascular alterations associated with DR, we used human retinal endothelial cells (HREC) treated with ADAM17 neutralizing antibodies and exposed to glucidic stress and streptozotocin-induced endothelial ADAM17 knockout mice. Evaluation of vascular permeability, vascular inflammation, and oxidative stress was performed. Loss of ADAM17 in endothelial cells markedly reduced oxidative stress evidenced by decreased levels of superoxide, 3-nitrotyrosine, and 4-hydroxynonenal and decreased leukocyte-endothelium adhesive interactions in vivo and in vitro. Reduced leukostasis was associated with decreased vascular permeability and was accompanied by downregulation of intercellular adhesion molecule-1 expression. Reduction in oxidative stress in HREC was associated with downregulation of NAD(P)H oxidase 4 (Nox4) expression. Our data suggest a role for endothelial ADAM17 in DR pathogenesis and identify ADAM17 as a potential new therapeutic target for DR.
| S-EPMC7073770 | biostudies-literature
Project description:The paucity of animal models exhibiting full pathology of diabetic retinopathy (DR) has impeded understanding of the pathogenesis of DR and the development of therapeutic interventions. Here, we investigated whether hyperhexosemic marmosets (Callithrix jacchus) develop characteristic retinal vascular lesions including macular edema (ME), a leading cause of vision loss in DR. Marmosets maintained on 30% galactose (gal)-rich diet for 2 years were monitored for retinal vascular permeability, development of ME, and morphological characteristics including acellular capillaries (AC) and pericyte loss (PL), vessel tortuosity, and capillary basement membrane (BM) thickness. Excess vascular permeability, increased number of AC and PL, vascular BM thickening, and increased vessel tortuosity were observed in the retinas of gal-fed marmosets. Optical coherence tomography (OCT) images revealed significant thickening of the retinal foveal and the juxtafoveal area, and histological analysis showed incipient microaneurysms in retinas of gal-fed marmosets. Findings from this study indicate that hyperhexosemia can trigger retinal vascular changes similar to those seen in human DR including ME and microaneurysms. The striking similarities between the marmoset retina and the human retina, and the exceptionally small size of the monkey, offer significant advantages to this primate model of DR.
| S-EPMC4477345 | biostudies-literature
Project description:AimTo detect the effect of connective tissue growth factor (CTGF) on the apoptosis in the diabetic retina with small interfering RNAs (siRNA) targeting CTGF.MethodsA total of 60 rats were divided into 6 groups including control group, diabetic 4, 8, 12, 16 weeks groups, and interference group. Diabetic rats were induced by intraperitoneal streptozotocin (STZ). Retinas were obtained from control, diabetic rats and diabetic rats of interference group treated by intravitreal injection of CTGFsiRNA to suppress the expression of CTGF mRNA. Retinal cells apoptosis was detected by Tunnel staining and mRNA expression of CTGF was analyzed by RT-PCR.ResultsThe levels of CTGF and the apoptosis in the retinas of diabetic rats were significantly higher than those in the controls. Apoptosis occurred at 4 weeks after a diabetic model being set up, became serious with the diabetes developing, while CTGF elevated at 8 weeks. The apoptosis cell counts increased to 25.8cells/mm(2) at 24weeks of diabetes. SiRNA-mediated inhibition of CTGF mRNA resulted in a significant decrease in apoptosis. Significant correlations were found between CTGF and apoptosis in the retina.ConclusionIt was suggested that CTGF might be involved in retinal cells apoptosis which is a characteristic of early diabetic retina. SiRNA targeting CTGF seems to have the advantage of ameliorating retinal cells apoptosis.
| S-EPMC3340772 | biostudies-literature