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Oxidized LDL, homocysteine, homocysteine thiolactone and advanced glycation end products act as pro-oxidant metabolites inducing cytokine release, macrophage infiltration and pro-angiogenic effect in ARPE-19 cells.

ABSTRACT: Age-related Macular Degeneration (AMD) is one of the major vision-threatening diseases of the eye. Oxidative stress is one of the key factors in the onset and progression of AMD. In this study, metabolites associated with AMD pathology more so at the systemic level namely, oxidized LDL (oxLDL), homocysteine (Hcy), homocysteine thiolactone (HCTL), advanced glycation end product (AGE) were evaluated for their pro-oxidant nature in a localized ocular environment based on in vitro studies in human retinal pigment epithelial cells (ARPE-19 cells). Human ARPE-19 cells were treated with pro-oxidants 50 ?g/mL oxLDL, 500 ?M Hcy, 500 nM HCTL, 100 ?g/mL AGE, 200 ?M H2O2 and 200 ?M H2O2 with and without pre-treatment of 5 mM N-acetyl cysteine (NAC). The cytokines IL-6, IL-8 and vascular endothelial growth factor (VEGF) secreted from ARPE-19 cells exposed to pro-oxidants were estimated by ELISA. In vitro angiogenesis assay was performed with conditioned media of the pro-oxidant treated ARPE-19 cells in Geltrex-Matrigel coated 96-well plate. The human acute monocytic leukemia cell line (THP-1) was differentiated into macrophages and its migration in response to conditioned media of ARPE-19 cells insulted with the pro-oxidants was studied by transwell migration assay. Western blot was performed to detect the protein expression of Bax, Bcl-2 and NF-?B to assess apoptotic changes. The compounds involved in the study showed a significant increase in reactive oxygen species (ROS) generation in ARPE-19 cells (oxLDL; Hcy; AGE: p < 0.001 and HCTL: p < 0.05). NAC pre-treatment significantly lowered the oxidative stress brought about by pro-oxidants as seen by lowered ROS and MDA levels in the cells. Treatment with pro-oxidants significantly increased the secretion of IL-6 (oxLDL: p < 0.05; Hcy, HCTL and AGE: p < 0.01) and IL-8 cytokines (oxLDL: p < 0.05; HCTL: p <. 001 and AGE: p < 0.01) in ARPE-19 cells. Serum samples of AMD patients (n = 23) revealed significantly higher IL-6 and IL-8 levels compared to control subjects (n = 23) (IL6: p < 0.01 and IL8: p < 0.05). The pro-oxidants also promoted VEGF secretion by ARPE-19 cells compared to untreated control (oxLDL: p < 0.001; Hcy: p < 0.01; HCTL and AGE: p < 0.05). In vitro angiogenesis assay showed that the conditioned media significantly increased the tube formation in RF/6A endothelial cells. Transwell migration assay revealed significant infiltration of macrophages in response to pro-oxidants. We further demonstrated that the pro-oxidants increased the Bax/Bcl-2 ratio and increased the NF-?B activation resulting in pro-apoptotic changes in ARPE-19 cells. Thus, oxLDL, Hcy, HCTL and AGE act as pro-oxidant metabolites in RPE that promote AMD through oxidative stress, inflammation, chemotaxis and neovascularization.

SUBMITTER: AnandBabu K

PROVIDER: S-EPMC6516731 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Oxidized LDL, homocysteine, homocysteine thiolactone and advanced glycation end products act as pro-oxidant metabolites inducing cytokine release, macrophage infiltration and pro-angiogenic effect in ARPE-19 cells.

AnandBabu Kannadasan K Sen Parveen P Angayarkanni Narayanasamy N

PloS one 20190514 5

Age-related Macular Degeneration (AMD) is one of the major vision-threatening diseases of the eye. Oxidative stress is one of the key factors in the onset and progression of AMD. In this study, metabolites associated with AMD pathology more so at the systemic level namely, oxidized LDL (oxLDL), homocysteine (Hcy), homocysteine thiolactone (HCTL), advanced glycation end product (AGE) were evaluated for their pro-oxidant nature in a localized ocular environment based on in vitro studies in human r ...[more]

PMID: 31086404

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Project description:Genetic or nutritional deficiencies in homocysteine (Hcy) metabolism lead to hyperhomocysteinemia (HHcy) and cause endothelial dysfunction, a hallmark of atherosclerosis. In addition to Hcy, related metabolites accumulate in HHcy but their role in endothelial dysfunction is unknown. Here, we examine how Hcy-thiolactone, N-Hcy-protein, and Hcy affect gene expression and molecular pathways in human umbilical vein endothelial cells. We used microarray technology, real-time quantitative polymerase chain reaction, and bioinformatic analysis with PANTHER, DAVID, and Ingenuity Pathway Analysis (IPA) resources. We identified 47, 113, and 30 mRNAs regulated by N-Hcy-protein, Hcy-thiolactone, and Hcy, respectively, and found that each metabolite induced a unique pattern of gene expression. Top molecular pathways affected by Hcy-thiolactone were chromatin organization, one-carbon metabolism, and lipid-related processes [-log(P value) = 20-31]. Top pathways affected by N-Hcy-protein and Hcy were blood coagulation, sulfur amino acid metabolism, and lipid metabolism [-log(P value)] = 4-11; also affected by Hcy-thiolactone, [-log(P value) = 8-14]. Top disease related to Hcy-thiolactone, N-Hcy-protein, and Hcy was 'atherosclerosis, coronary heart disease' [-log(P value) = 9-16]. Top-scored biological networks affected by Hcy-thiolactone (score = 34-40) were cardiovascular disease and function; those affected by N-Hcy-protein (score = 24-35) were 'small molecule biochemistry, neurological disease,' and 'cardiovascular system development and function'; and those affected by Hcy (score = 25-37) were 'amino acid metabolism, lipid metabolism,' 'cellular movement, and cardiovascular and nervous system development and function.' These results indicate that each Hcy metabolite uniquely modulates gene expression in pathways important for vascular homeostasis and identify new genes and pathways that are linked to HHcy-induced endothelial dysfunction and vascular disease.

Project description:Background and objectivesThe primary goals were to re-examine whether sevelamer carbonate (SC) reduces advanced glycation end products (AGEs) (methylglyoxal and carboxymethyllysine [CML]), increases antioxidant defenses, reduces pro-oxidants, and improves hemoglobin A1c (HbA1c) in patients with type 2 diabetes mellitus (T2DM) and diabetic kidney disease (DKD). Secondary goals examined albuminuria, age, race, sex, and metformin prescription.Design, setting, participants, & measurementsThis two-center, randomized, intention-to-treat, open-label study evaluated 117 patients with T2DM (HbA1c >6.5%) and stages 2-4 DKD (urinary albumin/creatinine ratio ≥200 mg/g) treated with SC (1600 mg) or calcium carbonate (1200 mg), three times a day, without changing medications or diet. Statistical analyses used linear mixed models adjusted for randomization levels. Preselected subgroup analyses of sex, race, age, and metformin were conducted.ResultsSC lowered serum methylglyoxal (95% confidence interval [CI], -0.72 to -0.29; P<0.001), serum CML (95% CI, -5.08 to -1.35; P≤0.001), and intracellular CML (95% CI, -1.63 to -0.28; P=0.01). SC increased anti-inflammatory defenses, including nuclear factor like-2 (95% CI, 0.58 to 1.29; P=0.001), AGE receptor 1 (95% CI, 0.23 to 0.96; P=0.001), NAD-dependent deacetylase sirtuin-1 (95% CI, 0.20 to 0.86; P=0.002), and estrogen receptor α (95% CI, 1.38 to 2.73; P ≤0.001). SC also decreased proinflammatory factors such as TNF receptor 1 (95% CI, -1.56 to -0.72; P≤0.001) and the receptor for AGEs (95% CI, -0.58 to 1.53; P≤0.001). There were no differences in HbA1c, GFR, or albuminuria in the overall group. Subanalyses showed that SC lowered HbA1c in women (95% CI, -1.71 to -0.27; P=0.01, interaction P=0.002), and reduced albuminuria in those aged <65 years (95% CI, -1.15 to -0.07; P=0.03, interaction P=0.02) and non-Caucasians (95% CI, -1.11 to -0.22; P=0.003, interaction P≤0.001), whereas albuminuria increased after SC and calcium carbonate in Caucasians.ConclusionsSC reduced circulating and cellular AGEs, increased antioxidants, and decreased pro-oxidants, but did not change HbA1c or the albumin/creatinine ratio overall in patients with T2DM and DKD. Because subanalyses revealed that SC may reduce HbA1c and albuminuria in some patients with T2DM with DKD, further studies may be warranted.

Dataset Information

Oxidized LDL, homocysteine, homocysteine thiolactone and advanced glycation end products act as pro-oxidant metabolites inducing cytokine release, macrophage infiltration and pro-angiogenic effect in ARPE-19 cells.

Publications

Oxidized LDL, homocysteine, homocysteine thiolactone and advanced glycation end products act as pro-oxidant metabolites inducing cytokine release, macrophage infiltration and pro-angiogenic effect in ARPE-19 cells.

Similar Datasets

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