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 μg/mL oxLDL, 500 μM Hcy, 500 nM HCTL, 100 μg/mL AGE, 200 μM H 2 O 2 and 200 μM H 2 O 2 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-κB 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 incr...

Permanent cystathionine-β-Synthase gene knockdown promotes inflammation and oxidative stress in immortalized human adipose-derived mesenchymal stem cells, enhancing their adipogenic capacity

“…CBS enzyme inhibition results in increased homocysteine in detrimental of cysthationine levels [ 32 , 33 ]. The pro-oxidant and pro-inflammatory effects of excess homocysteine have been demonstrated in in vitro and in vivo experiments [ [33] , [34] , [35] , [36] , [37] , [38] ]. Otherwise, similar to ASC52telo cells, breast tumor tissue displayed increased CBS, but very low CTH mRNA levels, resulting in increased intracellular levels of cysthationine, and in consequence preserving mitochondrial function and preventing oxidative and endoplasmic reticulum stress [ 39 ].…”

Paraoxonase 1 Phenotype and Protein N-Homocysteinylation in Patients with Rheumatoid Arthritis: Implications for Cardiovascular Disease

“…Protein N-homocysteinylation results in the incorporation of the additional thiol group which may render target protein more susceptible to oxidation. Indeed, oxidative stress is involved in detrimental effects of Hcy thiolactone in different experimental systems [ 55 , 56 , 57 , 58 ]. Thus, decomposing Hcy thiolactone may contribute to antioxidant activity of PON1.…”

Imbalance Between Oxidative Stress and Growth Factors in Human High Myopia

“…The VEGF that derives from RPE is involved in natural choroidal neovascularization regulation (Marneros et al, 2005), and takes care of retina microvasculature by two different ways: VEGFR2-facilitated vasculotrophism and by regulating complement proteins and, consequently, protecting against potential complement-mediated damage (Keir et al, 2017). Nevertheless, many experimental data have related oxidative stress, mainly chronic intracellular oxidative stress, to the persistent danger of RPE cells integrity (Plafker et al, 2012) via NF-kB signaling (AnandBabu et al, 2019). HM is related to RPE thinning (Fujiwara et al, 2009).…”