ABSTRACT
Retinal pigment epithelial (RPE) cells can undergo different forms of cell death, including autophagy-associated cell death during age-related macular degeneration (AMD). Failure of macrophages or dendritic cells (DCs) to engulf the different dying cells in the retina may result in the accumulation of debris and progression of AMD. ARPE-19 and primary human RPE cells undergo autophagy-associated cell death upon serum depletion and oxidative stress induced by hydrogen peroxide (H2O2). Autophagy was revealed by elevated light-chain-3 II (LC3-II) expression and electron microscopy, while autophagic flux was confirmed by blocking the autophago-lysosomal fusion using chloroquine (CQ) in these cells. The autophagy-associated dying RPE cells were engulfed by human macrophages, DCs and living RPE cells in an increasing and time-dependent manner. Inhibition of autophagy by 3-methyladenine (3-MA) decreased the engulfment of the autophagy-associated dying cells by macrophages, whereas sorting out the GFP-LC3-positive/autophagic cell population or treatment by the glucocorticoid triamcinolone (TC) enhanced it. Increased amounts of IL-6 and IL-8 were released when autophagy-associated dying RPEs were engulfed by macrophages. Our data suggest that cells undergoing autophagy-associated cell death engage in clearance mechanisms guided by professional and non-professional phagocytes, which is accompanied by inflammation as part of an in vitro modeling of AMD pathogenesis.
Subject(s)
Autophagy/drug effects , Culture Media, Serum-Free/pharmacology , Hydrogen Peroxide/pharmacology , Macrophages/drug effects , Phagocytosis/drug effects , Retinal Pigment Epithelium/drug effects , Adenine/analogs & derivatives , Adenine/pharmacology , Autophagy/genetics , Biomarkers/metabolism , Cell Line , Chloroquine/pharmacology , Coculture Techniques , Gene Expression , Genes, Reporter , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Humans , Interleukin-6/biosynthesis , Interleukin-6/metabolism , Interleukin-8/biosynthesis , Interleukin-8/metabolism , Macrophages/cytology , Macrophages/immunology , Macular Degeneration/genetics , Macular Degeneration/immunology , Macular Degeneration/pathology , Microtubule-Associated Proteins/genetics , Microtubule-Associated Proteins/immunology , Models, Biological , Oxidative Stress , Primary Cell Culture , Retinal Pigment Epithelium/immunology , Retinal Pigment Epithelium/pathology , Triamcinolone/pharmacologyABSTRACT
The aim of this study is to investigate the effect of sera obtained from patients of Crohn's disease treated by anti-TNF-alpha antibody (Infliximab) on the expression of endothelial nitric oxide synthase (eNOS) and vascular endothelial growth factor receptor-2 (VEGFR2) protein in human umbilical vein endothelial cells (HUVEC) cultured in vitro. HUVEC was cultured in the presence of sera derived from patients before and after treatment, or from healthy individuals. Effects of sera on the expression of eNOS and VEGFR2 were monitored by determination of mRNA and protein levels using real time quantitative PCR and Western blot analysis, respectively. The serum of Crohn's patients contained elevated levels of TNF-alpha (34±1.80 pg/mL), which resulted in a decrease in the protein level of eNOS in HUVEC with a simultaneous induction of VEGFR2. Infliximab treatment normalized the expression level of these proteins by decreasing TNF-alpha level, particularly in those cases when clinical healing was also recorded, and it also conferred restitution of the level of angiogenic cytokines. Results suggest that altered angiogenesis possibly contributes to the initiation and perpetuation of inflammatory processes in inflammatory bowel disease (IBD). Endothelial dysfunction, a selective feature of Crohn's disease is beneficially affected by intravascular TNF-alpha neutralization.