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Hypoxia-induced retinal neovascularization plays a central role in the pathogenesis of diabetic retinopathy. This study aimed to investigate whether hypoxia leads to the release of nuclear high mobility group box 1 (HMGB1) peptides from cultured retinal pigment epithelial ARPE-19 cells, to determine the effect of HMGB1 on angiogenic cytokine production and elucidate the involved signaling pathways. A chemical hypoxia mimetic agent, cobalt chloride, induced SIRT1 downregulation, HMGB1 nucleocytoplasmic relocation and extracellular release from ARPE-19 cells, implicating its autocrine function. Resveratrol treatment significantly reduced secretion of HMGB1 from ARPE-19 cells exposed to hypoxia. Cell proliferation and cell cycle analyses demonstrated that exogenous HMGB1 caused significant growth suppression and G1 cell cycle arrest in ARPE-19 cells. Morphological observations showed that HMGB1 enhanced adhesion, but suppressed migration of ARPE-19 cells. More intriguingly, HMGB1 up-regulated expression of angiofibrogenic factors in ARPE-19 cells, including VEGF, bFGF, TGF-β2, and CTGF. Signal profiling characterization indicated that HMGB1 triggered hyperphosphorylation of Akt, p38 MAPK, and NF-κB, but not that of ERK, JNK, and Smad2, whereas inhibition of PI3K, MAPK, or NF-κB significantly attenuated the HMGB1-driven cytokine overproduction in ARPE-19 cells. Functional neutralization with anti-TLR4 and -RAGE antibodies confirmed that both receptors were involved in the cytokine overproduction. In conclusion, chemically-mimicked hypoxia induced nucleocytoplasmic relocation and release of HMGB1 peptides, which in turn up-regulated the production of angiofibrogenic factors in RPE cells, thereby contributing to the pathogenesis of hypoxia-associated diabetic retinopathies. Conversely, blockades of intraocular HMGB1 bioavailability or signal activation may prevent angiofibrogenesis in development of diabetic retinopathy. [ABSTRACT FROM AUTHOR]

Methylglyoxal (MGO), a glycolytic metabolite, induces oxidative injury and apoptotic cell death that play a pathogenetic role in age-related macular degeneration (AMD). This study examined the impact of MGO on cell proliferation and autophagy flux in retinal pigment epithelium (RPE) ARPE-19 cells and elucidated the underlying mechanism. Short-term MGO exposure suppressed cell proliferation without induction of apoptotic cell death, increased production of reactive oxygen species, and potentiated H 2 O 2 -exhibited cytotoxicity in ARPE-19 cells. Conversely, pretreatment with N-acetylcysteine, a ROS scavenger, and aminoguanidine, an MGO blocker, prevented MGO-induced growth retardation. MGO significantly enhanced autophagy flux and increased intracellular accumulation of autophagosomes, which was functionally confirmed by addition of autophagy enhancer or inhibitors. Signaling kinetic observation indicated that MGO remarkably triggered phosphorylation of Akt, ERK1/2, p38 MAPK, and JNK1/2. Blockade of kinase activity demonstrated that the hyperphosphorylation of Akt, ERK1/2, JNK, and p38 MAPK were all involved in the MGO-enhanced autophagy and growth-arresting effect in ARPE-19 cells. Moreover, pretreatment with autophagic flux inhibitors including 3-methyladenine, bafilomycin A, and chloroquine effectively ameliorated MGO- but not H 2 O 2 -mediated ARPE-19 cytotoxicity. In conclusion, modulation of autophagy flux activity by using autophagic or kinase inhibitors may be an applicable modality to treat AMD. [ABSTRACT FROM AUTHOR]

Transforming growth factor-β2 (TGF-β2) has been implicated in the pathogenesis of proliferative vitreoretinopathy (PVR) and proliferative diabetic retinopathy (PDR), due to its ability to stimulate the overproduction of pro-angiogenic factors, such as vascular endothelial growth factor (VEGF), and remodeling of the extracellular matrix (ECM). Although intravitreal triamcinolone acetonide (TA) is clinically useful in the treatment of PVR and PDR, its molecular mechanism has yet to be fully elucidated. The present study investigated whether TA treatment altered TGF-β2-driven biological effects on the behavior of cultured human retinal pigment epithelial (RPE) cells, in order to determine which signaling pathway may be essential for the pharmacological action of TA. The R-50 human RPE cell line was treated with TA in the presence of TGF-β2, followed by analyses of cell viability and contraction using cell viability and collagen gel contraction assays. VEGF mRNA expression and protein production were measured using reverse transcription-quantitative PCR and ELISA, respectively. The phosphorylation status of signaling mediators and the protein expression of type I collagen (COL1A1), α-smooth muscle actin (α-SMA), and ECM-remodeling enzymes, including MMP-2 and MMP-9, were analyzed using western blotting. The gelatinolytic activity of MMPs was detected using gelatin zymography. TA treatment exhibited no prominent cytotoxicity but markedly antagonized TGF-β2-induced cytostatic effects on RPE cell viability and TGF-β2-enhanced contractility in collagen gels. In the context of TGF-β2-related signaling, TA significantly attenuated TGF-β2-elicited Smad2, extracellular-regulated kinase (ERK)1/2 and p38 mitogen-activated protein kinase (MAPK) phosphorylation. Moreover, TA markedly mitigated TGF-β2-induced VEGF upregulation through ablation of p38 signaling activity. TA also partially attenuated TGF-β2-elicted expression of COL1A1, α-SMA, MMP-2, and MMP-9, but only suppressed TGF-β2-induced MMP-9 gelatinolytic activity. Mechanistically, the MEK/ERK signaling pathway may have a critical role in the TGF-β2-induced upregulation of COL1A1, α-SMA and MMP-9. In conclusion, TA may be considered a useful therapeutic agent for treating TGF-β2-associated intraocular angiogenesis and tissue remodeling, the underlying mechanism of which may involve the ERK and p38 MAPK signaling pathways. [ABSTRACT FROM AUTHOR]

This study investigated the regulatory role of microRNA 100 (miR-100) in hydrogen peroxide (H2O2)-induced apoptosis of human retinal pigment epithelial ARPE-19 cells. H2O2 induced oxidative cell death of cultured ARPE-19 cells was measured by cytotoxicity assay. qRT-PCR was used to quantify cytosolic and extracellular contents of miR-100. Kinase and miR-100 inhibition treatments were applied to determine the regulatory signaling pathways involved in cell death regulation. H2O2 dose-dependently reduced viability of ARPE-19 cells and simultaneously upregulated miR-100 levels in both cytosolic and extracellular compartments. Western blotting detection indicated that H2O2 elicited hyperphosphorylation of PI3K/Akt, ERK1/2, JNK, p38 MAPK, and p65 NF-κB. Further kinase inhibition experiments demonstrated that PI3K, p38 MAPK, and NF-κB activities were involved in oxidative-stress-induced miR-100 upregulation in ARPE-19 cells, while blockade of PI3K, JNK, and NF-κB signaling significantly attenuated the oxidative cell death. Intriguingly, MiR-100 antagomir treatment exerted a cytoprotective effect against the H2O2-induced oxidative cell death through attenuating the oxidation-induced AMPK hyperphosphorylation, restoring cellular mTOR and p62/SQSTM1 levels and upregulating heme oxygenase-1 expression. These findings support that miR-100 at least in part mediates H2O2-induced cell death of ARPE-19 cells and can be regarded as a preventive and therapeutic target for retinal degenerative disease. [ABSTRACT FROM AUTHOR]

Dyslipidemia-associated diabetic retinopathy is featured by macular edema and retinal angiogenesis. This study investigated the in vitro lipotoxicity of free fatty acids and their modulatory roles in regulation of autophagy and angiogenic factor production in cultured human retinal pigment epithelium (RPE) ARPE-19 cells. ARPE-19 cells were exposed to monounsaturated oleic acid (OA), saturated palmitic acid (PA), or both. Cell viability, cell cycle distribution, migration, and autophagy of the treated cells were monitored. Angiogenic factor production was determined by RT-qPCR and ELISA. OA, but not PA, at doses higher than 500 μM significantly induced cytostasis and lipotoxicity in ARPE-19 cells. OA exposure not only markedly enhanced autophagy flux, but also enhanced cell migration, while PA suppressed motility of RPE cells. Meanwhile, OA stimulated de novo synthesis of angiogenic factors including VEGF and bFGF in ARPE-19 cells. Mechanistically, OA treatment stimulated not only AMPK/mTOR/p70S6K signaling, but also induced hyperphosphorylation of MAPK pathway mediators, including ERK, JNK and p38 MAPK, as well as NF-κB activation. Kinase inhibition assays showed that blockade of PI3K/Akt, MAPK and NF-κB pathways prevented the OA-upregulated VEGF transcription and its peptide release. Comparatively, only NF-κB inhibition significantly suppressed bFGF peptide release from ARPE-19 cells. Out findings support the OA-exhibited cytostasis, autophagy modulation and angiogenic factor production in RPE cells. This study sheds light on the interrelationship between metabolic disorder and retinopathy and provides molecular strategies for preventing and treating choroidal neovascularization in diabetic retinopathy. • Monounsaturated oleic acid, but not saturated palmitic acid induces lipotoxicity in ARPE-19 cells • Oleic acid enhances cell migration and de novo synthesis of angiogenic factors. • Oleic acid modulates autophagy flux via AMPK/mTOR/p70S6K signaling. • Oleic acid-activated MAPK and NF-κB pathways are involved in the VEGF upregulation. • Only NF-κB activity is crucial for the inducible bFGF release from ARPE-19 cells. [ABSTRACT FROM AUTHOR]

Abstract: Geldanamycin (GA), a benzoquinone ansamycin, was originally isolated as a natural product with anti-fungal activity. GA and its analogs, including 17-allylamino-demethoxy geldanamycin (17-AAG), are also known to block the function of a molecular chaperone, heat shock protein 90 (Hsp90). In light of their anti-tumor properties through direct cytotoxicity and anti-angiogenicity, GA has been previously demonstrated to suppress hypoxia-induced VEGF production in retinal pigment epithelium (RPE) cells, implicating its applicability in treating intraocular neovascularization. This study aimed at investigating the effectiveness of Hsp90 inhibitor treatment in suppressing proliferation of cultured human RPE cells and elucidating its underlying mechanism. Cultured RPE cells were treated with GA or 17-AAG and subjected for cell proliferation assay and cell cycle analysis. Expression of apoptotic regulators and survival signaling activity were monitored by Western blotting. The results showed that both GA and 17-AAG significantly inhibited RPE cell proliferation at micromolar levels. Treatment with GA and 17-AAG led to growth arrests in G1 and S phases, increased sub-G1 hypodipoid cell population, induced apoptotic cell death, and upregulated P53 and P21 expression, although the drug-induced Bcl-2 upregulation cannot prevent cell death. Additionally, GA and 17-AAG significantly suppressed constitutive contents of phosphorylated ERK1/2 and total Akt proteins, and completely abrogated wortmannin-sensitized Akt phosphorylation. In conclusion, GA and 17-AAG inhibit RPE cell proliferation and induce cytotoxicity, possibly through downregulating Akt- and ERK1/2-mediated signaling activities. They might potentially constitute a therapeutic agent for ocular disorders with RPE over proliferation, such as proliferative vitreoretinopathy. [Copyright &y& Elsevier]

Abstract: Statins, inhibitors of 3-hydroxy-3-methylglutaryl-coenzye A reductase, possess pleiotropic effects that have been extended to modulation of various cellular behaviors. This study aimed at examining whether atorvastatin (AVN) modulates cell growth, adhesion, migration, and contraction of cultured human retinal pigment epithelium (RPE) cells. The in vitro effects of AVN on human RPE cells was analyzed in terms of cell proliferation, cell cycle, cell adhesion, migration, and contraction assays. The modulatory effect of AVN on TGF-β2-triggered signaling was determined by Western blotting detection. AVN at submicromolar dose exhibited no prominent morphological alteration and cytotoxicity, whereas it elicited cytostatic effect at concentrations higher than 1 μM. Cell cycle analysis showed that AVN induced growth arrest in both G1 and G2/M phases. AVN at 1 μM or higher concentrations significantly suppressed RPE cell adhesion. Cell migration and 3D collagen contraction assays showed that AVN significantly suppressed RPE cell migration and contractility, respectively. Mechanistically, AVN treatment transiently up-regulated phosphorylation of Akt, ERK1/2, and p38 MAPK, whereas down-regulated that of JNK1. Intriguingly, AVN pretreatment prominently attenuated the TGF-β2-mediated non-Smad signaling, including Akt, ERK1/2, p38 MAPK, and JNK1 phosphorylation. Besides, it directly reduced constitutive level of myosin regulatory light chain peptide MYL9 and mitigated the TGF-β2-induced phosphorylation of myosin phosphatase-targeting subunit 1, MYPT1. These in vitro findings strongly suggest that AVN possesses pleiotropic function on RPE cells, including anti-proliferation, anti-adhesion, anti-migration as well as anti-contraction. In conclusion, AVN treatment may be considered a useful therapy for proliferative vitreoretinal diseases. [Copyright &y& Elsevier]