Dataset Information

Novel Roles of Chloroquine and Hydroxychloroquine in Graves' Orbitopathy Therapy by Targeting Orbital Fibroblasts.

ABSTRACT: CONTEXT:Graves' orbitopathy (GO) causes infiltrative exophthalmos by inducing excessive proliferation, adipogenesis, and glycosaminoglycan production in orbital fibroblasts (OFs). Interference with OF autophagy is a potential therapy for proptosis. OBJECTIVES:Here, we aimed to evaluate the effects of chloroquine (CQ) and hydroxychloroquine (HCQ), the autophagy inhibitors commonly used in clinical practice, on OFs. DESIGN/SETTING/PARTICIPANTS:OFs isolated from patients with GO (GO-OFs) or control individuals (non-GO-OFs) were cultured in proliferation medium (PM) or subjected to differentiation medium. OFs were treated with CQ or HCQ (0, 0.5, 2, and 10 ?M), and subsequently examined in vitro. MAIN OUTCOME MEASURES:CCK-8, EdU incorporation, and flow cytometry assays were used to assess cellular viability. Adipogenesis was assessed with Western blot analysis, real-time polymerase chain reaction (PCR) , and Oil Red O staining. Hyaluronan production was determined by real-time PCR and enzyme-linked immunosorbent assay. Autophagy flux was detected through red fluorescent protein (RFP)-green fluorescent protein (GFP)-LC3 fluorescence staining and Western blot analyses. RESULTS:CQ/HCQ halted proliferation and adipogenesis in GO-OFs in a concentration-dependent manner through blockage of autophagy, phenotypes that were not detected in non-GO-OFs. The inhibitory effect of CQ/HCQ on hyaluronan secretion of GO-OFs was also concentration dependent, mediated by downregulation of hyaluronan synthase 2 rather than hyaluronidases. Moreover, CQ (10 ?M) induced GO-OF apoptosis without aggravating oxidative stress. CONCLUSIONS:The antimalarials CQ/HCQ affect proliferation, adipogenesis, and hyaluronan generation in GO-OFs by inhibiting autophagy, providing evidence that they can be used to treat GO as autophagy inhibitors.

SUBMITTER: Guo Y

PROVIDER: S-EPMC7183395 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature

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Novel Roles of Chloroquine and Hydroxychloroquine in Graves' Orbitopathy Therapy by Targeting Orbital Fibroblasts.

Guo Yan Y Li Hai H Chen Xueying X Yang Huasheng H Guan Hongyu H He Xiaoying X Chen Yuxin Y Pokharel Sunil S Xiao Haipeng H Li Yanbing Y

The Journal of clinical endocrinology and metabolism 20200601 6

<h4>Context</h4>Graves' orbitopathy (GO) causes infiltrative exophthalmos by inducing excessive proliferation, adipogenesis, and glycosaminoglycan production in orbital fibroblasts (OFs). Interference with OF autophagy is a potential therapy for proptosis.<h4>Objectives</h4>Here, we aimed to evaluate the effects of chloroquine (CQ) and hydroxychloroquine (HCQ), the autophagy inhibitors commonly used in clinical practice, on OFs.<h4>Design/setting/participants</h4>OFs isolated from patients with ...[more]

PMID: 32249902

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Project description:PURPOSE:Chemokines are involved in the pathogenesis of various autoimmune diseases, including Graves' orbitopathy (GO), but comprehensive analyses of the dynamics of these cytokines and their receptors in such diseases remain lacking. In this study, we investigated the expressions of chemokines and their receptors during adipogenesis and inflammation in primary cultured orbital fibroblasts from patients with GO. METHODS:The messenger RNA (mRNA) expression levels of chemokines were compared between GO (n = 6) and non-GO (n = 5) orbital tissues by real-time polymerase chain reaction. After adipogenesis was induced in primary cultured orbital fibroblasts from patients with GO (n =5) and following stimulation with interleukin (IL)-1? and tumor necrosis factor (TNF)-?, the mRNA expression levels of chemokines and their receptors were analyzed. RESULTS:Chemokines were significantly downregulated in GO orbital tissues compared to non-GO orbital tissues (p < 0.05). Adipogenesis resulted in a strong increase in mRNA expression levels of chemokines and their receptors at an early stage (day 1); however, expression levels started to decrease thereafter and, eventually, decreased to below basal levels at the end of adipogenesis (day 10). Following stimulation with IL-1? and TNF-?, the mRNA expression levels of chemokines and their receptors increased, showing different responses to various proinflammatory cytokines. CONCLUSIONS:Chemokines were strongly upregulated in the early phase of adipogenesis before decreasing continuously until the end of adipogenesis. Also, overt mature GO tissues showed reduced mRNA expression of chemokines compared to controls, which might indicate the existence of a shorter window for effective medical inflammatory treatment. The heightened levels of chemokines and their receptors observed after stimulation with IL-1? and TNF-? suggest a crucial role of proinflammatory cytokines in the pathogenesis of GO and, further, support the idea that chemokines could be used as biomarkers of GO activity.

Project description:BackgroundThe proprotein convertase subtilisin/kexin type 9 (PCSK9) has been implicated in the pathogenesis of inflammatory diseases. We sought to investigate the role of PCSK9 in the pathogenesis of Graves' orbitopathy (GO) and whether it may be a legitimate target for treatment.MethodsThe PCSK9 was compared between GO (n=11) and normal subjects (n=7) in orbital tissue explants using quantitative real-time PCR, and in cultured interleukin-1β (IL-1β)-treated fibroblasts using western blot. Western blot was used to identify the effects of PCSK9 inhibition on IL-1β-induced pro-inflammatory cytokines production and signaling molecules expression as well as levels of adipogenic markers and oxidative stress-related proteins. Adipogenic differentiation was identified using Oil Red O staining. The plasma PCSK9 concentrations were compared between patients with GO (n=44) and healthy subjects (n=26) by ELISA.ResultsThe PCSK9 transcript level was higher in GO tissues. The depletion of PCSK9 blunted IL-1β-induced expression of intercellular adhesion molecule 1 (ICAM-1), IL-6, IL-8, and cyclooxygenase-2 (COX-2) in GO and non-GO fibroblasts. The levels of activated nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and phosphorylated forms of Akt and p38 were diminished when PCSK9 was suppressed in GO fibroblasts. Decreases in lipid droplets and attenuated levels of peroxisome proliferator-activated receptor gamma (PPARγ), CCAAT/enhancer-binding protein β (C/EBPβ), and leptin as well as hypoxia-inducible factor 1α (HIF-1α), manganese superoxide dismutase (MnSOD), thioredoxin (Trx), and heme oxygenase-1 (HO-1) were noted when PCSK9 was suppressed during adipocyte differentiation. The plasma PCSK9 level was significantly higher in GO patients and correlated with level of thyrotropin binding inhibitory immunoglobulin (TBII) and the clinical activity score (CAS).ConclusionsPCSK9 plays a significant role in GO. The PCSK9 inhibition attenuated the pro-inflammatory cytokines production, oxidative stress, and fibroblast differentiation into adipocytes. PCSK9 may serve as a therapeutic target and biomarker for GO.

Dataset Information

Novel Roles of Chloroquine and Hydroxychloroquine in Graves' Orbitopathy Therapy by Targeting Orbital Fibroblasts.

Publications

Novel Roles of Chloroquine and Hydroxychloroquine in Graves' Orbitopathy Therapy by Targeting Orbital Fibroblasts.

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