IP3R attenuates oxidative stress and inflammation damage in smoking-induced COPD by promoting autophagy

Tobacco smoking is one of the most important risk factors for chronic obstructive pulmonary disease (COPD). However, the most critical genes and proteins remain poorly understood. Therefore, we aimed to investigate these hub genes and proteins in tobacco smoke-induced COPD, together with the potential mechanism(s). Differentially expressed genes (DEGs) were analysed between smokers and patients with COPD. mRNA expression and protein expression of IP₃R were confirmed in patients with COPD and extracted smoke solution (ESS)-treated human bronchial epithelial (HBE) cells. Moreover, expression of oxidative stress, inflammatory cytokines and/or autophagy-related protein was tested when IP₃R was silenced or overexpressed in ESS-treated and/or 3-MA-treated cells. A total of 30 DEGs were obtained between patients with COPD and smoker samples. IP₃R was identified as one of the key targets in tobacco smoke-induced COPD. In addition, IP₃R was significantly decreased in patients with COPD and ESS-treated cells. Loss of IP₃R statistically increased expression of oxidative stress and inflammatory cytokines in ESS-treated HBE cells, and overexpression of IP₃R reversed the above functions. Furthermore, the autophagy-related proteins (Atg5, LC3 and Beclin1) were statistically decreased, and p62 was increased by silencing of IP₃R cells, while overexpression of IP₃R showed contrary results. Additionally, we detected that administration of 3-MA significantly reversed the protective effects of IP₃R overexpression on ESS-induced oxidative stress and inflammatory injury. Our results suggest that IP₃R might exert a protective role against ESS-induced oxidative stress and inflammation damage in HBE cells. These protective effects might be associated with promoting autophagy.     

Calcineurin suppresses AMPK-dependent cytoprotective autophagy in cardiomyocytes under oxidative stress

Calcineurin signalling plays a critical role in the pathogenesis of many cardiovascular diseases. Calcineurin has been proven to affect a series of signalling pathways and to exert a proapoptotic effect in cardiomyocytes. However, whether it is able to regulate autophagy remains largely unknown. Here, we report that prolonged oxidative stress-induced activation of calcineurin contributes to the attenuation of adaptive AMP-activated protein kinase (AMPK) signalling and inhibits autophagy in cardiomyocytes. Primary cardiomyocytes exhibited rapid formation of autophagosomes, microtubule-associated protein 1 light chain 3 (LC3) expression and phosphorylation of AMPK in response to hydrogen peroxide (H₂O₂) treatment. However, prolonged (12 h) H₂O₂ treatment attenuated these effects and was accompanied by a significant increase in calcineurin activity and apoptosis. Inhibition of calcineurin by FK506 restored AMPK function and LC3 expression, and decreased the extent of apoptosis caused by prolonged oxidative stress. In contrast, overexpression of the constitutively active form of calcineurin markedly attenuated the increase in LC3 induced by short-term (3 h) H₂O₂ treatment and sensitised cells to apoptosis. In addition, FK506 failed to induce autophagy and alleviate apoptosis in cardiomyocytes expressing a kinase-dead K45R AMPK mutant. Furthermore, inhibition of autophagy by 3-methylanine (3-MA) or by knockdown of the essential autophagy-related gene ATG7 abrogated the protective effect of FK506. These findings suggest a novel role of calcineurin in suppressing adaptive autophagy during oxidative stress by downregulating the AMPK signalling pathway. The results also provide insight into how altered calcineurin and autophagic signalling is integrated to control cell survival during oxidative stress and may guide strategies to prevent cardiac oxidative damage.     

Inhibition of inflammation and oxidative stress by an imidazopyridine derivative X22 prevents heart injury from obesity

Inflammation and oxidative stress plays an important role in the development of obesity‐related complications and cardiovascular disease. Benzimidazole and imidazopyridine compounds are a class of compounds with a variety of activities, including anti‐inflammatory, antioxidant and anti‐cancer. X22 is an imidazopyridine derivative we synthesized and evaluated previously for anti‐inflammatory activity in lipopolysaccharide‐stimulated macrophages. However, its ability to alleviate obesity‐induced heart injury via its anti‐inflammatory actions was unclear. This study was designed to evaluate the cardioprotective effects of X22 using cell culture studies and a high‐fat diet rat model. We observed that palmitic acid treatment in cardiac‐derived H9c2 cells induced a significant increase in reactive oxygen species, inflammation, apoptosis, fibrosis and hypertrophy. All of these changes were inhibited by treatment with X22. Furthermore, oral administration of X22 suppressed high‐fat diet‐induced oxidative stress, inflammation, apoptosis, hypertrophy and fibrosis in rat heart tissues and decreased serum lipid concentration. We also found that the anti‐inflammatory and anti‐oxidative actions of X22 were associated with Nrf2 activation and nuclear factor‐kappaB (NF‐κB) inhibition, respectively, both in vitro and in vivo. The results of this study indicate that X22 may be a promising cardioprotective agent and that Nrf2 and NF‐κB may be important therapeutic targets for obesity‐related complications.     

Neferine suppresses autophagy-induced inflammation,oxidative stress and adipocyte differentiation in Graves' orbitopathy

Previous studies in Graves’ orbitopathy (GO) patient-derived fibroblasts showed that inhibition of autophagy suppresses adipogenic differentiation. Autophagy activation is associated with inflammation, production of reactive oxygen species and fibrosis. Neferine is an alkaloid extracted from Nelumbo nucifera, which induces Nrf2 expression and inhibits autophagy. Here, we have elucidated the role of neferine on interleukin (IL)-13-induced autophagy using patient-derived orbital fibroblasts as an in vitro model of GO. GO patient-derived orbital fibroblasts were isolated and cultured to generate an in vitro model of GO. Autophagy was determined by Western blot detection of the markers such as Beclin-1, Atg-5 and LC3 and by immunofluorescence detection of autophagosome formation. Analysis of differentiation towards an adipogenic lineage was performed by Oil red O staining. The expression of inflammatory factors was detected by ELISA and semiquantitative RT-PCR. Neferine inhibited autophagy in GO orbital fibroblasts, as indicated by the suppression of IL-13-induced autophagosome formation, overexpression of autophagy markers, increased LC3-II/LC3-I levels and finally down-regulation of p62. Neferine suppressed IL-13-induced inflammation, ROS generation, fibrosis and adipogenic differentiation in GO patient-derived orbital fibroblasts. The anti-inflammatory, antioxidant and antiadipogenic effects of neferine were accompanied by the up-regulation of Nrf2. These results indicated that orbital tissue remodelling and inflammation in GO may be mediated by autophagy, and neferine suppressed autophagy-related inflammation and adipogenesis through a mechanism involving Nrf2.     

Autophagy regulates inflammation following oxidative injury in diabetes

T1D (type 1 diabetes) is an autoimmune disease characterized by lymphocytic infiltration, or inflammation in pancreatic islets called ‘insulitis.’ Comparatively speaking, T2D (type 2 diabetes) is traditionally characterized by insulin resistance and islet β cell dysfunction; however, a number of studies have clearly demonstrated that chronic tissue inflammation is a key contributing factor to T2D. The NLR (Nod-like receptor) family of innate immune cell sensors such as the NLRP3 inflammasome are implicated in leading to CASP1 activation and subsequent IL1B (interleukin 1, β) and IL18 secretion in T2D. Recent developments reveal a crucial role for the autophagy pathway under conditions of oxidative stress and inflammation. Increasingly, research on autophagy has begun to focus on its role in interacting with inflammatory processes, and thereby how it potentially affects the outcome of disease progression. In this review, we explore the pathophysiological pathways associated with oxidative stress and inflammation in T2D. We also explore how autophagy influences glucose homeostasis by modulating the inflammatory response. We will provide here a perspective on the current research between autophagy, inflammation and T2D.     

Autophagy of metallothioneins prevents TNF-induced oxidative stress and toxicity in hepatoma cells

Lysosomal membrane permeabilization (LMP) induced by oxidative stress has recently emerged as a prominent mechanism behind TNF cytotoxicity. This pathway relies on diffusion of hydrogen peroxide into lysosomes containing redox-active iron, accumulated by breakdown of iron-containing proteins and subcellular organelles. Upon oxidative lysosomal damage, LMP allows relocation to the cytoplasm of low mass iron and acidic hydrolases that contribute to DNA and mitochondrial damage, resulting in death by apoptosis or necrosis. Here we investigate the role of lysosomes and free iron in death of HTC cells, a rat hepatoma line, exposed to TNF following metallothionein (MT) upregulation. Iron-binding MT does not normally occur in HTC cells in significant amounts. Intracellular iron chelation attenuates TNF and cycloheximide (CHX)-induced LMP and cell death, demonstrating the critical role of this transition metal in mediating cytokine lethality. MT upregulation, combined with starvation-activated MT autophagy almost completely suppresses TNF and CHX toxicity, while impairment of both autophagy and MT upregulation by silencing of Atg7, and Mt1a and/or Mt2a, respectively, abrogates protection. Interestingly, MT upregulation by itself has little effect, while stimulated autophagy alone depresses cytokine toxicity to some degree. These results provide evidence that intralysosomal iron-catalyzed redox reactions play a key role in TNF and CHX-induced LMP and toxicity. The finding that chelation of intralysosomal iron achieved by autophagic delivery of MT, and to some degree probably of other iron-binding proteins as well, into the lysosomal compartment is highly protective provides a putative mechanism to explain autophagy-related suppression of death by TNF and CHX.     

Celecoxib prevents pressure overload‐induced cardiac hypertrophy and dysfunction by inhibiting inflammation,apoptosis and oxidative stress

To explore the effects of celecoxib on pressure overload‐induced cardiac hypertrophy (CH), cardiac dysfunction and explore the possible protective mechanisms. We surgically created abdominal aortic constrictions (AAC) in rats to induce CH. Rats with CH symptoms at 4 weeks after surgery were treated with celecoxib [2 mg/100 g body‐weight(BW)] daily for either 2 or 4 weeks. Survival rate, blood pressure and cardiac function were evaluated after celecoxib treatment. Animals were killed, and cardiac tissue was examined for morphological changes, cardiomyocyte apoptosis, fibrosis, inflammation and oxidative stress. Four weeks after AAC, rats had significantly higher systolic, diastolic and mean blood pressure, greater heart weight and enlarged cardiomyocytes, which were associated with cardiac dysfunction. Thus, the CH model was successfully established. Two weeks later, animals had impaired cardiac function and histopathological abnormalities including enlarged cardiomyocytes and cardiac fibrosis, which were exacerbated 2 weeks later. However, these pathological changes were remarkably prevented by the treatment of celecoxib, independent of preventing hypertension. Mechanistic studies revealed that celecoxib‐induced cardiac protection against CH and cardiac dysfunction was due to inhibition of apoptosis via the murine double mimute 2/P53 pathway, inhibition of inflammation via the AKT/mTOR/NF‐κB pathway and inhibition of oxidative stress via increases in nuclear factor E2‐related factor‐2‐mediated gene expression of multiple antioxidants. Celecoxib suppresses pressure overload‐induced CH by reducing apoptosis, inflammation and oxidative stress.     

Resveratrol attenuates vascular endothelial inflammation by inducing autophagy through the cAMP signaling pathway

Inflammation participates centrally in all stages of atherosclerosis (AS), which begins with inflammatory changes in the endothelium, characterized by expression of the adhesion molecules. Resveratrol (RSV) is a naturally occurring phytoalexin that can attenuate endothelial inflammation; however, the exact mechanisms have not been thoroughly elucidated. Autophagy refers to the normal process of cell degradation of proteins and organelles, and is protective against certain inflammatory injuries. Thus, we intended to determine the role of autophagy in the antiinflammatory effects of RSV in human umbilical vein endothelial cells (HUVECs). We found that RSV pretreatment reduced tumor necrosis factor α (TNF/TNFα)-induced inflammation and increased MAP1LC3B2 (microtubule-associated protein 1 light chain 3 β 2) expression and SQSTM1/p62 (sequestosome 1) degradation in a concentration-dependent manner. A bafilomycin A₁ (BafA1) challenge resulted in further accumulation of MAP1LC3B2 in HUVECs. Furthermore, autophagy inhibitors 3-methyladenine (3-MA), chloroquine as well as ATG5 and BECN1 siRNA significantly attenuated RSV-induced autophagy, which, subsequently, suppressed the downregulation of RSV-induced inflammatory factors expression. RSV also increased cAMP (cyclic adenosine monophosphate) content, the expression of PRKA (protein kinase A) and SIRT1 (sirtuin 1), as well as the activity of AMPK (AMP-activated protein kinase). RSV-induced autophagy in HUVECs was abolished in the presence of inhibitors of ADCY (adenylyl cyclase, KH7), PRKA (H-89), AMPK (compound C), or SIRT1 (nicotinamide and EX-527), as well as ADCY, PRKA, AMPK, and SIRT1 siRNA transfection, indicating that the effects of RSV on autophagy induction were dependent on cAMP, PRKA, AMPK and SIRT1. In conclusion, RSV attenuates endothelial inflammation by inducing autophagy, and the autophagy in part was mediated through the activation of the cAMP-PRKA-AMPK-SIRT1 signaling pathway.     

Sodium 5,6‐benzylidene‐L‐ascorbate induces oxidative stress,autophagy, and growth arrest in human colon cancer HT‐29 cells

Our previous studies have demonstrated the oxidative stress properties of sodium ascorbate (SAA) and its benzaldehyde derivative (SBA) on cancer cell lines, but the molecular mechanisms mediating their cytotoxicity remain unclear. In this study, we treated human colon cancer HT‐29 cells with SAA and SBA, and found a significant exposure time‐dependent increase of cytotoxicity in both treatments, with a higher cytotoxicity for 24 h with SAA (IC₅₀ = 5 mM) than SBA (IC₅₀ = 10 mM). A short‐term treatment of cells with 10 mM SAA for 2 h revealed a destabilization of the lysosomes and subsequent induction of cell death, whereas 10 mM SBA triggered a remarkable production of reactive oxidative species, phosphorylation of survival kinase AKT, expression of cyclin kinase‐dependent inhibitor p21, and induction of transient growth arrest. The crucial role of p21 mediating this cytotoxicity was confirmed by isogenic derivatives of the human colon carcinoma HCT116 cell lines (p21^+/+ and p21^?/?), and immunoprecipitation studies with p21 antibody. The SAA cytotoxicity was blocked by co‐incubation with catalase, whereas the SBA cytotoxicity and its subsequent growth arrest were abolished by N‐acetyl‐L‐cysteine (NAC), but was not affected by PI3K phosphorylation inhibitor LY294002, or catalase, suggesting two separated oxidative stress pathways were mediated by these two ascorbates. In addition, neither active caspase 3 nor apoptotic bodies but autophagic vacuoles associated with increased LC3‐II were found in SBA‐treated HT‐29 cells; implicating that SBA induced AKT phosphorylation‐autophagy and p21‐growth arrest in colon cancer HT‐29 cells through an NAC‐inhibitable oxidative stress pathway. J. Cell. Biochem. 111: 412–424, 2010. © 2010 Wiley‐Liss, Inc.     

Palmitoylation prevents sustained inflammation by limiting NLRP3 inflammasome activation through chaperone-mediated autophagy

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Alpha lipoic acid prevents doxorubicin‐induced nephrotoxicity by mitigation of oxidative stress,inflammation, and apoptosis in rats

This study aimed to evaluate the protective effects of alpha lipoic acid (ALA) against doxorubicin (DOX)‐induced nephrotoxicity in rats. A single dose of DOX (7.5 mg/kg i.v.) induced nephrotoxicity evidenced by significant elevations in kidney weight, kidney/body weight ratio, serum urea, creatinine, tumor necrosis factor alpha, and renal contents of malondialdehyde, nitric oxide, cyclooxygenase‐2, and caspase‐3. Also, it causes significant reduction in final body weight, serum albumin, renal contents of reduced glutathione and superoxide dismutase activity. Histopathological changes in the kidney tissue confirmed the nephrotoxic effect. In contrast, pretreatment with ALA (50 mg/kg, orally) for 14 days before DOX and for 7 days after DOX administration mitigated renal toxicity evidenced by greater improvement in the examined oxidative stress, inflammation, and apoptosis parameters. In conclusion, ALA had promising protective effects against DOX‐induced nephrotoxicity that might be attributed to its antioxidant, anti‐inflammatory, and antiapoptoic activities.     

Influence of metabolic syndrome on biomarkers of oxidative stress and inflammation in obese adults

Objective: Both obesity and the metabolic syndrome (MetS) have been independently linked with increased oxidative and inflammatory stress. This study tested the hypothesis that obesity with MetS is associated with greater oxidative and inflammatory burden compared with obesity alone. Research Methods and Procedures: Forty‐eight normal‐weight and 40 obese (20 without MetS; 20 with MetS) adults were studied. MetS was defined according to National Cholesterol Education Program Adult Treatment Panel III criteria. Plasma concentrations of oxidized low‐density lipoprotein, C‐reactive protein, tumor necrosis factor‐α, interleukin (IL)‐6, and IL‐18 were determined by enzyme immunoassay. Results: Plasma biomarkers of oxidative stress and inflammation were lowest in normal‐weight controls. Of note, obese MetS adults demonstrated significantly higher plasma concentrations of oxidized low‐density lipoprotein (62.3 ± 3.2 vs. 54.0 ± 4.0 U/L; p < 0.05), C‐reactive protein (3.0 ± 0.6 vs. 1.5 ± 0.3 mg/L; p < 0.01), tumor necrosis factor‐α (2.1 ± 0.1 vs. 1.6 ± 0.1 pg/mL; p < 0.05), IL‐6 (2.8 ± 0.4 vs. 1.4 ± 0.2 pg/mL; p < 0.01), and IL‐18 (253 ± 16 vs. 199 ± 16 pg/mL; p < 0.01), compared with obese adults without MetS. Discussion: These results suggest that MetS heightens oxidative stress and inflammatory burden in obese adults. Increased oxidative and inflammatory stress may contribute to the greater risk of coronary heart disease and cerebrovascular disease in obese adults with MetS.     

Dipeptidyl peptidase-4 inhibition prevents lung injury in mice under chronic stress via the modulation of oxidative stress and inflammation

Exposure to chronic psychosocial stress is a risk factor for various pulmonary diseases. In view of the essential role of dipeptidyl peptidase 4 (DPP4) in animal and human lung pathobiology, we investigated the role of DPP4 in stress-related lung injury in mice. Eight-week-old male mice were randomly divided into a non-stress group and a 2-week immobilization stress group. Non-stress control mice were left undisturbed. The mice subjected to immobilized stress were randomly assigned to the vehicle or the DPP4 inhibitor anagliptin for 2 weeks. Chronic stress reduced subcutaneous and inguinal adipose volumes and increased blood DPP4 levels. The stressed mice showed increased levels in the lungs of genes and/or proteins related to oxidative stress (p67^phox, p47^phox, p22^phox and gp91^phox), inflammation (monocyte chemoattractant protein-1, vascular cell adhesion molecule-1, and intracellular adhesion molecule-1), apoptosis (caspase-3, -8, -9), senescence (p16^INK4A, p21, and p53) and proteolysis (matrix metalloproteinase-2 to -9, cathepsin S/K, and tissue inhibitor of matrix metalloproteinase-1 and -2), and reduced levels of eNOS, Sirt1, and Bcl-2 proteins; and these effects were reversed by genetic and pharmacological inhibitions of DPP4. We then exposed human umbilical vein endothelial cells in vitro to hydrogen peroxide; anagliptin treatment was also observed to mitigate oxidative and inflammatory molecules in this setting. Anagliptin can improve lung injury in stressed mice, possibly by mitigating vascular inflammation, oxidative stress production, and proteolysis. DPP4 may become a new therapeutic target for chronic psychological stress-related lung disease in humans and animals.     

Chikungunya-induced cell death is limited by ER and oxidative stress-induced autophagy

It has been recognized that macroautophagy constitutes an important survival mechanism that allows both the maintenance of cellular homeostasis and the regulation of programmed cell death pathways (e.g., apoptosis). Although several pathogens have been described to induce autophagy, the prosurvival function of this process in infectious models remains poorly characterized. Our recent studies on chikungunya virus (CHIKV), the causative agent of major epidemics in India, Southeast Asia and southern Europe, reveal a novel mechanism by which autophagy limits the cytopathic effects of CHIKV by impinging upon virus-induced cell death pathways.     

Curcumin induces autophagy to protect vascular endothelial cell survival from oxidative stress damage

Our study first proposed that curcumin could protect human endothelial cells from the damage caused by oxidative stress via autophagy. Furthermore, our results revealed that curcumin causes some novel cellular mechanisms that promote autophagy as a protective effect. Pretreatment with curcumin remarkably improves the survival of human umbilical vein endothelial cells (HUVECs) from H₂O₂-induced viability loss, which specifically evokes an autophagic response. Exposed to H₂O₂, curcumin-treated HUVECs upregulate the level of microtubule-associated protein 1 light chain 3-II (LC3-II), the number of autophagosomes, and the degradation of p62. We show that this compound promotes BECN1 expression and inhibits the phosphatidylinositol 3-kinase (PtdIns3K)-AKT-mechanistic target of rapamycin (MTOR) signaling pathway. Curcumin can also reverse FOXO1 (a mediator of autophagy) nuclear localization along with causing an elevated level of cytoplasmic acetylation of FOXO1 and the interaction of acetylated FOXO1 and ATG7, under the circumstance of oxidative stress. Additionally, knockdown of FOXO1 by shRNA inhibits not only the protective effects that curcumin induced, but the autophagic process, from the quantity of LC3-II to the expression of RAB7. These results suggest that curcumin induces autophagy, indicating that curcumin has the potential for use as an autophagic-related antioxidant for prevention and treatment of oxidative stress. These data uncover a brand new protective mechanism involving FOXO1 as having a critical role in regulating autophagy in HUVECs, and suggest a novel role for curcumin in inducing a beneficial form of autophagy in HUVECs, which may be a potential multitargeted therapeutic avenue for the treatment of oxidative stress-related cardiovascular diseases.     

α-亚麻酸对糖尿病大鼠炎症介质和氧化应激的影响

目的:观察α-亚麻酸(ALA)对糖尿病大鼠体内炎症介质和氧化应激的影响,探讨ALA在糖尿病防治中的作用。方法:雄性SD大鼠高脂饮食喂养4周后,腹腔注射链脲佐菌素(STZ)30 mg/kg建立2型糖尿病(T2DM)模型。将大鼠随机分为3组(n=10):正常对照组、糖尿病模型组和ALA治疗组(500μg/kg.d)。4周后测定大鼠血清中肿瘤坏死因子(TNF-α)、可溶性P-选择素(sP-selectin)、可溶性细胞间黏附分子(sICAM-1)、一氧化氮(NO)、丙二醛(MDA)的含量以及超氧化物岐化酶(SOD)和过氧化氢酶(CAT)的活性。结果:与正常对照组相比,糖尿病大鼠血清中炎症介质TNF-α、sP-selectin和sICAM-1的含量增加,血清NO含量下降而MDA升高,同时抗氧化酶SOD和CAT的活性降低;ALA治疗可显著降低糖尿病大鼠血清中TNF-α、sP-selectin和sICAM-1的含量(与STZ+vehicle组相比,P<0.01),增加血清NO水平并减少MDA含量,升高抗氧化酶SOD和CAT的活性(与STZ+vehicle组相比,均P<0.05)。结论:ALA可显著降低糖尿病大鼠血清炎症介质的生成,减轻氧化应激水平,具有抗炎和抗氧化作用。提示ALA对糖尿病及糖尿病并发症的发生发展可能具有一定的防治作用。     

Dysregulated autophagy in the RPE is associated with increased susceptibility to oxidative stress and AMD

Autophagic dysregulation has been suggested in a broad range of neurodegenerative diseases including age-related macular degeneration (AMD). To test whether the autophagy pathway plays a critical role to protect retinal pigmented epithelial (RPE) cells against oxidative stress, we exposed ARPE-19 and primary cultured human RPE cells to both acute (3 and 24 h) and chronic (14 d) oxidative stress and monitored autophagy by western blot, PCR, and autophagosome counts in the presence or absence of autophagy modulators. Acute oxidative stress led to a marked increase in autophagy in the RPE, whereas autophagy was reduced under chronic oxidative stress. Upregulation of autophagy by rapamycin decreased oxidative stress-induced generation of reactive oxygen species (ROS), whereas inhibition of autophagy by 3-methyladenine (3-MA) or by knockdown of ATG7 or BECN1 increased ROS generation, exacerbated oxidative stress-induced reduction of mitochondrial activity, reduced cell viability, and increased lipofuscin. Examination of control human donor specimens and mice demonstrated an age-related increase in autophagosome numbers and expression of autophagy proteins. However, autophagy proteins, autophagosomes, and autophagy flux were significantly reduced in tissue from human donor AMD eyes and 2 animal models of AMD. In conclusion, our data confirm that autophagy plays an important role in protection of the RPE against oxidative stress and lipofuscin accumulation and that impairment of autophagy is likely to exacerbate oxidative stress and contribute to the pathogenesis of AMD.     

Dysregulated autophagy in the RPE is associated with increased susceptibility to oxidative stress and AMD

Autophagic dysregulation has been suggested in a broad range of neurodegenerative diseases including age-related macular degeneration (AMD). To test whether the autophagy pathway plays a critical role to protect retinal pigmented epithelial (RPE) cells against oxidative stress, we exposed ARPE-19 and primary cultured human RPE cells to both acute (3 and 24 h) and chronic (14 d) oxidative stress and monitored autophagy by western blot, PCR, and autophagosome counts in the presence or absence of autophagy modulators. Acute oxidative stress led to a marked increase in autophagy in the RPE, whereas autophagy was reduced under chronic oxidative stress. Upregulation of autophagy by rapamycin decreased oxidative stress-induced generation of reactive oxygen species (ROS), whereas inhibition of autophagy by 3-methyladenine (3-MA) or by knockdown of ATG7 or BECN1 increased ROS generation, exacerbated oxidative stress-induced reduction of mitochondrial activity, reduced cell viability, and increased lipofuscin. Examination of control human donor specimens and mice demonstrated an age-related increase in autophagosome numbers and expression of autophagy proteins. However, autophagy proteins, autophagosomes, and autophagy flux were significantly reduced in tissue from human donor AMD eyes and 2 animal models of AMD. In conclusion, our data confirm that autophagy plays an important role in protection of the RPE against oxidative stress and lipofuscin accumulation and that impairment of autophagy is likely to exacerbate oxidative stress and contribute to the pathogenesis of AMD.