Expression of miR-210-3p in the aqueous humor of patients with age-related cataracts and its effect on human lens epithelial cell injury induced by hydrogen peroxide / Expressão de miR-210-3p no humor aquoso de pacientes com catarata relacionada à idade e seu efeito na lesão de células epiteliais do cristalino humano induzida por peróxido de hidrogênio

ABSTRACT Purpose: The regulatory effect of microRNA on diseases has been confirmed. This study aimed to evaluate the expression of microRNA-210-3p in age-related cataracts and assess the effect of abnormal miR-210-3p expressions on H2O2-induced SAR01/04 cells. Methods: Reverse-transcription quantitative polymerase chain reaction method was performed to assess the levels of miR-210-3p in aqueous humor samples. Receiver operating characteristic analysis was employed to assess the discrimination ability of miR-210-3p between patients with age-related cataracts and healthy people, and Pearson correlation analysis was used to identify the correlation between miR-210-3p and oxidative stress indices such as superoxide dismutase, glutathione peroxidase, malonaldehyde. Cell counting kit-8 assay and Transwell assay were used to estimate the biological function of H2O2-induced age-related cataract cell model. The levels of oxidative stress indices such as superoxide dismutase, glutathione peroxidase, and malonaldehyde were measured to evaluate the degree of oxidative stress damage in the age-related cataract cell model. The relationship between miR-210-3p and its target gene was verified by luciferase reporter gene analysis. Results: The miR-210-3p expression was elevated in the aqueous humor of patients with age-related cataracts. A high miR-210-3p expression showed a high diagnostic value for age-related cataracts and was significantly associated with the level of oxidative stress markers in patients with age-related cataracts. The inhibition of miR-210-3p can reverse oxidative stress stimulation and adverse effects on H2O2-induced cell function. Conclusions: The results suggested that miR-210-3p could promote cell viability, cell migration, and oxidative stress by targeting autophagy-related gene 7 in in vitro age-related cataract cell model.

RESUMO Objetivo: O efeito regulador do microRNA em doenças tem sido confirmado, e este artigo tentou avaliar a expressão do microRNA-210-3p na catarata relacionada à idade e avaliar o efeito da expressão anormal do miR-210-3p em células SAR01/04 induzidas por H2O2. Métodos: O método de transcrição reversa seguida de reação em cadeia da polimerase (RT-PCR) quantitativa foi realizado para avaliar os níveis de miR-210-3p em amostras de humor aquoso. Análise de características operacionais do receptor foi feita para avaliar a capacidade de discriminação do miR-210-3p entre pacientes com catarata relacionada à idade e pessoas saudáveis. A análise de correlação de Pearson identificou a correlação do miR-210-3p e índices de estresse oxidativo, como superóxido dismutase, glutationa peroxidase, malonaldeído. O ensaio de contagem de células kit-8 (cck-8) e o ensaio no sistema Transwell foram utilizados para estimar a função biológica do formato de células de catarata relacionada com a idade induzida por H2O2. Os níveis de índices de estresse oxidativo como superóxido dismutase, glutationa peroxidase e malonaldeído foram detectados para avaliar o grau de dano do estresse oxidativo em formato de células de catarata relacionada à idade. A relação entre miR-210-3p e seu gene alvo foi verificada por análise do gene repórter luciferase. Resultados: A expressão miR-210-3p foi elevada no humor aquoso de pacientes com catarata relacionada à idade. A expressão miR-210-3p altamente expressiva mostrou alto valor diagnóstico para catarata relacionada à idade e foi significativamente associado ao nível de marcadores de estresse oxidativo em pacientes com catarata relacionada à idade. A inibição de miR-210-3p pode reverter a estimulação do estresse oxidativo e os efeitos adversos da função celular induzida por H2O2. Conclusões: Esses dados sugeriram que a expressão miR-210-3p poderia promover a viabilidade celular, migração celular e estresse oxidativo ao direcionar genes ATG 7 relacionados à autofagia em modelo in vitro de células de catarata relacionadas à idade.

Repopulation of autophagy-deficient stromal cells with autophagy-intact cells after repeated breeding in uterine mesenchyme-specific Atg7 knockout mice.

OBJECTIVE: Autophagy is highly active in ovariectomized mice experiencing hormone deprivation, especially in the uterine mesenchyme. Autophagy is responsible for the turnover of vasoactive factors in the uterus, which was demonstrated in anti-Müllerian hormone receptor type 2 receptor (Amhr2)-Cre-driven autophagy-related gene 7 (Atg7) knockout (Amhr-Cre/Atg7f/f mice). In that study, we uncovered a striking difference in the amount of sequestosome 1 (SQSTM1) accumulation between virgin mice and breeder mice with the same genotype. Herein, we aimed to determine whether repeated breeding changed the composition of mesenchymal cell populations in the uterine stroma. METHODS: All female mice used in this study were of the same genotype. Atg7 was deleted by Amhr2 promoter-driven Cre recombinase in the uterine stroma and myometrium, except for a triangular stromal region on the mesometrial side. Amhr-Cre/Atg7f/f female mice were divided into two groups: virgin mice with no mating history and aged between 11 and 12 months, and breeder mice with at least 6-month breeding cycles with multiple pregnancies and aged around 12 months. The uteri were used for Western blotting and immunofluorescence staining. RESULTS: SQSTM1 accumulation, representing Atg7 deletion and halted autophagy, was much higher in virgin mice than in breeders. Breeders showed reduced accumulation of several vasoconstrictive factors, which are potential autophagy targets, in the uterus, suggesting that the uterine stroma was repopulated with autophagy-intact cells during repeated pregnancies. CONCLUSION: Multiple pregnancies seem to have improved the uterine environment by replacing autophagy-deficient cells with autophagy-intact cells, providing evidence of cell mixing.

Lonicerin targets EZH2 to alleviate ulcerative colitis by autophagy-mediated NLRP3 inflammasome inactivation.

Aberrant activation of NLRP3 inflammasome in colonic macrophages strongly associates with the occurrence and progression of ulcerative colitis. Although targeting NLRP3 inflammasome has been considered to be a potential therapy, the underlying mechanism through which pathway the intestinal inflammation is modulated remains controversial. By focusing on the flavonoid lonicerin, one of the most abundant constituents existed in a long historical anti-inflammatory and anti-infectious herb Lonicera japonica Thunb., here we report its therapeutic effect on intestinal inflammation by binding directly to enhancer of zeste homolog 2 (EZH2) histone methyltransferase. EZH2-mediated modification of H3K27me3 promotes the expression of autophagy-related protein 5, which in turn leads to enhanced autophagy and accelerates autolysosome-mediated NLRP3 degradation. Mutations of EZH2 residues (His129 and Arg685) indicated by the dynamic simulation study have found to greatly diminish the protective effect of lonicerin. More importantly, in vivo studies verify that lonicerin dose-dependently disrupts the NLRP3-ASC-pro-caspase-1 complex assembly and alleviates colitis, which is compromised by administration of EZH2 overexpression plasmid. Thus, these findings together put forth the stage for further considering lonicerin as an anti-inflammatory epigenetic agent and suggesting EZH2/ATG5/NLRP3 axis may serve as a novel strategy to prevent ulcerative colitis as well as other inflammatory diseases.

[Hsa_circ_0006948 regulates the proliferation, migration and invasion in osteosarcoma by regulation of the expression of miR-490-3p target ATG7].

Objective: To investigate the effect of hsa_circ_0006948 (circ_0006948) on the proliferation, migration and invasion of osteosarcoma cells and the underlying mechanism. Methods: A total of 120 osteosarcoma tissues and 40 adjacent normal tissue samples were collected from patients admitted to the First People's Hospital of Shangqiu City from 2009 to 2015. Microarray analysis was performed to detect the differential expressions of circRNA in Saos-2 cell. The mRNA expressions of circ_0006948, microRNA (miR)-490-3p and autophagy-related protein 7 (ATG7) in osteosarcoma cells, NHOst cells, osteosarcoma tissues and adjacent tissues were detected by real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR). Cell clone formation assay was used to detect cell proliferation ability, Transwell assay was used to detect cell invasion ability, and cell scratch assay was used to detect cell migration ability. The interactions between circ_0006948 and miR-490-3p, miR-490-3p and ATG7 were detected by dual luciferase reporter gene assay. The correlation between miR-490-3p and ATG7 was analyzed by TargetScan database, and the expression levels of Bcl-2 and Bax proteins in cells were detected by western blot. Results: The mRNA expression levels of circ_0006948, miR-490-3p and ATG7 in SAOS-2 cells were significantly different from NHOst cells (P<0.01). The mRNA expression levels of circ_0006948, miR-490-3p and ATG7 in osteosarcoma tissues were significantly different from adjacent tissues (P<0.01). The numbers of cell clone, migration and mobility in circ_0006948-siRNA group were (32.78±1.76), (37.58±1.82) and (36.93±1.45)%, respectively, lower than (65.72±1.45), (78.63±1.93) and (65.32±1.74)% in the siRNA NC group (all P<0.01). The numbers of cell clone, migration and mobility in the miR-490-3p mimics group were (20.08±1.54), (30.24±1.78) and (21.15±1.68)%, respectively, lower than (60.36±1.83), (76.93±1.64) and (40.56±1.27)% in the mimics NC group (all P<0.01). The numbers of cell clone, migration and mobility in the miR-490-3p inhibitor+ siRNA NC group were (90.34±1.72), (120.89±2.34) and (70.83±1.93)%, respectively, higher than (61.27±1.73), (75.82±1.82) and (42.38±1.74)% in the inhibitor NC+ siRNA NC group (P<0.01). The numbers of cell clone, migration and mobility in the circ_0006948 siRNA+ miR-490-3p inhibitor group were (58.74±1.98), (73.46±1.04) and (40.35±1.72)%, respectively, lower than (90.34±1.72), (120.89±2.34) and (70.83±1.93)% in the miR-490-3p inhibitor+ siRNA NC group (P<0.01). The numbers of cell clone, migration and mobility in the ATG7 siRNA group were (20.56±1.87), (40.36±1.76) and (20.96±1.73)%, lower than (65.46±1.74), (90.87±2.32) and (40.87±2.03)% in the siRNA NC group (P<0.01). The absorbance of miR-490-3p mimics+ pcDNA-ATG7 group was 0.54±0.11, higher than (0.36±0.08) of miR-490-3p mimics group (P<0.05). The expression levels of Bax and Bcl-2 protein in Saos-2 cells of miR-490-3p mimics group were significantly different from mimics NC group (P<0.01). The protein expression levels of Bax and Bcl-2 in Saos-2 cells of miR-490-3p mimics + pcDNA-ATG7 group were significantly different from miR-490-3p mimics group (P<0.01). Conclusion: Circ_0006948 regulates ATG7 expression through miR-490-3p, therefore regulates the proliferation, migration and invasion of osteosarcoma cells.

Lonicerin targets EZH2 to alleviate ulcerative colitis by autophagy-mediated NLRP3 inflammasome inactivation

Aberrant activation of NLRP3 inflammasome in colonic macrophages strongly associates with the occurrence and progression of ulcerative colitis. Although targeting NLRP3 inflammasome has been considered to be a potential therapy, the underlying mechanism through which pathway the intestinal inflammation is modulated remains controversial. By focusing on the flavonoid lonicerin, one of the most abundant constituents existed in a long historical anti-inflammatory and anti-infectious herb

Value of serum autophagy-related protein 7 in diagnosis of HBV-related hepatocellular carcinoma / 临床肝胆病杂志

Objective To investigate the clinical significance of autophagy-related protein 7 (ATG7) in the diagnosis of HBV-related hepatocellular carcinoma (HBV-HCC) by measuring the expression level of serum ATG7 in patients with HBV-HCC. Methods A total of 50 patients with chronic hepatitis B (CHB) and 89 patients with HCC who were hospitalized in Mengchao Hepatobiliary Hospital of Fujian Medical University from June 2018 to December 2020 were enrolled, among whom 67 patients had HBV-HCC (HBV-HCC group) and 22 patients had no HBV-HCC (non-HBV-HCC group), and 20 healthy volunteers who underwent physical examination were enrolled as healthy control (HC) group. Demographic data and laboratory data including alpha-fetoprotein (AFP) were collected from each group, and ELISA was used to measure the serum level of ATG7. The receiver operating curve (ROC) was plotted for ATG7 and AFP used alone or in combination, and the area under the ROC curve (AUC) was compared. The Kruskal-Wallis H test was used for comparison of non-normally distributed continuous data between multiple groups, and the Mann-Whitney U test was used for comparison between two groups; the chi-square test was used for comparison of categorical data between groups; a Spearman correlation analysis was used to investigate correlation. Results The serum level of ATG7 was 22.88(19.79-23.04) ng/mL in the HBV-HCC group, 17.06(14.45-19.40) ng/mL in the non-HBV-HCC group, 19.21(16.65-20.82) ng/mL in the CHB group, and 13.82(8.70-17.82) ng/mL in the HC group, with a significant difference between groups ( χ 2 =65.144, P < 0.001). ATG7 had an AUC of 0.818 (95% confidence interval [ CI ]: 0.743-0.879) and AFP had an AUC of 0.777 (95% CI : 0.698-0.843) in the diagnosis of HBV-HCC, suggesting that ATG7 had a slightly higher AUC than AFP ( Z =0.852, P =0.394). ATG7 combined with AFP had an AUC of 0.859 (95% CI : 0.790-0.913) in the diagnosis of HBV-HCC, which was significantly higher than the AUC of ATG7 alone ( Z =2.192, P =0.028) and AFP alone ( Z =2.076, P =0.038). Conclusion ATG7 is a good marker for the diagnosis of HBV-HCC, and combined measurement of ATG7 and AFP can significantly improve the diagnostic rate for HBV-HCC.

Value of serum autophagy-related protein 7 in diagnosis of HBV-related hepatocellular carcinoma / 临床肝胆病杂志

Objective To investigate the clinical significance of autophagy-related protein 7 (ATG7) in the diagnosis of HBV-related hepatocellular carcinoma (HBV-HCC) by measuring the expression level of serum ATG7 in patients with HBV-HCC. Methods A total of 50 patients with chronic hepatitis B (CHB) and 89 patients with HCC who were hospitalized in Mengchao Hepatobiliary Hospital of Fujian Medical University from June 2018 to December 2020 were enrolled, among whom 67 patients had HBV-HCC (HBV-HCC group) and 22 patients had no HBV-HCC (non-HBV-HCC group), and 20 healthy volunteers who underwent physical examination were enrolled as healthy control (HC) group. Demographic data and laboratory data including alpha-fetoprotein (AFP) were collected from each group, and ELISA was used to measure the serum level of ATG7. The receiver operating curve (ROC) was plotted for ATG7 and AFP used alone or in combination, and the area under the ROC curve (AUC) was compared. The Kruskal-Wallis H test was used for comparison of non-normally distributed continuous data between multiple groups, and the Mann-Whitney U test was used for comparison between two groups; the chi-square test was used for comparison of categorical data between groups; a Spearman correlation analysis was used to investigate correlation. Results The serum level of ATG7 was 22.88(19.79-23.04) ng/mL in the HBV-HCC group, 17.06(14.45-19.40) ng/mL in the non-HBV-HCC group, 19.21(16.65-20.82) ng/mL in the CHB group, and 13.82(8.70-17.82) ng/mL in the HC group, with a significant difference between groups ( χ 2 =65.144, P < 0.001). ATG7 had an AUC of 0.818 (95% confidence interval [ CI ]: 0.743-0.879) and AFP had an AUC of 0.777 (95% CI : 0.698-0.843) in the diagnosis of HBV-HCC, suggesting that ATG7 had a slightly higher AUC than AFP ( Z =0.852, P =0.394). ATG7 combined with AFP had an AUC of 0.859 (95% CI : 0.790-0.913) in the diagnosis of HBV-HCC, which was significantly higher than the AUC of ATG7 alone ( Z =2.192, P =0.028) and AFP alone ( Z =2.076, P =0.038). Conclusion ATG7 is a good marker for the diagnosis of HBV-HCC, and combined measurement of ATG7 and AFP can significantly improve the diagnostic rate for HBV-HCC.

Autophagy Mediates Astrogenesis in Adult Hippocampal Neural Stem Cells.

Neural stem cells (NSCs) have the ability to self-renew and differentiate into neurons, oligodendrocytes, and astrocytes. Highly dynamic nature of NSC differentiation requires the intimate involvement of catabolic processes such as autophagy. Autophagy is a major intracellular degradation pathway necessary for cellular homeostasis and remodeling. Autophagy is important for mammalian development and its role in neurogenesis has recently drawn much attention. However, little is known about how autophagy is associated with differentiation of NSCs into other neural lineages. Here, we report that autophagy plays a critical role in differentiation of adult rat hippocampal neural stem (HCN) cells into astrocytes. During differentiation, autophagy flux peaked at early time points, and remained high. Pharmacological or genetic suppression of autophagy by stable knockdown of Atg7, LC3 or CRISPR-Cas9-mediated knockout (KO) of p62 impaired astrogenesis, while reintroduction of p62 recovered astrogenesis in p62 KO HCN cells. Taken together, our findings suggest that autophagy plays a key role in astrogenesis in adult NSCs.

Autophagy Mediates Astrogenesis in Adult Hippocampal Neural Stem Cells

Neural stem cells (NSCs) have the ability to self-renew and differentiate into neurons, oligodendrocytes, and astrocytes. Highly dynamic nature of NSC differentiation requires the intimate involvement of catabolic processes such as autophagy. Autophagy is a major intracellular degradation pathway necessary for cellular homeostasis and remodeling. Autophagy is important for mammalian development and its role in neurogenesis has recently drawn much attention. However, little is known about how autophagy is associated with differentiation of NSCs into other neural lineages. Here, we report that autophagy plays a critical role in differentiation of adult rat hippocampal neural stem (HCN) cells into astrocytes. During differentiation, autophagy flux peaked at early time points, and remained high. Pharmacological or genetic suppression of autophagy by stable knockdown of Atg7, LC3 or CRISPR-Cas9-mediated knockout (KO) of p62 impaired astrogenesis, while reintroduction of p62 recovered astrogenesis in p62 KO HCN cells. Taken together, our findings suggest that autophagy plays a key role in astrogenesis in adult NSCs.

Induction of autophagy by PI3K/MTOR and PI3K/MTOR/BRD4 inhibitors suppresses HIV-1 replication.

In this study, we investigated the effects of the dual phosphatidylinositol 3-kinase/mechanistic target of rapamycin (PI3K/MTOR) inhibitor dactolisib (NVP-BEZ235), the PI3K/MTOR/bromodomain-containing protein 4 (BRD4) inhibitor SF2523, and the bromodomain and extra terminal domain inhibitor JQ1 on the productive infection of primary macrophages with human immunodeficiency type-1 (HIV). These inhibitors did not alter the initial susceptibility of macrophages to HIV infection. However, dactolisib, JQ1, and SF2523 all decreased HIV replication in macrophages in a dose-dependent manner via degradation of intracellular HIV through autophagy. Macrophages treated with dactolisib, JQ1, or SF2523 displayed an increase in LC3B lipidation combined with SQSTM1 degradation without inducing increased cell death. LC3B-II levels were further increased in the presence of pepstatin A suggesting that these inhibitors induce autophagic flux. RNA interference for ATG5 and ATG7 and pharmacological inhibitors of autophagosome-lysosome fusion and of lysosomal hydrolases all blocked the inhibition of HIV. Thus, we demonstrate that the mechanism of PI3K/MTOR and PI3K/MTOR/BRD4 inhibitor suppression of HIV requires the formation of autophagosomes, as well as their subsequent maturation into autolysosomes. These data provide further evidence in support of a role for autophagy in the control of HIV infection and open new avenues for the use of this class of drugs in HIV therapy.

Di-retinoid-pyridinium-ethanolamine (A2E) Accumulation and the Maintenance of the Visual Cycle Are Independent of Atg7-mediated Autophagy in the Retinal Pigmented Epithelium.

Autophagy is an evolutionarily conserved catabolic mechanism that relieves cellular stress by removing/recycling damaged organelles and debris through the action of lysosomes. Compromised autophagy has been implicated in many neurodegenerative diseases, including retinal degeneration. Here we examined retinal phenotypes resulting from RPE-specific deletion of the autophagy regulatory gene Atg7 by generating Atg7(flox/flox);VMD2-rtTA-cre+ mice to determine whether autophagy is essential for RPE functions including retinoid recycling. Atg7-deficient RPE displayed abnormal morphology with increased RPE thickness, cellular debris and vacuole formation indicating that autophagy is important in maintaining RPE homeostasis. In contrast, 11-cis-retinal content, ERGs and retinal histology were normal in mice with Atg7-deficient RPE in both fasted and fed states. Because A2E accumulation in the RPE is associated with pathogenesis of both Stargardt disease and age-related macular degeneration (AMD) in humans, deletion of Abca4 was introduced into Atg7(flox/flox);VMD2-rtTA-cre+ mice to investigate the role of autophagy during A2E accumulation. Comparable A2E concentrations were detected in the eyes of 6-month-old mice with and without Atg7 from both Abca4(-/-) and Abca4(+/+) backgrounds. To identify other autophagy-related molecules involved in A2E accumulation, we performed gene expression array analysis on A2E-treated human RPE cells and found up-regulation of four autophagy related genes; DRAM1, NPC1, CASP3, and EIF2AK3/PERK. These observations indicate that Atg7-mediated autophagy is dispensable for retinoid recycling and A2E deposition; however, autophagy plays a role in coping with stress caused by A2E accumulation.

Autophagy induction by histone deacetylase inhibitors inhibits HIV type 1.

Histone deacetylase inhibitors (HDACi) are being evaluated in a "shock-and-kill" therapeutic approach to reverse human immunodeficiency virus type-1 (HIV) latency from CD4(+) T cells. Using this approach, HDACi have induced HIV RNA synthesis in latently infected cells from some patients. The hope is that the increase in viral production will lead to killing of the infected cell either by the virus itself or by the patient's immune system, a "sterilizing cure." Although administered within the context of combination antiretroviral therapy, the infection of bystander cells remains a concern. In this study, we investigated the effect of HDACi (belinostat, givinostat, panobinostat, romidepsin, and vorinostat) on the productive infection of macrophages. We demonstrate that the HDACi tested do not alter the initial susceptibility of macrophages to HIV infection. However, we demonstrate that HDACi decrease HIV release from macrophages in a dose-dependent manner (belinostat < givinostat < vorinostat < panobinostat < romidepsin) via degradation of intracellular HIV through the canonical autophagy pathway. This mechanism involves unc-51-like autophagy-activating kinase 1 (ULK1) and the inhibition of the mammalian target of rapamycin and requires the formation of autophagosomes and their maturation into autolysosomes in the absence of increased cell death. These data provide further evidence in support of a role for autophagy in the control of HIV infection and suggest that careful consideration of off-target effects will be essential if HDACi are to be a component of a multipronged approach to eliminate latently infected cells.

Inhibition of autophagic turnover in ß-cells by fatty acids and glucose leads to apoptotic cell death.

Autophagy, a cellular recycling process responsible for turnover of cytoplasmic contents, is critical for maintenance of health. Defects in this process have been linked to diabetes. Diabetes-associated glucotoxicity/lipotoxicity contribute to impaired ß-cell function and have been implicated as contributing factors to this disease. We tested the hypothesis that these two conditions affect ß-cell function by modulating autophagy. We report that exposure of ß-cell lines and human pancreatic islets to high levels of glucose and lipids blocks autophagic flux and leads to apoptotic cell death. EM analysis showed accumulation of autophagy intermediates (autophagosomes), with abundant engulfed cargo in palmitic acid (PA)- or glucose-treated cells, indicating suppressed autophagic turnover. EM studies also showed accumulation of damaged mitochondria, endoplasmic reticulum distention, and vacuolar changes in PA-treated cells. Pulse-chase experiments indicated decreased protein turnover in ß-cells treated with PA/glucose. Expression of mTORC1, an inhibitor of autophagy, was elevated in ß-cells treated with PA/glucose. mTORC1 inhibition, by treatment with rapamycin, reversed changes in autophagic flux, and cell death induced by glucose/PA. Our results indicate that nutrient toxicity-induced cell death occurs via impaired autophagy and is mediated by activation of mTORC1 in ß-cells, contributing to ß-cell failure in the presence of metabolic stress.

The essential autophagy gene ATG7 modulates organ fibrosis via regulation of endothelial-to-mesenchymal transition.

Pulmonary fibrosis is a progressive disease characterized by fibroblast proliferation and excess deposition of collagen and other extracellular matrix components. Although the origin of fibroblasts is multifactorial, recent data implicate endothelial-to-mesenchymal transition as an important source of fibroblasts. We report herein that loss of the essential autophagy gene ATG7 in endothelial cells (ECs) leads to impaired autophagic flux accompanied by marked changes in EC architecture, loss of endothelial, and gain of mesenchymal markers consistent with endothelial-to-mesenchymal transition. Loss of ATG7 also up-regulates TGFß signaling and key pro-fibrotic genes in vitro. In vivo, EC-specific ATG7 knock-out mice exhibit a basal reduction in endothelial-specific markers and demonstrate an increased susceptibility to bleomycin-induced pulmonary fibrosis and collagen accumulation. Our findings help define the role of endothelial autophagy as a potential therapeutic target to limit organ fibrosis, a condition for which presently there are no effective available treatments.

Deficiency of autophagy in dendritic cells protects against experimental autoimmune encephalomyelitis.

Dendritic cells (DCs) are the most potent antigen-presenting cells (APCs) in the immune system. DCs present antigens to CD8 and CD4 T cells in the context of class I or II MHC. Recent evidence suggests that autophagy, a conserved intracellular degradation pathway, regulates class II antigen presentation. In vitro studies have shown that deletion of autophagy-related genes reduced antigen presentation by APCs to CD4 T cells. In vivo studies confirmed these findings in the context of infectious diseases. However, the relevance of autophagy-mediated antigen presentation in autoimmunity remains to be elucidated. Here, we report that loss of autophagy-related gene 7 (Atg7) in DCs ameliorated experimental autoimmune encephalomyelitis (EAE), a CD4 T cell-mediated mouse model of multiple sclerosis, by reducing in vivo priming of T cells. In contrast, severity of hapten-induced contact hypersensitivity, in which CD8 T cells and NK cells play major roles, was unaffected. Administration of the autophagy-lysosomal inhibitor chloroquine, before EAE onset, delayed disease progression and, when administered after the onset, reduced disease severity. Our data show that autophagy is required in DCs for induction of EAE and suggest that autophagy might be a potential target for treating CD4 T cell-mediated autoimmune conditions.

Proteasome dysfunction activates autophagy and the Keap1-Nrf2 pathway.

The ubiquitin-proteasome system and autophagy are crucially important for proteostasis in cells. These pathways are interdependent, and dysfunction in either pathway causes accumulation of ubiquitin-positive aggregates, a hallmark of human pathological conditions. To elucidate in vivo compensatory action(s) against proteasomal dysfunction, we developed mice with reduced proteasome activity in their livers. The mutant mice exhibited severe liver damage, accompanied by formation of aggregates positive for ubiquitin and p62/Sqstm1, an adaptor protein for both selective autophagy and the anti-oxidative Keap1-Nrf2 pathway. These aggregates were selectively entrapped by autophagosomes, and pathological features of livers with impaired proteasome activity were exacerbated by simultaneous suppression of autophagy. In contrast, concomitant loss of p62/Sqstm1 had no apparent effect on the liver pathology though p62/Sqstm1 was indispensable for the aggregates formation. Furthermore, defective proteasome function led to transcriptional activation of the Nrf2, which served as a physiological adaptation. Our in vivo data suggest that cells contain networks of cellular defense mechanisms against defective proteostasis.