HIF1A Alleviates compression-induced apoptosis of nucleus pulposus derived stem cells via upregulating autophagy.
He Ruijun,Wang Zhe,Cui Min,Liu Sheng,Wu Wei,Chen Mo,Wu Yongchao,Qu Yanji,Lin Hui,Chen Sheng,Wang Baichuan,Shao Zengwu
Intervertebral disc degeneration (IDD) is the primary pathological mechanism that underlies low back pain. Overloading-induced cell death, especially endogenous stem cell death, is the leading factor that undermines intrinsic repair and aggravates IDD. Previous research has separately studied the effect of oxygen concentration and mechanical loading in IDD. However, how these two factors synergistically influence endogenous repair remains unclear. Therefore, we established and models to study the mechanisms by which hypoxia interacted with overloading-induced cell death of the nucleus pulposus derived stem cells (NPSCs). We found the content of HIF1A (hypoxia inducible factor 1 subunit alpha) and the number of NPSCs decreased with disc degeneration in both rats and human discs. Hence, we isolated this subpopulation from rat discs and treated them simultaneously with hypoxia and excessive mechanical stress. Our results demonstrated that hypoxia exerted protective effect on NPSCs under compression, partially through elevating macroautophagy/autophagy. Proteomics and knockdown experiments further revealed HIF1A-BNIP3-ATG7 axis mediated the increase in autophagy flux, in which HMOX1 and SLC2A1 were also involved. Moreover, HIF1A-overexpressing NPSCs exhibited stronger resistance to over-loading induced apoptosis . They also showed higher survival rates, along with elevated autophagy after being intra-disc transplanted into over-loaded discs. Jointly, both and experiments proved the anti-apoptotic effect of HIF1A on NPSCs under the excessive mechanical loading, suggesting that restoring hypoxia and manipulating autophagy is crucial to maintain the intrinsic repair and to retard disc degeneration. 3-MA: 3-methyladenine; ACAN: aggrecan; ATG7: autophagy related 7; BafA1: bafilomycin A; BAX: BCL2 associated X, apoptosis regulator; BECN1: beclin 1; BNIP3: BCL2 interacting protein 3; BNIP3L: BCL2 interacting protein 3 like; CASP3: caspase 3; CCK8: cell counting kit-8; CHT: chetomin; CMP: compression; CoCl: cobalt chloride; COL2A1: collagen type II alpha 1 chain; Ctrl: control; DAPI: 4,6-diamidino-2-phenylindole; DEP: differentially expressed protein; DiR: 1,1-dioctadecyl-3,3,3,3-tetramethyl indotricarbocyanine; ECM: extracellular matrix; FCM: flow cytometry; GD2: disialoganglioside GD 2; GFP: green fluorescent protein; GO: gene ontology; GSEA: gene set enrichment analysis; H&E: hematoxylin-eosin; HIF1A: hypoxia inducible factor 1 subunit alpha; HK2: hexokinase 2; HMOX1: heme oxygenase 1; HX: hypoxia mimicry; IDD: intervertebral disc degeneration; IF: immunofluorescence; IHC: immunohistochemistry; IVD: intervertebral disc; KEGG: kyoto encyclopedia of genes and genomes; LBP: low back pain; Lv: lentivirus; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MMP: mitochondrial membrane potential; NC: negative control; NIR: near-infrared; NP: nucleus pulposus; NPC: nucleus pulposus cell; NPSC: nucleus pulposus derived stem cell; NX: normoxia; PPI: protein-protein interactions; RFP: red fluorescent protein; SLC2A1/GLUT1: solute carrier family 2 member 1; SQSTM1/p62: sequestosome 1; TEK/TIE2: TEK receptor tyrosine kinase; TEM: transmission electron microscopy; TUBB: tubulin beta class I.
Hypoxia-Induced Autophagy Enhances Cisplatin Resistance in Human Bladder Cancer Cells by Targeting Hypoxia-Inducible Factor-1.
Mao Xiawa,Nanzhang ,Xiao Jiaquao,Wu Huifeng,Ding Kefeng
Journal of immunology research
Purpose:To investigate the effect of hypoxia on chemoresistance and the underlying mechanism in bladder cancer cells. Methods:BIU-87 bladder cancer cell line was treated with cisplatin under hypoxic and normoxic conditions and tested using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, flow cytometry, and Western blotting. All the data were expressed as mean ± standard error from three independent experiments and analyzed by multiple -tests. Results:Apoptosis of bladder cancer cells caused by cisplatin was attenuated in hypoxic conditions. Hypoxia enhanced autophagy caused by cisplatin. The autophagy inhibitor and HIF-1 inhibitor can reverse the chemoresistance in hypoxic condition. Apoptosis and autophagy of bladder cancer cells were downregulated by HIF-1 inhibitor YC-1. Hypoxia-induced autophagy enhanced chemoresistance to cisplatin via the HIF-1 signaling pathway. Conclusion:Resistance to cisplatin in BIU-87 bladder cancer cells under hypoxic conditions can be explained by activation of autophagy, which is regulated by HIF-1-associated signaling pathways. The hypoxia-autophagy pathway may be a target for improving the efficacy of cisplatin chemotherapy in bladder cancer.
TIGAR reduces smooth muscle cell autophagy to prevent pulmonary hypertension.
Yamanaka Ryoetsu,Hoshino Atsushi,Fukai Kuniyoshi,Urata Ryota,Minami Yoshito,Honda Sakiko,Fushimura Yohei,Hato Daichi,Iwai-Kanai Eri,Matoba Satoaki
American journal of physiology. Heart and circulatory physiology
TIGAR reduces smooth muscle cell autophagy to prevent pulmonary hypertension. 319: H1087-H1096, 2020. First published September 18, 2020; doi:10.1152/ajpheart.00314.2020.-Pulmonary arterial hypertension (PAH) is a refractory disease. Its prognosis remains poor; hence, establishment of novel therapeutic targets is urgent. TP53-induced glycolysis and apoptosis regulator (TIGAR) is a downstream target of p53 and exhibits functions inhibiting autophagy and reactive oxygen species (ROS). Recently, p53 was shown to suppress PAH progression. Because inhibition of autophagy and ROS is known to improve PAH, we examined the effect of TIGAR on PAH progression. We compared pulmonary hypertension (PH) development between TIGAR-deficient knockout (KO) and wild-type (WT) mice using a hypoxia-induced PH model. Human pulmonary artery smooth muscle cells (PASMCs) were used for in vitro experiments with small interfering RNA (siRNA) to investigate the possible molecular mechanisms. From the analysis of right ventricular pressure, right ventricular weight, and mortality rate, we concluded that the hypoxia-induced PH development was remarkably higher in TIGAR KO than in WT mice. Pathological investigation revealed that medial thickening of the pulmonary arterioles and cell proliferation were increased in TIGAR KO mice. Autophagy and ROS activity were also increased in TIGAR KO mice. TIGAR knockdown by siRNA increased cell proliferation and migration, exacerbated autophagy, and increased ROS generation during hypoxia. Autophagy inhibition by chloroquine and ROS inhibition by -acetylcysteine attenuated the proliferation and migration of PASMCs caused by TIGAR knockdown and hypoxia exposure. TIGAR suppressed the proliferation and migration of PASMCs via inhibiting autophagy and ROS and, therefore, improved hypoxia-induced PH. Thus, TIGAR might be a promising therapeutic target for PAH. Pulmonary arterial hypertension is a refractory disease. TP53-induced glycolysis and apoptosis regulator (TIGAR) is a downstream target of p53 and exhibits functions inhibiting autophagy and reactive oxygen species (ROS). By using TIGAR-deficient knockout mice and human pulmonary artery smooth muscle cells, we found that TIGAR suppressed the proliferation and migration of PASMCs via inhibiting autophagy and ROS and, therefore, improved hypoxia-induced PH. TIGAR will be a promising therapeutic target for PAH.
Baicalin inhibits Salmonella typhimurium-induced inflammation and mediates autophagy through TLR4/MAPK/NF-κB signalling pathway.
Zhang Ling,Sun Yuan,Xu Wei,Geng Yu,Su Yuhong,Wang Qiuning,Wang Jinli
Basic & clinical pharmacology & toxicology
Baicalin has been reported to protect mice against Salmonella typhimurium (S. typhimurium) infection, while its molecular mechanisms are unclear. In this study, multiplicity of infection (MOI) and observation time were measured. Cell viability and LDH levels were examined in RAW264.7 cells and H9 cells. RAW264.7 cells were stimulated with S typhimurium in the presence or absence of Baicalin, and the levels of pro-inflammatory cytokines were detected by enzyme-linked immunosorbent assay (ELISA). The changes in reactive oxygen species (ROS) production were determined by fluorescence microscopy and ELISA. The autophagy and TLR4/MAPK/NF-κB signalling pathway were examined by immunofluorescence microscopy, quantitative reverse transcription-polymerase chain reaction and Western blotting. The results indicated that MOI of 30 and duration of autophagy evident at 5 h were applicable to this study. Baicalin prevented death of macrophages, promoted bactericidal activity, decreased the levels of pro-inflammatory cytokines and ROS and reduced the changes of key biomarkers in autophagy and TLR4/MAPK/NF-κB signalling pathway infected by S typhimurium. TLR4-overexpressed cells, autophagy and TLR4/MAPK/NF-κB signalling pathway were activated by S typhimurium, which was suppressed by Baicalin. Our findings indicated that Baicalin exerts anti-inflammatory and cell-protective effects, and it mediates autophagy by down-regulating the activity of TLR4 infected by S typhimurium.
Dihydroartemisinin inhibits activation of the AIM2 inflammasome pathway and NF-κB/HIF-1α/VEGF pathway by inducing autophagy in A431 human cutaneous squamous cell carcinoma cells.
Wang Yajie,Li Zhijia,Teng Muzhou,Liu Junlin
International journal of medical sciences
The therapeutic effect of dihydroartemisinin (DHA) against cutaneous squamous cell carcinoma (cSCC) has been previously demonstrated; however, the underlying mechanism remains unclear. This study sought to verify the therapeutic effect of DHA against cSCC and explore its underlying mechanism in A431 cSCC cells. This study reported that DHA inhibited A431 cells proliferation in a time- and concentration-dependent manner and promoted A431 cells apoptosis. Moreover, DHA inhibited the invasion and migration of A431 cells. Mechanistically, DHA promoted autophagy and inhibited activation of the absent in melanoma 2 (AIM2) inflammasome pathway and NF-κB/HIF-1α/VEGF pathway. Treatment of A431 cells with the mTOR inhibitor, and autophagy promoter, rapamycin also inhibited these two pathways. In conclusion, DHA inhibited activation of the AIM2 inflammasome pathway and NF-κB/HIF-1α/VEGF pathway by promoting autophagy in A431 cells, thus accounting for its therapeutic effect. Induction of autophagy by DHA may be mediated by inhibiting the mTOR pathway and promoting reactive oxygen species production.
Melatonin prevents diabetes-associated cognitive dysfunction from microglia-mediated neuroinflammation by activating autophagy via TLR4/Akt/mTOR pathway.
Cui Yixin,Yang Mengmeng,Wang Yilin,Ren Jianmin,Lin Peng,Cui Chen,Song Jia,He Qin,Hu Huiqing,Wang Kexin,Sun Yu
FASEB journal : official publication of the Federation of American Societies for Experimental Biology
Cognitive dysfunction often occurs in diabetes mellitus patients. This study aimed to investigate the efficacy of melatonin (MLT) in improving diabetes-associated cognitive decline and the underlying mechanism involved. Type 2 diabetic mice and palmitic acid (PA)-stimulated BV-2 cells were treated by MLT, and the potential mechanisms among MLT, cognition, and autophagy were explored. The results showed that type 2 diabetic mice showed obvious learning and memory impairments in the Morris water maze test compared with normal controls, which could be ameliorated by MLT treatment. Meanwhile, MLT administration significantly improved neuroinflammation and regulated microglial apoptosis. Furthermore, autophagy inhibitor 3-methyladenine (3-MA) increased the microglial inflammation and apoptosis, indicating that the treatment effect of MLT was mediated by autophagy. Lastly, MLT treatment significantly decreased the levels of toll-like receptors 4 (TLR4), phosphorylated-protein kinase B (Akt), and phosphorylated-mechanistic target of rapamycin (mTOR), indicating that blocking TLR4/Akt/mTOR pathway might be an underlying basis for the anti-inflammatory and anti-apoptosis effects of MLT. Collectively, our study suggested that MLT could improve learning and memory in type 2 diabetic mice by activating autophagy via the TLR4/Akt/mTOR pathway, thereby inhibiting neuroinflammation and microglial apoptosis.
YiQiFuMai lyophilized injection attenuates particulate matter-induced acute lung injury in mice via TLR4-mTOR-autophagy pathway.
Xia Yuanli,S Dolgor,Jiang Siyu,Fan Ruiping,Wang Yumeng,Wang Yuwei,Tang Jiahui,Zhang Yuanyuan,He Rong Lucy,Yu Boyang,Kou Junping
Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie
Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are the serious diseases that are characterized by a severe inflammatory response of lung injuries and damage to the microvascular permeability, frequently resulting in death. YiQiFuMai (YQFM) lyophilized injection powder is a redeveloped preparation based on the well-known traditional Chinese medicine formula Sheng-Mai-San which is widely used in clinical practice in China, mainly for the treatment of microcirculatory disturbance-related diseases. However, there is little information about its role in ALI/ARDS. The aim of this study was to determine the protective effect of YQFM on particulate matter (PM)-induced ALI. The mice were intratracheally instilled with 50 mg/kg body weight of Standard Reference Material1648a (SRM1648a) in the PM-induced group. The mice in the YQFM group were given YQFM (three doses: 0.33, 0.67, and 1.34 g/kg) by tail vein injection 30 min after the intratracheal instillation of PM. The results showed that YQFM markedly reduced lung pathological injury and the lung wet/dry weight ratios induced by PM. Furthermore, we also found that YQFM significantly inhibited the PM-induced myeloperoxidase (MPO) activity in lung tissues, decreased the PM-induced inflammatory cytokines including interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α), reduced nitric oxide (NO) and total protein in bronchoalveolar lavage fluids (BALF), and effectively attenuated PM-induced increases lymphocytes in BALF. In addition, YQFM increased mammalian target of rapamycin (mTOR) phosphorylation and dramatically suppressed the PM-stimulated expression of toll-like receptor 4 (TLR4), MyD88, autophagy-related protein LC3Ⅱand Beclin 1 as well as autophagy. In conclusion, these findings indicate that YQFM had a critical anti-inflammatory effect due to its ability to regulate both TLR4-MyD88 and mTOR-autophagy pathways, and might be a possible therapeutic agent for PM-induced ALI.
MiR-205-3p protects human corneal epithelial cells from ultraviolet damage by inhibiting autophagy via targeting TLR4/NF-κB signaling.
Fu J-Y,Yu X-F,Wang H-Q,Lan J-W,Shao W-Q,Huo Y-N
European review for medical and pharmacological sciences
OBJECTIVE:MiRNA has been found to have therapeutic effect on corneal damage. This paper aimed to study the effect of miR-205-3p on corneal damage induced by ultraviolet (UV) radiation. MATERIALS AND METHODS:HCE cells were exposed to UV light and transfected. Quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) and Western blot were used to determine miRNA/mRNA and protein expression. CCK-8 assay, Edu incorporation experiment, and flow cytometry were used to separately measure cell activity, proliferation and apoptosis. LC3 puncta were researched by immunofluorescence experiment. TNF-α, IL-6 and IL-1β levels in cells were detected by enzyme-linked immunosorbent assay (ELISA) kit. MDA, SOD, and GSH-PX levels were measured using detection kits. Reactive oxygen species (ROS) level was reflected by detecting DCFH-DA density. Luciferase activity assay was performed to verify the regulating relationship between miR-205-3p and TLR4. RESULTS:UV radiation decreased HCE cell viability, proliferation, and increased HCE cell apoptosis and autophagy (all p < 0.01). When exposed UV radiation, the overexpression of miR-205-3p group elevated HCE cells viability, proliferation and weakened HCE cells apoptosis and autophagy (all p < 0.01). MiR-205-3p inhibited inflammation and oxidative stress in HCE cells induced by UV radiation (p < 0.01). MiR-205-3p directly inhibited TLR4 expression. The upregulation of TLR4 significantly reversed the effects of miR-205-3p on HCE cells phenotypes induced by UV radiation (p < 0.01). CONCLUSIONS:MiR-205-3p protected HCE cells from UV damage by inhibiting autophagy via targeting TLR4.
mycelium regulates autophagy of alveolar macrophages via TLR4/NF-κB signaling pathway.
Fu Juanhua,Lu Li,Wang Haining,Hou Yayi,Dou Huan
International journal of medical sciences
: mycelium (HSM) has potent anti-pulmonary fibrotic activities and has been proposed as an effective treatment for idiopathic pulmonary fibrosis. Macrophages are the main innate immune cells in the lung tissue, playing key roles in pulmonary fibrosis repair and homeostasis. Excessive macrophage autophagy plays a vital role in pulmonary fibrosis. The protective effect of HSM on macrophages of bleomycin (BLM)-induced pulmonary fibrotic mice remain unclear. : In this study, we collected lung tissue and bronchoalveolar lavage fluid (BALF) samples from pulmonary fibrotic mice. Meanwhile, alveolar macrophages were isolated and murine macrophage RAW264.7 cell line was cultured for further study of HSM autophagy. First, we found that HSM decreased the number of autophagosomes, as well as the levels of LC3B and ATG5, and increased the protein level of P62 during the development of pulmonary fibrosis. Meanwhile, HSM reduced alveolar macrophages infiltration into the BALF and inhibited their accumulation in the fibrotic lung tissue. Flow cytometry analysis showed that HSM administration inhibited the autophagy marker LC3B expression in CD11bCD11c alveolar macrophages in BLM-induced lung fibrosis without affecting CD11bCD11c interstitial macrophages. Transmission electron microscopy and JC-1 staining for mitochondrial membrane potential of alveolar macrophages also verified that the HSM significantly decreased autophagy in the alveolar macrophages of BLM-treated mice. , autophagosomes-lysosome fusion inhibitor chloroquine (CQ) was pre-incubated with RAW264.7 cells, and HSM reduced CQ-induced autophagosomes accumulation. TLR4 signaling inhibitor CLI095 reversed the above effects, suggesting HSM could reduce the cumulation of autophagosomes dependent on TLR4. Furthermore, lipopolysaccharide (LPS)-stimulated TLR4-related autophagy was significantly inhibited by HSM treatment. In addition, the protein expressions of TLR4 and phospho-NF-κB p65 were markedly inhibited in cells treated with HSM. : These results indicated that HSM could inhibit the autophagy of alveolar macrophages through TLR4/NF-κB signaling pathway to achieve anti-fibrotic effect.
miR‑760 mediates hypoxia-induced proliferation and apoptosis of human pulmonary artery smooth muscle cells via targeting TLR4.
Yang Yu-Zhong,Zhang Yun-Feng,Yang Lei,Xu Jing,Mo Xu-Ming,Peng Wei
International journal of molecular medicine
MicroRNAs (miRNAs) have a key role in the pathogenesis of pulmonary arterial hypertension (PAH), a disease characterized by enhanced proliferation and reduced apoptosis of pulmonary artery smooth muscle cells. In the present study, miR‑760 was demonstrated to be downregulated in PAH lung tissues compared with normal lung tissues, an effect that may be associated with the development of PAH. Hypoxia is an important stimulus for human pulmonary artery smooth muscle cell (hPASMC) proliferation and the occurrence of PAH. Therefore, the effect of miR‑760 in hypoxia‑treated and normal hPASMCs was investigated. Expression of exogenous miR‑760 decreased cell proliferation in hypoxia‑induced hPASMCs, and promoted cell apoptosis with an increase in the BCL2 associated X/BCL2 ratio and the expression levels of Caspase‑3 and Caspase‑9. In addition, overexpression of miR‑760 suppressed the migration of hPASMCs under hypoxic conditions. Furthermore, miR‑760 was demonstrated to mediate its anti‑proliferation effect via the regulation of toll‑like receptor 4 (TLR4), a direct target of miR‑760. The results revealed that knockdown of TLR4 restrained the hypoxia‑induced hPASMC proliferation and induced cell apoptosis. The present study uncovered a novel regulatory pathway involving miR‑760 and suggested that miR‑760 may be explored as a potential therapy for PAH in the future.
Expression and significance of TLR4 and HIF-1alpha in pancreatic ductal adenocarcinoma.
Zhang Jian-Jun,Wu He-Shui,Wang Lin,Tian Yuan,Zhang Jing-Hui,Wu Hai-Long
World journal of gastroenterology
AIM:To investigate the expression of toll-like receptor (TLR) 4, nuclear factor-kappaB (NF-kappaB) p65 and hypoxia-inducible transcription factor 1alpha (HIF-1alpha) in pancreatic ductal adenocarcinoma and their clinical significance. METHODS:The mRNA of TLR4 and HIF-1alpha were investigated by real-time polymerase chain reaction in 30 cases of pancreatic ductal adenocarcinoma and its adjacent tissues, and expression of TLR4, NF-kappaB p65 and HIF-1alpha protein were detected by immunohistochemistry in 65 cases of pancreatic ductal adenocarcinoma tissues and 38 cases of corresponding adjacent tissues. The relationship between TLR4 or HIF-1alpha and pathologic features, as well as the association between TLR4 and HIF-1alpha, were also analyzed. Kaplan-Meier method was used to assess the impact of expression of TLR4 and HIF-1alpha on survival of patients with pancreatic cancer. RESULTS:The relative quantification of TLR4 and HIF-1alpha mRNA in tumor tissues was 0.81 +/- 0.10 and 0.87 +/- 0.11, respectively, significantly higher than that in adjacent tissues (0.81 +/- 0.10 vs 0.70 +/- 0.16, P = 0.002; 0.87 +/- 0.11 vs 0.68 +/- 0.13, P = 0.000). The protein expression of TLR4, NF-kappaB p65 and HIF-1alpha in tumor tissues was 69.20%, 66.15% and 70.80%, respectively, being significantly higher than that in adjacent normal tissues (69.20% vs 39.50%, P = 0.003; 66.15% vs 31.58%, P = 0.001; 70.80% vs 36.80%, P = 0.001). There was no significant correlation between TLR4 or HIF-1alpha expression and the age, gender, tumor location, the degree of tumor differentiation in the patients (P > 0.05). However, there was significant correlation between the expression of TLR4 or HIF-1alpha and tumor size, lymph node metastasis, venous invasion and clinical staging (P < 0.05). The expression of TLR4 and HIF-1alpha had a significant impact on survival of patients with pancreatic adenocarcinoma. CONCLUSION:TLR4, NF-kappaB p65 and HIF-1alpha are overexpressed in pancreatic adenocarcinoma, TLR4 may be partly involved in up-regulating HIF-1alpha, and both synergestically promote development of pancreatic adenocarcinoma.
Melatonin activates autophagy via the NF-κB signaling pathway to prevent extracellular matrix degeneration in intervertebral disc.
Chen F,Liu H,Wang X,Li Z,Zhang J,Pei Y,Zheng Z,Wang J
Osteoarthritis and cartilage
OBJECTIVE:This study investigated whether melatonin alleviates intervertebral disc degeneration (IVDD) by promoting autophagy through inhibiting the NF-κB signaling pathway. METHODS:Magnetic resonance imaging (MRI), hematoxylin and eosin (H&E) staining and Safranin-O staining were used to measure disc degeneration in rat needle puncture IVDD models, and melatonin was injected intraperitoneally in the treated group to test its function. The expression of autophagy and extracellular matrix (ECM) degeneration related-markers were measured in the discs using immunohistochemistry. Transmission electron microscopy was used to evaluate the activation of autophagy in human nucleus pulposus (NP) tissues with different degenerated statuses. The expression of autophagy and disc degeneration related-markers were detected in NP cells by Western blot, RT-qPCR, and immunofluorescence analyses. NF-κB signaling pathway involvement was studied by lentivirus-mediated knockdown, Western blotting, and immunohistochemistry and immunofluorescence staining. RESULTS:Melatonin prevented IVDD development in vivo and in vitro. Compared to non-degenerated disc tissues, degenerated human NP tissues showed a decrease in the autophagy-specific marker LC3B and the numbers of autophagosomes and autolysosomes, whereas the p62 level was increased; similar results were observed in rat IVDD models, indicating a negative correlation between autophagy and IVDD. Furthermore, both in vivo and in vitro studies found that melatonin application induced autophagy and reduced ECM disc degradation. Melatonin was also shown to regulate autophagy by inhibiting the NF-κB signaling pathway in vivo and vitro. CONCLUSION:This study indicates that melatonin prevents IVDD by promoting autophagy, indicating its possible therapeutic potential for controlling the progression of IVDD.
Guizhi Fuling Wan reduces autophagy of granulosa cell in rats with polycystic ovary syndrome via restoring the PI3K/AKT/mTOR signaling pathway.
Liu Min,Zhu Hongqiu,Zhu Ying,Hu Xiaodan
Journal of ethnopharmacology
ETHNOPHARMACOLOGICAL RELEVANCE:Guizhi Fuling Wan (GFW) is a traditional Chinese medicine used to remove blood stasis and dissipate phlegm for treating gynecological diseases that was invented by Zhang Zhongjing in the Eastern Han dynasty. In recent years, GFW has been widely used to treat patients with polycystic ovary syndrome (PCOS). Clinical and animal studies have shown that it is effective in the treatment of PCOS, but its mechanism is unknown. Generally, it works by regulating autophagy via the PI3K/AKT/mTOR signaling pathway. AIM OF THE STUDY:This study investigated the effects and mechanism of GFW in PCOS rats with insulin resistance (IR) in order to provide better understanding of its observed clinical effects and a theoretical basis for the study of traditional Chinese medicine. MATERIALS AND METHODS:Eighty-four female Sprague-Dawley rats were randomly divided into seven groups (n = 12 per group): 1) control, 2) PCOS model, 3) low-dose GFW, 4) medium-dose GFW, 5) high-dose GFW, 6) metformin, and 7) medium-dose GFW plus LY294002. In all non-control groups, we induced PCOS through daily letrozole combined with intragastric high-fat emulsion for 21 days. After treatment, rats were sacrificed and serum follicle-stimulating hormone (FSH), testosterone (T), progesterone, luteinizing hormone (LH), 17β-estradiol, fasting insulin (FINS), and fasting plasma glucose levels were measured by enzyme-linked immunosorbent assay (ELISA). The LH/FSH ratios and HOMA-IR values were calculated. Ovarian morphology was observed by hematoxylin and eosin staining, and all follicles were counted under a microscope. MDC-positive vesicles were used as markers to detect autophagy, and the expression levels of p62, Beclin1, and LC3-II were examined by immunostaining. Western blotting was used to measure PI3K/AKT/mTOR pathway activation, granulosa cell apoptosis, and autophagy. RESULTS:Compared with the PCOS model group, GFW-treated rats had less atretic and cystic follicles, and more mature follicles and corpus lutea. The GFW-treated rats had lower serum T, LH, and FINS levels than the PCOS model group, as well as lower LH/FSH ratios and HOMA-IR values. GFW treatment resulted in significantly reduced levels of cleaved-Caspase-3, cleaved-Caspase-9, BAX, Beclin1, Atg5, and LC3-II. Phosphorylation of PI3K, AKT, and mTOR was significantly higher in GFW-treated rats compared with the PCOS model group. The phosphorylation of PI3K, AKT, and mTOR was decreased with the use of a PI3K antagonist. CONCLUSIONS:Our results indicate that GFW inhibited granulosa cell autophagy and promoted follicular development to attenuate ovulation disorder in PCOS-IR rats. This was associated with activation of the PI3K/AKT/mTOR signaling pathway.
Aconitine induces autophagy via activating oxidative DNA damage-mediated AMPK/ULK1 signaling pathway in H9c2 cells.
Wang Wenlin,Jiang Jialuo,Huang Yan,Peng Fu,Hu Tingting,Wu Jiayang,Pan Xiaoqi,Rao Chaolong
Journal of ethnopharmacology
ETHNOPHARMACOLOGICAL RELEVANCE:Aconitum species, with a medicinal history of 2000 years, was traditionally used in the treatment of rheumatism, arthritis, bruises, and pains. However, many studies have reported that Aconitum species can cause arrhythmia in experimental animals, resulting in myocardial fibrosis and cardiomyocyte damage. Cardiotoxicity is the main toxic effect of aconitine, but the detailed mechanism remains unclear. AIM OF THE STUDY:This study aimed to explore the effects and underlying mechanism of autophagy in H9c2 cardiomyocytes induced by aconitine. MATERIALS AND METHODS:H9c2 cells were incubated with different concentrations of aconitine for 24 h, and the intervention sections were pretreated with various inhibitors for 1 h. The effects of aconitine on the oxidative DNA damage, autophagy and viability of H9c2 cells were evaluated by flow cytometry, confocal microscopy, enzyme-linked immunosorbent assay and Western blot. RESULTS:In H9c2 cells, the cell viability declined, LDH release rate, the number of autophagosomes, protein expression levels of LC3 and Beclin-1 increased significantly after 24 h of aconitine incubation. The pretreatment of autophagy inhibitor 3-MA decreased markedly autophagosomes and protein expression levels of LC3 and Beclin-1, which suggested that aconitine could induce cell autophagy. The significant increase of ROS and 8-OHdG showed that aconitine could cause oxidative DNA damage through ROS accumulation. Meanwhile, treatment of aconitine dramatically increased AMPK and ULK1 phosphorylation, and Compound C inhibited AMPK and ULK1 phosphorylation, which proved that aconitine induced autophagy via AMPK activation mediated ULK1 phosphorylation. Antioxidant NAC significantly reduced LDH, ROS and 8-OHdG, inhibited the phosphorylation of AMPK and ULK1, and down-regulated autophagosomes and proteins expression levels of LC3 and Beclin-1. Consequently, the inhibition of oxidative DNA damage and AMPK/ULK1 signaling pathway alleviated the aconitine-induced autophagic death of H9c2 cells. CONCLUSIONS:These results showed that aconitine induces autophagy of H9c2 cardiomyocytes by activating AMPK/ULK1 signaling pathway mediated by oxidative DNA damage. The autophagy induced by aconitine in cardiomyocytes is dependent on the activation of the AMPK pathway, which may provide novel insights into the prevention of aconitine-related toxicity.
Fluoride regulates chondrocyte proliferation and autophagy via PI3K/AKT/mTOR signaling pathway.
Ma Lan,Zhang Ruixue,Li Demin,Qiao Tingting,Guo Xiaoying
Fluorine is an essential trace element for human health. However, excessive fluoride intake causes skeletal fluorosis which affects cartilage development. Fluoride inhibited chondrocyte proliferation which is the initial and critical step of endochondral ossification, but the underlying mechanism has not been clearly illustrated. Mammalian target of rapamycin (mTOR) is an important protein kinase which modulates various cellular processes and is believed to be a central regulator of chondrocyte proliferation and autophagy. In this study, we explored the effect of fluoride on the proliferation and autophagy of chondrocytes and the regulatory role of mTOR signaling pathway. Our results suggested that NaF inhibited the protein expressions of proliferating cell nuclear antigen (PCNA) and pS6 in cultured fetal rat tibias. Furthermore, NaF significantly downregulated the expressions of mTOR signaling pathway-related genes, including PI3K, AKT, mTOR, 4EBP1 and S6K1 in mouse ATDC5 chondrogenic cell line. We also found that NaF increased autophagy in ATDC5 cells. The mRNA and protein levels of autophagy-related genes LC3, Beclin1 and p62 were significantly changed after NaF treatment. Further studies demonstrated that MHY1485, a small-molecular mTOR activator, totally reversed fluoride-induced promotion of autophagy. MHY1485 also recovered the downregulation of proliferative chondrocytes markers Sox9 and Type Ⅱ Collagen (Col2a1) induced by fluoride in ATDC5 cells. Taken together, our result demonstrate that fluoride suppressed proliferation and facilitated autophagy via PI3K/AKT/mTOR signaling pathway in chondrogenesis.
Autophagy regulates hypoxia-induced osteoclastogenesis through the HIF-1α/BNIP3 signaling pathway.
Zhao Yi,Chen Gang,Zhang Wei,Xu Ning,Zhu Jun-Yi,Jia Jun,Sun Zhi-Jun,Wang Yi-Ning,Zhao Yi-Fang
Journal of cellular physiology
Previous studies have implicated that hypoxic stress could enhance osteoclast differentiation; however, the underlying mechanism remains poorly understood. Autophagy is a dynamic lysosomal degradation process that has emerged as an important regulator under hypoxic environment. In the present study, we demonstrate for the first time that autophagy regulates hypoxia-induced osteoclastogenesis in vitro. We found that exposure of RAW264.7 cells to hypoxia (0.2% oxygen) resulted in enhanced osteoclast differentiation, accompanied by the observation of several specific features of autophagy, including appearance of membranous vacuoles, formation of acidic vesicular organelles, cleavage and recruitment of microtubule-associated protein 1 light chain 3 (LC3) to autophagosomes, increase in autophagic flux, as well as up-regulation of autophagy-related gene (Atg) expression. Moreover, suppression of autophagy with DN-Atg5(K130R) or 3-methyladenine (3-MA) significantly attenuated the osteoclast differentiation under hypoxic conditions, indicating the functional significance of autophagy in hypoxia-induced osteoclastogenesis. The data also showed that the activation of autophagy under hypoxic conditions was caused by up-regulated expression of hypoxia-inducible factor-1α (HIF-1α)-dependent Bcl-2 adenovirus E1a 19 kDa interacting protein 3 (BNIP3). Importantly, knockdown of HIF-1α or BNIP3 obviously abrogated hypoxia-induced autophagy activation and osteoclastogenesis enhancement. Collectively, our results highlight the fact that autophagy is a pivotal regulator for hypoxia-induced osteoclast differentiation, which may provide new insight into the pathological processes of osteoclastogenesis under hypoxic stress and help develop new therapeutic strategies for abnormal osteoclastogenesis.
Upregulation of AMPK by 4-O-methylascochlorin promotes autophagy via the HIF-1α expression.
Seok Ji-Young,Jeong Yun-Jeong,Hwang Soon-Kyung,Kim Cheorl-Ho,Magae Junji,Chang Young-Chae
Journal of cellular and molecular medicine
4-O-methylascochlorin (MAC) is a derivative of ascochlorin, a prenyl-phenol compound antibiotic isolated from the fungus Ascochyta viciae. MAC induces caspase/poly (ADP-ribose) polymerase-mediated apoptosis in leukemia cells. However, the effects of MAC on autophagy in cancer cells and the underlying molecular mechanisms remain unknown. Here, we show that MAC induces autophagy in lung cancer cells. MAC significantly induced the expression of autophagy marker proteins including LC3-II, Beclin1, and ATG7. MAC promoted AMP-activated protein kinase (AMPK) phosphorylation and inhibited the phosphorylation of mammalian target of rapamycin (mTOR) and its downstream signalling proteins P70S6K and 4EBP1. The AMPK activator AICAR upregulated LC3-II expression through the AMPK/mTOR pathway similar to the effects of MAC. MAC-induced LC3-II protein expression was slightly reduced in AMPK siRNA transfected cells. MAC upregulated hypoxia-inducible factor-1α (HIF-1α) and BNIP3, which are HIF-1α-dependent autophagic proteins. Treatment with CoCl , which mimics hypoxia, induced autophagy similar to the effect of MAC. The HIF-1α inhibitor YC-1 and HIF-1α siRNA inhibited the MAC-induced upregulation of LC3-II and BNIP3. These results suggest that MAC induces autophagy via the AMPK/mTOR signalling pathway and by upregulating HIF-1α and BNIP3 protein expression in lung cancer cells.
Transition of autophagy and apoptosis in fibroblasts depends on dominant expression of HIF-1α or p53.
Journal of Zhejiang University. Science. B
It has been revealed that hypoxia is dynamic in hypertrophic scars; therefore, we considered that it may have different effects on hypoxia-inducible factor-1α (HIF-1α) and p53 expression. Herein, we aimed to confirm the presence of a teeterboard-like conversion between HIF-1α and p53, which is correlated with scar formation and regression. Thus, we obtained samples of normal skin and hypertrophic scars to identify the differences in HIF-1α and autophagy using immunohistochemistry and transmission electron microscopy. In addition, we used moderate hypoxia in vitro to simulate the proliferative scar, and silenced or gene expression or triggered overexpression to investigate the changes of HIF-1α and p53 expression, autophagy, apoptosis, and cell proliferation under this condition. HIF-1α, p53, and autophagy-related proteins were assayed using western blotting and immunofluorescence, whereas apoptosis was detected using flow cytometry analysis, and cell proliferation was detected using cell counting kit-8 (CCK-8) and 5-bromo-2'-deoxyuridine (BrdU) staining. Furthermore, immunoprecipitation was performed to verify the binding of HIF-1α and p53 to transcription cofactor p300. Our results demonstrated that, in scar tissue, HIF-1α expression increased in parallel with autophagosome formation. Under hypoxia, HIF-1α expression and autophagy were upregulated, whereas p53 expression and apoptosis were downregulated in vitro. HIF-1α knockdown downregulated autophagy, proliferation, and p300-bound HIF-1α, and upregulated p53 expression, apoptosis, and p300-bound p53. Meanwhile, p53 knockdown induced the opposite effects and enhanced HIF-1α, whereas p53 overexpression resulted in the same effects and reduced HIF-1α. Our results suggest a teeterboard-like conversion between HIF-1α and p53, which is linked with scar hyperplasia and regression.
Increased reactive oxygen species, metabolic maladaptation, and autophagy contribute to pulmonary arterial hypertension-induced ventricular hypertrophy and diastolic heart failure.
Rawat Dhawjbahadur K,Alzoubi Abdallah,Gupte Rakhee,Chettimada Sukrutha,Watanabe Makino,Kahn Andrea G,Okada Takao,McMurtry Ivan F,Gupte Sachin A
Hypertension (Dallas, Tex. : 1979)
Pulmonary arterial hypertension (PAH) is a debilitating and deadly disease with no known cure. Heart failure is a major comorbidity and a common cause of the premature death of patients with PAH. Increased asymmetrical right ventricular hypertrophy and septal wall thickening compress the left ventricular cavity and elicit diastolic heart failure. In this study, we used the Sugen5416/hypoxia/normoxia-induced PAH rat to determine whether altered pyridine nucleotide signaling in the failing heart contributes to 1) increased oxidative stress, 2) changes in metabolic phenotype, 3) autophagy, and 4) the PAH-induced failure. We found that increased reactive oxygen species, metabolic maladaptation, and autophagy contributed to the pathogenesis of right ventricular remodeling and hypertrophy that lead to left ventricular diastolic dysfunction. In addition, arterial elastance increased in PAH rats. Glucose-6-phosphate dehydrogenase is a major source of pyridine molecule (nicotinamide adenine dinucleotide phosphate), which is a substrate for nicotinamide adenine dinucleotide phosphate oxidases in the heart. Dehydroepiandrosterone, a 17-ketosteroid that reduces pulmonary hypertension and right ventricular hypertrophy, inhibited glucose-6-phosphate dehydrogenase, decreased oxidative stress, increased glucose oxidation and acetyl-coA, and reduced autophagy in the hearts of PAH rats. It also decreased arterial stiffness and improved left ventricular diastolic function. These findings demonstrate that pyridine nucleotide signaling, at least partly, mediates PAH-induced diastolic heart failure, and that reduction of glucose-6-phosphate dehydrogenase-derived nicotinamide adenine dinucleotide phosphate is beneficial to improve left ventricle diastolic function.
Glucagon-Like Peptide-1 Receptor Agonist Attenuates Autophagy to Ameliorate Pulmonary Arterial Hypertension through Drp1/NOX- and Atg-5/Atg-7/Beclin-1/LC3β Pathways.
Wu Yi-Chia,Wang Wei-Ting,Lee Su-Shin,Kuo Yur-Ren,Wang Ya-Chin,Yen Shih-Jung,Lee Mei-Yueh,Yeh Jwu-Lai
International journal of molecular sciences
Mitochondrial dysfunction is associated with cardiovascular diseases and diabetes. Pulmonary arterial hypertension (PAH) is characterized by pulmonary vascular remodeling, and the abnormal proliferation, apoptosis and migration of pulmonary arterial smooth muscle cells (PASMCs). The glucagon-like peptide-1 (GLP-1) receptor agonist, liraglutide, has been shown to prevent pulmonary hypertension in monocrotaline-exposed rats. The aim of this study was to investigate the effect of liraglutide on autophagy, mitochondrial stress and apoptosis induced by platelet-derived growth factor BB (PDGF-BB). PASMCs were exposed to PDGF-BB, and changes in mitochondrial morphology, fusion-associated protein markers, and reactive oxygen species (ROS) production were examined. Autophagy was assessed according to the expressions of microtubule-associated protein light chain 3 (LC3)-II, LC3 puncta and Beclin-1. Western blot analysis was used to assess apoptosis, mitochondrial stress and autophagy markers. Liraglutide significantly inhibited PDGF-BB proliferation, migration and motility in PASMCs. PDGF-BB-induced ROS production was mitigated by liraglutide. Liraglutide increased the expression of α-smooth muscle actin (α-SMA) and decreased the expression of p-Yes-associated protein (p-YAP), inhibited autophagy-related protein (Atg)-5, Atg-7, Beclin-1 and the formation of LC3-β and mitochondrial fusion protein dynamin-related (Drp)1. Therefore, liraglutide can mitigate the proliferation of PASMCs via inhibiting cellular Drp1/nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (NOX) pathways and Atg-5/Atg-7/Beclin-1/LC3β-dependent pathways of autophagy in PAH.
Piperlongumine attenuates vascular remodeling in hypoxic pulmonary hypertension by regulating autophagy.
Ye Wu,Tang Tingyu,Li Zhijun,Li Xuefang,Huang Qingdong
Journal of cardiology
OBJECTIVE:The aim of this study was to determine the therapeutic effect of piperlongumine on hypoxic pulmonary hypertension. METHODS:A hypoxic pulmonary hypertension rat model was constructed, primary rat pulmonary artery smooth muscle cells (PASMCs) were isolated, and the proliferation of PASMCs was measured by Cell Counting Kit‑8 assay. The expression of autophagic proteins microtubule-associated protein 1 light chain 3B (LC3B) and P62 were examined by western blot. Autophagic flux in PASMCs was detected by tandem mRFP-GFP-LC3 fluorescence analysis. RESULTS:Hypoxia-induced proliferation of PASMCs was significantly inhibited by piperlongumine exposure. Treatment with piperlongumine elevated LC3B II/LC3B I protein ratio and decreased the expression of P62 protein in both PASMCs and rat lung tissues. Tandem mRFP-GFP-LC3 fluorescence analysis showed that piperlongumine increased autophagic flux in PASMCs. Inhibition of autophagy using 3-methyladenine (3-MA) attenuated the inhibitory effect of piperlongumine on proliferation of PASMCs. Chronic hypoxia exposure led to a significant increase in rat right ventricle systolic pressure, right ventricular hypertrophy, wall thickness and area of pulmonary artery, and muscularization of pulmonary arterioles, which was obviously suppressed by administration of piperlongumine. 3-MA attenuated the alleviating effects of piperlongumine on pulmonary vascular remodeling. CONCLUSIONS:Piperlongumine attenuates vascular remodeling in hypoxic pulmonary hypertension by regulating autophagy. Piperlongumine treatment may serve as a promising therapy for hypoxic pulmonary hypertension.
Apelin inhibits the proliferation and migration of rat PASMCs via the activation of PI3K/Akt/mTOR signal and the inhibition of autophagy under hypoxia.
Zhang Hongyu,Gong Yongsheng,Wang Zhouguang,Jiang Liping,Chen Ran,Fan Xiaofang,Zhu Huanmian,Han Liping,Li Xiaokun,Xiao Jian,Kong Xiaoxia
Journal of cellular and molecular medicine
Apelin is highly expressed in the lungs, especially in the pulmonary vasculature, but the functional role of apelin under pathological conditions is still undefined. Hypoxic pulmonary hypertension is the most common cause of acute right heart failure, which may involve the remodeling of artery and regulation of autophagy. In this study, we determined whether treatment with apelin regulated the proliferation and migration of rat pulmonary arterial smooth muscle cells (SMCs) under hypoxia, and investigated the underlying mechanism and the relationship with autophagy. Our data showed that hypoxia activated autophagy significantly at 24 hrs. The addition of exogenous apelin decreased the level of autophagy and further inhibited pulmonary arterial SMC (PASMC) proliferation via activating downstream phosphatidylinositol-3-kinase (PI3K)/protein kinase B (Akt)/the mammalian target of Rapamycin (mTOR) signal pathways. The inhibition of the apelin receptor (APJ) system by siRNA abolished the inhibitory effect of apelin in PASMCs under hypoxia. This study provides the evidence that exogenous apelin treatment contributes to inhibit the proliferation and migration of PASMCs by regulating the level of autophagy.
Altered mTOR and Beclin-1 mediated autophagic activation during right ventricular remodeling in monocrotaline-induced pulmonary hypertension.
Deng Yan,Wu Weifeng,Guo Shenglan,Chen Yuming,Liu Chang,Gao Xingcui,Wei Bin
BACKGROUND:Right ventricular structure and function is a major predictor of outcomes in pulmonary hypertension (PH), yet the underlying mechanisms remain poorly understood. Growing evidence suggests the importance of autophagy in cardiac remodeling; however, its dynamics in the process of right ventricle(RV) remodeling in PH has not been fully explored. We sought to study the time course of cardiomyocyte autophagy in the RV in PH and determine whether mammalian target of rapamycin (mTOR) and Beclin-1 hypoxia-related pro-autophagic pathways are underlying mechanisms. METHODS:Rats were studied at 2, 4, and 6 weeks after subcutaneous injection of 60 mg/kg monocrotaline (MCT) (MCT-2 W, 4 W, 6 W) or vehicle (CON-2 W, 4 W, 6 W). Cardiac hemodynamics and RV function were assessed in rats. Autophagy structures and markers were assessed using transmission electron microscope, RT-qPCR, immunohistochemistry staining, and western blot analyses. Western blot was also used to quantify the expression of mTOR and Beclin-1 mediated pro-autophagy signalings in the RV. RESULTS:Two weeks after MCT injection, pulmonary artery systolic pressure increased and mild RV hypertrophy without RV dilation was observed. RV enlargement presented at 4 weeks with moderately decreased function, whereas typical characteristics of RV decompensation and failure occurred at 6 weeks thus demonstrating the progression of RV remodeling in the MCT model. A higher LC3 (microtubule- associated protein light chain 3) II/I ratio, upregulated LC3 mRNA and protein levels, as well as accumulation of autophagosomes in RV of MCT rats indicated autophagy induction. Autophagy activation was coincident with increased pulmonary artery systolic pressure. Pro-autophagy signaling pathways were activated in a RV remodeling stage-dependent manner since phospho-AMPK (adenosine monophosphate-activated protein kinase)-α were primarily upregulated and phospho-mTOR suppressed in the RV at 2 and 4 weeks post-MCT injection, whearas, BNIP3 (Bcl2-interacting protein 3) and beclin-1 expression were relatively low during these stages, they were significantly upregulated after 6 weeks in this model. CONCLUSIONS:Our findings provide evidence of sustained activation of autophagy in RV remodeling of MCT induced PH model, while pro-autophagic signaling pathways varied depending on the phase.
β-arrestin1 inhibits hypoxic injury-induced autophagy in human pulmonary artery endothelial cells via the Akt/mTOR signaling pathway.
Ning Hongjuan,Deng Jingyu,Chen Feng,Liu Yongfei,Kong Delei,Shan Lina,Zhang Zheng,Hu Taohong
The international journal of biochemistry & cell biology
Autophagy has been greatly implicated in injured endothelial cells during pulmonary arterial hypertension (PAH). β-arrestin1, a multifunctional cytoplasmic protein, has attracted considerable attention as an essential protective factor in PAH. However, its role in autophagy of injured pulmonary arterial endothelial cells (PAECs) remains to be determined. Here, we investigated the potential effects of β-arrestin1 on autophagy and apoptosis in human PAECs (hPAECs) under hypoxic stress. Hypoxic stimuli increases autophagy and decreases the level of β-arrestin1 in hPAECs. Furthermore, pathologic changes, namely increased proliferation, migration, and apoptosis resistance, are observed after hypoxia exposure. These are reversed after β-arrestin1 overexpression (β-arrestin1-OV) or treatment with 3-MA, an autophagy inhibitor. Finally, β-arrestin1 suppresses the increase in autophagy and apoptosis resistance of hypoxic hPAECs. Mechanistically, β-arrestin1 upregulates the activity of the Akt/mTOR signaling pathway and downregulates the expression of BNIP3 and Nix after hypoxic stress. Collectively, we have demonstrated, for the first time, that β-arrestin1 reduces excessive autophagy and apoptosis resistance by activating the Akt/mTOR axis in hypoxic hPAECs. This knowledge suggests a promising therapeutic target for PAH.
Mammalian target of rapamycin overexpression antagonizes chronic hypoxia-triggered pulmonary arterial hypertension via the autophagic pathway.
Li Lingxia,Wang Xiaochuang,Wang Lina,Qu Li,Zhu Xinye,Li Manxiang,Dang Xiaoyan,Li Ping,Gao Yanxia,Peng Zhuo,Pan Longfei,Wan Li
International journal of molecular medicine
Pulmonary arterial hypertension (PAH) is a progressive pulmonary vascular disorder with high morbidity and mortality, and is characterized by excessive growth of endothelial cells. Recently, the mammalian target of rapamycin (mTOR) has attracted increasing attention due to its potential as a therapeutic target against certain diseases associated with proliferative and metabolic abnormalities. However, the effect on mTOR on PAH has not yet been elucidated. In the present study, a marked downregulation of mTOR was observed in PAH patients. Following construction of a mouse model of PAH by chronic exposure to hypoxia, adenovirus-mediated upregulation of mTOR significantly attenuated right ventricular systolic pressure, right ventricular hypertrophy and wall thickness of pulmonary arterioles, indicating a protective effect of mTOR on PAH. Further analysis confirmed that mTOR overexpression inhibited autophagy triggered by hypoxia through blocking light chain 3 II expression and increasing p62 levels. In vitro, hypoxia enhanced the proliferation of human pulmonary artery endothelial cells (PAECs), which was markedly abrogated by mTOR overexpression. Of note, upregulation of mTOR inhibited the hypoxia-induced autophagy pathway, which contributed to cell proliferation, while silencing of autophagy by RNA interference with ATG5 significantly inhibited cell proliferation. In conclusion, the results of the present study suggested a potential protective effect of mTOR on the progression of PAH by suppressing PAEC proliferation through blocking the autophagic pathway. Therefore, the present study suggested that mTOR is a promising therapeutic agent against PAH.
Autophagic protein LC3B confers resistance against hypoxia-induced pulmonary hypertension.
Lee Seon-Jin,Smith Akaya,Guo Lanping,Alastalo Tero-Pekka,Li Molong,Sawada Hirofumi,Liu Xiaoli,Chen Zhi-Hua,Ifedigbo Emeka,Jin Yang,Feghali-Bostwick Carol,Ryter Stefan W,Kim Hong Pyo,Rabinovitch Marlene,Choi Augustine M K
American journal of respiratory and critical care medicine
RATIONALE:Pulmonary hypertension (PH) is a progressive disease with unclear etiology. The significance of autophagy in PH remains unknown. OBJECTIVES:To determine the mechanisms by which autophagic proteins regulate tissue responses during PH. METHODS:Lungs from patients with PH, lungs from mice exposed to chronic hypoxia, and human pulmonary vascular cells were examined for autophagy using electron microscopy and Western analysis. Mice deficient in microtubule-associated protein-1 light chain-3B (LC3B(-/-)), or early growth response-1 (Egr-1(-/-)), were evaluated for vascular morphology and hemodynamics. MEASUREMENTS AND MAIN RESULTS:Human PH lungs displayed elevated lipid-conjugated LC3B, and autophagosomes relative to normal lungs. These autophagic markers increased in hypoxic mice, and in human pulmonary vascular cells exposed to hypoxia. Egr-1, which regulates LC3B expression, was elevated in PH, and increased by hypoxia in vivo and in vitro. LC3B(-/-) or Egr-1(-/-), but not Beclin 1(+/-), mice displayed exaggerated PH during hypoxia. In vitro, LC3B knockdown increased reactive oxygen species production, hypoxia-inducible factor-1α stabilization, and hypoxic cell proliferation. LC3B and Egr-1 localized to caveolae, associated with caveolin-1, and trafficked to the cytosol during hypoxia. CONCLUSIONS:The results demonstrate elevated LC3B in the lungs of humans with PH, and of mice with hypoxic PH. The increased susceptibility of LC3B(-/-) and Egr-1(-/-) mice to hypoxia-induced PH and increased hypoxic proliferation of LC3B knockdown cells suggest adaptive functions of these proteins during hypoxic vascular remodeling. The results suggest that autophagic protein LC3B exerts a protective function during the pathogenesis of PH, through the regulation of hypoxic cell proliferation.
Effects of Bcl-2/Bcl-x Inhibitors on Pulmonary Artery Smooth Muscle Cells.
Rybka Vladyslava,Suzuki Yuichiro J,Shults Nataliia V
Antioxidants (Basel, Switzerland)
Pulmonary arterial hypertension (PAH) is a fatal disease without satisfactory therapeutic options. By the time patients are diagnosed with this disease, the remodeling of pulmonary arteries has already developed due to the abnormal growth of pulmonary vascular cells. Therefore, agents that reduce excess pulmonary vascular cells have therapeutic potential. Bcl-2 is known to function in an antioxidant pathway to prevent apoptosis. The present study examined the effects of inhibitors of the anti-apoptotic proteins Bcl-2 and Bcl-x. ABT-263 (Navitoclax), ABT-199 (Venetoclax), ABT-737, and Obatoclax, which all promoted the death of cultured human pulmonary artery smooth muscle cells. Further examinations using ABT-263 showed that Bcl-2/Bcl-x inhibition indeed promoted apoptotic programmed cell death. ABT-263-induced cell death was inhibited by antioxidants. ABT-263 also promoted autophagy; however, the inhibition of autophagy did not suppress ABT-263-induced cell death. This is in contrast to other previously studied drugs, including anthracyclines and proteasome inhibitors, which were found to mediate autophagy to induce cell death. The administration of ABT-263 to rats with PAH in vivo resulted in the reversal of pulmonary vascular remodeling. Thus, promoting apoptosis by inhibiting anti-apoptotic Bcl-2 and Bcl-x effectively kills pulmonary vascular smooth muscle cells and reverses pulmonary vascular remodeling.
NPS2390, a Selective Calcium-sensing Receptor Antagonist Controls the Phenotypic Modulation of Hypoxic Human Pulmonary Arterial Smooth Muscle Cells by Regulating Autophagy.
Journal of translational internal medicine
BACKGROUND AND OBJECTIVES:Calcium-sensing receptor (CaSR) is known to regulate hypoxia-induced pulmonary hypertension (HPH) and vascular remodeling via the phenotypic modulation of pulmonary arterial smooth muscle cells (PASMCs) in small pulmonary arteries. Moreover, autophagy is an essential modulator of VSMC phenotype. But it is not clear whether CaSR can regulate autophagy involving the phenotypic modulation under hypoxia. METHODS:The viability of human PASMCs was detected by cell cycle and BrdU. The expressions of proliferation protein, phenotypic marker protein, and autophagy protein in human PASMCs were determined by western blot. RESULTS:Our results showed that hypoxia-induced autophagy was considerable at 24 h. The addition of NPS2390 decreased the expression of autophagy protein and synthetic phenotype marker protein osteopontin and increased the expression of contractile phenotype marker protein SMA-ɑ and calponin via suppressing downstream PI3K/Akt/mTOR signal pathways. CONCLUSIONS:Our study demonstrates that treatment of NPS2390 was conducive to inhibit the proliferation and reverse phenotypic modulation of PASMCs by regulating autophagy levels.
Sphingosine-1-phosphate promotes pulmonary artery smooth muscle cells proliferation by stimulating autophagy-mediated E-cadherin/CDH1 down-regulation.
Zhai Cui,Feng Wei,Shi Wenhua,Wang Jian,Zhang Qianqian,Yan Xin,Wang Qingting,Li Shaojun,Liu Lu,Pan Yilin,Zhu Yanting,Chai Limin,Li Cong,Liu Pengtao,Chen Yuqian,Li Manxiang
European journal of pharmacology
It has been shown that sphingosine-1-phosphate (S1P) is elevated in patients with pulmonary arterial hypertension (PAH) and promotes the proliferation of pulmonary artery smooth muscle cells (PASMCs). Meanwhile, S1P has been found to induce the activation of autophagy in several types of human diseases including cancers. However, it is still unclear whether activation of autophagy mediates S1P-induced PASMCs proliferation, and detailed mechanisms responsible for these processes are indefinite. The aims of this study are to address these issues. S1P dose- and time-dependently reduced the expression of E-cadherin/CDH1 and stimulated PASMCs proliferation; this was accompanied with the elevation of TNF receptor-associated factor 2 (TRAF2), up-regulation and ubiquitination of BECN1 and the activation of autophagy. Prior silencing TRAF2 or BECN1 using siRNA or pre-incubation of cells with autophagy inhibitor chloroquine phosphate (CQ) suppressed S1P-induced autophagy activation and subsequent CDH1 degradation and further PASMCs proliferation. Taken together, our study indicates that S1P promotes the activation of autophagy by accelerating TRAF2-mediated BECN1 up-regulation and ubiquitination, which in turn results in CDH1 reduction and contributes to PASMCs proliferation.
Hypoxia-induced PINK1/Parkin-mediated mitophagy promotes pulmonary vascular remodeling.
Linqing Li,Yuhan Qin,Erfei Luo,Yong Qiao,Dong Wang,Chengchun Tang,Gaoliang Yan,Bo Liu
Biochemical and biophysical research communications
Pulmonary vascular remodeling (PVR) is not only the main pathophysiological feature of Pulmonary Artery Hypertension (PAH) but also the main reason for the progressive aggravation of PAH. Its central link is the excessive proliferation of pulmonary artery smooth muscle cells (PASMCs), which leads to the imbalance of proliferation/apoptosis, leads to the formation of PAH. At present, we found that hypoxia can up-regulate the expression of mitophagy protein PINK1/Parkin, induce the proliferation of PASMCs, and inhibit apoptosis. Knocking down PINK1 and/or Parkin, found that the proliferation of PASMCs was significantly inhibited compared with that of PINK1/Parkin, while the proliferation of cells under PINK1 Parkin was significantly lower than that of PINK1 Parkinor PINK1 Parkin. These results suggest that hypoxia can activate the PINK1/Parkin-mediated mitophagy pathway, induce the excessive proliferation of PASMCs, eventually lead to PVR, leading to HPH. Our team is further exploring which substances in HPH can induce mitotic response, which molecules specifically mediate the activation of mitotic pathways, and what role they play in the occurrence and development of HPH disease.
Ubiquitinated AIF is a major mediator of hypoxia-induced mitochondrial dysfunction and pulmonary artery smooth muscle cell proliferation.
Ma Cui,Wang Xiaoying,He Siyu,Zhang Lixin,Bai June,Qu Lihui,Qi Jing,Zheng Xiaodong,Zhu Xiangrui,Mei Jian,Guan Xiaoyu,Yuan Hao,Zhu Daling
Cell & bioscience
BACKGROUND:Excessive proliferation of pulmonary artery smooth muscle cells (PASMCs) is the main cause of hypoxic pulmonary hypertension (PH), and mitochondrial homeostasis plays a crucial role. However, the specific molecular regulatory mechanism of mitochondrial function in PASMCs remains unclear. METHODS:In this study, using the CCK8 assay, EdU incorporation, flow cytometry, Western blotting, co-IP, mass spectrometry, electron microscopy, immunofluorescence, Seahorse extracellular flux analysis and echocardiography, we investigated the specific involvement of apoptosis-inducing factor (AIF), a mitochondrial oxidoreductase in regulating mitochondrial energy metabolism and mitophagy in PASMCs. RESULTS:In vitro, AIF deficiency in hypoxia leads to impaired oxidative phosphorylation and increased glycolysis and ROS release because of the loss of mitochondrial complex I activity. AIF was also downregulated and ubiquitinated under hypoxia leading to the abnormal occurrence of mitophagy and autophagy through its interaction with ubiquitin protein UBA52. In vivo, treatment with the adeno-associated virus vector to overexpress AIF protected pulmonary vascular remodeling from dysfunctional and abnormal proliferation. CONCLUSIONS:Taken together, our results identify AIF as a potential therapeutic target for PH and reveal a novel posttranscriptional regulatory mechanism in hypoxia-induced mitochondrial dysfunction.
Sodium Tanshinone II Sulfonate A Ameliorates Hypoxia-Induced Pulmonary Hypertension.
Frontiers in pharmacology
BACKGROUND:Pulmonary hypertension (PH) remains a prevalent disease globally. Sodium tanshinone II sulfonate A (STS) has been used in clinical treatment of PH. AIMS:The aim of the present study was to investigate the effect of sodium STS treatment on hypoxia-induced PH and related mechanisms. METHODS:Male Sprague-Dawley rats were housed in a hypoxic chamber with an oxygen concentration of 10 ± 1% for 8 h a day over 21 days. Rats were treated with either STS (low-dose: 10 mg/kg or high-dose: 30 mg/kg) or LY294002 (which is an inhibitor of PI3K). Pulmonary arterial pressure (PAP) was measured, right ventricular hypertrophy parameters were monitored, lung edema parameters were measured, and pathological changes were observed by hematoxylin-eosin (HE) staining. Protein expressions of apoptosis, and PI3K/AKT/mTOR/autophagy pathways in rat lung tissue were examined by western blot. Levels of the pro-inflammatory factors IL-6, IL-8, TNF-α in lung tissues of rats were measured using an enzyme linked immunosorbent assay (ELISA). RESULTS:Results of our study demonstrate that persistent exposure to hypoxic conditions increased PAP, right ventricular hypertrophy, lung edema, parameters of lung vascular proliferation and decreased the ratio of Bax/Bcl-2. Furthermore, hypoxic conditions activated the PI3K/Akt/mTOR pathway, inhibited autophagy, and elevated abundance of inflammatory factors in rat lung tissue. Treatment with STS resulted in a dose-dependent decrease in PAP, right ventricular hypertrophy, lung edema, lung vascular proliferation and reversed hypoxia induced lung tissue protein expression and pro-inflammatory factors in rat lung tissue. In addition, hypoxia-induced increases in PAP, cardiac hypertrophy, and lung expression of the proteins PI3K/Akt/mTOR/autophagy pathway were partially reversed by treatment with LY294002. CONCLUSIONS:STS alleviates hypoxia-induced PH by promoting apoptosis, inhibiting PI3K/AKT/mTOR pathway, up-regulating autophagy, and inhibiting inflammatory responses.
REDD1 gene knockout alleviates vascular smooth muscle cell remodeling in pulmonary hypertension.
American journal of translational research
OBJECTIVES:Regulated in development and DNA damage responses 1 (REDD1) is an important transcription factor regulating mitochondria homeostasis, which is the important pathological alteration of pulmonary hypertension (PH). However, it is unclear whether REDD1 regulates the PASMCs mitochondria homeostasis by the similar mechanism in pulmonary arterial remodeling induced by hypoxia. METHODS:The global REDD1-knockout rats (REDD1-KO) on Sprague-Dawley background were used to generate a chronic hypoxia model of PH. Right ventricular hypertrophy and vascular remodeling were detected after exposure to hypoxia. Additionally, proliferation, apoptosis, migration, mitochondria homeostasis, and autophagy were performed and . RESULTS:The current research found that in human and experimental rats of PH, REDD1 expression is upregulated in the PASMCs. REDD1 gene knockout alleviated hypoxia PH and hemodynamic changes effectively and reversed hypoxic pulmonary vascular remodeling. In addition, REDD1 knockdown reduces the impairment of mitochondrial function caused by hypoxia in HPASMCs via autophagy inhibition, and this process may be regulated through the Parkin gene. Moreover, REDD1 knockdown can effectively inhibit the proliferation and migration of hypoxic PASMCs, and induce their apoptosis and . CONCLUSIONS:Our results suggested that REDD1 might be a potential target for improved pulmonary vascular remodeling in PH.
Dacomitinib, a new pan-EGFR inhibitor, is effective in attenuating pulmonary vascular remodeling and pulmonary hypertension.
Yu Xiufeng,Zhao Xijuan,Zhang Junting,Li YiYing,Sheng Ping,Ma Cui,Zhang Lixin,Hao XueWei,Zheng XiaoDong,Xing Yan,Qiao Hui,Qu Lihui,Zhu Daling
European journal of pharmacology
Accumulating evidence suggests that epidermal growth factor receptor (EGFR) plays a role in the progression of pulmonary arterial hypertension (PAH). Clinically-approved epidermal growth factor inhibitors such as gefitinib, erlotinib, and lapatinib have been explored for PAH. However, None of them were able to attenuate PAH. So, we explored the role of dacomitinib, a new pan-EGFR inhibitor, in PAH. Adult male Sprague-Dawley rats were used to study hypoxia- or monocrotaline-induced right ventricular remodeling as well as systolic function and hemodynamics using echocardiography and a pressure-volume admittance catheter. Morphometric analyses of lung vasculature and pressure-volume vessels were performed. Immunohistochemical staining, flow cytometry, and viability, as well as scratch-wound, and Boyden chamber migration assays were used to identify the roles of dacomitinib in pulmonary artery smooth muscle cells (PASMCs). The results revealed that dacomitinib has a significant inhibitory effect on the thickening of the media, adventitial collagen increased. Dacomitinib also has a significant role in attenuating pulmonary artery pressure and right ventricular hypertrophy. Additionally, dacomitinib inhibits hypoxia-induced proliferation, migration, autophagy and cell cycle progression through PI3K-AKT-mTOR signaling in PASMCs. Our study indicates that dacomitinib inhibited hypoxia-induced cell cycle progression, proliferation, migration, and autophagy of PASMCs, thereby attenuating pulmonary vascular remodeling and development of PAH via the PI3K-AKT-mTOR signaling pathway. Overall, dacomitinib may serve as new potential therapeutic for the treatment of PAH.
Paclitaxel alleviates monocrotaline-induced pulmonary arterial hypertension via inhibition of FoxO1-mediated autophagy.
Feng Wei,Wang Jian,Yan Xin,Zhai Cui,Shi Wenhua,Wang Qingting,Zhang Qianqian,Li Manxiang
Naunyn-Schmiedeberg's archives of pharmacology
It has been demonstrated that activation of autophagy is involved in the development of pulmonary arterial hypertension (PAH). Recent studies have shown that cytosolic forkhead box protein O1 (FoxO1) activates autophagy in cancer cells. Paclitaxel has been found to potentially reverse PAH progression. However, the role of FoxO1 and the effects of paclitaxel on autophagy in the pathogenesis of PAH remain unknown. PAH was generated by intraperitoneal injection of monocrotaline (MCT) to rats. The right ventricular systolic pressure (RVSP), the right ventricle hypertrophy index (RV/LV+S), and the percentage of medial wall thickness (%MT) were used to detect the development of PAH. Hematoxylin and eosin staining was performed to measure pulmonary vascular remodeling. The protein level, phosphorylation, and nucleus translocation of FoxO1 and the levels of LC3A, LC3B, and Beclin-1 were examined by immunoblotting. The results showed that in spite of reduced expression of FoxO1, elevated phosphorylation of FoxO1 caused most of FoxO1 accumulating in cytosolic fraction in MCT-PAH rats. Autophagy was also activated in the MCT-PAH group. In cultured rat pulmonary arterial smooth muscle cells (PASMCs), knockdown of FoxO1 markedly blocked autophagy activation, indicating that elevation of cytosolic FoxO1 stimulates autophagy activation. Treatment of PAH rats with paclitaxel reduced FoxO1 phosphorylation and increased FoxO1 nuclear accumulation, despite increased FoxO1 expression, therefore suppressed autophagy, finally reduced elevated RVSP, RV/LV+S, and %MT in MCT-induced PAH. Taken together, paclitaxel inhibits pulmonary vascular remodeling by FoxO1-mediated autophagy suppression, suggesting that paclitaxel might be a novel therapeutic agent for the prevention and treatment of PAH.
Activation of AMPK prevents monocrotaline-induced pulmonary arterial hypertension by suppression of NF-κB-mediated autophagy activation.
Zhai Cui,Shi Wenhua,Feng Wei,Zhu Yanting,Wang Jian,Li Shaojun,Yan Xin,Wang Qingting,Zhang Qianqian,Chai Limin,Li Cong,Liu Pengtao,Li Manxiang
AIMS:It has been shown that activation of autophagy is involved in the development of pulmonary arterial hypertension (PAH). Meanwhile, activation of nuclear factor-kappaB (NF-κB) has been found to induce autophagy in several types of human diseases including cancer and cardiac diseases. However, it is still unknown whether NF-κB mediates autophagy activation in PAH, and whether activation of adenosine monophosphate-activated protein kinase (AMPK) benefits PAH by modulation of NF-κB and autophagy. MAIN METHODS:Rat models of PAH were established by intraperitoneally injection of monocrotaline (MCT). The right ventricle systolic pressure (RVSP), right ventricular hypertrophy index (RVHI), and percentage of medial wall thickness (%MT) were performed to evaluate the development of PAH. The translocation of NF-κB p65 from cytosol to nucleus, the protein levels of LC3A, LC3B, and RND3 were determined by immunoblotting. Metformin was used to activate AMPK. KEY FINDINGS:NF-κB and autophagy were significantly activated in MCT-induced PAH rats, this was accompanied with the reduction of RND3. Pharmacological inhibition of NF-κB suppressed MCT-induced activation of autophagy and down-regulation of RND3 expression and reduced RVSP, RVHI, and %MT in MCT-induced PAH rats. In addition, activation of AMPK by metformin suppressed NF-κB-mediated autophagy activation and down-regulation of RND3 and therefore reduced RVSP, RVHI, and %MT in MCT-induced PAH. SIGNIFICANCE:NF-κB-induced autophagy activation and consequent down-regulation of RND3 contributes to the development of PAH in MCT-treated rats. Activation of AMPK prevents the development of PAH by targeting on NF-κB to suppress autophagy and vascular remodeling.
eIF2α promotes vascular remodeling via autophagy in monocrotaline-induced pulmonary arterial hypertension rats.
Drug design, development and therapy
PURPOSE:Eukaryotic initiation factor 2α (eIF2α) plays important roles in the proliferation and survival of pulmonary artery smooth muscle cells (PASMCs) in animal hypoxia-induced pulmonary hypertension models. However, the underlying mechanism remains unknown at large. Autophagy has been reported to play a key role in the vascular remodeling in pulmonary arterial hypertension (PAH). The purposes of this study are to determine the functions of eIF2α and autophagy in the vascular remodeling of the monocrotaline-induced PAH rats and to clarify the correlation between eIF2α and autophagy. METHODS:We established a rat model of monocrotaline-induced PAH, and we established a cell model of platelet derived growth factor (PDGF)-induced PASMCs proliferation. The vascular morphology and the expression of eIF2α, LC3B, and p62 were assessed in the pulmonary arterial tissue of Sprague-Dawleyrats and PDGF-induced PASMCs. RESULTS:Autophagy was significantly active in monocrotaline model group (MCT)-induced PAH rats, which obviously promotes vascular remodeling in MCT-induced PAH rats. Furthermore, the proliferation of PASMCs was induced by PDGF in vitro. The expression of LC3B, eIF2α was increased in the PDGF-induced PASMCs proliferation, and the expression of p62 was reduced in the PDGF-induced PASMCs proliferation. Moreover, eIF2α siRNA downregulated the expression of eIF2α and LC3B, and upregulated the expression of p62 in PDGF-induced PASMCs proliferation. eIF2α siRNA inhibited the PDGF-induced PASMCs proliferation. Finally, chloroquine can upregulate the protein expression of LC3B and p62, it also can inhibit proliferation in PDGF-induced PASMCs. CONCLUSION:Based on these observations, we conclude that eIF2α promotes the proliferation of PASMCs and vascular remodeling in monocrotaline-induced PAH rats through accelerating autophagy pathway.
Autophagy contributes to BMP type 2 receptor degradation and development of pulmonary arterial hypertension.
Gomez-Puerto Maria Catalina,van Zuijen Iris,Huang Christopher Jz,Szulcek Robert,Pan Xiaoke,van Dinther Maarten Ah,Kurakula Kondababu,Wiesmeijer Catharina C,Goumans Marie-Jose,Bogaard Harm-Jan,Morrell Nicholas W,Rana Amer Ahmed,Ten Dijke Peter
The Journal of pathology
Pulmonary arterial hypertension (PAH) is characterised by an increase in mean pulmonary arterial pressure which almost invariably leads to right heart failure and premature death. More than 70% of familial PAH and 20% of idiopathic PAH patients carry heterozygous mutations in the bone morphogenetic protein (BMP) type 2 receptor (BMPR2). However, the incomplete penetrance of BMPR2 mutations suggests that other genetic and environmental factors contribute to the disease. In the current study, we investigate the contribution of autophagy in the degradation of BMPR2 in pulmonary vascular cells. We demonstrate that endogenous BMPR2 is degraded through the lysosome in primary human pulmonary artery endothelial (PAECs) and smooth muscle cells (PASMCs): two cell types that play a key role in the pathology of the disease. By means of an elegant HaloTag system, we show that a block in lysosomal degradation leads to increased levels of BMPR2 at the plasma membrane. In addition, pharmacological or genetic manipulations of autophagy allow us to conclude that autophagy activation contributes to BMPR2 degradation. It has to be further investigated whether the role of autophagy in the degradation of BMPR2 is direct or through the modulation of the endocytic pathway. Interestingly, using an iPSC-derived endothelial cell model, our findings indicate that BMPR2 heterozygosity alone is sufficient to cause an increased autophagic flux. Besides BMPR2 heterozygosity, pro-inflammatory cytokines also contribute to an augmented autophagy in lung vascular cells. Furthermore, we demonstrate an increase in microtubule-associated protein 1 light chain 3 beta (MAP1LC3B) levels in lung sections from PAH induced in rats. Accordingly, pulmonary microvascular endothelial cells (MVECs) from end-stage idiopathic PAH patients present an elevated autophagic flux. Our findings support a model in which an increased autophagic flux in PAH patients contributes to a greater decrease in BMPR2 levels. Altogether, this study sheds light on the basic mechanisms of BMPR2 degradation and highlights a crucial role for autophagy in PAH. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.
Down-regulation of miR-204 attenuates endothelial-mesenchymal transition by enhancing autophagy in hypoxia-induced pulmonary hypertension.
Liu Ting,Zou Xiao-Zhou,Huang Ning,Ge Xiao-Yue,Yao Mao-Zhong,Liu Hong,Zhang Zheng,Hu Chang-Ping
European journal of pharmacology
Pulmonary arterial remodeling is a crucial cause of increased pulmonary artery pressure during pulmonary hypertension (PH). Recently, growing evidence has upheld the contribution of endothelial-mesenchymal transition (EndMT) to pulmonary arterial remodeling, but the underlying mechanisms remain largely unaddressed. miR-204 has been implicated in PH, being anti-proliferative and pro-apoptotic in pulmonary artery smooth muscles cells (PASMCs), but its role in EndMT is still unknown. Here we found that miR-204 was down-regulated by hypoxia in rat pulmonary arterial intima and human pulmonary artery endothelial cells (HPAECs), and its further down-regulation by using miR-204 inhibitor suppressed hypoxia-induced EndMT. Moreover, autophagy, evoked by hypoxia in rat pulmonary arterial intima and HPAECs, suppressed hypoxia-induced EndMT via p62-dependent degradation of Snail and Twist. Additionally, autophagy was regulated by miR-204 targeting ATG7. While down-regulation of miR-204 in PASMCs reportedly promoted monocrotaline-induced pulmonary arterial hypertension via increased cell proliferation, our data suggested an important, albeit dichotomous, role of miR-204 down-regulation in endothelial cells in the process of EndMT that it attenuated EndMT by enhancing autophagy, thereby ameliorating hypoxia-induced PH to some extent.
Puerarin prevents progression of experimental hypoxia-induced pulmonary hypertension via inhibition of autophagy.
Zhang Xiaodan,Liu Qi,Zhang Chen,Sheng Jiejing,Li Songlin,Li Wendi,Yang Xinying,Wang Xiaoying,He Siyu,Bai June,Zhu Daling
Journal of pharmacological sciences
Pulmonary arterial hypertension (PAH) is defined as elevation of mean pulmonary arterial pressure to ≥25 mmHg within the low pressure pulmonary circulatory system. PAH is characterized by obstructive vascular remodeling, partially due to excessive pulmonary arterial smooth muscle cell (PASMC) proliferation. Puerarin is a natural flavonoid isolated from the herb Radix puerariae, which has been widely used for the treatment of cardiovascular and cerebrovascular disorders and diabetes. However, how puerarin mediates autophagy in the progression of pulmonary vascular remodeling is unclear. In this study, we explored the effects of puerarin in a hypoxic pulmonary hypertension (PH) rat model using immunohistochemistry, and morphometric analyses of right ventricle. In addition, cell counting kit 8 assay, western blotting and flow cytometry were employed to test cell proliferation in PASMCs, and then autophagy was tested with mRFP-GFP-LC3 fluorescence microscopy and Western blot. We found that puerarin could alleviate hypoxia-induced PH in rats and improved pulmonary histopathology, and also reduced the expression of autophagy markers in vivo and in vitro. Moreover, puerarin also ameliorated hypoxia-induced PASMC proliferation in an autophagy-dependent manner. Overall, these findings demonstrated that puerarin could prevent hypoxia-induced PH in rats, possibly via reducing autophagy and suppressing cell proliferation.
Hypoxia-induced mitochondrial reactive oxygen species (mtROS) differentially regulates smooth muscle cell (SMC) proliferation of pulmonary and systemic vasculature.
Bhansali Shipra,Sohi Kamal,Dhawan Veena
BACKGROUND:Vascular remodeling plays a pivotal role in regulation of hypoxia-mediated pulmonary and systemic hypertension via the phenotypic modulation of smooth muscle cells (SMCs) of pulmonary and systemic arteries, respectively. Mitochondria serve as putative oxygen (O) sensors, and consequently, adaptations to hypoxia are mediated via HIF (hypoxia-inducible factors) activation, which impinges on mitochondrial function by suppressing the mitochondrial activity. Therefore, we explored the implication of hypoxia-mediated mitochondrial stress in pulmonary and systemic arterial remodeling. METHODS:The hypoxic (10% O) effect on human pulmonary artery and aortic SMCs was examined in vitro by cell viability assay, proliferation index, autophagy, and comet assays. Mitochondrial ROS (mtROS), membrane potential (MMP), and mitochondrial morphology were assessed using mitochondrial-selective fluorescent probes. Further, the cell cycle distribution was analyzed by flow cytometry using propidium iodide staining. RESULTS:Our data indicate no significant alterations in cell viability and active proliferation of hypoxic PASMCs; however, an excessive rise in mtROS production and disrupted MMP, accompanied by enhanced DNA damage and reduced autophagy was observed, highlighting the 'apoptosis resistance' phenotype in these cells. Conversely, in hypoxia-treated hASMCs, a modest rise in mtROS levels was associated with reduced DNA damage; followed by upregulated autophagy; increased S-phase DNA content and cell viability, depicting the cytoprotective effect of hypoxia-induced autophagy against mitochondrial damage in hASMCs. CONCLUSION:Our findings suggest that differential impact of mtROS on proliferative capacity may contribute to the variable hypoxic responses in pulmonary and systemic vasculature. Therefore, targeting mtROS may serve as an effective therapeutic strategy to prevent hypoxia-induced hypertension.
Knockdown of Hypoxia-Inducible Factor 1α (HIF-1α) Promotes Autophagy and Inhibits Phosphatidylinositol 3-Kinase (PI3K)/AKT/Mammalian Target of Rapamycin (mTOR) Signaling Pathway in Ovarian Cancer Cells.
Huang Jinling,Gao Likun,Li Bingshu,Liu Cheng,Hong Shasha,Min Jie,Hong Li
Medical science monitor : international medical journal of experimental and clinical research
BACKGROUND Ovarian cancer has the highest mortality rate among all female genital tumors because of its insidious onset and drug resistance. Hypoxia-inducible factor 1alpha (HIF-1alpha), one of the best-studied oncogenes, plays an important part in tumor adaptation to microenvironmental hypoxia and was found to be overexpressed in several malignancies, including ovarian cancer. Previous studies found that the effect of HIF-1alpha on cancers may be correlated with autophagy and some signaling pathways, such as PI3K/AKT/mTOR, in several tumors. However, the function and potential mechanism have not been clearly defined. MATERIAL AND METHODS The expression of HIF-1alpha in ovarian cancer tissues were detected by immunohistochemistry. HIF-1alpha was knocked down by siRNA transfection. Cell viability was examined by CCK8 and colony formation assay. Apoptosis and autophagy were detected with flow cytometry, transmission electron microscopy, and laser scanning confocal microscopy, respectively. The proteins related to autophagy and PI3K/AKT/mTOR were detected through Western blot analysis. RESULTS HIF-1alpha was expressed at higher levels in epithelial or metastatic ovarian cancer tissue than in normal fallopian tube tissue. When HIF-1alpha was knocked down by siRNA in A2780 and SKOV3 cells, the viability of ovarian cancer cells was weakened, but the apoptosis and autophagy were strengthened. Accordingly, autophagosome formation increased and the expression of autophagy-related proteins LC3 and P62 increased in HIF-1alpha knockdown cells. The PI3K/Akt/mTOR signaling pathway was also found to be inactivated in HIF-1alpha knockdown cells. CONCLUSIONS These findings show that knockdown of HIF-1alpha promoted autophagy and inhibited the PI3K/AKT/mTOR signaling pathway in ovarian cancer cells.
miR-4735-3p regulates phenotypic modulation of vascular smooth muscle cells by targeting HIF-1-mediated autophagy in intracranial aneurysm.
Gao Ge,Zhang Yang,Chao Yingjiu,Niu Chaoshi,Fu Xianming,Wei Jianjun
Journal of cellular biochemistry
Intracranial aneurysm (IA) is recognized as a lethal form of cerebrovascular disease mainly featured with a modulated phenotype of vascular smooth muscle cells (SMCs). It is generally believed that enhanced SMC proliferation and migration capabilities are the main characteristics in this process. In this study, we revealed that microRNA-4735 (miR-4735) participates in phenotypic modulation in a hypoxia-inducible factor-1 (HIF-1)-dependent manner of SMCs. miR-4735 targets the 3'-untranslated region of HIF-1. The downregulated expression of miR-4735 in IA tissues leads to elevated expression of HIF-1, which activates autophagy and promotes autophagy-mediated SMC proliferation and migration. Overexpression of miR-4735 suppressed HIF-1 expression and HIF-1-mediated autophagy, which led to impaired SMC proliferation and migration abilities. Forced expression of HIF-1 in miR-4735-overexpressed SMCs rescued the impaired SMC proliferation and migration abilities. In conclusion, miR-4735 plays an important role in phenotypic modulation in IA by regulating autophagy-promoted SMC proliferation and migration.