Autophagic degradation of MVBs in LSECs promotes Aldosterone induced-HSCs activation.

BACKGROUND AND AIMS: The important role of extracellular vesicles (EVs) in liver fibrosis has been confirmed. However, EVs derived from liver sinusoidal endothelial cells (LSECs) in the activation of hepatic stellate cells (HSCs) and liver fibrosis is still unclear. Our previous work demonstrated that Aldosterone (Aldo) may have the potential to regulate EVs from LSECs via autophagy pathway. Thus, we aim to investigate the role of Aldo in the regulation of EVs derived from LSECs. APPROACH AND RESULTS: Using an Aldo-continuous pumping rat model, we observed that Aldo-induced liver fibrosis and capillarization of LSECs. In vitro, transmission electron microscopy (TEM) revealed that stimulation of Aldo led to the upregulation of autophagy and degradation of multivesicular bodies (MVBs) in LSECs. Mechanistically, Aldo upregulated ATP6V0A2, which promoted lysosomal acidification and subsequent autophagy in LSECs. Inhibiting autophagy with si-ATG5 adeno-associated virus (AAV) in LSECs effectively mitigated Aldo-induced liver fibrosis in rats. RNA sequencing and nanoparticle tracking (NTA) analyses of EVs derived from LSECs indicated that Aldo result in a decrease in both the quantity and quality of EVs. We also observed a reduction in the protective miRNA-342-5P in EVs derived from Aldo-treated LSECs, which may play a critical role in HSCs activation. Target knockdown of EV secretion with si-RAB27a AAV in LSECs led to the development of liver fibrosis and HSC activation in rats. CONCLUSION: Aldo-induced Autophagic degradation of MVBs in LSECs promotes a decrease in the quantity and quality of EVs derived from LSECs, resulting in the activation of HSCs and liver fibrosis under hyperaldosteronism. Modulating the autophagy level of LSECs and their EV secretion may represent a promising therapeutic approach for treating liver fibrosis.

Author Correction: Autophagic degradation of caveolin-1 promotes liver sinusoidal endothelial cells defenestration.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Proteomic Profiling of LPS-Induced Macrophage-Derived Exosomes Indicates Their Involvement in Acute Liver Injury.

Exosomes are typically involved in cellular communication and signaling. Macrophages play a key role in lipopolysaccharide (LPS)-induced sepsis. However, the molecular comparison of exosomes derived from LPS-induced macrophage has not been well analyzed. The macrophage-exosomes are validated and the protein composition of those exosomes are investigated by isobaric tags for relative and absolute quantification (iTRAQ) mass spectrometry. A total of 5056 proteins are identified in macrophage-exosomes. We discovered 341 increased proteins and 363 reduced proteins in LPS-treated macrophage-exosomes compared with control exosomes. In addition, gene ontology analysis demonstrates that macrophage-exosomes proteins are mostly linked to cell, organelle, extracellular region, and membrane. The bioinformatics analysis also indicates that these proteins are mainly involved in cellular process, single-organism process, metabolic process, and biological regulation. Among these 341 upregulated proteins, Kyoto Encyclopedia of Genes and Genomes analysis reveals that 22 proteins are involved in the NOD-like receptor signaling pathway. Finally, hepatocytes can uptake macrophage-exosomes and subsequently NLRP3 inflammasome is activated in vitro and in vivo. These data emphasize the fundamental importance of macrophage-exosomes in sepsis-induced liver injury. Therefore, the iTRAQ proteomic strategy brings new insights into macrophage-derived exosomes. It may improve our understanding of macrophage-exosomes' functions and their possible use as therapeutic targets for sepsis.

Autophagic degradation of caveolin-1 promotes liver sinusoidal endothelial cells defenestration.

Autophagy, interacting with actin cytoskeleton and the NO-dependent pathway, may affect the phenotype and function of endothelial cells. Moreover, caveolin-1 (Cav-1), as a structure protein in liver sinusoidal endothelial cells (LSECs), is closely related to autophagy. Hence, we aim to explore the role of autophagic degradation of Cav-1 in LSECs defenestration. In vivo, we found the increase of autophagy in liver sinusoidal endothelium in human fibrotic liver. Furthermore, autophagy, degradation of Cav-1, and actin filament (F-actin) remodeling were triggered during the process of CCl4-induced LSECs defenestration; in contrast, autophagy inhibitor 3MA diminished the degradation of Cav-1 to maintain fenestrae and relieve CCl4-induced fibrosis. In vitro, during LSECs defenestration, the NO-dependent pathway was down-regulated through the reduction of the PI3K-AKT-MTOR pathway and initiation of autophagic degradation of Cav-1; while, these effects were aggravated by starvation. However, VEGF inhibited autophagic degradation of Cav-1 and F-actin remodeling to maintain LSECs fenestrae via activating the PI3K-AKT-MTOR pathway. Additionally, inhibiting autophagy, such as 3MA, bafilomycin, or ATG5-siRNA, could attenuate the depletion of Cav-1 and F-actin remodeling to maintain LSECs fenestrae and improve the NO-dependent pathway; in turn, eNOS-siRNA and L-NAME, for blocking the NO-dependent pathway, could elevate autophagic degradation of Cav-1 to aggravate defenestration. Finally, overexpressed Cav-1 rescued rapamycin-induced autophagic degradation of Cav-1 to maintain LSECs fenestrae; whereas knockdown of Cav-1 facilitated defenestration due to the activation of the AMPK-dependent autophagy. Consequently, autophagic degradation of Cav-1 promotes LSECs defenestration via inhibiting the NO-dependent pathway and F-actin remodeling.

Caveolin 1-related autophagy initiated by aldosterone-induced oxidation promotes liver sinusoidal endothelial cells defenestration.

Aldosterone, with pro-oxidation and pro-autophagy capabilities, plays a key role in liver fibrosis. However, the mechanisms underlying aldosterone-promoted liver sinusoidal endothelial cells (LSECs) defenestration remain unknown. Caveolin 1 (Cav1) displays close links with autophagy and fenestration. Hence, we aim to investigate the role of Cav1-related autophagy in LSECs defenestration. We found the increase of aldosterone/MR (mineralocorticoid receptor) level, oxidation, autophagy, and defenestration in LSECs in the human fibrotic liver, BDL or hyperaldosteronism models; while antagonizing aldosterone or inhibiting autophagy relieved LSECs defenestration in BDL-induced fibrosis or hyperaldosteronism models. In vitro, fenestrae of primary LSECs gradually shrank, along with the down-regulation of the NO-dependent pathway and the augment of the AMPK-dependent autophagy; these effects were aggravated by rapamycin (an autophagy activator) or aldosterone treatment. Additionally, aldosterone increased oxidation mediated by Cav1, reduced ATP generation, and subsequently induced the AMPK-dependent autophagy, leading to the down-regulation of the NO-dependent pathway and LSECs defenestration. These effects were reversed by MR antagonist spironolactone, antioxidants or autophagy inhibitors. Besides, aldosterone enhanced the co-immunoprecipitation of Cav1 with p62 and ubiquitin, and induced Cav1 co-immunofluorescence staining with LC3, ubiquitin, and F-actin in the perinuclear area of LSECs. Furthermore, aldosterone treatment increased the membrane protein level of Cav1, whereas decrease the cytoplasmic protein level of Cav1, indicating that aldosterone induced Cav1-related selective autophagy and F-actin remodeling to promote defenestration. Consequently, Cav1-related selective autophagy initiated by aldosterone-induced oxidation promotes LSECs defenestration via activating the AMPK-ULK1 pathway and inhibiting the NO-dependent pathway.

MicroRNA-21 Mediates Angiotensin II-Induced Liver Fibrosis by Activating NLRP3 Inflammasome/IL-1ß Axis via Targeting Smad7 and Spry1.

AIMS: Angiotensin II (AngII), a vasoconstrictive peptide of the renin-angiotensin system (RAS), promotes hepatic fibrogenesis and induces microRNA-21(mir-21) expression. Angiotensin-(1-7) [Ang-(1-7)] is a peptide of the RAS, which attenuates liver fibrosis. Recently, it was reported that the NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome participated in liver fibrosis. However, it remains unclear how mir-21 mediates AngII-induced NLRP3 inflammasome activation. We investigate the role of AngII-induced mir-21 in the regulation of NLRP3 inflammasome/IL-1ß axis in liver fibrosis. RESULTS: In vivo, circulating mir-21 was upregulated in patients with liver fibrosis and was positively correlated with liver fibrosis and oxidation. Treatment with Ang-(1-7) inhibited mir-21, NLRP3 inflammasome, and liver fibrosis after bile duct ligation (BDL) or AngII infusion. Inhibition of mir-21 suppressed the Smad7/Smad2/3/NOX4, Spry1/ERK/NF-κB pathway, NLRP3 inflammasome, and liver fibrosis induced by AngII infusion. In vitro, AngII upregulated mir-21 expression via targeting Smad7 and Spry1 in primary hepatic stellate cells (HSCs). In contrast, Ang-(1-7) suppressed mir-21 expression and oxidation induced by AngII. Overexpression of mir-21 promoted oxidation, and collagen production enhanced the effect of AngII on NLRP3 inflammasome activation via the Spry1/ERK/NF-κB, Smad7/Smad2/3/NOX4 pathways. However, downregulation of mir-21 exerted the opposite effects. Innovation and Conclusions: Mir-21 mediates AngII-activated NLRP3 inflammasome and resultant HSC activation via targeting Spry1 and Smad7. Ang-(1-7) protected against BDL or AngII infusion-induced hepatic fibrosis and inhibited mir-21 expression. Antioxid. Redox Signal. 27, 1-20.

Angiotensin-(1-7) Improves Liver Fibrosis by Regulating the NLRP3 Inflammasome via Redox Balance Modulation.

AIMS: Angiotensin II (Ang II) aggravates hepatic fibrosis by inducing NADPH oxidase (NOX)-dependent oxidative stress. Angiotensin-(1-7) [Ang-(1-7)], which counter-regulates Ang II, has been evidenced to protect against hepatic fibrosis. The NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome, being activated by reactive oxygen species (ROS), is identified as a novel mechanism of liver fibrosis. However, whether the NLRP3 inflammasome involves in regulation of Ang II-induced hepatic fibrosis remains unclear. This study investigates the different effects of the Ang II and Ang-(1-7) on collagen synthesis by regulating the NLRP3 inflammasome/Smad pathway via redox balance modulation. RESULTS: In vivo, Ang-(1-7) improved bile duct ligation-induced hepatic fibrosis, reduced H2O2 content, protein levels of NOX4, and the NLRP3 inflammasome, whereas it increased glutathione (GSH) and nuclear erythroid 2-related factor 2 (Nrf2) antioxidant response element (ARE). In vitro, Ang II treatment elevated NOX4 protein expression and ROS production in hepatic stellate cells (HSCs), whereas it inhibited GSH and Nrf2-ARE, resulting in the activation of the NLRP3 inflammasome in the mitochondria of HSCs. NLRP3 depletion inhibited Ang II-induced collagen synthesis. Furthermore, Ang II increased NLRP3 and pro-IL-1ß levels by activating the Toll-like receptor 4 (TLR4)/MyD88/NF-κB pathway. Treatment with antioxidants, NOX4 small interference RNA (siRNA), or Nrf2 activator inhibited Ang II-induced NLRP3 inflammasome activation and collagen synthesis. In contrast, the action of Ang-(1-7) opposed the effects of Ang II. INNOVATION AND CONCLUSIONS: Ang-(1-7) improved liver fibrosis by regulating NLRP3 inflammasome activation induced by Ang II-mediated ROS via redox balance modulation. Antioxid. Redox Signal. 24, 795-812.

[Losartan regulates oxidative stress via caveolin-1 and NOX4 in mice with ventilator- induced lung injury].

OBJECTIVE: To investigate the effect of losartan in regulating oxidative stress and the underlying mechanism in mice with ventilator-induced lung injury. METHODS: Thirty-six male C57 mice were randomly divided into control group, losartan treatment group, mechanical ventilation model group, and ventilation plus losartan treatment group. After the corresponding treatments, the lung injuries in each group were examined and the expressions of caveolin-1 and NOX4 in the lung tissues were detected. RESULTS: The mean Smith score of lung injury was significantly higher in mechanical ventilation model group (3.3) than in the control group (0.4), and losartan treatment group (0.3); the mean score was significantly lowered in ventilation plus losartan treatment group (2.3) compared with that in the model group (P<0.05). The expressions of caveolin-1 and NOX4 were significantly higher in the model group than in the control and losartan treatment groups (P<0.05) but was obviously lowered after losartan treatment (P<0.05). Co-expression of caveolin-1 and NOX4 in the lungs was observed in the model group, and was significantly decreased after losartan treatment. CONCLUSION: Losartan can alleviate ventilator-induced lung injury in mice and inhibit the expression of caveolin-1 and NOX4 and their interaction in the lungs.

[Aldosterone antagonist inhibits fibrosis-induced NOX4 protein expression in hepatic cells and tissues of rats].

OBJECTIVE: To investigate the inhibitory potential of aldosterone antagonist on NOX4 protein expression in hepatic fibrosis by using a rat model of carbon tetrachloride (CCl4)-induced hepatotoxicity. METHODS: Twenty-four male Wistar rats were randomly divided into three equal groups: fibrosis model group (receiving three subcutaneous injections per week of 2.5 ml/kg 40% CCl4); spironolactone (Sp)-treated fibrosis model group (receiving CCl4 regimen plus three injections per day of 20 mg/kg Sp in olive oil); negative-treatment fibrosis model group (receiving CCl4 regimen plus three injections per day of olive oil alone). Unmanipulated rats (receiving no CCl4 and no supplemental treatments) served as normal controls. After 4 weeks, liver histology was carried out to assess cytotoxicity (by hematoxylin-eosin staining), fibrosis (by Masson staining and METAVIR scoring), and NOX4 protein expression (by immunohistochemistry). In addition, in vitro analyses of immortalized rat hepatic stellate cells, HSC-T6, were performed to evaluate dose-response (10-9, 10-7 and 10-5 mol/L) and time-response (6, 12 and 24 h) of aldosterone agonist (Ald) and an aldosterone antagonist, eplerenone (EPLE). Effects on NOX4 protein expression were evaluated by western blotting. RESULTS: The fibrosis model group showed significantly more fibrosis than the normal control group (16.060 +/- 0.300 vs. 2.471 +/- 0.160, P = 0.000]; however, the Sp-treated fibrosis model group showed significantly less CCl4-induced fibrosis (5.761 +/- 0.152 vs. model: 16.060 +/- 0.300, P = 0.000). The fibrosis model group also showed significantly higher NOX4 protein expression in liver tissues than the normal control group (7.231 +/- 0.211 vs. 1.350 +/- 0.252, P = 0.000), and the Sp-treated fibrosis model tissues showed significantly less CCl4-induced up-regulated NOX4 protein expression (4.270 +/- 0.242 vs. model: 7.231 +/- 0.211, P = 0.000]. Ald induced up-regulated NOX4 protein expression in HSC-T6 cells in dose- and concentration-dependent manners, with the peak expression being induced by the 10-5 mol/L concentration and 24 h exposure. The Ald-treated cells expressed significantly more NOX4 protein than the untreated control cells (0.710 +/- 0.011 vs. 0.316 +/- 0.015, P = 0.000]. and the EPLE-treated cells showed significantly less Ald-induced up-regulated NOX4 expression (0.615 +/- 0.014 vs. 0.710 +/- 0.011, P = 0.000]. CONCLUSION: Aldosterone antagonists inhibit the fibrosis-induced NOX4 protein expression in rat hepatic cells.

[Inhibitory effect of angiotensin (1-7) on hepatic sinusoid angiogenesis in bile duct ligation-induced hepatic fibrosis of rats].

OBJECTIVE: To explore the inhibitory effect of angiotensin (1-7) on hepatic sinusoid angiogenesis using a rat model of hepatic fibrosis. METHODS: Eighteen male Wistar rats were randomly divided into three equal groups for sham operation (untreated/uninduced control group), bile duct ligation (BDL) (untreated model group), or BDL with angiotensin (1-7) treatment (treated model group). Histological analysis was used to assess the liver fibrosis score, by hematoxylin-eosin staining, and the level of fibrosis, by Masson's trichrome staining. Immunohistochemistry, western blotting, and immunofluorescence were used to assess the expression of the angiogenesis markers vWF, VEGFA, and CD31. RESULTS: Compared with the untreated/uninduced control group, the untreated BDL model group showed remarkably higher fibrosis score, area of the type I collagen expression, and expression levels of vWF, VEGFA, and CD31. However, the angiotensin (1-7)-treatment protected against the BLD-related changes, as evidenced by decreased robustness and down-regulation of the corresponding indicators. Moreover, the expression level of VEGFA was highly correlated to the expression level of vWF (r = 0.956, P = 0.000). CONCLUSION: BDL-induced hepatic fibrosis is accompanied by significant increases in angiogenesis-related factors, but angiotensin (1-7) treatment may inhibit hepatic sinusoid angiogenesis during the liver fibrosis process.

[Angiotensin (1-7) inhibits angiotensin II-stimulated expression of connective tissue growth factor mRNA in hepatic stellate cells].

To explore the angiotensin peptide [Ang (1-7)]-mediated inhibition of Ang II in human hepatic stellate cells (HSCs) and determine the involvement of the ACE2-Ang (1-7)-Mas axis. The human HSC line, LX2, was used in all experiments, and divided into control (unstimulated) and Ang II-stimulated (10-6 mol/L) groups. The Ang II-stimulated cells were further divided among several pre-treatment (prior to Ang II) groups: ROCK-inhibited (Y27632 blocking agent, 10-6 mol/L); irbesartan-inhibited (AT-1 receptor antagonist, 10-6 mol/L); and Mas receptor-inhibited (A779 Mas receptor antagonist, 10-6 mol/L). To explore the potential inhibitory effects of various Ang family members, the Ang II-stimulated and pre-treated LX2 cells were exposed to Ang (1-7) (10-6 mol/L) for 24 h. Western blot, reverse transcription-polymerase chain reaction (RT-PCR), and QuantiGene assay were used to assess changes in protein and mRNA expression levels of RhoA, ROCK, and connective tissue growth factor (CTGF). Compared with the control group, Ang II-stimulated cells showed significantly increased levels of RhoA protein (0.337+/-0.074 vs. 0.870+/-0.093), ROCK2 mRNA (0.747+/-0.061 vs. 0.368+/-0.023), and CTGF mRNA (0.262+/-0.007 vs. 0.578+/-0.028) (all, P less than 0.01). Pre-treatment with irbesartan or Y27632 eliminated these responses. Ang (1-7) inhibited the Ang II-stimulated up-regulation of RhoA, ROCK, and CTGF. Ang (1-7) can inhibit the Ang II-stimulated up-regulation of RhoA, ROCK and CTGF in hepatic stellate cells, indicating that the ACE2-Ang (1-7)-Mas axis, an important branch of the renin-Ang-aldosterone system is involved in the occurrence and development of liver fibrosis.

[Spironolactone inhibits hepatic sinusoid angiogenesis in rats with hepatic fibrosis].

OBJECTIVE: To investigate the inhibitory effects of spironolactone against hepatic sinusoid angiogenesis in rats with hepatic fibrosis. METHODS: Twenty-four male Wistar rats were randomly divided into sham-operated group, bile duct ligation (BDL) group, and BDL+SP group in which the rats received daily spironolactone injection (20 mg/kg) the day after BDL. Four weeks after the operation, the rats were sacrificed for examination of liver histology using Masson staining and the expression of vascular endothelial growth factor A (VEGF-A) mRNA in the liver using real-time quantitative PCR. Immunohistochemistry was used to detect the expression of von Willebrand factor (vWF) in the hepatic tissues. RESULTS: Spironolactone significantly inhibited liver fibrogenesis in rats after BDL (METAVIR liver fibrosis scores 2.84∓0.44 vs 19.73∓3.54, P=0.00). Real-time PCR and immunohistochemistry showed that compared with BDL group, spironolactone treatment significantly inhibited the expression of VEGF-A mRNA (0.71∓0.12 vs 1.75∓0.15, P=0.00) and vWF (1.15∓0.09 vs 3.08∓0.17, P=0.00) in the liver. The expression of VEGF-A mRNA was highly correlated with the expression of vWF (r=0.890, P=0.000). CONCLUSION: Spironolactone can inhibit hepatic sinusoid angiogenesis in rats with BDL-induced hepatic fibrosis by inhibiting the expression of VEGF-A.

[Prolonged angiotensin-(1-7) infusion inhibits hepatic fibrosis in rats with bile duct ligation].

OBJECTIVE: To observe the inhibitory effect of angiotensin-(1-7) on liver fibrosis induced by bile duct ligation in rats. METHODS: Eighteen Wistar rats were randomized into 3 groups and subject to sham operation, bile duct ligation (BDL), or BDL with angiotensin-(1-7) treatment. An osmotic minipump was implanted intraperitoneally for administration of saline in the sham-operated and BDL groups and angiotensin-(1-7) (25 µg·kg(-1)·h(-1)) in angiotensin-(1-7) treatment group. After a 4-week treatments, the fibrosis score, Masson staining, and hydroxyproline assay were used to evaluate the level of liver fibrosis in the rats, and immunohistochemistry was used to detect expression of α-smooth muscle actin (α-SMA) in the liver tissue. RESULTS: Compared with BDL group, a 4-week treatment with angiotensin-(1-7) following BDL significantly reduced the fibrosis score (2.33±0.52 vs 5.17±0.75), hydroxyproline content (0.36±0.03 vs 0.52±0.04) and α-SMA expression (54.11±17.55 vs 191.84±31.72) in the liver tissue of the rats (P<0.01). CONCLUSION: Prolonged infusion of angiotensin-(1-7) inhibit the formation of hepatic fibrosis in rats following bile duct ligation.