Inhibition of OGFOD1 by FG4592 confers neuroprotection by activating unfolded protein response and autophagy after ischemic stroke.

BACKGROUND: Acute ischemic stroke is a common neurological disease with a significant financial burden but lacks effective drugs. Hypoxia-inducible factor (HIF) and prolyl hydroxylases (PHDs) participate in the pathophysiological process of ischemia. However, whether FG4592, the first clinically approved PHDs inhibitor, can alleviate ischemic brain injury remains unclear. METHODS: The infarct volumes and behaviour tests were first analyzed in mice after ischemic stroke with systemic administration of FG4592. The knockdown of HIF-1α and pretreatments of HIF-1/2α inhibitors were then used to verify whether the neuroprotection of FG4592 is HIF-dependent. The targets predicting and molecular docking methods were applied to find other targets of FG4592. Molecular, cell biological and gene knockdown methods were finally conducted to explore the potential neuroprotective mechanisms of FG4592. RESULTS: We found that the systemic administration of FG4592 decreased infarct volume and improved neurological defects of mice after transient or permanent ischemia. Meanwhile, FG4592 also activated autophagy and inhibited apoptosis in peri-infarct tissue of mice brains. However, in vitro and in vivo results suggested that the neuroprotection of FG4592 was not classical HIF-dependent. 2-oxoglutarate and iron-dependent oxygenase domain-containing protein 1 (OGFOD1) was found to be a novel target of FG4592 and regulated the Pro-62 hydroxylation in the small ribosomal protein s23 (Rps23) with the help of target predicting and molecular docking methods. Subsequently, the knockdown of OGFOD1 protected the cell against ischemia/reperfusion injury and activated unfolded protein response (UPR) and autophagy. Moreover, FG4592 was also found to activate UPR and autophagic flux in HIF-1α independent manner. Blocking UPR attenuated the neuroprotection, pro-autophagy effect and anti-apoptosis ability of FG4592. CONCLUSION: This study demonstrated that FG4592 could be a candidate drug for treating ischemic stroke. The neuroprotection of FG4592 might be mediated by inhibiting alternative target OGFOD1, which activated the UPR and autophagy and inhibited apoptosis after ischemic injury. The inhibition of OGFOD1 is a novel therapy for ischemic stroke.

Specific detection and deletion of the sigma-1 receptor widely expressed in neurons and glial cells in vivo.

The chaperon protein sigma-1 receptor (S1R) has been discovered over 40 years ago. Recent pharmacological studies using S1R exogenous ligands demonstrated a promising therapeutical potential of targeting the S1R in several neurological disorders. Although intensive in vitro studies have revealed S1Rs are mainly residing at the membrane of the endoplasmic reticulum (ER), the cell-specific in vivo expression pattern of S1Rs is still unclear, mainly because of the lack of a reliable detection method which also prevented a comprehensive functional analysis. Here, first, we identified a highly specific antibody using S1R knockout (KO) mice and established an immunohistochemical protocol involving a 1% sodium dodecyl sulphate (SDS) antigen retrieval step. Second, we characterized the S1R expression in the mouse brain and can demonstrate that the S1R is widely expressed: in principal neurons, interneurons and all glial cell types. In addition, unlike reported in previous studies, we showed that the S1R expression in astrocytes is not colocalized with the astrocytic cytoskeleton protein GFAP. Thus, our results raise concerns over previously reported S1R properties. Finally, we generated a Cre-dependent S1R conditional KO mouse (S1R flox) to study cell-type-specific functions of the S1R. As a proof of concept, we successfully ablated S1R expressions in neurons or microglia employing neuronal and microglial Cre-expressing mice, respectively. In summary, we provide powerful tools to cell-specifically detect, delete and functionally characterize S1R in vivo.

Extracellular Vesicle-Mediated Delivery of Circular RNA SCMH1 Promotes Functional Recovery in Rodent and Nonhuman Primate Ischemic Stroke Models.

BACKGROUND: Stroke is a leading cause of adult disability that can severely compromise the quality of life of patients, yet no effective medication currently exists to accelerate rehabilitation. A variety of circular RNA (circRNA) molecules are known to function in ischemic brain injury. Lentivirus-based expression systems have been widely used in basic studies of circRNAs, but safety issues with such delivery systems have limited exploration of the potential therapeutic roles for circRNAs. METHODS: Circular RNA SCMH1 (circSCMH1) was screened from the plasma of patients with acute ischemic stroke by using circRNA microarrays. Engineered rabies virus glycoprotein-circSCMH1-extracellular vesicles were generated to selectively deliver circSCMH1 to the brain. Nissl staining was used to examine infarct size. Behavioral tasks were performed to evaluate motor functions in both rodent and nonhuman primate ischemic stroke models. Golgi staining and immunostaining were used to examine neuroplasticity and glial activation. Proteomic assays and RNA-sequencing data combined with transcriptional profiling were used to identify downstream targets of circSCMH1. RESULTS: CircSCMH1 levels were significantly decreased in the plasma of patients with acute ischemic stroke, offering significant power in predicting stroke outcomes. The decreased levels of circSCMH1 were further confirmed in the plasma and peri-infarct cortex of photothrombotic stroke mice. Beyond demonstrating proof-of-concept for an RNA drug delivery technology, we observed that circSCMH1 treatment improved functional recovery after stroke in both mice and monkeys, and we discovered that circSCMH1 enhanced the neuronal plasticity and inhibited glial activation and peripheral immune cell infiltration. CircSCMH1 binds mechanistically to the transcription factor MeCP2 (methyl-CpG binding protein 2), thereby releasing repression of MeCP2 target gene transcription. CONCLUSIONS: Rabies virus glycoprotein-circSCMH1-extracellular vesicles afford protection by promoting functional recovery in the rodent and the nonhuman primate ischemic stroke models. Our study presents a potentially widely applicable nucleotide drug delivery technology and demonstrates the basic mechanism of how circRNAs can be therapeutically exploited to improve poststroke outcomes.

Identification of microRNA-9 linking the effects of childhood maltreatment on depression using amygdala connectivity.

Childhood maltreatment (CM) is regarded as an important risk factor for major depressive disorder (MDD). However, the neural links corresponding to the process of early CM experience producing brain alterations and then leading to depression later remain unclear. To explore the neural basis of the effects of CM on MDD and the potential role of microRNA-9 (miR-9) in these processes, we recruited 40 unmedicated MDD patients and 34 healthy controls (HCs) to complete resting-state fMRI scans and peripheral blood miR-9 tests. The neural substrates of CM, miR-9, and depression, as well as their interactive effects on intrinsic amygdala functional connectivity (AFC) networks were investigated in MDD patients. Two-step mediation analysis was separately employed to explore whether AFC strength mediates the association among CM severity, miR-9 levels, and depression. A support vector classifier (SVC) model of machine learning was used to distinguish MDD patients from HCs. MDD patients showed higher miR-9 levels that were negatively correlated with CM scores and depressive severity. Overlapping effects of CM, miR-9, and depressive severity on bilateral AFC networks in MDD patients were primarily located in the prefrontal-striatum pathway and limbic system. The connection of amygdala to prefrontal-limbic circuits could mediate the effects of CM severity on the miR-9 levels, as well as the impacts of miR-9 levels on the severity of depression in MDD patients. Furthermore, the SVC model, which integrated miR-9 levels, CM severity, and AFC strength in prefrontal-limbic regions, had good power in differentiating MDD patients from HCs (accuracy 85.1%). MiR-9 may play a crucial role in the process of CM experience-produced brain changes targeting prefrontal-limbic regions and that subsequently leads to depression. The present neuroimaging-epigenetic results provide new insight into our understanding of MDD pathophysiology.

CircDYM ameliorates depressive-like behavior by targeting miR-9 to regulate microglial activation via HSP90 ubiquitination.

Circular RNAs (circRNAs), highly expressed in the central nervous system, are involved in various regulatory processes and implicated in some pathophysiology. However, the potential role of circRNAs in psychiatric diseases, particularly major depressive disorder (MDD), remains largely unknown. Here, we demonstrated that circular RNA DYM (circDYM) levels were significantly decreased both in the peripheral blood of patients with MDD and in the two depressive-like mouse models: the chronic unpredictable stress (CUS) and lipopolysaccharide (LPS) models. Restoration of circDYM expression significantly attenuated depressive-like behavior and inhibited microglial activation induced by CUS or LPS treatment. Further examination indicated that circDYM functions as an endogenous microRNA-9 (miR-9) sponge to inhibit miR-9 activity, which results in a downstream increase of target-HECT domain E3 ubiquitin protein ligase 1 (HECTD1) expression, an increase of HSP90 ubiquitination, and a consequent decrease of microglial activation. Taken together, the results of our study demonstrate the involvement of circDYM and its coupling mechanism in depression, providing translational evidence that circDYM may be a novel therapeutic target for depression.

Circular RNA TLK1 Aggravates Neuronal Injury and Neurological Deficits after Ischemic Stroke via miR-335-3p/TIPARP.

Circular RNAs (circRNAs) are expressed at high levels in the brain and are involved in various CNS diseases. However, the potential role of circRNAs in ischemic stroke-associated neuronal injury remains largely unknown. Here, we investigated the important functions of circRNA TLK1 (circTLK1) in this process. The levels of circTLK1 were significantly increased in brain tissues in a mouse model of focal cerebral ischemia and reperfusion. Knockdown of circTLK1 significantly decreased infarct volumes, attenuated neuronal injury, and improved neurological deficits. Furthermore, circTLK1 functioned as an endogenous miR-335-3p sponge to inhibit miR-335-3p activity, resulting in the increase of 2,3,7,8-tetrachlorodibenzo-p-dioxin-inducible poly (ADP-ribose) polymerase expression and a subsequent exacerbation of neuronal injury. Clinical studies confirmed increased levels of circTLK1 in the plasma of patients with acute ischemic stroke (59 males and 12 females). Our findings reveal a detrimental role of circTLK1 in ischemic brain injury.SIGNIFICANCE STATEMENT The extent of neuronal injury after brain ischemia is a primary factor determining stroke outcomes. However, the molecular switches that control the death of ischemic neurons are poorly understood. While our previous studies indicated the involvement of circRNAs in ischemic stroke, the potential role of circRNAs in neuronal injury remains largely unknown. The levels of circTLK1 were significantly increased in the brain tissue and plasma isolated from animal models of ischemic stroke and patients. Knockdown of circTLK1 significantly decreased infarct volumes, attenuated neuronal injury, and improved subsequent long-term neurological deficits. To our knowledge, these results provide the first definitive evidence that circTLK1 is detrimental in ischemic stroke.

Circulating Circular RNAs as Biomarkers for the Diagnosis and Prediction of Outcomes in Acute Ischemic Stroke.

Background and Purpose- Circular RNAs (CircRNAs) show promise as stroke biomarkers because of their participation in various pathophysiological processes associated with acute ischemic stroke (AIS) and stability in peripheral blood. Methods- A circRNA microarray was used to identify differentially expressed circulating circRNAs in a discovery cohort (3 versus 3). Validation (36 versus 36) and replication (200 versus 100) were performed in independent cohorts by quantitative polymerase chain reaction. Platelets, lymphocytes, and granulocytes were separated from blood to examine the origins of circRNAs. Results- There were 3 upregulated circRNAs in Chinese population-based AIS patients compared with healthy controls. The combination of 3 circRNAs resulted in an area under the curve of 0.875, corresponding to a specificity of 91% and a sensitivity of 71.5% in AIS diagnosis. Furthermore, the combination of change rate in 3 circRNAs within the first 7 days of treatment showed an area under the curve of 0.960 in predicting stroke outcome. There was significant increase in lymphocytes and granulocytes for circPDS5B (circular RNA PDS5B) and only in granulocytes for circCDC14A (circular RNA CDC14A) in AIS patients compared with healthy controls. Conclusions- Three circRNAs could serve as biomarkers for AIS diagnosis and prediction of stroke outcomes. The elevated levels of circPDS5B and circCDC14A after stroke might be because of increased levels in lymphocytes and granulocytes.

Correction to: Role of PUMA in the methamphetamine-induced migration of microglia.

In the original publication of this article, wrong western blot images were inadvertently included in Fig. 2 and Fig. 3. The corrected figures are shown below. The authors declare that these amendments do not change the results or conclusions of their paper, and apologize for this oversight.

Circular RNA DLGAP4 Ameliorates Ischemic Stroke Outcomes by Targeting miR-143 to Regulate Endothelial-Mesenchymal Transition Associated with Blood-Brain Barrier Integrity.

Circular RNAs (circRNAs) are highly expressed in the CNS and regulate physiological and pathophysiological processes. However, the potential role of circRNAs in stroke remains largely unknown. Here, we show that the circRNA DLGAP4 (circDLGAP4) functions as an endogenous microRNA-143 (miR-143) sponge to inhibit miR-143 activity, resulting in the inhibition of homologous to the E6-AP C-terminal domain E3 ubiquitin protein ligase 1 expression. circDLGAP4 levels were significantly decreased in the plasma of acute ischemic stroke patients (13 females and 13 males) and in a mouse stroke model. Upregulation of circDLGAP4 expression significantly attenuated neurological deficits and decreased infarct areas and blood-brain barrier damage in the transient middle cerebral artery occlusion mouse stroke model. Endothelial-mesenchymal transition contributes to blood-brain barrier disruption and circDLGAP4 overexpression significantly inhibited endothelial-mesenchymal transition by regulating tight junction protein and mesenchymal cell marker expression. Together, the results of our study are illustrative of the involvement of circDLGAP4 and its coupling mechanism in cerebral ischemia, providing translational evidence that circDLGAP4 serves as a novel therapeutic target for acute cerebrovascular protection.SIGNIFICANCE STATEMENT Circular RNAs (circRNAs) are involved in the regulation of physiological and pathophysiological processes. However, whether circRNAs are involved in ischemic injury, particularly cerebrovascular disorders, remains largely unknown. Here, we demonstrate a critical role for circular RNA DLGAP4 (circDLGAP4), a novel circular RNA originally identified as a sponge for microRNA-143 (miR-143), in ischemic stroke outcomes. Overexpression of circDLGAP4 significantly attenuated neurological deficits and decreased infarct areas and blood-brain barrier damage in the transient middle cerebral artery occlusion mouse stroke model. To our knowledge, this is the first report describing the efficacy of circRNA injection in an ischemic stroke model. Our investigation suggests that circDLGAP4 may serve as a novel therapeutic target for acute ischemic injury.

CircRNA-012091/PPP1R13B-mediated Lung Fibrotic Response in Silicosis via Endoplasmic Reticulum Stress and Autophagy.

Silicosis is a progressive fibrotic disease of lung tissue caused by long-term inhalation of SiO2. However, relatively few studies of the direct effects of SiO2 on lung fibroblasts have been performed. PPP1R13B is a major member of the apoptosis-stimulating proteins of the p53 family, but its role in pulmonary fibrosis is unclear. To elucidate the role of PPP1R13B in the pathological process of silicosis, we explored the molecular mechanisms related to PPP1R13B and the functional effects of proliferation and migration of fibroblasts. Through lentivirus transfection, Western blotting, and fluorescent in situ hybridization experiments, we found that SiO2 downregulated circRNA-012091 (circ-012091) expression in lung fibroblasts and induced upregulation of downstream PPP1R13B. Transfection of L929 cells with PPP1R13B CRISPR NIC plasmid inhibited the upregulation of endoplasmic reticulum stress (ERS) and autophagy-related protein expression in lung fibroblasts treated with SiO2, and induced decreases in cell proliferation, migration, and viability. Transfection of L929 cells with the PPP1R13B CRISPR ACT plasmid induced increases in cell proliferation, migration, and viability. In addition, the ERS inhibitor salubrinal and the autophagy inhibitor 3-methyladenine inhibited the increased migration of L929 cells transfected with the PPP1R13B CRISPR ACT plasmid. These results suggest that PPP1R13B regulated by circ-012091 promotes the proliferation and migration of lung fibroblasts through ERS and autophagy, and plays a crucial role in the development of pulmonary fibrosis in silicosis.

Role of PUMA in the methamphetamine-induced migration of microglia.

In this study, we demonstrated that PUMA was involved in the microglial migration induced by methamphetamine. PUMA expression was examined by western blotting and immunofluorescence staining. BV2 and HAPI cells were pretreated with a sigma-1R antagonist and extracellular signal-regulated kinase (ERK), mitogen-activated protein kinase (MAPK), c-Jun N-terminal protein kinase (JNK), and phosphatidylinositol-3 kinase (PI3K)/Akt inhibitors, and PUMA expression was detected by western blotting. The cell migration in BV2 and HAPI cells transfected with a lentivirus encoding red fluorescent protein (LV-RFP) was also examined using a wound-healing assay and nested matrix model and cell migration assay respectively. The molecular mechanisms of PUMA in microglial migration were validated using a siRNA approach. The exposure of BV2 and HAPI cells to methamphetamine increased the expression of PUMA, reactive oxygen species (ROS), the MAPK and PI3K/Akt pathways and the downstream transcription factor signal transducer and activator of transcription 3 (STAT3) pathways. PUMA knockdown in microglia transfected with PUMA siRNA attenuated the increased cell migration induced by methamphetamine, thereby implicating PUMA in the migration of BV2 and HAPI cells. This study demonstrated that methamphetamine-induced microglial migration involved PUMA up-regulation. Targeting PUMA could provide insights into the development of a potential therapeutic approach for the alleviation of microglia migration induced by methamphetamine.

Non-coding RNAs and Autophagy.

Autophagy is an important metabolic pathway of cells. Cells degrade harmful intracellular components with the aid of autophagy to maintain a healthy state. In recent decades, the study of non-coding RNA in the regulation of autophagy has been a hot area. Mounting evidence indicates that many ncRNAs are involved in the dynamic process of autophagy, and further studies were undertaken to dissect the detailed cellular and molecular mechanisms underlying this process. In this chapter, we mainly summarized the regulation of different non-coding RNAs in autophagy as well as the detailed mechanisms. Based on these findings, we also discussed the roles of non-coding RNAs in the diagnosis, treatment, and prognosis of diseases with an emphasis on their use as potential biomarkers and therapeutic targets for different diseases.

Metalloprotease Adam10 suppresses epilepsy through repression of hippocampal neuroinflammation.

BACKGROUND: Mice with pilocarpine-induced temporal lobe epilepsy (TLE) are characterized by intense hippocampal neuroinflammation, a prominent pathological hallmark of TLE that is known to contribute to neuronal hyperexcitability. Recent studies indicate that Adam10, a member of a disintegrin and metalloproteinase domain-containing protein (Adam) family, has been involved in the neuroinflammation response. However, it remains unclear whether and how Adam10 modulates neuroinflammation responses in the context of an epileptic brain or whether Adam10 affects epileptogenesis via the neuroinflammation pathway. METHODS: Adult male C57BL/6J mice were subjected to intraperitoneal injection of pilocarpine to induce TLE. Adeno-associated viral (AAV) vectors carrying Adam10 (AAV-Adam10) or lentiviral vectors carrying short hairpin RNA, which is specific to the mouse Adam10 mRNA (shRNA-Adam10), were bilaterally injected into the hippocampus to induce overexpression or knockdown of Adam10, respectively. The specific anti-inflammatory agent minocycline was administered following status epilepticus (SE) to block hippocampal neuroinflammation. Continuous video EEG recording was performed to analyze epileptic behavior. Western blot, immunofluorescence staining, and ELISA were performed to determine Adam10 expression as well as hippocampal neuroinflammation. RESULTS: In this study, we demonstrate that overexpression of Adam10 in the hippocampus suppresses neuroinflammation and reduces seizure activity in TLE mice, whereas knockdown of Adam10 exacerbates hippocampal neuroinflammation and increases seizure activity. Furthermore, increased seizure activity in Adam10 knockdown TLE mice is dependent on hippocampal neuroinflammation. CONCLUSION: These results suggest that Adam10 suppresses epilepsy through repression of hippocampal neuroinflammation. Our findings provide new insights into the Adam10 regulation of development of epilepsy via the neuroinflammation pathway and identify a potential therapeutic target for epilepsy.

Involvement of PUMA in pericyte migration induced by methamphetamine.

Mounting evidence indicates that methamphetamine causes blood-brain barrier damage, with emphasis on endothelial cells. The role of pericytes in methamphetamine-induced BBB damage remains unknown. Our study demonstrated that methamphetamine increased the migration of pericytes from the endothelial basement membrane. However, the detailed mechanisms underlying this process remain poorly understood. Thus, we examined the molecular mechanisms involved in methamphetamine-induced pericyte migration. The results showed that exposure of C3H/10T1/2 cells and HBVPs to methamphetamine increased PUMA expression via activation of the sigma-1 receptor, MAPK and Akt/PI3K pathways. Moreover, methamphetamine treatment resulted in the increased migration of C3H/10T1/2 cells and HBVPs. Knockdown of PUMA in pericytes transduced with PUMA siRNA attenuated the methamphetamine-induced increase in cell migration through attenuation of integrin and tyrosine kinase mechanisms, implicating a role of PUMA in the migration of C3H/10T1/2 cells and HBVPs. This study has demonstrated that methamphetamine-mediated pericytes migration involves PUMA up-regulation. Thus, targeted studies of PUMA could provide insights to facilitate the development of a potential therapeutic approach for alleviation of methamphetamine-induced pericyte migration.

Effect of methamphetamine on the fasting blood glucose in methamphetamine abusers.

Methamphetamine is a popular psychostimulant worldwide which causes neurotoxicity and neuroinflammation. Although previous studies have characterized potential associations between addictive drugs and fasting blood glucose, the influence of methamphetamine on the blood glucose is still largely unknown. The present study was designed to investigate the change of fasting blood glucose of methamphetamine abusers and to confirm the impairment of liver and kidney. Fasting blood glucose was significantly decreased in methamphetamine abusers and in a high-fat diet mouse model with methamphetamine treatment discontinuation. Serum level of ALT, creatine kinase and creatinine were increased in methamphetamine abusers. Serum level of ALT and AST were increased in a high-fat diet mouse model after methamphetamine injection, but there was no significant difference in the anatomy of the liver and kidney in high-fat diet treated mice with or without methamphetamine. The levels of ALT and creatinine were also increased in the methamphetamine abusers. This study demonstrated that the level of glucose was decreased in methamphetamine abusers and in high-fat diet-fed mice after methamphetamine treatment discontinuation. The effect of methamphetamine on the levels of blood glucose may provide the evidence that methamphetamine abusers should be keep energy balance due to the low blood glucose.

An Increase of Sigma-1 Receptor in the Penumbra Neuron after Acute Ischemic Stroke.

BACKGROUND: Penumbra salvage from infarction by early reperfusion within the time window is the target of acute ischemic stroke therapies. Although the penumbral imaging is potently usable in clinic trial, additional work needs to be performed to advancing the field with better-defined, evaluated, and validated imaging measures. METHODS: Mice were subjected to permanent stroke by right middle cerebral artery (MCA) occlusion. Multimodel magnetic resonance imaging (MRI) method was assessed to define the penumbra as that brain region in which the perfusion and diffusion-weighted MR images are mismatched (perfusion-weighted imaging [PWI]-diffusion-weighted imaging [DWI] mismatch). MRI measurements were performed at 1 hour after MCA occlusion (MCAO). Sigma-1 receptor expression was assessed by immunoblotting and immunostaining in PWI-DWI-defined penumbra and core compared with sham or contralateral slice. Penumbral sigma-1 receptor identified the correlation with the neuron, astrocyte, and microglia by immuno-colocalization. RESULTS: Sigma-1 receptor was significantly upregulated in penumbra or peri-infarct compared with sham and core tissue at 1 hour and 24 hours after MCAO. There was a colocalization of sigma-1 receptor and neuron in penumbra at 1 hour after stroke. Sigma-1 receptor is specifically increased in ischemic penumbral neuron at 1 hour after MCAO. CONCLUSIONS: Sigma-1 receptor may act as an endogenous marker of penumbra after acute ischemic stroke.

MCPIP1 mediates silica-induced cell migration in human pulmonary fibroblasts.

Silicosis is a systemic disease caused by inhaling silicon dioxide (SiO2). Phagocytosis of SiO2 in the lungs initiates an inflammatory cascade that results in fibroblast proliferation and migration followed by fibrosis. According to previous data from our laboratory, monocyte chemotactic protein-1 (MCP-1) plays a critical role in fibroblast proliferation and migration in conventional two-dimensional (2D) monolayer cultures. The present study aimed to explore the downstream cascade of MCP-1 in both 2D and three-dimensional (3D) cell culture models of silicosis. Experiments using primary cultured adult human pulmonary fibroblasts (HPF-a) demonstrated the following: 1) SiO2 treatment induces expression of MCP-1-induced protein (MCPIP1) in a time- and dose-dependent manner in both 2D and 3D cultures; 2) the MAPK and phosphatidylinositol-3-kinase (PI3K)/Akt pathways are involved in SiO2-induced MCPIP1 expression; and 3) MCPIP1 induction mediates the SiO2-induced increase in cell migration in both 2D and 3D cultures. The effect of MCP-1 in silicosis occurs mainly through MCPIP1, which, in turn, mediates the observed SiO2-induced increase in pulmonary fibroblast migration. However, the time frame for MCPIP1 induction differed between 2D and 3D cultures, indicating that, compared with conventional 2D cell culture systems, 3D culture may be useful for analyses of fibroblast physiology under conditions that more closely resemble in vivo environments. Our study determined the link between fibroblast-derived MCPIP1 and SiO2-induced cell migration, and this finding provides novel evidence of the potential of MCPIP1 in the development of novel therapeutic strategies for silicosis.

iNOS Induces Vascular Endothelial Cell Migration and Apoptosis Via Autophagy in Ischemia/Reperfusion Injury.

BACKGROUND/AIMS: Inducible nitric oxide synthase (iNOS) plays a crucial role in ischemia/reperfusion (I/R). Autophagy is involved in irreversible cell injury and death under extreme conditions. However, whether iNOS mediates myocardial ischemia/reperfusion (I/R) injury in endothelial cells via autophagy remains ill-defined. In this study, we examined whether I/R-mediated up-regulation of iNOS is critical in the modulation of cell migration and apoptosis via autophagy in human umbilical vein endothelial cells (HUVECs). METHODS: iNOS expression was detected in HUVECs using Western blotting analyses and immunocytochemistry. An in vitro scratch assay was performed to detect cell migration. The autophagy markers ATG5, LC3B and BECN were detected using Western blotting analysis and adenovirus-mRFP-GFP-LC3. The pharmacological inhibitor of autophagy 3-MA was also applied to confirm the role of autophagy in I/R. RESULTS: I/R induced the expression of iNOS, which subsequently increased the migration and apoptosis of HUVECs and was associated with the up-regulation of autophagy. The iNOS specific inhibitor L-NAME abolished I/R-induced autophagy, while L-NAME and 3-MA both attenuated cell apoptosis and migration induced by I/R. CONCLUSION: These findings suggested that I/R-induced iNOS regulates migration and apoptosis in HUVECs via autophagy, which indicates a new therapeutic strategy for individuals with I/R injury.

Role of MCPIP1 in the Endothelial-Mesenchymal Transition Induced by Silica.

BACKGROUND: Silicosis is characterized by the accumulation of fibroblasts and the excessive deposition of extracellular matrix. Fibroblast generation via endothelial-mesenchymal transition (EndMT) is one process responsible for this accumulation of fibroblasts. However, the mechanisms underlying EndMT remain unknown. METHODS: Human umbilical vein endothelial cells (HUVECs) were exposed to SiO2 (50 µg/cm2). Specific endothelial and mesenchymal markers were evaluated using immunofluorescence and western blot analysis. Functional changes were evaluated by analyzing cell migration and proliferation. LC3-adenovirus transfections were performed, and changes in autophagy were measured using a marker of autophagy. RESULTS: SiO2 induced decreases in the endothelial cell-specific markers in HUVECs while dramatically increasing mesenchymal cell product levels and mesenchymal functions. Although MCPIP1 expression increased in parallel with the increase in specific mesenchymal cell products, the MCPIP1 expression level was not consistent with the observed decrease in specific endothelial marker expression. Autophagy mediated the effects of MCPIP1, as rapamycin and 3-MA enhanced and attenuated the effect of SiO2 on HUVECs, respectively. MAPKs and the PI3K/Akt pathway were involved in the regulation of MCPIP1 by SiO2, and Pyk2 and MLC-2 mediated cell migration. CONCLUSION: Our findings reveal a new potential function of MCPIP1, suggesting a possible mechanism of fibrosis in pulmonary silicosis.

Neogambogic acid prevents silica-induced fibrosis via inhibition of high-mobility group box 1 and MCP-1-induced protein 1.

BACKGROUND: Silicosis is a systemic disease caused by inhaling silicon dioxide (SiO2); early stages are characterized by alveolar inflammation, and later stages are characterized by progressive lung fibrosis. Mounting evidence indicates that high-mobility group box 1 (HMGB1) is involved in pulmonary fibrosis. Whether neogambogic acid (NGA) inhibits macrophage and fibroblast activation induced by SiO2 by targeting HMGB1 remains unclear. METHODS AND RESULTS: Experiments using cultured mouse macrophages (RAW264.7 cells) demonstrated that SiO2 treatment induces the expression of HMGB1 in a time- and dose-dependent manner via mitogen-activated protein kinases (MAPKs) and the phosphatidylinositol 3-kinase (PI3K)/Akt pathway; in turn, this expression causes macrophage apoptosis and fibroblast activation. Pretreating macrophages with NGA inhibited the HMGB1 expression induced by SiO2 and attenuated both macrophage apoptosis and fibroblast activation. Moreover, NGA directly inhibited MCP-1-induced protein 1 (MCPIP1) expression, as well as markers of fibroblast activation and migration induced by SiO2. Furthermore, the effects of NGA on macrophages and fibroblasts were confirmed in vivo by exposing mice to SiO2. CONCLUSION: NGA can prevent SiO2-induced macrophage activation and apoptosis via HMGB1 inhibition and SiO2-induced fibrosis via the MCPIP1 pathway. Targeting HMGB1 and MCPIP1 with NGA could provide insights into the potential development of a therapeutic approach for alleviating the inflammation and fibrosis induced by SiO2.