p90RSK Inhibition Ameliorates TGF-ß1 Signaling and Pulmonary Fibrosis by Inhibiting Smad3 Transcriptional Activity.

BACKGROUND/AIMS: Idiopathic pulmonary fibrosis (IPF) is a specific form of progressive and chronic interstitial lung disease of unknown cause. IPF is characterized by excessive deposition of extracellular matrix (ECM) and destructive pathological remodeling due to epithelial-to-mesenchymal transition (EMT). Eventually, lung interstitium thickens and stiffens and breathing becomes difficult. It has been well established that the transforming growth factor-ß1 (TGF-ß1)/Smad signaling pathway plays a critical role in the pathogenesis of pulmonary fibrosis. TGF-ß1-mediated activation of mitogen activated protein kinase (MAPK) family affects Smad signaling. p90RSK is a serine/threonine kinase and is activated by the extracellular signal-regulated kinase (ERK) signaling pathway. However, the roles played by p90RSK in TGF-ß1 signaling and the pathogenesis of pulmonary fibrosis remain unknown. METHODS: We investigated whether p90RSK regulates the pathogenesis of pulmonary fibrosis using in vitro and in vivo systems and Western blotting, real-time quantitative PCR, transcriptional activity assays and immunofluorescence studies. RESULTS: Pharmacological inhibition of p90RSK by FMK or inhibition of p90RSK with adenoviral vector encoding a dominant negative form of p90RSK suppressed TGF-ß1-induced ECM accumulation and EMT in lung epithelial cells and fibroblasts. Interestingly, FMK significantly inhibited TGF-ß1-induced Smad3 nuclear translocation and smad binding element-dependent transcriptional activity, but not Smad3 phosphorylation. Furthermore, in a mouse model of bleomycin-induced lung fibrosis, FMK ameliorated pulmonary fibrosis. CONCLUSION: These findings indicate that p90RSK plays critical roles in pulmonary fibrosis, which suggests it be viewed as a novel therapeutic target for the treatment of lung fibrosis.

C/EBP homologous protein deficiency inhibits statin-induced myotoxicity.

It has been well established that HMG-CoA reductase inhibitors (statins) cause adverse side effects in skeletal muscle ranging from mild to fatal myotoxicity upon dose, drug interaction, and exercise. However, the underlying mechanisms by which statins induce myotoxicity have not been fully addressed. Recent reports showed that statins induce endoplasmic reticulum (ER) stress and cell death in immune cells and myoblasts in vitro. Therefore, the goal of study is to investigate the molecular mechanism by which statins induce skeletal muscle cell death and myopathy via the regulation of ER stress. Biochemical data showed that TUDCA, an ER stress inhibitor, inhibited atorvastatin- and simvastatin-induced protein cleavages of PARP-1 and caspase-3, respectively. Actually, statin treatment activated marker proteins of unfolded protein responses (UPR) including ATF6, CHOP, and spliced XBP1 and these responses were inhibited by TUDCA. In addition, statin treatment induced mRNA levels of UPR marker genes, suggesting that statins activate ER stress in a transcriptional regulation. The physiological relevance of ER stress in statin-induced myopathy was demonstrated in a mouse model of myopathy, in which instillation of simvastatin and atorvastatin led to myopathy. Notably, the reduction of muscular endurance in response to statin instillation was significantly improved in TUDCA treating group compared to vehicle control group. Moreover, CHOP deficiency mice showed restoration of statin-induced reduction of muscular endurance, suggesting that statin induces myopathy via ER stress and in a CHOP-dependent manner. Taken together, these findings indicate that statins specifically induce myopathy in an ER stress-dependent manner, suggesting the therapeutic potential of ER stress regulation in preventing adverse effects of statin.

FMK, an Inhibitor of p90RSK, Inhibits High Glucose-Induced TXNIP Expression via Regulation of ChREBP in Pancreatic ß Cells.

Hyperglycemia is the major characteristic of diabetes mellitus, and a chronically high glucose (HG) level causes ß-cell glucolipotoxicity, which is characterized by lipid accumulation, impaired ß-cell function, and apoptosis. TXNIP (Thioredoxin-interacting protein) is a key mediator of diabetic ß-cell apoptosis and dysfunction in diabetes, and thus, its regulation represents a therapeutic target. Recent studies have reported that p90RSK is implicated in the pathogenesis of diabetic cardiomyopathy and nephropathy. In this study, we used FMK (a p90RSK inhibitor) to determine whether inhibition of p90RSK protects ß-cells from chronic HG-induced TXNIP expression and to investigate the molecular mechanisms underlying the effect of FMK on its expression. In INS-1 pancreatic ß-cells, HG-induced ß-cell dysfunction, apoptosis, and ROS generation were significantly diminished by FMK. In contrast BI-D1870 (another p90RSK inhibitor) did not attenuate HG-induced TXNIP promoter activity or TXNIP expression. In addition, HG-induced nuclear translocation of ChREBP and its transcriptional target molecules were found to be regulated by FMK. These results demonstrate that HG-induced pancreatic ß-cell dysfunction resulting in HG conditions is associated with TXNIP expression, and that FMK is responsible for HG-stimulated TXNIP gene expression by inactivating the regulation of ChREBP in pancreatic ß-cells. Taken together, these findings suggest FMK may protect against HG-induced ß-cell dysfunction and TXNIP expression by ChREBP regulation in pancreatic ß-cells, and that FMK is a potential therapeutic reagent for the drug development of diabetes and its complications.

Structural insight into the interaction between the Hox and HMGB1 and understanding of the HMGB1-enhancing effect of Hox-DNA binding.

The Hox DNA binding domain, the homeodomain, plays critical roles in genetic control of development and cell fate determination. The variable regulatory functions of Hox proteins are accomplished by binding to target DNA sequences and collaborating protein partners that includes human high mobility group B1 (HMGB1). To better understand the interaction between Hox and HMGB1 and the facilitation of Hox-DNA binding by HMGB1, we solved the solution structure of the homeodomain of Hox including the N-terminal arm region (Hoxc9DBD hereafter). In addition, the details of the interaction between these two proteins, as well as DNA binding of the Hox-HMGB1 complex, were investigated by NMR, ITC, and EMSA. The results suggest that binding of the HMGB1 A-box to Hoxc9DBD makes the loop-1 (loop preceding helix-2 of Hoxc9DBD) more access to DNA backbone, which facilitate Hox-DNA binding with enhanced affinity.

Activated protein C prevents methylglyoxal-induced endoplasmic reticulum stress and cardiomyocyte apoptosis via regulation of the AMP-activated protein kinase signaling pathway.

Previous epidemiological studies have shown that methylglyoxal (MGO) levels are highly regulated in diabetic cardiovascular diseases. We have also previously reported that MGO mediates ER stress and apoptosis in cardiomyocytes. Furthermore, activated protein C (APC) has recently been shown to play a protective role against ER stress, as well as a cardioprotective role against ischemia and reperfusion injury by augmenting the AMP-activated protein kinase (AMPK) signaling pathway. Therefore, we hypothesized that APC protects against MGO-induced cardiomyocyte apoptosis through the inhibition of ER stress. Our results showed that APC inhibited MGO-induced cardiomyocyte apoptosis and ER stress-related gene expression. Additionally, APC inhibited MGO-induced Ca2+ mobilization and the generation of reactive oxygen species. In contrast, inhibitors of AMPK signaling abolished the cytoprotective effects of APC. Collectively, these data depict a pivotal role for AMPK signaling in inhibiting ER stress responses via the activation of APC during MGO-induced cardiomyocyte apoptosis. Thus, APC may be a potential novel therapeutic target for the management of diabetic cardiovascular complications such as diabetic cardiomyopathy.

CHOP deficiency inhibits methylglyoxal-induced endothelial dysfunction.

Epidemiological studies suggested that diabetic patients are susceptible to develop cardiovascular complications along with having endothelial dysfunction. It has been suggested that methylglyoxal (MGO), a glycolytic metabolite, has more detrimental effects on endothelial dysfunction rather than glucose itself. Here, we investigated the molecular mechanism by which MGO induces endothelial dysfunction via the regulation of ER stress. Biochemical data showed that 4-PBA significantly inhibited MGO-induced protein cleavages of PARP-1 and caspase-3. In addition, it was found that high glucose-induced endothelial apoptosis was enhanced in the presence of GLO1 inhibitor, suggesting the role of endogenous MGO in high glucose-induced endothelial dysfunction. MGO-induced endothelial apoptosis was significantly diminished by the depletion of CHOP with si-RNA against human CHOP, but not by SP600125, a specific inhibitor of JNK. The physiological relevance of this signaling pathway was demonstrated in CHOP deficiency mouse model, in which instillation of osmotic pump containing MGO led to aortic endothelial dysfunction. Notably, the aortic endothelial dysfunction response to MGO infusion was significantly improved in CHOP deficiency mice compared to littermate control. Taken together, these findings indicate that MGO specifically induces endothelial dysfunction in a CHOP-dependent manner, suggesting the therapeutic potential of CHOP inhibition in diabetic cardiovascular complications.

CHOP deficiency prevents methylglyoxal-induced myocyte apoptosis and cardiac dysfunction.

Epidemiological studies indicate that methylglyoxal (MGO) plasma levels are closely linked to diabetes and the exacerbation of diabetic cardiovascular complications. Recently, it was established that endoplasmic reticulum (ER) stress importantly contributes to the pathogenesis of diabetes and its cardiovascular complications. The objective of this study was to explore the mechanism by which diabetes instigates cardiomyocyte apoptosis and cardiac dysfunction via MGO-mediated myocyte apoptosis. Intriguingly, the MGO activated unfolded protein response pathway accompanying apoptotic events, such as cleavages of PARP-1 and caspase-3. In addition, Western blot analysis revealed that MGO-induced myocyte apoptosis was inhibited by depletion of CHOP with siRNA against Ddit3, the gene name for rat CHOP. To investigate the physiologic roles of CHOP in vivo, glucose tolerance and cardiac dysfunction were assessed in CHOP-deficient mice. No significant difference was observed between CHOP KO and littermate naïve controls in terms of the MGO-induced impairment of glucose tolerance. In contrast, myocyte apoptosis, inflammation, and cardiac dysfunction were significantly diminished in CHOP KO compared with littermate naïve controls. These results showed that CHOP is the key signal for myocyte apoptosis and cardiac dysfunction induced by MGO. These findings suggest a therapeutic potential of CHOP inhibition in the management of diabetic cardiovascular complications including diabetic cardiomyopathy.

CHIP Haploinsufficiency Exacerbates Hepatic Steatosis via Enhanced TXNIP Expression and Endoplasmic Reticulum Stress Responses.

TXNIP is a critical regulator of glucose homeostasis, fatty acid synthesis, and cholesterol accumulation in the liver, and it has been reported that metabolic diseases, such as obesity, atherosclerosis, hyperlipidemia, type 2 diabetes, and nonalcoholic fatty liver disease (NAFLD), are associated with endoplasmic reticulum (ER) stress. Because CHIP, an E3 ligase, was known to be involved in regulating tissue injury and inflammation in liver, its role in regulating ER stress-induced NAFLD was investigated in two experimental NAFLD models, a tunicamycin (TM)-induced and other diet-induced NAFLD mice models. In the TM-induced NAFLD model, intraperitoneal injection of TM induced liver steatosis in both CHIP+/+ and CHIP+/- mice, but it was severely exacerbated in CHIP+/- mice compared to CHIP+/+ mice. Key regulators of ER stress and de novo lipogenesis were also enhanced in the livers of TM-inoculated CHIP+/- mice. Furthermore, in the diet-induced NAFLD models, CHIP+/- mice developed severely impaired glucose tolerance, insulin resistance and hepatic steatosis compared to CHIP+/+ mice. Interestingly, CHIP promoted ubiquitin-dependent degradation of TXNIP in vitro, and inhibition of TXNIP was further found to alleviate the inflammation and ER stress responses increased by CHIP inhibition. In addition, the expression of TXNIP was increased in mice deficient in CHIP in the TM- and diet-induced models. These findings suggest that CHIP modulates ER stress and inflammatory responses by inhibiting TXNIP, and that CHIP protects against TM- or HF-HS diet-induced NAFLD and serves as a potential therapeutic means for treating liver diseases.

Recombinant human KAI1/CD82 attenuates M1 macrophage polarization on LPS-stimulated RAW264.7 cells via blocking TLR4/JNK/NF-κB signal pathway.

KAI1/CD82, a membrane tetraspanin protein, can prevent various cancers and retinal disorders through its anti-angiogenic and anti-metastatic capacity. However, little is known about its anti-inflammatory effect and molecular mechanism. Therefore, the present study aimed to inLPSvestigate effect of a recombinant protein of the large extracellular domain of human KAI1 (Gly 111-Leu 228, rhKAI1) on lipopolysaccharides (LPS)-stimulated RAW264.7 macrophage-like cells and mouse bone marrow-derived macrophages (BMDM) and to identify its underlying mechanism. Our data showed that rhKAI1 suppressed expression levels of classically macrophages (M1) phenotyperelated surface markers F4/80+CD86+ in LPS-stimulated BMDM and RAW264.7 cells. In addition, LPS markedly increased mRNA expression and release levels of pro-inflammatory cytokines and mediators such as interleukin (IL)-1ß, IL-6, tumor necrosis factor-α, cyclooxygenase-2, nitric oxide and prostaglandin E2, whereas these increases were substantially down-regulated by rhKAI1. Furthermore, LPS strongly increased expression of NF-κB p65 in the nuclei and phosphorylation of ERK, JNK, and p38 MAPK. However, nuclear translocation of NF-κB p65 and phosphorylation of JNK were greatly reversed in the presence of rhKAI1. Especially, rhKAI1 markedly suppressed expression of toll-like receptor (TLR4) and prevented binding of LPS with TLR4 through molecular docking predict analysis. Importantly, Glu 214 of rhKAI1 residue strongly interacted with Lys 360 of TLR4 residue, with a binding distance of 2.9 Å. Taken together, these findings suggest that rhKAI1 has an anti-inflammatory effect on LPS-polarized macrophages by interacting with TLR4 and down-regulating the JNK/NF-κB signaling pathway. [BMB Reports 2023; 56(6): 359-364].

Shear stress induces monocyte/macrophage-mediated inflammation by upregulating cell-surface expression of heat shock proteins.

The loss of endothelial cells is associated with the accumulation of monocytes/macrophages underneath the surface of the arteries, where cells are prone to mechanical stimulation, such as shear stress. However, the impact of mechanical stimuli on monocytic cells remains unclear. To assess whether mechanical stress affects monocytic cell function, we examined the expression of inflammatory molecules and surface proteins, whose levels changed following shear stress in human THP-1 cells. Shear stress increased the inflammatory chemokine CCL2, which enhanced the migration of monocytic cells and tumor necrosis factor (TNF)-α and interleukin (IL)- 1ß at transcriptional and protein levels. We identified that the surface levels of heat shock protein 70 (HSP70), HSP90, and HSP105 increased using mass spectrometry-based proteomics, which was confirmed by western blot analysis, flow cytometry, and immunofluorescence. Treatment with HSP70/HSP105 and HSP90 inhibitors suppressed the expression and secretion of CCL2 and monocytic cell migration, suggesting an association between HSPs and inflammatory responses. We also demonstrated the coexistence and colocalization of increased HSP90 immunoreactivity and CD68 positive cells in atherosclerotic plaques of ApoE deficient mice fed a high-fat diet and human femoral artery endarterectomy specimens. These results suggest that monocytes/macrophages affected by shear stress polarize to a pro-inflammatory phenotype and increase surface protein levels involved in inflammatory responses. The regulation of the abovementioned HSPs upregulated on the monocytes/macrophages surface may serve as a novel therapeutic target for inflammation due to shear stress.

Pilot study of a mobile application-based intervention to induce changes in neural activity in the frontal region and behaviors in children with attention deficit hyperactivity disorder and/or intellectual disability.

Children with neurodevelopmental disorders, such as attention deficit hyperactivity disorder (ADHD) and intellectual disability (ID), need early intervention and continuous treatment. We aimed to investigate the feasibility and acceptability of mobile application-based interventions in children with ADHD and ID in supporting attention and cognitive function. Twenty-six children with ADHD and/or ID with attention and cognition difficulties were recruited. Participants completed a 12-week mobile application-based intervention. To assess whether digital intervention improved attention and cognitive function, we used the Comprehensive Attention Test (CAT), Cambridge Neuropsychological Tests Automated Battery (CANTAB), and electroencephalography (EEG) to examine direct changes in children's behavior and neural activity. Clinicians and parents assessed changes using the Behavior Rating Inventory of Executive Function, Second Edition (BRIEF-2), Korean version of the ADHD Rating Scale (K-ARS), Clinical Global Impression-Improvement Scale, and parental questionnaires. The intervention induced changes in neural activities on EEG and behavior but there were no significant changes in CAT and CANTAB results. Relative theta and alpha power were significantly lower post-intervention in the eyes-open (EO) condition of EEG recording and these changes were mainly observed in the frontal regions of the brain. Parental reports using the BRIEF-2 and K-ARS noted significant improvements in executive function, attention, and hyperactivity-impulsivity. In addition, the clinical impression improved in 60% of participants. These results provide evidence that a mobile application-based intervention has the benefit of supporting children with ADHD and/or ID. Digital intervention could change neural activity and improve children's attention and cognitive function. Given our findings, we suggested that mobile application-based digital therapeutics may have great potential for helping children with neurodevelopmental disorders who need continuous treatment.

CD82 attenuates TGF-ß1-mediated epithelial-mesenchymal transition by blocking smad-dependent signaling in ARPE-19 cells.

In retinal pigment epithelial (RPE) cells, transforming growth factor-beta (TGF-ß) plays a critical role in epithelial-mesenchymal transition (EMT), which contributes to various fibrotic retinal disorders. In the present study, we investigated the effect of recombinant human cluster of differentiation 82 (rhCD82), a tumor metastasis suppressor, on TGF-ß-induced EMT in the human RPE cell line APRE-19. The results show that TGF-ß1 significantly enhanced cell migration, invasion and the expression of EMT-mediate factors in ARPE-19 cells. However, rhCD82 markedly inhibited cell mobility and the expression of epithelial marker, zonula occludens-1, as well as increased the expression of mesenchymal markers, such as vimentin and α-smooth muscle actin in TGF-ß1-treated APRE-19 cells. In addition, TGF-ß1 upregulated the phosphorylation of Smad, extracellular signal regulated kinase (ERK) and glycogen synthase kinase-3ß (GSK-3ß), but only phosphorylation of Smad was suppressed by rhCD82. Noteworthy, rhCD82 greatly suppressed the expression of TGF-ß receptor I (TGFRI), TGFRII and integrins in TGF-ß1-treated APRE-19 cells. In particular, the result of molecular docking analysis and structural modeling show that rhCD82 partially interacts with the TGF-ß1 binding sites of TGFRI, TGFRII, integrin ß1 and integrin αv. Taken together, this finding suggested that rhCD82 suppressed TGF-ß1-induced EMT of RPE by blocking of Smad-dependent pathway, which is caused by rhCD82 interaction with TGFRs and integrins, suggesting new insight into CD82 as a potential therapeutic strategy in fibrotic retinal disorders.

Efficacy of low dose and short duration defibrotide prophylaxis for hepatic veno-occlusive disease after autologous haematopoietic stem cell transplantation.

Hepatic veno-occlusive disease (VOD) is a serious systemic endothelial complication after stem cell transplantation. Defibrotide is under investigation as a prophylactic agent for VOD; however, high costs limit its utility. We evaluated the prophylactic efficacy of a low-dose defibrotide regimen for VOD. We retrospectively enrolled 147 paediatric patients who underwent autologous haematopoietic stem cell transplantation (HSCT; 69 with defibrotide prophylaxis and 78 historical controls) at the Yonsei Cancer Center in Seoul, Korea, between March 2013 and Feb 2020. Low-dose defibrotide (12.5 mg/kg/day) was administered from D-3 to D+10 after HSCT. The most common diagnosis in the cohort was brain tumour (N = 86). VOD developed in 10 (12.8%) and 3 (4.3%) patients in the control and prophylaxis groups, respectively (P = 0.071). In the second HSCT group, VOD incidence was significantly lower in the prophylaxis group [2.9% (1/35)] than in the control group (28.6%, 6/21, P = 0.005). VOD severity was significantly higher in the control group than in the prophylaxis group (P = 0.006). Three VOD-related mortalities occurred in the control group, whereas no VOD-related mortality occurred in the prophylaxis group. In conclusion, low-dose defibrotide prophylaxis is a promising and economical strategy for preventing VOD, especially in second-round HSCT.

"Reading the Mind in the Eyes Test": Translated and Korean Versions.

OBJECTIVE: The Reading the Mind in the Eyes Test (RMET) was developed by using Caucasian eyes, which may not be appropriate to be used in Korean. The aims of the present study were 1) to develop a Korean version of the RMET (K-RMET) by using Korean eye stimuli and 2) to examine the psychometric properties of the Korean-translated version of the RMET and the K-RMET. METHODS: Thirty-six photographs of Korean eyes were selected. A total of 196 (101 females) healthy subjects were asked to take the Korean-translated version of the RMET and K-RMET. To assess internal consistency reliability, Cronbach's alpha coefficients were computed, and test-retest reliability was assessed by the intraclass correlation coefficient (ICC) and Bland-Altman plots. Confirmatory factor analysis (CFA) and item analysis were also conducted. RESULTS: Internal consistency, measured by Cronbach's alpha, was 0.542 for the Korean-translated version of the RMET, and 0.540 for the K-RMET. Test-retest reliability (n=25), measured by the ICC, was 0.787 for the Korean-translated version of the RMET, and 0.758 for the K-RMET. In CFA, the assumed single and 3-factor model fit indices were not good in the both types of RMETs. There was difficulty in discrimination in nine items of the Korean-translated version of the RMET and 10 items of the K-RMET. CONCLUSION: The psychometric properties of both the Korean-translated version of the RMET and the K-RMET are acceptable. Both tests are applicable to the clinical population, as well as the general population in Korea.

Sodium/glucose Co-Transporter 2 Inhibitor, Empagliflozin, Alleviated Transient Expression of SGLT2 after Myocardial Infarction.

BACKGROUND AND OBJECTIVES: Large clinical studies of sodium/glucose cotransporter 2 (SGLT2) inhibitors have shown a significant beneficial effect on heart failure-associated hospitalization and cardiovascular events. As SGLT2 is known to be absent in heart cells, improved cardiovascular outcomes are thought to be accounted for by the indirect effects of the drug. We sought to confirm whether such benefits were mediated through SGLT2 expressed in the heart using myocardial infarction (MI) model. METHODS: Mice pre-treated with empagliflozin (EMPA), an SGLT2 inhibitor, showed a significantly reduced infarct size compared with the vehicle group three days post-MI. Interestingly, we confirmed SGLT2 localized in the infarct zone. The sequential changes of SGLT2 expression after MI were also evaluated. RESULTS: One day after MI, SGLT2 transiently appeared in the ischemic areas in the vehicle group and increased until 72 hours. The appearance of SGLT2 was delayed and less in amount compared with the vehicle group. Additionally, there was a significant difference in metabolites, including glucose and amino acids in the ¹H nuclear magnetic resonance analysis between groups. CONCLUSIONS: Our work demonstrates that SGLT2 is transiently expressed in heart tissue early after MI and EMPA may directly operate on SGLT2 to facilitate metabolic substrates shifts.

Ex vivo expansion of regulatory T cell and T helper 2 cells using a hematosphere culture.

Regulatory T cells (T-reg) are important components of immune system required to understand the mechanistic details of cancer immunity and autoimmune diseases. However, reliable and efficient methods of regulatory T cell expansion have not been established yet. Here, we show that a human peripheral blood mononuclear cell (PBMNC) derived blood-born hematosphere (BBHS) culture without cytokine treatment increased T-reg and T helper 2 cell (Th2) populations in the cell suspension. We found that the isolated cells suspended around the hematospheres expressed T helper cell markers. Importantly, the Foxp3+/CD25+ T-reg and IL-4+/CD25+ Th2 cell populations in the cell suspension were greatly expanded during hematosphere culture. Through ELISA analysis of the supernatant, we showed that secretion of Th2-related cytokines such as IL-5, IL-10, and IL-13 also increased. Taken together, hematosphere culture is a good method for ex vivo expansion of T-reg and Th2, which can provide therapeutic benefits for the treatment of immune disorders.

Metformin ameliorates bile duct ligation-induced acute hepatic injury via regulation of ER stress.

Cholestasis is a condition in which the bile duct becomes narrowed or clogged by a variety of factors and bile acid is not released smoothly. Bile acid-induced liver injury is facilitated by necrotic cell death, neutrophil infiltration, and inflammation. Metformin, the first-line treatment for type 2 diabetes, is known to reduce not only blood glucose but also inflammatory responses. In this study, we investigated the effects of metformin on liver injury caused by cholestasis with bile acid-induced hepatocyte injury. Static bile acid-induced liver injury is thought to be related to endoplasmic reticulum (ER) stress, inflammatory response, and chemokine expression. Metformin treatment reduced liver injury caused by bile acid, and it suppressed ER stress, inflammation, chemokine expression, and neutrophil infiltration. Similar results were obtained in mouse primary hepatocytes exposed to bile acid. Hepatocytes treated with tauroursodeoxycholic acid, an ER stress inhibitor, showed inhibition of ER stress, as well as reduced levels of inflammation and cell death. These results suggest that metformin may protect against liver injury by suppressing ER stress and inflammation and reducing chemokine expression. [BMB Reports 2020; 53(6): 311-316].

Anti-malarial Drugs Reduce Vascular Smooth Muscle Cell Proliferation via Activation of AMPK and Inhibition of Smad3 Signaling.

OBJECTIVE: The aim of this study was to investigate the effects of 2 anti-malarial drugs, chloroquine (CQ) and hydroxychloroquine (HCQ), on inhibition of vascular smooth muscle cell (VSMC) proliferation both in vivo and in vitro via Adenosine monophosphate-activated protein kinase (AMPK) activation. METHODS: Protein and mRNA levels were determined by western blot analysis and real-time reverse transcription-polymerase chain reaction in primary rat VSMCs treated with CQ and HCQ, respectively. Cell proliferation was measured by flow cytometry and cell counting. Mice carotid arteries were ligated and treated with CQ or HCQ every other day for 3 weeks. Pathological changes of carotid arteries were visualized by both microscopy and fluorescence microscopy. RESULTS: CQ and HCQ increase AMPK phosphorylation in VSMCs. Both CQ and HCQ decrease platelet-derived growth factor-induced VSMC proliferation and cell cycle progression in an AMPK-dependent manner. In addition, CQ and HCQ inhibit Smad3 phosphorylation and VSMC proliferation induced by transforming growth factor-ß1. Moreover, CQ and HCQ diminished neointimal proliferation in a mouse model of carotid artery ligation-induced neointima formation. CONCLUSION: The results demonstrated that CQ and HCQ inhibit cell proliferation and cell cycle progression in VSMCs via the AMPK-dependent signaling pathway. Carotid artery ligation-induced intima thickness was reduced in mouse arteries treated with CQ or HCQ, suggesting a role for antimalarial drugs in treating atherosclerosis and restenosis.

PAR-1 is a novel mechano-sensor transducing laminar flow-mediated endothelial signaling.

Recent studies have indicated that protease-activated receptor-1 (PAR-1) is involved in cytoprotective and anti-inflammatory responses in endothelial cells (ECs). However, the role of PAR-1 in laminar flow-mediated atheroprotective responses remains unknown. Herein, we investigated whether PAR-1 regulates laminar flow-mediated mechanotransduction in ECs. Confocal analysis showed that PAR-1 was internalized into early endosomes in response to laminar flow. In addition, flow cytometry analysis showed that cell surface expression of PAR-1 was reduced by laminar flow, suggesting that PAR-1 was activated in response to laminar flow. Depletion of PAR-1 using human PAR-1 siRNA inhibited unidirectional laminar flow-mediated actin stress fiber formation and cellular alignment as well as atheroprotective gene expressions in HUVECs. Moreover, PAR-1 knockdown inhibited laminar flow-stimulated eNOS phosphorylation, and inhibited the phosphorylations of Src, AMPK, ERK5 and HDAC5. Furthermore, PAR-1 depletion inhibited laminar flow-mediated anti-inflammatory responses as demonstrated by reduced TNFα-induced VCAM-1 expression and by monocyte adhesion to HUVECs, and prevented laminar flow-mediated anti-apoptotic response. An investigation of the role of PAR-1 in vasomotor modulation using mouse aortic rings revealed that acetylcholine-induced vasorelaxation was diminished in PAR-1 deficient mice compared to littermate controls. Taken together, these findings suggest that PAR-1 be viewed as a novel pharmacologic target for the treatment of vascular diseases, including atherosclerosis.

Fluvastatin inhibits AGE-induced cell proliferation and migration via an ERK5-dependent Nrf2 pathway in vascular smooth muscle cells.

Advanced glycation endproduct (AGE)-induced vascular smooth muscle cell (VSMC) proliferation and reactive oxygen species (ROS) production are emerging as important mechanisms of diabetic vasculopathy, but little is known about the molecular mechanism responsible for the antioxidative effects of statins on AGEs. It has been reported that statins exert pleiotropic effects on the cardiovascular system due to decreases in AGE-induced cell proliferation, migration, and vascular inflammation. Thus, in the present study, the authors investigated the molecular mechanism by which statins decrease AGE-induced cell proliferation and VSMC migration. In cultured VSMCs, statins upregulated Nrf2-related antioxidant gene, NQO1 and HO-1, via an ERK5-dependent Nrf2 pathway. Inhibition of ERK5 by siRNA or BIX02189 (a specific ERK5 inhibitor) reduced the statin-induced upregulations of Nrf2, NQO1, and HO-1. Furthermore, fluvastatin was found to significantly increase ARE promoter activity through ERK5 signaling, and to inhibit AGE-induced VSMC proliferation and migration as determined by MTT assay, cell counting, FACS analysis, a wound scratch assay, and a migration chamber assay. In addition, AGE-induced proliferation was diminished in the presence of Ad-CA-MEK5α encoding a constitutively active mutant form of MEK5α (an upstream kinase of ERK5), whereas depletion of Nrf2 restored statin-mediated reduction of AGE-induced cell proliferation. Moreover, fluvastatin suppressed the protein expressions of cyclin D1 and Cdk4, but induced p27, and blocked VSMC proliferation by regulating cell cycle. These results suggest statin-induced activation of an ERK5-dependent Nrf2 pathway reduces VSMC proliferation and migration induced by AGEs, and that the ERK5-Nrf2 signal module be viewed as a potential therapeutic target of vasculopathy in patients with diabetes and complications of the disease.