Sunitinib induces cardiotoxicity through modulating oxidative stress and Nrf2-dependent ferroptosis in vitro and in vivo.

SUN, a multi-targeted tyrosine kinase inhibitor, exerts cardiotoxicity which hinders its clinical use. It is necessary to elucidate molecular mechanism of SUN-induced cardiotoxicity. To elucidate molecular mechanism of SUN-induced cardiotoxicity and whether it is related to Nrf2-dependent ferroptosis, in vitro model with H9c2 cells derived from rat heart tissue and in vivo model (C57BL/6J male mouse) were used in the present study. In vivo model was established by oral treatment of SUN at dose of 10, 20, 40 mg/kg for 14 days. Body weight, ECG, plasma enzyme activities, histology staining were performed to evaluate heart function. Western-blot was performed to analyze the level of ferroptosis-related proteins. In vitro results indicated that SUN markedly induced ferroptosis embodied as collapsed MMP, accumulated iron and elevated ROS. In vivo results showed that SUN significantly impaired cardiac function. Abnormal electrocardiogram, increased serum CK and lactate LDH levels were significantly observed in SUN groups. Histology staining showed that SUN caused structural injuries and fibrosis deposition. Moreover, SUN increased the level of MDA and Fe2+ content, decreased the level of GSH. Both in vitro and in vivo experiments indicated that SUN reduced the expression of Nrf2, HO-1, NQO1, GPX4 and FTH1, enhanced the TfR expression. This study suggested that oxidative stress and Nrf2-dependent ferroptosis played a vital role in SUN-induced cardiotoxicity.

Exploration and Validation of Potential Biomarkers and Therapeutic Targets in Ferroptosis of Asthma.

Purpose: Asthma is a chronic inflammatory airway disease involving multiple mechanisms, of which ferroptosis is a form of programmed cell death. Recent studies have shown that ferroptosis may play a crucial role in the pathogenesis of asthma, but no specific ferroptosis gene has been found in asthma, and the exact mechanism is still unclear. The present study aimed to screen ferroptosis genes associated with asthma and find therapeutic targets, in order to contribute a new clue for the diagnosis and therapy of asthma. Methods: Ferroptosis-related differentially expressed genes (FR-DEGs) in asthma were selected by the GSE41861, GSE43696 and ferroptosis datasets. Next, the FR-DEGs were subjected by GO and KEGG enrichment, and the mRNA-miRNA network was constructed. Then, GSEA and GSVA enrichment analysis and Immune infiltration analysis were performed, followed by targeted drug prediction. Finally, the expression of FR-DEGs was confirmed using GSE63142 dataset and RT-PCR assay. Results: We found 13 FR-DEGs by the GSE41861, GSE43696 and ferroptosis database. Functional enrichment analysis revealed that the 13 FR-DEGs were enriched in oxidative stress, immune response, ferroptosis, lysosome, necrosis, apoptosis etc. Moreover, our results revealed the mRNA-miRNA network of the FR-DEGs and identified candidate drugs. Also, immune infiltration revealed that ELAVL1, CREB5, CBR1 and NR1D2 are associated with the immune cells and may be potential targets in asthma. Finally, 10 FR-DEGs were validated by the GSE63142 database. It was verified that 7 FR-DEGs were differentially expressed by collecting asthma patients and healthy controls. Conclusion: This study ultimately identified 7 FR-DEGs for the diagnosis and therapy of asthma. These 7 FR-DEGs contribute to oxidative stress and immune responses. This study provides potential therapeutic targets and biomarkers for asthma patients, shedding further light on the pathogenesis of asthma as well as providing new insights into the treatment of asthma.

Predictors of COVID-19 Severity in Elderly Patients Infected by Omicron in China, 18 December 2022-5 February 2023.

Purpose: To analyze the clinical characteristics and prognosis of patients hospitalized with non-severe, severe pneumonia and death in Omicron COVID-19. Patients and Methods: We collected clinical data from 118 patients with COVID-19 in China from 18 December, 2022 and 5 February, 2023. According to the outcome, the patients were divided into non-severe group, severe group and death group. Subsequently, we statistically analyzed the general condition, clinical manifestations, laboratory parameters, NLR, MLR, PLR and HALP of these groups. We also retrospectively analyzed the possible factors affecting the prognostic regression of patients with COVID-19. Results: A total of 118 COVID-19 patients were enrolled in this study, including 64 non-severe patients, 38 severe patients and 16 death patients. Compared with the non-severe group, T lymphocytes, B lymphocytes, Th1, Th2, Th17, Treg cells, IgA, IgG, IgM in the severe and death groups decreased more significantly (P<0.05). The levels of myocardial markers, ALT, AST, BUN, Cr, D-dimer, fibrinogen, NLR, MLR and PLR in the severe and death groups were significantly higher than those in the non-severe group (P<0.05). The level of HALP was significantly lower than that of non-severe group (P<0.05). MLR is not only an independent risk factor for the transition from non-severe to severe disease, but also an independent risk factor for predicting the possibility of death in COVID-19 patients. Conclusion: The analysis of COVID-19 patients in China showed that severe patients were older, more likely to have related complications, lower lymphocyte count, liver and kidney function disorder, glucose and lipid metabolism disorders, myocardial injury, and abnormal coagulation function, suggesting the need for early anticoagulant therapy. In addition, NLR, MLR, PLR and HALP can be used as biomarkers to evaluate the severity and prognosis of COVID-19 patients.

Utilization of the GOAL questionnaire as a standardized screening tool for obstructive sleep apnea.

The purpose of our study was to evaluate the application value of the GOAL questionnaire in screening obstructive sleep apnea (OSA) and to compare it with the other three questionnaires in sleep clinics. A cross-sectional study was conducted in 436 patients who had undergone nocturnal polysomnography in the sleep unit of the First Hospital of Shanxi Medical University between September 2021 and May 2022, and all patients completed the four questionnaires (GOAL questionnaire, STOP-Bang questionnaire, NoSAS score and No-Apnea score) truthfully, and the patients were divided into 3 groups: AHI ≥ 5 events/h group, AHI ≥ 15 events/h group and AHI ≥ 30 events/h group. The predictive effect of the questionnaire on different AHI cut-off values was calculated, and performance of four questionnaires was assessed by the discriminatory ability. This study ultimately included 410 patients, and there were statistically significant differences in gender, age, BMI, neck circumference, clinical symptoms, hypertension, diabetes, AHI, and minimum oxygen saturation between OSA and non-OSA groups (P < 0.05). The AUC for No-Apnea score was 0.79, the AUC for STOP-Bang questionnaire was 0.86, the AUC for NoSAS score was 0.81, and the AUC for GOAL questionnaire was 0.77. These four questionnaires were effective in screening OSA when AHI ≥ 15 events/h. Similar to No-Apnea score, STOP-Bang score and NoSAS score, GOAL questionnaire has a good predictive value for OSA, which is a questionnaire suitable for primary health-care centers and clinics.

Tetramisole is a new IK1 channel agonist and exerts IK1 -dependent cardioprotective effects in rats.

Cardiac ischemia, hypoxia, arrhythmias, and heart failure share the common electrophysiological changes featured by the elevation of intracellular Ca2+ (Ca2+ overload) and inhibition of the inward rectifier potassium (IK1 ) channel. IK1 channel agonists have been considered a new type of anti-arrhythmia and cardioprotective agents. We predicted using a drug repurposing strategy that tetramisole (Tet), a known anthelminthic agent, was a new IK1 channel agonist. The present study aimed to experimentally identify the above prediction and further demonstrate that Tet has cardioprotective effects. Results of the whole-cell patch clamp technique showed that Tet at 1-100 µmol/L enhanced IK1 current, hyperpolarized resting potential (RP), and shortened action potential duration (APD) in isolated rat cardiomyocytes, while without effects on other ion channels or transporters. In adult Sprague-Dawley (SD) rats in vivo, Tet showed anti-arrhythmia and anticardiac remodeling effects, respectively, in the coronary ligation-induced myocardial infarction model and isoproterenol (Iso, i.p., 3 mg/kg/day, 10 days) infusion-induced cardiac remodeling model. Tet also showed anticardiomyocyte remodeling effect in Iso (1 µmol/L) infused adult rat ventricular myocytes or cultured H9c2 (2-1) cardiomyocytes. Tet at 0.54 mg/kg in vivo or 30 µmol/L in vitro showed promising protections on acute ischemic arrhythmias, myocardial hypertrophy, and fibrosis. Molecular docking was performed and identified the selective binding of Tet with Kir2.1. The cardioprotection of Tet was associated with the facilitation of IK1 channel forward trafficking, deactivation of PKA signaling, and inhibition of intracellular calcium overload. Enhancing IK1 may play dual roles in anti-arrhythmia and antiventricular remodeling mediated by restoration of Ca2+ homeostasis.

Establishment of a Suspension MDBK Cell Line in Serum-Free Medium for Production of Bovine Alphaherpesvirus-1.

The Madin-Darby bovine kidney (MDBK) cell line is currently used for the production of bovine alphaherpesvirus-1 (BoHV-1) vaccine. For the purpose of vaccine manufacturing, suspension cells are preferred over adherent ones due to simplified sub-cultivation and an easier scale-up process, both of which could significantly reduce production cost. This study aimed to establish a procedure for the culture of BoHV-1 in the suspended MDBK cell line in serum-free medium. We screened several commercially available serum-free media and chose ST503 for subsequent experiments. We successfully adapted the adherent MDBK cells to suspended growth in ST503 in the absence of serum. The maximum density of suspension-adapted MDBK cells could reach 2.5 × 107 cells/mL in ST503 medium with optimal conditions. The average size of suspension-adapted cells increased to 18 ± 1 µm from 16 ± 1 µm. Moreover, we examined tumorigenicity of the suspended cells and found no sign of tumorigenicity post adaptation. Next, we developed a protocol for the culture of BoHV-1 in the cell line described above and found that ultrasonic treatment could facilitate virus release and enhance virus yield by 11-fold, with the virus titer reaching 8.0 ± 0.2 log10TCID50/mL. Most importantly, the prototype inactivated BoHV-1 vaccine we generated using the suspension cultures of MDBK cells induced neutralizing antibodies to a titer comparable to that of the commercial inactivated BoHV-1 vaccine. Overall, we established and optimized a protocol for the production of inactivated BoHV-1 vaccine in MDBK cells adapted for suspension culture, which provides insights for future large-scale manufacturing of BoHV-1 vaccine.

Targeted inhibition of ß-catenin alleviates airway inflammation and remodeling in asthma via modulating the profibrotic and anti-inflammatory actions of transforming growth factor-ß1.

BACKGROUND: TGF-ß1 is a key cytokine involved in both airway inflammation and airway remodeling in asthma because of its anti-inflammatory and profibrotic effect. In our previous study, we found that knockdown of cytosolic ß-catenin alleviated the profibrogenic effect of TGF-ß1 without influencing its anti-inflammatory effect. However, the exact role of targeting ß-catenin in asthma is not yet fully demonstrated. In the present study, we investigated the effect and mechanism of targeting ß-catenin in OVA-challenged asthmatic rats with airway inflammation and remodeling features. METHODS: We integrated experimental asthma model and asthma related cell model to explore the effect of targeting ß-catenin on airway inflammation and remodeling of asthma. RESULTS: Blocking ß-catenin with ICG001, a small molecule inhibitor of ß-catenin/TCF via binding to cAMP-response elementbinding protein, attenuated airway inflammation by increasing levels of anti-inflammation cytokines IL-10, IL-35 and decreasing levels of T helper (Th)2 cells and Th17 cytokine. Suppressing ß-catenin by ICG001 inhibited airway remodeling via reducing the level of TGF-ß1 and the expressions of Snail, MMP-7, MMP-9 and, up-regulating expression of E-cadherin, down-regulating expressions of α-SMA and Fn. Inhibition of ß-catenin with ICG001 suppressed TGF-ß1 induced proliferation and activation of CCC-REPF-1, blocked TGF-ß1 induced epithelial-mesenchymal transition (EMT) of RLE-6TN. CONCLUSION: Blockade of ß-catenin/TCF not only prevents TGF-ß1 induced EMT and profibrogenic effects involved in pathological remodeling of airway, but also alleviates airway inflammation in asthma by balancing pro-inflammatory and anti-inflammatory cytokine. In conclusion, targeting ß-catenin specifically via inhibition of ß-catenin/TCF might be a new therapeutic strategy for asthma.The reviews of this paper are available via the supplemental material section.

1α, 25-dihydroxyvitamin D3 inhibits transforming growth factor ß1-induced epithelial-mesenchymal transition via ß-catenin pathway.

BACKGROUND: The transforming growth factor ß1 (TGF-ß1)-induced epithelial-mesenchymal transition (EMT) has been proven associated with the pathogenesis of asthmatic airway remodeling, in which the Wnt/ß-catenin pathway plays an important role, notably with regard to TGF-ß1. Recent studies have shown that 1α, 25-dihydroxyvitamin D3(1α, 25(OH)2D3) inhibits TGF-ß1-induced EMT, although the underlying mechanism have not yet been fully elucidated. METHODS: Alveolar epithelial cells were exposed to 1α, 25(OH)2D3, ICG-001, or a combination of both, followed by stimulation with TGF-ß1. The protein expression of E-cadherin, α-smooth muscle actin, fibronectin, and ß-catenin was analyzed by western blotting and immunofluorescence analysis. The mRNA transcript of Snail was analyzed using RT-qPCR, and matrix metalloproteinase 9 (MMP-9) activity was analyzed by gelatin zymogram. The activity of the Wnt/ß-catenin signaling pathway was analyzed using the Top/Fop flash reporters. RESULTS: Both 1α, 25(OH)2D3 and ICG-001 blocked TGF-ß1-induced EMT in alveolar epithelial cells. In addition, the Top/Fop Flash reporters showed that 1α, 25(OH)2D3 suppressed the activity of the Wnt/ß-catenin pathway and reduced the expression of target genes, including MMP-9 and Snail, in synergy with ICG-001. CONCLUSION: 1α, 25(OH)2D3 synergizes with ICG-001 and inhibits TGF-ß1-induced EMT in alveolar epithelial cells by negatively regulating the Wnt/ß-catenin signaling pathway.

Redirecting TGF-ß Signaling through the ß-Catenin/Foxo Complex Prevents Kidney Fibrosis.

TGF-ß is a key profibrotic factor, but targeting TGF-ß to prevent fibrosis also abolishes its protective anti-inflammatory effects. Here, we investigated the hypothesis that we can redirect TGF-ß signaling by preventing downstream profibrotic interaction of ß-catenin with T cell factor (TCF), thereby enhancing the interaction of ß-catenin with Foxo, a transcription factor that controls differentiation of TGF-ß induced regulatory T cells (iTregs), and thus, enhance anti-inflammatory effects of TGF-ß In iTregs derived from EL4 T cells treated with recombinant human TGF-ß1 (rhTGF-ß1) in vitro, inhibition of ß-catenin/TCF transcription with ICG-001 increased Foxp3 expression, interaction of ß-catenin and Foxo1, binding of Foxo1 to the Foxp3 promoter, and Foxo transcriptional activity. Moreover, the level of ß-catenin expression positively correlated with the level of Foxo1 binding to the Foxp3 promoter and Foxo transcriptional activity. T cell fate mapping in Foxp3gfp Ly5.1/5.2 mice revealed that coadministration of rhTGF-ß1 and ICG-001 further enhanced the expansion of iTregs and natural Tregs observed with rhTGF-ß1 treatment alone. Coadministration of rhTGF-ß1 with ICG-001 also increased the number of Tregs and reduced inflammation and fibrosis in the kidney fibrosis models of unilateral ureteric obstruction and ischemia-reperfusion injury. Notably, ICG-001 prevented the fibrosis in distant organs (lung and liver) caused by rhTGF-ß1. Together, our results show that diversion of ß-catenin from TCF- to Foxo-mediated transcription inhibits the ß-catenin/TCF-mediated profibrotic effects of TGF-ß while enhancing the ß-catenin/Foxo-mediated anti-inflammatory effects. Targeting ß-catenin/Foxo may be a novel therapeutic strategy in the treatment of fibrotic diseases that lead to organ failure.

Bacillus Calmette-Guerin alleviates airway inflammation and remodeling by preventing TGF-ß1 induced epithelial-mesenchymal transition.

Bacillus Calmette-Guerin (BCG) is a potent agent for the prevention of tuberculosis. Current studies have regarded BCG as an immunomodulator. However, there is little information on whether it can be used to inhibit airway inflammation and airway remodeling caused by asthma. Therefore, in this study, we investigate the role of epithelial-mesenchymal transition (EMT) in airway inflammation and airway remodeling as well as the possible therapeutic mechanism of BCG for the treatment of asthma. Wistar rats were sensitized and challenged by ovalbumin for 2 weeks or 8 weeks. BCG was subcutaneously administered daily before every ovalbumin challenge to determine its therapeutic effects. The 2 weeks model group showed extensive eosinophilia, chronic inflammatory responses, bronchial wall thickening, airway epithelium damage, increased levels of transforming growth factor ß 1 (TGF-ß1) in both bronchoalveolar lavage fluid and sera, decreased expression of epithelial marker E-cadherin, and increased expressions of mesenchymal markers α-smooth muscle actin (α-SMA) and Fibronectin (Fn). Except for inflammatory responses, all responses were more significant in the 8 weeks model group which displayed characteristics of airway remodeling including subepithelial fibrosis, smooth muscle hypertrophy, and goblet cell hyperplasia. When compared with the model groups, BCG administration inhibited airway inflammation and airway remodeling, decreased TGF-ß1 levels, upregulated expression of E-cadherin, and downregulated expression of α-SMA and Fn. The present study suggests for the first time that increased secretion of TGF- ß1 induced by asthmatic chronic inflammation may result in EMT, which is one of the most important mechanisms of airway inflammation and airway remodeling seen with asthma. BCG alleviates airway inflammation and airway remodeling by preventing TGF-ß1 induced EMT, therefore BCG may be a new therapy for treating asthma.

α3 Integrin of Cell-Cell Contact Mediates Kidney Fibrosis by Integrin-Linked Kinase in Proximal Tubular E-Cadherin Deficient Mice.

Loss of E-cadherin marks a defect in epithelial integrity and polarity during tissue injury and fibrosis. Whether loss of E-cadherin plays a causal role in fibrosis is uncertain. α3ß1 Integrin has been identified to complex with E-cadherin in cell-cell adhesion, but little is known about the details of their cross talk. Herein, E-cadherin gene (Cdh1) was selectively deleted from proximal tubules of murine kidney by Sglt2Cre. Ablation of E-cadherin up-regulated α3ß1 integrin at cell-cell adhesion. E-cadherin-deficient proximal tubular epithelial cell displayed enhanced transforming growth factor-ß1-induced α-smooth muscle actin (α-SMA) and vimentin expression, which was suppressed by siRNA silencing of α3 integrin, but not ß1 integrin. Up-regulation of transforming growth factor-ß1-induced α-SMA was mediated by an α3 integrin-dependent increase in integrin-linked kinase (ILK). Src phosphorylation of ß-catenin and consequent p-ß-catenin-Y654/p-Smad2 transcriptional complex underlies the transcriptional up-regulation of ILK. Kidney fibrosis after unilateral ureteric obstruction or ischemia reperfusion was increased in proximal tubule E-cadherin-deficient mice in comparison to that of E-cadherin intact control mice. The exacerbation of fibrosis was explained by the α3 integrin-dependent increase of ILK, ß-catenin nuclear translocation, and α-SMA/proximal tubular-specific Cre double positive staining in proximal tubular epithelial cell. These studies delineate a nonconventional integrin/ILK signaling by α3 integrin-dependent Src/p-ß-catenin-Y654/p-Smad2-mediated up-regulation of ILK through which loss of E-cadherin leads to kidney fibrosis.

Japonicone A inhibits the growth of non-small cell lung cancer cells via mitochondria-mediated pathways.

Japonicone A, which is a natural product isolated from the aerial part of Inula japonica Thunb., has a wide range of clinical applications, including anti-inflammation and anti-oxidation. This study investigated the effects of japonicone A on the growth of non-small cell lung cancer (NSCLC) cell lines. The results showed that japonicone A significantly inhibited the growth of NSCLC cell lines in a dose- and time-dependent manner. This product also blocked cell cycle progression at S phase and induced mitochondrial-related apoptosis by upregulating Bax, cleaved caspase-9, cleaved caspase-3, and cleaved poly(ADP-ribose) polymerase (PARP) protein levels and by downregulating Bcl-2, cyclin D1, CDC25A, and CDK2 protein levels. In vivo, japonicone A suppressed tumor growth via the same mechanism as that observed in vitro. In conclusion, our study is the first to report that japonicone A has an inhibitory effect on the growth of NSCLC cells, indicating that japonicone A administration is a potential therapeutic approach for future NSCLC treatments.

Baicalin inhibits PDGF-induced proliferation and migration of airway smooth muscle cells.

Airway smooth muscle (ASM) cell proliferation and migration play important roles in airway remodeling in asthma. In vitro platelet-derived growth factor (PDGF) induced ASM cell proliferation and migration. Baicalin is one of flavonoid extracts from Scutellaria baicalensis, which has an anti-asthma effect. However, little is known about its role in PDGF-induced proliferation and migration in rat ASM (RASM) cells. In this study, we aimed to investigate the effects of baicalin on PDGF-induced RASM cell proliferation and migration. We also identified the signaling pathway by which baicalin influences RASM cell proliferation and migration. In the current study, we demonstrated that baicalin suppressed PDGF-induced RASM cell proliferation, arrested PDGF-induced cell-cycle progression. It also suppressed PDGF-induced RASM cell migration. Furthermore, baicalin suppressed PDGF-induced expression of phosphorylated p38, ERK1/2 and JNK in RASM cells. In summary, our study is the first to show that baicalin pretreatment can significantly inhibit PDGF-induced RASM cell proliferation and migration by suppressing the MAPK signaling pathway, and baicalin may be a useful chemotherapeutic agent for asthma.

[Therapeutic effects of Bacille Calmette-Guerin vaccine on asthma airway remodeling in rats and its mechanism].

OBJECTIVE: To explore the therapeutic effects and mechanism of Bacillus Calmette-Guerin (BCG) vaccine on airway remodeling in rats. METHODS: Forty male Wistar rats were randomly divided into 5 groups of control, asthma,BCG vaccine, dexamethasone and BCG vaccine plus dexamethasone (n = 8 each). The animals were then sensitized and challenged by ovalbum in to establish the asthmatic model. A subcutaneous injection of BCG vaccine 0.025 mg was administered for the BCG vaccine group and an intraperitoneal injection of dexamethasone 0.5 mg/kg for the dexamethasone group.In BCG vaccine plus dexamethasone group, the rats received a subcutaneous injection of BCG vaccine (0.025 mg) plus an intraperitoneal injection of dexamethasone (0.25 mg/kg). All treatments were offered at half an hour pre-atomization. The control rats received an aerosol inhalation of normal saline instead of ovalbum. The parameters of airway morphological changes and the degree of airway remodeling were analyzed with computer graphics. The levels of transforming growth factor beta 1 (TGF-ß1) in bronchoalveolar lavage fluid (BALF) and the expressions of E-cadherin, α-smooth muscle actin (α-SMA) and fibronectin in lung tissue and sera were detected. RESULTS: In asthmatic rats, the thickness of airway wall and smooth muscle were more significant than those of the control group ((95.01 ± 0.48), (43.86 ± 0.51) vs (25.96 ± 0.42), (15.14 ± 0.18) µm). Compared with the control group, the levels of TGF-ß1 in BALF and sera were significantly higher ((10.05 ± 0.26), (75.67 ± 1.17) vs (1.53 ± 0.18), (22.24 ± 0.35) µg/L), the expression of E-cadherin significantly decreased (0.26 ± 0.03 vs 0.45 ± 0.04), while α-SMA and fibronectin significantly increased (0.54 ± 0.06,0.56 ± 0.06 vs 0.32 ± 0.04, 0.35 ± 0.06) (all P < 0.01); Notably, compared with the asthmatic group, the thickness of airway wall and smooth muscle ((58.46 ± 2.43),(49.51 ± 1.44), (49.63 ± 1.42) and (25.84 ± 0.54), (25.44 ± 0.40), (25.62 ± 1.17) µm) significantly decreased by the treatments of BCG vaccine, dexamethasone or BCG vaccine plus dexamethasone, the levels of TGF-ß1 in BALF and sera ((3.42 ± 0.18), (3.27 ± 0.34), (3.39 ± 0.26) and (37.61 ± 0.22), (35.65 ± 0.49), (36.22 ± 0.71) µg/L) significantly decreased, the expressions of E-cadherin (0.29 ± 0.04, 0.32 ± 0.04, 0.31 ± 0.03) significantly increased and α-SMA and fibronectin (0.40 ± 0.06, 0.35 ± 0.06, 0.40 ± 0.05 and 0.47 ± 0.03, 0.43 ± 0.06, 0.47 ± 0.04) significantly declined (all P < 0.01). Western blot showed the similar results. CONCLUSIONS: BCG vaccine alleviates airway epithelial cell injury and epithelial mesenchymal transition induced by TGF-ß1 through immunoregulation. It also reduces asthmatic airway remodeling with a combination of dexamethasone.

Matrix metalloproteinases contribute to kidney fibrosis in chronic kidney diseases.

Matrix metalloproteinases (MMPs) are members of the neutral proteinase family. They were previously thought to be anti-fibrotic because of their ability to degrade and remodel of extracellular matrix. However, recent studies have shown that MMPs are implicated in initiation and progression of kidney fibrosis through tubular cell epithelial-mesenchymal transition (EMT) as well as activation of resident fibroblasts, endothelial-mesenchymal transition (EndoMT) and pericyte-myofibroblast transdifferentiation. Interstitial macrophage infiltration has also been shown to correlate with the severity of kidney fibrosis in various chronic kidney diseases. MMPs secreted by macrophages, especially MMP-9, has been shown by us to be profibrotic by induction of tubular cells EMT. EMT is mainly induced by transforming growth factor-ß (TGF-ß). However, MMP-9 was found by us and others to be up-regulated by TGF-ß1 in kidney tubular epithelial cells and secreted by activated macrophages, resulting in EMT and ultimately kidney fibrosis. Therefore, MMP-9 may serve as a potential therapeutic target to prevent kidney fibrosis in chronic kidney disease. This review, by a particular focus on EMT, seeks to provide a comprehensive understanding of MMPs, especially MMP-9, in kidney fibrosis.

Matrix metalloproteinase-9 of tubular and macrophage origin contributes to the pathogenesis of renal fibrosis via macrophage recruitment through osteopontin cleavage.

A pro-fibrotic role of matrix metalloproteinase-9 (MMP-9) in tubular cell epithelial-mesenchymal transition (EMT) is well established in renal fibrosis; however studies from our group and others have demonstrated some previously unrecognized complexity of MMP-9 that has been overlooked in renal fibrosis. Therefore, the aim of this study was to determine the expression pattern, origin and the exact mechanism underlying the contribution of MMP-9 to unilateral ureteral obstruction (UUO), a well-established model of renal fibrosis via MMP-9 inhibition. Renal MMP-9 expression in BALB/c mice with UUO was examined on day 1, 3, 5, 7, 9, 11 and 14. To inhibit MMP-9 activity, MMP-2/9 inhibitor or MMP-9-neutralizing antibody was administered daily for 4 consecutive days from day 0-3, 6-9 or 10-13 and tissues harvested at day 14. In UUO, there was a bi-phasic early- and late-stage upregulation of MMP-9 activity. Interestingly, tubular epithelial cells (TECs) were the predominant source of MMP-9 during early stage, whereas TECs, macrophages and myofibroblasts produced MMP-9 during late-stage UUO. Early- and late-stage inhibition of MMP-9 in UUO mice significantly reduced tubular cell EMT and renal fibrosis. Moreover, MMP-9 inhibition caused a significant reduction in MMP-9-cleaved osteopontin and macrophage infiltration in UUO kidney. Our in vitro study showed MMP-9-cleaved osteopontin enhanced macrophage transwell migration and MMP-9 of both primary TEC and macrophage induced tubular cell EMT. In summary, our result suggests that MMP-9 of both TEC and macrophage origin may directly or indirectly contribute to the pathogenesis of renal fibrosis via osteopontin cleavage, which, in turn further recruit macrophage and induce tubular cell EMT. Our study also highlights the time dependency of its expression and the potential of stage-specific inhibition strategy against renal fibrosis.

Association of ß-catenin with P-Smad3 but not LEF-1 dissociates in vitro profibrotic from anti-inflammatory effects of TGF-ß1.

Transforming growth factor ß1 (TGF-ß1) is known to be both anti-inflammatory and profibrotic. Cross-talk between TGF-ß/Smad and Wnt/ß-catenin pathways in epithelial-mesenchymal transition (EMT) suggests a specific role for ß-catenin in profibrotic effects of TGF-ß1. However, no such mechanistic role has been demonstrated for ß-catenin in the anti-inflammatory effects of TGF-ß1. In the present study, we explored the role of ß-catenin in the profibrotic and anti-inflammatory effects of TGF-ß1 by using a cytosolic, but not membrane, ß-catenin knockdown chimera (F-TrCP-Ecad) and the ß-catenin/CBP inhibitor ICG-001. TGF-ß1 induced nuclear Smad3/ß-catenin complex, but not ß-catenin/LEF-1 complex or TOP-flash activity, during EMT of C1.1 (renal tubular epithelial) cells. F-TrCP-Ecad selectively degraded TGF-ß1-induced cytoplasmic ß-catenin and blocked EMT of C1.1 cells. Both F-TrCP-Ecad and ICG-001 blocked TGF-ß1-induced Smad3/ß-catenin and Smad reporter activity in C1.1 cells, suggesting that TGF-ß1-induced EMT depends on ß-catenin binding to Smad3, but not LEF-1 downstream of Smad3, through canonical Wnt. In contrast, in J774 macrophages, the ß-catenin level was low and was not changed by interferon-γ (IFN-γ) or lipopolysaccharide (LPS) with or without TGF-ß1. TGF-ß1 inhibition of LPS-induced TNF-α and IFN-γ-stimulated inducible NO synthase (iNOS) expression was not affected by F-TrCP-Ecad, ICG-001 or by overexpression of wild-type ß-catenin in J774 cells. Inhibition of ß-catenin by either F-TrCP-Ecad or ICG-001 abolished LiCl-induced TOP-flash, but not TGF-ß1-induced Smad reporter, activity in J774 cells. These results demonstrate for the first time that ß-catenin is required as a co-factor of Smad in TGF-ß1-induced EMT of C1.1 epithelial cells, but not in TGF-ß1 inhibition of macrophage activation. Targeting ß-catenin may dissociate the TGF-ß1 profibrotic and anti-inflammatory effects.

E-cadherin/ß-catenin complex and the epithelial barrier.

E-Cadherin/ß-catenin complex plays an important role in maintaining epithelial integrity and disrupting this complex affect not only the adhesive repertoire of a cell, but also the Wnt-signaling pathway. Aberrant expression of the complex is associated with a wide variety of human malignancies and disorders of fibrosis resulting from epithelial-mesenchymal transition. These associations provide insights into the complexity that is likely responsible for the fibrosis/tumor suppressive action of E-cadherin/ß-catenin.

Inhibition of allergic airway inflammation by antisense-induced blockade of STAT6 expression.

BACKGROUND: The signal transducer and activator of transcription 6 (STAT6) expression in lung epithelial cells plays a pivotal role in asthma pathogenesis. Activation of STAT6 expression results in T helper cell type 2 (Th2) cell differentiation leading to Th2-mediated IgE production, development of allergic airway inflammation and hyperreactivity. Therefore, antagonizing the expression and/or the function of STAT6 could be used as a mode of therapy for allergic airway inflammation. METHODS: In this study, we synthesized a 20-mer phosphorothioate antisense oligonucleotide (ASODN) overlapping the translation starting site of STAT6 and constructed STAT6 antisense RNA (pANTI-STAT6), then transfected them into murine spleen lymphocytes and analyzed the effects of antagonizing STAT6 function in vitro and in a murine model of asthma. RESULTS: In vitro, we showed suppression of STAT6 expression and interleukin (IL)-4 production of lymphocytes by STAT6 ASODN. This effect was more prominent when cells were cultured with pANTI-STAT6. In a murine model of asthma associated with allergic pulmonary inflammation in ovalbumin (OVA)-sensitized mice, local intranasal administration of fluorescein isothiocyanate (FITC)-labeled STAT6 ASODN to DNA uptake in lung cells was accompanied by a reduction of intracellular STAT6 expression. Such intrapulmonary blockade of STAT6 expression abrogated signs of lung inflammation, infiltration of eosinophils and Th2 cytokine production. CONCLUSION: These data suggest a critical role of STAT6 in the pathogenesis of asthma and the use of local delivery of STAT6 ASODN as a novel approach for the treatment of allergic airway inflammation such as in asthma.

Macrophage matrix metalloproteinase-9 mediates epithelial-mesenchymal transition in vitro in murine renal tubular cells.

As a rich source of pro-fibrogenic growth factors and matrix metalloproteinases (MMPs), macrophages are well-placed to play an important role in renal fibrosis. However, the exact underlying mechanisms and the extent of macrophage involvement are unclear. Tubular cell epithelial-mesenchymal transition (EMT) is an important contributor to renal fibrosis and MMPs to induction of tubular cell EMT. The aim of this study was to investigate the contribution of macrophages and MMPs to induction of tubular cell EMT. The murine C1.1 tubular epithelial cell line and primary tubular epithelial cells were cultured in activated macrophage-conditioned medium (AMCM) derived from lipopolysaccharide-activated J774 macrophages. MMP-9, but not MMP-2 activity was detected in AMCM. AMCM-induced tubular cell EMT in C1.1 cells was inhibited by broad-spectrum MMP inhibitor (GM6001), MMP-2/9 inhibitor, and in AMCM after MMP-9 removal by monoclonal Ab against MMP-9. AMCM-induced EMT in primary tubular epithelial cells was inhibited by MMP-2/9 inhibitor. MMP-9 induced tubular cell EMT in both C1.1 cells and primary tubular epithelial cells. Furthermore, MMP-9 induced tubular cell EMT in C1.1 cells to an extent similar to transforming growth factor-beta. Transforming growth factor-beta-induced tubular cell EMT in C1.1 cells was inhibited by MMP-2/9 inhibitor. Our in vitro study provides evidence that MMPs, specifically MMP-9, secreted by effector macrophages can induce tubular cell EMT and thereby contribute to renal fibrosis.