Urban airborne PM2.5 induces pulmonary fibrosis through triggering glycolysis and subsequent modification of histone lactylation in macrophages.

Airborne fine particulate matter (PM2.5) can cause pulmonary inflammation and even fibrosis, however, the underlying molecular mechanisms of the pathogenesis of PM2.5 exposure have not been fully appreciated. In the present study, we explored the dynamics of glycolysis and modification of histone lactylation in macrophages induced by PM2.5-exposure in both in vivo and in vitro models. Male C57BL/6 J mice were anesthetized and administrated with PM2.5 by intratracheal instillation once every other day for 4 weeks. Mouse RAW264.7 macrophages and alveolar epithelial MLE-12 cells were treated with PM2.5 for 24 h. We found that PM2.5 significantly increased lactate dehydrogenase (LDH) activities and lactate contents, and up-regulated the mRNA expression of key glycolytic enzymes in the lungs and bronchoalveolar lavage fluids of mice. Moreover, PM2.5 increased the levels of histone lactylation in both PM2.5-exposed lungs and RAW264.7 cells. The pro-fibrotic cytokines secreted from PM2.5-treated RAW264.7 cells triggered epithelial-mesenchymal transition (EMT) in MLE-12 cells through activating transforming growth factor-ß (TGF-ß)/Smad2/3 and VEGFA/ERK pathways. In contrast, LDHA inhibitor (GNE-140) pretreatment effectively alleviated PM2.5-induced pulmonary inflammation and fibrosis via inhibiting glycolysis and subsequent modification of histone lactylation in mice. Thus, our findings suggest that PM2.5-induced glycolysis and subsequent modification of histone lactylation play critical role in the PM2.5-associated pulmonary fibrosis.

Ultra-sensitive analysis of exhaled biomarkers in ozone-exposed mice via PAI-TOFMS assisted with machine learning algorithms.

Ground-level ozone ranks sixth among common air pollutants. It worsens lung diseases like asthma, emphysema, and chronic bronchitis. Despite recent attention from researchers, the link between exhaled breath and ozone-induced injury remains poorly understood. This study aimed to identify novel exhaled biomarkers in ozone-exposed mice using ultra-sensitive photoinduced associative ionization time-of-flight mass spectrometry and machine learning. Distinct ion peaks for acetonitrile (m/z 42, 60, and 78), butyronitrile (m/z 70, 88, and 106), and hydrogen sulfide (m/z 35) were detected. Integration of tissue characteristics, oxidative stress-related mRNA expression, and exhaled breath condensate free-radical analysis enabled a comprehensive exploration of the relationship between ozone-induced biological responses and potential biomarkers. Under similar exposure levels, C57BL/6 mice exhibited pulmonary injury characterized by significant inflammation, oxidative stress, and cardiac damage. Notably, C57BL/6 mice showed free radical signals, indicating a distinct susceptibility profile. Immunodeficient non-obese diabetic Prkdc-/-/Il2rg-/- (NPI) mice exhibited minimal biological responses to pulmonary injury, with little impact on the heart. These findings suggest a divergence in ozone-induced damage pathways in the two mouse types, leading to alterations in exhaled biomarkers. Integrating biomarker discovery with comprehensive biopathological analysis forms a robust foundation for targeted interventions to manage health risks posed by ozone exposure.

Downregulation of nutrition sensor GCN2 in macrophages contributes to poor wound healing in diabetes.

Poor skin wound healing, which is common in patients with diabetes, is related to imbalanced macrophage polarization. Here, we find that nutrition sensor GCN2 (general control nonderepressible 2) and its downstream are significantly upregulated in human skin wound tissue and mouse skin wound macrophages, but skin wound-related GCN2 expression and activity are significantly downregulated by diabetes and hyperglycemia. Using wound healing models of GCN2-deleted mice, bone marrow chimeric mice, and monocyte-transferred mice, we show that GCN2 deletion in macrophages significantly delays skin wound healing compared with wild-type mice by altering M1 and M2a/M2c polarization. Mechanistically, GCN2 inhibits M1 macrophages via OXPHOS-ROS-NF-κB pathway and promotes tissue-repairing M2a/M2c macrophages through eukaryotic translation initiation factor 2 (eIF2α)-hypoxia-inducible factor 1α (HIF1α)-glycolysis pathway. Importantly, local supplementation of GCN2 activator halofuginone efficiently restores wound healing in diabetic mice with re-balancing M1 and M2a/2c polarization. Thus, the decreased macrophage GCN2 expression and activity contribute to poor wound healing in diabetes and targeting GCN2 improves wound healing in diabetes.

Defining newly formed and tissue-resident bone marrow-derived macrophages in adult mice based on lysozyme expression.

Tissue-resident macrophages are derived from different precursor cells and display different phenotypes. Reconstitution of the tissue-resident macrophages of inflamed or damaged tissues in adults can be achieved by bone marrow-derived monocytes/macrophages. Using lysozyme (Lysm)-GFP-reporter mice, we found that alveolar macrophages (AMs), Kupffer cells, red pulp macrophages (RpMacs), and kidney-resident macrophages were Lysm-GFP-, whereas all monocytes in the fetal liver, adult bone marrow, and blood were Lysm-GFP+. Donor-derived Lysm-GFP+ resident macrophages gradually became Lysm-GFP- in recipients and developed gene expression profiles characteristic of tissue-resident macrophages. Thus, Lysm may be used to distinguish newly formed and long-term surviving tissue-resident macrophages that were derived from bone marrow precursor cells in adult mice under pathological conditions. Furthermore, we found that Irf4 might be essential for resident macrophage differentiation in all tissues, while cytokine and receptor pathways, mTOR signaling pathways, and fatty acid metabolic processes predominantly regulated the differentiation of RpMacs, Kupffer cells, and kidney macrophages, respectively. Deficiencies in ST2, mechanistic target of rapamycin (mTOR) and fatty acid-binding protein 5 (FABP5) differentially impaired the differentiation of tissue-resident macrophages from bone marrow-derived monocytes/macrophages in the lungs, liver, and kidneys. These results indicate that a combination of shared and unique signaling pathways coordinately shape tissue-resident macrophage differentiation in various tissues.

Dimethylarginine dimethylaminohydrolase 1 protects PM2.5 exposure-induced lung injury in mice by repressing inflammation and oxidative stress.

BACKGROUND: Airborne fine particulate matter with aerodynamic diameter ≤ 2.5 µm (PM2.5) pollution is associated with the prevalence of respiratory diseases, including asthma, bronchitis and chronic obstructive pulmonary disease. In patients with those diseases, circulating asymmetric dimethylarginine (ADMA) levels are increased, which contributes to airway nitric oxide deficiency, oxidative stress and inflammation. Overexpression of dimethylarginine dimethylaminohydrolase 1 (DDAH1), an enzyme degrading ADMA, exerts protective effects in animal models. However, the impact of DDAH1/ADMA on PM2.5-induced lung injury has not been investigated. METHODS: Ddah1-/- and DDAH1-transgenic mice, as well as their respective wild-type (WT) littermates, were exposed to either filtered air or airborne PM2.5 (mean daily concentration ~ 50 µg/m3) for 6 months through a whole-body exposure system. Mice were also acutely exposed to 10 mg/kg PM2.5 and/or exogenous ADMA (2 mg/kg) via intratracheal instillation every other day for 2 weeks. Inflammatory response, oxidative stress and related gene expressions in the lungs were examined. In addition, RAW264.7 cells were exposed to PM2.5 and/or ADMA and the changes in intracellular oxidative stress and inflammatory response were determined. RESULTS: Ddah1-/- mice developed more severe lung injury than WT mice after long-term PM2.5 exposure, which was associated with greater induction of pulmonary oxidative stress and inflammation. In the lungs of PM2.5-exposed mice, Ddah1 deficiency increased protein expression of p-p65, iNOS and Bax, and decreased protein expression of Bcl-2, SOD1 and peroxiredoxin 4. Conversely, DDAH1 overexpression significantly alleviated lung injury, attenuated pulmonary oxidative stress and inflammation, and exerted opposite effects on those proteins in PM2.5-exposed mice. In addition, exogenous ADMA administration could mimic the effect of Ddah1 deficiency on PM2.5-induced lung injury, oxidative stress and inflammation. In PM2.5-exposed macrophages, ADMA aggravated the inflammatory response and oxidative stress in an iNOS-dependent manner. CONCLUSION: Our data revealed that DDAH1 has a marked protective effect on long-term PM2.5 exposure-induced lung injury.

Exploring breath biomarkers in BLM-induced pulmonary fibrosis mice with associative ionization time-of-flight mass spectrometry.

Pulmonary fibrosis (PF) is a common but fatal disease that threatens human health worldwide. Developing effective diagnostic methods is of great importance for the early detection of PF in patients. In this study, bleomycin (BLM) was used in mice to mimic idiopathic pulmonary fibrosis (IPF). The exhaled breath of BLM-induced PF, PF plus DDAH1 overexpression, and healthy control mice were analyzed in real-time using a newly developed associative ionization time-of-flight mass spectrometry method (AI-TOFMS), which is uniquely sensitive, especially to oxygenated volatile organic compounds (VOCs). Multivariate data analyses and discriminant modeling analyses revealed that four exhaled compounds, i.e., acrolein, ethanol, nitric oxide, and ammonia, had a strong correlation with PF symptoms. An Orthogonal Partial Least Square Discriminant Analysis (OPLS-DA) score plot showed an excellent separation between these three groups. The area under the receiver operating characteristic (ROC) curve for these four compounds distinguished PF mice from healthy controls at 0.989. In addition, the degrees of acute inflammation and fibrosis were assessed with Hematoxylin and Eosin (H&E) staining and Masson's Trichrome staining. Finally, combined with pathological characteristics and mRNA expression levels, the formation of the above-mentioned volatile compounds was explored. The obtained experimental results indicated that these four breath compounds, acrolein, ethanol, nitric oxide, and ammonia, were potential exhaled biomarkers for pulmonary fibrosis.

Adipose-derived stem cells therapy effectively attenuates PM2.5-induced lung injury.

BACKGROUND: The adverse health effects of fine particulate matter (PM2.5) exposure are associated with marked inflammatory responses. Adipose-derived stem cells (ADSCs) have immunosuppressive effects, and ADSC transplantation could attenuate pulmonary fibrosis in different animal disease models. However, whether ADSCs affect PM2.5-induced lung injury has not been investigated. METHOD: C57BL/6 mice were exposed to PM2.5 every other day via intratracheal instillation for 4 weeks. After that, the mice received tail vein injections of ADSCs every 2 weeks. RESULTS: ADSC transplantation significantly attenuated systemic and pulmonary inflammation, cardiac dysfunction, fibrosis, and cell death in PM2.5-exposed mice. RNA-sequencing results and bioinformatic analysis suggested that the downregulated differentially expressed genes (DEGs) were mainly enriched in inflammatory and immune pathways. Moreover, ADSC transplantation attenuated PM2.5-induced cell apoptosis and pyroptosis in the lungs and hearts. CONCLUSION: ADSCs protect against PM2.5-induced adverse health effects through attenuating pulmonary inflammation and cell death. Our findings suggest that ADSC transplantation may be a potential therapeutic approach for severe air pollution-associated diseases.

Tempol ameliorates polycystic ovary syndrome through attenuating intestinal oxidative stress and modulating of gut microbiota composition-serum metabolites interaction.

Polycystic ovary syndrome (PCOS) is a complex endocrine and metabolic disorder, which is often accompanied by oxidative stress. Tempol, a superoxide dismutase mimetic, protects against several diseases caused by oxidative stress. However, the effect of tempol on PCOS has not been investigated. The present study demonstrated the alleviation of ovarian dysfunction and glucose tolerance in dehydroepiandrosterone (DHEA)-induced PCOS rats treated with tempol. Tempol significantly reduced the intestinal oxidative stress in PCOS rats without affecting the ovarian redox rate. The 16S rDNA sequencing of the intestinal microbiome and non-targeted metabolomics analysis indicated significant differences in gut microbiota composition and serum metabolite profiles between the control and PCOS rats, and most of these differences were reduced after tempol intervention. Tempol alters the gut microbiome by increasing the abundance of genus Ruminococcus_1 and by decreasing the abundance of Ruminococcus_2, Staphylococcus, Ideonella, and Corynebnacterium genera. Tempol also attenuates the reduction of serum bile acid and stachyose levels in PCOS rats, and the serum stachyose level was significantly correlated with the abundance of 15 genera, particularly Ruminococcus_1 and Ruminococcus_2. Moreover, stachyose administration improved ovarian dysfunction in PCOS rats. Thus, our data indicate that tempol ameliorates PCOS phenotype by reducing intestinal oxidative stress, restoring gut dysbiosis, and modulating the interaction between gut microbiota and host metabolite. Therefore, tempol intervention is a potential therapeutic approach for PCOS.

Metformin protects against PM2.5-induced lung injury and cardiac dysfunction independent of AMP-activated protein kinase α2.

Fine particulate matter (PM2.5) airborne pollution increases the risk of respiratory and cardiovascular diseases. Although metformin is a well-known antidiabetic drug, it also confers protection against a series of diseases through the activation of AMP-activated protein kinase (AMPK). However, whether metformin affects PM2.5-induced adverse health effects has not been investigated. In this study, we exposed wild-type (WT) and AMPKα2-/- mice to PM2.5 every other day via intratracheal instillation for 4 weeks. After PM2.5 exposure, the AMPKα2-/- mice developed more severe lung injury and cardiac dysfunction than were developed in the WT mice; however the administration of metformin was effective in attenuating PM2.5-induced lung injury and cardiac dysfunction in both the WT and AMPKα2-/- mice. In the PM2.5-exposed mice, metformin treatment resulted in reduced systemic and pulmonary inflammation, preserved left ventricular ejection fraction, suppressed induction of pulmonary and myocardial fibrosis and oxidative stress, and increased levels of mitochondrial antioxidant enzymes. Moreover, pretreatment with metformin significantly attenuated PM2.5-induced cell death and oxidative stress in control and AMPKα2-depleted BEAS-2B and H9C2 cells, and was associated with preserved expression of mitochondrial antioxidant enzymes. These data support the notion that metformin protects against PM2.5-induced adverse health effects through a pathway that appears independent of AMPKα2. Our findings suggest that metformin may also be a novel drug for therapies that treat air pollution associated disease.

The effect of exposure time and concentration of airborne PM2.5 on lung injury in mice: A transcriptome analysis.

The association between airborne fine particulate matter (PM2.5) concentration and the risk of respiratory diseases has been well documented by epidemiological studies. However, the mechanism underlying the harmful effect of PM2.5 has not been fully understood. In this study, we exposed the C57BL/6J mice to airborne PM2.5 for 3 months (mean daily concentration ~50 or ~110 µg/m3, defined as PM2.5-3L or PM2.5-3H) or 6 months (mean daily concentration ~50 µg/m3, defined as PM2.5-6L) through a whole-body exposure system. Histological and biochemical analysis revealed that PM2.5-3H exposure caused more severe lung injury than did PM2.5-3L, and the difference was greater than that of PM2.5-6L vs PM2.5-3L exposure. With RNA-sequencing technique, we found that the lungs exposed with different concentration of PM2.5 have distinct transcriptional profiles. PM2.5-3H exposure caused more differentially expressed genes (DEGs) in lungs than did PM2.5-3L or PM2.5-6L. The DEGs induced by PM2.5-3L or PM2.5-6L exposure were mainly enriched in immune pathways, including Hematopoietic cell lineage and Cytokine-cytokine receptor interaction, while the DEGs induced by PM2.5-3H exposure were mainly enriched in cardiovascular disease pathways, including Hypertrophic cardiomyopathy and Dilated cardiomyopathy. In addition, we found that upregulation of Cd5l and reduction of Hspa1 and peroxiredoxin-4 was associated with PM2.5-induced pulmonary inflammation and oxidative stress. These results may provide new insight into the cytotoxicity mechanism of PM2.5 and help to development of new strategies to attenuate air pollution associated respiratory disease.

Indirect effect of PM1 on endothelial cells via inducing the release of respiratory inflammatory cytokines.

A large number of epidemiological studies have shown that increased cardiovascular morbidity and mortality are associated with exposure to high concentrations of PM2.5. One of the ways that PM2.5 affects the cardiovascular system is through systemic inflammation. Inflammatory cytokines such as TNF-α, IL-1ß, IL-6, and IL-8 stimulate endothelial cells, which leads to endothelial dysfunction. Compared with PM2.5, PM1 is smaller in size, has a larger surface area and absorbs more toxic substances such as heavy metals, organic compounds, and black carbon. However, the effect of PM1 on human health is less studied. Here, we used BEAS-2B cells and differentiated THP-1 cells to simulate epithelial cells and macrophages in the lung, respectively. The indirect effect of PM1 on endothelial cells was studied with a coculture model consisting of two cell lines (BEAS-2B cells and macrophages) in the top compartment and one cell line, human umbilical vein endothelial cells (EA.hy926), in the bottom compartment of a transwell plate. The results showed that PM1 could promote the release of inflammatory cytokines, including TNF-α and IL-6, from BEAS-2B cells and macrophages. In addition, PM1 upregulated ICAM-1 expression in EA.hy926 cells through TNF-α/NF-κB signaling pathways, promoting the adhesion of endothelial cells and monocytes, a key event in the initiation of atherosclerosis.

GCN2 deficiency protects against high fat diet induced hepatic steatosis and insulin resistance in mice.

Nonalcoholic fatty liver disease (NAFLD) is characterized by hepatic lipid deposition and oxidative stress. It has been demonstrated that general control nonderepressible 2 (GCN2) is required to maintain hepatic fatty acid homeostasis under conditions of amino acid deprivation. However, the impact of GCN2 on the development of NAFLD has not been investigated. In this study, we used Gcn2-/- mice to investigate the effect of GCN2 on high fat diet (HFD)-induced hepatic steatosis. After HFD feeding for 12 weeks, Gcn2-/- mice were less obese than wild-type (WT) mice, and Gcn2-/- significantly attenuated HFD-induced liver dysfunction, hepatic steatosis and insulin resistance. In the livers of the HFD-fed mice, GCN2 deficiency resulted in higher levels of lipolysis genes, lower expression of genes related to FA synthesis, transport and lipogenesis, and less induction of oxidative stress. Furthermore, we found that knockdown of GCN2 attenuated, whereas overexpression of GCN2 exacerbated, palmitic acid-induced steatosis, oxidative & ER stress, and changes of peroxisome proliferator-activated receptor gamma (PPARγ), fatty acid synthase (FAS) and metallothionein (MT) expression in HepG2 cells. Collectively, our data provide evidences that GCN2 deficiency protects against HFD-induced hepatic steatosis by inhibiting lipogenesis and reducing oxidative stress. Our findings suggest that strategies to inhibit GCN2 activity in the liver may provide a novel approach to attenuate NAFLD development.

AMPKα2 deficiency exacerbates long-term PM2.5 exposure-induced lung injury and cardiac dysfunction.

Previous studies have demonstrated that long-term exposure to fine particulate matter (PM2.5) increases the risk of respiratory and cardiovascular diseases. As a metabolic sensor, AMP-activated protein kinase (AMPK) is a promising target for cardiovascular disease. However, the impact of AMPK on the adverse health effects of PM2.5 has not been investigated. In this study, we exposed wild-type (WT) and AMPKα2-/- mice to either airborne PM2.5 (mean daily concentration ~64 µg/m3) or filtered air for 6 months through a whole-body exposure system. After exposure, AMPKα2-/- mice developed severe lung injury and left ventricular dysfunction. In the PM2.5-exposed lungs and hearts, loss of AMPKα2 resulted in higher levels of fibrotic genes, more collagen deposition, lower levels of peroxiredoxin 5 (Prdx5), and greater induction of oxidative stress and inflammation than observed in the lungs and hearts of WT mice. In PM2.5-exposed BEAS-2B and H9C2 cells, inhibition of AMPK activity significantly decreased cell viability and Prdx5 expression, and increased the intracellular ROS and p-NF-κB levels. Collectively, our results provide the first direct evidence that AMPK has a marked protective effect on the adverse health effects induced by long-term PM2.5 exposure. Our findings suggest that strategies to increase AMPK activity may provide a novel approach to attenuate air pollution associated disease.

GCN2 deficiency ameliorates doxorubicin-induced cardiotoxicity by decreasing cardiomyocyte apoptosis and myocardial oxidative stress.

The clinical use of doxorubicin for cancer therapy is limited by its cardiotoxicity, which involves cardiomyocyte apoptosis and oxidative stress. Previously, we showed that general control nonderepressible 2 (GCN2), an eukaryotic initiation factor 2α (eIF2α) kinase, impairs the ventricular adaptation to chronic pressure overload by affecting cardiomyocyte apoptosis. However, the impact of GCN2 on Dox-induced cardiotoxicity has not been investigated. In the present study, we treated wild type (WT) and Gcn2-/- mice with four intraperitoneal injections (5 mg/kg/week) to induce cardiomyopathy. After Dox treatment, Gcn2-/- mice developed less contractile dysfunction, myocardial fibrosis, apoptosis, and oxidative stress compared with WT mice. In the hearts of the Dox-treated mice, GCN2 deficiency attenuated eIF2α phosphorylation and induction of its downstream targets, activating transcription factor 4 (ATF4) and C/EBP homologous protein (CHOP), and preserved the expression of anti-apoptotic factor Bcl-2 and mitochondrial uncoupling protein-2(UCP2). Furthermore, we found that GCN2 knockdown attenuated, whereas GCN2 overexpression exacerbated, Dox-induced cell death, oxidative stress and reduction of Bcl-2 and UCP2 expression through the eIF2α-CHOP-dependent pathway in H9C2 cells. Collectively, our data provide solid evidence that GCN2 has a marked effect on Dox induced myocardial apoptosis and oxidative stress. Our findings suggest that strategies to inhibit GCN2 activity in cardiomyocyte may provide a novel approach to attenuate Dox-related cardiotoxicity.

YAP promotes breast cancer metastasis by repressing growth differentiation factor-15.

The transcriptional co-activator Yes-associated protein (YAP) has been implicated as an oncogene and is found to promote breast cancer metastasis. However, the pro-metastatic mechanism of YAP remains unclear. Here, we demonstrated that YAP functions as a transcriptional repressor of growth differentiation factor-15 (GDF15), a divergent member of the transforming growth factor superfamily, in several breast cancer cell lines. Functionally, knockdown of YAP decreased, whereas knockdown of GDF15 increased, the metastatic potential of breast cancer cells. More than that, the reduced metastasis in YAP-depleted cells could be reversed by simultaneous knockdown of GDF15. Mechanistically, the repressive effect of YAP on GDF15 requires its transcriptional factor TEAD (TEA domain family). In addition, YAP recruits polycomb repressive complex 2 (PRC2) to tri-methylate histone H3 lysine 27 in the promoter region of GDF15. Co-immunoprecipitation experiments demonstrated that YAP and enhancer of zeste 2 PRC2 subunit (EZH2) physically interact with each other. In conclusion, our data reveal that YAP promotes metastasis of breast cancer cells by repressing GDF15 transcription and present a novel molecular mechanism underlying the pro-metastasis function of YAP oncoprotein, with the implication of a therapeutic avenue for breast cancer treatment.

TMT-Based Quantitative Proteomics Analysis Reveals Airborne PM2.5-Induced Pulmonary Fibrosis.

Epidemiological and experimental studies have documented that long-term exposure to fine particulate matter (PM2.5) increases the risk of respiratory diseases. However, the details of the underlying mechanism remain unclear. In this study, male C57BL/6 mice were exposed to ambient PM2.5 (mean daily concentration ~64 µg/m³) for 12 weeks through a "real-world" airborne PM2.5 exposure system. We found that PM2.5 caused severe lung injury in mice as evidenced by histopathological examination. Then, tandem mass tag (TMT) labeling quantitative proteomic technology was performed to analyze protein expression profiling in the lungs from control and PM2.5-exposed mice. A total of 32 proteins were differentially expressed in PM2.5-exposed lungs versus the controls. Among these proteins, 24 and 8 proteins were up- and down-regulated, respectively. Gene ontology analysis indicated that PM2.5 exerts a toxic effect on lungs by affecting multiple biological processes, including oxidoreductase activity, receptor activity, and protein binding. Furthermore, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that extracellular matrix (ECM)⁻receptor interaction, phagosome, small cell lung cancer, and phosphatidylinositol 3-kinase(PI3K)-protein kinase B (Akt) signaling pathways contribute to PM2.5-induced pulmonary fibrosis. Taken together, these results provide a comprehensive proteomics analysis to further understanding of the molecular mechanisms underlying PM2.5-elicited pulmonary disease.

Dimethylarginine Dimethylaminohydrolase 1 Deficiency Induces the Epithelial to Mesenchymal Transition in Renal Proximal Tubular Epithelial Cells and Exacerbates Kidney Damage in Aged and Diabetic Mice.

AIMS: Asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase, is mainly degraded by dimethylarginine dimethylaminohydrolase (DDAH). Emerging evidence suggests that plasma ADMA accumulation and DDAH1 activity/expression reduction are linked to chronic kidney disease (CKD) pathology, but the mechanisms remain largely unknown. Here, we examined the role of ADMA/DDAH1 in the epithelial-mesenchymal transition (EMT) of tubular epithelial cells (TECs), an important mechanism for the pathogenesis of renal fibrosis. RESULTS: In HK-2 cells, DDAH1 expression was reduced by aldosterone treatment, and overexpression of DDAH1 significantly attenuated aldosterone-induced EMT. More interestingly, DDAH1 deficiency resulted in EMT-related changes in primary TECs via increasing oxidative stress, impairing adenosine monophosphate-activated kinase (AMPK) signaling, and downregulating of peroxiredoxin 5 (Prdx5). However, those effects could not be mimicked by increasing the ADMA concentration. After regular feeding for 24 months or inducing type 2 diabetes, Ddah1-/- mice had higher serum creatinine levels than wild-type (WT) mice. In the kidneys of the aged or diabetic mice, loss of DDAH1 resulted in more interstitial fibrosis, more collagen deposition, and greater induction of EMT-related changes and oxidative stress than in the WT kidneys. Innovation and Conclusion: Our results provide the first direct evidence that the DDAH1 has a marked effect on kidney fibrosis and oxidative stress induced by aging or diabetes. Our findings suggest that strategies to increase DDAH1 activity in TECs may provide a novel approach to attenuate CKD development. Antioxid. Redox Signal. 27, 1347-1360.

DDAH1 plays dual roles in PM2.5 induced cell death in A549 cells.

BACKGROUND: Dimethylarginine dimethylaminohydrolase 1 (DDAH1) is an enzyme that can degrade asymmetric dimethylarginine (ADMA), an endogenous nitric oxide synthase (NOS) inhibitor. Emerging evidence suggests that alterations in the ADMA-DDAH1 pathway are involved in environmental pollution induced airway inflammation. However, the role of DDAH1 in protection against cytotoxicity of ambient airborne particulate matter is unclear. METHODS: We examined the influence of DDAH1 expression on oxidative stress and cell apoptosis in human type II alveolar epithelial A549 cells exposed to PM2.5 (particulate matter with an aerodynamic diameter less than 2.5µM). RESULTS: We found that PM2.5 exposure for 48h significantly decreased DDAH1 expression. However, knockdown of DDAH1 prior to PM2.5 exposure actually attenuated the cytotoxicity of PM2.5. Cytoprotection in DDAH1 deficient cells was due to increased reactive oxygen species, activation of PI3K-AKT and mitogen-activated protein kinase (MAPK) pathways, subsequent activation of nuclear factor erythroid-2-related factor 2 (Nrf2) and this caused a subsequent reduction in PM2.5 induced oxidative stress relative to control. DDAH1 depletion also repressed the induction of inducible NOS (iNOS) in PM2.5-exposed cells and knockdown of iNOS protected cells against PM2.5 induced cell death. Interestingly, overexpression of DDAH1 also exerted a protective effect against the cytotoxicity of PM2.5 and this was associated with a reduction in oxidative stress and upregulation of the anti-apoptotic protein Bcl-2. CONCLUSIONS: Our data indicate that DDAH1 plays dual roles in protection against cytotoxicity of PM2.5 exposure, apparently by limiting PM2.5 induced oxidative stress. GENERAL SIGNIFICANCE: Our findings reveal new insights into the role(s) of the DDAH1/ADMA in pulmonary protection against airborne pollutants. This article is part of a Special Issue entitled Air Pollution, edited by Wenjun Ding, Andrew J. Ghio and Weidong Wu.

S-nitrosylation of PDE5 increases its ubiquitin-proteasomal degradation.

Phosphodiesterase type 5 (PDE5) expression is upregulated in human failing heart, and overexpression of PDE5 in transgenic mice exacerbates stress-induced left-ventricular dysfunction, suggesting that increased PDE5 expression might contribute to the development of congestive heart failure. However, the underlying mechanisms for increased PDE5 expression are not totally understood. In the present study, we found that PDE5 activity and expression were regulated by S-nitrosylation, a covalent modification of cysteine residues by nitric oxide (NO). S-nitrosylation of PDE5 occurs at Cys220, which is located in the GAFA domain. Upon S-nitrosylation, PDE5 exhibits reduced activity and degradation via the ubiquitin-proteasome system. The decrease in PDE5 expression induced by NO could be blunted by mutation of Cys220 or the phosphorylation site of PDE5 (S102), as well as by pretreatment with H2O2. Conversely, decreased NO bioavailability by nitric oxide synthase (NOS) inhibitors or knockout of NOS3 increased PDE5 expression in cardiomyocytes. Collectively, to the best of our knowledge, our data demonstrate for the first time that S-nitrosylation is one of the mechanisms for PDE5 degradation. This novel regulatory mechanism probably accounts for the increase in PDE5 in the failing heart and other diseases in which NO bioavailability is decreased by oxidative stress.