Epigenetic Reprogramming Drives Epithelial Disruption in Chronic Obstructive Pulmonary Disease.

Chronic obstructive pulmonary disease (COPD) remains a major public health challenge that contributes greatly to mortality and morbidity worldwide. Although it has long been recognized that the epithelium is altered in COPD, there has been little focus on targeting it to modify the disease course. Therefore, mechanisms that disrupt epithelial cell function in patients with COPD are poorly understood. In this study, we sought to determine whether epigenetic reprogramming of the cell-cell adhesion molecule E-cadherin, encoded by the CDH1 gene, disrupts epithelial integrity. By reducing these epigenetic marks, we can restore epithelial integrity and rescue alveolar airspace destruction. We used differentiated normal and COPD-derived primary human airway epithelial cells, genetically manipulated mouse tracheal epithelial cells, and mouse and human precision-cut lung slices to assess the effects of epigenetic reprogramming. We show that the loss of CDH1 in COPD is due to increased DNA methylation site at the CDH1 enhancer D through the downregulation of the ten-eleven translocase methylcytosine dioxygenase (TET) enzyme TET1. Increased DNA methylation at the enhancer D region decreases the enrichment of RNA polymerase II binding. Remarkably, treatment of human precision-cut slices derived from patients with COPD with the DNA demethylation agent 5-aza-2'-deoxycytidine decreased cell damage and reduced air space enlargement in the diseased tissue. Here, we present a novel mechanism that targets epigenetic modifications to reverse the tissue remodeling in human COPD lungs and serves as a proof of concept for developing a disease-modifying target.

TRPM7 channels regulate breathing during sleep in obesity by acting peripherally in the carotid bodies.

Sleep-disordered breathing (SDB) affects over 50% of obese individuals. Exaggerated hypoxic chemoreflex is a cardinal trait of SDB in obesity. We have shown that leptin acts in the carotid bodies (CB) to augment chemoreflex and that leptin activates the transient receptor potential melastatin 7 (TRPM7) channel. However, the effect of leptin-TRPM7 signalling in CB on breathing and SDB has not been characterized in diet-induced obesity (DIO). We hypothesized that leptin acts via TRPM7 in the CB to increase chemoreflex leading to SDB in obesity. DIO mice were implanted with EEG/EMG electrodes and transfected with Leprb short hairpin RNA (shRNA) or Trpm7 shRNA vs. control shRNA in the CB area bilaterally. Mice underwent a full-polysomnography and metabolic studies at baseline and after transfection. Ventilatory responses to hypoxia and hypercapnia were assessed during wakefulness. Leprb and Trpm7 were upregulated and their promoters were demethylated in the CB of DIO mice. Leprb knockdown in the CB did not significantly affect ventilation. Trpm7 knockdown in the CB stimulated breathing during sleep in normoxia. These effects were not driven by changes in CB chemosensitivity or metabolism. Under sustained hypoxia, Trpm7 shRNA in the CB augmented ventilation during sleep, but decreased oxyhaemoglobin saturation. We conclude that the suppression of TRPM7 in the CB improved sleep-related hypoventilation and that the respiratory effects of CB TRPM7 channels in obesity are independent of leptin. TRPM7 signalling in the CB could be a therapeutic target for the treatment of obesity-related SDB. KEY POINTS: The leptin-TRPM7 axis in the carotid bodies may play an important role in the pathogenesis of sleep-disordered breathing. TRPM7 channels regulate breathing during sleep by acting peripherally in the carotid bodies. Suppression of TRPM7 signalling in the carotid bodies improves the obesity-induced hypoventilation in mice. Pharmacological blockade of TRPM7 channels in the carotid bodies could be a therapy for sleep-disordered breathing in obesity.

Leptin Induces Epigenetic Regulation of Transient Receptor Potential Melastatin 7 in Rat Adrenal Pheochromocytoma Cells.

Obesity elevates the plasma level of leptin, which has been associated with hypertension. Our recent studies in mice demonstrated that leptin increases blood pressure by activating the carotid sinus nerve, which transmits the chemosensory input from carotid bodies (CBs) to the medullary centers, and that the effect of leptin is mediated via Trpm7 (TRP [transient receptor potential] melastatin 7) channels in CB glomus cells. We also found that Trpm7 overexpression and Trpm7 promoter demethylation in CBs correlate positively with the hyperleptinemia and leptin receptor overexpression in CBs. Hence, we postulated that leptin epigenetically regulates Trpm7 expression in CBs. We addressed our hypothesis by using rat adrenal pheochromocytoma (PC12) cells as a model of CB glomus cells. PC12 cells expressing LEPRb (long, active form of leptin receptor) showed dramatic induction of the promoter activity and expression of Trpm7 upon leptin treatment. The increased Trpm7 expression coincided with the reduction of CpG site-specific methylation and trimethylation of H3K27 (H3 [histone 3] K27 [lysine 27]) and the increase of acetylation of H3K27 and trimethylation of H3K4 (H3 lysine 4) at the Trpm7 promoter. The inhibitor of STAT3 (signal transducer and activator of transcription 3) signaling, SD1008, reversed the leptin-induced Trpm7 promoter activity via modulations of the binding of pSTAT3 (phosphorylated STAT3) and DNMT3B (DNA methyltransferase 3B) and modifications of H3K27 and H3K4 at the Trpm7 promoter. Our results suggest that leptin-activated pSTAT3 epigenetically regulates the transcription of Trpm7 through DNA methylation and histone modifications. Because epigenetic changes are reversible, targeting epigenetic modifications of Trpm7 may serve as a new therapeutic approach for the treatment of hypertension in obesity.

Prenatal exposure to mercury and precocious puberty: a prospective birth cohort study.

STUDY QUESTION: Is in utero exposure to mercury associated with the risk of precocious puberty? SUMMARY ANSWER: Prenatal exposure to high levels of mercury was associated with increased risk of precocious puberty, which was strengthened by concomitant maternal cardiometabolic conditions and adverse birth outcomes. WHAT IS KNOWN ALREADY: The developing fetus is sensitive to mercury, a well-known endocrine disruptor which impacts the endocrine and reproductive system. STUDY DESIGN, SIZE, DURATION: This study included 1512 mother-child pairs from the Boston Birth Cohort, a longitudinal cohort which recruited at birth and followed prospectively up to 21 years of age. PARTICIPANTS/MATERIALS, SETTING, METHODS: Mother-child pairs, from a predominantly urban minority population, were enrolled from 2002 to 2013. Prenatal exposure was assessed by maternal mercury concentration in red blood cells (RBCs) collected at 1-3 days after delivery. Precocious puberty was defined based on International Classification of Disease codes. Cox proportional hazards models were applied to the association between maternal mercury concentrations and the risk of precocious puberty. MAIN RESULTS AND THE ROLE OF CHANCE: The median (interquartile range) of maternal mercury concentrations among children with and without precocious puberty were 3.4 (1.9-4.6) µg/l and 2.0 (1.0-3.7) µg/l, respectively. Compared to those in the lowest tertile for mercury, the highest tertile was associated with increased risk of precocious puberty, with an adjusted hazard ratio (HR) of 2.41, 95% CI: 1.16-5.03. In addition, concomitant maternal cardiometabolic conditions and adverse birth outcomes strengthened the effects of mercury on the risk of precocious puberty. The highest risk of precocious puberty was observed among children who had adverse birth outcomes and whose mothers had high RBC-mercury concentrations along with cardiometabolic conditions, with an HR of 4.76 (95% CI: 1.66-13.60) compared to children with favorable profiles of all three risk factors. LIMITATIONS, REASONS FOR CAUTION: Precocious puberty was defined based on medical records, not on a direct assessment, which may have led to underdiagnosis and the inability to make a subclassification. The study included a predominately urban, low-income, minority population and as such our findings may not be widely generalizable. WIDER IMPLICATIONS OF THE FINDINGS: Prenatal Hg exposure was associated with an increased risk of precocious puberty. This risk was strengthened by concomitant maternal cardiometabolic conditions during pregnancy and adverse birth outcomes. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by the NIH/National Institute of Environmental Health Sciences, NIH/Eunice Kennedy Shriver National Institute of Child Health and Human Development and the Health Resources and Services Administration of the U.S. Department of Health and Human Services. The authors declare no conflicts of interest. TRIAL REGISTRATION NUMBER: N/A.

A Nonlinear Relation Between Maternal Red Blood Cell Manganese Concentrations and Child Blood Pressure at Age 6-12 y: A Prospective Birth Cohort Study.

BACKGROUND: Although manganese (Mn) is an essential trace element and a common component of most multivitamins on the market, an adverse effect on blood pressure (BP) has been reported in adults. In addition, the longitudinal relation between prenatal Mn status and childhood BP is still unknown. OBJECTIVE: This study investigated the association between prenatal Mn concentrations and risk of elevated BP at ages 3-12 y. METHOD: The analyses included 1268 mother-child dyads who were enrolled at birth and followed prospectively at the Boston Medical Center. Maternal RBC Mn concentrations were measured by inductively coupled plasma mass spectrometry, using RBCs collected within 1-3 d after delivery (reflecting late-pregnancy Mn exposure). Child elevated BP was defined as systolic or diastolic BP ≥90th percentile for a given age, sex and height. Multivariate logistic regression models were conducted. Path analysis was applied to mediation estimation. RESULTS: The median (IQR) maternal RBC Mn concentration was 37.5 (29.2-48.5) µg/L. The rate of child elevated BP at ages 3-12 y was 25%. Both the lowest and highest quartiles of maternal RBC Mn concentrations were associated with higher risk of elevated BP among children aged 6-12 y (OR: 1.52; 95% CI: 1.04, 2.21 and OR: 1.65; 95% CI: 1.13, 2.40, respectively) compared with those in the second and third quartiles. Gestational age and fetal growth mediated the association between low maternal RBC Mn (first quartile) and child elevated BP, explaining 25% of the association, but not for high (fourth quartile) maternal RBC Mn concentrations. No association was found between maternal RBC Mn concentrations and BP among children aged 3-5 y. CONCLUSION: We found a nonlinear association between maternal RBC Mn concentrations and elevated BP among children aged 6-12 y from a high-risk, predominantly minority population. Our findings warrant further investigation.

Genome-wide association study identifies a novel maternal gene × stress interaction associated with spontaneous preterm birth.

BACKGROUND: Maternal stress is potentially a modifiable risk factor for spontaneous preterm birth (sPTB). However, epidemiologic findings on the maternal stress-sPTB relationship have been inconsistent. METHODS: To investigate whether the maternal stress-sPTB associations may be modified by genetic susceptibility, we performed genome-wide gene × stress interaction analyses in 1490 African-American women from the Boston Birth cohort who delivered term (n = 1033) or preterm (n = 457) infants. Genotyping was performed using Illumina HumanOmni 2.5 array. Replication was performed using data from the NICHD genomic and Proteomic Network (GPN) for PTB research. RESULTS: rs35331017, a T-allele insertion/deletion polymorphism in the protein-tyrosine phosphatase receptor Type D (PTPRD) gene, was the top hit that interacted significantly with maternal lifetime stress on risk of sPTB (PG × E = 4.7 × 10-8). We revealed a dose-responsive association between degree of stress and risk of sPTB in mothers carrying the insertion/insertion genotype, but an inverse association was observed in mothers carrying the heterozygous or deletion/deletion genotypes. This interaction was replicated in African-American (PG × E = 0.088) and Caucasian mothers (PG × E = 0.023) from the GPN study. CONCLUSION: We demonstrated a significant maternal PTPRD × stress interaction on sPTB risk. This finding, if further confirmed, may provide new insight into individual susceptibility to stress-induced sPTB. IMPACT: This was the first preterm study to demonstrate a significant genome-wide gene-stress interaction in African Americans, specifically, PTPRD gene variants can interact with maternal perceived stress to affect risk of spontaneous preterm birth. The PTPRD × maternal stress interaction was demonstrated in African Americans and replicated in both African Americans and Caucasians from the GPN study. Our findings highlight the importance of considering genetic susceptibility in assessing the role of maternal stress on spontaneous preterm birth.

Leptin Induces Hypertension Acting on Transient Receptor Potential Melastatin 7 Channel in the Carotid Body.

RATIONALE: Obesity leads to resistant hypertension and mechanisms are poorly understood, but high plasma levels of leptin have been implicated. Leptin increases blood pressure acting both centrally in the dorsomedial hypothalamus and peripherally. Sites of the peripheral hypertensive effect of leptin have not been identified. We previously reported that leptin enhanced activity of the carotid sinus nerve, which transmits chemosensory input from the carotid bodies (CBs) to the medullary centers, and this effect was abolished by nonselective blockers of Trp (transient receptor potential) channels. We searched our mouse CB transcriptome database and found that the Trpm7 (transient receptor potential melastatin 7) channel was the most abundant Trp channel. OBJECTIVE: To examine if leptin induces hypertension acting on the CB Trpm7. METHODS AND RESULTS: C57BL/6J (n=79), leptin receptor (LepRb) deficient db/db mice (n=22), and LepRb-EGFP (n=4) mice were used. CB Trpm7 and LepRb gene expression was determined and immunohistochemistry was performed; CB glomus cells were isolated and Trpm7-like current was recorded. Blood pressure was recorded continuously in (1) leptin-treated C57BL/6J mice with intact and denervated CB; (2) leptin-treated C57BL/6J mice, which also received a nonselective Trpm7 blocker FTY720 administered systemically or topically to the CB area; (3) leptin-treated C57BL/6J mice transfected with Trpm7 small hairpin RNA to the CB, and (4) Leprb deficient obese db/db mice before and after Leprb expression in CB. Leptin receptor and Trpm7 colocalized in the CB glomus cells. Leptin induced a nonselective cation current in these cells, which was inhibited by Trpm7 blockers. Leptin induced hypertension in C57BL/6J mice, which was abolished by CB denervation, Trpm 7 blockers, and Trpm7 small hairpin RNA applied to CBs. Leprb overexpression in CB of Leprb-deficient db/db mice demethylated the Trpm7 promoter, increased Trpm7 gene expression, and induced hypertension. CONCLUSIONS: We conclude that leptin induces hypertension acting on Trmp7 in CB, which opens horizons for new therapy.

Biological sex affects vaccine efficacy and protection against influenza in mice.

Biological sex affects adaptive immune responses, which could impact influenza infection and vaccine efficacy. Infection of mice with 2009 H1N1 induced antibody responses, CD4+ T cell and CD8+ T cell memory responses that were greater in females than males; both sexes, however, were equally protected against secondary challenge with an H1N1 drift variant virus. To test whether greater antibody in females is sufficient for protection against influenza, males and females were immunized with an inactivated H1N1 vaccine that induced predominantly antibody-mediated immunity. Following vaccination, females had greater antibody responses and protection against challenge with an H1N1 drift variant virus than males. Antibody derived from vaccinated females was better at protecting both naïve males and females than antibody from males, and this protection was associated with increased antibody specificity and avidity to the H1N1 virus. The expression of Tlr7 was greater in B cells from vaccinated females than males and was associated with reduced DNA methylation in the Tlr7 promoter region, higher neutralizing antibody, class switch recombination, and antibody avidity in females. Deletion of Tlr7 reduced sex differences in vaccine-induced antibody responses and protection following challenge and had a greater impact on responses in females than males. Taken together, these data illustrate that greater TLR7 activation and antibody production in females improves the efficacy of vaccination against influenza.

Role of Isocitrate Dehydrogenase 2 on DNA Hydroxymethylation in Human Airway Smooth Muscle Cells.

Global DNA hydroxymethylation mediated by the TET (ten-eleven translocation) enzyme was induced in allergen-induced airway hyperresponsiveness in mouse lung tissues and specifically in isolated airway smooth muscle (ASM) cells. TET is an α-ketoglutarate (α-KG)-dependent enzyme, and the production of α-KG is catalyzed by IDH (isocitrate dehydrogenase). However, the role of IDH in the regulation of DNA hydroxymethylation in ASM cells is unknown. In comparison with nonasthmatic cells, asthmatic ASM cells exhibited higher TET activity and IDH2 (but not IDH-1 or IDH-3) gene expression levels. We modified the expression of IDH2 in ASM cells from humans with asthma by siRNA and examined the α-KG levels, TET activity, global DNA hydroxymethylation, cell proliferation, and expression of ASM phenotypic genes. Inhibition of IDH2 in asthmatic ASM cells decreased the α-KG levels, TET activity, and global DNA hydroxymethylation, and reversed the aberrant ASM phenotypes (including decreased cell proliferation and ASM phenotypic gene expression). Specifically, asthmatic cells transfected with siRNA against IDH2 showed decreased 5hmC (5-hydroxymethylcytosine) levels at the TGFB2 (transforming growth factor-ß2) promoter determined by oxidative bisulfite sequencing. Taken together, our findings reveal that IDH2 plays an important role in the epigenetic regulation of ASM phenotypic changes in asthmatic ASM cells, suggesting that IDH2 is a potential therapeutic target for reversing the abnormal phenotypes seen in asthma.

DNA methylation and adiposity phenotypes: an epigenome-wide association study among adults in the Strong Heart Study.

BACKGROUND: Elevated adiposity is often posited by medical and public health researchers to be a risk factor associated with cardiovascular disease, diabetes, and other diseases. These health challenges are now thought to be reflected in epigenetic modifications to DNA molecules, such as DNA methylation, which can alter gene expression. METHODS: Here we report the results of three Epigenome Wide Association Studies (EWAS) in which we assessed the differential methylation of DNA (obtained from peripheral blood) associated with three adiposity phenotypes (BMI, waist circumference, and impedance-measured percent body fat) among American Indian adult participants in the Strong Heart Study. RESULTS: We found differential methylation at 8264 CpG sites associated with at least one of our three response variables. Of the three adiposity proxies we measured, waist circumference had the highest number of associated differentially methylated CpGs, while percent body fat was associated with the lowest. Because both waist circumference and percent body fat relate to physiology, we focused interpretations on these variables. We found a low degree of overlap between these two variables in our gene ontology enrichment and Differentially Methylated Region analyses, supporting that waist circumference and percent body fat measurements represent biologically distinct concepts. CONCLUSIONS: We interpret these general findings to indicate that highly significant regions of the genome (DMR) and synthesis pathways (GO) in waist circumference analyses are more likely to be associated with the presence of visceral/abdominal fat than more general measures of adiposity. Our findings confirmed numerous CpG sites previously found to be differentially methylated in association with adiposity phenotypes, while we also found new differentially methylated CpG sites and regions not previously identified.

Can social support during pregnancy affect maternal DNA methylation? Findings from a cohort of African-Americans.

BACKGROUND: While stress and the absence of social support during pregnancy have been linked to poor health outcomes, the underlying biological mechanisms are unclear. METHODS: We examined whether adverse experiences during pregnancy alter DNA methylation (DNAm) in maternal epigenomes. Analyses included 250 African-American mothers from the Boston Birth Cohort. Genome-wide DNAm profiling was performed in maternal blood collected after delivery, using the Infinium HumanMethylation450 Beadchip. Linear regression models, with adjustment of pertinent covariates, were applied. RESULTS: While self-reported maternal psychosocial lifetime stress and stress during pregnancy was not associated with DNAm alterations, we found that absence of support from the baby's father was significantly associated with maternal DNAm changes in TOR3A, IQCB1, C7orf36, and MYH7B and that lack of support from family and friends was associated with maternal DNA hypermethylation on multiple genes, including PRDM16 and BANKL. CONCLUSIONS: This study provides intriguing results suggesting biological embedding of social support during pregnancy on maternal DNAm, warranting additional investigation, and replication.

Leptin acts in the carotid bodies to increase minute ventilation during wakefulness and sleep and augment the hypoxic ventilatory response.

KEY POINTS: Leptin is a potent respiratory stimulant. A long functional isoform of leptin receptor, LepRb , was detected in the carotid body (CB), a key peripheral hypoxia sensor. However, the effect of leptin on minute ventilation (VE ) and the hypoxic ventilatory response (HVR) has not been sufficiently studied. We report that LepRb is present in approximately 74% of the CB glomus cells. Leptin increased carotid sinus nerve activity at baseline and in response to hypoxia in vivo. Subcutaneous infusion of leptin increased VE and HVR in C57BL/6J mice and this effect was abolished by CB denervation. Expression of LepRb in the carotid bodies of LepRb deficient obese db/db mice increased VE during wakefulness and sleep and augmented the HVR. We conclude that leptin acts on LepRb in the CBs to stimulate breathing and HVR, which may protect against sleep disordered breathing in obesity. ABSTRACT: Leptin is a potent respiratory stimulant. The carotid bodies (CB) express the long functional isoform of leptin receptor, LepRb , but the role of leptin in CB has not been fully elucidated. The objectives of the current study were (1) to examine the effect of subcutaneous leptin infusion on minute ventilation (VE ) and the hypoxic ventilatory response to 10% O2 (HVR) in C57BL/6J mice before and after CB denervation; (2) to express LepRb in CB of LepRb -deficient obese db/db mice and examine its effects on breathing during sleep and wakefulness and on HVR. We found that leptin enhanced carotid sinus nerve activity at baseline and in response to 10% O2 in vivo. In C57BL/6J mice, leptin increased VE from 1.1 to 1.5 mL/min/g during normoxia (P < 0.01) and from 3.6 to 4.7 mL/min/g during hypoxia (P < 0.001), augmenting HVR from 0.23 to 0.31 mL/min/g/Δ FIO2 (P < 0.001). The effects of leptin on VE and HVR were abolished by CB denervation. In db/db mice, LepRb expression in CB increased VE from 1.1 to 1.3 mL/min/g during normoxia (P < 0.05) and from 2.8 to 3.2 mL/min/g during hypoxia (P < 0.02), increasing HVR. Compared to control db/db mice, LepRb transfected mice showed significantly higher VE throughout non-rapid eye movement (20.1 vs. -27.7 mL/min respectively, P < 0.05) and rapid eye movement sleep (16.5 vs 23.4 mL/min, P < 0.05). We conclude that leptin acts in CB to augment VE and HVR, which may protect against sleep disordered breathing in obesity.

Estrogen down regulates COMT transcription via promoter DNA methylation in human breast cancer cells.

Catechol-O-methyltransferase (COMT) acts as a 'gate-keeper' to prevent DNA damage during estrogen metabolism. Both experimental and epidemiological studies suggest the role of COMT in pathogenesis of human breast cancer (BCa). It was previously reported that inhibition of COMT enzyme activity in estradiol-treated human breast epithelial carcinoma-derived MCF-7 cells caused increased oxidative DNA damage and formation of mutagenic depurinating adducts. To improve our understanding of factors influencing estrogen metabolism in BCa, it requires a mechanistic study illustrating the regulation of this 'gate-keeper'. We investigated the epigenetic mechanisms underlying decreased COMT transcription in MCF-7 cells exposed to 17ß-estradiol (E2) and the phytoestrogen, genistein (GEN). CpG site-specific methylation at promoters for both soluble (S) and membrane-bound (MB) COMT transcripts were assessed. Both E2 and GEN induced CpG site-specific methylation within the distal promoter of MB-COMT. In addition, ChIP analysis showed that there was increased binding of DNMT3B, MBD2 and HDAC1 within this promoter. These epigenetic changes were associated with decreased COMT transcript levels. Interestingly, sulforaphane, an antioxidant commonly found in cruciferous vegetables, was able to reverse the estrogen-induced epigenetic changes and gene silencing of COMT. Our data provide a new insight in epigenetically targeting COMT transcription. Since reactive estrogen metabolites may contribute to breast cancer, our findings may help in developing prevention and/or intervention strategies for human BCa.

Aberrant DNA Methylation of Phosphodiesterase [corrected] 4D Alters Airway Smooth Muscle Cell Phenotypes.

Airway hyperresponsiveness (AHR) is a hallmark feature in asthma characterized by exaggerated airway contractile response to stimuli due to increased airway sensitivity and chronic airway remodeling. We have previously shown that allergen-induced AHR in mice is associated with aberrant DNA methylation in the lung genome, suggesting that AHR could be epigenetically regulated, and these changes might predispose the animals to asthma. Previous studies demonstrated that overexpression of phosphodiesterase 4D (PDE4D) is associated with increased AHR. However, epigenetic regulation of this gene in asthmatic airway smooth muscle cells (ASMCs) has not been examined. In this study, we aimed to examine the relationship between epigenetic regulation of PDE4D and ASMC phenotypes. We identified CpG site-specific hypomethylation at PDE4D promoter in human asthmatic ASMCs. We next used methylated oligonucleotides to introduce CpG site-specific methylation at PDE4D promoter and examined its effect on ASMCs. We showed that PDE4D methylation decreased cell proliferation and migration of asthmatic ASMCs. We further elucidated that methylated PDE4D decreased PDE4D expression in asthmatic ASMCs, increased cAMP level, and inhibited the aberrant increase in Ca(2+) level. Moreover, PDE4D methylation reduced the phosphorylation level of downstream effectors of Ca(2+) signaling, including myosin light chain kinase and p38. Taken together, our findings demonstrate that gene-specific epigenetic changes may predispose ASMCs to asthma through alterations in cell phenotypes. Modulation of ASMC phenotypes by methylated PDE4D oligonucleotides can reverse the aberrant ASMC functions to normal phenotypes. This has provided new insight to the development of novel therapeutic options for this debilitative disease.

A Short-Term Fasting in Neonates Induces Breathing Instability and Epigenetic Modification in the Carotid Body.

The respiratory control system is not fully developed in newborn, and data suggest that adequate nutrition is important for the development of the respiratory control system. Infants need to be fed every 2-4 h to maintain appropriate energy levels, but a skip of feeding can occur due to social economical reasons or mild sickness of infants. Here, we asked questions if a short-term fasting (1) alters carotid body (CB) chemoreceptor activity and integrated function of the respiratory control system; (2) causes epigenetic modification within the respiratory control system. Mouse pups (

Differential methylation of the arsenic (III) methyltransferase promoter according to arsenic exposure.

Inorganic arsenic is methylated in the body by arsenic (III) methyltransferase (AS3MT). Arsenic methylation is thought to play a role in arsenic-related epigenetic phenomena, including aberrant DNA and histone methylation. However, it is unclear whether the promoter of the AS3MT gene, which codes for AS3MT, is differentially methylated as a function of arsenic exposure. In this study, we evaluated AS3MT promoter methylation according to exposure, assessed by urinary arsenic excretion in a stratified random sample of 48 participants from the Strong Heart Study who had urine arsenic measured at baseline and DNA available from 1989 to 1991 and 1998-1999. For this study, all data are from the 1989-1991 visit. We measured AS3MT promoter methylation at its 48 CpG loci by bisulphite sequencing. We compared mean  % methylation at each CpG locus by arsenic exposure group using linear regression adjusted for study centre, age and sex. A hypomethylated region in the AS3MT promoter was associated with higher arsenic exposure. In vitro, arsenic induced AS3MT promoter hypomethylation, and it increased AS3MT expression in human peripheral blood mononuclear cells. These findings may suggest that arsenic exposure influences the epigenetic regulation of a major arsenic metabolism gene.

Carotid body denervation improves hyperglycemia in obese mice.

The carotid bodies (CBs) have been implicated in glucose abnormalities in obesity via elevation of activity of the sympathetic nervous system. Obesity-induced hypertension is mediated by insulin receptor (INSR) signaling and by leptin, which binds to the leptin receptor (LEPRb) in CB and activates transient receptor potential channel subfamily M member 7 (TRPM7). We hypothesize that in mice with diet-induced obesity, hyperglycemia, glucose intolerance, and insulin resistance will be attenuated by the CB denervation (carotid sinus nerve dissection, CSND) and by knockdown of Leprb, Trpm7, and Insr gene expression in CB. In series of experiments in 75 male diet-induced obese (DIO) mice, we performed either CSND (vs. sham) surgeries or shRNA-induced suppression of Leprb, Trpm7, or Insr gene expression in CB, followed by blood pressure telemetry, intraperitoneal glucose tolerance and insulin tolerance tests, and measurements of fasting plasma insulin, leptin, corticosterone, glucagon and free fatty acids (FFAs) levels, hepatic expression of gluconeogenesis enzymes phosphoenolpyruvate carboxykinase (PEPCK) and glucose 6-phosphatase (G-6-Pase) mRNA and liver glycogen levels. CSND decreased blood pressure, fasting blood glucose levels and improved glucose tolerance without any effect on insulin resistance. CSND did not affect any hormone levels and gluconeogenesis enzymes, but increased liver glycogen level. Genetic knockdown of CB Leprb, Trpm7, and Insr had no effect on glucose metabolism. We conclude that CB contributes to hyperglycemia of obesity, probably by modulation of the glycogen-glucose equilibrium. Diabetogenic effects of obesity on CB in mice do not occur via activation of CB Leprb, Trpm7, and Insr.NEW & NOTEWORTHY This paper provides first evidence that carotid body denervation abolishes hypertension and improves fasting blood glucose levels and glucose tolerance in mice with diet-induced obesity. Furthermore, we have shown that this phenomenon is associated with increased liver glycogen content, whereas insulin sensitivity and enzymes of gluconeogenesis were not affected.

Epigenetic alterations by DNA methylation in house dust mite-induced airway hyperresponsiveness.

Asthma is one of the most prevalent chronic lung diseases, affecting 235 million individuals around the world, with its related morbidity and mortality increasing steadily over the last 20 years. Exposure to the environmental allergen, house dust mite (HDM), results in airway inflammation with a variable degree of airway obstruction. Although there has been much experimental work in the past using HDM challenge models to understand mechanistic details in allergic inflammation and airway hyperresponsiveness (AHR), there has been no study on reprogramming of lung or airways mediated through epigenetic mechanisms in response to an acute HDM exposure. Male mice, 6 weeks of age, were administrated HDM extracts or saline at Days 1, 14, and 21. Exposure of mice to HDM extracts caused significant airway inflammation and increased AHR. These HDM-challenged mice also exhibited a change in global DNA methylation as compared with saline-exposed (control) mice. Next, by employing methylation-sensitive restriction fingerprinting, we identified a set of genes, showing aberrant methylation status, associated with the HDM-induced AHR. These candidate genes are known to be involved in cAMP signaling (pde4 d), Akt-signaling (akt1 s1), ion transport (tm6 sf1, pom121l2, and slc8a3), and fatty acid metabolism (acsl3). Slc8a3 and acsl3 were down-regulated, whereas pde4 d, akt1 s1, tm6 sf1, and pom121l2 were up-regulated in the mice exposed to HDM. Hence, our results suggest that HDM exposure induces a series of aberrant methylated genes that are potentially important for the development of allergic AHR.

Exercise-primed extracellular vesicles improve cell-matrix adhesion and chondrocyte health.

Extracellular vesicles (EVs) have been suggested to transmit the health-promoting effects of exercise throughout the body. Yet, the mechanisms by which beneficial information is transmitted from extracellular vesicles to recipient cells are poorly understood, precluding a holistic understanding of how exercise promotes cellular and tissue health. In this study, using articular cartilage as a model, we introduced a network medicine paradigm to simulate how exercise facilitates communication between circulating EVs and chondrocytes, the cells resident in articular cartilage. Using the archived small RNA-seq data of EV before and after aerobic exercise, microRNA regulatory network analysis based on network propagation inferred that circulating EVs activated by aerobic exercise perturb chondrocyte-matrix interactions and downstream cellular aging processes. Building on the mechanistic framework identified through computational analyses, follow up experimental studies interrogated the direct influence of exercise on EV-mediated chondrocyte-matrix interactions. We found that pathogenic matrix signaling in chondrocytes was abrogated in the presence of exercise-primed EVs, restoring a more youthful phenotype, as determined by chondrocyte morphological profiling and evaluation of chondrogenicity. Epigenetic reprograming of the gene encoding the longevity protein, α-Klotho, mediated these effects. These studies provide mechanistic evidence that exercise transduces rejuvenation signals to circulating EVs, endowing EVs with the capacity to ameliorate cellular health even in the presence of an unfavorable microenvironmental signals.

Age-related matrix stiffening epigenetically regulates α-Klotho expression and compromises chondrocyte integrity.

Extracellular matrix stiffening is a quintessential feature of cartilage aging, a leading cause of knee osteoarthritis. Yet, the downstream molecular and cellular consequences of age-related biophysical alterations are poorly understood. Here, we show that epigenetic regulation of α-Klotho represents a novel mechanosensitive mechanism by which the aged extracellular matrix influences chondrocyte physiology. Using mass spectrometry proteomics followed by a series of genetic and pharmacological manipulations, we discovered that increased matrix stiffness drove Klotho promoter methylation, downregulated Klotho gene expression, and accelerated chondrocyte senescence in vitro. In contrast, exposing aged chondrocytes to a soft matrix restored a more youthful phenotype in vitro and enhanced cartilage integrity in vivo. Our findings demonstrate that age-related alterations in extracellular matrix biophysical properties initiate pathogenic mechanotransductive signaling that promotes Klotho promoter methylation and compromises cellular health. These findings are likely to have broad implications even beyond cartilage for the field of aging research.