Smooth muscle expression of RNA editing enzyme ADAR1 controls vascular integrity and progression of atherosclerosis.

Mapping the genomic architecture of complex disease has been predicated on the understanding that genetic variants influence disease risk through modifying gene expression. However, recent discoveries have revealed that a significant burden of disease heritability in common autoinflammatory disorders and coronary artery disease is mediated through genetic variation modifying post-transcriptional modification of RNA through adenosine-to-inosine (A-to-I) RNA editing. This common RNA modification is catalyzed by ADAR enzymes, where ADAR1 edits specific immunogenic double stranded RNA (dsRNA) to prevent activation of the double strand RNA (dsRNA) sensor MDA5 ( IFIH1 ) and stimulation of an interferon stimulated gene (ISG) response. Multiple lines of human genetic data indicate impaired RNA editing and increased dsRNA sensing to be an important mechanism of coronary artery disease (CAD) risk. Here, we provide a crucial link between observations in human genetics and mechanistic cell biology leading to progression of CAD. Through analysis of human atherosclerotic plaque, we implicate the vascular smooth muscle cell (SMC) to have a unique requirement for RNA editing, and that ISG induction occurs in SMC phenotypic modulation, implicating MDA5 activation. Through culture of human coronary artery SMCs, generation of a conditional SMC specific Adar1 deletion mouse model on a pro-atherosclerosis background, and with incorporation of single cell RNA sequencing cellular profiling, we further show that Adar1 controls SMC phenotypic state, is required to maintain vascular integrity, and controls progression of atherosclerosis and vascular calcification. Through this work, we describe a fundamental mechanism of CAD, where cell type and context specific RNA editing and sensing of dsRNA mediates disease progression, bridging our understanding of human genetics and disease causality.

In utero exposure to diesel exhaust air pollution promotes adverse intrauterine conditions, resulting in weight gain, altered blood pressure, and increased susceptibility to heart failure in adult mice.

Exposure to fine particulate air pollution (PM2.5) is strongly associated with cardiovascular morbidity and mortality. Exposure to PM2.5 during pregnancy promotes reduced birthweight, and the associated adverse intrauterine conditions may also promote adult risk of cardiovascular disease. Here, we investigated the potential for in utero exposure to diesel exhaust (DE) air pollution, a major source of urban PM2.5, to promote adverse intrauterine conditions and influence adult susceptibility to disease. We exposed pregnant female C57Bl/6J mice to DE (≈300 µg/m³ PM2.5, 6 hrs/day, 5 days/week) from embryonic day (E) 0.5 to 17.5. At E17.5 embryos were collected for gravimetric analysis and assessed for evidence of resorption. Placental tissues underwent pathological examination to assess the extent of injury, inflammatory cell infiltration, and oxidative stress. In addition, some dams that were exposed to DE were allowed to give birth to pups and raise offspring in filtered air (FA) conditions. At 10-weeks of age, body weight and blood pressure were measured. At 12-weeks of age, cardiac function was assessed by echocardiography. Susceptibility to pressure overload-induced heart failure was then determined after transverse aortic constriction surgery. We found that in utero exposure to DE increases embryo resorption, and promotes placental hemorrhage, focal necrosis, compaction of labyrinth vascular spaces, inflammatory cell infiltration and oxidative stress. In addition, we observed that in utero DE exposure increased body weight, but counterintuitively reduced blood pressure without any changes in baseline cardiac function in adult male mice. Importantly, we observed these mice to have increased susceptibility to pressure-overload induced heart failure, suggesting this in utero exposure to DE 'reprograms' the heart to a heightened susceptibility to failure. These observations provide important data to suggest that developmental exposure to air pollution may strongly influence adult susceptibility to cardiovascular disease.

In utero and early life exposure to diesel exhaust air pollution increases adult susceptibility to heart failure in mice.

BACKGROUND: Fine particulate air pollution (PM2.5) is a global health concern, as exposure to PM2.5 has consistently been found to be associated with increased cardiovascular morbidity and mortality. Although adult exposure to traffic related PM2.5, which is largely derived from diesel exhaust (DE), has been associated with increased cardiac hypertrophy, there are limited investigations into the potential effect of in utero and early life exposure on adult susceptibility to heart disease. In this study, we investigate the effect of in utero and early life exposure to DE on adult susceptibility to heart failure. METHODS: Female C57BL/6 J mice were exposed to either filtered air (FA) or DE for 3 weeks (≈ 300 µg/m3 PM2.5 for 6 hours/day, 5 days/week) and then introduced to male breeders for timed matings. Female mice were exposed to either FA or DE throughout pregnancy and until offspring were 3 weeks of age. Offspring were then transferred to either FA or DE for an additional 8 weeks of exposure. At 12 weeks of age, male offspring underwent a baseline echocardiographic assessment, followed by a sham or transverse aortic constriction (TAC) surgery to induce pressure overload. Following sacrifice three weeks post surgery, ventricles were processed for histology to assess myocardial fibrosis and individual cardiomyocyte hypertrophy. mRNA from lung tissue was isolated to measure expression of inflammatory cytokines IL6 and TNFα. RESULTS: We observed that mice exposed to DE during in utero and early life development have significantly increased susceptibility to cardiac hypertrophy, systolic failure, myocardial fibrosis, and pulmonary congestion following TAC surgery compared to FA control, or adult DE exposed mice. In utero and early life DE exposure also strongly modified the inflammatory cytokine response in the adult lung. CONCLUSIONS: We conclude that exposure to diesel exhaust air pollution during in utero and early life development in mice increases adult susceptibility to heart failure. The results of this study may imply that the effects of air pollution on cardiovascular disease in human populations may be strongly mediated through a 'fetal origins' of adult disease pathway. Further investigations on this potential pathway of disease are warranted.

Inhalation of diesel exhaust does not exacerbate cardiac hypertrophy or heart failure in two mouse models of cardiac hypertrophy.

BACKGROUND: Strong associations have been observed between exposure to fine ambient particulate matter (PM2.5) and adverse cardiovascular outcomes. In particular, exposure to traffic related PM2.5 has been associated with increases in left ventricular hypertrophy, a strong risk factor for cardiovascular mortality. As much of traffic related PM2.5 is derived from diesel exhaust (DE), we investigated the effects of chronic DE exposure on cardiac hypertrophy and heart failure in the adult mouse by exposing mice to DE combined with either of two mouse models of cardiac hypertrophy: angiotensin II infusion or pressure overload induced by transverse aortic banding. METHODS: Wild type male C57BL/6 J mice were either infused with angiotensin II (800 ng/kg/min) via osmotic minipump implanted subcutaneously for 1 month, or underwent transverse aortic banding (27 gauge needle 1 week for observing acute reactions, 26 gauge needle 3 months or 6 months for observing chronic reactions). Vehicle (saline) infusion or sham surgery was used as a control. Shortly after surgery, mice were transferred to our exposure facility and randomly assigned to either diesel exhaust (300 or 400 µg/m(3)) or filtered air exposures. After reaching the end of designated time points, echocardiography was performed to measure heart structure and function. Gravimetric analysis was used to measure the ventricular weight to body weight ratio. We also measured heart rate by telemetry using implanted ambulatory ECG monitors. RESULTS: Both angiotensin II and transverse aortic banding promoted cardiac hypertrophy compared to vehicle or sham controls. Transverse aortic banding for six months also promoted heart failure in addition to cardiac hypertrophy. In all cases, DE failed to exacerbate the development of hypertrophy or heart failure when compared to filtered air controls. Prolonged DE exposure also led to a decrease in average heart rate. CONCLUSIONS: Up to 6-months of DE exposure had no effect on cardiac hypertrophy and heart function induced by angiotensin II stimulation or pressure overload in adult C57BL/6 J mice. This study highlights the potential importance of particle constituents of ambient PM2.5 to elicit cardiotoxic effects. Further investigations on particle constituents and cardiotoxicity are warranted.

Glutathione (GSH) and the GSH synthesis gene Gclm modulate plasma redox and vascular responses to acute diesel exhaust inhalation in mice.

CONTEXT: Inhalation of fine particulate matter (PM2.5) is associated with acute pulmonary inflammation and impairments in cardiovascular function. In many regions, PM2.5 is largely derived from diesel exhaust (DE), and these pathophysiological effects may be due in part to oxidative stress resulting from DE inhalation. The antioxidant glutathione (GSH) is important in limiting oxidative stress-induced vascular dysfunction. The rate-limiting enzyme in GSH synthesis is glutamate cysteine ligase and polymorphisms in its catalytic and modifier subunits (GCLC and GCLM) have been shown to influence vascular function and risk of myocardial infarction in humans. OBJECTIVE: We hypothesized that compromised de novo synthesis of GSH in Gclm⁻/⁺ mice would result in increased sensitivity to DE-induced lung inflammation and vascular effects. MATERIALS AND METHODS: WT and Gclm⁻/⁺ mice were exposed to DE via inhalation (300 µg/m³) for 6 h. Neutrophil influx into the lungs, plasma GSH redox potential, vascular reactivity of aortic rings and aortic nitric oxide (NO•) were measured. RESULTS: DE inhalation resulted in mild bronchoalveolar neutrophil influx in both genotypes. DE-induced effects on plasma GSH oxidation and acetylcholine (ACh)-relaxation of aortic rings were only observed in Gclm⁻/⁺ mice. Contrary to our hypothesis, DE exposure enhanced ACh-induced relaxation of aortic rings in Gclm⁻/⁺ mice. DISCUSSION AND CONCLUSION: THESE data support the hypothesis that genetic determinants of antioxidant capacity influence the biological effects of acute inhalation of DE. However, the acute effects of DE on the vasculature may be dependent on the location and types of vessels involved. Polymorphisms in GSH synthesis genes are common in humans and further investigations into these potential gene-environment interactions are warranted.

The glutathione synthesis gene Gclm modulates amphiphilic polymer-coated CdSe/ZnS quantum dot-induced lung inflammation in mice.

Quantum dots (QDs) are unique semi-conductor fluorescent nanoparticles with potential uses in a variety of biomedical applications. However, concerns exist regarding their potential toxicity, specifically their capacity to induce oxidative stress and inflammation. In this study we synthesized CdSe/ZnS core/shell QDs with a tri-n-octylphosphine oxide, poly(maleic anhydride-alt-1-tetradecene) (TOPO-PMAT) coating and assessed their effects on lung inflammation in mice. Previously published in vitro data demonstrated these TOPO-PMAT QDs cause oxidative stress resulting in increased expression of antioxidant proteins, including heme oxygenase, and the glutathione (GSH) synthesis enzyme glutamate cysteine ligase (GCL). We therefore investigated the effects of these QDs in vivo in mice deficient in GSH synthesis (Gclm +/- and Gclm -/- mice). When mice were exposed via nasal instillation to a TOPO-PMAT QD dose of 6 µg cadmium (Cd) equivalents/kg body weight, neutrophil counts in bronchoalveolar lavage fluid (BALF) increased in both Gclm wild-type (+/+) and Gclm heterozygous (+/-) mice, whereas Gclm null (-/-) mice exhibited no such increase. Levels of the pro-inflammatory cytokines KC and TNFα increased in BALF from Gclm +/+ and +/- mice, but not from Gclm -/- mice. Analysis of lung Cd levels suggested that QDs were cleared more readily from the lungs of Gclm -/- mice. There was no change in matrix metalloproteinase (MMP) activity in any of the mice. However, there was a decrease in whole lung myeloperoxidase (MPO) content in Gclm -/- mice, regardless of treatment, relative to untreated Gclm +/+ mice. We conclude that in mice TOPO-PMAT QDs have in vivo pro-inflammatory properties, and the inflammatory response is dependent on GSH synthesis status. Because there is a common polymorphism in humans that influences GCLM expression, these findings imply that humans with reduced GSH synthesis capabilities may be more susceptible to the pro-inflammatory effects of QDs.

Glutathione (GSH) and the GSH synthesis gene Gclm modulate vascular reactivity in mice.

Oxidative stress has been implicated in the development of vascular disease and in the promotion of endothelial dysfunction via the reduction in bioavailable nitric oxide (NO()). Glutathione (GSH) is a tripeptide thiol antioxidant that is utilized by glutathione peroxidase (GPx) to scavenge reactive oxygen species such as hydrogen peroxide and phospholipid hydroperoxides. Relatively frequent single-nucleotide polymorphisms (SNPs) within the 5' promoters of the GSH synthesis genes GCLC and GCLM are associated with impaired vasomotor function, as measured by decreased acetylcholine-stimulated coronary artery dilation, and with increased risk of myocardial infarction. Although the influence of genetic knockdown of GPx on vascular function has been investigated in mice, no work to date has been published on the role of genetic knockdown of GSH synthesis genes on vascular reactivity. We therefore investigated the effects of targeted disruption of Gclm in mice and the subsequent depletion of GSH on vascular reactivity, NO() production, aortic nitrotyrosine protein modification, and whole-genome transcriptional responses as measured by DNA microarray. Gclm(-/+) and Gclm(-/-) mice had 72 and 12%, respectively, of wild-type (WT) aortic GSH content. Gclm(-/+) mice had a significant impairment in acetylcholine (ACh)-induced relaxation in aortic rings as well as increased aortic nitrotyrosine protein modification. Surprisingly, Gclm(-/-) aortas showed enhanced relaxation compared to Gclm(-/+) aortas, as well as increased NO() production. Although aortic rings from Gclm(-/-) mice had enhanced ACh relaxation, they had a significantly increased sensitivity to phenylephrine (PE)-induced contraction. Alternatively, the PE response of Gclm(-/+) aortas was nearly identical to that of their WT littermates. To examine the role of NO() or other potential endothelium-derived factors in differentially regulating vasomotor activity, we incubated aortic rings with the NO() synthase inhibitor L-NAME or physically removed the endothelium before PE treatment. L-NAME treatment and endothelium removal enhanced PE-induced contraction in WT and Gclm(-/+) mice, but this effect was severely diminished in Gclm(-/-) mice, indicating a potentially unique role for GSH in mediating vessel contraction. Whole-genome assessment of aortic mRNA in Gclm(-/-) and WT mice revealed altered expression of genes within the canonical Ca(2+) signaling pathway, which may have a role in mediating these observed functional effects. These findings provide additional evidence that the de novo synthesis of GSH can influence vascular reactivity and provide insights regarding possible mechanisms by which SNPs within GCLM and GCLC influence the risk of developing vascular diseases in humans.

Heterozygosity in the glutathione synthesis gene Gclm increases sensitivity to diesel exhaust particulate induced lung inflammation in mice.

CONTEXT: Inhalation of ambient fine particulate matter (PM2.5) is associated with adverse respiratory and cardiovascular effects. A major fraction of PM2.5 in urban settings is diesel exhaust particulate (DEP), and DEP-induced lung inflammation is likely a critical event mediating many of its adverse health effects. Oxidative stress has been proposed to be an important factor in PM2.5-induced lung inflammation, and the balance between pro- and antioxidants is an important regulator of this inflammation. An important intracellular antioxidant is the tripeptide thiol glutathione (GSH). Glutamate cysteine ligase (GCL) carries out the first step in GSH synthesis. In humans, relatively common genetic polymorphisms in both the catalytic (Gclc) and modifier (Gclm) subunits of GCL have been associated with increased risk for lung and cardiovascular diseases. OBJECTIVE: This study was aimed to determine the effects of Gclm expression on lung inflammation following DEP exposure in mice. MATERIALS AND METHODS: We exposed Gclm wild type, heterozygous, and null mice to DEP via intranasal instillation and assessed lung inflammation as determined by neutrophils and inflammatory cytokines in lung lavage, inflammatory cytokine mRNA levels in lung tissue, as well as total lung GSH, Gclc, and Gclm protein levels. RESULTS: The Gclm heterozygosity was associated with a significant increase in DEP-induced lung inflammation when compared to that of wild type mice. DISCUSSION AND CONCLUSION: This finding indicates that GSH synthesis can mediate DEP-induced lung inflammation and suggests that polymorphisms in Gclm may be an important factor in determining adverse health outcomes in humans following inhalation of PM2.5.

DIESEL particulate exposed macrophages alter endothelial cell expression of eNOS, iNOS, MCP1, and glutathione synthesis genes.

There is considerable debate regarding inhaled diesel exhaust particulate (DEP) causing impairments in vascular reactivity. Although there is evidence that inhaled particles can translocate from the lung into the systemic circulation, it has been suggested that inflammatory factors produced in the lung following macrophage particle engulfment also pass into the circulation. To investigate these differing hypotheses, we used in vitro systems to model each exposure. By using a direct exposure system and a macrophage-endothelial cell co-culture model, we compared the effects of direct DEP exposure and exposure to inflammatory factors produced by DEP-treated macrophages, on endothelial cell mRNA levels for eNOS, iNOS, endothelin-1, and endothelin-converting-enzyme-1. As markers of oxidative stress, we measured the effects of DEP treatment on glutathione (GSH) synthesis genes and on total GSH. In addition, we analyzed the effect of DEP treatment on monocyte chemo-attractant protein-1. Direct DEP exposure increased endothelial GCLC and GCLM as well as total GSH in addition to increased eNOS, iNOS, and Mcp1 mRNA. Alternatively, inflammatory factors released from DEP-exposed macrophages markedly up-regulated endothelial iNOS and Mcp1 while modestly down-regulating eNOS. These data support both direct exposure to DEP and the release of inflammatory cytokines as explanations for DEP-induced impairments in vascular reactivity.