Accelerated epigenetic age as a biomarker of cardiovascular sensitivity to traffic-related air pollution.

BACKGROUND: Accelerated epigenetic age has been proposed as a biomarker of increased aging, which may indicate disruptions in cellular and organ system homeostasis and thus contribute to sensitivity to environmental exposures. METHODS: Using 497 participants from the CATHGEN cohort, we evaluated whether accelerated epigenetic aging increases cardiovascular sensitivity to traffic-related air pollution (TRAP) exposure. We used residential proximity to major roadways and source apportioned air pollution models as measures of TRAP exposure, and chose peripheral arterial disease (PAD) and blood pressure as outcomes based on previous associations with TRAP. We used Horvath epigenetic age acceleration (AAD) and phenotypic age acceleration (PhenoAAD) as measures of age acceleration, and adjusted all models for chronological age, race, sex, smoking, and socioeconomic status. RESULTS: We observed significant interactions between TRAP and both AAD and PhenoAAD. Interactions indicated that increased epigenetic age acceleration elevated associations between proximity to roadways and PAD. Interactions were also observed between AAD and gasoline and diesel source apportioned PM2.5. CONCLUSION: Epigenetic age acceleration may be a biomarker of sensitivity to air pollution, particularly for TRAP in urban cohorts. This presents a novel means by which to understand sensitivity to air pollution and provides a molecular measure of environmental sensitivity.

Role of research in public health.

Environmental health research is at a promising stage for more precisely identifying gene-environment components of disease. Simplistic models and reductionist approaches that have been the norm both in toxicology and in clinical medicine are beginning to be replaced with a more holistic or systems biology approach. We are slowly moving to an understanding that the time between an exposure and its consequence as a diagnosed disease is a time during which many different biochemical changes are occurring and a time during which many biomarkers of disease progression could be identified and used. With such information in hand, clinicians would be able to intervene early in disease progression. With such information, environmental health researchers and policy makers could more reliably identify which elements in our environment need to be controlled or reduced and which populations need the most protection. With such information, the incidence of many human diseases could be dramatically reduced.

Evaluating DNA methylation age on the Illumina MethylationEPIC Bead Chip.

DNA methylation age (DNAm age) has become a widely utilized epigenetic biomarker for the aging process. The Horvath method for determining DNAm age is perhaps the most widely utilized and validated DNA methylation age assessment measure. Horvath DNAm age is calculated based on methylation measurements at 353 loci, present on Illumina's 450k and 27k DNA methylation microarrays. With increasing use of the more recently developed Illumina MethylationEPIC (850k) microarray, it is worth revisiting this aging measure to evaluate estimation differences due to array design. Of the requisite 353 loci, 17 are missing from the 850k microarray. Similarly, an alternate, 71 loci DNA methylation age assessment measure created by Hannum et al. is missing 6 requisite loci. Using 17 datasets with 27k, 450k, and/or 850k methylation data, we compared each sample's epigenetic age estimated from all 353 loci required by the Horvath DNAm age calculator, and using only the 336 loci available on the 850k array. In 450k/27k data, removing loci not on the 850k array resulted in underestimation of Horvath's DNAm age. Underestimation of Horvath DNAm age increased from ages 0 to ~20, remaining stable thereafter (mean deviation = -3.46 y, SD = 1.13 for individuals ≥20 years). Underestimation of Horvath's DNAm age by the reduced 450k/27k data was similar to the underestimation observed in the 850k data indicating it is driven by missing probes. In analogous examination of Hannum's DNAm age, the magnitude and direction of epigenetic age misestimation varied with chronological age. In conclusion, inter-array deviations in DNAm age estimations may be largely driven by missing probes between arrays, despite default probe imputation procedures. Though correlations and associations based on Horvath's DNAm age may be unaffected, researchers should exercise caution when interpreting results based on absolute differences in DNAm age or when mixing samples assayed on different arrays.

A novel approach for measuring residential socioeconomic factors associated with cardiovascular and metabolic health.

Individual-level characteristics, including socioeconomic status, have been associated with poor metabolic and cardiovascular health; however, residential area-level characteristics may also independently contribute to health status. In the current study, we used hierarchical clustering to aggregate 444 US Census block groups in Durham, Orange, and Wake Counties, NC, USA into six homogeneous clusters of similar characteristics based on 12 demographic factors. We assigned 2254 cardiac catheterization patients to these clusters based on residence at first catheterization. After controlling for individual age, sex, smoking status, and race, there were elevated odds of patients being obese (odds ratio (OR)=1.92, 95% confidence intervals (CI)=1.39, 2.67), and having diabetes (OR=2.19, 95% CI=1.57, 3.04), congestive heart failure (OR=1.99, 95% CI=1.39, 2.83), and hypertension (OR=2.05, 95% CI=1.38, 3.11) in a cluster that was urban, impoverished, and unemployed, compared with a cluster that was urban with a low percentage of people that were impoverished or unemployed. Our findings demonstrate the feasibility of applying hierarchical clustering to an assessment of area-level characteristics and that living in impoverished, urban residential clusters may have an adverse impact on health.

The Role of the Epigenome in Translating Neighborhood Disadvantage Into Health Disparities.

The possible causal role of the environment in health disparities is not well understood, even though it has been a national priority for many years. Progress to investigate the relationship between genetics, environmental exposures, and health outcomes has been hampered by the lack of analytical tools to quantify the combined or cumulative effect of multiple chemical and non-chemical stressors on gene expression. The studies cited here provide a strong rationale for using epigenomic analysis to assess cumulative risk from multiple environmental exposures over the life course. The environment-specific "imprints" on the genome, coupled with transcriptomics and metabolomics, can be used to advance our understanding of the relationship between neighborhood disadvantage and health disparities.

Epigenome: biosensor of cumulative exposure to chemical and nonchemical stressors related to environmental justice.

Understanding differential disease susceptibility requires new tools to quantify the cumulative effects of environmental stress. Evidence suggests that social, physical, and chemical stressors can influence disease through the accumulation of epigenetic modifications. Geographically stable epigenetic alterations could identify plausible mechanisms for health disparities among the disadvantaged and poor. Relations between neighborhood-specific epigenetic markers and disease would identify the most appropriate targets for medical and environmental intervention. Complex interactions among genes, the environment, and disease require the examination of how epigenetic changes regulate susceptibility to environmental stressors. Progress in understanding disparities in disease susceptibility may depend on assessing the cumulative effect of environmental stressors on genetic substrates. We highlight key concepts regarding the interface between environmental stress, epigenetics, and chronic disease.

Laboratory to community: chemoprevention is the answer.

In the current issue, Johnson and colleagues present exciting results, using biomarkers involved in aflatoxin B1 (AFB1)-induced hepatocarcinogenesis, as an example of a conceptual framework to target mechanisms of action in developing chemopreventive agents. Their innovative approach offers considerable promise for a field that has long been neglected. Proof-of-principle was demonstrated using a synthetic triterpenoid (CDDO-Im), which activates Nrf2 signal transduction pathway, inhibits formation of AFB1-induced DNA adducts and neoplastic hepatic foci, and alters the expression of genes associated with aflatoxin-mediated toxicity.

p38 mitogen-activated protein kinase interacts with vinculin at focal adhesions during fatty acid-stimulated cell adhesion.

Arachidonic acid stimulates cell adhesion by activating α2ß1 integrins in a process that depends on protein kinases, including p38 mitogen activated protein kinase. Here, we describe the interaction of cytoskeletal components with key signaling molecules that contribute to the spreading of, and morphological changes in, arachidonic acid-treated MDA-MB-435 human breast carcinoma cells. Arachidonic acid-treated cells showed increased attachment and spreading on collagen type IV, as measured by electric cell-substrate impedance sensing. Fatty acid-treated cells displayed short cortical actin filaments associated with an increased number of ß1 integrin-containing pseudopodia, whereas untreated cells displayed elongated stress fibers and fewer clusters of ß1 integrins. Confocal microscopy of arachidonic acid-treated cells showed that vinculin and phospho-p38 both appeared enriched in pseudopodia and at the tips of actin filaments, and fluorescence ratio imaging indicated the increase was specific for the phospho-(active) form of p38. Immunoprecipitates of phospho-p38 from extracts of arachidonic acid-treated cells contained vinculin, and GST-vinculin fusion proteins carrying the central region of vinculin bound phospho-p38, whereas fusion proteins expressing the terminal portions of vinculin did not. These data suggest that phospho-p38 associates with particular domains on critical focal adhesion proteins that are involved in tumor cell adhesion and spreading, and that this association can be regulated by factors in the tumor microenvironment.

Plasminogen activator inhibitor 1 RNAi suppresses gastric cancer metastasis in vivo.

Cancer metastasis remains the primary cause of pain, suffering, and death in cancer patients, and even the most current therapeutic strategies have not been highly successful in preventing or inhibiting metastasis. In most patients with scirrhous gastric cancer (one of the most aggressive of diffuse-type gastric cancer), recurrence occurs even after potentially curative resection, most frequently in the form of peritoneal metastasis. Given that the occurrence of diffuse-type gastric cancers has been increasing, the development of new strategies to combat metastasis of this disease is critically important. Plasminogen activator inhibitor-1 (PAI-1) is a critical factor in cancer progression; thus, PAI-1 RNAi may be an effective therapy against cancer metastasis. In the present study, we used an RNAi technique to reduce PAI-1 expression in an in vivo model system for gastric cancer metastasis. Ex vivo plasmid transfection and adenovirus infection were tested as mechanisms to incorporate specific PAI-1 RNAi vectors into human gastric carcinoma cells. Both approaches significantly decreased peritoneal tumor growth and the formation of bloody ascites in the mouse model, suggesting that this approach may provide a new, effective strategy for inhibiting cancer metastasis.

Neighborhood-specific epigenome analysis: the pathway forward to understanding gene-environment interactions.

Morbidity and mortality associated with complex diseases are expected to increase as the population ages and the number of Americans living in poverty continues to expand. Therefore, improved translation of research findings into clinical practice and public health policy must become a priority. This commentary emphasizes the need for a new research model that accommodates the complex nature of disease etiology.

Discovering how environmental exposures alter genes could lead to new treatments for chronic illnesses.

Emerging research demonstrates that diet, pollution, and other environmental triggers can alter both the function and expression of human genes and lead to a heightened disease risk. These environment-gene interactions can cause so-called epigenetic changes in gene expression-patterns of which genes are switched "on" or "off"-that may account for the rising mortality from chronic diseases in industrialized nations. In this paper, we call for a new transdisciplinary approach to public health that would examine how environmental exposures, both physical and social, influence gene expression and a person's susceptibility to chronic disease. This initiative could lead to new ways to prevent and treat such illnesses.

Arachidonic acid stimulates formation of a novel complex containing nucleolin and RhoA.

Arachidonic acid (AA) stimulates cell adhesion through a p38 mitogen activated protein kinase-mediated RhoA signaling pathway. Here we report that a proteomic screen following AA-treatment identified nucleolin, a multifunctional nucleolar protein, in a complex with the GTPase, RhoA, that also included the Rho kinase, ROCK. AA-stimulated cell adhesion was inhibited by expression of nucleolin-targeted shRNA and formation of the multiprotein complex was blocked by expression of dominant-negative RhoA. AA-treatment also induced ROCK-dependent serine phosphorylation of nucleolin and translocation of nucleolin from the nucleus to the cytoplasm, where it appeared to co-localize with RhoA. These data suggest the existence of a new signaling pathway through which the location and post-translational state of nucleolin are modulated.

Lysine 63-linked ubiquitination is important for arachidonic acid-induced cellular adhesion and migration.

Arachidonic acid, a dietary cis-polyunsaturated fatty acid, stimulates adhesion and migration of human cancer cells on the extracellular matrix by activation of intracellular signaling pathways. Polyubiquitin chains bearing linkages through different lysine residues convey distinct structural and functional information that is important for signal transduction. We investigated whether ubiquitination was required for arachidonic acid-induced cellular adhesion and migration of MDA-MB-435 cells on collagen type IV. An E1 (ubiquitin-activating enzyme) inhibitor, PYR-431, completely abrogated arachidonic acid-stimulated adhesion. Additionally, expression of a lysine null mutant ubiquitin prevented activation of cellular adhesion. Cells expressing ubiquitin in which lysine 63 (K63) was mutated to arginine (K63R) were unable to adhere to collagen upon exposure to arachidonic acid. When K63 was the only lysine present, the cells retained the ability to adhere, indicating that K63-linked ubiquitin is both necessary and sufficient. Moreover, K63-linked ubiquitin was required for the induction of cell migration by arachidonic acid. The ubiquitin mutants and PYR-431 did not prevent arachidonic acid-induced phosphorylation of TGF-ß activated kinase-1 (TAK1) and p38 MAPK, suggesting K63-linked ubiquitination occurs downstream of MAPK. These novel findings are the first to demonstrate a role for K63-linked ubiquitination in promoting cell adhesion and migration.

Finding synergy: reducing disparities in health by modifying multiple determinants.

Although researchers acknowledge that health disparities have multiple determinants, most recommendations for reducing inequities focus on a single approach. We suggest integrating 2 approaches for reducing disparities: improving access to primary care and updating and more vigorously enforcing consumer and environmental protection laws. This strategy could reduce the main causes of disparities, such as chronic diseases and injuries; win public and policymaker support; and provide a cost-effective start for achieving equity. Most of the scientific knowledge needed to implement this strategy exists, thus years of additional research would not be needed. Developing targeted regulatory and health care policies to reduce deaths from chronic diseases and injuries would be a major step forward in eliminating health disparities in the United States.

Arachidonic acid stimulates cell adhesion through a novel p38 MAPK-RhoA signaling pathway that involves heat shock protein 27.

Rho GTPases are critical components of cellular signal transduction pathways. Both hyperactivity and overexpression of these proteins have been observed in human cancers and have been implicated as important factors in metastasis. We previously showed that dietary n-6 fatty acids increase cancer cell adhesion to extracellular matrix proteins, such as type IV collagen. Here we report that in MDA-MB-435 human melanoma cells, arachidonic acid activates RhoA, and inhibition of RhoA signaling with either C3 exoenzyme or dominant negative Rho blocked arachidonic acid-induced cell adhesion. Inhibition of the Rho kinase (ROCK) with either small molecule inhibitors or ROCK II-specific small interfering RNA (siRNA) blocked the fatty acid-induced adhesion. However, unlike other systems, inhibition of ROCK did not block the activation of p38 mitogen-activated protein kinase (MAPK); instead, Rho activation depended on p38 MAPK activity and the presence of heat shock protein 27 (HSP27), which is phosphorylated downstream of p38 after arachidonic acid treatment. HSP27 associated with p115RhoGEF in fatty acid-treated cells, and this association was blocked when p38 was inhibited. Furthermore, siRNA knockdown of HSP27 blocked the fatty acid-stimulated Rho activity. Expression of dominant negative p115-RhoGEF or p115RhoGEF-specific siRNA inhibited both RhoA activation and adhesion on type IV collagen, whereas a constitutively active p115RhoGEF restored the arachidonic acid stimulation in cells in which the p38 MAPK had been inhibited. These data suggest that n-6 dietary fatty acids stimulate a set of interactions that regulates cell adhesion through RhoA and ROCK II via a p38 MAPK-dependent association of HSP27 and p115RhoGEF.