Implementation of multiomic mass spectrometry approaches for the evaluation of human health following environmental exposure.

Omics analyses collectively refer to the possibility of profiling genetic variants, RNA, epigenetic markers, proteins, lipids, and metabolites. The most common analytical approaches used for detecting molecules present within biofluids related to metabolism are vibrational spectroscopy techniques, represented by infrared, Raman, and nuclear magnetic resonance (NMR) spectroscopies and mass spectrometry (MS). Omics-based assessments utilizing MS are rapidly expanding and being applied to various scientific disciplines and clinical settings. Most of the omics instruments are operated by specialists in dedicated laboratories; however, the development of miniature portable omics has made the technology more available to users for field applications. Variations in molecular information gained from omics approaches are useful for evaluating human health following environmental exposure and the development and progression of numerous diseases. As MS technology develops so do statistical and machine learning methods for the detection of molecular deviations from personalized metabolism, which are correlated to altered health conditions, and they are intended to provide a multi-disciplinary overview for researchers interested in adding multiomic analysis to their current efforts. This includes an introduction to mass spectrometry-based omics technologies, current state-of-the-art capabilities and their respective strengths and limitations for surveying molecular information. Furthermore, we describe how knowledge gained from these assessments can be applied to personalized medicine and diagnostic strategies.

Lipid mediators of inhalation exposure-induced pulmonary toxicity and inflammation.

Many inhalation exposures induce pulmonary inflammation contributing to disease progression. Inflammatory processes are actively regulated via mediators including bioactive lipids. Bioactive lipids are potent signaling molecules involved in both pro-inflammatory and resolution processes through receptor interactions. The formation and clearance of lipid signaling mediators are controlled by multiple metabolic enzymes. An imbalance of these lipids can result in exacerbated and sustained inflammatory processes which may result in pulmonary damage and disease. Dysregulation of pulmonary bioactive lipids contribute to inflammation and pulmonary toxicity following exposures. For example, inhalation of cigarette smoke induces activation of pro-inflammatory bioactive lipids such as sphingolipids, and ceramides contributing to chronic obstructive pulmonary disease. Additionally, exposure to silver nanoparticles causes dysregulation of inflammatory resolution lipids. As inflammation is a common consequence resulting from inhaled exposures and a component of numerous diseases it represents a broadly applicable target for therapeutic intervention. With new appreciation for bioactive lipids, technological advances to reliably identify and quantify lipids have occurred. In this review, we will summarize, integrate, and discuss findings from recent studies investigating the impact of inhaled exposures on pro-inflammatory and resolution lipids within the lung and their contribution to disease. Throughout the review current knowledge gaps in our understanding of bioactive lipids and their contribution to pulmonary effects of inhaled exposures will be presented. New methods being employed to detect and quantify disruption of pulmonary lipid levels following inhalation exposures will be highlighted. Lastly, we will describe how lipid dysregulation could potentially be addressed by therapeutic strategies to address inflammation.

Angiogenic potential of skeletal muscle derived extracellular vesicles differs between oxidative and glycolytic muscle tissue in mice.

Skeletal muscle fibers regulate surrounding endothelial cells (EC) via secretion of numerous angiogenic factors, including extracellular vesicles (SkM-EV). Muscle fibers are broadly classified as oxidative (OXI) or glycolytic (GLY) depending on their metabolic characteristics. OXI fibers secrete more pro-angiogenic factors and have greater capillary densities than GLY fibers. OXI muscle secretes more EV than GLY, however it is unknown whether muscle metabolic characteristics regulate EV contents and signaling potential. EVs were isolated from primarily oxidative or glycolytic muscle tissue from mice. MicroRNA (miR) contents were determined and endothelial cells were treated with OXI- and GLY-EV to investigate angiogenic signaling potential. There were considerable differences in miR contents between OXI- and GLY-EV and pathway analysis identified that OXI-EV miR were predicted to positively regulate multiple endothelial-specific pathways, compared to GLY-EV. OXI-EV improved in vitro angiogenesis, which may have been mediated through nitric oxide synthase (NOS) related pathways, as treatment of endothelial cells with a non-selective NOS inhibitor abolished the angiogenic benefits of OXI-EV. This is the first report to show widespread differences in miR contents between SkM-EV isolated from metabolically different muscle tissue and the first to demonstrate that oxidative muscle tissue secretes EV with greater angiogenic signaling potential than glycolytic muscle tissue.

Compositional variations in metal nanoparticle components of welding fumes impact lung epithelial cell toxicity.

Welding fumes contain harmful metals and gas by-products associated with development of lung dysfunction, asthma, bronchitis, and lung cancer. Two prominent welding fume particulate metal components are nanosized iron (Fe) and manganese (Mn) which might induce oxidative stress and inflammation resulting in pulmonary injury. Welding fume toxicity may be dependent upon metal nanoparticle (NP) components. To examine toxicity of welding fume NP components, a system was constructed for controlled and continuous NP generation from commercial welding and customized electrodes with varying proportions of Fe and Mn. Aerosols generated consisted of nanosized particles and were compositionally consistent with each electrode. Human alveolar lung A459 epithelial cells were exposed to freshly generated metal NP mixtures at a target concentration of 100 µg/m3 for 6 hr and then harvested for assessment of cytotoxicity, generation of reactive oxygen species (ROS), and alterations in the expression of genes and proteins involved in metal regulation, inflammatory responses, and oxidative stress. Aerosol exposures decreased cell viability and induced increased ROS production. Assessment of gene expression demonstrated variable up-regulation in cellular mechanisms related to metal transport and storage, inflammation, and oxidative stress based upon aerosol composition. Specifically, interleukin-8 (IL-8) demonstrated the most robust changes in both transcriptional and protein levels after exposure. Interleukin-8 has been determined to serve as a primary cytokine mediating inflammatory responses induced by welding fume exposures in alveolar epithelial cells. Overall, this study demonstrated variations in cellular responses to metal NP mixtures suggesting compositional variations in NP content within welding fumes may influence inhalation toxicity.

Pulmonary and neurological health effects associated with exposure to representative composite manufacturing emissions and corresponding alterations in circulating metabolite profiles.

Cured-in-place pipe (CIPP) technology is increasingly being utilized to repair aging and damaged pipes, however, there are concerns associated with the public health hazards of emissions. CIPP installation involves the manufacture of a new plastic composite pipe at the worksite and includes multiple variable components including resin material, curing methods, and operational conditions. We hypothesize styrene-based composite manufacturing emissions (CMEs) will induce greater pulmonary inflammatory responses and oxidative stress, as well as neurological toxicity compared with nonstyrene CMEs. Further, these CME-toxicological responses will be sex- and time-dependent. To test the hypothesis, representative CMEs were generated using a laboratory curing chamber and characterized using thermal desorption-gas chromatography-mass spectrometry and photoionization detector. Styrene was released during staying, isothermal curing, and cooling phases of the process and peaked during the cooling phase. Male and female C57BL6/J mice were utilized to examine alterations in pulmonary responses and neurotoxicity 1 day and 7 days following exposure to air (controls), nonstyrene-CMEs, or styrene-CMEs. Serum styrene metabolites were increased in mice exposed to styrene-CMEs. Metabolic and lipid profiling revealed alterations related to CIPP emissions that were resin-, time-, and sex-dependent. Exposure to styrene-CMEs resulted in an influx of lymphocytes in both sexes. Expression of inflammatory and oxidative stress markers, including Tnfα, Vcam1, Ccl2, Cxcl2, Il6, Cxcl1, Tgfß1, Tgmt2, and Hmox1, displayed alterations following exposure to emissions. These changes in pulmonary and neurological markers of toxicity were dependent on resin type, sex, and time. Overall, this study demonstrates resin-specific differences in representative CMEs and alterations in toxicity endpoints, which can potentially inform safer utilization of composite manufacturing processes.

Regulatory Significance of Plastic Manufacturing Air Pollution Discharged into Terrestrial Environments and Real-Time Sensing Challenges.

Cured-in-place-pipe (CIPP) is an onsite plastic manufacturing technology used in the U.S. and has not been evaluated for regulatory compliance with federal air pollution laws. The practice involves the discharge of manufacturing waste into the environment. The study goal was to estimate the magnitude of volatile organic compounds (VOCs) discharged into the atmosphere for styrene and nonstyrene composite manufacture and examine low-cost air monitoring sensor reliability. Time-resolved emission analysis revealed that VOC emission was not only isolated to the thermal curing period but also occurred before and after curing. In addition to the styrene monomer, other gas-phase hazardous air pollutants regulated under the Clean Air Act were also emitted. Based on typical CIPP installations, 0.9 to 16.6 U.S. tons of emitted VOCs were estimated for styrene CIPPs, and 0.09 to 1.6 U.S. tons of emitted VOCs were estimated for nonstyrene CIPPs. Because the number and size of CIPPs manufactured in a single community can vary, the total air pollution burden will significantly differ across communities. Low-cost VOC sensors commonly utilized near CIPP manufacturing activities did not accurately quantify styrene and should not be relied upon for that purpose. Up to several thousand-fold detection differences were observed. Regulatory evaluation of CIPP air pollution and PID sensor reliability assessments are recommended.

Enhanced silver nanoparticle-induced pulmonary inflammation in a metabolic syndrome mouse model and resolvin D1 treatment.

BACKGROUND: Metabolic syndrome (MetS) exacerbates susceptibility to inhalation exposures such as particulate air pollution, however, the mechanisms responsible remain unelucidated. Previously, we determined a MetS mouse model exhibited exacerbated pulmonary inflammation 24 h following AgNP exposure compared to a healthy mouse model. This enhanced response corresponded with reduction of distinct resolution mediators. We hypothesized silver nanoparticle (AgNP) exposure in MetS results in sustained pulmonary inflammation. Further, we hypothesized treatment with resolvin D1 (RvD1) will reduce exacerbations in AgNP-induced inflammation due to MetS. RESULTS: To evaluate these hypotheses, healthy and MetS mouse models were exposed to vehicle (control) or AgNPs and a day later, treated with resolvin D1 (RvD1) or vehicle (control) via oropharyngeal aspiration. Pulmonary lung toxicity was evaluated at 3-, 7-, 14-, and 21-days following AgNP exposure. MetS mice exposed to AgNPs and receiving vehicle treatment, demonstrated exacerbated pulmonary inflammatory responses compared to healthy mice. In the AgNP exposed mice receiving RvD1, pulmonary inflammatory response in MetS was reduced to levels comparable to healthy mice exposed to AgNPs. This included decreases in neutrophil influx and inflammatory cytokines, as well as elevated anti-inflammatory cytokines. CONCLUSIONS: Inefficient resolution may contribute to enhancements in MetS susceptibility to AgNP exposure causing an increased pulmonary inflammatory response. Treatments utilizing specific resolution mediators may be beneficial to individuals suffering MetS following inhalation exposures.

Modulation of Pulmonary Toxicity in Metabolic Syndrome Due to Variations in Iron Oxide Nanoparticle-Biocorona Composition.

Nanoparticles (NPs) interact with biomolecules by forming a biocorona (BC) on their surface after introduction into the body and alter cell interactions and toxicity. Metabolic syndrome (MetS) is a prevalent condition and enhances susceptibility to inhaled exposures. We hypothesize that distinct NP-biomolecule interactions occur in the lungs due to MetS resulting in the formation of unique NP-BCs contributing to enhanced toxicity. Bronchoalveolar lavage fluid (BALF) was collected from healthy and MetS mouse models and used to evaluate variations in the BC formation on 20 nm iron oxide (Fe3O4) NPs. Fe3O4 NPs without or with BCs were characterized for hydrodynamic size and zeta potential. Unique and differentially associated proteins and lipids with the Fe3O4 NPs were identified through proteomic and lipidomic analyses to evaluate BC alterations based on disease state. A mouse macrophage cell line was utilized to examine alterations in cell interactions and toxicity due to BCs. Exposures to 6.25, 12.5, 25, and 50 µg/mL of Fe3O4 NPs with BCs for 1 h or 24 h did not demonstrate overt cytotoxicity. Macrophages increasingly associated Fe3O4 NPs following addition of the MetS BC compared to the healthy BC. Macrophages exposed to Fe3O4 NPs with a MetS-BC for 1 h or 24 h at a concentration of 25 µg/mL demonstrated enhanced gene expression of inflammatory markers: CCL2, IL-6, and TNF-α compared to Fe3O4 NPs with a healthy BC. Western blot analysis revealed activation of STAT3, NF-κB, and ERK pathways due to the MetS-BC. Specifically, the Jak/Stat pathway was the most upregulated inflammatory pathway following exposure to NPs with a MetS BC. Overall, our study suggests the formation of distinct BCs due to NP exposure in MetS, which may contribute to exacerbated inflammatory effects and susceptibility.

Emergency responder and public health considerations for plastic sewer lining chemical waste exposures in indoor environments.

The cured-in-place pipe (CIPP) manufacturing process is used to repair buried pipes, and its waste commonly discharged into the air can enter nearby buildings. Exposure can prompt illness and the need for medical care. A mass balance model was applied to estimate indoor styrene concentrations due to intrusion of CIPP emissions through plumbing under different bathroom ventilation conditions. To better understand building contamination and recommend emergency response actions, calculations to estimate chemical intrusion through plumbing were developed. Field reports and study calculations showed that contractor-applied external pressures during plastic manufacture have and can displace plumbing trap water seals. Modeled styrene vapor concentrations that entered the building (1, 300, 1000 ppm) were similar to those measured at CIPP worksites. Modeling revealed that in some cases, bathroom exhaust fan operation during a CIPP project may increase indoor styrene concentrations due to enhanced entrainment of styrene-laden air from the sink and toilet. However, styrene concentrations decreased with increasing air leakage across the bathroom door due to reduced suction from the plumbing system. CIPP waste discharge should be treated as a hazardous material release and can pose a threat to human health. Immediate building evacuation, respiratory protection, provision of medical assistance, source elimination, and building decontamination are recommended.

Bystander chemical exposures and injuries associated with nearby plastic sewer pipe manufacture: public health practice and lessons.

Cured-in-place pipes (CIPPs) are plastic liners manufactured inside existing damaged sanitary sewer, storm sewer, and water pipes that extend the service life of host pipes. This process often is conducted in neighborhoods and near roadways. Before, during, and after plastic manufacture, waste materials that include volatile materials are released into the air. Emissions from this manufacturing process can affect outdoor air quality and indoor air quality for buildings connected to the sewer system. We identified key issues and solicited stakeholder feedback to estimate and manage public health risks of CIPP-generated chemical air pollution. A work group representing 13 U.S. agencies and public health associations provided feedback and prioritized public health issues for action. To mitigate potential public and occupational health risks, additional testing and public health educational efforts were recommended. An improved understanding of CIPP chemical exposure pathways, as well as stakeholder needs and interests, is essential.

Disruption of pulmonary resolution mediators contribute to exacerbated silver nanoparticle-induced acute inflammation in a metabolic syndrome mouse model.

Pre-existing conditions modulate sensitivity to numerous xenobiotic exposures such as air pollution. Specifically, individuals suffering from metabolic syndrome (MetS) demonstrate enhanced acute inflammatory responses following particulate matter inhalation. The mechanisms associated with these exacerbated inflammatory responses are unknown, impairing interventional strategies and our understanding of susceptible populations. We hypothesize MetS-associated lipid dysregulation influences mediators of inflammatory resolution signaling contributing to increased acute pulmonary toxicity. To evaluate this hypothesis, healthy and MetS mouse models were treated with either 18-hydroxy eicosapentaenoic acid (18-HEPE), 14-hydroxy docosahexaenoic acid (14-HDHA), 17-hydroxy docosahexaenoic acid (17-HDHA), or saline (control) via intraperitoneal injection prior to oropharyngeal aspiration of silver nanoparticles (AgNP). In mice receiving saline treatment, AgNP exposure resulted in an acute pulmonary inflammatory response that was exacerbated in MetS mice. A targeted lipid assessment demonstrated 18-HEPE, 14-HDHA, and 17-HDHA treatments altered lung levels of specialized pro-resolving lipid mediators (SPMs). 14-HDHA and 17-HDHA treatments more efficiently reduced the exacerbated acute inflammatory response in AgNP exposed MetS mice as compared to 18-HEPE. This included decreased neutrophilic influx, diminished induction of inflammatory cytokines/chemokines, and reduced alterations in SPMs. Examination of SPM receptors determined baseline reductions in MetS mice compared to healthy as well as decreases due to AgNP exposure. Overall, these results demonstrate AgNP exposure disrupts inflammatory resolution, specifically 14-HDHA and 17-HDHA derived SPMs, in MetS contributing to exacerbated acute inflammatory responses. Our findings identify a potential mechanism responsible for enhanced susceptibility in MetS that can be targeted for interventional therapeutic approaches.

Extracellular vesicles released from stress-induced prematurely senescent myoblasts impair endothelial function and proliferation.

NEW FINDINGS: What is the central question of this study? What is the impact of stress-induced premature senescence on skeletal muscle myoblast-derived extracellular vesicles (EVs) and myoblast-endothelial cell crosstalk? What is the main finding and its importance? Hydrogen peroxide treatment of human myoblasts induced stress-induced premature senescence (SIPS) and increased the release of exosome-sized EVs (30-150 nm in size) five-fold compared to untreated controls. Treatment of SIPS myoblast-derived EVs on endothelial cells increased senescence markers and decreased proliferation. Gene expression analysis of SIPS myoblast-derived EVs revealed a four-fold increase in senescence factor transforming growth factor-ß. These results highlight potential mechanisms by which senescence imparts deleterious effects on the cellular microenvironment. ABSTRACT: Cellular senescence contributes to numerous diseases through the release of pro-inflammatory factors as part of the senescence-associated secretory phenotype (SASP). In skeletal muscle, resident muscle progenitor cells (satellite cells) express markers of senescence with advancing age and in response to various pathologies, which contributes to reduced regenerative capacities in vitro. Satellite cells regulate their microenvironment in part through the release of extracellular vesicles (EVs), but the effect of senescence on EV signaling is unknown. Primary human myoblasts were isolated following biopsies of the vastus lateralis from young healthy subjects. Hydrogen peroxide (H2 O2 ) treatment was used to achieve stress-induced premature senescence (SIPS) of myoblasts. EVs secreted by myoblasts with and without H2 O2 treatment were isolated, analysed and used to treat human umbilical vein endothelial cells (HUVECs) to assess senescence and angiogenic impact. H2 O2 treatment of primary human myoblasts in vitro increased markers of senescence (ß-galactosidase and p21Cip1 ), decreased proliferation and increased exosome-like EV (30-150 nm) release approximately five-fold. In HUVECs, EV treatment from H2 O2 -treated myoblasts increased markers of senescence (ß-galactosidase and transforming growth factor ß), decreased proliferation and impaired HUVEC tube formation. Analysis of H2 O2 -treated myoblast-derived EV mRNA revealed a nearly four-fold increase in transforming growth factor ß expression. Our novel results highlight the impact of SIPS on myoblast communication and identify a VasoMyo Crosstalk by which SIPS myoblast-derived EVs impair endothelial cell function in vitro.

Exacerbation of Nanoparticle-Induced Acute Pulmonary Inflammation in a Mouse Model of Metabolic Syndrome.

Nanotechnology has the capacity to revolutionize numerous fields and processes, however, exposure-induced health effects are of concern. The majority of nanoparticle (NP) safety evaluations have been performed utilizing healthy models and have demonstrated the potential for pulmonary toxicity. A growing proportion of individuals suffer diseases that may enhance their susceptibility to exposures. Specifically, metabolic syndrome (MetS) is increasingly prevalent and is a risk factor for the development of chronic diseases including type-2 diabetes, cardiovascular disease, and cancer. MetS is a combination of conditions which includes dyslipidemia, obesity, hypertension, and insulin resistance. Due to the role of lipids in inflammatory signaling, we hypothesize that MetS-associated dyslipidemia may modulate NP-induced immune responses. To examine this hypothesis, mice were fed either a control diet or a high-fat western diet (HFWD) for 14-weeks. A subset of mice were treated with atorvastatin for the final 7-weeks to modulate lipids. Mice were exposed to silver NPs (AgNPs) via oropharyngeal aspiration and acute toxicity endpoints were evaluated 24-h post-exposure. Mice on the HFWD demonstrated MetS-associated alterations such as increased body weight and cholesterol compared to control-diet mice. Cytometry analysis of bronchoalveolar lavage fluid (BALF) demonstrated exacerbation of AgNP-induced neutrophilic influx in MetS mice compared to healthy. Additionally, enhanced proinflammatory mRNA expression and protein levels of monocyte chemoattractant protein-1, macrophage inflammatory protein-2, and interleukin-6 were observed in MetS mice compared to healthy following exposure. AgNP exposure reduced mRNA expression of enzymes involved in lipid metabolism, such as arachidonate 5-lipoxygenase and arachidonate 15-lipoxygenase in both mouse models. Exposure to AgNPs decreased inducible nitric oxide synthase gene expression in MetS mice. An exploratory lipidomic profiling approach was utilized to screen lipid mediators involved in pulmonary inflammation. This assessment indicates the potential for reduced levels of lipids mediators of inflammatory resolution (LMIR) in the MetS model compared to healthy mice following AgNP exposure. Statin treatment inhibited enhanced inflammatory responses as well as alterations in LMIR observed in the MetS model due to AgNP exposure. Taken together our data suggests that MetS exacerbates the acute toxicity induced by AgNPs exposure possibly via a disruption of LMIR leading to enhanced pulmonary inflammation.

Neuromelanin Modulates Heterocyclic Aromatic Amine-Induced Dopaminergic Neurotoxicity.

Heterocyclic aromatic amines (HAAs) are mutagens and potential human carcinogens. Our group and others have demonstrated that HAAs may also produce selective dopaminergic neurotoxicity, potentially relevant to Parkinson's disease (PD). The goal of this study was to elucidate mechanisms of HAA-induced neurotoxicity through examining a translational biochemical weakness of common PD models. Neuromelanin is a pigmented byproduct of dopamine metabolism that has been debated as being both neurotoxic and neuroprotective in PD. Importantly, neuromelanin is known to bind and potentially release dopaminergic neurotoxicants, including HAAs (eg, ß-carbolines such as harmane). Binding of other HAA subclasses (ie, aminoimidazoaazarenes) to neuromelanin has not been investigated, nor has a specific role for neuromelanin in mediating HAA-induced neurotoxicity been examined. Thus, we investigated the role of neuromelanin in modulating HAA-induced neurotoxicity. We characterized melanin from Sepia officinalis and synthetic dopamine melanin, proposed neuromelanin analogs with similar biophysical properties. Using a cell-free assay, we demonstrated strong binding of harmane and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) to neuromelanin analogs. To increase cellular neuromelanin, we transfected SH-SY5Y neuroblastoma cells with tyrosinase. Relative to controls, tyrosinase-expressing cells exhibited increased neuromelanin levels, cellular HAA uptake, cell toxicity, and oxidative damage. Given that typical cellular and rodent PD models form far lower neuromelanin levels than humans, there is a critical translational weakness in assessing HAA-neurotoxicity. The primary impacts of these results are identification of a potential mechanism by which HAAs accumulate in catecholaminergic neurons and support for the need to conduct neurotoxicity studies in systems forming neuromelanin.

Factors secreted from high glucose treated endothelial cells impair expansion and differentiation of human skeletal muscle satellite cells.

KEY POINTS: Cellular communication occurs between endothelial cells and skeletal muscle satellite cells and is mitogenic for both cell types under normal conditions. Skeletal muscle atrophy and endothelial cell dysfunction occur in tandem in cardiovascular disease, type II diabetes and ageing. The present study investigated how induction of endothelial cell dysfunction via high glucose treatment impacts growth and differentiation of human skeletal muscle satellite cells in vitro. Secreted factors from high glucose treated endothelial cells impaired satellite cell expansion and differentiation via decreased proliferation and dysregulation of p38 mitogen-activated protein kinase in satellite cells committed to myogenesis. These findings highlight a novel potential role for endothelial cells in the development and pathology of skeletal muscle atrophy, which is common in patients with endothelial dysfunction related pathologies. ABSTRACT: Cross-talk between endothelial cells (ECs) and skeletal muscle satellite cells (MuSC) has been identified as an important regulator of cellular functions in both cell types. In healthy conditions, EC secreted factors promote MuSC growth and differentiation. Endothelial and satellite cell dysfunction occur in tandem in many disease states; however, no data exist examining the impact of dysfunctional EC signalling on satellite cells. Therefore, the present study aimed to evaluate the effect that factors secreted from high glucose (HG) treated ECs have on the growth and differentiation of human satellite cells (HMuSC) using a conditioned medium (CM) cell culture model. Satellite cells were isolated from human skeletal muscle and grown in CM from normal or HG treated human umbilical vein ECs (HUVECs). Satellite cells grown in CM from HG treated HUVECs reduced growth (25%), differentiation (25%) and myonuclear fusion (35%). These responses were associated with increased superoxide (50%) and inflammatory cytokines (25-50%) in HG treated HUVECs and HG-CM. Decreased expansion of HG-CM treated HMuSCs was driven by a decrease in proliferation. Impaired gene expression and protein content of myogenic differentiation factors were preceded by decreased phosphorylation of p38 mitogen-activated protein kinase in HMuSC treated with CM from HG treated HUVECs. The results obtained in the present study are the first to show that factors secreted from HG treated ECs cause impairments in human muscle satellite cell growth and differentiation in vitro, highlighting endothelial cell health and secretion as a potential target for treating vascular disease-associated skeletal muscle dysfunction.

Advanced Glycation End-Products Suppress Mitochondrial Function and Proliferative Capacity of Achilles Tendon-Derived Fibroblasts.

Debilitating cases of tendon pain and degeneration affect the majority of diabetic individuals. The high rate of tendon degeneration persists even when glucose levels are well controlled, suggesting that other mechanisms may drive tendon degeneration in diabetic patients. The purpose of this study was to investigate the impact of advanced glycation end-products on tendon fibroblasts to further our mechanistic understanding of the development and progression of diabetic tendinopathy. We proposed that advanced glycation end-products would induce limitations to mitochondrial function and proliferative capacity in tendon-derived fibroblasts, restricting their ability to maintain biosynthesis of tendon extracellular matrix. Using an in-vitro cell culture system, rat Achilles tendon fibroblasts were treated with glycolaldehyde-derived advanced glycation end-products (0, 50, 100, and 200 µg/ml) for 48 hours in normal glucose (5.5 mM) and high glucose (25 mM) conditions. We demonstrate that tendon fibroblasts treated with advanced glycation end-products display reduced ATP production, electron transport efficiency, and proliferative capacity. These impairments were coupled with alterations in mitochondrial DNA content and expression of genes associated with extracellular matrix remodeling, mitochondrial energy metabolism, and apoptosis. Our findings suggest that advanced glycation end-products disrupt tendon fibroblast homeostasis and may be involved in the development and progression of diabetic tendinopathy.

Early Proteome Shift and Serum Bioactivity Precede Diesel Exhaust-induced Impairment of Cardiovascular Recovery in Spontaneously Hypertensive Rats.

Single circulating factors are often investigated to explain air pollution-induced cardiovascular dysfunction, yet broader examinations of the identity and bioactivity of the entire circulating milieu remain understudied. The purpose of this study was to determine if exposure-induced cardiovascular dysfunction can be coupled with alterations in both serum bioactivity and the circulating proteome. Two cohorts of Spontaneously Hypertensive Rats (SHRs) were exposed to 150 or 500 µg/m3 diesel exhaust (DE) or filtered air (FA). In Cohort 1, we collected serum 1 hour after exposure for proteomics analysis and bioactivity measurements in rat aortic endothelial cells (RAECs). In Cohort 2, we assessed left ventricular pressure (LVP) during stimulation and recovery from the sympathomimetic dobutamine HCl, one day after exposure. Serum from DE-exposed rats had significant changes in 66 serum proteins and caused decreased NOS activity and increased VCAM-1 expression in RAECs. While rats exposed to DE demonstrated increased heart rate at the start of LVP assessments, heart rate, systolic pressure, and double product fell below baseline in DE-exposed rats compared to FA during recovery from dobutamine, indicating dysregulation of post-exertional cardiovascular function. Taken together, a complex and bioactive circulating milieu may underlie air pollution-induced cardiovascular dysfunction.

An Integrative Proteomic/Lipidomic Analysis of the Gold Nanoparticle Biocorona in Healthy and Obese Conditions.

Introduction: When nanoparticles (NPs) enter a physiological environment, a coating of biomolecules or biocorona (BC) forms on the surface. Formation of the NP-BC is dependent on NP properties, the physiological environment, and time. The BC influences NP properties and biological interactions such as cellular internalization, immune responses, biodistribution, and others, leading to pharmacological and toxicological consequences. To date, examination of the NP-BC has focused primarily on protein components and healthy conditions. Therefore, we evaluated the protein and lipid content of BCs that formed on physicochemically distinct gold nanoparticles (AuNPs) under healthy and obese conditions. A comprehensive understanding of the NP-BC is necessary for the translation of in vitro toxicity assessments to clinical applications. Materials and Methods: AuNPs with two coatings (poly-N-vinylpyrrolidone [PVP] or citrate) and diameters (20 or 100 nm) were incubated in pooled human serum, and an integrated proteomic/lipidomic approach was used to evaluate BC composition. Macrophages were utilized to evaluate differential immune responses due to variations in the AuNP-BC. Results: AuNPs form distinct BCs based on physicochemical properties and the surrounding environment, with the obese BC containing more proteins and fewer lipids than the healthy BC. Differential macrophage inflammatory responses were observed based on AuNP properties and BC composition. Discussion and Conclusion: Overall, these findings demonstrate that AuNP size and coating, as well as physiological environment, influence the protein and lipid composition of the BC, which impacts cellular responses following exposure. These findings demonstrate that incorporation of BCs representing distinct physiological conditions may enhance the translatability of nanosafety in vitro studies.

Biocorona formation contributes to silver nanoparticle induced endoplasmic reticulum stress.

Prior research has demonstrated cells exposed to silver nanoparticles (AgNPs) undergo endoplasmic reticulum (ER) stress leading to cellular apoptosis and toxicity, however, the fundamental mechanism underlying AgNP-induced ER stress is unknown. We hypothesize the biophysical interactions between AgNPs and adsorbed proteins lead to misfolded proteins to elicit an ER stress response. Our investigation examined rat aortic endothelial cells (RAEC) exposed to 20 or 100 nm AgNPs with or without a biocorona (BC) consisting of bovine serum albumin (BSA), high density lipoprotein (HDL) or fetal bovine serum (FBS) to form a complex BC. The presence of a BC consisting of BSA or FBS proteins significantly reduced uptake of 20 nm and 100 nm AgNPs in RAEC. Western blot analysis indicated robust activation of the IREα and PERK pathways in RAEC exposed to 20 nm despite the reduction in uptake by the presence of a BC. This was not observed for the 100 nm AgNPs. Hyperspectral darkfield microscopy qualitatively confirmed that the preformed BC was maintained following uptake by RAEC. Transmission electron microscopy demonstrated a size dependent effect on the sub-cellular localization of AgNPs. Overall, these results suggest that AgNP size, surface area and BC formation governs the induction of ER stress and alterations in intracellular trafficking.

Altered formation of the iron oxide nanoparticle-biocorona due to individual variability and exercise.

Nanoparticles (NPs), introduced into a biological environment, accumulate a coating of biomolecules or biocorona (BC). Although the BC has toxicological and pharmacological consequences, the effects of inter-individual variability and exercise on NP-BC formation are unknown. We hypothesized that NPs incubated in plasma form distinct BCs between individuals, and exercise causes additional intra-individual alterations. 20 nm iron oxide (Fe3O4) NPs were incubated in pre- or post-exercise plasma ex vivo, and proteomics was utilized to evaluate BC components. Analysis demonstrated distinct BC formation between individuals, while exercise was found to enhance NP-BC complexity. Abundance differences of NP-BC proteins were determined between individuals and resulting from exercise. Differential human macrophage response was identified due to NP-BC variability. These findings demonstrate that individuals form unique BCs and that exercise influences NP-biomolecule interactions. An understanding of NP-biomolecule interactions is necessary for elucidation of mechanisms responsible for variations in human responses to NP exposures and/or nano-based therapies.