Aryl hydrocarbon receptor activation-mediated vascular toxicity of ambient fine particulate matter: contribution of polycyclic aromatic hydrocarbons and osteopontin as a biomarker.

BACKGROUND: Exposure to ambient fine particulate matter (PM2.5) is associated with vascular diseases. Polycyclic aromatic hydrocarbons (PAHs) in PM2.5 are highly hazardous; however, the contribution of PM2.5-bound PAHs to PM2.5-associated vascular diseases remains unclear. The ToxCast high-throughput in vitro screening database indicates that some PM2.5-bound PAHs activate the aryl hydrocarbon receptor (AhR). The present study investigated whether the AhR pathway is involved in the mechanism of PM2.5-induced vascular toxicity, identified the PAH in PM2.5 that was the major contributor of AhR activation, and identified a biomarker for vascular toxicity of PM2.5-bound PAHs. RESULTS: Treatment of vascular smooth muscle cells (VMSCs) with an AhR antagonist inhibited the PM2.5-induced increase in the cell migration ability; NF-κB activity; and expression of cytochrome P450 1A1 (CYP1A1), 1B1 (CYP1B1), interleukin-6 (IL-6), and osteopontin (OPN). Most PM2.5-bound PAHs were extracted into the organic fraction, which drastically enhanced VSMC migration and increased mRNA levels of CYP1A1, CYP1B1, IL-6, and OPN. However, the inorganic fraction of PM2.5 moderately enhanced VSMC migration and only increased IL-6 mRNA levels. PM2.5 increased IL-6 secretion through NF-κB activation; however, PM2.5 and its organic extract increased OPN secretion in a CYP1B1-dependent manner. Inhibiting CYP1B1 activity and silencing OPN expression prevented the increase in VSMC migration ability caused by PM2.5 and its organic extract. The AhR activation potencies of seven PM2.5-bound PAHs, reported in the ToxCast database, were strongly correlated with their capabilities of enhancing the migration ability of VSMCs. Benzo(k)fluoranthene (BkF) contributed the most to the AhR agonistic activity of ambient PM2.5-bound PAHs. The association between PM2.5-induced vascular toxicity, AhR activity, and OPN secretion was further verified in mice; PM2.5-induced intimal hyperplasia in pulmonary small arteries and OPN secretion were alleviated in mice with low AhR affinity. Finally, urinary concentrations of 1-hydroxypyrene, a major PAH metabolite, were positively correlated with plasma OPN levels in healthy humans. CONCLUSIONS: The present study offers in vitro, animal, and human evidences supporting the importance of AhR activation for PM2.5-induced vascular toxicities and that BkF was the major contributor of AhR activation. OPN is an AhR-dependent biomarker of PM2.5-induced vascular toxicity. The AhR activation potency may be applied in the risk assessment of vascular toxicity in PAH mixtures.

Echocardiographic parameters and indices in 23 healthy Maltese dogs.

BACKGROUND: Echocardiography is a primary tool used by veterinarians to evaluate heart diseases. In recent years, various studies have targeted standard echocardiographic values for different breeds. Reference data are currently lacking in Maltese dogs and it is important to fill this gap as this breed is predisposed to myxomatous mitral valve disease, which is a volume overload disease. OBJECTIVES: To establish the normal echocardiographic parameters for Maltese dogs. METHODS: In total, 23 healthy Maltese dogs were involved in this study. Blood pressure measurements, thoracic radiography, and complete transthoracic echocardiography were performed. The effects of body weight, age and sex were evaluated, and the correlations between weight and linear and volumetric dimensions were calculated by regression analysis. RESULTS: The mean vertebral heart size was 9.1 ± 0.4. Aside from the ejection fraction, fractional shortening, and the left atrial to aorta root ratio, all the other echocardiographic parameters were significantly correlated with weight. CONCLUSION: This study describes normal echocardiographic parameters that may be useful in the echocardiographic evaluation of Maltese dogs.

Ambient Particulate Matter Induces Vascular Smooth Muscle Cell Phenotypic Changes via NOX1/ROS/NF-κB Dependent and Independent Pathways: Protective Effects of Polyphenols.

Epidemiological studies have demonstrated an association between ambient particulate matter (PM) exposure and vascular diseases. Here, we observed that treatment with ambient PM increased cell migration ability in vascular smooth muscle cells (VSMCs) and pulmonary arterial SMCs (PASMCs). These results suggest that VSMCs and PASMCs transitioned from a differentiated to a synthetic phenotype after PM exposure. Furthermore, treatment with PM increased intracellular reactive oxygen species (ROS), activated the NF-κB signaling pathway, and increased the expression of proinflammatory cytokines in VSMCs. Using specific inhibitors, we demonstrated that PM increased the migration ability of VSMCs via the nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase 1 (NOX1)/ROS-dependent NF-κB signaling pathway, which also partially involved in the induction of proinflammatory cytokines. Finally, we investigated whether nature polyphenolic compounds prevent PM-induced migration and proinflammatory cytokines secretion in VSMCs. Curcumin, resveratrol, and gallic acid prevented PM2.5-induced migration via the ROS-dependent NF-κB signaling pathway. However, honokiol did not prevent PM2.5-induced migration or activation of the ROS-dependent NF-κB signaling pathway. On the other hand, all polyphenols prevented PM2.5-induced cytokines secretion. These data indicated that polyphenols prevented PM-induced migration and cytokine secretion via blocking the ROS-dependent NF-κB signaling pathway in VSMCs. However, other mechanisms may also contribute to PM-induced cytokine secretion.

Identification of ambient fine particulate matter components related to vascular dysfunction by analyzing spatiotemporal variations.

Exposure to ambient fine particulate matter (PM2.5) has been associated with vascular diseases in epidemiological studies. We have demonstrated previously that exposure to ambient PM2.5 caused pulmonary vascular remodeling in mice and increased vascular smooth muscle cells (VSMCs) viability. Here, we further demonstrated that exposure of mice to ambient PM2.5 increased urinary 8­hydroxy­2'­deoxyguanosine (8-OHdG) and cytokines concentrations in the broncheoalveolar lavage. The objective of the present study was to identify the PM2.5 components related to vascular dysfunction. Exposure to PM2.5 collected from various areas and seasons in Taiwan significantly increased viability, oxidative stress, and inflammatory cytokines secretion in VSMCs. The mass concentrations of benz[a]anthracene (BaA), benzo[e]pyrene (BeP), perylene, dibenzo[a,e]pyrene, molybdenum, zinc (Zn), vanadium (V), and nickel in the PM2.5 were significantly associated with increased viability of VSMCs. These components, except BaA and BeP, also were significantly associated with chemokine (CC motif) ligand 5 (CCL5) concentrations in the VSMCs. The effects of V and Zn on cell viability and CCL5 expression, respectively, were verified. In addition, the mass concentrations of sulfate and manganese (Mn) in PM2.5 were significantly correlated with increased oxidative stress; this correlation was also confirmed. After extraction, the inorganic fraction of PM2.5 increased cell viability and oxidative stress, but the organic fraction of PM2.5 increased only cell viability, which was inhibited by an aryl hydrocarbon receptor antagonist. These data suggest that controlling the emission of Zn, V, Mn, sulfate, and PAHs may prevent the occurrence of PM2.5-induced vascular diseases.

Persistent elevation of blood pressure by ambient coarse particulate matter after recovery from pulmonary inflammation in mice.

Exposure to ambient particulate matter (PM) is associated with hypertension and cardiovascular diseases. Recently, we reported that exposure to fine and coarse PM caused pulmonary inflammation and pulmonary small arterial remodeling in mice, and osteopontin (OPN) level was elevated following PM exposure. However, in the present study, cotreatment with 5-methoxytryptophan for 4 weeks partially reduced coarse PM-induced pulmonary inflammation without reducing pulmonary OPN secretion or recovery from pulmonary arterial remodeling in mice. Persistent vascular dysfunction may lead to vascular remodeling. Therefore, we further compared the relationship between coarse PM-induced inflammation and vascular dysfunction by exposing mice to PM before and after cessation of PM exposure. Oropharyngeal aspiration of PM for 8 weeks induced pulmonary inflammation and pulmonary small artery remodeling in mice, as well as increased serum C-reactive protein and OPN concentrations and systolic blood pressure (SBP). After the cessation of PM exposure for another 8 weeks, lung inflammation had recovered and vascular remodeling had partially recovered. Elevation of OPN, metalloproteinases (MMPs), and cytokines in bronchioalveolar lavage were significantly reduced. However, PM-induced systemic responses did not recover after the cessation of PM exposure. Notably, not only serum OPN and SBP remained significantly elevated; also, serum endothelin-1, MMP-9, and keratinocyte-derived chemokine concentrations were significantly increased after cessation of PM exposure for another 8 weeks. These data suggested that systemic inflammation and systemic vascular dysfunction might be important in PM-induced elevation of SBP. Furthermore, SBP elevation was persistent after cessation of PM exposure for 8 weeks.

Identification of osteopontin as a biomarker of human exposure to fine particulate matter.

Ambient particulate matter (PM) exposure is associated with pulmonary and cardiovascular diseases; however, there is scant research linking data on animal and human cells. The objective of this study was to investigate these associations. Vascular remodeling plays a crucial role in both pulmonary and cardiovascular diseases. Therefore, we conducted a transcriptomic analysis using vascular smooth muscle cells (VSMCs) to identify potential regulators or markers of PM exposure. We demonstrated that fine and coarse PM increased VSMC proliferation in mice. We conducted a genome-wide cDNA microarray analysis, followed by a pathway analysis of VSMCs treated with coarse PM for durations of 24, 48, and 72 h. Sixteen genes were discovered to be time-dependently upregulated and involved in VSMC proliferation. Osteopontin (OPN) is indicated as one of the regulators of these upregulated genes. Both fine and coarse PM from industrial and urban areas significantly increased OPN expression in VSMCs and macrophages. Moreover, oropharyngeal instillation of fine and coarse PM for 8 weeks increased the VSMCs in the pulmonary arteries of mice. OPN level was consistently increased in the lung tissues, bronchoalveolar lavage fluid, and serum of mice. Moreover, we analyzed the plasma OPN levels of 72 healthy participants recruited from the studied metropolitan area. Each participant wore a personal PM2.5 sampler to assess their PM2.5 exposure over a 24 h period. Our results indicate that personal exposure to fine PM is positively correlated with plasma OPN level in young adults. The data obtained in this study suggest that exposure to fine and coarse PM may cause pulmonary vascular lesions in humans and that OPN level may be a biomarker of PM exposure in humans.

Author Correction: Geometric Control of Cell Migration.

A correction has been published and is appended to both the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Development of an in Vitro-Based Risk Assessment Framework for Predicting Ambient Particulate Matter-Bound Polycyclic Aromatic Hydrocarbon-Activated Toxicity Pathways.

Polycyclic aromatic hydrocarbons (PAHs) are widely distributed throughout the atmosphere as mixtures attached to ambient particulate matter (PM). PAHs usually elicit similar toxicological pathways but do so with varying levels of efficacy. In this study, we utilized high-throughput screening (HTS) in vitro data of PAHs to predict health risks associated with coarse and fine PM. PM samples with 22 PAH compounds obtained from residential areas close to industrial parks in central Taiwan were analyzed. On the basis of the PM-bound PAH concentrations and their activities reported in HTS assays, we developed a probabilistic model for estimating cumulative exposure of humans to PAHs. Activity-to-exposure ratio (AER) values were calculated to compare relative risks of activating the aryl hydrocarbon receptor (AhR), nuclear factor erythroid 2-related factor 2 (Nrf2), and tumor suppressor gene (p53) when children or adults were exposed to fine or coarse PM in different seasons. On the basis of AER values, the risk of fine PM exposure was relatively higher than the risk of exposure to coarse PM in pathway activation. Children as a susceptible population had a risk of the activating AhR pathway greater than that of adults. Particularly higher risks were observed in winter than in summer. Among three pathways, AhR was the most sensitive one activated by exposure to PAHs. In addition, the activation of the AhR, Nrf2, and p53 pathways was compared by in vitro reporter assays with and without the pre-extraction of PAHs from PM. Our proposed novel approach accounts for mixture toxicities in characterizing in vitro pathway-based risks via inhalation exposure to ambient PAHs.

Interleukin-24 as a target cytokine of environmental aryl hydrocarbon receptor agonist exposure in the lung.

Exposure to environmental aryl hydrocarbon receptor (AhR) agonists, such as halogenated aromatic hydrocarbons and polycyclic aromatic hydrocarbons (PAHs), has great impacts on the development of various lung diseases. As emerging molecular targets for AhR agonists, cytokines may contribute to the inflammatory or immunotoxic effects of environmental AhR agonists. However, general cytokine expression may not specifically indicate environmental AhR agonist exposure. By comparing cytokine and chemokine expression profiles in human lung adenocarcinoma cell line CL5 treated with AhR agonists and the non-AhR agonist polychlorinated biphenyl (PCB) 39, we identified a target cytokine of environmental AhR agonist exposure of in the lungs. Thirteen cytokine and chemokine genes were altered in the AhR agonists-treated cells, but none were altered in the PCB39-treated cells. Interleukin (IL)-24 was the most highly induced gene among AhR-modulated cytokines. Cotreatment with AhR antagonist completely prevented IL-24 induction by AhR agonists in the CL5 cells. Knockdown AhR expression with short-hairpin RNA (shRNA) significantly reduced benzo[a]pyrene (BaP)-induced IL-24 mRNA levels. We further confirmed that gene transcription, but not mRNA stability, was involved in IL-24 upregulation by BaP. Particulate matter (PM) in the ambient air contains some PAHs and is reported to activate AhR. Oropharyngeal aspiration of PM significantly increased IL-24 levels in lung epithelia and in bronchoalveolar lavage fluid of mice 4weeks after treatment. Thus, our data suggests that IL-24 is a pulmonary exposure target cytokine of environmental AhR agonists.

Decoupling the Functional Pleiotropy of Stem Cell Factor by Tuning c-Kit Signaling.

Most secreted growth factors and cytokines are functionally pleiotropic because their receptors are expressed on diverse cell types. While important for normal mammalian physiology, pleiotropy limits the efficacy of cytokines and growth factors as therapeutics. Stem cell factor (SCF) is a growth factor that acts through the c-Kit receptor tyrosine kinase to elicit hematopoietic progenitor expansion but can be toxic when administered in vivo because it concurrently activates mast cells. We engineered a mechanism-based SCF partial agonist that impaired c-Kit dimerization, truncating downstream signaling amplitude. This SCF variant elicited biased activation of hematopoietic progenitors over mast cells in vitro and in vivo. Mouse models of SCF-mediated anaphylaxis, radioprotection, and hematopoietic expansion revealed that this SCF partial agonist retained therapeutic efficacy while exhibiting virtually no anaphylactic off-target effects. The approach of biasing cell activation by tuning signaling thresholds and outputs has applications to many dimeric receptor-ligand systems.

Involvement of the cytokine-IDO1-AhR loop in zinc oxide nanoparticle-induced acute pulmonary inflammation.

Zinc oxide nanoparticles (ZnONPs) are widely used in our daily life, such as in sunscreens and electronic nanodevices. However, pulmonary exposure to ZnONPs causes acute pulmonary inflammation, which is considered as an initial event for various respiratory diseases. Thus, elucidation of the underlying cellular mechanisms of ZnONPs can help us in predicting their potential effects in respiratory diseases. In this study, we observed that ZnONPs increased proinflammatory cytokines, accompanied with an increased expression of aryl hydrocarbon receptor (AhR) and its downstream target cytochrome P450 1A1 (CYP1A1) in macrophages in vitro and in mouse lung epithelia in vivo. Moreover, zinc nitrate, but not silica or titanium dioxide nanoparticles (NPs), had similar effects on macrophages, indicating that the zinc element or ion released from ZnONPs is likely responsible for the activation of the AhR pathway. Cotreatment with an AhR antagonist or AhR knockout reduced ZnONPs-induced cytokine secretion in macrophages or mice, respectively. Furthermore, kynurenine (KYN), an endogenous AhR agonist and a tryptophan metabolite catalyzed by indoleamine 2,3-dioxygenase (IDO), was increased in the serums of mice that aspirated ZnONPs. Consistently, ZnONPs increased IDO1 expression in lung cells in vitro and in vivo. Finally, AhR knockout reduced ZnONPs-induced pulmonary inflammation, cytokine secretion and KYN production in mice, suggesting that AhR activation is involved in ZnONPs-induced cytokine secretion and pulmonary inflammation. In summary, we demonstrated that the pulmonary exposure of ZnONPs stimulated the cytokine-IDO1-AhR loop in the lungs, which has been implied to play roles in immune dysfunctions.

Durable antitumor responses to CD47 blockade require adaptive immune stimulation.

Therapeutic antitumor antibodies treat cancer by mobilizing both innate and adaptive immunity. CD47 is an antiphagocytic ligand exploited by tumor cells to blunt antibody effector functions by transmitting an inhibitory signal through its receptor signal regulatory protein alpha (SIRPα). Interference with the CD47-SIRPα interaction synergizes with tumor-specific monoclonal antibodies to eliminate human tumor xenografts by enhancing macrophage-mediated antibody-dependent cellular phagocytosis (ADCP), but synergy between CD47 blockade and ADCP has yet to be demonstrated in immunocompetent hosts. Here, we show that CD47 blockade alone or in combination with a tumor-specific antibody fails to generate antitumor immunity against syngeneic B16F10 tumors in mice. Durable tumor immunity required programmed death-ligand 1 (PD-L1) blockade in combination with an antitumor antibody, with incorporation of CD47 antagonism substantially improving response rates. Our results highlight an underappreciated contribution of the adaptive immune system to anti-CD47 adjuvant therapy and suggest that targeting both innate and adaptive immune checkpoints can potentiate the vaccinal effect of antitumor antibody therapy.

Quantum dots induced interferon beta expression via TRIF-dependent signaling pathways by promoting endocytosis of TLR4.

Quantum dots (QDs) are nano-sized semiconductors. Previously, intratracheal instillation of QD705s induces persistent inflammation and remodeling in the mouse lung. Expression of interferon beta (IFN-ß), involved in tissue remodeling, was induced in the mouse lung. The objective of this study was to understand the mechanism of QD705 induced interferon beta (IFN-ß) expression. QD705-COOH and QD705-PEG increased IFN-ß and IP-10 mRNA levels during day 1 to 90 post-exposure in mouse lungs. QD705-COOH increased IFN-ß expression via Toll/interleukin-1 receptor domain-containing adapter protein (TRIF) dependent Toll-like receptor (TLR) signaling pathways in macrophages RAW264.7. Silencing TRIF expression with siRNA or co-treatment with a TRIF inhibitor tremendously abolished QD705s-induced IFN-ß expression. Co-treatment with a TLR4 inhibitor completely prevented IFN-ß induction by QD705-COOH. QD705-COOH readily entered cells, and co-treatment with either inhibitors of endocytosis or intracellular TLRs prevented IFN-ß induction. Thus, activation of the TRIF dependent TLRs pathway by promoting endocytosis of TLR4 is one of the mechanisms for immunomodulatory effects of nanoparticles.

Cytoskeletal architecture and cell motility remain unperturbed in mouse embryonic fibroblasts from Plk3 knockout mice.

Polo-like kinase 3 (Plk3) is best known for its involvement in cell cycle checkpoint regulation following exposure to cytotoxicants or induction of DNA damage. Yet, Plk3 has also been implicated in roles beyond those of cellular responses to DNA damage. Here, we have investigated the proposition, suggested by the Plk literature, that Plk3 regulates cytoskeletal architecture and cell functions mediated by the cytoskeleton. To this end, we have assayed mouse embryonic fibroblasts (MEFs) generated from both Plk3 knockout and wild-type mice. In particular, we asked whether Plk3 is involved in actin fiber and microtubule integrity, cell migration, cell attachment, and/or cell invasion. Our results demonstrate that functional Plk3 is not critical for the regulation of cytoskeletal integrity, cell morphology, cell adhesion, or motility in MEFs.

Engineering Dynamic Biointerfaces.

Recent advances in dynamic biointerfaces enable spatiotemporal control over cell position and migration after attachment using substrates that employ chemical, optical, thermal, or electrical triggers. This review focuses on flexible and accessible methods for the fabrication of cellular arrays or co cultures for fundamental studies of cell biology or regenerative medicine.

Physiologically based pharmacokinetic modeling of zinc oxide nanoparticles and zinc nitrate in mice.

Zinc oxide nanoparticles (ZnO NPs) have been widely used in consumer products, therapeutic agents, and drug delivery systems. However, the fate and behavior of ZnO NPs in living organisms are not well described. The purpose of this study was to develop a physiologically based pharmacokinetic model to describe the dynamic interactions of (65)ZnO NPs in mice. We estimated key physicochemical parameters of partition coefficients and excretion or elimination rates, based on our previously published data quantifying the biodistributions of 10 nm and 71 nm (65)ZnO NPs and zinc nitrate ((65)Zn(NO3)2) in various mice tissues. The time-dependent partition coefficients and excretion or elimination rates were used to construct our physiologically based pharmacokinetic model. In general, tissue partition coefficients of (65)ZnO NPs were greater than those of (65)Zn(NO3)2, particularly the lung partition coefficient of 10 nm (65)ZnO NPs. Sensitivity analysis revealed that 71 nm (65)ZnO NPs and (65)Zn(NO3)2 were sensitive to excretion and elimination rates in the liver and gastrointestinal tract. Although the partition coefficient of the brain was relative low, it increased time-dependently for (65)ZnO NPs and (65)Zn(NO3)2. The simulation of (65)Zn(NO3)2 was well fitted with the experimental data. However, replacing partition coefficients of (65)ZnO NPs with those of (65)Zn(NO3)2 after day 7 greatly improved the fitness of simulation, suggesting that ZnO NPs might decompose to zinc ion after day 7. In this study, we successfully established a potentially predictive dynamic model for slowly decomposed NPs. More caution is suggested for exposure to (65)ZnO NPs <10 nm because those small (65)ZnO NPs tend to accumulate in the body for a relatively longer time than 71 nm (65)ZnO NPs and (65)Zn(NO3)2 do.

Cell-Substrate Interactions Feedback to Direct Cell Migration along or against Morphological Polarization.

In response to external stimuli, cells polarize morphologically into teardrop shapes prior to moving in the direction of their blunt leading edge through lamellipodia extension and retraction of the rear tip. This textbook description of cell migration implies that the initial polarization sets the direction of cell migration. Using microfabrication techniques to control cell morphologies and the direction of migration without gradients, we demonstrate that after polarization, lamelipodia extension and attachment can feedback to change and even reverse the initial morphological polarization. Cells do indeed migrate faster in the direction of their morphologically polarization. However, feedback from subsequent lamellipodia extension and attachment can be so powerful as to induce cells to reverse and migrate against their initial polarization, albeit at a slower speed. Constitutively active mutants of RhoA show that RhoA stimulates cell motility when cells are guided either along or against their initial polarization. Cdc42 activation and inhibition, which results in loss of directional motility during chemotaxis, only reduces the speed of migration without altering the directionality of migration on the micropatterns. These results reveal significant differences between substrate directed cell migration and that induced by chemotactic gradients.

Cell shape dependent regulation of nuclear morphology.

Recent studies suggest that actin filaments are essential in how a cell controls its nuclear shape. However, little is known about the relative importance of membrane tension in determining nuclear morphology. In this study, we used adhesive micropatterned substrates to alter the cellular geometry (aspect ratio, size, and shape) that allowed direct membrane tension or without membrane lateral contact with the nucleus and investigate nuclear shape remodeling and orientation on a series of rectangular shapes. Here we showed that at low cell aspect ratios the orientation of the nucleus was regulated by actin filaments while cells with high aspect ratios can maintain nuclear shape and orientation even when actin polymerization was blocked. A model adenocarcinoma cell showed similar behavior in the regulation of nuclear shape in response to changes in cell shape but actin filaments were essential in maintaining cell shape. Our results highlight the two distinct mechanisms to regulate nuclear shape through cell shape control and the difference between fibroblasts and a model cancerous cell in cell adhesion and cell shape control.