Early detection of pulmonary arterial hypertension through [18F] positron emission tomography imaging with a vascular endothelial receptor small molecule.

The objective of this study is to provide a positron emission tomography (PET) imaging modality targeting vascular endothelial growth factor receptors (VEGFR) for the early noninvasive detection and assessment of pulmonary arterial hypertension (PAH) severity. To validate the effectiveness of the [18F]VEGFR PET tracer, we utilized a monocrotaline (MCT)-induced PAH rat model. Molecular optical imaging, using a Cy5.5-conjugated VEGFR targeting agent, was employed to demonstrate the uptake of the agent at pulmonary arterioles, correlating with the onset and progression of PAH. Histological examinations of the MCT-PAH rat lung revealed a significant correlation between VEGFR2 expression and the pathogenesis of PAH. Molecular optical imaging demonstrated heightened uptake of the Cy5.5-conjugated VEGFR targeting agent at pulmonary arterioles, corresponding with the onset and progression of PAH. [18F]VEGFR PET showed increased lung uptake detectable in early-stage PAH before increase in pulmonary artery pressures, and this uptake correlated with increased PAH severity. Moreover, when compared to [18F]FDG PET, [18F]VEGFR PET exhibited markedly lower background cardiac signal, enhancing imaging sensitivity for lung abnormalities. Our study provides a compelling evidence for the potential utility of the innovative [18F]VEGFR PET tracer, in non-invasively detecting early signs of PAH, and monitoring its progression. The observed correlations between VEGFR2 expression, molecular optical imaging results, and [18F]VEGFR PET findings support the use of this tracer for early detection, and assessment of PAH severity. The lower background cardiac signal observed with [18F]VEGFR PET further enhances its imaging sensitivity, emphasizing its potential clinical significance.

NADPH and xanthine oxidases control induction of inflammatory mediator expression by organic dust in the lung.

Exposure to organic dust in animal and agricultural farms and the ensuing lung inflammation are linked to the development of respiratory diseases. We found previously that elevated production of reactive oxygen species (ROS) by aqueous poultry organic dust extract (hereafter referred to as dust extract) mediates induction of proinflammatory mediators in airway epithelial cells. In the present study, we investigated whether ROS generated by NADPH oxidases (NOX) and xanthine oxidase (XO) controls induction of inflammatory mediators by dust extract and the underlying mechanisms in bronchial epithelial cells. Using chemical inhibitors and siRNA targeted knockdown, we found that NOX1, NOX2, NOX4, and XO-derived ROS regulates induction of proinflammatory mediator levels. Like airway epithelial cells in vitro, NOX inhibitor VAS2870 reduced keratinocyte chemoattractant (KC), IL-6, and TNF-α production and 4-hydroxynonenal (4-HNE) staining induced by dust extract in mouse lungs. VAS2870 inhibition of proinflammatory mediators was associated with reduced NFκB and Stat3 activation indicating that NOX generated ROS activates NFκB and Stat3 to induce proinflammatory gene expression. Dust extract increased the membrane association of p47phox in airway epithelial cells indicating NOX2 activation but had no effect on NOX2 protein levels. In summary, our studies have shown that NOX and XO generated ROS control organic dust induction of proinflammatory mediators in airway epithelial cells via NFκB and Stat3 activation.

HnRNPK and lysine specific histone demethylase-1 regulates IP-10 mRNA stability in monocytes.

Altered mRNA metabolism is a feature of many inflammatory diseases. Post transcriptional regulation of interferon-γ-inducible protein (IP)-10 has been uncharacterized in diabetes conditions. RNA-affinity capture method and RNA immuno-precipitation revealed S100b treatment increased the binding of heterogeneous nuclear ribonucleoprotein (hnRNP)K to the IP-10 3'UTR and increased IP-10 mRNA accumulation. Luciferase activity assay using reporter plasmids showed involvement of IP-10 3'UTR. Knocking down of hnRNPK destabilized S100b induced IP-10 mRNA accumulation. S100b promoted the translocation of hnRNPK from nucleus to the cytoplasm and this was confirmed by phosphomimetic S284/353D mutant and non-phosphatable S284/353A hnRNPK mutant. S100b treatment demethylates hnRNPK at Lys219 by Lysine Specific Demethylase (LSD)-1. HnRNPKK219I, a demethylation defective mutant increased IP-10 mRNA stability. Apparently, triple mutant hnRNPKK219I/S284D/353D promoted IP-10 mRNA stability. Interestingly, knocking down LSD-1 abolished S100b induced IP-10 mRNA accumulation. These observations show for the first time that IP-10 mRNA stability is dynamically regulated by Lysine demethylation of hnRNPK by LSD-1. These results indicate that hnRNPK plays an important role in IP-10 mRNA stability induced by S100b which could exacerbate monocyte activation, relevant to the pathogenesis of diabetic complications like atherosclerosis.

Bacterial extracellular vesicles isolated from organic dust induce neutrophilic inflammation in the lung.

Inhalation of organic dust is an occupational hazard leading to the development of respiratory symptoms and respiratory diseases. Bioaerosols from concentrated animal feeding operations are rich in bacteria and could carry bacterial extracellular vesicles (EVs) that could induce lung inflammation. It is not known if organic dust contains bacterial EVs and whether they modulate lung inflammation. Herein, we show that poultry organic dust contains bacterial EVs (dust EVs) that induce lung inflammation. Treatment of airway epithelial cells, THP-1-monocytes and -macrophages with dust EVs rapidly induced IL-8, IL-6, ICAM-1, proIL-1ß, and TNF-α levels. In airway epithelial cells, induction of inflammatory mediators was due to increased mRNA levels and NF-κB activation. Induction of inflammatory mediators by dust EVs was not inhibited by polymyxin B. Single and repeated treatments of mice with dust EVs increased lung KC, IL-6, and TNF-α levels without significantly altering IL-17A levels. Increases in cytokines were associated with enhanced neutrophil infiltration into the lung. Repeated treatments of mice with dust EVs increased lung mean linear intercept and increased collagen deposition around airways indicating lung remodeling. Peribronchial cell infiltrates and airway epithelial thickening were also observed in treated mice. Because bacterial EVs are nanometer-sized particles, they can reach and accumulate in the bronchiolar and alveolar regions causing lung injury leading to the development of respiratory diseases. Our studies have provided new evidence for the presence of bacterial EVs in organic dust and for their role as one of the causative agents of organic dust-induced lung inflammation and lung injury.

Organic dust induces inflammatory gene expression in lung epithelial cells via ROS-dependent STAT-3 activation.

Exposure to dust in agricultural and animal environments, known as organic dust, is associated with the development of respiratory symptoms and respiratory diseases. Inflammation is a key feature of lung pathologies associated with organic dust exposure, and exposure to organic dust induces the expression of several immune and inflammatory mediators. However, information on transcription factors and cellular and molecular mechanisms controlling the production of immune and inflammatory mediators induced by organic dust is limited. In this study, we have identified STAT-3 as an important transcription factor controlling the induction of expression of immune and inflammatory mediators by poultry dust extracts in airway epithelial cells and in mouse lungs and delineated the cellular pathway for STAT-3 activation. Poultry dust extract activated STAT-3 phosphorylation in Beas2B and normal human bronchial epithelial cells and in mouse lungs. Chemical inhibition and siRNA knockdown of STAT-3 suppressed induction of immune and inflammatory mediator expression. Antioxidants suppressed the increase of STAT-3 phosphorylation induced by poultry dust extract indicating that oxidative stress [elevated reactive oxygen species (ROS) levels] is important for the activation. Chemical inhibition and siRNA knockdown experiments demonstrated that STAT-3 activation is dependent on the activation of nonreceptor tyrosine-protein kinase 2 (TYK2) and epidermal growth factor receptor (EGFR) tyrosine kinases. Our studies show that poultry dust extract controls the induction of immune and inflammatory mediator expression via a cellular pathway involving oxidative stress-mediated STAT-3 activation by TYK2 and EGFR tyrosine kinases.

Organic dust inhibits surfactant protein expression by reducing thyroid transcription factor-1 levels in human lung epithelial cells.

Exposure to organic dust is a risk factor for the development of respiratory diseases. Surfactant proteins (SP) reduce alveolar surface tension and modulate innate immune responses to control lung inflammation. Therefore, changes in SP levels could contribute to the development of organic-dust-induced respiratory diseases. Because information on the effects of organic dust on SP levels is lacking, we studied the effects of dust from a poultry farm on SP expression. We found that dust extract reduced SP-A and SP-B mRNA and protein levels in H441 human lung epithelial cells by inhibiting their promoter activities, but did not have any effect on SP-D protein levels. Dust extract also reduced SP-A and SP-C levels in primary human alveolar epithelial cells. The inhibitory effects were not due to LPS or protease activities present in dust extract or mediated via oxidative stress, but were dependent on a heat-labile factor(s). Thyroid transcription factor-1, a key transcriptional activator of SP expression, was reduced in dust-extract-treated cells, indicating that its down-regulation mediates inhibition of SP levels. Our study implies that down-regulation of SP levels by organic dust could contribute to the development of lung inflammation and respiratory diseases in humans.

Proteases and oxidant stress control organic dust induction of inflammatory gene expression in lung epithelial cells.

BACKGROUND: Persistant inflammatory responses to infectious agents and other components in organic dust underlie lung injury and development of respiratory diseases. Organic dust components responsible for eliciting inflammation and the mechanisms by which they cause lung inflammation are not fully understood. We studied the mechanisms by which protease activities in poultry dust extracts and intracellular oxidant stress induce inflammatory gene expression in A549 and Beas2B lung epithelial cells. METHODS: The effects of dust extracts on inflammatory gene expression were analyzed by quantitative polymerase chain reaction (qPCR), enzyme linked immunosorbent (ELISA) and western blot assays. Oxidant stress was probed by dihydroethidium (DHE) labeling, and immunostaining for 4-hydroxynonenal (4-HNE). Effects on interleukin-8 (IL-8) promoter regulation were determined by transient transfection assay. RESULTS: Dust extracts contained trypsin and elastase activities, and activated protease activated receptor (PAR)-1 and -2. Serine protease inhibitors and PAR-1 or PAR-2 knockdown suppressed inflammatory gene induction. Dust extract induction of IL-8 gene expression was associated with increased DHE-fluorescence and 4-HNE staining, and antioxidants suppressed inflammatory gene induction. Protease inhibitors and antioxidants suppressed protein kinase C and NF-κB activation and induction of IL-8 promoter activity in cells exposed to dust extract. CONCLUSIONS: Our studies demonstrate that proteases and intracellular oxidants control organic dust induction of inflammatory gene expression in lung epithelial cells. Targeting proteases and oxidant stress may serve as novel approaches for the treatment of organic dust induced lung diseases. This is the first report on the involvement of oxidant stress in the induction of inflammatory gene expression by organic dust.

Gene expression profiling of the effects of organic dust in lung epithelial and THP-1 cells reveals inductive effects on inflammatory and immune response genes.

The intensification and concentration of animal production operations expose workers to high levels of organic dusts in the work environment. Exposure to organic dusts is a risk factor for the development of acute and chronic respiratory symptoms and diseases. Lung epithelium plays important roles in the control of immune and inflammatory responses to environmental agents to maintain lung health. To better understand the effects of organic dust on lung inflammatory responses, we characterized the gene expression profiles of A549 alveolar and Beas2B bronchial epithelial and THP-1 monocytic cells influenced by exposure to poultry dust extract by DNA microarray analysis using Illumina Human HT-12 v4 Expression BeadChip. We found that A549 alveolar and Beas2B bronchial epithelial and THP-1 cells responded with unique changes in the gene expression profiles with regulation of genes encoding inflammatory cytokines, chemokines, and other inflammatory proteins being common to all the three cells. Significantly induced genes included IL-8, IL-6, IL-1ß, ICAM-1, CCL2, CCL5, TLR4, and PTGS2. Validation by real-time qRT-PCR, ELISA, Western immunoblotting, and immunohistochemical staining of lung sections from mice exposed to dust extract validated DNA microarray results. Pathway analysis indicated that dust extract induced changes in gene expression influenced functions related to cellular growth and proliferation, cell death and survival, and cellular development. These data show that a broad range of inflammatory mediators produced in response to poultry dust exposure can modulate lung immune and inflammatory responses. This is the first report on organic dust induced changes in expression profiles in lung epithelial and THP-1 monocytic cells.

The Advanced Lipoxidation end Product Precursor Malondialdehyde Induces IL-17E Expression and Skews Lymphocytes to the th17 Subset.

Malondialdehyde (MDA) is a highly reactive endogenous product of thromboxane synthesis in the prostagland and lipid peroxidation by reactive oxygen species. Elevated MDA levels occur in diabetes and atherosclerotic plaques. The aim of this study was to examine the molecular mechanisms of MDA-induced IL-17E cytokine expression and its effect on T-cell differentiation. Real-time PCR, RT-PCR and ELISA were used to assess the expression of IL-17 family cytokines in Jurkat T-cells and human peripheral blood lymphocytes (PBLCs) from diabetic subjects. Luciferase reporter assays were used for the promoter activation study. Pharmacological inhibitors were used for signaling pathway experiments. FACS analyses were used to measure the Th1, Th2 and Th17 subset levels. MDA induced significant (2- to 3-fold; p < 0.01) generation of IL-17E mRNA in a dose- and time-dependent manner in Jurkat T-cells and PBLCs. Elevated IL-17E mRNA levels were found in the lymphocytes from diabetic subjects. The increased IL-17E protein and mRNA levels correlate well with serum MDA levels from diabetic patients. Transient transfection of plasmid containing the minimum IL-17E promoter region (pIL-17E-Luc) showed a significant (2-fold; p < 0.01) increase in luciferase activity. Pretreatment of lymphocytes with pharmacological inhibitors showed the involvement of antioxidant, NF-ƙB, p38MAPK, PKC and ERK signaling pathways. Quantification of the Th1, Th2 and Th17 cell population in PBLCs via FACS analyses revealed an increase in the Th17 subset. These results show that MDA transcriptionally upregulates the expression of IL-17E in lymphocytes and alters lymphocyte differentiation towards the pathogenic Th17 subset.

High glucose driven expression of pro-inflammatory cytokine and chemokine genes in lymphocytes: molecular mechanisms of IL-17 family gene expression.

High glucose is an independent risk factor that alters the expression pattern of cytokines/chemokine leading to leukocyte activation in diabetes. Fluctuation of cytokine milieu in lymphocytes may lead to differentiation into a particular subset. Our objectives were to profile high glucose induced inflammatory gene expression in lymphocytes, to examine in vivo relevance in diabetes and to identify the key transcription factors and signaling pathways involved. Cytokine gene arrays and T-helper (Th1/Th2/Th17) cytokine profiler RT(2)-PCR arrays used for cytokine expression profiling followed by validation using Real Time-qPCR and relative RT-PCR in Jurkat T-lymphocytes, peripheral blood lymphocytes (PBLCs) from normal and diabetes subjects. Luciferase reporter plasmid, pharmacological inhibitors and mutant plasmids were used for promoter activation and signaling pathway studies. High glucose induced gene profiling in Jurkat T-lymphocytes showed significantly increased expression of 64 proinflammatory genes including IL-6 and IL-17A and most of these genes were Nuclear Factor (NF)-κB and AP-1 regulated. RT(2)-PCR array results suggested the transcriptional activation of IL-17 and its downstream signaling in Jurkat T-lymphocytes upon high glucose treatment. Candidate genes like Interleukin (IL)-17A, IL-17E IL-17F and IL-6 were up-regulated in both Jurkat T-lymphocytes and PBLCs from normal and diabetes subjects. This high glucose induced cytokine expression was due to promoter activation. Pharmacology inhibitor studies showed the involvement of NF-κB, protein kinase-C, p38 Mitogen activated protein kinase; Janus activated kinase-signal transducer and activator of transcription and extracellular regulated kinase signaling pathways. Further, high glucose treatment increased the adhesion of lymphocytes to human umbilical vein endothelial cells. These results show that IL-17 cytokines are induced by high glucose via key signaling pathways leading to lymphocyte activation and relevant to the pathogenesis of diabetic complications like atherosclerosis.