Identifying biomarkers of drug use recurrence using wearable device technologies and phone applications.

BACKGROUND: Identifying predictors of drug use recurrence (DUR) is critical to combat the addiction epidemic. Wearable devices and phone-based applications for obtaining self-reported assessments in the patient's natural environment (e.g., ecological momentary assessment; EMA) have been used in various healthcare settings. However, the utility of combining these technologies to predict DUR in substance use disorder (SUD) has not yet been explored. This study investigates the combined use of wearable technologies and EMA as a potential mechanism for identifying physiological/behavioral biomarkers of DUR. METHODS: Participants, recruited from an SUD treatment program, were provided with a commercially available wearable device that continuously monitors biometric signals (e.g., heart rate/variability [HR/HRV], sleep characteristics). They were also prompted daily to complete an EMA via phone-based application (EMA-APP) that included questionnaires regarding mood, pain, and craving. RESULTS: Seventy-seven participants are included in this pilot study (34 participants experienced a DUR during enrollment). Wearable technologies revealed that physiological markers were significantly elevated in the week prior to DUR relative to periods of sustained abstinence (p<0.001). Results from the EMA-APP revealed that those who experienced a DUR reported greater difficulty concentrating, exposure to triggers associated with substance use, and increased isolation the day prior to DUR (p<0.001). Compliance with study procedures during the DUR week was lower than any other period of measurement (p<0.001). CONCLUSIONS: These results suggest that data acquired via wearable technologies and the EMA-APP may serve as a method of predicting near-term DUR, thereby potentially prompting intervention before drug use occurs.

Flower lose, a cell fitness marker, predicts COVID-19 prognosis.

Risk stratification of COVID-19 patients is essential for pandemic management. Changes in the cell fitness marker, hFwe-Lose, can precede the host immune response to infection, potentially making such a biomarker an earlier triage tool. Here, we evaluate whether hFwe-Lose gene expression can outperform conventional methods in predicting outcomes (e.g., death and hospitalization) in COVID-19 patients. We performed a post-mortem examination of infected lung tissue in deceased COVID-19 patients to determine hFwe-Lose's biological role in acute lung injury. We then performed an observational study (n = 283) to evaluate whether hFwe-Lose expression (in nasopharyngeal samples) could accurately predict hospitalization or death in COVID-19 patients. In COVID-19 patients with acute lung injury, hFwe-Lose is highly expressed in the lower respiratory tract and is co-localized to areas of cell death. In patients presenting in the early phase of COVID-19 illness, hFwe-Lose expression accurately predicts subsequent hospitalization or death with positive predictive values of 87.8-100% and a negative predictive value of 64.1-93.2%. hFwe-Lose outperforms conventional inflammatory biomarkers and patient age and comorbidities, with an area under the receiver operating characteristic curve (AUROC) 0.93-0.97 in predicting hospitalization/death. Specifically, this is significantly higher than the prognostic value of combining biomarkers (serum ferritin, D-dimer, C-reactive protein, and neutrophil-lymphocyte ratio), patient age and comorbidities (AUROC of 0.67-0.92). The cell fitness marker, hFwe-Lose, accurately predicts outcomes in COVID-19 patients. This finding demonstrates how tissue fitness pathways dictate the response to infection and disease and their utility in managing the current COVID-19 pandemic.

Molecular Analysis of ZNF71 KRAB in Non-Small-Cell Lung Cancer.

Our previous study found that zinc finger protein 71 (ZNF71) mRNA expression was associated with chemosensitivity and its protein expression was prognostic of non-small-cell lung cancer (NSCLC). The Krüppel associated box (KRAB) transcriptional repression domain is commonly present in human zinc finger proteins, which are linked to imprinting, silencing of repetitive elements, proliferation, apoptosis, and cancer. This study revealed that ZNF71 KRAB had a significantly higher expression than the ZNF71 KRAB-less isoform in NSCLC tumors (n = 197) and cell lines (n = 117). Patients with higher ZNF71 KRAB expression had a significantly worse survival outcome than patients with lower ZNF71 KRAB expression (log-rank p = 0.04; hazard ratio (HR): 1.686 [1.026, 2.771]), whereas ZNF71 overall and KRAB-less expression levels were not prognostic in the same patient cohort. ZNF71 KRAB expression was associated with epithelial-to-mesenchymal transition (EMT) in both patient tumors and cell lines. ZNF71 KRAB was overexpressed in NSCLC cell lines resistant to docetaxel and paclitaxel treatment compared to chemo-sensitive cell lines, consistent with its association with poor prognosis in patients. Therefore, ZNF71 KRAB isoform is a more effective prognostic factor than ZNF71 overall and KRAB-less expression for NSCLC. Functional analysis using CRISPR-Cas9 and RNA interference (RNAi) screening data indicated that a knockdown/knockout of ZNF71 did not significantly affect NSCLC cell proliferation in vitro.

Intermittent Lipopolysaccharide Exposure Significantly Increases Cortical Infarct Size and Impairs Autophagy.

Globally, stroke is a leading cause of death and disability. Traditional risk factors like hypertension, diabetes, and obesity do not fully account for all stroke cases. Recent infection is regarded as changes in systemic immune signaling, which can increase thrombosis formation and other stroke risk factors. We have previously shown that administration of lipopolysaccharide (LPS) 30-minutes prior to stroke increases in infarct volume. In the current study, we found that animals intermittently exposed to LPS have larger cortical infarcts when compared to saline controls. To elucidate the mechanism behind this phenomenon, several avenues were investigated. We observed significant upregulation of tumor necrosis factor-alpha (TNF-α) mRNA, especially in the ipsilateral hemisphere of both saline and LPS exposed groups compared to sham surgery animals. We also observed significant reductions in expression of genes involved in autophagy in the ipsilateral hemisphere of LPS stroke animals. In addition, we assessed DNA methylation of autophagy genes and observed a significant increase in the ipsilateral hemisphere of LPS stroke animals. Intermittent exposure to LPS increases cortical infarct volume, downregulates autophagy genes, and induces hypermethylation of the corresponding CpG islands. These data suggest that intermittent immune activation may deregulate epigenetic mechanisms and promote neuropathological outcomes after stroke.

Hypoxia-Inducible Factor α Subunits Regulate Tie2-Expressing Macrophages That Influence Tumor Oxygen and Perfusion in Murine Breast Cancer.

Tie2-expressing monocytes/macrophages (TEMs) are a distinct subset of proangiogenic monocytes selectively recruited to tumors in breast cancer. Because of the hypoxic nature of solid tumors, we investigated if oxygen, via hypoxia-inducible transcription factors HIF-1α and HIF-2α, regulates TEM function in the hypoxic tumor microenvironment. We orthotopically implanted PyMT breast tumor cells into the mammary fat pads of syngeneic LysMcre, HIF-1α fl/fl /LysMcre, or HIF-2α fl/fl /LysMcre mice and evaluated the tumor TEM population. There was no difference in the percentage of tumor macrophages among the mouse groups. In contrast, HIF-1α fl/fl /LysMcre mice had a significantly smaller percentage of tumor TEMs compared with control and HIF-2α fl/fl /LysMcre mice. Proangiogenic TEMs in macrophage HIF-2α-deficient tumors presented significantly more CD31+ microvessel density but exacerbated hypoxia and tissue necrosis. Reduced numbers of proangiogenic TEMs in macrophage HIF-1α-deficient tumors presented significantly less microvessel density but tumor vessels that were more functional as lectin injection revealed more perfusion, and functional electron paramagnetic resonance analysis revealed more oxygen in those tumors. Macrophage HIF-1α-deficient tumors also responded significantly to chemotherapy. These data introduce a previously undescribed and counterintuitive prohypoxia role for proangiogenic TEMs in breast cancer which is, in part, suppressed by HIF-2α.

Stress-induced Norepinephrine Downregulates CCL2 in Macrophages to Suppress Tumor Growth in a Model of Malignant Melanoma.

Psychological stressors have been implicated in the progression of various tumor types. We investigated a role for stress in tumor immune cell chemotaxis in the B16F10 mouse model of malignant melanoma. We exposed female mice to 6-hour periods of restraint stress (RST) for 7 days, then implanted B16F10 malignant melanoma tumor cells and continued the RST paradigm for 14 additional days. We determined serum corticosterone and liver catecholamine concentrations in these mice. To evaluate the tumor microenvironment, we performed IHC and examined cytokine expression profiles using ELISA-based analysis of tumor homogenates. We found that tumors in mice subjected to RST grew significantly slower, had reduced tumor C-C motif ligand 2 (CCL2), and contained fewer F4/80-positive macrophages than tumors from unstressed mice. We observed a concomitant increase in norepinephrine among the RST mice. An in vitro assay confirmed that norepinephrine downregulates CCL2 production in both mouse and human macrophages, and that pretreatment with the pan-ß-adrenergic receptor inhibitor nadolol rescues this activity. Furthermore, RST had no effect on tumor growth in transgenic CCL2-deficient mice. This study suggests that stress reduces malignant melanoma by reducing recruitment of tumor-promoting macrophages by CCL2.

Constitutive AKT Activity Predisposes Lung Fibrosis by Regulating Macrophage, Myofibroblast and Fibrocyte Recruitment and Changes in Autophagy.

The etiology and pathogenesis of pulmonary fibrosis is poorly understood. We and others reported that M-CSF/CSF-1, M-CSF-R and downstream AKT activation plays an important role in lung fibrosis in mice models and in IPF patients. To understand potential molecular pathways used by M-CSF-R activation to direct lung fibrosis, we used a novel transgenic mouse model that expresses a constitutively-active form of AKT, myristoylated AKT (Myr-Akt), driven by the c-fms (M-CSF-R) promoter. We were particularly interested in the basal immune state of the lungs of these Myr-Akt mice to assess M-CSF-R-related priming for lung fibrosis. In support of a priming effect, macrophages isolated from the lungs of unchallenged Myr-Akt mice displayed an M2-tropism, enhanced co-expression of M-CSF-R and α-SMA, reduced autophagy reflected by reduced expression of the key autophagy genes Beclin-1, MAP1-Lc3a(Lc3a), and MAP1-Lc3b(Lc3b), and increased p62/STSQM1 expression compared with littermate WT mice. Furthermore, Myr-Akt mice had more basal circulating fibrocytes than WT mice. Lastly, upon bleomycin challenge, Myr-Akt mice showed enhanced collagen deposition, increased F4/80+ and CD45+ cells, reduced autophagy genes Beclin-1, Lc3a, and Lc3b expression, and a shorter life-span than WT littermates. These data provide support that M-CSF-R/AKT activation may have a priming effect which can predispose lung tissue to pulmonary fibrosis.

Circulating extracellular vesicle content reveals de novo DNA methyltransferase expression as a molecular method to predict septic shock.

Extracellular vesicles (EVs) are mRNA-containing cell fragments shed into circulation during pathophysiological events. DNA methyltransferases (DNMT1, DNMT3A, and DNMT3B) regulate gene expression by modifying DNA methylation and altering transcription. Sepsis is a systemic insult resulting in vascular dysfunction, which can lead to shock and death. We analysed plasma from ICU patients for circulating EV numbers, defined as particles isolated from 1 mL plasma at 21,000xg, and DNMTs mRNA content as prognostic markers of septic shock. Compared to plasma from critically ill patients with or without sepsis, plasma from septic shock patients contained more EVs per mL, expressed as total DNMTs mRNAs over 5 days, and more individual DNMT mRNAs at each day. A comparison of EV-DNMT1 (maintenance methylation) with EV-DNMT3A+DNMT3B (de novo methylation) expression correlated highly with severity, and EVs from septic shock patients carried more total DNMT mRNAs and more DNMT3A+DNMT3B mRNAs than control or sepsis EVs. Total plasma EVs also correlated with sepsis severity. EV-DNMT mRNAs load, when coupled with total plasma EV number, may be a novel method to diagnose septic shock upon ICU admittance and offer opportunities to more precisely intervene with standard therapy or other targeted interventions to regulate EV release and/or specific DNMT activity.

Of mice and men: correlations between microRNA-17∼92 cluster expression and promoter methylation in severe bronchopulmonary dysplasia.

We previously demonstrated that decreased miR-17∼92 cluster expression was 1) present in lungs from human infants who died with bronchopulmonary dysplasia (BPD); 2) inversely correlated with DNA methyltransferase (DNMT) expression and promoter methylation; and 3) correlated with a subsequent diagnosis of BPD at 36 wk gestational age. We tested the hypothesis that plasma miR-17 levels would be lowest in infants who ultimately develop severe BPD. Secondly, we utilized our well-characterized murine model of severe BPD that combines perinatal inflammation with postnatal hyperoxia to test the hypothesis that alterations in lung miR-17∼92, DNMT, and promoter methylation in our model would mirror our findings in tissues from premature human infants. Plasma was obtained during the first 5 days of life from premature infants born ≤32 wk gestation. Lung tissues were harvested from mice exposed to maternal inflammation and neonatal hyperoxia for 14 days after birth. miR-17∼92 cluster expression and DNA methyltransferase expression were measured by qRT-PCR, and promoter methylation was assessed by Methyl-Profiler assay. Plasma miR-17 levels are significantly lower in the first week of life in human infants who develop severe BPD compared with mild or moderate BPD. Data from our severe BPD murine model reveal that lung miR-17∼92 cluster expression is significantly attenuated, and levels inversely correlated with DNMT expression and miR-17∼92 cluster promoter methylation. Collectively, our data support a plausible role for epigenetically altered miR-17∼92 cluster in the pathogenesis of severe BPD.

Behavioral abnormalities in mice lacking mesenchyme-specific Pten.

Phosphatase and tensin homolog (Pten) is a negative regulator of cell proliferation and growth. Using a Cre-recombinase approach with Lox sequences flanking the fibroblast-specific protein 1 (Fsp1 aka S100A4; a mesenchymal marker), we probed sites of expression using a ß-galactosidase Rosa26(LoxP) reporter allele; the transgene driving deletion of Pten (exons 4-5) was found throughout the brain parenchyma and pituitary, suggesting that deletion of Pten in Fsp1-positive cells may influence behavior. Because CNS-specific deletion of Pten influences social and anxiety-like behaviors and S100A4 is expressed in astrocytes, we predicted that loss of Pten in Fsp1-expressing cells would result in deficits in social interaction and increased anxiety. We further predicted that environmental enrichment would compensate for genetic deficits in these behaviors. We conducted a battery of behavioral assays on Fsp1-Cre;Pten(LoxP/LoxP) male and female homozygous knockouts (Pten(-/-)) and compared their behavior to Pten(LoxP/LoxP) (Pten(+/+)) conspecifics. Despite extensive physical differences (including reduced hippocampal size) and deficits in sensorimotor function, Pten(-/-) mice behaved remarkably similar to control mice on nearly all behavioral tasks. These results suggest that the social and anxiety-like phenotypes observed in CNS-specific Pten(-/-) mice may depend on neuronal Pten, as lack of Pten in Fsp1-expressing cells of the CNS had little effect on these behaviors.

Center of excellence for mobile sensor data-to-knowledge (MD2K).

Mobile sensor data-to-knowledge (MD2K) was chosen as one of 11 Big Data Centers of Excellence by the National Institutes of Health, as part of its Big Data-to-Knowledge initiative. MD2K is developing innovative tools to streamline the collection, integration, management, visualization, analysis, and interpretation of health data generated by mobile and wearable sensors. The goal of the big data solutions being developed by MD2K is to reliably quantify physical, biological, behavioral, social, and environmental factors that contribute to health and disease risk. The research conducted by MD2K is targeted at improving health through early detection of adverse health events and by facilitating prevention. MD2K will make its tools, software, and training materials widely available and will also organize workshops and seminars to encourage their use by researchers and clinicians.

Attenuation of miR-17∼92 Cluster in Bronchopulmonary Dysplasia.

RATIONALE: Bronchopulmonary dysplasia remains a significant cause of neonatal morbidity; however, the identification of novel targets to predict or prevent the development of bronchopulmonary dysplasia remains elusive. Proper microRNA (miR)-17∼92 cluster is necessary for normal lung development, and alterations in expression are reported in other pulmonary diseases. The overall hypothesis for our work is that altered miR-17∼92 cluster expression contributes to the molecular pathogenesis of bronchopulmonary dysplasia. OBJECTIVES: The current studies tested the hypothesis that alterations in miR-17∼92 cluster and DNA methyltransferase expression are present in bronchopulmonary dysplasia. METHODS: miR-17∼92 cluster expression, promoter methylation, and DNA methyltransferase expression were determined in autopsy lung samples obtained from premature infants who died with bronchopulmonary dysplasia, or from term/near-term infants who died from nonrespiratory causes. Expression of miR-17∼92 cluster members miR-17 and -19b was measured in plasma samples collected in the first week of life from a separate cohort of preterm infants at a second institution in whom bronchopulmonary dysplasia was diagnosed subsequently. MEASUREMENTS AND MAIN RESULTS: Autopsy tissue data indicated that miR-17∼92 expression is significantly lower in bronchopulmonary dysplasia lungs and is inversely correlated with promoter methylation and DNA methyltransferase expression when compared with that of control subjects without bronchopulmonary dysplasia. Plasma sample analyses indicated that miR-17 and -19b expression was decreased in infants who subsequently developed bronchopulmonary dysplasia. CONCLUSIONS: Our data are the first to demonstrate altered expression of the miR-17∼92 cluster in bronchopulmonary dysplasia. The consistency between our autopsy and plasma findings further support our working hypothesis that the miR-17∼92 cluster contributes to the molecular pathogenesis of bronchopulmonary dysplasia.

The mannose-6-phosphate analogue, PXS64, inhibits fibrosis via TGF-ß1 pathway in human lung fibroblasts.

Idiopathic pulmonary fibrosis (IPF) is a chronic disease characterised by a progressive decline in lung function which can be attributed to excessive scarring, inflammation and airway remodelling. Mannose-6-phosphate (M6P) is a strong inhibitor of fibrosis and its administration has been associated with beneficial effects in tendon repair surgery as well as nerve repair after injury. Given this promising therapeutic approach we developed an improved analogue of M6P, namely PXS64, and explored its anti-fibrotic effects in vitro. Normal human lung fibroblasts (NHLF) and human lung fibroblast 19 cells (HF19) were exposed to active recombinant human TGF-ß1 to induce increases in fibrotic markers. rhTGF-ß1 increased constitutive protein levels of fibronectin and collagen in the NHLF cells, whereas HF19 cells showed increased levels of fibronectin, collagen as well as αSMA (alpha smooth muscle actin). PXS64 demonstrated a robust inhibitory effect on all proteins analysed. IPF patient fibroblasts treated with PXS64 presented an improved phenotype in terms of their morphological appearance, as well as a decrease in fibrotic markers (collagen, CTGF, TGF-ß3, tenascin C, αSMA and THBS1). To explore the cell signalling pathways involved in the anti-fibrotic effects of PXS64, proteomics analysis with iTRAQ labelling was performed and the data demonstrated a specific antagonistic effect on the TGF-ß1 pathway. This study shows that PXS64 effectively inhibits the production of extracellular matrix, as well as myofibroblast differentiation during fibrosis. These results suggest that PXS64 influences tissue remodelling by inhibiting TGF-ß1 signalling in NHLF and HF19 cell lines, as well as in IPF patient fibroblasts. Thus PXS64 is a potential candidate for preclinical application in pulmonary fibrosis.

Supernatants from stored red blood cell (RBC) units, but not RBC-derived microvesicles, suppress monocyte function in vitro.

BACKGROUND: We have previously shown that critically ill children transfused with red blood cells (RBCs) of longer storage durations have more suppressed monocyte function after transfusion compared to children transfused with fresher RBCs and that older stored RBCs directly suppress monocyte function in vitro, through unknown mechanisms. We hypothesized that RBC-derived microvesicles (MVs) were responsible for monocyte suppression. STUDY DESIGN AND METHODS: To determine the role of stored RBC unit-derived MVs, we cocultured monocytes with supernatants, isolated MVs, or supernatants that had been depleted of MVs from prestorage leukoreduced RBCs that had been stored for either 7 or 30 days. Isolated MVs were characterized by electron microscopy and flow cytometry. Monocyte function after coculture experiments was measured by cytokine production after stimulation with lipopolysaccharide (LPS). RESULTS: Monocyte function was suppressed after exposure to supernatants from 30-day RBC units compared to monocytes cultured in medium alone (LPS-induced tumor necrosis factor-α production, 17,611 ± 3,426 vs. 37,486 ± 5,598 pg/mL; p = 0.02). Monocyte function was not suppressed after exposure to MV fractions. RBC supernatants that had been depleted of MVs remained immunosuppressive. Treating RBC supernatants with heat followed by RNase (to degrade protein-bound RNA) prevented RBC supernatant-induced monocyte suppression. CONCLUSION: Our findings implicate soluble mediators of stored RBC-induced monocyte suppression outside of MV fractions and suggest that extracellular protein-bound RNAs (such as microRNA) may play a role in transfusion-related immunomodulation.

MicroRNA-133a engineered mesenchymal stem cells augment cardiac function and cell survival in the infarct heart.

: Cardiovascular disease is the number 1 cause of morbidity and mortality in the United States. The most common manifestation of cardiovascular disease is myocardial infarction (MI), which can ultimately lead to congestive heart failure. Cell therapy (cardiomyoplasty) is a new potential therapeutic treatment alternative for the damaged heart. Recent preclinical and clinical studies have shown that mesenchymal stem cells (MSCs) are a promising cell type for cardiomyoplasty applications. However, a major limitation is the poor survival rate of transplanted stem cells in the infarcted heart. miR-133a is an abundantly expressed microRNA (miRNA) in the cardiac muscle and is downregulated in patients with MI. We hypothesized that reprogramming MSCs using miRNA mimics (double-stranded oligonucleotides) will improve survival of stem cells in the damaged heart. MSCs were transfected with miR-133a mimic and antagomirs, and the levels of miR-133a were measured by quantitative real-time polymerase chain reaction. Rat hearts were subjected to MI and MSCs transfected with miR-133a mimic or antagomir were implanted in the ischemic hearts. Four weeks after MI, cardiac function, cardiac fibrosis, miR-133a levels, and apoptosis-related genes (Apaf-1, Caspase-9, and Caspase-3) were measured in the heart. We found that transfecting MSCs with miR-133a mimic improves survival of MSCs as determined by the MTT assay. Similarly, transplantation of miR-133a mimic transfected MSCs in rat hearts subjected to MI led to a significant increase in cell engraftment, cardiac function, and decreased fibrosis when compared with MSCs only or MI groups. At the molecular level, quantitative real-time polymerase chain reaction data demonstrated a significant decrease in expression of the proapoptotic genes; Apaf-1, caspase-9, and caspase-3 in the miR-133a mimic transplanted group. Furthermore, luciferase reporter assay confirmed that miR-133a is a direct target for Apaf-1. Overall, bioengineering of stem cells through miRNAs manipulation could potentially improve the therapeutic outcome of patients undergoing stem cell transplantation for MI.

Involvement of tumor macrophage HIFs in chemotherapy effectiveness: mathematical modeling of oxygen, pH, and glutathione.

The four variables, hypoxia, acidity, high glutathione (GSH) concentration and fast reducing rate (redox) are distinct and varied characteristics of solid tumors compared to normal tissue. These parameters are among the most significant factors underlying the metabolism and physiology of solid tumors, regardless of their type or origin. Low oxygen tension contributes to both inhibition of cancer cell proliferation and therapeutic resistance of tumors; low extracellular pH, the reverse of normal cells, mainly enhances tumor invasion; and dysregulated GSH and redox potential within cancer cells favor their proliferation. In fact, cancer cells under these microenvironmental conditions appreciably alter tumor response to cytotoxic anti-cancer treatments. Recent experiments measured the in vivo longitudinal data of these four parameters with tumor development and the corresponding presence and absence of tumor macrophage HIF-1α or HIF-2α in a mouse model of breast cancer. In the current paper, we present a mathematical model-based system of (ordinary and partial) differential equations to monitor tumor growth and susceptibility to standard chemotherapy with oxygen level, pH, and intracellular GSH concentration. We first show that our model simulations agree with the corresponding experiments, and then we use our model to suggest treatments of tumors by altering these four parameters in tumor microenvironment. For example, the model qualitatively predicts that GSH depletion can raise the level of reactive oxygen species (ROS) above a toxic threshold and result in inhibition of tumor growth.

Macrophage colony-stimulating factor augments Tie2-expressing monocyte differentiation, angiogenic function, and recruitment in a mouse model of breast cancer.

Reports demonstrate the role of M-CSF (CSF1) in tumor progression in mouse models as well as the prognostic value of macrophage numbers in breast cancer patients. Recently, a subset of CD14+ monocytes expressing the Tie2 receptor, once thought to be predominantly expressed on endothelial cells, has been characterized. We hypothesized that increased levels of CSF1 in breast tumors can regulate differentiation of Tie2- monocytes to a Tie2+ phenotype. We treated CD14+ human monocytes with CSF1 and found a significant increase in CD14+/Tie2+ positivity. To understand if CSF1-induced Tie2 expression on these cells improved their migratory ability, we pre-treated CD14+ monocytes with CSF1 and used Boyden chemotaxis chambers to observe enhanced response to angiopoietin-2 (ANG2), the chemotactic ligand for the Tie2 receptor. We found that CSF1 pre-treatment significantly augmented chemotaxis and that Tie2 receptor upregulation was responsible as siRNA targeting Tie2 receptor abrogated this effect. To understand any augmented angiogenic effect produced by treating these cells with CSF1, we cultured human umbilical vein endothelial cells (HUVECs) with conditioned supernatants from CSF1-pre-treated CD14+ monocytes for a tube formation assay. While supernatants from CSF1-pre-treated TEMs increased HUVEC branching, a neutralizing antibody against the CSF1R abrogated this activity, as did siRNA against the Tie2 receptor. To test our hypothesis in vivo, we treated PyMT tumor-bearing mice with CSF1 and observed an expansion in the TEM population relative to total F4/80+ cells, which resulted in increased angiogenesis. Investigation into the mechanism of Tie2 receptor upregulation on CD14+ monocytes by CSF1 revealed a synergistic contribution from the PI3 kinase and HIF pathways as the PI3 kinase inhibitor LY294002, as well as HIF-1α-deficient macrophages differentiated from the bone marrow of HIF-1αfl/fl/LysMcre mice, diminished CSF1-stimulated Tie2 receptor expression.

MicroRNA 17-92 cluster mediates ETS1 and ETS2-dependent RAS-oncogenic transformation.

The ETS-family transcription factors Ets1 and Ets2 are evolutionarily conserved effectors of the RAS/ERK signaling pathway, but their function in Ras cellular transformation and biology remains unclear. Taking advantage of Ets1 and Ets2 mouse models to generate Ets1/Ets2 double knockout mouse embryonic fibroblasts, we demonstrate that deletion of both Ets1 and Ets2 was necessary to inhibit HrasG12V induced transformation both in vitro and in vivo. HrasG12V expression in mouse embryonic fibroblasts increased ETS1 and ETS2 expression and binding to cis-regulatory elements on the c-Myc proximal promoter, and consequently induced a robust increase in MYC expression. The expression of the oncogenic microRNA 17-92 cluster was increased in HrasG12V transformed cells, but was significantly reduced when ETS1 and ETS2 were absent. MYC and ETS1 or ETS2 collaborated to increase expression of the oncogenic microRNA 17-92 cluster in HrasG12V transformed cells. Enforced expression of exogenous MYC or microRNA 17-92 rescued HrasG12V transformation in Ets1/Ets2-null cells, revealing a direct function for MYC and microRNA 17-92 in ETS1/ETS2-dependent HrasG12V transformation.

Methodological challenges in utilizing miRNAs as circulating biomarkers.

MicroRNAs (miRNAs) have emerged as important regulators in the post-transcriptional control of gene expression. The discovery of their presence not only in tissues but also in extratissular fluids, including blood, urine and cerebro-spinal fluid, together with their changes in expression in various pathological conditions, has implicated these extracellular miRNAs as informative biomarkers of disease. However, exploiting miRNAs in this capacity requires methodological rigour. Here, we report several key procedural aspects of miRNA isolation from plasma and serum, as exemplified by research in cardiovascular and pulmonary diseases. We also highlight the advantages and disadvantages of various profiling methods to determine the expression levels of plasma- and serum-derived miRNAs. Attention to such methodological details is critical, as circulating miRNAs become diagnostic tools for various human diseases.