Clay B. Marsh

Detection of microRNA Expression in Human Peripheral Blood Microvesicles

Background MicroRNAs (miRNA) are small non-coding RNAs that regulate translation of mRNA and protein. Loss or enhanced expression of miRNAs is associated with several diseases, including cancer. However, the identification of circulating miRNA in healthy donors is not well characterized. Microvesicles, also known as exosomes or microparticles, circulate in the peripheral blood and can stimulate cellular signaling. In this study, we hypothesized that under normal healthy conditions, microvesicles contain miRNAs, contributing to biological homeostasis. Methodology/Principal Findings Microvesicles were isolated from the plasma of normal healthy individuals. RNA was isolated from both the microvesicles and matched mononuclear cells and profiled for 420 known mature miRNAs by real-time PCR. Hierarchical clustering of the data sets indicated significant differences in miRNA expression between peripheral blood mononuclear cells (PBMC) and plasma microvesicles. We observed 71 miRNAs co-expressed between microvesicles and PBMC. Notably, we found 33 and 4 significantly differentially expressed miRNAs in the plasma microvesicles and mononuclear cells, respectively. Prediction of the gene targets and associated biological pathways regulated by the detected miRNAs was performed. The majority of the miRNAs expressed in the microvesicles from the blood were predicted to regulate cellular differentiation of blood cells and metabolic pathways. Interestingly, a select few miRNAs were also predicted to be important modulators of immune function. Conclusions This study is the first to identify and define miRNA expression in circulating plasma microvesicles of normal subjects. The data generated from this study provides a basis for future studies to determine the predictive role of peripheral blood miRNA signatures in human disease and will enable the definition of the biological processes regulated by these miRNA.

Acquired Weakness, Handgrip Strength, and Mortality in Critically Ill Patients

RATIONALE ICU-acquired paresis (ICUAP) is common in survivors of critical illness. There is significant associated morbidity, including prolonged time on the ventilator and longer hospital stay. However, it is unclear whether ICUAP is independently associated with mortality, as sicker patients are more prone and existing studies have not adjusted for this. OBJECTIVES To test the hypothesis that ICUAP is independently associated with increased mortality. Secondarily, to determine if handgrip dynamometry is a concise measure of global strength and is independently associated with mortality. METHODS A prospective multicenter cohort study was conducted in intensive care units (ICU) of five academic medical centers. Adults requiring at least 5 days of mechanical ventilation without evidence of preexisting neuromuscular disease were followed until awakening and were then examined for strength. MEASUREMENTS AND MAIN RESULTS We measured global strength and handgrip dynamometry. The primary outcome was in-hospital mortality and secondary outcomes were hospital and ICU-free days, ICU readmission, and recurrent respiratory failure. Subjects with ICUAP (average MRC score of < 4) had longer hospital stays and required mechanical ventilation longer. Handgrip strength was lower in subjects with ICUAP and had good test performance for diagnosing ICUAP. After adjustment for severity of illness, ICUAP was independently associated with hospital mortality (odds ratio [OR], 7.8; 95% confidence interval [CI], 2.4-25.3; P = 0.001). Separately, handgrip strength was independently associated with hospital mortality (OR, 4.5; 95% CI, 1.5-13.6; P = 0.007). CONCLUSIONS ICUAP is independently associated with increased hospital mortality. Handgrip strength is also independently associated with poor hospital outcome and may serve as a simple test to identify ICUAP. Clinical trial registered with www.clinicaltrials.gov (NCT00106665).

MicroRNA regulation of a cancer network: Consequences of the feedback loops involving miR-17-92, E2F, and Myc

The transcription factors E2F and Myc participate in the control of cell proliferation and apoptosis, and can act as oncogenes or tumor suppressors depending on their levels of expression. Positive feedback loops in the regulation of these factors are predicted—and recently shown experimentally—to lead to bistability, which is a phenomenon characterized by the existence of low and high protein levels (“off” and “on” levels, respectively), with sharp transitions between levels being inducible by, for example, changes in growth factor concentrations. E2F and Myc are inhibited at the posttranscriptional step by members of a cluster of microRNAs (miRs) called miR-17-92. In return, E2F and Myc induce the transcription of miR-17-92, thus forming a negative feedback loop in the interaction network. The consequences of the coupling between the E2F/Myc positive feedback loops and the E2F/Myc/miR-17-92 negative feedback loop are analyzed using a mathematical model. The model predicts that miR-17-92 plays a critical role in regulating the position of the off–on switch in E2F/Myc protein levels, and in determining the on levels of these proteins. The model also predicts large-amplitude protein oscillations that coexist with the off steady state levels. Using the concept and model prediction of a “cancer zone,” the oncogenic and tumor suppressor properties of miR-17-92 is demonstrated to parallel the same properties of E2F and Myc.

Implementing genomic medicine in the clinic: the future is here

Although the potential for genomics to contribute to clinical care has long been anticipated, the pace of defining the risks and benefits of incorporating genomic findings into medical practice has been relatively slow. Several institutions have recently begun genomic medicine programs, encountering many of the same obstacles and developing the same solutions, often independently. Recognizing that successful early experiences can inform subsequent efforts, the National Human Genome Research Institute brought together a number of these groups to describe their ongoing projects and challenges, identify common infrastructure and research needs, and outline an implementation framework for investigating and introducing similar programs elsewhere. Chief among the challenges were limited evidence and consensus on which genomic variants were medically relevant; lack of reimbursement for genomically driven interventions; and burden to patients and clinicians of assaying, reporting, intervening, and following up genomic findings. Key infrastructure needs included an openly accessible knowledge base capturing sequence variants and their phenotypic associations and a framework for defining and cataloging clinically actionable variants. Multiple institutions are actively engaged in using genomic information in clinical care. Much of this work is being done in isolation and would benefit from more structured collaboration and sharing of best practices.Genet Med 2013:15(4):258–267

Glucose variability and mortality in patients with sepsis

Objective:Treatment and prevention of hyperglycemia has been advocated for subjects with sepsis. Glucose variability, rather than the glucose level, has also been shown to be an important factor associated with in-hospital mortality, in general, critically ill patients. Our objective was to determine the association between glucose variability and hospital mortality in septic patients and the expression of glucose variability that best reflects this risk. Design:Retrospective, single-center cohort study. Setting:Academic, tertiary care hospital. Patients:Adult subjects hospitalized for >1 day, with a diagnosis of sepsis were included. Interventions:None. Measurements:Glucose variability was calculated for all subjects as the average and standard deviation of glucose, the mean amplitude of glycemic excursions, and the glycemic lability index. Hospital mortality was the primary outcome variable. Logistic regression was used to determine the odds of hospital death in relation to measures of glucose variability after adjustment for important covariates. Main results:Of the methods used to measure glucose variability, the glycemic lability index had the best discrimination for mortality (area under the curve = 0.67, p < 0.001). After adjustment for confounders, including the number of organ failures and the occurrence of hypoglycemia, there was a significant interaction between glycemic lability index and average glucose level, and the odds of hospital mortality. Higher glycemic lability index was not independently associated with mortality among subjects with average glucose levels above the median for the cohort. However, subjects with increased glycemic lability index, but lower average glucose values had almost five-fold increased odds of hospital mortality (odds ratio = 4.73, 95% confidence interval = 2.6-8.7) compared with those with lower glycemic lability index. Conclusions:Glucose variability is independently associated with hospital mortality in septic patients. Strategies to reduce glucose variability should be studied to determine whether they improve the outcomes of septic patients.

Norepinephrine Up-regulates the Expression of Vascular Endothelial Growth Factor, Matrix Metalloproteinase (MMP)-2, and MMP-9 in Nasopharyngeal Carcinoma Tumor Cells

Recent studies using ovarian cancer cells have shown that the catecholamine hormones norepinephrine (norepi) and epinephrine (epi) may influence cancer progression by modulating the expression of matrix metalloproteinases (MMP) and vascular endothelial growth factor (VEGF). The purpose of this study is to determine if the stress hormone norepi can influence the expression of MMP-2, MMP-9, and VEGF in nasopharyngeal carcinoma (NPC) tumors by using three NPC tumor cell lines. The NPC cell lines HONE-1, HNE-1, and CNE-1 were treated with norepi. The effects of norepi on MMP-2, MMP-9, and VEGF synthesis were measured by ELISA; functional MMP activity was measured by the invasive potential of the cells using a membrane invasion culture system whereas functional activity of VEGF was analyzed using a human umbilical vein endothelial cell tube formation assay. Norepi treatment increased MMP-2, MMP-9, and VEGF levels in culture supernatants of HONE-1 cells, which could be inhibited by the beta-blocker propranolol. Norepi induced the invasiveness of all NPC cell lines in a dose-dependent manner, which was blocked by CMT-3, an MMP inhibitor, and propranolol. Norepi stimulated the release of functional angiogenic VEGF by HONE-1 cells as well. Finally, HONE-1 cells were shown to express beta-adrenergic receptors as did seven of seven NPC biopsies examined. The data suggest that catecholamine hormones produced by the sympathetic-adrenal medullary axis may affect NPC tumor progression, in part, through modulation of key angiogenic cytokines.

MicroRNA-126 inhibits invasion in non-small cell lung carcinoma cell lines

Crk is a member of a family of adaptor proteins that are involved in intracellular signal pathways altering cell adhesion, proliferation, and migration. Increased expression of Crk has been described in lung cancer and associated with increased tumor invasiveness. MicroRNAs (miRNAs) are a family of small non-coding RNAs (approximately 21-25 nt long) that are capable of targeting genes for either degradation of mRNA or inhibition of translation. Crk is a predicted putative target gene for miR-126. Over-expression of miR126 in a lung cancer cell line resulted in a decrease in Crk protein without any alteration in the associated mRNA. These lung cancer cells exhibit a decrease in adhesion, migration, and invasion. Decreased cancer cell invasion was also evident following targeted knockdown of Crk. MiR-126 alters lung cancer cell phenotype by inhibiting adhesion, migration, and invasion and the effects on invasion may be partially mediated through Crk regulation.

Body mass index is independently associated with hospital mortality in mechanically ventilated adults with acute lung injury

Objective:To determine the association between body mass index (BMI) and hospital mortality for critically ill adults. Design:Retrospective cohort study. Setting:One-hundred six intensive care units (ICUs) in 84 hospitals. Patients:Mechanically ventilated adults (n = 1,488) with acute lung injury (ALI) included in the Project IMPACT database between December 1995 and September 2001. Interventions:None. Measurements and Main Results:Over half of the cohort had a BMI above the normal range. Unadjusted analyses showed that BMI was higher among subjects who survived to hospital discharge vs. those who did not (p < .0001). ICU and hospital mortality rates were lower in higher BMI categories. After risk-adjustment, BMI was independently associated with hospital mortality (p < .0001) when modeled as a continuous variable. The adjusted odds were highest at the lowest BMIs and then declined to a minimum between 35 and 40 kg/m2. Odds increased after the nadir but remained below those seen at low BMIs. With use of a categorical designation, BMI was also independently associated with hospital mortality (p = .0055). The adjusted odds were highest for the underweight BMI group (adjusted odds ratio [OR], 1.94; 95% confidence interval [CI], 1.05–3.60) relative to the normal BMI group. As in the analysis using the continuous BMI variable, the odds of hospital mortality were decreased for the groups with higher BMIs (overweight adjusted OR, 0.72; 95% CI, 0.51–1.02; obese adjusted OR, 0.67; 95% CI, 0.46–0.97; severely obese adjusted OR, 0.78; 95% CI, 0.44–1.38). Differences in the use of heparin prophylaxis mediated some of the protective effect of severe obesity. Conclusions:BMI was associated with risk-adjusted hospital mortality among mechanically ventilated adults with ALI. Lower BMIs were associated with higher odds of death, whereas overweight and obese BMIs were associated with lower odds.

Macrophage microvesicles induce macrophage differentiation and miR-223 transfer.

Microvesicles are small membrane-bound particles comprised of exosomes and various-sized extracellular vesicles. These are released by several cell types. Microvesicles have a variety of cellular functions from communication to mediating growth and differentiation. Microvesicles contain proteins and nucleic acids. Previously, we showed that plasma microvesicles contain microRNAs (miRNAs). Based on our previous report, the majority of peripheral blood microvesicles are derived from platelets, while mononuclear phagocytes, including macrophages, are the second most abundant population. Here, we characterized macrophage-derived microvesicles and explored their role in the differentiation of naive monocytes. We also identified the miRNA content of the macrophage-derived microvesicles. We found that RNA molecules contained in the macrophage-derived microvesicles were transported to target cells, including mono cytes, endothelial cells, epithelial cells, and fibroblasts. Furthermore, we found that miR-223 was transported to target cells and was functionally active. Based on our observations, we hypothesize that microvesicles bind to and activate target cells. Furthermore, we find that microvesicles induce the differentiation of macrophages. Thus, defining key components of this response may identify novel targets to regulate host defense and inflammation.

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.