Metabolomics Analysis of Mesenchymal Stem Cell (MSC) Therapy in a Phase I Clinical Trial of Septic Shock: An Exploratory Study.

Sepsis is the result of an uncontrolled host inflammatory response to infection that may lead to septic shock with multiorgan failure and a high mortality rate. There is an urgent need to improve early diagnosis and to find markers identifying those who will develop septic shock and certainly a need to develop targeted treatments to prevent septic shock and its high mortality. Herein, we explore metabolic alterations due to mesenchymal stromal cell (MSC) treatment of septic shock. The clinical findings for this study were already reported; MSC therapy was well-tolerated and safe in patients in this phase I clinical trial. In this exploratory metabolomics study, 9 out of 30 patients received an escalating dose of MSC treatment, while 21 patients were without MSC treatment. Serum metabolomics profiling was performed to detect and characterize metabolite changes due to MSC treatment and to help determine the sample size needed for a phase II clinical trial and to define a metabolomic response to MSC treatment. Serum metabolites were measured using 1H-NMR and HILIC-MS at times 0, 24 and 72 h after MSC infusion. The results demonstrated the significant impact of MSC treatment on serum metabolic changes in a dose- and time-dependent manner compared to non-MSC-treated septic shock patients. This study suggests that plasma metabolomics can be used to assess the response to MSC therapy and that treatment-related metabolomics effects can be used to help determine the sample size needed in a phase II trial. As this study was not powered to detect outcome, how the treatment-induced metabolomic changes described in this study of MSC-treated septic shock patients are related to outcomes of septic shock in the short and long term will need to be explored in a larger adequately powered phase II clinical trial.

Single-cell transcriptomic atlas of lung microvascular regeneration after targeted endothelial cell ablation.

We sought to define the mechanism underlying lung microvascular regeneration in a model of severe acute lung injury (ALI) induced by selective lung endothelial cell ablation. Intratracheal instillation of DT in transgenic mice expressing human diphtheria toxin (DT) receptor targeted to ECs resulted in ablation of >70% of lung ECs, producing severe ALI with near complete resolution by 7 days. Using single-cell RNA sequencing, eight distinct endothelial clusters were resolved, including alveolar aerocytes (aCap) ECs expressing apelin at baseline and general capillary (gCap) ECs expressing the apelin receptor. At 3 days post-injury, a novel gCap EC population emerged characterized by de novo expression of apelin, together with the stem cell marker, protein C receptor. These stem-like cells transitioned at 5 days to proliferative endothelial progenitor-like cells, expressing apelin receptor together with the pro-proliferative transcription factor, Foxm1, and were responsible for the rapid replenishment of all depleted EC populations by 7 days post-injury. Treatment with an apelin receptor antagonist prevented ALI resolution and resulted in excessive mortality, consistent with a central role for apelin signaling in EC regeneration and microvascular repair. The lung has a remarkable capacity for microvasculature EC regeneration which is orchestrated by newly emergent apelin-expressing gCap endothelial stem-like cells that give rise to highly proliferative, apelin receptor-positive endothelial progenitors responsible for the regeneration of the lung microvasculature.

Circulating miR-206 and Wnt-signaling are associated with cardiovascular complications and a history of preeclampsia in women.

Women with a history of preeclampsia (PE) have increased risk of cardiovascular disease (CVD) later in life. However, the molecular determinants underlying this risk remain unclear. We sought to understand how circulating miRNA levels are affected by prior PE, and related to biological pathways underpinning cardiovascular disease. RNA sequencing was used to profile plasma levels of 2578 miRNAs in a retrospective study of women with a history of PE or normotensive pregnancy, in two independent cohorts with either acute coronary syndrome (ACS) (n = 17-18/group) or no ACS (n = 20/group). Differential miRNA alterations were assessed in relation to a history of PE (within each cohort) or ACS (across cohorts), and compared with miRNAs previously reported to be altered during PE. A history of PE was associated with altered levels of 30 and 20 miRNAs in the ACS and non-ACS cohorts, respectively, whereas ACS exposure was associated with alterations in 259 miRNAs. MiR-206 was identified at the intersection of all comparisons relating to past/current PE and ACS exposure, and has previously been implicated in atherogenic activities related to hepatocytes, vascular smooth muscle cells and macrophages. Integration of all differentially altered miRNAs with their predicted and experimentally validated targets in silico revealed a number of highly targeted genes with potential atherogenic functions (including NFAT5, CCND2 and SMAD2), and one significantly enriched KEGG biological pathway (Wnt signaling) that was shared between all exposure groups. The present study provides novel insights into miRNAs, target genes and biological pathways that may underlie the long-term cardiovascular sequelae of PE.

Systematic review of microRNA biomarkers in acute coronary syndrome and stable coronary artery disease.

The aim of this systematic review was to assess dysregulated miRNA biomarkers in coronary artery disease (CAD). Dysregulated microRNA (miRNAs) have been shown to be linked to cardiovascular pathologies including CAD and may have utility as diagnostic and prognostic biomarkers. We compared miRNAs identified in acute coronary syndrome (ACS) compared with stable CAD and control populations. We conducted a systematic search of controlled vocabulary and free text terms related to ACS, stable CAD and miRNA in Biosis Previews (OvidSP), The Cochrane Library (Wiley), Embase (OvidSP), Global Health (OvidSP), Medline (PubMed and OvidSP), Web of Science (Clarivate Analytics), and ClinicalTrials.gov which yielded 7370 articles. Of these, 140 original articles were appropriate for data extraction. The most frequently reported miRNAs in any CAD (miR-1, miR-133a, miR-208a/b, and miR-499) are expressed abundantly in the heart and play crucial roles in cardiac physiology. In studies comparing ACS cases with stable CAD patients, miR-21, miR-208a/b, miR-133a/b, miR-30 family, miR-19, and miR-20 were most frequently reported to be dysregulated in ACS. While a number of miRNAs feature consistently across studies in their expression in both ACS and stable CAD, when compared with controls, certain miRNAs were reported as biomarkers specifically in ACS (miR-499, miR-1, miR-133a/b, and miR-208a/b) and stable CAD (miR-215, miR-487a, and miR-502). Thus, miR-21, miR-133, and miR-499 appear to have the most potential as biomarkers to differentiate the diagnosis of ACS from stable CAD, especially miR-499 which showed a correlation between the level of their concentration gradient and myocardial damage. Although these miRNAs are potential diagnostic biomarkers, these findings should be interpreted with caution as the majority of studies conducted predefined candidate-driven assessments of a limited number of miRNAs (PROSPERO registration: CRD42017079744).

Effects of Mesenchymal Stem Cell Treatment on Systemic Cytokine Levels in a Phase 1 Dose Escalation Safety Trial of Septic Shock Patients.

OBJECTIVES: Cellular Immunotherapy for Septic Shock is the first-in-human clinical trial evaluating allogeneic mesenchymal stem/stromal cells in septic shock patients. Here, we sought to determine whether plasma cytokine profiles may provide further information into the safety and biological effects of mesenchymal stem/stromal cell treatment, as no previous study has conducted a comprehensive analysis of circulating cytokine levels in critically ill patients treated with mesenchymal stem/stromal cells. DESIGN: Phase 1 dose-escalation trial. PATIENTS: The interventional cohort (n = 9) of septic shock patients received a single dose of 0.3, 1.0, or 3.0 million mesenchymal stem/stromal cells/kg body weight (n = 3 per dose). The observational cohort received no mesenchymal stem/stromal cells (n = 21). INTERVENTIONS: Allogeneic bone marrow-derived mesenchymal stem/stromal cells. MEASUREMENTS AND MAIN RESULTS: Serial plasma samples were collected at study baseline prior to mesenchymal stem/stromal cell infusion (0 hr), 1 hour, 4 hours, 12 hours, 24 hours, and 72 hours after mesenchymal stem/stromal cell infusion/trial enrollment. Forty-nine analytes comprised mostly of cytokines along with several biomarkers were measured. We detected no significant elevations in a broad range of pro-inflammatory cytokines and biomarkers between the interventional and observational cohorts. Stratification of the interventional cohort by mesenchymal stem/stromal cell dose further revealed patient-specific and dose-dependent perturbations in cytokines, including an early but transient dampening of pro-inflammatory cytokines (e.g., interleukin-1ß, interleukin-2, interleukin-6, interleukin-8, and monocyte chemoattractant protein 1), suggesting that mesenchymal stem/stromal cell treatment may alter innate immune responses and underlying sepsis biology. CONCLUSIONS: A single infusion of up to 3 million cells/kg of allogeneic mesenchymal stem/stromal cells did not exacerbate elevated cytokine levels in plasma of septic shock patients, consistent with a safe response. These data also offer insight into potential biological mechanisms of mesenchymal stem/stromal cell treatment and support further investigation in larger randomized controlled trials.

Circulating MicroRNAs Implicate Multiple Atherogenic Abnormalities in the Long-Term Cardiovascular Sequelae of Preeclampsia.

BACKGROUND: Women who have had preeclampsia (PE) are at increased risk for premature cardiovascular disease (CVD). The underlying pathophysiology of this risk remains unclear, but potentially involves subclinical vascular damage or dysfunction. Alterations in the levels of circulating microRNAs may be implicated, as they are known to play pervasive roles in vascular biology. We investigated whether levels of circulating microRNAs are altered between women with premature acute coronary syndrome (ACS), with and without a history of PE. METHODS: Women with premature ACS (age ≤ 55 years) were categorized based on a prior history of PE or normotensive pregnancy. Relative plasma levels of 372 microRNAs were initially assessed by polymerase chain reaction array in a subset of subjects (n = 12-13/group) matched for age, chronic hypertension, dyslipidemia, and smoking status. Candidate microRNAs were then validated in a larger cohort of ACS patients (n = 176). RESULTS: MicroRNAs previously linked to angiogenesis (miR-126-3p), inflammation (miR-146a-5p), and cholesterol metabolism (miR-122-5p) were significantly decreased in women with prior PE compared to women with prior normotensive pregnancy (P = 0.002, 0.017, and 0.009, respectively), even after adjustment for chronic hypertension. CONCLUSIONS: Circulating levels of miR-126-3p, -146a-5p, and -122-5p were significantly decreased in women with premature ACS who reported prior PE compared to those with prior normotensive pregnancy. These data provide novel insight into potential pathways that may contribute to the increased risk of CVD following PE.

Systematic Assessment of Strategies for Lung-targeted Delivery of MicroRNA Mimics.

There is considerable interest in the use of synthetic miRNA mimics (or inhibitors) as potential therapeutic agents in pulmonary vascular disease; however, the optimal delivery method to achieve high efficiency, selective lung targeting has not been determined. Here, we sought to investigate the relative merits of different lung-targeted strategies for delivering miRNA mimics in rats. Methods: Tissue levels of a synthetic miRNA mimic, cel-miR-39-3p (0.5 nmol in 50 µL invivofectamine/PBS vehicle) were compared in male rats (n=3 rats/method) after delivery by commonly used lung-targeting strategies including intratracheal liquid instillation (IT-L), intratracheal aerosolization with (IT-AV) or without ventilator assistance (IT-A), intranasal liquid instillation (IN-L) and intranasal aerosolization (IN-A). Intravenous (IV; via jugular vein), intraperitoneal (IP) and subcutaneous (SC) delivery served as controls. Relative levels of cel-miR-39 were quantified by RT-qPCR. Results: At 2 h post delivery, IT-L showed the highest lung mimic level, which was significantly higher than levels achieved by all other methods (from ~10- to 10,000-fold, p<0.05). Mimic levels remained detectable in the lung 24 h after delivery, but were 10- to 100-fold lower. The intrapulmonary distribution of cel-miR-39 was comparable when delivered as either a liquid or aerosol, with evidence of mimic distribution to both the left and right lung lobes and penetration to distal regions. All lung-targeted strategies showed lung-selective mimic uptake, with mimic levels 10- to 100-fold lower in heart and 100- to 10,000-fold lower in liver, kidney and spleen. In contrast, IV, SC and IP routes showed comparable or higher mimic levels in non-pulmonary tissues. Conclusions: miRNA uptake in the lungs differed markedly by up to 4 orders of magnitude, demonstrating that the choice of delivery strategy could have a significant impact on potential therapeutic outcomes in preclinical investigations of miRNA-based drug candidates.

High circulating angiopoietin-2 levels exacerbate pulmonary inflammation but not vascular leak or mortality in endotoxin-induced lung injury in mice.

BACKGROUND: Elevated plasma levels of angiopoietin-2 (ANGPT2) have been reported in patients with acute lung injury (ALI); however, it remains unclear whether this increase contributes to, or just marks, the underlying vasculopathic inflammation and leak associated with ALI. Here we investigated the biological consequences of inducing high circulating levels of ANGPT2 in a mouse model of endotoxin-induced ALI. METHODS: Transgenic mice (ANGPT2OVR) with elevated circulating levels of ANGPT2, achieved through conditional hepatocyte-specific overexpression, were examined from 3 to 72 hours following lipopolysaccharide (LPS)-induced ALI. An aptamer-based inhibitor was used to neutralise the effects of circulating ANGPT2 in LPS-exposed ANGPT2OVR mice. RESULTS: Total cells, neutrophils and macrophages, as well as inflammatory cytokines, were significantly higher in bronchoalveolar lavage (BAL) of ANGPT2OVR versus littermate controltTA mice at 48 hours and 6 hours post-LPS, respectively. In contrast, LPS-induced vascular leak, evidenced by total BAL protein levels and lung wet/dry ratio, was unchanged between ANGPT2OVR and controlstTA, while BAL levels of IgM and albumin were decreased in ANGPT2OVR mice between 24 hours and 48 hours suggesting a partial attenuation of vascular leak. There was no significant difference in LPS-induced mortality between ANGPT2OVR and controlstTA. An ANGPT2-neutralising aptamer partially attenuated alveolar cell infiltration while exacerbating vascular leak in LPS-exposed ANGPT2OVR mice, supported by underlying time-dependent changes in the lung transcriptional profiles of multiple genes linked to neutrophil recruitment/adhesion and endothelial integrity. CONCLUSIONS: Our findings suggest that high circulating ANGPT2 potentiates endotoxin-induced lung inflammation but may also exert other pleiotropic effects to help fine-tune the vascular response to lung injury.

Systemic delivery of MicroRNA mimics with polyethylenimine elevates pulmonary microRNA levels, but lacks pulmonary selectivity.

Reversing pathologic alterations in vascular microRNA (miRNA) expression represents a potential therapeutic strategy for pulmonary hypertension. While polyethylenimine (PEI) has previously been shown to be an effective vehicle for vascular lung-directed delivery of plasmid DNA, it remains unclear whether this utility is generalizable to miRNAs. Here we show that despite elevated lung levels, the intravenous infusion of PEI-miRNA mimic complexes fails to provide lung-selective delivery in rats.

Cellular Immunotherapy for Septic Shock. A Phase I Clinical Trial.

RATIONALE: In septic animal models mesenchymal stem (stromal) cells (MSCs) modulate inflammation, enhance tissue repair and pathogen clearance, and reduce death. OBJECTIVES: To conduct a phase I dose escalation trial of MSCs in septic shock with the primary objective of examining the safety and tolerability of MSCs. METHODS: We enrolled nine participants within 24 hours of admission to the ICU. A control cohort of 21 participants was enrolled before starting the MSC interventional cohort to characterize expected adverse events (AEs) and to serve as a comparator for the intervention cohort. Three separate MSC dose cohorts, with three participants per cohort, received a single intravenous dose of 0.3, 1.0, and 3.0 × 106 cells/kg. A prespecified safety plan monitored participants for the occurrence of AEs; cytokines were collected at prespecified time points. MEASUREMENTS AND MAIN RESULTS: Ages of participants in the interventional versus observational cohorts were median of 71 (range, 38-91) and 61 (range, 23-95). Acute Physiology and Chronic Health Evaluation scores were median of 25 (range, 11-28) and 26 (range, 17-32). MSC doses ranged from 19 to 250 million cells. There were no prespecified MSC infusion-associated or serious unexpected AEs, nor any safety or efficacy signals for the expected AEs or the measured cytokines between the interventional and observational cohorts. CONCLUSIONS: The infusion of freshly cultured allogenic bone marrow-derived MSCs, up to a dose of 3 million cells/kg (250 million cells), into participants with septic shock seems safe. Clinical trial registered with www.clinicaltrials.gov (NCT02421484).

Clinical Features and Outcomes of Acute Coronary Syndrome in Women With Previous Pregnancy Complications.

BACKGROUND: Women with previous cardiometabolic complications of pregnancy experience double the risk of cardiovascular disease. However, few data exist on the clinical effect of these complications at the time of an acute coronary syndrome (ACS). The objective of this work was to compare risk factors, clinical features, and outcomes among women with premature ACS with or without previous pregnancy complications (gestational diabetes and/or hypertensive disorders of pregnancy). METHODS: Data were obtained from a multicentre cohort of individuals hospitalized with premature ACS. A total of 251 parous women were included and provided obstetric history and blood samples. They were followed for the development of major adverse cardiac events at 12 months. RESULTS: At presentation with ACS, women with a previous pregnancy complication (38%) were slightly younger than were women without such complications (47.4 ± 6.2 vs 49.1 ± 5.6 years; P = 0.002). They also had more traditional atherosclerotic risk factors. Specifically, women with previous preeclampsia were more likely to have chronic hypertension and an elevated ratio of soluble fms-like tyrosine kinase:placental growth factor. There was no between-group difference in Global Registry of Acute Coronary Events (GRACE) score or troponin tertile but there was a trend toward higher risk of ST-elevation myocardial infarction in women who had a previous pregnancy complication (odds ratio, 1.80; 95% confidence interval, 1.00-3.23; P = 0.05). There was also an increased risk of recurrent ACS at 12 months in women with previous preeclampsia (hazard ratio, 6.79; 95% confidence interval, 1.37-33.63; P = 0.02). CONCLUSIONS: Among a cohort of women with ACS, previous pregnancy complications were associated with more severe disease and poorer outcome.

Identification of MicroRNA-124 as a Major Regulator of Enhanced Endothelial Cell Glycolysis in Pulmonary Arterial Hypertension via PTBP1 (Polypyrimidine Tract Binding Protein) and Pyruvate Kinase M2.

BACKGROUND: Pulmonary arterial hypertension (PAH) is characterized by abnormal growth and enhanced glycolysis of pulmonary artery endothelial cells. However, the mechanisms underlying alterations in energy production have not been identified. METHODS: Here, we examined the miRNA and proteomic profiles of blood outgrowth endothelial cells (BOECs) from patients with heritable PAH caused by mutations in the bone morphogenetic protein receptor type 2 (BMPR2) gene and patients with idiopathic PAH to determine mechanisms underlying abnormal endothelial glycolysis. We hypothesized that in BOECs from patients with PAH, the downregulation of microRNA-124 (miR-124), determined with a tiered systems biology approach, is responsible for increased expression of the splicing factor PTBP1 (polypyrimidine tract binding protein), resulting in alternative splicing of pyruvate kinase muscle isoforms 1 and 2 (PKM1 and 2) and consequently increased PKM2 expression. We questioned whether this alternative regulation plays a critical role in the hyperglycolytic phenotype of PAH endothelial cells. RESULTS: Heritable PAH and idiopathic PAH BOECs recapitulated the metabolic abnormalities observed in pulmonary artery endothelial cells from patients with idiopathic PAH, confirming a switch from oxidative phosphorylation to aerobic glycolysis. Overexpression of miR-124 or siRNA silencing of PTPB1 restored normal proliferation and glycolysis in heritable PAH BOECs, corrected the dysregulation of glycolytic genes and lactate production, and partially restored mitochondrial respiration. BMPR2 knockdown in control BOECs reduced the expression of miR-124, increased PTPB1, and enhanced glycolysis. Moreover, we observed reduced miR-124, increased PTPB1 and PKM2 expression, and significant dysregulation of glycolytic genes in the rat SUGEN-hypoxia model of severe PAH, characterized by reduced BMPR2 expression and endothelial hyperproliferation, supporting the relevance of this mechanism in vivo. CONCLUSIONS: Pulmonary vascular and circulating progenitor endothelial cells isolated from patients with PAH demonstrate downregulation of miR-124, leading to the metabolic and proliferative abnormalities in PAH ECs via PTPB1 and PKM1/PKM2. Therefore, the manipulation of this miRNA or its targets could represent a novel therapeutic approach for the treatment of PAH.

Lack of elevation in plasma levels of pro-inflammatory cytokines in common rodent models of pulmonary arterial hypertension: questions of construct validity for human patients.

Translational research depends on the relevance of animal models and how well they replicate human disease. Here, we investigated plasma levels of three important pro-inflammatory cytokines (TNFα, IL-6, and MCP-1), known to be elevated in human pulmonary arterial hypertension (PAH), and systematically assessed their levels in PAH patients compared to five different rodent models of pulmonary hypertension (PH). A consistent immunoassay platform (Luminex xMAP) and source (Millipore) was used to measure all specimens. PAH patients (n = 29) exhibited significant elevations in all three cytokines (median [IQR] pg/mL; TNFα, 7.0 [4.8-11.7]; IL-6, 9.2 [3.8-17.2]; MCP-1, 109 [65-142]) versus healthy participants (n = 20) (median [IQR] pg/mL; TNFα, 3.0 [2.0-3.6]; IL-6, 1.7 [0.5-7.2]; MCP-1, 79 [49-93]. In contrast, mice with PH established after three weeks of hypoxia (n = 18) or SU5416 plus hypoxia (n = 20) showed no significant change in their plasma cytokine levels versus controls (n = 16), based on three to four independent experiments per group. Similarly, plasma cytokine levels were not elevated in rats with PH established three weeks after monocrotaline (n = 23), eight weeks after SU5416 alone (n = 10) or six to eight weeks after SU5416 plus hypoxia (n = 21) versus controls (n = 36 rats), based on three to eight independent experiments per group. Positive biologic control specimens from sepsis patients (n = 9), cecal-ligation and puncture (CLP)-induced septic mice (n = 6), and lipopolysaccharide-induced septic rats (n = 4) showed robust elevations in all three cytokines. This study suggests that animal models commonly used for the development of novel diagnostic and therapeutic approaches for PAH may have limited construct validity with respect to markers of systemic immune activation seen in human patients.

Assessment of Circulating LncRNAs Under Physiologic and Pathologic Conditions in Humans Reveals Potential Limitations as Biomarkers.

Long non-coding RNAs (lncRNA) are a new class of regulatory molecules with diverse cellular functions. Recent reports have suggested that extracellular lncRNAs are detectable in human plasma and may serve as biomarkers. Here, we sought to investigate circulating lncRNAs as potential biomarkers for pulmonary arterial hypertension (PAH). Eighty-four lncRNAs, representing some of the most abundant and functionally relevant candidates identified in cellular studies, were assessed via RT-qPCR in plasma from PAH and healthy subjects. However, despite preamplification, the majority of lncRNAs were surprisingly undetectable or sporadically detectable, and showed no differential changes. Systematic characterization of plasma/RNA quality and technical performance via internal and external controls revealed no evidence of RNA degradation or RT-qPCR inhibition, and most lncRNAs were robustly detectable in pulmonary tissue. In plasma, lncRNA levels were the lowest among several different RNA species examined, and this was generalizable to other chronic and acute vascular conditions including coronary artery disease, acute coronary syndrome, and septic shock. In addition, two of three previously reported circulating lncRNA biomarker candidates were not detectable in any of the plasma samples. This study reveals new insight on the relative levels of lncRNAs in circulation, which has important implications for their potential development as biomarkers.

Customized Internal Reference Controls for Improved Assessment of Circulating MicroRNAs in Disease.

BACKGROUND: Altered levels of circulating extracellular miRNA in plasma and serum have shown promise as non-invasive biomarkers of disease. However, unlike the assessment of cellular miRNA levels for which there are accepted housekeeping genes, analogous reference controls for normalization of circulating miRNA are lacking. Here, we provide an approach to identify and validate circulating miRNA reference controls on a de novo basis, and demonstrate the advantages of these customized internal controls in different disease settings. Importantly, these internal controls overcome key limitations of external spike-in controls. METHODS: Using a global RT-qPCR screen of 1066 miRNAs in plasma from pulmonary hypertension patients (PAH) and healthy subjects as a case example, we identified a large pool of initial candidate miRNAs that were systematically ranked according to their plasma level stability using a predefined algorithm. The performance of the top candidates was validated against multiple comparators, and in a second independent cohort of PAH and control subjects. The broader utility of this approach was demonstrated in a completely different disease setting with 372 miRNAs screened in plasma from septic shock patients and healthy controls. RESULTS: Normalization of data with specific internal reference controls significantly reduced the overall variation in circulating miRNA levels between subjects (relative to raw data), provided a more balanced distribution of up- and down-regulated miRNAs, replicated the results obtained by the benchmark geometric averaging of all detected miRNAs, and outperformed the commonly used external spike-in strategy. CONCLUSIONS: We demonstrate the feasibility of identifying circulating reference controls that can reduce extraneous technical variations, and improve the assessment of disease-related changes in plasma miRNA levels. This study provides a novel conceptual framework that addresses a critical and previously unmet need if circulating miRNAs are to advance as reliable diagnostic tools in medicine.

Discordant Regulation of microRNA Between Multiple Experimental Models and Human Pulmonary Hypertension.

BACKGROUND: The dysregulation of microRNA (miRNA) is known to contribute to the pathobiology of pulmonary arterial hypertension (PAH). However, the relationships between changes in tissue and circulating miRNA levels associated with different animal models and human pulmonary hypertension (PH) have not been defined. METHODS: A set of miRNAs that have been causally implicated in PH, including miR-17, -21, -130b, -145, -204, -424, and -503, were measured by reverse transcription-quantitative polymerase chain reaction in the plasma, lung, and right ventricle of three of the most common rodent models of PH: the rat monocrotaline and SU5416 plus chronic hypoxia (SuHx) models and the mouse chronic hypoxia model. Plasma miRNA levels were also evaluated in a cohort of patients with PAH and healthy subjects. RESULTS: Several miRNA showed PH model-dependent perturbations in plasma and tissue levels; however, none of these were conserved across all three experimental models. Principle component analysis of miR expression changes in plasma revealed distinct clustering between rodent models, and SuHx-triggered PH showed the greatest similarity to human PAH. Changes in the plasma levels of several miRNA also correlated with changes in tissue expression. In particular, miR-424 was concordantly increased (1.3- to 1.5-fold, P < .05) in the plasma, lung, and right ventricle of hypoxic mice and in the plasma of patients with PAH. CONCLUSIONS: miRNAs with established etiologic roles in PH showed context-dependent changes in tissue and circulating levels, which were not consistent across rodent models and human PAH. This suggests different miRNA-dependent mechanisms may contribute to experimental and clinical PH, complicating potential diagnostic and therapeutic applications amenable to these miRNAs.

A versatile endoribonuclease mimic made of DNA: characteristics and applications of the 8-17 RNA-cleaving DNAzyme.

Enzymes play a crucial role in all living organisms by accelerating the rates of a myriad of biochemical reactions that are necessary to sustain life. Although the vast majority of known enzymes are made of protein, in recent years it has become increasingly apparent that other molecular formats, like nucleic acids, can also serve in this capacity. DNAzymes (also known as deoxyribozymes) are synthetic enzymes made of short, single strands of deoxyribonucleic acid. These DNA-based enzymes offer the prospect of significant commercial utility, because they are exceptionally stable and can be produced very easily and inexpensively. The study of one particular DNAzyme, known as "8-17", has enhanced our understanding of DNAzyme-mediated catalysis. Moreover, the function of 8-17 has been regarded with special importance because it can catalyze sequence-specific cleavage of RNA, a reaction that has broad implications in biotechnology and biomedical fields. In this review, we explore the creation, characterization, and application of the 8-17 RNA-cleaving DNAzyme.

A genotype-to-phenotype map of in vitro selected RNA-cleaving DNAzymes: implications for accessing the target phenotype.

Herein, we describe a case study into the population dynamics of in vitro selection, using RNA-cleaving DNAzymes as a model system. We sought to understand how the composition of the population can change over time in response to different levels of selection pressure, and how well these changes are correlated with selection of the target phenotype. The model population is composed of 857 DNAzyme clones representing 215 discrete sequence classes, which had previously been identified from two parallel selection experiments, conducted under an increasingly stringent, or permissive and constant selection time pressure. In this report, we determined the principal phenotypic properties (i.e. k(obs), maximum cleavage yield and PCR efficiency) from a sample of 58 clones representing 46 different major and minor sequence classes from various rounds of each selection experiment. Interestingly, a positive correlation between the catalytic rate constant and the corresponding frequency and temporal position of a given DNAzyme was not consistently observed; however, the strength of the correlation was qualitatively higher under conditions of more stringent selection time pressure. These results suggest that the selective sampling paradigm on which in vitro selection is based, may underestimate the true functional capacity of any given random-sequence library.

Biologically inspired synthetic enzymes made from DNA.

In cells, DNA typically consists of two antiparallel strands arranged in a double-helical structure, which is central to its fundamental role in storing and transmitting genetic information. In laboratories, however, DNA can be readily synthesized as a single-stranded polymer that can adopt many other types of structures, including some that have been shown to catalyze chemical transformations. These catalytic DNA molecules are commonly referred to as DNAzymes, or deoxyribozymes. Thus far, DNAzymes have not been found in cells, but hundreds of structural and functional variations have been created in the laboratory. This alternative catalytic platform has piqued the curiosity of many researchers, including those who seek to exploit them in potential applications ranging from analytical tools to therapeutic agents. In this review, we explore the unconventional role of DNA as a biologically inspired synthetic enzyme.