Evidence of a redox imbalance in the MCT-treated rat lung.

<p>Despite an increase in glutathione recycling, as indicated by increases in the 5-oxoproline metabolite, both reduced and oxidized glutathione are significantly decreased in the pre-PH (A), indicative of increased oxidative stress. The ratio between pre-PH and control metabolites demonstrate significant increases (red boxes) gamma-glutamyl amino acids (B). Activation of the gamma-glutamyl cycle is usually associated with an increased inflammatory response (N = 10, p<0.05).</p

Disruption of arginine metabolism in the MCT-treated rat lung.

<p>The metabolic fate of arginine is complex being involved in NO signaling, the urea cycle, proliferation, and matrix remodeling (center pathway diagram). There is significantly increased urea production in the pre-PH lung indicative of increased arginase activity. Aspartate and fumarate, which are involved in arginine biosynthesis, are significantly increased in the pre-PH lung as is arginine itself. Polyamine metabolites are significantly increased in the pre-PH lung, indicative of increased cellular proliferation while the significant increase in proline pathway metabolites is suggestive of extracellular matrix remodeling (N = 10, p<0.05).</p

Biomarkers of cellular damage are increased in the MCT-treated rat lung.

<p>Phospholipid degradation and membrane remodeling markers (ethanolamine and glycerophosphoethanolamine) are significantly increased in the pre-PH lung. Methylhistidines (1-methylhhistidine and N-acetyl-3-histidine), produced by methylation of actin and myosin in muscle, are indicative of muscle protein breakdown and therefore muscular damage. Significant increases in mitochondrial membrane degradation (2-stearoylglycerophosphoglycerol) and the breakdown product of mitochondrially-encoded/synthesized proteins (N-formylmethionine) reflect mitochondrial damage. (N = 10, p<0.05).</p

A shift in the balance omega 6 and omega 3 fatty acids and increased pro-inflammatory eicosanoid production in the MCT-treated rat lung.

<p>The ratio between pre-PH and control metabolites show both significant increases (red boxes) for omega 6, omega 3 fatty acids and eicosanoids and a trending decrease (light green box) for stearidonate (A). The pro-inflammatory prostaglandins E2, D2, J2 and leukotriene B5 are also significantly increased in the pre-PH lung (N = 10, p<0.05, B).</p

Carnitine homeostasis is altered in the MCT-treated rat lung.

<p>Significant decreases in conjugated acyl carnitines such as palmitoylcarnitine, stearoylcarnitine and oleoylcarnitine indicate that there is disrupted fatty acid transport to mitochondria in the lungs of pre-PH rats (N = 10, p<0.5).</p

Evidence for a glycolytic switch in the MCT-treated rat lung.

<p>Data for control lung are represented in grey boxes and data for the pre-PH lung are represented in blue boxes. Quantities are in arbitrary units (N = 10, p<0.05). MCT-treated animals have higher levels of glucose, glucose-6-phosphate, glucose-1-phosphate, fructose, fructose-6-phosphate, 6-phospho gluconate, ribulose 5-phosphate, sedoheptulose-7-phosphate, pyruvate and lactate. These metabolic data are consistent with an upregulation of glycolytic or glucose dependent pathways in the lungs of pre-PH rats.</p

Inflammatory metabolites are increased in the MCT-treated rat lung.

<p>Inflammation, via INF-gamma or TNF-alpha, activates indoleamine-2,3-dioxygenase (IDO) or tryptophan-2,3-dioxygenase (TDO) that degrade tryptophan to kynurenine/kynurenate (center pathway). There is a significant increase in tryptophan, kynurenine, kynurenate and serotonin in the pre-PH lung suggestive of increased inflammation. (N = 10, p<0.05).</p

Differentially Expressed Plasma MicroRNAs and the Potential Regulatory Function of Let-7b in Chronic Thromboembolic Pulmonary Hypertension

<div><p>Chronic thromboembolic pulmonary hypertension (CTEPH) is a progressive disease characterized by misguided thrombolysis and remodeling of pulmonary arteries. MicroRNAs are small non-coding RNAs involved in multiple cell processes and functions. During CTEPH, circulating microRNA profile endued with characteristics of diseased cells could be identified as a biomarker, and might help in recognition of pathogenesis. Thus, in this study, we compared the differentially expressed microRNAs in plasma of CTEPH patients and healthy controls and investigated their potential functions. Microarray was used to identify microRNA expression profile and qRT-PCR for validation. The targets of differentially expressed microRNAs were identified <i>in silico</i>, and the Gene Ontology database and Kyoto Encyclopedia of Genes and Genomes pathway database were used for functional investigation of target gene profile. Targets of let-7b were validated by fluorescence reporter assay. Protein expression of target genes was determined by ELISA or western blotting. Cell migration was evaluated by wound healing assay. The results showed that 1) thirty five microRNAs were differentially expressed in CTEPH patients, among which, a signature of 17 microRNAs, which was shown to be related to the disease pathogenesis by <i>in silico</i> analysis, gave diagnostic efficacy of both sensitivity and specificity >0.9. 2) Let-7b, one of the down-regulated anti-oncogenic microRNAs in the signature, was validated to decrease to about 0.25 fold in CTEPH patients. 3) ET-1 and TGFBR1 were direct targets of let-7b. Altering let-7b level influenced ET-1 and TGFBR1 expression in pulmonary arterial endothelial cells (PAECs) as well as the migration of PAECs and pulmonary arterial smooth muscle cells (PASMCs). These results suggested that CTEPH patients had aberrant microRNA signature which might provide some clue for pathogenesis study and biomarker screening. Reduced let-7b might be involved in the pathogenesis of CTEPH by affecting ET-1 expression and the function of PAECs and PASMCs.</p></div

ET-1 and TGFBR1 were direct target genes of let-7b.

<p>EGFP reporter assay was applied for target validation in HEK 293 cells. Oligonucleotide mimics and negative control (NC) of let-7b were co-transfected with reporter plasmids and mutated controls, respectively. (<b>A</b>) <b>&</b> (<b>B</b>) Predicted duplex of let-7b and its target region of TGFBR1 and ET-1 with experimental mutation in seed sequences respectively. The number indicates the region position in its 3′-UTRs. The mutated nucleotide was shown in red, and the original nucleotide was its complementary one. (<b>C</b>) <b>&</b> (<b>D</b>) let-7b down-regulated EGFP expression through the predicted seed sequences in 3′-UTRs of ET-1 and TGFBR1. <i>P</i> value was calculated by two-sample Kolmogorov-Smirnov test. * <i>P</i><0.05.</p

Let-7b regulated PAECs and PASMCs migration.

<p>Human PAECs or PASMCs were transfected with a let-7b antagonist lentivirus or let-7b mimics, and the migration was evaluated by wound healing assays. Pictures were taken at 0 h, 12 h, 24 h, and 48 h. The wounded area was expressed as the percentage of recovery. (<b>A</b>) Let-7b mimics suppressed PAECs migration, and its antagonist promoted TGF-β induced PAECs migration at 24 h (n = 5). (<b>B</b>).Let-7b mimics suppressed PASMCs migration, and its antagonist promoted TGF-β induced PASMCs migration at 24 h (n = 5). <i>P</i> value was calculated by two-way ANOVA, and Post Hoc Test was done by Student-Newman-Keuls method. *** <i>P</i><0.001. * <i>P</i><0.05.</p