miR-200 family expression during normal and abnormal lung development due to congenital diaphragmatic hernia at the later embryonic stage in the nitrofen rat model.

INTRODUCTION: Congenital diaphragmatic hernia (CDH) is a life-threatening disease associated with pulmonary hypoplasia. CDH occurs approximately 1 in every 2000-3000 live births, and the pathophysiology is unknown. MicroRNAs are short, non-coding RNAs that control gene expression through post-transcriptional regulation. Based on our previous work, we hypothesized that the miR-200 family is differentially expressed in normal and abnormal lung development. We aimed to examine the expression of the miR-200 family during normal and hypoplastic lung development due to CDH. METHODS: We performed reverse transcriptase polymerase chain reaction (RT-qPCR) and fluorescent in situ hybridization (FISH) to study the expression levels and distribution of the miR-200 family members on embryonic day 21 (E21) rat control and nitrofen-induced hypoplastic CDH lungs. RESULTS: RT-qPCR showed up-regulation of miR-200a in hypoplastic CDH lungs. FISH showed contrasting expression patterns for miR- 200a, miR-200c, and miR-429 between control and hypoplastic CDH lungs, while we could not detect miR-141 in control and hypoplastic CDH lungs. CONCLUSION: We demonstrate a specific expression pattern of miR-200 family members in hypoplastic CDH lungs different from control lungs. This study suggests that disruption of miR-200 family expression plays a role in the pathogenesis of pulmonary hypoplasia associated with CDH.

Prenatal microRNA miR-200b Therapy Improves Nitrofen-induced Pulmonary Hypoplasia Associated With Congenital Diaphragmatic Hernia.

OBJECTIVE: We aimed to evaluate the use of miR-200b as a prenatal transplacental therapy in the nitrofen rat model of abnormal lung development and congenital diaphragmatic hernia (CDH). BACKGROUND: Pulmonary hypoplasia (PH) and pulmonary hypertension determine mortality and morbidity in CDH babies. There is no safe medical prenatal treatment available. We previously discovered that higher miR-200b is associated with better survival in CDH babies. Here, we investigate the role of miR-200b in the nitrofen rat model of PH and CDH and evaluate its use as an in vivo prenatal therapy. METHODS: We profiled miR-200b expression during nitrofen-induced PH using RT-qPCR and in situ hybridization in the nitrofen rat model of PH and CDH. The effects of nitrofen on downstream miR-200b targets were studied in bronchial lung epithelial cells using a SMAD luciferase assay, Western blotting and Immunohistochemistry. We evaluated miR-200b as a lung growth promoting therapy ex vivo and in vivo using lung explant culture and transplacental prenatal therapy in the nitrofen rat model. RESULTS: We show that late lung hypoplasia in CDH is associated with (compensatory) upregulation of miR-200b in less hypoplastic lungs. Increasing miR-200b abundance with mimics early after nitrofen treatment decreases SMAD-driven TGF-ß signaling and rescues lung hypoplasia both in vitro and in vivo. Also, prenatal miR-200b therapy decreases the observed incidence of CDH. CONCLUSIONS: Our data indicate that miR-200b improves PH and decreases the incidence of CDH. Future studies will further exploit this newly discovered prenatal therapy for lung hypoplasia and CDH.

Unique Tracheal Fluid MicroRNA Signature Predicts Response to FETO in Patients With Congenital Diaphragmatic Hernia.

OBJECTIVE AND BACKGROUND: Our objective was to determine the fetal in vivo microRNA signature in hypoplastic lungs of human fetuses with severe isolated congenital diaphragmatic hernia (CDH) and changes in tracheal and amniotic fluid of fetuses undergoing fetoscopic endoluminal tracheal occlusion (FETO) to reverse severe lung hypoplasia due to CDH. METHODS: We profiled microRNA expression in prenatal human lungs by microarray analysis. We then validated this signature with real-time quantitative polymerase chain reaction in tracheal and amniotic fluid of CDH patients undergoing FETO. We further explored the role of miR-200b using semiquantitative in situ hybridization and immunohistochemistry for TGF-ß2 in postnatal lung sections. We investigated miR-200b effects on TGF-ß signaling using a SMAD-luciferase reporter assay and Western blotting for phospho-SMAD2/3 and ZEB-2 in cultures of human bronchial epithelial cells. RESULTS: CDH lungs display an increased expression of 2 microRNAs: miR-200b and miR-10a as compared to control lungs. Fetuses undergoing FETO display increased miR-200 expression in their tracheal fluid at the time of balloon removal. Future survivors of FETO display significantly higher miR-200 expression than those with a limited response. miR-200b was expressed in bronchial epithelial cells and vascular endothelial cells. TGF-ß2 expression was lower in CDH lungs. miR-200b inhibited TGF-ß-induced SMAD signaling in cultures of human bronchial epithelial cells. CONCLUSIONS: Human fetal hypoplastic CDH lungs have a specific miR-200/miR-10a signature. Survival after FETO is associated with increased miR-200 family expression. miR-200b overexpression in CDH lungs results in decreased TGF-ß/SMAD signaling.

MicroRNA-200b regulates distal airway development by maintaining epithelial integrity.

miR-200b plays a role in epithelial-to-mesenchymal transition (EMT) in cancer. We recently reported abnormal expression of miR-200b in the context of human pulmonary hypoplasia in congenital diaphragmatic hernia (CDH). Smaller lung size, a lower number of airway generations, and a thicker mesenchyme characterize pulmonary hypoplasia in CDH. The aim of this study was to define the role of miR-200b during lung development. Here we show that miR-200b-/- mice have abnormal lung function due to dysfunctional surfactant, increased fibroblast-like cells and thicker mesenchyme in between the alveolar walls. We profiled the lung transcriptome in miR-200b-/- mice, and, using Gene Ontology analysis, we determined that the most affected biological processes include cell cycle, apoptosis and protein transport. Our results demonstrate that miR-200b regulates distal airway development through maintaining an epithelial cell phenotype. The lung abnormalities observed in miR-200b-/- mice recapitulate lung hypoplasia in CDH.

Effects of oxidized and glycated low-density lipoproteins on transcription and secretion of plasminogen activator inhibitor-1 in vascular endothelial cells.

Plasminogen activator inhibitor-1 (PAI-1) is the major physiological inhibitor of fibrinolysis. Elevated levels of PAI-1 were frequently detected in patients with coronary artery disease (CAD) or diabetes. Low-density lipoprotein (LDL) is a classical risk factor of CAD. Oxidation and glycation increase the atherogenecity of LDL. Previous studies demonstrated that oxidized LDL (oxLDL) or glycated LDL (gly-LDL) increased the release of PAI-1 from endothelial cells (ECs). The present study examined the effects of oxLDL and gly-LDL on the transcription, expression, secretion, and subcellular distribution of PAI-1 in cultured human ECs. Treatment with LDL significantly increased the promoter activity, mRNA level, and the release of PAI-1 from ECs by two- to threefold compared to controls. Oxidation or glycation significantly enhanced the effects of LDL on PAI-1 production in ECs compared to LDL (four- to fivefold vs. controls). No significant differences were detected between the effects of oxLDL and gly-LDL. Abundant PAI-1 antigens were detected in the perinuclear region of ECs and overlapped with giantin, a marker of Golgi apparatus. Treatment with brefeldin A disturbed the stack structure of Golgi apparatus and almost completely inhibited the release of PAI-1 from ECs induced by the lipoproteins and at basal conditions. The results suggest that oxidation and glycation enhanced the effects of LDL on the production of PAI-1 in ECs through increasing the transcription of PAI-1. Intact Golgi apparatus is required for PAI-1 generation from ECs induced by LDL or its modified forms.

Impact of simvastatin on hemostatic and fibrinolytic regulators in Type 2 diabetes mellitus.

Simvastatin, a widely used 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase inhibitor, effectively reduced cardiac death and ischemic events in patients with coronary heart disease (CHD) and diabetes mellitus (DM). The mechanism of cardiovascular benefits of statins in DM remains unclear. We examined how simvastatin influences the levels of several in vivo markers for coagulation and fibrinolysis in 26 Type 2 DM patients. The diabetic patients received 20 mg/day of simvastatin up to 12 months. The levels of total cholesterol (TC), low density lipoprotein-cholesterol (LDL-c) and triglycerides in peripheral circulation of patients were significantly reduced after > or =6 weeks of simvastatin treatment. Levels of prothrombin fragment 1+2 (F1+2), factor VII, plasminogen activator inhibitor-1 (PAI-1) and tissue factor pathway inhibitor (TFPI) antigens, but not tissue plasminogen activator (tPA) antigen, in the pre-simvastatin plasmas of the diabetic patients were significantly higher than the levels found in plasmas of healthy subjects. Significant reductions in F1+2 and PAI-1 levels were evident > or =6 weeks after the diabetic patients received simvastatin. Levels of total tPA, TFPI, FVII, hemoglobin A1c, fasting blood glucose, and insulin in the diabetic patients' plasma were not significantly altered by simvastatin treatment. Positive correlations were found between PAI-1 versus TC, PAI-1 versus LDL-c, and FVII versus triglycerides in the plasmas of simvastatin-treated patients. The results suggest that simvastatin reduces in vivo prothrombinase activity and PAI-1 levels in type 2 DM patients. These actions may contribute to the protective properties of simvastatin against ischemic events in diabetic patients.

Whole and fractionated yellow pea flours modulate insulin, glucose, oxygen consumption, and the caecal microbiome in Golden Syrian hamsters.

The objective was to evaluate the effects of whole and fractionated yellow peas on circulating lipids, glucose and insulin levels, energy expenditure, and body composition, as well as to assess their prebiotic actions in Golden Syrian hamsters. Forty-five hamsters consumed a hypercholesterolemic diet for 28 days, then were randomly assigned to 1 of 3 groups: control (CON), whole pea flour (WPF), and fractionated pea flour (hulls only) (FPF). WPF and FPF were incorporated into the diets, replacing 10% of the cornstarch. WPF and FPF feeding produced negligible effects on circulating cholesterol and triglyceride levels. However, both WPF (56.76 ± 9.22 pmol·L⁻¹, p = 0.002) and FPF (89.27 ± 19.82 pmol·L⁻¹, p = 0.032) reduced circulating insulin levels compared with the CON group (131.70 ± 17.70 pmol·L⁻¹). Moreover, FPF decreased (p = 0.03) circulating glucose levels (6.26 ± 0.51 mmol·L⁻¹) compared with CON (8.27 ± 0.81 mmol·L⁻¹). Energy expenditure analysis revealed that hamsters consuming WPF demonstrated a higher (p = 0.036) oxygen consumption (2.00 ± 0.31 mL O2·g⁻¹ lean body mass) vs. the CON group (1.56 ± 0.089 mL O2·g⁻¹ lean body mass). Analysis of caecal digesta showed that WPF produced shifts in the abundance of microbial taxa with the most predominant changes occurring within the phylum Firmicutes. Yellow peas and their constituents should be investigated as future functional food ingredients that help prevent and manage lifestyle-related diseases such as diabetes and obesity.

Effects of losartan on urinary secretion of extracellular matrix and their modulators in type 2 diabetes mellitus patients with microalbuminuria.

PURPOSE: Angiotensin II receptor Type 1 antagonists postpone the development of nephropathy in type 2 diabetes mellitus (DM). We hypothesize that Losartan may ameliorate renal function in diabetic patients through the regulation on the generation of transforming growth factor (TGF)-beta and fibrinolytic regulators. METHODS: Twenty-two type 2 DM patients with microalbuminuria were treated with 50-100 mg/day of Losartan for 6 months. Urinary secretion of TGF-, plasminogen activator inhibitor-1 (PAI-1), tissue and urokinase plasminogen activators (tPA and uPA) fibronectin, collagen IV and plasma levels of TGF-beta, PAI-1, tPA and uPA of the patients before and after the treatment were analyzed using enzyme-linked immunoabosorbance assay. RESULTS: Losartan effectively reduced arterial blood pressure and urinary albumin excretion. The levels of TGF-beta in urine, but not in plasma, were reduced after 2, 4 and 6 months of the treatment (-32% to -48%, P < 0.05 or 0.01). Urinary or plasma levels of PAI-1, tPA or uPA, and urinary secretion of fibronectin or collagen IV were not significantly altered by Losartan treatment. Urinary levels of collagen IV positively correlated with uPA, and that of fibronectin negatively correlated with PAI-1 in the patients (P < 0.01). Urinary TGF-beta negatively correlated uPA in urine of the patients (P < 0.01). CONCLUSION: Losartan reduced urinary excretion of TGF-beta and albumin in type 2 DM patients with microalbuminuria. Fibrinolytic regulators and TGF-beta are implicated in the regulation of ECM turnover in kidneys of the patients with diabetic nephropathy.