Plasma microRNA signature associated with retinopathy in patients with type 2 diabetes.

Diabetic retinopathy (DR) is a leading cause of vision loss and disability. Effective management of DR depends on prompt treatment and would benefit from biomarkers for screening and pre-symptomatic detection of retinopathy in diabetic patients. MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression which are released in the bloodstream and may serve as biomarkers. Little is known on circulating miRNAs in patients with type 2 diabetes (T2DM) and DR. Here we show that DR is associated with higher circulating miR-25-3p (P = 0.004) and miR-320b (P = 0.011) and lower levels of miR-495-3p (P < 0.001) in a cohort of patients with T2DM with DR (n = 20), compared with diabetic subjects without DR (n = 10) and healthy individuals (n = 10). These associations persisted significant after adjustment for age, gender, and HbA1c. The circulating levels of these miRNAs correlated with severity of the disease and their concomitant evaluation showed high accuracy for identifying DR (AUROC = 0.93; P < 0.001). Gene ontology analysis of validated targets revealed enrichment in pathways such as regulation of metabolic process (P = 1.5 × 10-20), of cell response to stress (P = 1.9 × 10-14), and development of blood vessels (P = 2.7 × 10-14). Pending external validation, we anticipate that these miRNAs may serve as putative disease biomarkers and highlight novel molecular targets for improving care of patients with diabetic retinopathy.

High dose rosuvastatin increases ABCA1 transporter in human atherosclerotic plaques in a cholesterol-independent fashion.

BACKGROUND: ATP-binding cassette A1 (ABCA1) and G1 (ABCG1) mediate cholesterol efflux from lipid-laden macrophages, thus promoting anti-atherosclerotic outcomes. The mechanism(s) linking treatment with statins and ABCA1/ABCG1 in human atherosclerosis are not fully understood and require further investigation. Therefore, we studied whether short-term treatment with low- or high-dose rosuvastatin may affect ABCA1 and ABCG1 expression in human atherosclerotic plaques. METHODS: Seventy patients with severe stenosis of the internal carotid artery were randomized to receive low (10 mg/day) or high (40 mg/day) dose rosuvastatin for 12 weeks before elective endarterectomy. As controls, we analyzed a reference group of 10 plaques from subjects with hypercholesterolemia but not receiving statin treatment and an additional set of 11 plaques collected from normocholesterolemic patients. On atherosclerotic plaques, ABCA1 and ABCG1 expression was evaluated at RNA level by qPCR and at protein level by immunoblotting and immunohistochemistry. RESULTS: Both rosuvastatin doses were associated with lower plaque ABCA1 mRNA levels and with a trend toward reduction for ABCG1. However, ABCA1 protein was paradoxically higher in patients treated with high-dose rosuvastatin and was associated with lower levels of miR-33b-5p, a microRNA known as a regulator of ABCA1. Multivariate analyses showed that the effect is cholesterol-independent. Finally, no effects were found for ABCG1 protein. CONCLUSIONS: High-dose rosuvastatin increases macrophage ABCA1 protein levels in human atherosclerotic plaque despite mRNA reduction in a mechanism unrelated to plasma cholesterol reduction and potentially involving miR-33b-5p. This pathway may reflect an additional feature contributing to the anti-atherosclerotic effect for high-dose rosuvastatin. TRIAL REGISTRATION: ISRCTN16590640.

MicroRNA profiling in migraine without aura: pilot study.

BACKGROUND AND PURPOSE: MicroRNAs (miRNAs) are short, non-coding RNAs whose deregulation has been shown in several human diseases, including pain states and diseases associated with increased cardiovascular (CV) risk. This study aimed at identifying differentially expressed circulating miRNAs in patients with 'migraine without aura' (MO), a pain condition whose link with CV risk remains debated. METHODS: Fifteen female MO patients and 13 matching healthy controls underwent a circulating microRNA expression profiling. MiR-22, miR-26a, miR-26b, miR-27b, miR-29b, let-7b, miR-181a, miR-221, miR-30b, and miR-30e were selected for validation by quantitative real-time polymerase chain reaction. RESULTS: In migraineurs versus controls, four miRNAs were differentially expressed: miR-27b was significantly up-regulated (q < 0.004), while miR-181a, let-7b, and miR-22 were significantly down-regulated (q ≤ 0.01). MiR-22 and let-7b down-regulation was also confirmed in circulating blood monocytes. A logistic regression model based on microRNA expression profile showed a high accuracy for identifying migraine (AUC of ROC curve: 0.956; P < 0.001). CONCLUSION: A specific circulating miRNAs profile is associated with migraine without aura. Remarkably, the same miRNAs are known to be modulated in the setting of atherosclerosis and stroke in humans. This study represents a first step towards further characterization of MO diagnosis/pathophysiology, also in relation to its link with cardiovascular risk.

Plasma exosome microRNA profiling unravels a new potential modulator of adiponectin pathway in diabetes: effect of glycemic control.

CONTEXT: Type 2 diabetes is a chronic disease characterized by inadequate ß-cell response to the progressive insulin resistance. MicroRNAs (miRNAs) are short, endogenous, noncoding RNAs representing a class of powerful gene expression modulators. Previous population studies observed a modulation of circulating miRNAs in diabetic patients; however, few data are presently available on miRNA modulation in diabetic patients naïve to pharmacological treatment as well as the effect of glycemic control on this. OBJECTIVE: We aimed at studying circulating miRNA expression in diabetic patients naïve to treatment and at investigating the influence on this of glycemic control. DESIGN: This was a case-control study. PARTICIPANTS: Eighteen treatment-naïve diabetic patients with poor metabolic control and 12 control patients participated in the study. MAIN OUTCOME MEASURES: Wide miRNA expression profiling was performed, and the expression of miRNAs found to be dysregulated was then validated by quantitative RT-PCR. Finally, algorithm-identified putative miRNA targets were evaluated by quantitative RT-PCR and ELISA. RESULTS: In diabetic patients, microarray analysis showed that four miRNAs are increased, whereas 21 miRNAs are decreased. Quantitative RT-PCR validation confirmed the significant up-regulation of miR-326 (P = .004) and down-regulation of let-7a (P < .001) and let-7f (P = .003). Notably, an inverse negative correlation was found between circulating miR-326 and its putative target adiponectin (p = -0.479, P = .009). After 12 months of antidiabetic treatment, quantitative RT-PCR data analysis showed that miR-326 levels were unaffected, whereas the levels of let-7a and let-7f were significantly increased. CONCLUSIONS: Treatment-naïve, poorly controlled diabetic patients show a significant dysregulation of miRNAs involved in the regulation of the adiponectin pathway, a phenomenon that may be reversed, at least in part, by improved glycemic control.

Overexpression of microRNA-145 in atherosclerotic plaques from hypertensive patients.

BACKGROUND: MicroRNAs (miRNAs) are endogenous, non-coding, short, single-stranded RNAs and represent a new class of gene regulators. Recent evidence supports a role for miRNAs in cardiovascular pathophysiology and atherosclerosis development. We have previously demonstrated that miR-145 is widely expressed in human atherosclerotic lesions and its downregulation has been correlated with vascular smooth muscle cell dedifferentiation, a cardinal step in the development of atherosclerosis. However, no evidences are available at this time about modulation of miR-145 in the setting of hypertension. Thus, the aim of this study was to investigate the expression of miR-145 in complicated hypertension. MATERIALS AND METHODS: Atherosclerotic plaques were obtained from 22 patients undergoing carotid endarterectomy for high-grade internal carotid artery stenosis. Plaques were subdivided into hypertension (n = 15) and control (n = 7) groups according to the presence or absence of hypertension (as defined by blood pressure > 140/90 mmHg or current antihypertensive treatment). In study plaques, miR-145 values were evaluated using real-time PCR. The level of induction has been tested by using ΔΔ cycle threshold method. RESULTS: We found that miR-145 was significantly more expressed in atherosclerotic plaques of hypertensive patients than in control plaques (1.201 ± 0.260 vs 0.483 ± 0.148 fold induction ± SE; p = 0.026). Moreover, a post-hoc analysis showed that treatment with angiotensin receptor blockers may be associated with the maximum increase in miR-145 levels, although these data did not show any statistical significance probably due to the limited sample size. CONCLUSIONS: To the best of our knowledge, this study is the first demonstration that hypertension may upregulate miR-145 expression in human atherosclerotic plaques. Future investigations will be necessary to establish the molecular readout of miR-145 upregulation in atherosclerotic lesions in hypertension.

Assessing LINE-1 retrotransposition activity in neuroblastoma cells exposed to extremely low-frequency pulsed magnetic fields.

Mobile genetic elements represent an important source of mutation and genomic instability, and their activity can be influenced by several chemical and physical agents. In this research we address the question whether exposure to extremely low-frequency pulsed magnetic fields (EMF-PMF) could affect the mobility of the human LINE-1(RP) retrotransposon. To this purpose, an in vitro retrotransposition assay was used on human neuroblastoma BE(2) cells exposed for 48h to 1mT, 50Hz PMF, or sham-exposed. Moreover, since it is well known that retrotransposition causes DNA double-strand breaks (DSB), an estimation of γ-H2AX foci, which is a marker of DNA DSB, was carried out on PMF- and sham-exposed samples. The results show that PMF-exposed cells had a lower number of both retrotransposition events and DNA DSB compared with sham-exposed samples. These results suggest that exposure to PMF can interfere with retrotransposition activity by inducing a decrease of retrotransposition events.

LINE-1 retrotransposition in human neuroblastoma cells is affected by oxidative stress.

Long interspersed element-1s (LINE-1 or L1s) are abundant retrotransposons that occur in mammalian genomes and that can cause insertional mutagenesis and genomic instability. L1 activity is generally repressed in most cells and tissues but has been found in some embryonic cells and, in particular, in neural progenitors. Moreover, L1 retrotransposition can be induced by several DNA-damaging agents. We have carried out experiments to verify whether L1 retrotransposition is affected by oxidative DNA damage, which plays a role in a range of human diseases, including cancer and inflammatory and neurodegenerative disease. To this purpose, BE(2)C neuroblastoma cells, which are thought to represent embryonic precursors of sympathetic neurons, have been treated with hydrogen peroxide and subjected to an in vitro retrotransposition assay involving an episomal L1(RP) element tagged with enhanced green fluorescent protein. Our results indicate that hydrogen peroxide treatment induces an increase in the retrotransposition of transiently transfected L1(RP) and an increase in the expression of endogenous L1 transcripts. An increase of γ-H2AX foci and changes in the mRNA levels of MRE11, RAD50, NBN and ERCC1 (all involved in DNA repair) have also been found. Thus, oxidative stress can cause L1 dysregulation.

Effect of extremely low frequency magnetic field exposure on DNA transposition in relation to frequency, wave shape and exposure time.

PURPOSE: To examine the effect of extremely low frequency magnetic field (ELF-MF) exposure on transposon (Tn) mobility in relation to the exposure time, the frequency and the wave shape of the field applied. MATERIALS AND METHODS: Two Escherichia coli model systems were used: (1) Cells unable to express ß-galactosidase (LacZ(-)), containing a mini-transposon Tn10 element able to give ability to express ß-galactosidase (LacZ(+)) upon its transposition; therefore in these cells transposition activity can be evaluated by analysing LacZ(+) clones; (2) cells carrying Fertility plasmid (F(+)), and a Tn5 element located on the chromosome; therefore in these cells transposition activity can be estimated by a bacterial conjugation assay. Cells were exposed to sinusoidal (SiMF) or pulsed-square wave (PMF) magnetic fields of various frequencies (20, 50, 75 Hz) and for different exposure times (15 and 90 min). RESULTS: Both mini-Tn10 and Tn5 transposition decreased under SiMF and increased under PMF, as compared to sham exposure control. No significant difference was found between frequencies and between exposure times. CONCLUSIONS: ELF-MF exposure affects transposition activity and the effects critically depend on the wave shape of the field, but not on the frequency and the exposure time, at least in the range observed.

Evaluation of LINE-1 mobility in neuroblastoma cells by in vitro retrotransposition reporter assay: FACS analysis can detect only the tip of the iceberg of the inserted L1 elements.

Long Interspersed Nuclear Elements (L1) are retroelements generally repressed in most differentiated somatic cells. Their activity has been observed in some undifferentiated and tumour cells and could be involved in tumour onset and progression. Growing evidences show that the L1 activation can occur in neuronal precursor cells during differentiation process. Neuroblastoma is a tumour originating from neuronal precursor cells, and, although the molecular basis of its progression is still poorly understood, the implication of L1 activation has not yet been investigated. In this study L1 mobility in neuroblastoma BE(2)C cells was assessed using the in vitro retrotransposition assay consisting in an episomal EGFP-tagged L1(RP) element, whose mobility can be evaluated by cytofluorimetric analysis (FACS) of EGFP expression. FACS results have shown a low retrotransposition activity. To detect L1(RP) integrated in transcriptionally repressed genomic sites, both a cell treatment with a stimulator of reporter gene promoter, and a quantitative Real-Time PCR analysis were performed. A retrotransposition activity ten and one thousand times that of FACS was found, respectively. These results point out that the real rate of L1 retrotransposition events in tumour cells might be considerably higher than that reported so far by evaluating only the reporter gene expression.

Synergic effect of retinoic acid and extremely low frequency magnetic field exposure on human neuroblastoma cell line BE(2)C.

The aim of the present study was to assess whether exposure to a sinusoidal extremely low frequency magnetic field (ELF-MF; 50 Hz, 1 mT) can affect proliferation and differentiation in the human neuroblastoma cell line BE(2)C, which is representative of high risk neuroblastomas. Cells were subjected to ELF-MF exposure in the presence or absence of a neuronal differentiating agent (all-trans-retinoic acid, ATRA) for 24-72 h. In each experiment, ELF-MF-exposed samples were compared to sham-exposed samples. Cells exposed to ELF-MF combined with retinoic treatment showed a decreased cellular proliferation and an increased proportion of G(0)/G(1) phase cells compared to cells exposed to either treatment alone. Moreover, ELF-MF- and ATRA-treated cells showed more differentiated morphological traits (a higher neurite number/cell, an increased neurite length), together with a significant increase of mRNA levels of p21(WAF1/CIP1) and cdk5 genes, both involved in neuronal differentiation. In addition, the expression of cyp19 gene, which is involved both in neuronal differentiation and stress response, was evaluated; cyp19 gene expression was enhanced by ATRA treatment and significantly enhanced further by ELF-MF exposure combined with ATRA. In conclusion, our data suggest that ELF-MF exposure can strengthen ATRA effects on neuroblastoma cells.

Extremely low frequency magnetic field exposure affects DnaK and GroEL expression in E. coli cells with impaired heat shock response.

In our earlier experiments, we found that extremely low frequency magnetic fields (ELF-MF) affect heat shock protein (HSP) expression in wild type Escherichia coli cells. In the present work we investigate the ability of ELF-MF exposure to trigger an increase of DnaK and GroEL protein levels also in E. coli cells not exhibiting the classic heat shock response (HSR) when subjected to a 42 degrees C heat stress. We find that these cells, although lacking a HSR to heat shock treatment, show an enhancement of DnaK and GroEL protein levels after 30 or 90 min sinusoidal ELF-MF exposure (50 Hz, 1 mT). This result suggests that the HSP induction pathway triggered by ELF-MF exposure could be different from that elicited by heat shock treatment.