MicroRNAs mediate the senescence-associated decline of NRF2 in endothelial cells.

Oxidative stress predisposes to several aging-associated diseases, such as cardiovascular diseases and cancer. In aging, increase in the production of reactive oxygen species is typically accompanied with a decline in adaptive stress responses to oxidative stress. The decline is primarily due to a decrease in antioxidant production. Nuclear factor E2-Related Factor 2 (NRF2) is a key transcription factor regulating oxidative and electrophilic stress responses, but it has also been shown to play a role in the regulation of cell metabolism. NRF2 expression declines in aging, but the mechanisms remain unclear. In this study, we show that microRNAs (miRNAs) that are abundant in old endothelial cells decrease NRF2 expression by direct targeting of NRF2 mRNA. The effect is reversed by miRNA inhibition. The senescence-associated downregulation of NRF2 decreases endothelial glycolytic activity and stress tolerance both of which are restored after reinstating NRF2. Manipulation of the senescence-associated miRNA levels affects the glycolytic activity and stress tolerance consistently with the NRF2 results. We conclude that senescence-associated miRNAs are involved in the decline of NRF2 expression, thus contributing to the repression of adaptive responses during cell senescence.

NRF2 regulates endothelial glycolysis and proliferation with miR-93 and mediates the effects of oxidized phospholipids on endothelial activation.

Phospholipids, such as 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (PAPC), are the major components of cell membranes. Their exposure to reactive oxygen species creates oxidized phospholipids, which predispose to the development of chronic inflammatory diseases and metabolic disorders through endothelial activation and dysfunction. Although the effects of oxidized PAPC (oxPAPC) on endothelial cells have been previously studied, the underlying molecular mechanisms evoking biological responses remain largely unknown. Here, we investigated the molecular mechanisms of oxPAPC function with a special emphasis on NRF2-regulated microRNAs (miRNAs) in human umbilical vein endothelial cells (HUVECs) utilizing miRNA profiling, global run-on sequencing (GRO-seq), genome-wide NRF2 binding model, and RNA sequencing (RNA-seq) with miRNA overexpression and silencing. We report that the central regulators of endothelial activity, KLF2 for quiescence, PFKFB3 for glycolysis, and VEGFA, FOXO1 and MYC for growth and proliferation, are regulated by transcription factor NRF2 and the NRF2-regulated miR-106b∼25 cluster member, miR-93, in HUVECs. Mechanistically, oxPAPC was found to induce glycolysis and proliferation NRF2-dependently, and oxPAPC-dependent induction of the miR-106b∼25 cluster was mediated by NRF2. Additionally, several regulatory loops were established between NRF2, miR-93 and the essential regulators of healthy endothelium, collectively implying that NRF2 controls the switch between the quiescent and the proliferative endothelial states together with miR-93.

MicroRNA Profiling Reveals Distinct Profiles for Tissue-Derived and Cultured Endothelial Cells.

Endothelial plasticity enables the cells to switch their phenotype according to the surrounding vascular microenvironment. MicroRNAs (miRNAs) are small noncoding RNAs that control endothelial plasticity. The objective of this study was to investigate the differences in miRNA profiles of tissue-derived cells and cultured endothelial cells. To this end, miRNA expression was profiled from freshly isolated tissue-derived human vascular endothelial cells and endothelial cells cultured until cellular senescence using miRNA sequencing. In addition, the data was searched for putative novel endothelial miRNAs and miRNA isoforms. The data analysis revealed a striking change in endothelial miRNA profile as the cells adapted from tissue to cell culture environment and the overall miRNA expression decreased significantly in cultured compared to tissue-derived endothelial cells. In addition to changes in mechanosensitive miRNA expression, alterations in senescence-associated and endothelial-to-mesenchymal-transition-associated miRNAs were observed in aging cells. Collectively, the data illustrates the adaptability of endothelial cell miRNA expression that mirrors prevailing cellular environment.

Nitro-Oleic Acid Regulates Endothelin Signaling in Human Endothelial Cells.

Nitro-fatty acids are reactive signaling mediators that are formed when unsaturated fatty acids react with nitric oxide or nitric oxide-derived species. Nitro-fatty acids can modify specific signaling pathways via post-translational modifications of Cys residues in key regulatory proteins. One of the signaling cascades activated by nitro-fatty acids is the Keap1-Nrf2 pathway. We have previously studied the effects of nitro-oleic acid (OA-NO2) on the human endothelial cell transcriptome. We observed that endothelin receptor B [ET-B (gene name EDNRB)], the receptor mediating the vasodilatory effects of endothelin-1 (ET-1) is induced by OA-NO2 Inasmuch as ET-1 is one of the key regulators of vascular tone, we chose to examine in more detail the effect of OA-NO2 on endothelin signaling in human endothelial cells. Nrf2 was found to regulate the OA-NO2-induced transcription of ET-B in human and mouse endothelial cells. Furthermore, chromatin immunoprecipitation analysis revealed that OA-NO2 increased the binding of Nrf2 to an antioxidant response element in the enhancer region of the EDNRB gene. In addition, we show that the overexpression of both OA-NO2 and Nrf2 substantially decreased and that Nrf2 silencing increased the ET-1 concentration in the culture media of endothelial cells. The change in the extracellular ET-1 concentration was dependent on ET-B receptor expression. These data suggest that OA-NO2 modulates endothelin signaling by increasing Nrf2-dependent expression of the ET-B receptor in endothelial cells, which in turn mediates the decrease in extracellular ET-1 concentration. Based on these results, we propose that OA-NO2 and Nrf2 may alleviate the vasoconstrictive effects of ET-1 by removing it from the circulation.

The Effects of Sequence Variation on Genome-wide NRF2 Binding--New Target Genes and Regulatory SNPs.

Transcription factor binding specificity is crucial for proper target gene regulation. Motif discovery algorithms identify the main features of the binding patterns, but the accuracy on the lower affinity sites is often poor. Nuclear factor E2-related factor 2 (NRF2) is a ubiquitous redox-activated transcription factor having a key protective role against endogenous and exogenous oxidant and electrophile stress. Herein, we decipher the effects of sequence variation on the DNA binding sequence of NRF2, in order to identify both genome-wide binding sites for NRF2 and disease-associated regulatory SNPs (rSNPs) with drastic effects on NRF2 binding. Interactions between NRF2 and DNA were studied using molecular modelling, and NRF2 chromatin immunoprecipitation-sequence datasets together with protein binding microarray measurements were utilized to study binding sequence variation in detail. The binding model thus generated was used to identify genome-wide binding sites for NRF2, and genomic binding sites with rSNPs that have strong effects on NRF2 binding and reside on active regulatory elements in human cells. As a proof of concept, miR-126-3p and -5p were identified as NRF2 target microRNAs, and a rSNP (rs113067944) residing on NRF2 target gene (Ferritin, light polypeptide, FTL) promoter was experimentally verified to decrease NRF2 binding and result in decreased transcriptional activity.

MicroRNA profiling of pericardial fluid samples from patients with heart failure.

AIMS: Multicellular organisms maintain vital functions through intercellular communication. Release of extracellular vesicles that carry signals to even distant target organs is one way of accomplishing this communication. MicroRNAs can also be secreted from the cells in exosomes and act as paracrine signalling molecules. In addition, microRNAs have been implicated in the pathogenesis of a large number of diseases, including cardiovascular diseases, and are considered as promising candidate biomarkers due to their relative stability and easy quantification from clinical samples. Pericardial fluid contains hormones secreted by the heart and is known to reflect the cardiac function. In this study, we sought to investigate whether pericardial fluid contains microRNAs and if so, whether they could be used to distinguish between different cardiovascular pathologies and disease stages. METHODS AND RESULTS: Pericardial fluid was collected from heart failure patients during open-heart surgery. MicroRNA profiles of altogether 51 patients were measured by quantitative real-time PCR (qPCR) using Exiqon human panels I and II. On the average, 256 microRNAs were detected per sample, and 70 microRNAs out of 742 profiled microRNAs were detected in every sample. The five most abundant microRNAs in pericardial fluid were miR-21-5p, miR-451a, miR-125b-5p, let-7b-5p and miR-16-5p. No specific signatures for cardiovascular pathologies or clinically assessed heart failure stages could be detected from the profiles and, overall, microRNA profiles of the samples were found to be very similar despite the heterogeneity in the study population. CONCLUSION: Measured microRNA profiles did not separate the samples according to the clinical features of the patients. However, several previously identified heart failure marker microRNAs were detected. The pericardial fluid microRNA profile appeared to be a result of an active and selective secretory process indicating that microRNAs may act as paracrine signalling factors by mediating the local crosstalk between cardiac cells.

The Keap1-Nrf2 pathway: Mechanisms of activation and dysregulation in cancer.

The Keap1-Nrf2 pathway is the major regulator of cytoprotective responses to oxidative and electrophilic stress. Although cell signaling pathways triggered by the transcription factor Nrf2 prevent cancer initiation and progression in normal and premalignant tissues, in fully malignant cells Nrf2 activity provides growth advantage by increasing cancer chemoresistance and enhancing tumor cell growth. In this graphical review, we provide an overview of the Keap1-Nrf2 pathway and its dysregulation in cancer cells. We also briefly summarize the consequences of constitutive Nrf2 activation in cancer cells and how this can be exploited in cancer gene therapy.

Oxidative stress-regulated lentiviral TK/GCV gene therapy for lung cancer treatment.

Nuclear factor erythroid-2 related factor 2 (Nrf2) is a transcription factor that regulates protection against a wide variety of toxic insults to cells, including cytotoxic cancer chemotherapeutic drugs. Many lung cancer cells harbor a mutation in either Nrf2 or its inhibitor Keap1 resulting in permanent activation of Nrf2 and chemoresistance. In this study, we sought to examine whether this attribute could be exploited in cancer suicide gene therapy by using a lentiviral (LV) vector expressing herpes simplex virus thymidine kinase (HSV-TK/GCV) under the regulation of antioxidant response element (ARE), a cis-acting enhancer sequence that binds Nrf2. In human lung adenocarcinoma cells in which Nrf2 is constitutively overexpressed, ARE activity was found to be high under basal conditions. In this setting, ARE-HSV-TK was more effective than a vector in which HSV-TK expression was driven by a constitutively active promoter. In a mouse xenograft model of lung cancer, suicide gene therapy with LV-ARE-TK/GCV was effective compared with LV-PGK-TK/GCV in reducing tumor size. We conclude that ARE-regulated HSV-TK/GCV therapy offers a promising approach for suicide cancer gene therapy in cells with high constitutive ARE activity, permitting a greater degree of therapeutic targeting to those cells.

Electrophilic nitro-fatty acids activate NRF2 by a KEAP1 cysteine 151-independent mechanism.

Nitro-fatty acids (NO(2)-FAs) are electrophilic signaling mediators formed in vivo via nitric oxide (NO)- and nitrite (NO(2)(-))-dependent reactions. Nitro-fatty acids modulate signaling cascades via reversible covalent post-translational modification of nucleophilic amino acids in regulatory proteins and enzymes, thus altering downstream signaling events, such as Keap1-Nrf2-antioxidant response element (ARE)-regulated gene expression. In this study, we investigate the molecular mechanisms by which 9- and 10-nitro-octadec-9-enoic acid (OA-NO(2)) activate the transcription factor Nrf2, focusing on the post-translational modifications of cysteines in the Nrf2 inhibitor Keap1 by nitroalkylation and its downstream responses. Of the two regioisomers, 9-nitro-octadec-9-enoic acid was a more potent ARE inducer than 10-nitro-octadec-9-enoic acid. The most OA-NO(2)-reactive Cys residues in Keap1 were Cys(38), Cys(226), Cys(257), Cys(273), Cys(288), and Cys(489). Of these, Cys(273) and Cys(288) accounted for ∼50% of OA-NO(2) reactions in a cellular milieu. Notably, Cys(151) was among the least OA-NO(2)-reactive of the Keap1 Cys residues, with mutation of Cys(151) having no effect on net OA-NO(2) reaction with Keap1 or on ARE activation. Unlike many other Nrf2-activating electrophiles, OA-NO(2) enhanced rather than diminished the binding between Keap1 and the Cul3 subunit of the E3 ligase for Nrf2. OA-NO(2) can therefore be categorized as a Cys(151)-independent Nrf2 activator, which in turn can influence the pattern of gene expression and therapeutic actions of nitroalkenes.