Franziska Bollmann

Vascular Dysfunction in Experimental Diabetes Is Improved by Pentaerithrityl Tetranitrate but Not Isosorbide-5-Mononitrate Therapy

OBJECTIVE Diabetes is associated with vascular oxidative stress, activation of NADPH oxidase, and uncoupling of nitric oxide (NO) synthase (endothelial NO synthase [eNOS]). Pentaerithrityl tetranitrate (PETN) is an organic nitrate with potent antioxidant properties via induction of heme oxygenase-1 (HO-1). We tested whether treatment with PETN improves vascular dysfunction in the setting of experimental diabetes. RESEARCH DESIGN AND METHODS After induction of hyperglycemia by streptozotocin (STZ) injection (60 mg/kg i.v.), PETN (15 mg/kg/day p.o.) or isosorbide-5-mononitrate (ISMN; 75 mg/kg/day p.o.) was fed to Wistar rats for 7 weeks. Oxidative stress was assessed by optical methods and oxidative protein modifications, vascular function was determined by isometric tension recordings, protein expression was measured by Western blotting, RNA expression was assessed by quantitative RT-PCR, and HO-1 promoter activity in stable transfected cells was determined by luciferase assays. RESULTS PETN, but not ISMN, improved endothelial dysfunction. NADPH oxidase and serum xanthine oxidase activities were significantly reduced by PETN but not by ISMN. Both organic nitrates had minor effects on the expression of NADPH oxidase subunits, eNOS and dihydrofolate reductase (Western blotting). PETN, but not ISMN, normalized the expression of GTP cyclohydrolase-1, extracellular superoxide dismutase, and S-glutathionylation of eNOS, thereby preventing eNOS uncoupling. The expression of the antioxidant enzyme, HO-1, was increased by STZ treatment and further upregulated by PETN, but not ISMN, via activation of the transcription factor NRF2. CONCLUSIONS In contrast to ISMN, the organic nitrate, PETN, improves endothelial dysfunction in diabetes by preventing eNOS uncoupling and NADPH oxidase activation, thereby reducing oxidative stress. Thus, PETN therapy may be suited to treat patients with cardiovascular complications of diabetes.

Resveratrol post-transcriptionally regulates pro-inflammatory gene expression via regulation of KSRP RNA binding activity

Resveratrol shows beneficial effects in inflammation-based diseases like cancer, cardiovascular and chronic inflammatory diseases. Therefore, the molecular mechanisms of the anti-inflammatory resveratrol effects deserve more attention. In human epithelial DLD-1 and monocytic Mono Mac 6 cells resveratrol decreased the expression of iNOS, IL-8 and TNF-α by reducing mRNA stability without inhibition of the promoter activity. Shown by pharmacological and siRNA-mediated inhibition, the observed effects are SIRT1-independent. Target-fishing and drug responsive target stability experiments showed selective binding of resveratrol to the RNA-binding protein KSRP, a central post-transcriptional regulator of pro-inflammatory gene expression. Knockdown of KSRP expression prevented resveratrol-induced mRNA destabilization in human and murine cells. Resveratrol did not change KSRP expression, but immunoprecipitation experiments indicated that resveratrol reduces the p38 MAPK-related inhibitory KSRP threonine phosphorylation, without blocking p38 MAPK activation or activity. Mutation of the p38 MAPK target site in KSRP blocked the resveratrol effect on pro-inflammatory gene expression. In addition, resveratrol incubation enhanced KSRP-exosome interaction, which is important for mRNA degradation. Finally, resveratrol incubation enhanced its intra-cellular binding to the IL-8, iNOS and TNF-α mRNA. Therefore, modulation of KSRP mRNA binding activity and, thereby, enhancement of mRNA degradation seems to be the common denominator of many anti-inflammatory effects of resveratrol.

Heme Oxygenase-1 Induction and Organic Nitrate Therapy: Beneficial Effects on Endothelial Dysfunction, Nitrate Tolerance, and Vascular Oxidative Stress

Organic nitrates are a group of very effective anti-ischemic drugs. They are used for the treatment of patients with stable angina, acute myocardial infarction, and chronic congestive heart failure. A major therapeutic limitation inherent to organic nitrates is the development of tolerance, which occurs during chronic treatment with these agents, and this phenomenon is largely based on induction of oxidative stress with subsequent endothelial dysfunction. We therefore speculated that induction of heme oxygenase-1 (HO-1) could be an efficient strategy to overcome nitrate tolerance and the associated side effects. Indeed, we found that hemin cotreatment prevented the development of nitrate tolerance and vascular oxidative stress in response to chronic nitroglycerin therapy. Vice versa, pentaerithrityl tetranitrate (PETN), a nitrate that was previously reported to be devoid of adverse side effects, displayed tolerance and oxidative stress when the HO-1 pathway was blocked pharmacologically or genetically by using HO-1+/– mice. Recently, we identified activation of Nrf2 and HuR as a principle mechanism of HO-1 induction by PETN. With the present paper, we present and discuss our recent and previous findings on the role of HO-1 for the prevention of nitroglycerin-induced nitrate tolerance and for the beneficial effects of PETN therapy.

qRT-PCR: a method and its difficulties

Today, quantitative reverse transcription polymerase chain reaction (qRT-PCR) is the favored method to quantify gene expression in molecular biology and clinical studies. qRTPCR analyses can be performed in a high-throughput manner and are very sensitive and specific, cost-effective, and reproducible. Even though this technique is easy to handle, the executer has to pay attention to some critical points. Right from the beginning of proper data generation, it is important to store the samples correctly and to prepare pure and intact RNA. It is, for example, more difficult to obtain good-quality RNA from paraffin-embedded samples than of frozen tissues (Takano et al. 2010). There are RNA protection and preparation kits available that help to maintain the integrity of RNA. Subsequently, an effective reverse transcription reaction (RT) and a specific primer design are indispensable for a high PCR efficiency. For a clear survey, Udvardis group posted 11 golden rules of performing qRT-PCR analyses and gave a concise support from sample collection to data analysis (Udvardi et al. 2008). Before performing qRT-PCR analyses, the choice between two detection methods has to be made: nonprobe-based and probe-based assays. Nonprobe-based analyses use an intercalating dye like SYBR® green or ethidium bromide for the target detection. The dye intercalates into all double stranded DNAs formed during the PCR reaction. To exclude the detection of unspecific PCR products, it is necessary to analyze the PCR products further bymelting curves or gel electrophoresis. Themajor advantage of nonprobe-based analyses even in high throughput experiments is the lower investment, because each optimized PCR can easily be converted into a quantitative PCR. In contrast, probe-based detection needs an additional oligonucleotide that binds internal to the amplified target sequence and contains a quencher and reporter. The probe has to be designed for every target sequence, and this, in turn, guarantees a higher specificity for the detection system. Several different probe structures can be used for this method: TaqMan®, molecular beacons, or Scorpion Primers. A crucial point of qRT-PCR analyses is the normalization of the results that, improperly done, can have a profound influence on study conclusions (Ferguson et al. 2010). Even though normalization is a highly discussed topic, mostly neither editors nor reviewer demands on the proof of a suitable data evaluation. Current used normalization strategies range from standardization of sample size, like tissue weight or RNA amount, to internal and external standards. When normalizing with external standards, the absolute copy number of the target transcripts can be determined. The standard curve can either be generated from diluted plasmid DNA (Li and Wang 2000) or from in vitro-transcribed RNAs (Workenhe et al. 2008). The quantification is based on the assumption that plasmid DNA and cDNA [reverse transcribed frommessenger RNA (mRNA)] have the same amplifications rates. In contrast to the external control, it is also possible to use coamplified internal controls. This normalization strategy to one or more internal controls is commonly used (Vandesompele et al. 2002). The principle of this method is based on the quantification of the mRNA of an internal reference gene that undergoes the same procedures and conditions as the mRNA of interest. Using this kind of normalization, it is absolutely essential to search for a reference gene whose expression is Franziska Bollmann, Ingrid Casper, and Jenny Henke contributed equally to this editorial.

Tristetraprolin regulation of interleukin-22 production

Interleukin (IL)-22 is a STAT3-activating cytokine displaying characteristic AU-rich elements (ARE) in the 3′-untranslated region (3′-UTR) of its mRNA. This architecture suggests gene regulation by modulation of mRNA stability. Since related cytokines undergo post-transcriptional regulation by ARE-binding tristetraprolin (TTP), the role of this destabilizing protein in IL-22 production was investigated. Herein, we demonstrate that TTP-deficient mice display augmented serum IL-22. Likewise, IL-22 mRNA was enhanced in TTP-deficient splenocytes and isolated primary T cells. A pivotal role for TTP is underscored by an extended IL-22 mRNA half-life detectable in TTP-deficient T cells. Luciferase-reporter assays performed in human Jurkat T cells proved the destabilizing potential of the human IL-22-3′-UTR. Furthermore, overexpression of TTP in HEK293 cells substantially decreased luciferase activity directed by the IL-22-3′-UTR. Transcript destabilization by TTP was nullified upon cellular activation by TPA/A23187, an effect dependent on MEK1/2 activity. Accordingly, IL-22 mRNA half-life as determined in TPA/A23187-stimulated Jurkat T cells decreased under the influence of the MEK1/2 inhibitor U0126. Altogether, data indicate that TTP directly controls IL-22 production, a process counteracted by MEK1/2. The TTP-dependent regulatory pathway described herein likely contributes to the role of IL-22 in inflammation and cancer and may evolve as novel target for pharmacological IL-22 modulation.

Endothelial dysfunction in tristetraprolin-deficient mice is not caused by enhanced tumor necrosis factor-α expression.

Background: Chronic inflammatory diseases are associated with increased cardiovascular mortality due to accelerated atherosclerosis. Results: Chronic inflammation in tristetraprolin (TTP)-deficient mice leads to endothelial dysfunction, which is related to enhanced Nox2-dependent reactive oxygen species production but independent from TNF-α. Conclusion: Inflammation-related oxidative stress is an important mediator in inflammation-driven atherogenesis. Significance: In inflammatory diseases oxidative stress seems to be a major cause of cardiovascular events. Cardiovascular events are important co-morbidities in patients with chronic inflammatory diseases like rheumatoid arthritis. Tristetraprolin (TTP) regulates pro-inflammatory processes through mRNA destabilization and therefore TTP-deficient mice (TTP−/− mice) develop a chronic inflammation resembling human rheumatoid arthritis. We used this mouse model to evaluate molecular signaling pathways contributing to the enhanced atherosclerotic risk in chronic inflammatory diseases. In the aorta of TTP−/− mice we observed elevated mRNA expression of known TTP targets like tumor necrosis factor-α (TNF-α) and macrophage inflammatory protein-1α, as well as of other pro-atherosclerotic mediators, like Calgranulin A, Cathepsin S, and Osteopontin. Independent of cholesterol levels TTP−/− mice showed a significant reduction of acetylcholine-induced, nitric oxide-mediated vasorelaxation. The endothelial dysfunction in TTP−/− mice was associated with increased levels of reactive oxygen and nitrogen species (RONS), indicating an enhanced nitric oxide inactivation by RONS in the TTP−/− animals. The altered RONS generation correlates with increased expression of NADPH oxidase 2 (Nox2) resulting from enhanced Nox2 mRNA stability. Although TNF-α is believed to be a central mediator of inflammation-driven atherosclerosis, genetic inactivation of TNF-α neither improved endothelial function nor normalized Nox2 expression or RONS production in TTP−/− animals. Systemic inflammation caused by TTP deficiency leads to endothelial dysfunction. This process is independent of cholesterol and not mediated by TNF-α solely. Thus, other mediators, which need to be identified, contribute to enhanced cardiovascular risk in chronic inflammatory diseases.

Anti-Inflammatory and Anti-Thrombotic Effects of the Fungal Metabolite Galiellalactone in Apolipoprotein E-Deficient Mice

Patients suffering from chronic inflammatory diseases have an increased mortality risk resulting from cardiovascular disorders due to enhanced atherosclerotic and thrombotic events. Until now, it is not completely understood in which way an abnormal expression of pro-inflammatory mediators contributes to this elevated cardiovascular risk, but there is a need for new drugs that on the one hand suppress the expression of pro-inflammatory mediators and on the other hand inhibit arterial platelet adhesion. Thus, we analyzed the anti-inflammatory and anti-thrombotic capacity of the fungal metabolite Galiellalactone in atherosclerosis-prone apolipoprotein E-deficient mice. Treatment of the mice with Galiellalactone lowered the inflammatory expression profile and improved blood clotting times, as well as platelet adhesion to the injured common carotid artery. The results indicate that administration of Galiellalactone is able to reduce the extent of inflammation and arterial platelet adhesion in this mouse model.

T Cell-Specific Overexpression of TGFß1 Fails to Influence Atherosclerosis in ApoE-Deficient Mice

Clinical data have indicated a negative correlation between plasma TGFß1 concentrations and the extent of atherosclerosis and have thus led to the hypothesis that the pleiotropic cytokine may have anti-atherogenic properties. T-cells are currently discussed to significantly participate in atherogenesis, but the precise role of adaptive immunity in atherogenesis remains to be elucidated. TGFß1 is known to strongly modulate the function of T-cells, however, inhibition of TGFß1 signalling in T-cells of atherosclerosis-prone knock-out mice failed to unequivocally clarify the role of the cytokine for the development of atherosclerosis. In the present study, we thus tried to specify the role of TGFß1 in atherogenesis by using the murine CD2-TGFß1 transgenic strain which represents a well characterized model of T-cell specific TGFß1 overexpression. The CD2-TGFß1 transgenic mice were crossed to ApoE knock-out mice and quantity and quality of atherosclerosis regarding number of macrophages, smooth muscle cells, CD3 positive T-cells and collagen was analyzed in CD2-TGFß1 ApoE double mutants as well as non-transgenic ApoE controls on both normal and atherogenic diet of a duration of 8, 16 or 24 weeks, respectively. In all experimental groups investigated, we failed to detect any influence of TGFß1 overexpression on disease. Total number of CD3-positive T-lymphocytes was not significantly different in atherosclerotic lesions of CD2-TGFß1 ApoE−/− females and isogenic ApoE−/− controls, even after 24 weeks on the atherogenic diet. The synopsis of these data and our previous study on TGFß1 overexpressing macrophages suggests that potential effects of TGFß1 on atherosclerosis are most probably mediated by macrophages rather than T-cells.

Differentially Tolerized Mouse Antigen Presenting Cells Share a Common miRNA Signature Including Enhanced mmu-miR-223-3p Expression Which Is Sufficient to Imprint a Protolerogenic State

Dendritic cells (DCs) are pivotal for the induction and maintenance of antigen-specific tolerance and immunity. miRNAs mediate post-transcriptional gene regulation and control in part the differentiation and stimulation-induced immunogenic function of DCs. However, the relevance of miRNAs for the induction and maintenance of a tolerogenic state of DCs has scarcely been highlighted yet. We differentiated mouse bone marrow cells to conventional/myeloid DCs or to tolerogenic antigen presenting cells (APCs) by using a glucocorticoid (dexamethasone) or interleukin-10, and assessed the miRNA expression patterns of unstimulated and LPS-stimulated cell populations by array analysis and QPCR. Differentially tolerized mouse APCs convergingly down-regulated a set of miRNA species at either state of activation as compared with the corresponding control DC population (mmu-miR-9-5p, mmu-miR-9-3p, mmu-miR-155-5p). These miRNAs were also upregulated in control DCs in response to stimulation. In contrast, miRNAs that were convergingly upregulated in both tolerized APC groups at stimulated state (mmu-miR-223-3p, mmu-miR-1224-5p) were downregulated in control DCs in response to stimulation. Overexpression of mmu-miR-223-3p in DCs was sufficient to prevent stimulation-associated acquisition of potent T cell stimulatory capacity. Overexpression of mmu-miR-223-3p in a DC line resulted in attenuated expression of known (Cflar, Rasa1, Ras) mRNA targets of this miRNA species shown to affect pathways that control DC activation. Taken together, we identified sets of miRNAs convergingly regulated in differentially tolerized APCs, which may contribute to imprint stimulation-resistant tolerogenic function as demonstrated for mmu-miR-223-3p. Knowledge of miRNAs with protolerogenic function enables immunotherapeutic approaches aimed to modulate immune responses by regulating miRNA expression.

Erratum: Tristetraprolin regulation of interleukin-22 production.

Scientific Reports 5 Article number: 15112; 10.1038/srep15112 published online: October212015; updated: December182015.