Angelika Pfanne

Development of Long Noncoding RNA-Based Strategies to Modulate Tissue Vascularization

Background Long noncoding ribonucleic acids (lncRNAs) are a subclass of regulatory noncoding ribonucleic acids for which expression and function in human endothelial cells and angiogenic processes is not well studied. Objectives The authors discovered hypoxia-sensitive human lncRNAs via next-generation ribonucleic acid sequencing and microarray approaches. To address their functional importance in angiogenic processes, several endothelial lncRNAs were characterized for their angiogenic characteristics in vitro and ex vivo. Methods Ribonucleic acid sequencing and microarray-derived data showed specific endothelial lncRNA expression changes after hypoxia. Validation experiments confirmed strong hypoxia-dependent activation of 2 intergenic lncRNAs: LINC00323 and MIR503HG. Results Silencing of these lncRNA transcripts led to angiogenic defects, including repression of growth factor signaling and/or the key endothelial transcription factor GATA2. Endothelial loss of these hypoxia-driven lncRNAs impaired cell-cycle control and inhibited capillary formation. The potential clinical importance of these endothelial lncRNAs to vascular structural integrity was demonstrated in an ex vivo model of human induced pluripotent stem cell–based engineered heart tissue. Conclusions The authors report an expression atlas of human hypoxia-sensitive lncRNAs and identified 2 lncRNAs with important functions to sustain endothelial cell biology. LncRNAs hold great promise to serve as important future therapeutic targets of cardiovascular disease.

Mitochondrial long noncoding RNAs as blood based biomarkers for cardiac remodeling in patients with hypertrophic cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is a hereditary heart disease with a high risk for sudden cardiac death in young people. As a subtype, hypertrophic obstructive cardiomyopathy (HOCM) additionally has a left ventricular outflow gradient, showing stronger symptoms and requires a different treatment compared with hypertrophic nonobstructive cardiomyopathy (HNCM). In this study our aim was to investigate the regulation of mitochondrial and cardiac remodeling associated long noncoding RNAs (lncRNAs) in blood of patients affected with HOCM and HNCM. We included 28 HNCM, 57 HOCM, and 26 control inviduals. Already known mitochondrial and cardiac remodeling associated lncRNAs uc004cos.4, uc004coz.1, uc004cov.4, uc011mfi.2, uc022bqw.1, uc022bqs.1, and uc022bqu.1 were amplified in serum of these patients and correlated with clinical parameters. Long noncoding RNAs uc004cov.4 and uc022bqu.1 were significantly increased in patients with HOCM but not in patients with HNCM. With the use of receiver operator characteristic (ROC) curve analysis, lncRNAs uc004cov.4 and uc022bqu.1 were able to identify HOCM patients. In our study we evidenced that the specific mitochondrial long noncoding RNAs uc004cov.4 and uc022bqu.1 were upregulated in patients with HOCM and they were also able to identify HOCM and could be developed as useful clinical biomarkers in the future.

MicroRNA-Based Therapy of GATA2-Deficient Vascular Disease

Background: The transcription factor GATA2 orchestrates the expression of many endothelial-specific genes, illustrating its crucial importance for endothelial cell function. The capacity of this transcription factor in orchestrating endothelial-important microRNAs (miRNAs/miR) is unknown. Methods: Endothelial GATA2 was functionally analyzed in human endothelial cells in vitro. Endogenous short interfering RNA–mediated knockdown and lentiviral-based overexpression were applied to decipher the capacity of GATA2 in regulating cell viability and capillary formation. Next, the GATA2-dependent miR transcriptome was identified by using a profiling approach on the basis of quantitative real-time polymerase chain reaction. Transcriptional control of miR promoters was assessed via chromatin immunoprecipitation, luciferase promoter assays, and bisulfite sequencing analysis of sites in proximity. Selected miRs were modulated in combination with GATA2 to identify signaling pathways at the angiogenic cytokine level via proteome profiler and enzyme-linked immunosorbent assays. Downstream miR targets were identified via bioinformatic target prediction and luciferase reporter gene assays. In vitro findings were translated to a mouse model of carotid injury in an endothelial GATA2 knockout background. Nanoparticle-mediated delivery of proangiogenic miR-126 was tested in the reendothelialization model. Results: GATA2 gain- and loss-of-function experiments in human umbilical vein endothelial cells identified a key role of GATA2 as master regulator of multiple endothelial functions via miRNA-dependent mechanisms. Global miRNAnome-screening identified several GATA2-regulated miRNAs including miR-126 and miR-221. Specifically, proangiogenic miR-126 was regulated by GATA2 transcriptionally and targeted antiangiogenic SPRED1 and FOXO3a contributing to GATA2-mediated formation of normal vascular structures, whereas GATA2 deficiency led to vascular abnormalities. In contrast to GATA2 deficiency, supplementation with miR-126 normalized vascular function and expression profiles of cytokines contributing to proangiogenic paracrine effects. GATA2 silencing resulted in endothelial DNA hypomethylation leading to induced expression of antiangiogenic miR-221 by GATA2-dependent demethylation of a putative CpG island in the miR-221 promoter. Mechanistically, a reverted GATA2 phenotype by endogenous suppression of miR-221 was mediated through direct proangiogenic miR-221 target genes ICAM1 and ETS1. In a mouse model of carotid injury, GATA2 was reduced, and systemic supplementation of miR-126–coupled nanoparticles enhanced miR-126 availability in the carotid artery and improved reendothelialization of injured carotid arteries in vivo. Conclusions: GATA2-mediated regulation of miR-126 and miR-221 has an important impact on endothelial biology. Hence, modulation of GATA2 and its targets miR-126 and miR-221 is a promising therapeutic strategy for treatment of many vascular diseases.

Deregulation of microRNA-181c in cerebrospinal fluid of patients with clinically isolated syndrome is associated with early conversion to relapsing-remitting multiple sclerosis.

Background: MiRNA-181c, miRNA-633 and miRNA-922 have been reported to be deregulated in multiple sclerosis. Objectives: To investigate the association between miRNA-181c, miRNA-633 and miRNA-922 and conversion from clinically isolated syndrome (CIS) to relapsing–remitting multiple sclerosis (RRMS); and to compare microRNAs in cerebrospinal fluid (CSF) and serum with regard to dysfunction of the blood–CSF barrier. Methods: CSF and serum miRNA-181c, miRNA-633 and miRNA-922 were retrospectively determined by quantitative real-time polymerase chain reaction in CIS patients with (CIS-RRMS) and without (CIS-CIS) conversion to RRMS within 1 year. Results: Thirty of 58 CIS patients developed RRMS. Cerebrospinal fluid miRNA-922, serum miRNA-922 and cerebrospinal fluid miRNA-181c were significantly higher in CIS-RRMS compared to CIS-CIS (P=0.027, P=0.048, P=0.029, respectively). High levels of cerebrospinal fluid miRNA-181c were independently associated with conversion from CIS to RRMS in multivariate Cox regression analysis (hazard ratio 2.99, 95% confidence interval 1.41–6.34, P=0.005). A combination of high cerebrospinal fluid miRNA-181c, younger age and more than nine lesions on magnetic resonance imaging showed the highest specificity (96%) and positive predictive value (94%) for conversion from CIS to RRMS. MiRNA-181c was higher in serum than in cerebrospinal fluid (P <0.001), while miRNA-633 and miRNA-922 were no different in cerebrospinal fluid and serum. Cerebrospinal fluid/serum albumin quotients did not correlate with microRNAs in cerebrospinal fluid (all P>0.711). Conclusions: Cerebrospinal fluid miRNA-181c might serve as a biomarker for early conversion to RRMS. Moreover, our data suggest an intrathecal origin of microRNAs detected in the cerebrospinal fluid.

Circulating microRNA‐132 levels improve risk prediction for heart failure hospitalization in patients with chronic heart failure

Non‐coding microRNAs (miRNAs) are critically involved in cardiovascular pathophysiology. Since they are measurable in most body fluids, they have been proposed as circulating biomarkers. We examined the prognostic value of a specific candidate miRNA in a large cohort of patients with chronic heart failure (HF) enrolled in a multicentre clinical trial.

Identification of miR-126 as a new regulator of skin ageing

Movie S2 This is similar to movie 1, but with a more direct interaction. This is to indicate the heterogeneous nature of the nerve interactions with keratinocytes Movie S3 A 3D resolution of nerve fiber innervation of keratinocytes showing synaptic boutonlike structure penetrating and exiting keratinocytes; corresponds to figure 2d Movie S4 A bottom to top Z projection of the sample from figure 2d and Movie 3, indicating that the nerve fiber could be within one or more keratinocytes Movie S5 A 3D resolution of nerve fiber innervation of keratinocytes showing nerve actin filaments merging with keratinocyte actin filaments; corresponds to figure 2e and figure S5

MicroRNA 628-5p as a Novel Biomarker for Cardiac Allograft Vasculopathy.

Background Cardiac allograft vasculopathy (CAV) remains the leading cause of morbidity and mortality after orthotopic heart transplantation (OHT). Because of its clinically silent progression and lack of symptoms, detection is often difficult and invasive coronary angiography is performed routinely. To date, there are no established noninvasive biomarkers available for prediction of CAV in transplanted patients. MicroRNAs (miRNAs) are highly conserved, small noncoding RNA molecules that negatively regulate gene expression. As they are detectable in peripheral blood, recent studies have suggested miRNAs as biomarkers for various cardiovascular diseases. Thus, we hypothesized that circulating miRNAs may serve as noninvasive biomarkers for CAV. Methods To determine the regulation of circulating miRNAs, we performed miRNA profiling studies in plasma samples of OHT patients with confirmed high-degree CAV and a matched control group consisting of patients without any signs of CAV at least 5 years after OHT. Candidate miRNAs were verified by quantitative reverse transcriptase polymerase chain reaction. Results Microarray analysis revealed 5 candidate miRNAs (miR-34a, miR-98, miR-155, miR-204, miR-628-5p) that were differentially regulated in plasma samples of patients with CAV and therefore were selected for verification by quantitative reverse transcriptase polymerase chain reaction. In CAV patients, plasma levels of miR-628-5p and miR-155 were significantly increased (P = 0.001 and P = 0.028, respectively). A miR628-5p value above 1.336 was able to predict CAV with a sensitivity of 72% and a specificity of 83%. Conclusions For the first time, the present study identifies the circulating miRNA miR-628-5p as a novel potential biomarker of CAV in patients after OHT.

Inhibition of miRNA-212/132 improves the reprogramming of fibroblasts into induced pluripotent stem cells by de-repressing important epigenetic remodelling factors

MicroRNAs (miRNAs) repeatedly have been demonstrated to play important roles in the generation of induced pluripotent stem cells (iPSCs). To further elucidate the molecular mechanisms underlying transcription factor-mediated reprogramming we have established a model, which allows for the efficient screening of whole libraries of miRNAs modulating the generation of iPSCs from murine embryonic fibroblasts. Applying this model, we identified 14 miRNAs effectively inhibiting iPSC generation, including miR-132 and miR-212. Intriguingly, repression of these miRNAs during iPSC generation also resulted in significantly increased reprogramming efficacy. MiRNA target evaluation by qRT-PCR, Western blot, and luciferase assays revealed two crucial epigenetic regulators, the histone acetyl transferase p300 as well as the H3K4 demethylase Jarid1a (KDM5a) to be directly targeted by both miRNAs. Moreover, we demonstrated that siRNA-mediated knockdown of either p300 or Jarid1a recapitulated the miRNA effects and led to a significant decrease in reprogramming efficiency. Thus, conducting a full library miRNA screen we here describe a miRNA family, which markedly reduces generation of iPSC and upon inhibition in turn enhances reprogramming. These miRNAs, at least in part, exert their functions through repression of the epigenetic modulators p300 and Jarid1a, highlighting these two molecules as an endogenous epigenetic roadblock during iPSC generation.

MicroRNAs regulating superoxide dismutase 2 are new circulating biomarkers of heart failure

Although several risk factors such as infarct size have been identified, the progression of heart failure (HF) remains difficult to predict in clinical practice. Using an experimental rat model of post-myocardial infarction (MI), we previously identified 45 proteins differentially modulated during HF by proteomic analysis. This study sought to identify microRNAs (miRNAs) able to regulate these proteins and to test their relevance as biomarkers for HF. In silico bioinformatical analysis selected 13 miRNAs related to the 45 proteins previously identified. These miRNAs were analyzed in the rat and in cohorts of patients phenotyped for left ventricular remodeling (LVR). We identified that 3 miRNAs, miR-21-5p, miR-23a-3p and miR-222-3p, and their target Mn superoxide dismutase (SOD2) were significantly increased in LV and plasma of HF-rats. We found by luciferase activity a direct interaction of miR-222-3p with 3′UTR of SOD2. Transfection of human cardiomyocytes with miR-222-3p mimic or inhibitor induced respectively a decrease and an increase of SOD2 expression. Circulating levels of the 3 miRNAs and their target SOD2 were associated with high LVR post-MI in REVE-2 patients. We demonstrated for the first time the potential of microRNAs regulating SOD2 as new circulating biomarkers of HF.

miR-625-3p is upregulated in CD8+ T cells during early immune reconstitution after allogeneic stem cell transplantation

Alloreactive CD8+ T-cells mediate the curative graft-versus-leukaemia effect, the anti-viral immunity and graft-versus-host-disease (GvHD) after allogeneic stem cell transplantation (SCT). Thus, immune reconstitution with CD8+ T-cells is critical for the outcome of patients after allogeneic SCT. Certain miRNAs such as miR-146a or miR-155 play an important role in the regulation of post-transplant immunity in mice. While some miRNAs e.g. miR-423 or miR-155 are regulated in plasma or full blood during acute GvHD also in man, the relevance and expression profile of miRNAs in T-cells after allogeneic SCT is unknown. miR-625-3p has recently been described to be overexpressed in colorectal malignancies where it promotes migration, invasion and apoptosis resistance. Since similar regulative functions in cancer and T-cells have been described for an increasing number of miRNAs, we assumed a role for the cancer-related miR-625-3p also in T-cells. Here, we studied miR-625-3p expression selectively in CD8+ T-cells both in vitro and during immune reconstitution after allogeneic SCT in man. T-cell receptor stimulation lead to miR-625-3p upregulation in human CD8+ T-cells in vitro. Maintenance of elevated miR-625-3p expression levels was dependent on ongoing T-cell proliferation and was abrogated by withdrawal of interleukin 2 or the mTOR inhibitor rapamycin. Finally, miR-625-3p expression was analyzed in human CD8+ T-cells purified from 137 peripheral blood samples longitudinally collected from 74 patients after allogeneic SCT. miR-625-3p expression was upregulated on day 25 and on day 45, i.e. during the early phase of CD8+ T-cell reconstitution after allogeneic SCT and subsequently declined with completion of CD8+ T-cell reconstitution until day 150. In conclusion, this study has shown for the first time that miR-625-3p is regulated in CD8+ T-cells during proliferation in vitro and during early immune reconstitution after allogeneic SCT in vivo. These results warrant further studies to identify the targets and function of miR-625-3p in CD8+ T-cells and to analyze its predictive value for an effective immune reconstitution.