The plasma miRNome and venous thromboembolism in high-grade glioma: miRNA Sequencing of a nested case-control cohort.

Patients with high-grade gliomas are at high risk of venous thromboembolism (VTE). MicroRNAs (miRNAs) are small non-coding RNAs with multiple roles in tumour biology, haemostasis and platelet function. Their association with VTE risk in high-grade glioma has not been comprehensively mapped so far. We thus conducted a nested case-control study within 152 patients with WHO grade IV glioma that had been part of a prospective cohort study on VTE risk factors. At inclusion a single blood draw was taken, and patients were thereafter followed for a maximum of 2 years. During that time, 24 patients (16%) developed VTE. Of the other 128 patients, we randomly selected 24 age- and sex-matched controls. After quality control, the final group size was 21 patients with VTE during follow-up and 23 without VTE. Small RNA next-generation sequencing of plasma was performed. We observed that hsa-miR-451a was globally the most abundant miRNA. Notably, 51% of all miRNAs showed a correlation with platelet count. The analysis of miRNAs differentially regulated in VTE patients-with and without platelet adjustment-identified potential VTE biomarker candidates such as has-miR-221-3p. Therewith, we here provide one of the largest and deepest peripheral blood miRNA datasets of high-grade glioma patients so far, in which we identified first VTE biomarker candidates that can serve as the starting point for future research.

MicroRNA-30d-5p-A Potential New Therapeutic Target for Prevention of Ischemic Cardiomyopathy after Myocardial Infarction.

(1) Background and Objective: MicroRNAs (miRs) are biomarkers for assessing the extent of cardiac remodeling after myocardial infarction (MI) and important predictors of clinical outcome in heart failure. Overexpression of miR-30d-5p appears to have a cardioprotective effect. The aim of the present study was to demonstrate whether miR-30d-5p could be used as a potential therapeutic target to improve post-MI adverse remodeling. (2) Methods and Results: MiR profiling was performed by next-generation sequencing to assess different expression patterns in ischemic vs. healthy myocardium in a rat model of MI. MiR-30d-5p was significantly downregulated (p < 0.001) in ischemic myocardium and was selected as a promising target. A mimic of miR-30d-5p was administered in the treatment group, whereas the control group received non-functional, scrambled siRNA. To measure the effect of miR-30d-5p on infarct area size of the left ventricle, the rats were randomized and treated with miR-30d-5p or scrambled siRNA. Histological planimetry was performed 72 h and 6 weeks after induction of MI. Infarct area was significantly reduced at 72 h and at 6 weeks by using miR-30d-5p (72 h: 22.89 ± 7.66% vs. 35.96 ± 9.27%, p = 0.0136; 6 weeks: 6.93 ± 4.58% vs. 12.48 ± 7.09%, p = 0.0172). To gain insight into infarct healing, scratch assays were used to obtain information on cell migration in human umbilical vein endothelial cells (HUVECs). Gap closure was significantly faster in the mimic-treated cells 20 h post-scratching (12.4% more than the scrambled control after 20 h; p = 0.013). To analyze the anti-apoptotic quality of miR-30d-5p, the ratio between phosphorylated p53 and total p53 was evaluated in human cardiomyocytes using ELISA. Under the influence of the miR-30d-5p mimic, cardiomyocytes demonstrated a decreased pp53/total p53 ratio (0.66 ± 0.08 vs. 0.81 ± 0.17), showing a distinct tendency (p = 0.055) to decrease the apoptosis rate compared to the control group. (3) Conclusion: Using a mimic of miR-30d-5p underlines the cardioprotective effect of miR-30d-5p in MI and could reduce the risk for development of ischemic cardiomyopathy.

Circulating microRNAs in young individuals with long-duration type 1 diabetes in comparison with healthy controls.

MicroRNAs (miRNAs) are short non-coding RNAs that are involved in post-transcriptional control of gene expression and might be used as biomarkers for diabetes-related complications. The aim of this case-control study was to explore potential differences in circulating miRNAs in young individuals with long-duration type 1 diabetes (T1D) compared to healthy controls, and how identified miRNAs are expressed across different tissues. Twelve adolescents, age 15.0-17.9 years, with T1D duration of more than 8 years (mean 11.1 years), were enrolled from the Swedish diabetes quality registry. An age-matched control group was recruited. Circulating miRNAs (n = 187) were analyzed by quantitative PCR. We observed that 27 miRNAs were upregulated and one was downregulated in T1D. Six of these miRNAs were tissue-enriched (blood cells, gastrointestinal, nerve, and thyroid tissues). Six miRNAs with the largest difference in plasma, five up-regulated (hsa-miR-101-3p, hsa-miR-135a-5p, hsa-miR-143-3p, hsa-miR-223-3p and hsa-miR-410-3p (novel for T1D)) and one down-regulated (hsa-miR-495-3p), with P-values below 0.01, were selected for further in-silico analyses. AKT1, VEGFA and IGF-1 were identified as common targets. In conclusion, 28 of the investigated miRNAs were differently regulated in long-duration T1D in comparison with controls. Several associations with cancer were found for the six miRNAs with the largest difference in plasma.

Comprehensive mRNA-sequencing-based characterization of three HEK-293 cell lines during an rAAV production process for gene therapy applications.

Human embryonal kidney cells (HEK-293) are the most common host cells used for transient recombinant adeno-associated virus (rAAV) production in pharmaceutical industry. To better cover the expected gene therapy product demands in the future, different traditional strategies such as cell line sub-cloning and/or addition of chemical substances to the fermentation media have been used to maximize titers and improve product quality. A more effective and advanced approach to boost yield can be envisaged by characterizing the transcriptome of different HEK-293 cell line pedigrees with distinct rAAV productivity patterns to subsequently identify potential gene targets for cell engineering. In this work, the mRNA expression profile of three HEK-293 cell lines, resulting in various yields during a fermentation batch process for rAAV production, was investigated to gain basic insight into cell variability and eventually to identify genes that correlate with productivity. Mock runs using only transfection reagents were performed in parallel as a control. It finds significant differences in gene regulatory behaviors between the three cell lines at different growth and production stages. The evaluation of these transcriptomics profiles combined with collected in-process control parameters and titers shed some light on potential cell engineering targets to maximize transient production of rAAV in HEK-293 cells.

Independent human mesenchymal stromal cell-derived extracellular vesicle preparations differentially attenuate symptoms in an advanced murine graft-versus-host disease model.

BACKGROUND AIMS: Extracellular vesicles (EVs) harvested from conditioned media of human mesenchymal stromal cells (MSCs) suppress acute inflammation in various disease models and promote regeneration of damaged tissues. After successful treatment of a patient with acute steroid-refractory graft-versus-host disease (GVHD) using EVs prepared from conditioned media of human bone marrow-derived MSCs, this study focused on improving the MSC-EV production for clinical application. METHODS: Independent MSC-EV preparations all produced according to a standardized procedure revealed broad immunomodulatory differences. Only a proportion of the MSC-EV products applied effectively modulated immune responses in a multi-donor mixed lymphocyte reaction (mdMLR) assay. To explore the relevance of such differences in vivo, at first a mouse GVHD model was optimized. RESULTS: The functional testing of selected MSC-EV preparations demonstrated that MSC-EV preparations revealing immunomodulatory capabilities in the mdMLR assay also effectively suppress GVHD symptoms in this model. In contrast, MSC-EV preparations, lacking such in vitro activities, also failed to modulate GVHD symptoms in vivo. Searching for differences of the active and inactive MSC-EV preparations, no concrete proteins or miRNAs were identified that could serve as surrogate markers. CONCLUSIONS: Standardized MSC-EV production strategies may not be sufficient to warrant manufacturing of MSC-EV products with reproducible qualities. Consequently, given this functional heterogeneity, every individual MSC-EV preparation considered for the clinical application should be evaluated for its therapeutic potency before administration to patients. Here, upon comparing immunomodulating capabilities of independent MSC-EV preparations in vivo and in vitro, we found that the mdMLR assay was qualified for such analyses.

Transcriptomic landscapes of effective and failed liver regeneration in humans.

Background & Aims: Although extensive experimental evidence on the process of liver regeneration exists, in humans, validation is largely missing. However, liver regeneration is critically affected by underlying liver disease. Within this project, we aimed to systematically assess early transcriptional changes during liver regeneration in humans and further assess how these processes differ in people with dysfunctional liver regeneration. Methods: Blood samples of 154 patients and intraoperative tissue samples of 46 patients undergoing liver resection were collected and classified with regard to dysfunctional postoperative liver regeneration. Of those, a matched cohort of 21 patients were used for RNA sequencing. Samples were assessed for circulating cytokines, gene expression dynamics, intrahepatic neutrophil accumulation, and spatial transcriptomics. Results: Individuals with dysfunctional liver regeneration demonstrated an aggravated transcriptional inflammatory response with higher intracellular adhesion molecule-1 induction. Increased induction of this critical leukocyte adhesion molecule was associated with increased intrahepatic neutrophil accumulation and activation upon induction of liver regeneration in individuals with dysfunctional liver regeneration. Comparing baseline gene expression profiles in individuals with and without dysfunctional liver regeneration, we found that dual-specificity phosphatase 4 (DUSP4) expression, a known critical regulator of intracellular adhesion molecule-1 expression in endothelial cells, was markedly reduced in patients with dysfunctional liver regeneration. Mimicking clinical risk factors for dysfunctional liver regeneration, we found liver sinusoidal endothelial cells of two liver disease models to have significantly reduced baseline levels of DUSP4. Conclusions: Exploring the landscape of early transcriptional changes of human liver regeneration, we observed that people with dysfunctional regeneration experience overwhelming intrahepatic inflammation. Subclinical liver disease might account for DUSP4 reduction in liver sinusoidal endothelial cells, which ultimately primes the liver for an aggravated inflammatory response. Impact and implications: Using a unique human biorepository, focused on liver regeneration (LR), we explored the landscape of circulating and tissue-level alterations associated with both functional and dysfunctional LR. In contrast to experimental animal models, people with dysfunctional LR demonstrated an aggravated transcriptional inflammatory response, higher intracellular adhesion molecule-1 (ICAM-1) induction, intrahepatic neutrophil accumulation and activation upon induction of LR. Although inflammatory responses appear rapidly after liver resection, people with dysfunctional LR have exaggerated inflammatory responses that appear to be related to decreased levels of LSEC DUSP4, challenging existing concepts of post-resectional LR.

Identification of circulating microRNA patterns in patients in psoriasis and psoriatic arthritis.

OBJECTIVE: miRNAs are small non-coding RNAs that control gene expression. Specific intra- and extracellular miRNA signatures have been identified in various diseases. Whether certain miRNA signatures are associated with psoriasis (PsO) and PsA is currently unknown. We aimed to search for circulating miRNA signatures associated with PsO and PsA patients. METHODS: Expression of miRNAs was analysed by reverse transcription quantitative real-time PCR (RT-qPCR) in the serum of PsA, PsO patients and healthy controls. Demographic and disease-specific characteristics and imaging data from hand MRI were recorded. In the discovery phase, 192 miRNA assays were analysed in 48 samples (PsA, PsO, controls: each N = 16). For validation, 17 selected miRNAs were measured in the total population. RESULTS: A total of 141 patients and controls were analysed (51 PsA, 40 PsO, 50 controls). In the discovery phase 51 miRNAs in PsO and 64 miRNAs in PsA were down- or upregulated compared with controls, with 33 miRNAs being changed in both (adj. P < 0.05). The 17 top candidates from discovery were assessed in the validation phase, 9 of them discriminated PsA and PsO from controls [area under the curve (AUC) ≥0.70, all P < 0.05]. Four miRNAs (miR-19b-3p, miR-21-5p, miR-92a-3p and let-7b-5p) were significantly differently regulated between PsO and PsA. A combination of these miRNAs increased the AUC to 0.92 in multivariate regression model to discriminate PsO and PsA. CONCLUSION: miRNA signatures in PsA and PsO patients differ from controls. Nine miRNAs were differentially regulated in PsA and PsO patients, five of them previously reported to be involved in bone and cartilage metabolism, indicating an intimate association of psoriatic inflammation and bone/cartilage changes.

Exosomal miRNAs from Prostate Cancer Impair Osteoblast Function in Mice.

Prostate cancer (PCa) is the most frequent malignancy in older men with a high propensity for bone metastases. Characteristically, PCa causes osteosclerotic lesions as a result of disrupted bone remodeling. Extracellular vesicles (EVs) participate in PCa progression by conditioning the pre-metastatic niche. However, how EVs mediate the cross-talk between PCa cells and osteoprogenitors in the bone microenvironment remains poorly understood. We found that EVs derived from murine PCa cell line RM1-BM increased metabolic activity, vitality, and cell proliferation of osteoblast precursors by >60%, while significantly impairing mineral deposition (-37%). The latter was further confirmed in two complementary in vivo models of ossification. Accordingly, gene and protein set enrichments of osteoprogenitors exposed to EVs displayed significant downregulation of osteogenic markers and upregulation of proinflammatory factors. Additionally, transcriptomic profiling of PCa-EVs revealed the abundance of three microRNAs, miR-26a-5p, miR-27a-3p, and miR-30e-5p involved in the suppression of BMP-2-induced osteogenesis in vivo, suggesting the critical role of these EV-derived miRNAs in PCa-mediated suppression of osteoblast activity. Taken together, our results indicate the importance of EV cargo in cancer-bone cross-talk in vitro and in vivo and suggest that exosomal miRNAs may contribute to the onset of osteosclerotic bone lesions in PCa.

Longitudinal Changes of Circulating miRNAs During Bisphosphonate and Teriparatide Treatment in an Animal Model of Postmenopausal Osteoporosis.

MicroRNAs regulate bone homeostasis, and circulating microRNAs have been proposed as novel bone biomarkers. The effect of anti-osteoporotic treatment on circulating microRNAs has not been described in detail. Therefore, we performed a comprehensive analysis of microRNA serum levels in ovariectomized (OVX) and sham-operated (SHAM) rats over 12 weeks of antiresorptive or osteoanabolic treatment. Forty-two Sprague Dawley rats underwent SHAM surgery (n = 10) or ovariectomy (n = 32). After 8 weeks, OVX rats were randomized to antiresorptive treatment with zoledronate (n = 11), osteoanabolic treatment with teriparatide (n = 11), or vehicle treatment (n = 10). Serum samples were collected at weeks 8, 12, 16, and 20 after surgery. A total of 91 microRNAs were analyzed by RT-qPCR in serum samples collected at week 20. Based on the results, 29 microRNAs were selected for longitudinal analysis at all four study time points. Changes in bone mineral density and microstructure were followed up by in vivo micro-CT and ex vivo nano-CT. Ovariectomy resulted in the loss of trabecular bone, which was reversed by osteoanabolic and antiresorptive treatment. Differential expression analysis identified 11 circulating miRNAs that were significantly regulated after treatment. For example, miR-107 and miR-31-5p increased in vehicle-treated OVX animals, whereas they decreased during teriparatide treatment. Additional miRNAs were identified that showed significant correlations to bone microstructure or bone miRNA expression, including miR-203a-3p, which exhibited a significant negative correlation to vertebral and tibial trabecular bone volume fraction (%). Longitudinal analysis confirmed eight microRNAs with significant changes in serum over time that were prevented by teriparatide and zoledronate treatment (miR-34a-5p, miR-31-5p, miR-30d-3p, miR-378a-5p) or teriparatide treatment only (miR-375-3p, miR-183-5p, miR-203a-3p, miR-203b-3p). Gene target network analysis identified WNT and Notch signaling as the main signaling pathways controlled by these miRNAs. Thus, ovariectomy results in time-dependent deregulation of circulating miRNAs compared with SHAM animals. Anti-osteoporotic treatments can rescue this effect, showing that bone-related miRNAs might act as novel biomarkers for treatment monitoring. © 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Dysregulation of tissue and serum microRNAs in organ transplant recipients with cutaneous squamous cell carcinomas.

Micro RNAs (miRNAs) are considered as promising biomarkers for skin cancer. By next generation sequencing (NGS), we measured and compared miRNA expression profiles of tumor, peri-lesional, and control tissue of immunosuppressed organ transplant recipients (OTR), suffering from localized cutaneous squamous cell carcinoma (cSCC). Out of 490 miRNAs detected in cSCC tissue, 23 significantly regulated miRNAs were selected for quantitative targeted analysis in serum samples of our patients and control sera from OTR without cSCC. Two onco-miRNAs (mir-1290, mir-1246), previously identified as crucial drivers for tumor initiation and cancer progression, were significantly and exclusively upregulated in both tumor tissue and serum. This finding suggests that miRNA dysregulation in cSCC tissue is reflected in serum even in patients without advanced disease and highly differentiated cSCCs.

microRNA-146a controls age-related bone loss.

Bone loss is one of the consequences of aging, leading to diseases such as osteoporosis and increased susceptibility to fragility fractures and therefore considerable morbidity and mortality in humans. Here, we identify microRNA-146a (miR-146a) as an essential epigenetic switch controlling bone loss with age. Mice deficient in miR-146a show regular development of their skeleton. However, while WT mice start to lose bone with age, animals deficient in miR-146a continue to accrue bone throughout their life span. Increased bone mass is due to increased generation and activity of osteoblasts in miR-146a-deficient mice as a result of sustained activation of bone anabolic Wnt signaling during aging. Deregulation of the miR-146a target genes Wnt1 and Wnt5a parallels bone accrual and osteoblast generation, which is accompanied by reduced development of bone marrow adiposity. Furthermore, miR-146a-deficient mice are protected from ovariectomy-induced bone loss. In humans, the levels of miR-146a are increased in patients suffering fragility fractures in comparison with those who do not. These data identify miR-146a as a crucial epigenetic temporal regulator which essentially controls bone homeostasis during aging by regulating bone anabolic Wnt signaling. Therefore, miR-146a might be a powerful therapeutic target to prevent age-related bone dysfunctions such as the development of bone marrow adiposity and osteoporosis.

Impact of Anticoagulation and Sample Processing on the Quantification of Human Blood-Derived microRNA Signatures.

Blood-derived microRNA signatures have emerged as powerful biomarkers for predicting and diagnosing cardiovascular disease, cancer, and metabolic disorders. Platelets and platelet-derived microvesicles are a major source of microRNAs. We have previously shown that the inappropriate anticoagulation and storage of blood samples causes substantial platelet activation that is associated with the release of platelet-stored molecules into the plasma. However, it is currently unclear if circulating microRNA levels are affected by artificial platelet activation due to suboptimal plasma preparation. To address this issue, we used a standardized RT-qPCR test for 12 microRNAs (thrombomiR®, TAmiRNA GmbH, Vienna, Austria) that have been associated with cardiovascular and thrombotic diseases and were detected in platelets and/other hematopoietic cells. Blood was prevented from coagulating with citrate-theophylline-adenosine-dipyridamole (CTAD), sodium citrate, or ethylenediaminetetraacetic acid (EDTA) and stored for different time periods either at room temperature or at 4 °C prior to plasma preparation and the subsequent quantification of microRNAs. We found that five microRNAs (miR-191-5p, miR-320a, miR-21-5p, miR-23a-3p, and miR-451a) were significantly increased in the EDTA plasma. Moreover, we observed a time-dependent increase in plasma microRNAs that was most pronounced in the EDTA blood stored at room temperature for 24 h. Furthermore, significant correlations between microRNA levels and plasma concentrations of platelet-stored molecules pointed towards in vitro platelet activation. Therefore, we strongly recommend to (i) use CTAD as an anticoagulant, (ii) process blood samples as quickly as possible, and (iii) store blood samples at 4 °C whenever immediate plasma preparation is not feasible to generate reliable data on blood-derived microRNA signatures.

MicroRNA Expression Profile Changes after Cardiopulmonary Bypass and Ischemia/Reperfusion-Injury in a Porcine Model of Cardioplegic Arrest.

Identification of microRNAs (miRNA) associated with cardiopulmonary bypass, cardiac arrest and subsequent myocardial ischemia/reperfusion may unravel novel therapeutic targets and biomarkers. The primary aim of the present study was to investigate the effects of cardiopulmonary bypass and temperature of cardioplegic arrest on myocardial miRNA profile in pigs' left ventricular tissue. We employed next-generation sequencing to analyse miRNA profiles in the following groups: (1) hearts were arrested with antegrade warm St Thomas Hospital No. 2 (STH2) cardioplegia (n = 5; STH2-warm, 37 °C) and (2) cold STH2 (n = 6; STH2-cold, 4 °C) cardioplegia. Sixty min of ischemia was followed by 60 min of on-pump reperfusion with an additional 90 min of off-pump reperfusion. In addition, two groups without cardiac arrest (off-pump and on-pump group; n = 3, respectively) served as additional controls. STH2-warm and STH2-cold cardioplegia revealed no hemodynamic differences. In contrast, coronary venous creatine kinase-myocardial band (CK-MB) levels were significantly lower in pigs receiving STH2-warm cardioplegia (p < 0.05). Principal component analysis revealed that cardiopulmonary bypass and cardioplegic arrest markedly affected miRNAs in left ventricular tissue. Accordingly, ssc-miR-122, ssc-miR-10a-5p, ssc-miR-193a-3p, ssc-miR-499-3p, ssc-miR-374a-5p, ssc-miR-345-5p, ssc-miR-142-3p, ssc-miR-424-5p, ssc-miR-545-3p, ssc-miR-30b-5p, ssc-miR-145-5p, ssc-miR-374b-5p and ssc-miR-139-3p were differently regulated by cardiopulmonary bypass (false discovery rate (FDR) < 0.05 versus off-pump group). However, only ssc-miR-451 was differently expressed between STH2-warm and STH2-cold (FDR < 0.05). These data demonstrate for the first time that cardiopulmonary bypass and temperature of cardioplegic solution affected the expression of miRNAs in left ventricular tissue. In conclusion, specific miRNAs are potential therapeutic targets for limiting ischemia-reperfusion injury in patients undergoing cardiac surgery.

Combined proteomics/miRNomics of dendritic cell immunotherapy-treated glioblastoma patients as a screening for survival-associated factors.

Glioblastoma is the most prevalent and aggressive brain cancer. With a median overall survival of ~15-20 months under standard therapy, novel treatment approaches are desperately needed. A recent phase II clinical trial with a personalized immunotherapy based on tumor lysate-charged dendritic cell (DC) vaccination, however, failed to prolong survival. Here, we investigated tumor tissue from trial patients to explore glioblastoma survival-related factors. We followed an innovative approach of combining mass spectrometry-based quantitative proteomics (n = 36) with microRNA sequencing plus RT-qPCR (n = 38). Protein quantification identified, e.g., huntingtin interacting protein 1 (HIP1), retinol-binding protein 1 (RBP1), ferritin heavy chain (FTH1) and focal adhesion kinase 2 (FAK2) as factor candidates correlated with a dismal prognosis. MicroRNA analysis identified miR-216b, miR-216a, miR-708 and let-7i as molecules potentially associated with favorable tissue characteristics as they were enriched in patients with a comparably longer survival. To illustrate the utility of integrated miRNomics and proteomics findings, focal adhesion was studied further as one example for a pathway of potential general interest. Taken together, we here mapped possible drivers of glioblastoma outcome under immunotherapy in one of the largest DC vaccination tissue analysis cohorts so far-demonstrating usefulness and feasibility of combined proteomics/miRNomics approaches. Future research should investigate agents that sensitize glioblastoma to (immuno)therapy-potentially building on insights generated here.

Anti-CD3 Antibody Treatment Reduces Scar Formation in a Rat Model of Myocardial Infarction.

: Introduction: Antibody treatment with anti-thymocyte globulin (ATG) has been shown to be cardioprotective. We aimed to evaluate which single anti-T-cell epitope antibody alters chemokine expression at a level similar to ATG and identified CD3, which is a T-cell co-receptor mediating T-cell activation. Based on these results, the effects of anti-CD3 antibody treatment on angiogenesis and cardioprotection were tested in vitro and in vivo. METHODS: Concentrations of IL-8 and MCP-1 in supernatants of human peripheral blood mononuclear cell (PBMC) cultures following distinct antibody treatments were evaluated by Enzyme-linked Immunosorbent Assay (ELISA). In vivo, anti-CD3 antibodies or vehicle were injected intravenously in rats subjected to acute myocardial infarction (AMI). Chemotaxis and angiogenesis were evaluated using tube and migration assays. Intracellular pathways were assessed using Western blot. Extracellular vesicles (EVs) were quantitatively evaluated using fluorescence-activated cell scanning, exoELISA, and nanoparticle tracking analysis. Also, microRNA profiles were determined by next-generation sequencing. RESULTS: Only PBMC stimulation with anti-CD3 antibody led to IL-8 and MCP-1 changes in secretion, similar to ATG. In a rat model of AMI, systemic treatment with an anti-CD3 antibody markedly reduced infarct scar size (27.8% (Inter-quartile range; IQR 16.2-34.9) vs. 12.6% (IQR 8.3-27.2); p < 0.01). The secretomes of anti-CD3 treated PBMC neither induced cardioprotective pathways in cardiomyocytes nor pro-angiogenic mechanisms in human umbilical vein endothelial cell (HUVECs) in vitro. While EVs quantities remained unchanged, PBMC incubation with an anti-CD3 antibody led to alterations in EVs miRNA expression. CONCLUSION: Treatment with an anti-CD3 antibody led to decreased scar size in a rat model of AMI. Whereas cardioprotective and pro-angiogenetic pathways were unaltered by anti-CD3 treatment, qualitative changes in the EVs miRNA expression could be observed, which might be causal for the observed cardioprotective phenotype. We provide evidence that EVs are a potential cardioprotective treatment target. Our findings will also provide the basis for a more detailed analysis of putatively relevant miRNA candidates.

MicroRNA Profiling Reveals Distinct Signatures in Degenerative Rotator Cuff Pathologies.

MicroRNAs (miRNAs) have emerged as key regulators orchestrating a wide range of inflammatory and fibrotic diseases. However, the role of miRNAs in degenerative shoulder joint disorders is poorly understood. The aim of this explorative case-control study was to identify pathology-related, circulating miRNAs in patients with chronic rotator cuff tendinopathy and degenerative rotator cuff tears (RCT). In 2017, 15 patients were prospectively enrolled and assigned to three groups based on the diagnosed pathology: (i) no shoulder pathology, (ii) chronic rotator cuff tendinopathy, and (iii) degenerative RCTs. In total, 14 patients were included. Venous blood samples ("liquid biopsies") were collected from each patient and serum levels of 187 miRNAs were determined. Subsequently, the change in expression of nine candidate miRNAs was verified in tendon biopsy samples, collected from patients who underwent arthroscopic shoulder surgery between 2015 and 2018. Overall, we identified several miRNAs to be progressively deregulated in sera from patients with either chronic rotator cuff tendinopathy or degenerative RCTs. Importantly, for the several of these miRNAs candidates repression was also evident in tendon biopsies harvested from patients who were treated for a supraspinatus tendon tear. As similar expression profiles were determined for tendon samples, the newly identified systemic miRNA signature has potential as novel diagnostic or prognostic biomarkers for degenerative rotator cuff pathologies. © 2019 The Authors. Journal of Orthopaedic Research® published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society. Inc. J Orthop Res 38:202-211, 2020.

MicroRNA levels in bone and blood change during bisphosphonate and teriparatide therapy in an animal model of postmenopausal osteoporosis.

MicroRNAs control the activity of a variety of genes that are pivotal to bone metabolism. Therefore, the clinical utility of miRNAs as biomarkers and drug targets for bone diseases certainly merits further investigation. This study describes the use of an animal model of postmenopausal osteoporosis to generate a comprehensive dataset on miRNA regulation in bone tissue and peripheral blood during bone loss and specifically anti-resorptive and osteo-anabolic treatment. Forty-two Sprague-Dawley rats were randomized to SHAM surgery (n=10) or ovariectomy (OVX, n=32). Eight weeks after surgery, OVX animals were further randomized to anti-resorptive treatment with zoledronate (n=11), osteo-anabolic treatment with teriparatide (n=11), or vehicle treatment (n=10). After 12 weeks of treatment, bone and serum samples were used for microRNA analysis using next-generation sequencing (NGS), mRNA levels using RT-qPCR, and bone microarchitecture analysis using nanoCT. Ovariectomy resulted in loss of trabecular bone, which was fully rescued using osteo-anabolic treatment, and partially rescued using anti-resorptive treatment. NGS revealed that both, anti-resorptive and anabolic treatment had a significant impact on miRNA levels in bone tissue and serum: out of 426 detected miRNAs, 46 miRNAs were regulated by teriparatide treatment an d 10 by zoledronate treatment (p-adj.<0.1). Interestingly, teriparatide and zoledronate treatment were able to revert miRNA changes in tissue and serum of untreated OVX animals, such as the up-regulation of miR-203a-3p, a known osteo-inhibitory miRNA. We confirmed previously established mechanisms of miR-203a by analyzing its direct target Dlx5 in femoral head. Our data reveal a significant effect of ovariectomy-induced bone loss, as well as the two major types of anti-osteoporotic treatment on miRNA transcription in femoral head tissue. These changes are associated with altered activity of target genes relevant to bone formation, such as Dlx5. The observed effects of bone loss and treatment response on miRNA levels in bone are also reflected in the peripheral blood, suggesting the possibility of minimally-invasive monitoring of bone-derived miRNAs using liquid biopsies.

St Thomas' Hospital polarizing blood cardioplegia improves hemodynamic recovery in a porcine model of cardiopulmonary bypass.

OBJECTIVE: Cardiac surgery demands highly effective cardioprotective regimens. We previously demonstrated improved cardioprotection with "polarized" compared with "depolarized" arrest. This study uses a clinically relevant porcine model of cardiopulmonary bypass to compare the efficacy of blood-based St Thomas' Hospital polarizing cardioplegia (STH-Pol-B) with blood-based St Thomas' Hospital hyperkalemic cardioplegia (STH2-B). METHODS: Pigs were monitored and subjected to normothermic cardiopulmonary bypass, cardiac arrest via antegrade cold (4°C) blood cardioplegia (STH2-B, control group: n = 6 or STH-Pol-B, study group: n = 7), and global ischemia (60 minutes) followed by on-pump reperfusion (60 minutes) and subsequent off-pump reperfusion (90 minutes). At termination, tissue samples were taken for analysis of high-energy phosphates, ultrastructure, and microRNAs. The primary endpoint of this study was creatine kinase-muscle/brain release during reperfusion. RESULTS: Creatine kinase-muscle/brain was comparable in both groups. After pigs were weaned from cardiopulmonary bypass, hemodynamic parameters such as mean arterial pressure (P = .007), left ventricular systolic pressure (P < .001), external heart work (P = .012), stroke volume (P = .015), as well as dp/dtmax (P = .027), were improved with polarizing cardioplegia. Wedge pressure was significantly lower in the study group (P < .01). Energy charge was comparable between groups. MicroRNA-708-5p was significantly lower (P = .019) and microRNA-122 expression significantly (P = .046) greater in STH-Pol-B hearts. CONCLUSIONS: Polarized cardiac arrest offers similar myocardial protection and enhances functional recovery in a porcine model of cardiopulmonary bypass. Differential expression of microRNAs may indicate possible new ischemia-reperfusion markers. These results confirm the noninferiority and potential of polarized versus depolarized arrest.

Combining laser microdissection and microRNA expression profiling to unmask microRNA signatures in complex tissues.

Neglecting tissue heterogeneity during the analysis of microRNA (miRNA) levels results in average signals from an unknown mixture of different cell types that are difficult to interpret. Here we demonstrate the technical requirements needed to obtain high-quality, quantitative miRNA expression information from tumor tissue compartments obtained by laser microdissection (LMD). Furthermore, we show the significance of disentangling tumor tissue heterogeneity by applying the newly developed protocols for combining LMD of tumor tissue compartments with RT-qPCR analysis to reveal compartment-specific miRNA expression signatures. An important advantage of this strategy is that the miRNA signature can be directly linked to histopathology. In summary, combining LMD and RT-qPCR is a powerful approach for spatial miRNA expression analysis in complex tissues, enabling discovery of disease mechanisms, biomarkers and drug candidates.

Biotransformation of the Mycotoxin Zearalenone to its Metabolites Hydrolyzed Zearalenone (HZEN) and Decarboxylated Hydrolyzed Zearalenone (DHZEN) Diminishes its Estrogenicity In Vitro and In Vivo.

Zearalenone (ZEN)-degrading enzymes are a promising strategy to counteract the negative effects of this mycotoxin in livestock. The reaction products of such enzymes need to be thoroughly characterized before technological application as a feed additive can be envisaged. Here, we evaluated the estrogenic activity of the metabolites hydrolyzed zearalenone (HZEN) and decarboxylated hydrolyzed zearalenone (DHZEN) formed by hydrolysis of ZEN by the zearalenone-lactonase Zhd101p. ZEN, HZEN, and DHZEN were tested in two in vitro models, the MCF-7 cell proliferation assay (0.01-500 nM) and an estrogen-sensitive yeast bioassay (1-10,000 nM). In addition, we compared the impact of dietary ZEN (4.58 mg/kg) and equimolar dietary concentrations of HZEN and DHZEN on reproductive tract morphology as well as uterine mRNA and microRNA expression in female piglets (n = 6, four weeks exposure). While ZEN increased cell proliferation and reporter gene transcription, neither HZEN nor DHZEN elicited an estrogenic response, suggesting that these metabolites are at least 50-10,000 times less estrogenic than ZEN in vitro. In piglets, HZEN and DHZEN did not increase vulva size or uterus weight. Moreover, RNA transcripts altered upon ZEN treatment (EBAG9, miR-135a-5p, miR-187-3p and miR-204-5p) were unaffected by HZEN and DHZEN. Our study shows that both metabolites exhibit markedly reduced estrogenicity in vitro and in vivo, and thus provides an important basis for further evaluation of ZEN-degrading enzymes.