Posttranscriptional Regulation of 14q32 microRNAs by RNA Binding Proteins CIRBP and HADHB during Vascular Regeneration after Ischemia:Posttranscriptional regulation of 14q32 microRNAs

After induction of ischemia in mice, 14q32 microRNAs are regulated in three distinct temporal patterns. These expression patterns, as well as basal expression levels, are independent of the microRNA genes’ order in the 14q32 locus. This implies that posttranscriptional processing is a major determinant of 14q32 microRNA expression. Therefore, we hypothesized that RNA binding proteins (RBPs) regulate posttranscriptional processing of 14q32, and we aimed to identify these RBPs. To identify proteins responsible for this posttranscriptional regulation, we used RNA pull-down SILAC mass spectrometry (RP-SMS) on selected precursor microRNAs. We observed differential binding of cold-inducible RBP (CIRBP) and hydroxyacyl-CoA dehydrogenase trifunctional multienzyme complex subunit beta (HADHB) to the precursors of late-upregulated miR-329-3p and unaffected miR-495-3p. Immunohistochemical staining confirmed expression of both CIRBP and HADHB in the adductor muscle of mice. Expression of both CIRBP and HADHB was upregulated after hindlimb ischemia in mice. Using RBP immunoprecipitation experiments, we showed specific binding of CIRBP to pre-miR-329 but not to pri-miR-329. Finally, using CRISPR/Cas9, we generated HADHB−/− 3T3 cells, which display reduced expression of miR-329 and miR-495 but not their precursors. These data suggest a novel role for CIRBP and HADHB in posttranscriptional regulation of 14q32 microRNAs. Keywords: microRNA, 14q32, microRNA cluster, miR-329, miR-495, HADHB, CIRBP, RNA binding proteins, ischemia, hindlimb ischemia mode

Circulating Small Non-coding RNAs as Biomarkers for Recovery After Exhaustive or Repetitive Exercise

Circulating microRNAs have proven to be reliable biomarkers, due to their high stability, both in vivo in the circulation, and ex vivo during sample preparation and storage. Small nucleolar RNAs (snoRNAs) are a different type of small non-coding RNAs that can also be reliably measured in plasma, but have only been studied sporadically. In this study, we aimed to identify RNA-biomarkers that can distinguish between different exercise regimes and that entail clues about muscle repair and recovery after prolonged exhaustive endurance exercise. We compared plasma microRNA profiles between two cohorts of elite cyclists, subjected to two different types of exercise regimes, as well as a cohort of patients with peripheral artery disease (PAD) that were scheduled to undergo lower limb amputation, due to critical limb ischemia. In elite athletes, muscle tissue recovers quickly even after exhaustive exercise, whereas in PAD patients, recovery is completely impaired. Furthermore, we measured levels of a specific group of snoRNAs in the plasma of both elite cyclists and PAD patients. Using a multiplex qPCR screening, we detected a total of 179 microRNAs overall, of which, on average, 161 microRNAs were detected per sample. However, only 30 microRNAs were consistently expressed in all samples. Of these, two microRNAs, miR-29a-3p and miR193a-5p, that responded differently two different types of exercise, namely exhaustive exercise and non-exhaustive endurance exercise. Using individual rt/qPCR, we also identified a snoRNA, SNORD114.1, which was significantly upregulated in plasma in response to endurance exercise. Furthermore, two microRNAs, miR-29a-3p and miR-495-3p, were significantly differentially expressed in athletes compared to PAD patients, but only following exercise. We suggest that these two microRNAs could function as markers of impaired muscle repair and recovery. In conclusion, microRNAs miR-29a-3p and miR-193a-5p may help us distinguish between repeated exhaustive and non-exhaustive endurance exercise. MicroRNA miR-29a-3p, as well as miR-495-3p, may further mark impaired muscle recovery in patients with severe critical limb ischemia. Furthermore, we showed for the first time that a circulating snoRNA, SNORD114.1, is regulated in response to exercise and may be used as biomarker

Atorvastatin pleiotropically decreases intraplaque angiogenesis and intraplaque haemorrhage by inhibiting ANGPT2 release and VE-Cadherin internalization

Funding This work was supported by a grant from the European Union, MSCA joint doctoral project MoGlyNet [675527].Peer reviewedPublisher PD

Phosphorylcholine Monoclonal Antibody Therapy Decreases Intraplaque Angiogenesis and Intraplaque Hemorrhage in Murine Vein Grafts

Funding: This work was supported by the European Union Program Grant CVDIMMUNE [037227], CARDIMMUN [601728] and Marie Sklodowska Curie Actions joint doctoral project MoGlyNet [675527]. Acknowledgments: We would like to thank Raghed Halawani with help of quantifying histological images.Peer reviewedPublisher PD

Metabolic Alterations in Cardiopulmonary Vascular Dysfunction

Cardiovascular diseases (CVD) are the leading cause of death worldwide. CVD comprise a range of diseases affecting the functionality of the heart and blood vessels, including acute myocardial infarction (AMI) and pulmonary hypertension (PH). Despite their different causative mechanisms, both AMI and PH involve narrowed or blocked blood vessels, hypoxia, and tissue infarction. The endothelium plays a pivotal role in the development of CVD. Disruption of the normal homeostasis of endothelia, alterations in the blood vessel structure, and abnormal functionality are essential factors in the onset and progression of both AMI and PH. An emerging theory proposes that pathological blood vessel responses and endothelial dysfunction develop as a result of an abnormal endothelial metabolism. It has been suggested that, in CVD, endothelial cell metabolism switches to higher glycolysis, rather than oxidative phosphorylation, as the main source of ATP, a process designated as the Warburg effect. The evidence of these alterations suggests that understanding endothelial metabolism and mitochondrial function may be central to unveiling fundamental mechanisms underlying cardiovascular pathogenesis and to identifying novel critical metabolic biomarkers and therapeutic targets. Here, we review the role of the endothelium in the regulation of vascular homeostasis and we detail key aspects of endothelial cell metabolism. We also describe recent findings concerning metabolic endothelial cell alterations in acute myocardial infarction and pulmonary hypertension, their relationship with disease pathogenesis and we discuss the future potential of pharmacological modulation of cellular metabolism in the treatment of cardiopulmonary vascular dysfunction. Although targeting endothelial cell metabolism is still in its infancy, it is a promising strategy to restore normal endothelial functions and thus forestall or revert the development of CVD in personalized multi-hit interventions at the metabolic level

RP105 deficiency attenuates early atherosclerosis via decreased monocyte influx in a CCR2 dependent manner

AbstractObjective: Toll like receptor 4 (TLR4) plays a key role in inflammation and previously it was established that TLR4 deficiency attenuates atherosclerosis. RadioProtective 105 (RP105) is a structural homolog of TLR4 and an important regulator of TLR4 signaling, suggesting that RP105 may also be an important effector in atherosclerosis. We thus aimed to determine the role of RP105 in atherosclerotic lesion development using RP105 deficient mice on an atherosclerotic background. Methods and results: Atherosclerosis was induced in Western-type diet fed low density lipoprotein receptor deficient (LDLr−/−) and LDLr/RP105 double knockout (LDLr−/−/RP105−/−) mice by means of perivascular carotid artery collar placement. Lesion size was significantly reduced by 58% in LDLr−/−/RP105−/− mice, and moreover, plaque macrophage content was markedly reduced by 40%. In a model of acute peritonitis, monocyte influx was almost 3-fold reduced in LDLr−/−/RP105−/− mice (P = 0.001), while neutrophil influx remained unaltered, suggestive of an altered migratory capacity of monocytes upon deletion of RP105. Interestingly, in vitro stimulation of monocytes with LPS induced a downregulation of CCR2, a chemokine receptor crucially involved in monocyte influx to atherosclerotic lesions, which was more pronounced in LDLr−/−/RP105−/− monocytes as compared to LDLr−/− monocytes. Conclusion: We here show that RP105 deficiency results in reduced early atherosclerotic plaque development with a marked decrease in lesional macrophage content, which may be due to disturbed migration of RP105 deficient monocytes resulting from CCR2 downregulation