NfκB signaling dynamics and their target genes differ between mouse blood cell types and induce distinct cell behavior.

Cells can use signaling pathway activity over time (ie, dynamics) to control cell fates. However, little is known about the potential existence and function of signaling dynamics in primary hematopoietic stem and progenitor cells (HSPCs). Here, we use time-lapse imaging and tracking of single murine HSPCs from green fluorescent protein-p65/H2BmCherry reporter mice to quantify their nuclear factor κB (NfκB) activity dynamics in response to tumor necrosis factor α and interleukin 1ß. We find response dynamics to be heterogeneous between individual cells, with cell type-specific dynamics distributions. Transcriptome sequencing of single cells physically isolated after live dynamics quantification shows activation of different target gene programs in cells with different dynamics. Finally, artificial induction of oscillatory NfκB activity causes changes in granulocyte/monocyte progenitor behavior. Thus, HSPC behavior can be influenced by signaling dynamics, which are tightly regulated during hematopoietic differentiation and enable cell type-specific responses to the same signaling inputs.

Regulation of the one carbon folate cycle as a shared metabolic signature of longevity.

The metabolome represents a complex network of biological events that reflects the physiologic state of the organism in health and disease. Additionally, specific metabolites and metabolic signaling pathways have been shown to modulate animal ageing, but whether there are convergent mechanisms uniting these processes remains elusive. Here, we used high resolution mass spectrometry to obtain the metabolomic profiles of canonical longevity pathways in C. elegans to identify metabolites regulating life span. By leveraging the metabolomic profiles across pathways, we found that one carbon metabolism and the folate cycle are pervasively regulated in common. We observed similar changes in long-lived mouse models of reduced insulin/IGF signaling. Genetic manipulation of pathway enzymes and supplementation with one carbon metabolites in C. elegans reveal that regulation of the folate cycle represents a shared causal mechanism of longevity and proteoprotection. Such interventions impact the methionine cycle, and reveal methionine restriction as an underlying mechanism. This comparative approach reveals key metabolic nodes to enhance healthy ageing.

miR-127-3p Is an Epigenetic Activator of Myofibroblast Senescence Situated within the MicroRNA-Enriched Dlk1-Dio3‒Imprinted Domain on Mouse Chromosome 12.

During wound healing, fibroblasts differentiate into nonproliferative contractile myofibroblasts, contribute to skin repair, and eventually undergo apoptosis or become senescent. MicroRNAs are post-transcriptional regulators of gene expression networks that control cell fate and survival and may also regulate senescence. In this study, we determined the regulated microRNAs in myofibroblasts isolated from wounds and analyzed their role in senescent myofibroblast formation. Transcriptome profiling showed that a 200 kilobase pair region of the Dlk1-Dio3‒imprinted domain on mouse chromosome 12 encodes for most of the upregulated microRNAs in the entire genome of mouse myofibroblasts. Among those, miR-127-3p induced a myofibroblast-like phenotype associated with a block in proliferation. Molecular analysis revealed that miR-127-3p induced a prolonged cell cycle arrest with unique molecular features of senescence, including the activation of the senescence-associated ß-galactosidase, increase in p53 and p21 levels, inhibition of lamin B1, proliferation factors, and the production of senescence-associated inflammatory and extracellular matrix‒remodeling components. Hence, miR-127-3p emerges as an epigenetic activator regulating the transition from repair to remodeling during skin wound healing but may also induce age-related defects, pathological scarring, and fibrosis, all linked to myofibroblast senescence.

Respiratory chain inactivation links cartilage-mediated growth retardation to mitochondrial diseases.

In childhood, skeletal growth is driven by transient expansion of cartilage in the growth plate. The common belief is that energy production in this hypoxic tissue mainly relies on anaerobic glycolysis and not on mitochondrial respiratory chain (RC) activity. However, children with mitochondrial diseases causing RC dysfunction often present with short stature, which indicates that RC activity may be essential for cartilage-mediated skeletal growth. To elucidate the role of the mitochondrial RC in cartilage growth and pathology, we generated mice with impaired RC function in cartilage. These mice develop normally until birth, but their later growth is retarded. A detailed molecular analysis revealed that metabolic signaling and extracellular matrix formation is disturbed and induces cell death at the cartilage-bone junction to cause a chondrodysplasia-like phenotype. Hence, the results demonstrate the overall importance of the metabolic switch from fetal glycolysis to postnatal RC activation in growth plate cartilage and explain why RC dysfunction can cause short stature in children with mitochondrial diseases.

Detection of annexin A8 antibodies in serum of patients with antiphospholipid syndrome.

INTRODUCTION: Antibodies specific for annexin A8 (AnxA8) have not been investigated in patients suffering from antiphospholipid syndrome (APS) yet. The aim of this study was to compare the presence of AnxA8 antibodies in serum of APS patients with that of age-matched healthy controls and to investigate whether AnxA8 antibodies are potential biomarkers for APS. MATERIALS AND METHODS: We enrolled 22 APS patients and 22 healthy controls in this case-control study. We used sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunoblot to investigate the presence of AnxA8 antibodies, and we applied enzyme-linked immunosorbent assay to investigate the presence of cardiolipin (CL) and beta-2-glycoprotein I (ß2GPI) antibodies. RESULTS: The serum of 9/22 APS patients showed AnxA8 IgG isotype antibody reactivity compared to serum of 2/22 healthy controls (P = 0.034). When we also included weak immunoblot signals, 12/22 APS patients exhibited AnxA8 IgG isotype antibody reactivity compared to 3/22 healthy controls (P = 0.005). We also investigated the presence of AnxA8 IgM isotype antibodies in the serum of APS patients but found no statistically significant difference between the APS patient group and healthy control group (P = 0.500). We further investigated the presence of ß2GPI and CL IgG and IgM isotype antibodies. AnxA8 IgG isotype antibodies were present in APS patients in a similar frequency as the APS "criteria" antibody against CL (P = 0.764). CONCLUSION: We demonstrated that AnxA8 IgG isotype antibodies are potential biomarkers for the diagnosis of APS.

Mimicking Angiogenesis in vitro: Three-dimensional Co-culture of Vascular Endothelial Cells and Perivascular Cells in Collagen Type I Gels.

Angiogenesis defines the process of formation of new vascular structures form existing blood vessels, involved during development, repair processes like wound healing but also linked to pathological changes. During angiogenic processes, endothelial cells build a vascular network and recruit perivascular cells to form mature, stable vessels. Endothelial cells and perivascular cells secret and assemble a vascular basement membrane and interact via close cell-cell contacts. To mimic these processes in vitro we have developed a versatile three-dimensional culture system where perivascular cells (PVC) are co-cultured with human umbilical cord vascular endothelial cells (HUVEC) in a collagen type I gel. This co-culture system can be used to determine biochemical and cellular processes during neoangiogenic events with a wide range of analyses options.

Detection of multiple annexin autoantibodies in a patient with recurrent miscarriages, fulminant stroke and seronegative antiphospholipid syndrome.

Anti-phospholipid syndrome (APS) is one of the main causes for recurrent miscarriages. The diagnosis of APS is based on the occurrence of clinical symptoms such as thrombotic events or obstetric complications as well as the detection of antiphospholipid antibodies directed against ß2-glycoprotein I and cardiolipin, or a positive lupus anticoagulant assay. However, there is a subpopulation of patients with clinical symptoms of APS, but the lack of serological markers (seronegative APS). In addition, a large proportion of patients with unexplained recurrent miscarriages exist. These cases may be attributed, at least in part, to a seronegative APS.The presence of autoantibodies against annexins is potentially associated with APS. Here we used immunoassays and immunoblots to detect autoantibodies directed against annexin A1-5, and A8, respectively, in a patient with a seronegative APS and a history of six recurrent pregnancy losses and fulminant stroke. We found strong IgM isotype antibody reactivity directed against annexin A2 and annexin A8, and moderate to weak IgM isotype antibody reactivity directed against annexin A1, A3, and A5. Further studies will evaluate the diagnostic value of IgM isotype antibodies against annexin A1-A5, and A8 for seronegative APS and recurrent miscarriages.

miR-126-3p Promotes Matrix-Dependent Perivascular Cell Attachment, Migration and Intercellular Interaction.

microRNAs (miRNAs) can regulate the interplay between perivascular cells (PVC) and endothelial cells (EC) during angiogenesis, but the relevant PVC-specific miRNAs are not yet defined. Here, we identified miR-126-3p and miR-146a to be exclusively upregulated in PVC upon interaction with EC, determined their influence on the PVC phenotype and elucidate their molecular mechanisms of action. Specifically the increase of miR-126-3p strongly promoted the motility of PVC on the basement membrane-like composite and stabilized networks of EC. Subsequent miRNA target analysis showed that miR-126-3p inhibits SPRED1 and PLK2 expression, induces ERK1/2 phosphorylation and stimulates TLR3 expression to modulate cell-cell and cell-matrix contacts of PVC. Gain of expression experiments in vivo demonstrated that miR-126-3p stimulates PVC coverage of newly formed vessels and transform immature into mature, less permeable vessels. In conclusion we showed that miR-126-3p regulates matrix-dependent PVC migration and intercellular interaction to modulate vascular integrity. Stem Cells 2016;34:1297-1309.