Loss of VHL in mesenchymal progenitors of the limb bud alters multiple steps of endochondral bone development

Adaptation to low oxygen tension (hypoxia) is a critical event during development. The transcription factors Hypoxia Inducible Factor-1α (HIF-1α) and HIF-2α are essential mediators of the homeostatic responses that allow hypoxic cells to survive and differentiate. Von Hippel Lindau protein (VHL) is the E3 ubiquitin ligase that targets HIFs to the proteasome for degradation in normoxia. We have previously demonstrated that the transcription factor HIF-1α is essential for survival and differentiation of growth plate chondrocytes, whereas HIF-2α is not necessary for fetal growth plate development. We have also shown that VHL is important for endochondral bone development, since loss of VHL in chondrocytes causes severe dwarfism. In this study, in order to expand our understanding of the role of VHL in chondrogenesis, we conditionally deleted VHL in mesenchymal progenitors of the limb bud, i.e. in cells not yet committed to the chondrocyte lineage. Deficiency of VHL in limb bud mesenchyme does not alter the timely differentiation of mesenchymal cells into chondrocytes. However, it causes structural collapse of the cartilaginous growth plate as a result of impaired proliferation, delayed terminal differentiation, and ectopic death of chondrocytes. This phenotype is associated to delayed replacement of cartilage by bone. Notably, loss of HIF-2α fully rescues the late formation of the bone marrow cavity in VHL mutant mice, though it does not affect any other detectable abnormality of the VHL mutant growth plates. Our findings demonstrate that VHL regulates bone morphogenesis as its loss considerably alters size, shape and overall development of the skeletal elements

The HIF Signaling Pathway in Osteoblasts Directly Modulates Erythropoiesis through the Production of EPO

SummaryOsteoblasts are an important component of the hematopoietic microenvironment in bone. However, the mechanisms by which osteoblasts control hematopoiesis remain unknown. We show that augmented HIF signaling in osteoprogenitors results in HSC niche expansion associated with selective expansion of the erythroid lineage. Increased red blood cell production occurred in an EPO-dependent manner with increased EPO expression in bone and suppressed EPO expression in the kidney. In contrast, inactivation of HIF in osteoprogenitors reduced EPO expression in bone. Importantly, augmented HIF activity in osteoprogenitors protected mice from stress-induced anemia. Pharmacologic or genetic inhibition of prolyl hydroxylases1/2/3 in osteoprogenitors elevated EPO expression in bone and increased hematocrit. These data reveal an unexpected role for osteoblasts in the production of EPO and modulation of erythropoiesis. Furthermore, these studies demonstrate a molecular role for osteoblastic PHD/VHL/HIF signaling that can be targeted to elevate both HSCs and erythroid progenitors in the local hematopoietic microenvironment.PaperCli

MiR-155 has a protective role in the development of non-alcoholic hepatosteatosis in mice

Hepatic steatosis is a global epidemic that is thought to contribute to the pathogenesis of type 2 diabetes. MicroRNAs (miRs) are regulators that can functionally integrate a range of metabolic and inflammatory pathways in liver. We aimed to investigate the functional role of miR-155 in hepatic steatosis. Male C57BL/6 wild-type (WT) and miR-155−/− mice were fed either normal chow or high fat diet (HFD) for 6 months then lipid levels, metabolic and inflammatory parameters were assessed in livers and serum of the mice. Mice lacking endogenous miR-155 that were fed HFD for 6 months developed increased hepatic steatosis compared to WT controls. This was associated with increased liver weight and serum VLDL/LDL cholesterol and alanine transaminase (ALT) levels, as well as increased hepatic expression of genes involved in glucose regulation (Pck1, Cebpa), fatty acid uptake (Cd36) and lipid metabolism (Fasn, Fabp4, Lpl, Abcd2, Pla2g7). Using miRNA target prediction algorithms and the microarray transcriptomic profile of miR-155−/− livers, we identified and validated that Nr1h3 (LXRα) as a direct miR-155 target gene that is potentially responsible for the liver phenotype of miR-155−/− mice. Together these data indicate that miR-155 plays a pivotal role regulating lipid metabolism in liver and that its deregulation may lead to hepatic steatosis in patients with diabetes

An innovative strategy for the identification of novel genes or microRNAs involved in cardiac protection

Despite the recent advances in cardiovascular therapies, cardiac diseases are still the leading cause of morbidity and mortality in developed countries and for this reason it is of pivotal importance to discover new therapeutic and prevention targets. The aim of this thesis is to implement a novel strategy to define new factors, either secreted proteins or microRNAs that protect myocardial cells from either ischemic or toxic damage. The protective activity of discovered factors will be validated in further studies in vivo in different models of myocardial damage. The first step of the work is to clone two sub-libraries of secreted proteins and microRNAs in an adeno-associated viral vector (AAV) backbone plasmid (pZac2.1 and pAAV-MCS) containing the CMV promoter, that allows transgene expression into mammalian cells, and the ITR sequences, essential for viral genome packaging. So far many AAV serotypes have been recognized with specific capabilities to transduce different cell types. The AAV vectors are good candidates for gene therapy because of their promising features: these vectors retain less then 10% of the viral genome, do not express viral proteins, permitting a long persistence in vivo without either immunogenic or inflammatory response and are very efficient for in vivo transduction of many fully differentiated tissues, such as retina, neurons, heart and muscle. Cells are transduced at high multiplicity of infection and mixing of different rAAV preparation results in the simultaneous expression of gene combinations in vivo. After the cloning, we obtained two different libraries to begin the selection procedure. Starting from pools of cDNAs and microRNAs cloned in an AAV backbone, we produced the corresponding pools of viruses and used the preparation to transduce cardiomyocytes. The serotype 9 was used for these experiments because it was shown that it has the highest tropism for cardiomyocytes in vitro and in vivo. Afterwards the cells were treated with a toxic chemical drug. The DNA of the surviving cells was extracted and analyzed by PCR to determine if any of the transduced genes carried by the AAV vectors was enriched; theoretically the cells that were transduced with a considerable amount of the protective factor should have a survival advantage and contain a considerable amount of the specific DNA sequence. Similarly, neonatal mice were infected with the same preparations of AAV vectors and an ischemic-like damage to the heart was induced by chemicals. Hearts were retrieved and DNA of the vectors retained by the living cells amplified in order to detect if any of the factors carried by the AAV pools had a positive effect on myocardium. In parallel we decided to exploit also another strategy, transfecting a more permissive cardiac cell line with each interesting factor individually and inducing a toxic damage with a chemical drug. The viability and apoptotic rate of the cells treated with different factors was measured with high-throughput assays. Two types of cells were used for the selection procedures: rat primary cardiomyocytes obtained from neonatal rats and HL-1, a murine cell line from mouse atrial cardiac myocytes that maintains the differentiated phenotype in culture, mainly used for the high-throughput screening. The mice employed for the in vivo part of this experiment belonged to the C57 strain. To produce the toxic damage we used doxorubicin in the cells, a pro-apoptotic anthacycline drug, while isoproterenol in vivo, a synthetic catecholamine that stimulates both beta1 and beta adrenergic receptors, producing a widely accepted ischemic-like myocardial damage. The proper dose to be used in each cell type has been determined prior to the selection experiments. 1) The selection strategy was preliminarily validated in vitro in HEK 293T cells with a pool of AAV virus coding for 5 random proteins and a reporter gene, the green fluorescent protein (GFP). Cells were infected with the preparation of AAV2 virus and subsequently the ones with fluorescent phenotype were divided from the non-fluorescent cells with a fluorescence-activated cell sorter. The DNA of the nuclei of cells from the different populations was amplified by PCR and the analyses showed an enrichment in the GFP cDNA in the positive fractions. 2) Neonatay rat cardiomyocytes cell coltures were transduced with AAV serotype 9. Each viral vector preparation contained 10 different genes or 28/12 microRNAs. The day after transduction the cells were treated with drugs for 24 hours and then stained with a fluorescent Annexin-V, that marks cells in apoptosis. With a fluorescence-activated cells sorter, the apoptotic cells are divided from the living ones. Only the DNA from the nuclei was extracted from each fraction of cells and analyzed through PCR to measure any appreciable difference in plasmid composition. 3) HL-1 cells were transfected with the most promising genes identified in step 2. The transfection was performed one plasmid at a time in a 96-well plate. The cells were subsequently treated with doxorubicin at the appropriate dose for 24 hours and analyzed in a plate reader with a luminescence-based assay that measures the viability of the cells in terms of ATP production. In this way, the protective potential of genes encoding for proteins or microRNA of unknown function can be analyzed in a short time. 4) Neonatal mice were intraperitoneally injected with the AAV9 preparations. After three weeks, the animals were treated with isoproterenol in order to induce an ischemic-like damage. Ten days later, the hearts of the mice were retrieved and DNA extracted. A PCR was used for amplifying the transduced plasmids in order to identify if one of them was preferentially retained because of its protective action on the myocardial muscle. A single cycle of infection-damage-extraction of DNA from cardiomyocytes or hearts in most cases was not sufficient to identify a clear enrichment of one or more protective factors. In future developments of this project cDNA and microRNAs retrieved will be recloned in AAV plasmids to produce a new set of vectors carrying different proportions of the original genes. This new preparation will be used for further rounds of in vitro selection until the identification of a clear enrichment of one or more protective factors. The final goal of this project is the in vivo and in vitro molecular characterization of the newly identified anti-apoptotic or pro-survival factors

Lithium treatment extends human lifespan: findings from the UK Biobank

Lithium is a nutritional trace element that is also used pharmacologically for the management of bipolar and related psychiatric disorders. Recent studies have shown that lithium supplementation can extend health and lifespan in different animal models. Moreover, nutritional lithium uptake from drinking water was repeatedly found to be positively correlated with human longevity. By analyzing a large observational aging cohort (UK Biobank, n = 501,461 individuals) along with prescription data derived from the National Health Services (NHS), we here find therapeutic supplementation of lithium linked to decreased mortality (p = 0.0017) of individuals diagnosed with affective disorders. Subsequent multivariate survival analyses reveal lithium to be the strongest factor in regards to increased survival effects (hazard ratio = 0.274 [0.119-0.634 CI 95%, p = 0.0023]), corresponding to 3.641 times lower (95% CI 1.577-8.407) chances of dying at a given age for lithium users compared to users of other anti-psychotic drugs. While these results may further support the use of lithium as a geroprotective supplement, it should be noted that doses applied within the UK Biobank/NHS setting require close supervision by qualified medical professionals.ISSN:1945-458

The Diabetes Gene JAZF1 Is Essential for the Homeostatic Control of Ribosome Biogenesis and Function in Metabolic Stress

The ability of pancreatic β-cells to respond to increased demands for insulin during metabolic stress critically depends on proper ribosome homeostasis and function. Excessive and long-lasting stimulation of insulin secretion can elicit endoplasmic reticulum (ER) stress, unfolded protein response, and β-cell apoptosis. Here we show that the diabetes susceptibility gene JAZF1 is a key transcriptional regulator of ribosome biogenesis, global protein, and insulin translation. JAZF1 is excluded from the nucleus, and its expression levels are reduced upon metabolic stress and in diabetes. Genetic deletion of Jazf1 results in global impairment of protein synthesis that is mediated by defects in ribosomal protein synthesis, ribosomal RNA processing, and aminoacyl-synthetase expression, thereby inducing ER stress and increasing β-cell susceptibility to apoptosis. Importantly, JAZF1 function and its pleiotropic actions are impaired in islets of murine T2D and in human islets exposed to metabolic stress. Our study identifies JAZF1 as a central mediator of metabolic stress in β-cells.ISSN:2666-3864ISSN:2211-124

Macrophage deficiency of miR‐21 promotes apoptosis, plaque necrosis, and vascular inflammation during atherogenesis

Abstract Atherosclerosis, the major cause of cardiovascular disease, is a chronic inflammatory disease characterized by the accumulation of lipids and inflammatory cells in the artery wall. Aberrant expression of microRNAs has been implicated in the pathophysiological processes underlying the progression of atherosclerosis. Here, we define the contribution of miR‐21 in hematopoietic cells during atherogenesis. Interestingly, we found that miR‐21 is the most abundant miRNA in macrophages and its absence results in accelerated atherosclerosis, plaque necrosis, and vascular inflammation. miR‐21 expression influences foam cell formation, sensitivity to ER‐stress‐induced apoptosis, and phagocytic clearance capacity. Mechanistically, we discovered that the absence of miR‐21 in macrophages increases the expression of the miR‐21 target gene, MKK3, promoting the induction of p38‐CHOP and JNK signaling. Both pathways enhance macrophage apoptosis and promote the post‐translational degradation of ABCG1, a transporter that regulates cholesterol efflux in macrophages. Altogether, these findings reveal a major role for hematopoietic miR‐21 in atherogenesis