Methylglyoxal accumulation de-regulates HoxA5 expression,thereby impairing angiogenesis in glyoxalase 1 knock-down mouse aortic endothelial cells

Impaired angiogenesis leads to long-term complications and is a major contributor of the high morbidity in patients with Diabetes Mellitus (DM). Methylglyoxal (MGO) is a glycolysis byproduct that accumulates in DM and is detoxified by the Glyoxalase 1 (Glo1). Several studies suggest that MGO contributes to vascular complications through mechanisms that remain to be elucidated. In this study we have clarified for the first time the molecular mechanism involved in the impairment of angiogenesis induced by MGO accumulation.Angiogenesis was evaluated in mouse aortic endothelial cells isolated from Glo1-knockdown mice (Glo1KD MAECs) and their wild-type littermates (WT MAECs). Reduction in Glo1 expression led to an accumulation of MGO and MGO-modified proteins and impaired angiogenesis of Glo1KD MAECs. Both mRNA and protein levels of the anti-angiogenic HoxA5 gene were increased in Glo1KD MAECs and its silencing improved both their migration and invasion. Nuclear NF-?B-p65 was increased 2.5-fold in the Glo1KD as compared to WT MAECs. Interestingly, NF-?B-p65 binding to HoxA5 promoter was also 2-fold higher in Glo1KD MAECs and positively regulated HoxA5 expression in MAECs. Consistent with these data, both the exposure to a chemical inhibitor of Glo1 “SpBrBzGSHCp2” (GI) and to exogenous MGO led to the impairment of migration and the increase of HoxA5 mRNA and NF-?B-p65 protein levels in microvascular mouse coronary endothelial cells (MCECs).This study demonstrates, for the first time, that MGO accumulation increases the antiangiogenic factor HoxA5 via NF-?B-p65, thereby impairing the angiogenic ability of endothelial cells.     

Type I interferons and MAVS signaling are necessary for tissue resident memory CD8+ T cell responses to RSV infection

Respiratory syncytial virus (RSV) can cause bronchiolitis and viral pneumonia in young children and the elderly. Lack of vaccines and recurrence of RSV infection indicate the difficulty in eliciting protective memory immune responses. Tissue resident memory T cells (T_RM) can confer protection from pathogen re-infection and, in human experimental RSV infection, the presence of lung CD8⁺ T_RM cells correlates with a better outcome. However, the requirements for generating and maintaining lung T_RM cells during RSV infection are not fully understood. Here, we use mouse models to assess the impact of innate immune response determinants in the generation and subsequent expansion of the T_RM cell pool during RSV infection. We show that CD8⁺ T_RM cells expand independently from systemic CD8⁺ T cells after RSV re-infection. Re-infected MAVS and MyD88/TRIF deficient mice, lacking key components involved in innate immune recognition of RSV and induction of type I interferons (IFN-α/β), display impaired expansion of CD8⁺ T_RM cells and reduction in antigen specific production of granzyme B and IFN-γ. IFN-α treatment of MAVS deficient mice during primary RSV infection restored T_RM cell expansion upon re-challenge but failed to recover T_RM cell functionality. Our data reveal how innate immunity, including the axis controlling type I IFN induction, instructs and regulates CD8⁺ T_RM cell responses to RSV infection, suggesting possible mechanisms for therapeutic intervention.     

SIRT6 stabilization and cytoplasmic localization in macrophages regulates acute and chronic inflammation in mice

Acute and chronic inflammations are key homeostatic events in health and disease. Sirtuins (SIRTs), a family of NAD-dependent protein deacylases, play a pivotal role in the regulation of these inflammatory responses. Indeed, SIRTs have anti-inflammatory effects through a myriad of signaling cascades, including histone deacetylation and gene silencing, p65/RelA deacetylation and inactivation, and nucleotide‑binding oligomerization domain, leucine rich repeat, and pyrin domain‑containing protein 3 inflammasome inhibition. Nevertheless, recent findings show that SIRTs, specifically SIRT6, are also necessary for mounting an active inflammatory response in macrophages. SIRT6 has been shown to positively regulate tumor necrosis factor alpha (TNFα) secretion by demyristoylating pro-TNFα in the cytoplasm. However, how SIRT6, a nuclear chromatin-binding protein, fulfills this function in the cytoplasm is currently unknown. Herein, we show by Western blot and immunofluorescence that in macrophages and fibroblasts there is a subpopulation of SIRT6 that is highly unstable and quickly degraded via the proteasome. Upon lipopolysaccharide stimulation in Raw 264.7, bone marrow, and peritoneal macrophages, this population of SIRT6 is rapidly stabilized and localizes in the cytoplasm, specifically in the vicinity of the endoplasmic reticulum, promoting TNFα secretion. Furthermore, we also found that acute SIRT6 inhibition dampens TNFα secretion both in vitro and in vivo, decreasing lipopolysaccharide-induced septic shock. Finally, we tested SIRT6 relevance in systemic inflammation using an obesity-induced chronic inflammatory in vivo model, where TNFα plays a key role, and we show that short-term genetic deletion of SIRT6 in macrophages of obese mice ameliorated systemic inflammation and hyperglycemia, suggesting that SIRT6 plays an active role in inflammation-mediated glucose intolerance during obesity.     

GCAT|Panel,a comprehensive structural variant haplotype map of the Iberian population from high-coverage whole-genome sequencing

The combined analysis of haplotype panels with phenotype clinical cohorts is a common approach to explore the genetic architecture of human diseases. However, genetic studies are mainly based on single nucleotide variants (SNVs) and small insertions and deletions (indels). Here, we contribute to fill this gap by generating a dense haplotype map focused on the identification, characterization, and phasing of structural variants (SVs). By integrating multiple variant identification methods and Logistic Regression Models (LRMs), we present a catalogue of 35 431 441 variants, including 89 178 SVs (≥50 bp), 30 325 064 SNVs and 5 017 199 indels, across 785 Illumina high coverage (30x) whole-genomes from the Iberian GCAT Cohort, containing a median of 3.52M SNVs, 606 336 indels and 6393 SVs per individual. The haplotype panel is able to impute up to 14 360 728 SNVs/indels and 23 179 SVs, showing a 2.7-fold increase for SVs compared with available genetic variation panels. The value of this panel for SVs analysis is shown through an imputed rare Alu element located in a new locus associated with Mononeuritis of lower limb, a rare neuromuscular disease. This study represents the first deep characterization of genetic variation within the Iberian population and the first operational haplotype panel to systematically include the SVs into genome-wide genetic studies.     

Hyperkalemic periodic paralysis M1592V mutation modifies activation in human skeletal muscle Na+ channel

Mutations in thehuman skeletal muscle Na⁺ channelunderlie the autosomal dominant disease hyperkalemic periodic paralysis (HPP). Muscle fibers from affected individuals exhibit sustained Na⁺ currents thought to depolarizethe sarcolemma and thus inactivate normalNa⁺ channels. We expressed humanwild-type or M₁₅₉₂V mutant-subunits with the ₁-subunitin Xenopus laevis oocytes and recordedNa⁺ currents using two-electrodeand cut-open oocyte voltage-clamp techniques. The most prominentfunctional difference betweenM₁₅₉₂V mutant and wild-typechannels is a 5- to 10-mV shift in the hyperpolarized direction of thesteady-state activation curve. The shift in the activation curve forthe mutant results in a larger overlap with the inactivation curve thanthat observed for wild-type channels. Accordingly, the current throughM₁₅₉₂V channels displays a larger noninactivating component than does that through wild-type channels atmembrane potentials near 40 mV. The functional properties of theM₁₅₉₂V mutant resemble those ofthe previously characterized HPPT₇₀₄M mutant. Both clinicallysimilar phenotypes arise from mutations located at a distance from theputative voltage sensor of the channel.

     

Nitric oxide synthase activity in brain tissues from llama fetuses submitted to hypoxemia.

The fetal llama (Lama glama; a species adapted to live in chronic hypoxia in the highlands of the Andes) did not increase cerebral blood flow and reduce the brain oxygen uptake during hypoxemia. Although nitric oxide (NO) is a normal mediator in the regulation of vascular tone and synaptic transmission, NO overproduction by hypoxemia could produce neuronal damage. We hypothesized that nitric oxide synthase (NOS) activity is either maintained or reduced in the central nervous system of the llama fetuses submitted to chronic hypoxemia. Approximately 85% of the Ca(2+)-dependent NOS activity was soluble, at least 12% was associated with the mitochondrial fraction, and less than 5% remains associated with microsomes. To understand the role of NO in chronic hypoxemia, we determined the effect of 24-h hypoxemia on NOS activity in the central nervous system. No changes in activity or the subcellular distribution of NOS activity in brain tissues after hypoxemia were found. We proposed that the lack of changes in NOS activity in the llama under hypoxemia could be a cytoprotective mechanism inherent to the llama, against possible toxic effects of NO.     

Selective decrease in paracellular conductance of tight junctions: role of the first extracellular domain of claudin-5

Claudin-5 is a protein component of many endothelial tight junctions, including those at the blood-brain barrier, a barrier that limits molecular exchanges between the central nervous system and the circulatory system. To test the contribution of claudin-5 to this barrier function of tight junctions, we expressed murine claudin-5 in Madin-Darby canine kidney II cells. The result was a fivefold increase in transepithelial resistance in claudin-5 transductants and a reduction in conductance of monovalent cations. However, the paracellular flux of neither neutral nor charged monosaccharides was significantly changed in claudin-5 transductants compared to controls. Therefore, expression of claudin-5 selectively decreased the permeability to ions. Additionally, site-directed mutations of particular amino acid residues in the first extracellular domain of claudin-5 altered the properties of the tight junctions formed in response to claudin-5 expression. In particular, the conserved cysteines were crucial: mutation of either cysteine abolishted the ability of claudin-5 to increase transepithelial resistance, and mutation of Cys(64) strikingly increased the paracellular flux of monosaccharides. These new insights into the functions of claudin-5 at the molecular level in tight junctions may account for some aspects of the blood-brain barrier's selective permeability.     

Angiopoietin decoy secreted at tumor site impairs tumor growth and metastases by inducing local inflammation and altering neoangiogenesis

The extracellular domain of the receptor tyrosine kinase Tie2/TEK (exTEK) has been used as an angiopoietin decoy to study the role of angiopoietins in the tumor–host interactions, using a syngeneic model of experimental metastases and subcutaneous tumor. Soluble exTEK secreted by transfected tumor cells inhibited HUVECs from forming tubes in Matrigel. ExTEK-transfected C26 colon carcinoma and TS/A mammary tumor cells displayed reduced growth rate when injected subcutaneously, and reduced ability to form experimental metastases when injected intravenously. Immunohistochemical analysis of tumors and metastases showed increased leukocytes infiltration and signs of inflammation in exTEK-secreting compared to parental tumor, as well as impairment in neo-vessel growth and organization. However, while neoangiogenesis eventually rescued in the subcutis, it failed to organize in the experimental metastases of exTEK-secreting tumor, contributing to the hampering of metastatic growth and to increased mice survival. The reactive infiltrate of C26TEK contained a different percentage of leukocytes and was responsible for the tumor inhibition. In fact, leukopenia induced by -irradiation of recipient mice or injection into interferon gamma (IFN-) gene knockout (GKO) mice resulted in reduced mouse survival and an increased number of lung metastases. On the other hand, interleukin (IL)-12 treatment prolonged the survival of mice bearing subcutaneous C26TEK but not of those bearing lung metastases, suggesting that IL-12 could exert further antiangiogenic effects at the site where the tumor can restore neoangiogenesis. These results show in vivo that reduced angiopoietin availability at the tumor site induces a local inflammatory response and impairment of neoangiogenesis which act synergistically to limit tumor growth and metastasis.Abbreviations AEC amino-ethylcarbazole - ELISA enzyme-linked immunosorbent assay - HRP horseradish peroxidase - HUVEC human umbilical vascular endothelial cell - i.v. intravenous - s.c. subcutaneous - TBS Tris-HCl buffered solution     

Differences in nocturnal basal and rhythmic prolactin secretion in untreated compared to treated HIV-infected men are associated with CD4+ T-lymphocytes

The existence of decreased hypothalamic dopaminergic tone in HIV-infected men has been suggested. In a cross-sectional study, we determined 12 h nocturnal basal and pulsatile prolactin (PRL) release levels (by blood sampling every 10 min) and their correlation with CD4+ T cells in seven volunteer HIV-negative, healthy men (group 1), and 21 normoprolactinemic, euthyroid, HIV-infected men divided into 3 groups (each group = 7): (i) group 2, asymptomatic HIV-infected stage A1 men, untreated; (ii) group 3, AIDS stage C3 without active opportunistic infections, untreated; and (iii) group 4, previously stage C3 after at least 6 months of successful highly active antiretroviral therapy. Serum PRL was measured by radioimmunoanalysis and the results were analysed by waveform-independent deconvolution analysis. CD4+ T lymphocytes were measured by flow cytometry and viral load by a nucleic acid sequence-based amplification assay. No differences were detected in the first two groups. In the third group, however, 100% of prolactin secretion was found to be pulsatile with a shorter secretory burst duration (P = 0.04), and a greater circulating half-life and pulse amplitude (P < or = 0.04). Group 4 had the greatest basal prolactin secretion (P < or = 0.04), and a shorter secretory burst duration (P = 0.04 vs group 2), circulating half-life (P = 0.01 vs group 3) and intersecretory burst interval (P = 0.06 vs group 1). PRL approximate entropy was similar among all groups. Linear correlations existed between CD4+ T cell counts and PRL secretory burst half duration (r = 0.62, P = 0.002) and amplitude (r = -0.63, P = 0.001), and in circulating serum half-life (r = - 0.61, P = 0.002) in HIV-infected groups. Viral load showed no correlations. It is suggested that differential changes in nocturnal prolactin secretion among HIV-infected men occurred while maintaining the normal coordinate feedback and/or feedforward control within the lactotropic axis. These changes may represent an adaptative mechanism to sustain, by different means, the maximal physiologic PRL production to stimulate the highest cellular immune response and/or reconstitution in attempting to survive.