Nestinþ cells direct inflammatory cell migration in atherosclerosis

Atherosclerosis is a leading death cause. Endothelial and smooth muscle cells participate in atherogenesis, but it is unclear whether other mesenchymal cells contribute to this process. Bone marrow (BM) nestinþ cells cooperate with endothelial cells in directing monocyte egress to bloodstream in response to infections. However, it remains unknown whether nestinþ cells regulate inflammatory cells in chronic inflammatory diseases, such as atherosclerosis. Here, we show that nestinþ cells direct inflammatory cell migration during chronic inflammation. In Apolipoprotein E (ApoE) knockout mice fed with high-fat diet, BM nestinþ cells regulate the egress of inflammatory monocytes and neutrophils. In the aorta, nestinþ stromal cells increase B30 times and contribute to the atheroma plaque. Mcp1 deletion in nestinþ cells—but not in endothelial cells only— increases circulating inflammatory cells, but decreases their aortic infiltration, delaying atheroma plaque formation and aortic valve calcification. Therefore, nestin expression marks cells that regulate inflammatory cell migration during atherosclerosis.Pro-CNIC FoundationSevero Ochoa Center of Excellence award SEV-2015-0505 to CNICWellcome Trust and MRC to the Cambridge Stem Cell InstituteMinisterio de Economía y Competitividad (RETIC Grant RD12/0042/0028 to V.A.; SAF2012-40127 to J.M-G.; Plan Nacional Grant SAF-2011-30308, Ramón y Cajal Program Grant RYC-2009-04703 and Spanish Cell Therapy Network TerCel to S.M-F.)Marie Curie Career Integration Program Grant (FP7-PEOPLE-2011-294096)ConSEPOC-Comunidad de Madrid Grant (S2010/BMD-2542)National Health Institute Blood and Transplant (United Kingdom)Horizon2020 (ERC-2014-CoG-64765)Horizon2020 (ERC-2014-CoG-64765

Regulation of oxygen sensing by ion channels

O2 sensing is of critical importance for cell survival and adaptation of living organisms to changing environments or physiological conditions. O2-sensitive ion channels are major effectors of the cellular responses to hypoxia. These channels are preferentially found in excitable neurosecretory cells (glomus cells of the carotid body, cells in the neuroepithelial bodies of the lung, and neonatal adrenal chromaffin cells), which mediate fast cardiorespiratory adjustments to hypoxia. O2- sensitive channels are also expressed in the pulmonary and systemic arterial smooth muscle cells where they participate in the vasomotor responses to low O2 tension (particularly in hypoxic pulmonary vasoconstriction). The mechanisms underlying O2 sensing and how the O2 sensors interact with the ion channels remain unknown. Recent advances in the field give different support to the various current hypotheses. Besides the participation of ion channels in acute O2 sensing, they also contribute to the gene program developed under chronic hypoxia. Gene expression of T-type calcium channels is upregulated by hypoxia through the same hypoxiainducible factor-dependent signaling pathway utilized by the classical O2-regulated genes. Alteration of acute or chronic O2 sensing by ion channels could participate in the pathophysiology of human diseases, such as sudden infant death syndrome or primary pulmonary hypertension

Circulating miR-320a as a Predictive Biomarker for Left Ventricular Remodelling in STEMI Patients Undergoing Primary Percutaneous Coronary Intervention

Restoration of epicardial coronary blood flow, achieved by early reperfusion with primary percutaneous coronary intervention (PPCI), is the guideline recommended to treat patients with ST-segment-elevation myocardial infarction (STEMI). However, despite successful blood restoration, increasing numbers of patients develop left ventricular adverse remodelling (LVAR) and heart failure. Therefore, reliable prognostic biomarkers for LVAR in STEMI are urgently needed. Our aim was to investigate the role of circulating microRNAs (miRNAs) and their association with LVAR in STEMI patients following the PPCI procedure. We analysed the expression of circulating miRNAs in blood samples of 56 patients collected at admission and after revascularization (at 3, 6, 12 and 24 h). The associations between miRNAs and left ventricular end diastolic volumes at 6 months were estimated to detect LVAR. miRNAs were also analysed in samples isolated from peripheral blood mononuclear cells (PBMCs) and human myocardium of failing hearts. Kinetic analysis of miRNAs showed a fast time-dependent increase in miR-133a, miR-133b, miR-193b, miR-499, and miR-320a in STEMI patients compared to controls. Moreover, the expression of miR-29a, miR-29b, miR-324, miR-208, miR-423, miR-522, and miR-545 was differentially expressed even before PPCI in STEMI. Furthermore, the increase in circulating miR-320a and the decrease in its expression in PBMCs were significantly associated with LVAR and correlated with the expression of miR-320a in human failing myocardium from ischaemic origin. In conclusion, we determined the time course expression of new circulating miRNAs in patients with STEMI treated with PPCI and we showed that miR-320a was positively associated with LVAR

Role of VHL, HIF1A and SDH on the expression of miR-210: Implications for tumoral pseudo-hypoxic fate

The hypoxia-inducible factor 1a (HIF-1a) and its microRNA target, miR-210, are candidate tumor-drivers of metabolic reprogramming in cancer. Neuroendocrine neoplasms such as paragangliomas (PGLs) are particularly appealing for understanding the cancer metabolic adjustments because of their associations with deregulations of metabolic enzymes, such as succinate dehydrogenase (SDH), and the von Hippel Lindau (VHL) gene involved in HIF-1α stabilization. However, the role of miR-210 in the pathogenesis of SDH-related tumors remains an unmet challenge. Herein is described an in vivo genetic analysis of the role of VHL, HIF1A and SDH on miR-210 by using knockout murine models, siRNA gene silencing, and analyses of human tumors. HIF-1a knockout abolished hypoxia-induced miR-210 expression in vivo but did not alter its constitutive expression in paraganglia. Normoxic miR-210 levels substantially increased by complete, but not partial, VHL silencing in paraganglia of knockout VHL-mice and by over-expression of p76del-mutated pVHL. Similarly, VHLmutated PGLs, not those with decreased VHL-gene/mRNA dosage, over-expressed miR-210 and accumulate HIF-1a in most tumor cells. Ablation of SDH activity in SDHD-null cell lines or reduction of the SDHD or SDHB protein levels elicited by siRNA-induced gene silencing did not induce miR-210 whereas the presence of SDH mutations in PGLs and tumor-derived cell lines was associated with mild increase of miR-210 and the presence of a heterogeneous, HIF-1a-positive and HIF-1a-negative, tumor cell population. Thus, activation of HIF-1a is likely an early event in VHLdefective PGLs directly linked to VHL mutations, but it is a late event favored but not directly triggered by SDHx mutations. This combined analysis provides insights into the mechanisms of HIF-1a/miR-210 regulation in normal and tumor tissues potentially useful for understanding the pathogenesis of cancer and other diseases sharing similar underpinnings.Instituto de Salud Carlos III FIS PI11/929Red Temática de Investigación Cooperativa en Cáncer RD12/0036/0015 Instituto de Salud Carlos III (ISCIII)Ministerio de Economía y Competitividad y Fondo Europeo de Desarrollo RegionalFondo Europeo de Desarrollo Regional (FEDER) (CIBERONC

TRP Channels: Current Perspectives in the Adverse Cardiac Remodeling

Calcium is an important second messenger required not only for the excitation-contraction coupling of the heart but also critical for the activation of cell signaling pathways involved in the adverse cardiac remodeling and consequently for the heart failure. Sustained neurohumoral activation, pressure-overload, or myocardial injury can cause pathologic hypertrophic growth of the heart followed by interstitial fibrosis. The consequent heart’s structural and molecular adaptation might elevate the risk of developing heart failure and malignant arrhythmia. Compelling evidences have demonstrated that Ca2+ entry through TRP channels might play pivotal roles in cardiac function and pathology. TRP proteins are classified into six subfamilies: TRPC (canonical), TRPV (vanilloid), TRPM (melastatin), TRPA (ankyrin), TRPML (mucolipin), and TRPP (polycystin), which are activated by numerous physical and/or chemical stimuli. TRP channels participate to the handling of the intracellular Ca2+ concentration in cardiac myocytes and are mediators of different cardiovascular alterations. This review provides an overview of the current knowledge of TRP proteins implication in the pathologic process of some frequent cardiac diseases associated with the adverse cardiac remodeling such as cardiac hypertrophy, fibrosis, and conduction alteration.Spanish Ministry of Economy and Competitiveness BFU2016–74932-C2Institute of Carlos III PI15/00203; PI16/00259; CB16/11/00431Andalusia Government PI-0313-201

Regulación de la expresión génica de canales iónicos por hipoxia crónica en células cromafines

El objetivo general de este proyecto fue el estudio de la regulación por hipoxia de la expresión de genes que codifican canales de calcio tipo T y canales maxi-K en células cromafines. En el caso de los canales de calcio tipo T, los objetivos específicos fueron: 1. Caracterización molecular y funcional de los canales de calcio tipo T expresados en células PC12 mediante el empleo de técnicas de biología molecular y electrofisiología. 2. Estudio de la regulación por hipoxia de la expresión de estos canales en células PC12 y caracterización molecular y funcional de la respuesta celular a la disminución de oxígeno. 3. Análisis de la ruta de transducción de señales implicada en la regulación génica por hipoxia de los canales de calcio tipo T. 4. Determinación del papel del calcio extracelular en la regulación de la expresión de los canales de calcio tipo T por hipoxia. En el caso de los canales de potasio dependientes de voltaje y calcio maxi-K los objetivos específicos fueron: 1. Caracterización molecular de los canales maxi-K en células cromafines y PC12. 2. Estudio del efecto de la hipoxia sobre la regulación de la expresión de los genes que codifican canales maxi-K, tanto la subunidad principal α como las subunidades auxiliares β. 3. Caracterización funcional con técnicas de electrofisiología del cambio de excitabilidad asociado a la regulación de la expresión génica por hipoxia de los canales maxi-K. 4. Estudio de la ruta de transducción de señales responsable de la regulación de la expresión de estos canales en hipoxia crónica. Las principales conclusiones alcanzadas en este estudio son las que se exponen a continuación: 1. La línea celular PC12 expresa canales de calcio tipo T funcionalmente activos, siendo la subunidad α1H la que más se expresa en estas células. 2. Cuando estas células se exponen a bajas concentraciones de oxígeno se produce una sobreexpresión de la subunidad α1H de los canales de calcio tipo T. Este incremento se detecta con técnicas de biología molecular y se traduce en un mayor número de canales funcionalmente activos. 3.Los genes que codifican canales de calcio dependientes de voltaje del tipo HVA, (CaL, CaN, y CaP/Q) no se regulan por exposición a hipoxia crónica en células PC12. 4. La expresión de la subunidad α1H en hipoxia crónica se regula por el factor de transcripción HIF-2α y no depende de calcio extracelular. 5. La expresión de la subunidad α1H se regula por despolarización de la membrana celular. La exposición de las células a alto potasio extracelular da lugar a un descenso del nivel de mRNA de la subunidad α1H. La regulación por alto potasio, a diferencia de la regulación por hipoxia, depende de calcio extracelular. 6. La subunidad β2 de los canales maxi-K es la subunidad auxiliar que más se expresa en células cromafines y PC12. 7. La expresión de la subunidad β2 se regula en células cromafines de la médula adrenal de animales mantenidos en hipoxia crónica. Este efecto también se observa en células PC12 expuestas a hipoxia. 8. La regulación de la expresión de la subunidad β2 en el animal completo depende del tiempo de exposición a hipoxia. En células PC12, también depende del tiempo así como de la dosis de oxígeno empleada. 9. El efecto represor de la hipoxia sobre la expresión de la subunidad β2 no se reproduce en cultivos primarios de células cromafines de rata. 10. En células PC12, la familia de factores de transcripción HIF no parece ser responsable de la regulación por hipoxia de la subunidad β2. Sin embargo, la regulación se ejerce a nivel transcripcional y depende de calcio y de la síntesis de novo de proteínas celulares. 11. La exposición de células cromafines a alto potasio extracelular produce una disminución de la subunidad β2 del mismo rango que el que se produce cuando las células se exponen a hipoxia. Este efecto también depende de calcio extracelular. 12. La regulación de la subunidad β2 por hipoxia crónica se bloquea por Cheletythrine cloride y Calmidazolium, inhibidores de la PKC y la calmodulina, respectivamente

TRPC and TRPV Channels’ Role in Vascular Remodeling and Disease

Transient receptor potentials (TRPs) are non-selective cation channels that are widely expressed in vascular beds. They contribute to the Ca2+ influx evoked by a wide spectrum of chemical and physical stimuli, both in endothelial and vascular smooth muscle cells. Within the superfamily of TRP channels, different isoforms of TRPC (canonical) and TRPV (vanilloid) have emerged as important regulators of vascular tone and blood flow pressure. Additionally, several lines of evidence derived from animal models, and even from human subjects, highlighted the role of TRPC and TRPV in vascular remodeling and disease. Dysregulation in the function and/or expression of TRPC and TRPV isoforms likely regulates vascular smooth muscle cells switching from a contractile to a synthetic phenotype. This process contributes to the development and progression of vascular disorders, such as systemic and pulmonary arterial hypertension, atherosclerosis and restenosis. In this review, we provide an overview of the current knowledge on the implication of TRPC and TRPV in the physiological and pathological processes of some frequent vascular diseases

Potential tamoxifen repurposing to combat infections by multidrug-resistant Gram-negative bacilli

The development of new strategic therapies for multidrug-resistant bacteria, like the use of non-antimicrobial approaches and/or drugs repurposed to be used as monotherapies or in combination with clinically relevant antibiotics, has become urgent. A therapeutic alternative for infections by multidrug-resistant Gram-negative bacilli (MDR-GNB) is immune system modulation to improve the infection clearance. We showed that immunocompetent mice pretreated with tamoxifen at 80 mg/kg/d for three days and infected with Acinetobacter baumannii, Pseudomonas aeruginosa, or Escherichia coli in peritoneal sepsis models showed reduced release of the monocyte chemotactic protein-1 (MCP-1) and its signaling pathway interleukin-18 (IL-18), and phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2). This reduction of MCP-1 induced the reduction of migration of inflammatory monocytes and neutrophils from the bone marrow to the blood. Indeed, pretreatment with tamoxifen in murine peritoneal sepsis models reduced the bacterial load in tissues and blood, and increased mice survival from 0% to 60–100%. Together, these data show that tamoxifen presents therapeutic efficacy against MDR A. baumannii, P. aeruginosa, and E. coli in experimental models of infection and may be a new candidate to be repurposed as a treatment for GNB infections.Instituto de Salud Carlos III , CP15/00132; PI16/01378; PI19/01453; RD16/0016/000

A cholinergic neuroskeletal interface promotes bone formation during postnatal growth and exercise.

The autonomic nervous system is a master regulator of homeostatic processes and stress responses. Sym pathetic noradrenergic nerve fibers decrease bone mass, but the role of cholinergic signaling in bone has remained largely unknown. Here, we describe that early postnatally, a subset of sympathetic nerve fibers un dergoes an interleukin-6 (IL-6)-induced cholinergic switch upon contacting the bone. A neurotrophic depen dency mediated through GDNF-family receptor-a2 (GFRa2) and its ligand, neurturin (NRTN), is established between sympathetic cholinergic fibers and bone-embedded osteocytes, which require cholinergic innerva tion for their survival and connectivity. Bone-lining osteoprogenitors amplify and propagate cholinergic signals in the bone marrow (BM). Moderate exercise augments trabecular bone partly through an IL-6-depen dent expansion of sympathetic cholinergic nerve fibers. Consequently, loss of cholinergic skeletal innerva tion reduces osteocyte survival and function, causing osteopenia and impaired skeletal adaptation to mod erate exercise. These results uncover a cholinergic neuro-osteocyte interface that regulates skeletogenesis and skeletal turnover through bone-anabolic effects