K+ channels expression in hypertension after arterial injury, and effect of selective Kv1.3 blockade with PAP-1 on intimal hyperplasia formation

Producción CientíficaK+ channels are central to vascular pathophysiology. Previous results demonstrated that phenotypic modulation associates with a change in Kv1.3 to Kv1.5 expression, and that Kv1.3 blockade inhibits proliferation of VSMCs cultures. Purpose: To explore whether the Kv1.3 to Kv1.5 switch could be a marker of the increased risk of intimal hyperplasia in essential hypertension and whether systemic treatment with Kv1.3 blockers can prevent intimal hyperplasia after endoluminal lesion . Methods: Morphometric and immunohistochemical analysis were performed in arterial segments following arterial injury and constant infusion of the Kv1.3 blocker PAP-1 during 28 days. Differential expression of K+ channel genes was studied in VSMC from hypertensive (BPH) and normotensive (BPN) mice, both in control and after endoluminal lesion. Finally, the migration and proliferation rate of BPN and BPH VSMCs was explored in vitro. Results: Changes in mRNA expression led to an increased Kv1.3/Kv1.5 ratio in BPH VSMC. Consistent with this, arterial injury in BPH mice induced a higher degree of luminal stenosis, (84±4 % vs. 70±5 % in BPN, p<0.01), although no differences in migration and proliferation rate were observed in cultured VSMCs. The in vivo proliferative lesions were significantly decreased upon PAP-1 systemic infusion (18± 6 % vs. 58±20 % with vehicle, p<0.05). Conclusions: Hypertension leads to a higher degree of luminal stenosis in our arterial injury model, that correlates with a decreased expression of Kv1.5 channels. Kv1.3 blockers decreased in vitro VSMCs proliferation, migration, and in vivo intimal hyperplasia formation, pointing to Kv1.3 channels as promising therapeutical targets against restenosis.La versión original del artículo contiene un error. El gráfico de la página 505 es incorrecto. La corrección del mismo se encuentra en el segundo fichero "Erratum to: K+ Channels Expression in Hypertension After Arterial Injury, and Effect of Selective Kv1.3 Blockade with PAP-1 on Intimal Hyperplasia Formation".Ministerio de Economía, Industria y Competitividad (project RD12/0042/0006)Fondo de Investigación en Salud - Instituto Carlos III (project PI11/00225)VALTEC 09-1-0042Ministerio de Ciencia, Innovación y Universidades (grant BFU2010-15898)Junta de Castilla y León (grant VA094A11-2

Ionic immune suppression within the tumour microenvironment limits T cell effector function.

Tumours progress despite being infiltrated by tumour-specific effector T cells. Tumours contain areas of cellular necrosis, which are associated with poor survival in a variety of cancers. Here, we show that necrosis releases intracellular potassium ions into the extracellular fluid of mouse and human tumours, causing profound suppression of T cell effector function. Elevation of the extracellular potassium concentration ([K+]e) impairs T cell receptor (TCR)-driven Akt-mTOR phosphorylation and effector programmes. Potassium-mediated suppression of Akt-mTOR signalling and T cell function is dependent upon the activity of the serine/threonine phosphatase PP2A. Although the suppressive effect mediated by elevated [K+]e is independent of changes in plasma membrane potential (Vm), it requires an increase in intracellular potassium ([K+]i). Accordingly, augmenting potassium efflux in tumour-specific T cells by overexpressing the potassium channel Kv1.3 lowers [K+]i and improves effector functions in vitro and in vivo and enhances tumour clearance and survival in melanoma-bearing mice. These results uncover an ionic checkpoint that blocks T cell function in tumours and identify potential new strategies for cancer immunotherapy

Insulin Promotes Glycogen Storage and Cell Proliferation in Primary Human Astrocytes

In the human brain, there are at least as many astrocytes as neurons. Astrocytes are known to modulate neuronal function in several ways. Thus, they may also contribute to cerebral insulin actions. Therefore, we examined whether primary human astrocytes are insulin-responsive and whether their metabolic functions are affected by the hormone.Commercially available Normal Human Astrocytes were grown in the recommended medium. Major players in the insulin signaling pathway were detected by real-time RT-PCR and Western blotting. Phosphorylation events were detected by phospho-specific antibodies. Glucose uptake and glycogen synthesis were assessed using radio-labeled glucose. Glycogen content was assessed by histochemistry. Lactate levels were measured enzymatically. Cell proliferation was assessed by WST-1 assay.We detected expression of key proteins for insulin signaling, such as insulin receptor β-subunit, insulin receptor substrat-1, Akt/protein kinase B and glycogen synthase kinase 3, in human astrocytes. Akt was phosphorylated and PI-3 kinase activity increased following insulin stimulation in a dose-dependent manner. Neither increased glucose uptake nor lactate secretion after insulin stimulation could be evidenced in this cell type. However, we found increased insulin-dependent glucose incorporation into glycogen. Furthermore, cell numbers increased dose-dependently upon insulin treatment.This study demonstrated that human astrocytes are insulin-responsive at the molecular level. We identified glycogen synthesis and cell proliferation as biological responses of insulin signaling in these brain cells. Hence, this cell type may contribute to the effects of insulin in the human brain

Nitric oxide-mediated mitochondrial impairment in neural cells: a role for glucose metabolism in neuroprotection

Producción CientíficaNitric oxide (žNO) is a highly diffusible, short-lived physiological messenger present in the central nervous system (CNS) (Garthwaite et al., 1988) that is synthesised by a family of nitric oxide synthases (NOSs) which catalyze the conversion of arginine to citrulline and žNO (Bredt and Snyder, 1990; Knowles and Moncada, 1994). All CNS cells synthesise žNO (Murphy et al., 1993). Neurones produce žNO by calcium-dependent activation of neuronal, constitutive NOS (nNOS or NOS1), whereas glial cells synthesise žNO in a calcium-independent way that requires previous transcriptional induction of NOS (inducible NOS, iNOS or NOS2) (Galea et al., 1992; Simmons and Murphy, 1992). Astrocytes also synthesise žNO through NNOS activity (Murphy et al., 1990, 1991; Agullo´ andGarcı´a, 1992a,b). A third isoform of NOS (endothelial NOS, eNOS or NOS3) is associated with brain vasculature. In general, žNO participates in the transduction pathway leading to elevations in intracellular cyclic Ł Corresponding author: Dr. Juan P. Bolan˜os, Departamento de Bioquı´mica y Biologı´a Molecular, Universidad de Salamanca, Edificio Departamental, Plaza Doctores de la Reina s=n, 37007 Salamanca, Spain. Tel.: C34-923-294526; Fax: C34-923-294579; E-mail: [email protected] GMP levels (Bredt and Snyder, 1989; Knowles et al., 1989) and therefore participates in cyclic GMP functions (Wang and Robinson, 1997). However, an increasing body of evidence is now arising to suggest that žNO and its most active metabolite, the peroxynitrite anion (ONOO ), may be involved in the regulation of brain energy metabolism. This chapter will specifically focus on the mechanisms involving žNO and ONOO -mediated interference with brain mitochondrial energy production and the modulating role of glutathione in cell energy metabolism. Finally, we discuss recent evidence that strongly suggests the importance of cell glucose utilisation in maintaining glutathione homeostasis and hence in preventing nitric oxide-mediated mitochondrial impairment.Este trabajo forma parte del proyecto de investigación: C.I.C.Y.T. (SAF98-0127), FEDER (1FD97-1118) and Fundación Ramón Arece

Association of circulating microRNAs with coronary artery disease and usefulness for reclassification of healthy individuals: the REGICOR Study

Risk prediction tools cannot identify most individuals at high coronary artery disease (CAD) risk. Oxidized low-density lipoproteins (oxLDLs) and microRNAs are actively involved in atherosclerosis. Our aim was to examine the association of CAD and oxLDLs-induced microRNAs, and to assess the microRNAs predictive capacity of future CAD events. Human endothelial and vascular smooth muscle cells were treated with oxidized/native low-density lipoproteins, and microRNA expression was analyzed. Differentially expressed and CAD-related miRNAs were examined in serum samples from (1) a case-control study with 476 myocardial infarction (MI) patients and 487 controls, and (2) a case-cohort study with 105 incident CAD cases and 455 randomly-selected cohort participants. MicroRNA expression was analyzed with custom OpenArray plates, log rank tests and Cox regression models. Twenty-one microRNAs, two previously undescribed (hsa-miR-193b-5p and hsa-miR-1229-5p), were up- or down-regulated upon cell treatment with oxLDLs. One of the 21, hsa-miR-122-5p, was also upregulated in MI cases (fold change = 4.85). Of the 28 CAD-related microRNAs tested, 11 were upregulated in MI cases -1 previously undescribed (hsa-miR-16-5p)-, and 1/11 was also associated with CAD incidence (adjusted hazard ratio = 0.55 (0.35-0.88)) and improved CAD risk reclassification, hsa-miR-143-3p. We identified 2 novel microRNAs modulated by oxLDLs in endothelial cells, 1 novel microRNA upregulated in AMI cases compared to controls, and one circulating microRNA that improved CAD risk classification.This work was supported by the Spain’s Ministry of Science and Innovation (Madrid, Spain), co-financed with European Union European Regional Development Funds –ERDF- (FIS-CP12/03287, FIS-14/00449, FIS-PI081327, INTRASALUD PI11/01801, PI15/00064, IJCI-2016-29393 to DdG-C, CIBERCV (CB16/11/00229, 00246, 00403), CIBERESP CB06/02/0029, CIBEROBN CB06/03/0028); the Spain’s ministry of Economy and Competiveness (Madrid, Spain) (BFU2016-75360-R); the BBVA Foundation (Bilbao, Spain) (PR-16-BIO-CAR-0041); the Health Departament of the Generalitat de Catalunya (Barcelona, Spain) through the Agència de Gestió d’Ajuts Universitaris de Recerca de Catalunya (AGAUR) (Barcelona, Spain) (2017SGR222), the Strategic Plan for research and health innovation (PERIS) (Barcelona, Spain) (SLT006/17/00234, SLT002/16/00145, SLT006/17/00029 to IRD); and by the Junta de Castilla y León (Valladolid, Spain) (VA114P17). CIBERs of Pathophysiology of Obesity and Nutrition (CIBEROBN), Cardiovascular Diseases (CIBERCV), and Epidemiology (CIBERESP) are initiatives of the Instituto de Salud Carlos III, Madrid, Spain

Tungstate-targeting of BKαβ1 channels tunes ERK phosphorylation and cell proliferation in human vascular smooth muscle

Despite the substantial knowledge on the antidiabetic, antiobesity and antihypertensive actions of tungstate, information on its primary target/s is scarce. Tungstate activates both the ERK1/2 pathway and the vascular voltage- and Ca2+-dependent large-conductance BKαβ1 potassium channel, which modulates vascular smooth muscle cell (VSMC) proliferation and function, respectively. Here, we have assessed the possible involvement of BKαβ1 channels in the tungstate-induced ERK phosphorylation and its relevance for VSMC proliferation. Western blot analysis in HEK cell lines showed that expression of vascular BKαβ1 channels potentiates the tungstate-induced ERK1/2 phosphorylation in a Gi/o protein-dependent manner. Tungstate activated BKαβ1 channels upstream of G proteins as channel activation was not altered by the inhibition of G proteins with GDPβS or pertussis toxin. Moreover, analysis of Gi/o protein activation measuring the FRET among heterologously expressed Gi protein subunits suggested that tungstate-targeting of BKαβ1 channels promotes G protein activation. Single channel recordings on VSMCs from wild-type and β1-knockout mice indicated that the presence of the regulatory β1 subunit was essential for the tungstate-mediated activation of BK channels in VSMCs. Moreover, the specific BK channel blocker iberiotoxin lowered tungstate-induced ERK phosphorylation by 55% and partially reverted (by 51%) the tungstate-produced reduction of platelet-derived growth factor (PDGF)-induced proliferation in human VSMCs. Our observations indicate that tungstate-targeting of BKαβ1 channels promotes activation of PTX-sensitive Gi proteins to enhance the tungstate-induced phosphorylation of ERK, and inhibits PDGF-stimulated cell proliferation in human vascular smooth muscle.This work was supported by grants from the Spanish Ministry of Economy and Competitiveness (SAF2012-31089 to JMFF, SAF2012-38140 to MAV, BFU2013-45867-R to JRLL), FEDER Funds, Ministry of Science and Innovation (BFU 2008-00769 to JG, BFU2010-15898 to MTPG), Instituto de Salud Carlos III (RIC RD12/0042/0006, RD12/0042/0014, Red HERACLES) and Junta de Castilla y León (VA094A11-2 to JRLL) and European Community (FP7-People-CIG-321721 to RK)