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Producción Científica, Objectives: Restenosis after vessel angioplasty due to dedifferentiation of the vascular smooth muscle cells (VSMCs) limits the success of surgical treatment of vascular occlusions. Type 2 diabetes (T2DM) has a major impact on restenosis, with patients exhibiting more aggressive forms of vascular disease and poorer outcomes after surgery. Kv1.3 channels are critical players in VSMC proliferation. Kv1.3 blockers inhibit VSMCs MEK/ERK signalling and prevent vessel restenosis. We hypothesize that dysregulation of microRNAs (miR) play critical roles in adverse remodelling, contributing to Kv1.3 blockers efficacy in T2DM VSMCs. Methods and results: We used clinically relevant in vivo models of vascular risk factors (VRF) and vessels and VSMCs from T2DM patients. Results: Human T2DM vessels showed increased remodelling, and changes persisted in culture, with augmented VSMCs migration and proliferation. Moreover, there were downregulation of PI3K/AKT/mTOR and upregulation of MEK/ERK pathways, with increased miR-126 expression. The inhibitory effects of Kv1.3 blockers on remodelling were significantly enhanced in T2DM VSMCs and in VRF model. Finally, miR-126 overexpression confered “diabetic” phenotype to non-T2DM VSMCs by downregulating PI3K/AKT axis. Conclusions: miR-126 plays crucial roles in T2DM VSMC metabolic memory through activation of MEK/ERK pathway, enhancing the efficacy of Kv1.3 blockers in the prevention of restenosis in T2DM patients., Ministerio de Economía, Industria y Competitividad (grant BFU2016-75360-R and SAF2016-77871-C2-1-R), Junta de Castilla y León (grants VA114P17; VA172P20 and CLU-2019-02), Novo Nordisk Foundation (grant 34366), Swedish Research Council (grants 2017–00860 and 2020-01145), Swedish Heart and Lung Foundation (grant 20200322)

Producción Científica, Objectives: Restenosis after vessel angioplasty due to dedifferentiation of the vascular smooth muscle cells (VSMCs) limits the success of surgical treatment of vascular occlusions. Type 2 diabetes (T2DM) has a major impact on restenosis, with patients exhibiting more aggressive forms of vascular disease and poorer outcomes after surgery. Kv1.3 channels are critical players in VSMC proliferation. Kv1.3 blockers inhibit VSMCs MEK/ERK signalling and prevent vessel restenosis. We hypothesize that dysregulation of microRNAs (miR) play critical roles in adverse remodelling, contributing to Kv1.3 blockers efficacy in T2DM VSMCs. Methods and results: We used clinically relevant in vivo models of vascular risk factors (VRF) and vessels and VSMCs from T2DM patients. Results: Human T2DM vessels showed increased remodelling, and changes persisted in culture, with augmented VSMCs migration and proliferation. Moreover, there were downregulation of PI3K/AKT/mTOR and upregulation of MEK/ERK pathways, with increased miR-126 expression. The inhibitory effects of Kv1.3 blockers on remodelling were significantly enhanced in T2DM VSMCs and in VRF model. Finally, miR-126 overexpression confered “diabetic” phenotype to non-T2DM VSMCs by downregulating PI3K/AKT axis. Conclusions: miR-126 plays crucial roles in T2DM VSMC metabolic memory through activation of MEK/ERK pathway, enhancing the efficacy of Kv1.3 blockers in the prevention of restenosis in T2DM patients., Ministerio de Economía, Industria y Competitividad (grant BFU2016-75360-R and SAF2016-77871-C2-1-R), Junta de Castilla y León (grants VA114P17; VA172P20 and CLU-2019-02), Novo Nordisk Foundation (grant 34366), Swedish Research Council (grants 2017–00860 and 2020-01145), Swedish Heart and Lung Foundation (grant 20200322)

Producción Científica, Objectives: Restenosis after vessel angioplasty due to dedifferentiation of the vascular smooth muscle cells (VSMCs) limits the success of surgical treatment of vascular occlusions. Type 2 diabetes (T2DM) has a major impact on restenosis, with patients exhibiting more aggressive forms of vascular disease and poorer outcomes after surgery. Kv1.3 channels are critical players in VSMC proliferation. Kv1.3 blockers inhibit VSMCs MEK/ERK signalling and prevent vessel restenosis. We hypothesize that dysregulation of microRNAs (miR) play critical roles in adverse remodelling, contributing to Kv1.3 blockers efficacy in T2DM VSMCs. Methods and results: We used clinically relevant in vivo models of vascular risk factors (VRF) and vessels and VSMCs from T2DM patients. Results: Human T2DM vessels showed increased remodelling, and changes persisted in culture, with augmented VSMCs migration and proliferation. Moreover, there were downregulation of PI3K/AKT/mTOR and upregulation of MEK/ERK pathways, with increased miR-126 expression. The inhibitory effects of Kv1.3 blockers on remodelling were significantly enhanced in T2DM VSMCs and in VRF model. Finally, miR-126 overexpression confered “diabetic” phenotype to non-T2DM VSMCs by downregulating PI3K/AKT axis. Conclusions: miR-126 plays crucial roles in T2DM VSMC metabolic memory through activation of MEK/ERK pathway, enhancing the efficacy of Kv1.3 blockers in the prevention of restenosis in T2DM patients., Ministerio de Economía, Industria y Competitividad (grant BFU2016-75360-R and SAF2016-77871-C2-1-R), Junta de Castilla y León (grants VA114P17; VA172P20 and CLU-2019-02), Novo Nordisk Foundation (grant 34366), Swedish Research Council (grants 2017–00860 and 2020-01145), Swedish Heart and Lung Foundation (grant 20200322)

Producción Científica, Objectives: Restenosis after vessel angioplasty due to dedifferentiation of the vascular smooth muscle cells (VSMCs) limits the success of surgical treatment of vascular occlusions. Type 2 diabetes (T2DM) has a major impact on restenosis, with patients exhibiting more aggressive forms of vascular disease and poorer outcomes after surgery. Kv1.3 channels are critical players in VSMC proliferation. Kv1.3 blockers inhibit VSMCs MEK/ERK signalling and prevent vessel restenosis. We hypothesize that dysregulation of microRNAs (miR) play critical roles in adverse remodelling, contributing to Kv1.3 blockers efficacy in T2DM VSMCs. Methods and results: We used clinically relevant in vivo models of vascular risk factors (VRF) and vessels and VSMCs from T2DM patients. Results: Human T2DM vessels showed increased remodelling, and changes persisted in culture, with augmented VSMCs migration and proliferation. Moreover, there were downregulation of PI3K/AKT/mTOR and upregulation of MEK/ERK pathways, with increased miR-126 expression. The inhibitory effects of Kv1.3 blockers on remodelling were significantly enhanced in T2DM VSMCs and in VRF model. Finally, miR-126 overexpression confered “diabetic” phenotype to non-T2DM VSMCs by downregulating PI3K/AKT axis. Conclusions: miR-126 plays crucial roles in T2DM VSMC metabolic memory through activation of MEK/ERK pathway, enhancing the efficacy of Kv1.3 blockers in the prevention of restenosis in T2DM patients., Ministerio de Economía, Industria y Competitividad (grant BFU2016-75360-R and SAF2016-77871-C2-1-R), Junta de Castilla y León (grants VA114P17; VA172P20 and CLU-2019-02), Novo Nordisk Foundation (grant 34366), Swedish Research Council (grants 2017–00860 and 2020-01145), Swedish Heart and Lung Foundation (grant 20200322)

Introduction: Camphorated p-chlorophenol is a chlorophenolic derivative commonly used as an intra-oral medication in endodontics. Scientific reports on its effects in arterial vascular smooth muscle and local blood flow regulation are scarce. Objective: To determine the effect of camphorated p-chlorophenol on the contractile dynamics of arterial vascular smooth muscle. Method: An experimental and preclinical research was conducted with the use of 14 aortic rings of Wistar rats. The rings were placed in an organ bath and preactivated with noradrenaline, and the tension developed by the vascular smooth muscle at different time intervals was recorded after induction of camphorated p-chlorophenol. Results: Most of the vascular smooth muscle (51.4%) relaxed with the use of camphorated p-chlorophenol. The greatest decrease in vascular tone occurred between the third and fifth minute after use the drug. Wilcoxon rank tests showed significant differences between tension observed at baseline and those recorded at the different time intervals studied. Conclusions: Camphorated p-chlorophenol, induces in vitro, relax the arterial smooth muscle through a pharmacomechanical excitation-contraction link, which increases according to the time., Introdução: O cânfora-clorofenol é um derivado clorofenólico comumente utilizado como medicamento intracanal em Endodontia. Relatórios científicos sobre seus efeitos no músculo liso vascular arterial e na regulação do fluxo sanguíneo local são escassos. Objetivo: Determinar o efeito da cânfora-clorofenol na dinâmica contrátil do músculo liso vascular arterial ao longo do tempo. Método: Foi realizada investigação experimental pré-clínica com 14 anéis aórticos obtidos de ratos Wistar. Os anéis foram colocados em banho de órgãos e pré-ativados com norepinefrina, em seguida, a tensão desenvolvida pela musculatura lisa vascular foi registrada após a adição de cânfora-clorofenol em diferentes intervalos de tempo. Resultados: 51,4% dos músculos lisos vasculares estavam relaxados pela ação do cânfora-clorofenol. A maior diminuição do tônus vascular ocorreu entre o terceiro e o quinto minuto após a adição do medicamento. Os testes de Wilcoxon das faixas com sinais mostraram diferenças significativas entre a tensão base inicial e a registrada nos diferentes intervalos de tempo estudados. Conclusões: O cânfora-clorofenol induz, in vitro, relaxamento da musculatura lisa arterial por meio de um acoplamento excitação-contração do tipo farmacomecânico, que aumenta em função do tempo, Introducción: El p-clorofenol alcanforado es un derivado clorofenólico de uso común como medicación intraconducto en Endodoncia. Son escasos los reportes científicos sobre sus efectos en la musculatura lisa vascular arterial y la regulación del flujo sanguíneo local. Objetivo: Determinar el efecto del p-clorofenol alcanforado sobre la dinámica contráctil del músculo liso vascular arterial en el tiempo. Método: Se realizó una investigación experimental preclínica utilizando 14 anillos de aorta obtenidos de ratas Wistar. Los anillos se colocaron en baño de órganos y se preactivaron con noradrenalina, registrándose luego la tensión desarrollada por el músculo liso vascular tras la adición de p-clorofenol alcanforado durante diferentes intervalos de tiempo. Resultados: El 51,4 % de la musculatura lisa vascular se relajó por la acción del p-clorofenol alcanforado. El mayor descenso del tono vascular se produjo entre el tercer y quinto minuto de añadido el medicamento. Las pruebas de Wilcoxon de los rangos con signos evidenciaron diferencias significativas entre la tensión base inicial y la registrada en los diferentes intervalos de tiempo estudiados. Conclusiones: el p-clorofenol alcanforado, induce in vitro, relajación del músculo liso arterial a través de un acoplamiento excitación-contracción de tipo farmacomecánico, la cual se incrementa en función del tiempo.

Introduction: In Dentistry, p-chlorophenol (4-chlorophenol) is one of the most widely used derivatives of chlorophenols. It is used as an antibacterial agent in root canal disinfection during pulp-radicular treatment. There are few scientific reports on its effects on vascular smooth musculature and the regulation of local blood flow.Objective: To determine the effect of 4-chlorophenol on vascular smooth muscle of abdominal aorta from Wistar rats.Material and Methods: A preclinical experimental research was carried out using 30 abdominal aortic rings (upper portion) obtained from adult Wistar rats. The preparations of about 5 mm were placed in an organ bath, recording the tension developed by the vascular smooth muscle after the addition of 4-chlorophenol at different concentrations and during different time intervals.Results: The results demonstrate that 4-Chlorophenol induced vasorelaxation after the preactivation of the vascular smooth muscle of the abdominal aortic rings, which increased during the entire study time and with increased drug concentration. There were significant differences among average tension values registered at different intervals of time in relation to the initial base tension.Conclusions: It is concluded that in vitro, p-chlorophenol induced relaxation of abdominal aorta vascular smooth muscle in Wistar rats., Introducción: Uno de los derivados de los clorofenoles más utilizado en Estomatología, lo constituye el p-clorofenol (4-clorofenol), empleado como agente antibacteriano en la desinfección del conducto radicular durante el tratamiento pulporradicular. Son escasos los reportes científicos sobre sus efectos en la musculatura lisa vascular arterial y la regulación del flujo sanguíneo local.Objetivo: Determinar el efecto del 4-clorofenol sobre el músculo liso vascular arterial.Método: Se realizó una investigación experimental preclínica, utilizando 30 anillos de aorta abdominal (porción superior) obtenidos de ratas Wistar adultas. Las preparaciones de unos 5 mm se colocaron en baño de órganos, registrándose la tensión desarrollada por el músculo liso vascular tras la adición de 4-clorofenol en diferentes concentraciones y durante diferentes intervalos de tiempo.Resultados: El 4-clorofenol, tras la preactivación del musculo liso vascular de anillos de aorta abdominal, indujo vasorelajación, la que se incrementó durante todo el tiempo de estudio y al aumento de la concentración del medicamento. Existieron diferencias significativas entre los valores de tensión promedios registrados en los diferentes intervalos de tiempo con los de la tensión base inicial.Conclusiones: El p-clorofenol, indujo “in vitro”, relajación del músculo liso vascular arterial de ratas Wistar.

Producción Científica, The dynamic properties of the actin cytoskeleton in smooth muscle cells play an important role in a number of cardiovascular disease states. The state of actin does not only mediate mechanical stability and contractile function but can also regulate gene expression via myocardin related transcription factors (MRTFs). These transcriptional co-activators regulate genes encoding contractile and cytoskeletal proteins in smooth muscle. Regulation of small non-coding microRNAs (miRNAs) by actin polymerization may mediate some of these effects. MiRNAs are short non-coding RNAs that modulate gene expression by post-transcriptional regulation of target messenger RNA. In this study we aimed to determine a profile of miRNAs that were 1) regulated by actin/MRTF-A, 2) associated with the contractile smooth muscle phenotype and 3) enriched in muscle cells., The Swedish Research Council (grant 2012-2197), The Crafoord Foundation (grant 20150629), Instituto de Salud Carlos III (grant RD12/0042/0006), Ministerio de Economía, Industria y Competitividad (grant BFU2013-45867-R)

Producción Científica, K+ channels encoded by the ether-a-go-go related gene (ERG1 or KCNH2) are important determinants of the cardiac action potential. Expression of both cardiac isoforms (ERG1a and ERG1b) were identified in murine portal vein and distinctive voltage-gated K+ currents were recorded from single myocytes. The aim of the present study was to ascertain the expression and functional impact of ERG channels in murine arteries., British Heart Foundation (projects PG/06/057/20864 and PG/11/93/21943), Medical Research Council (project MR/K019074/1), Ministerio de Economía, Industria y Competitividad (grants BFU2013-45867-R and BFU2016-75360)

El término Diabetes Mellitus (DM) engloba a un conjunto de entidades nosológicas que cursan con alteraciones en el metabolismo de los carbohidratos, lípidos y proteínas que tienen en común la existencia de hiperglucemia debido a un fallo en la función de la insulina como hormona reguladora. Este situación puede estar suscitada primariamente por un déficit real de insulina por insuficiencia de las células beta pancreáticas que normalmente la producen o bien por una disminución de la sensibilidad a la misma de los tejidos diana de esta hormona (Tuomi et al., 2014)., Máster en Investigación Biomédica

El término Diabetes Mellitus (DM) engloba a un conjunto de entidades nosológicas que cursan con alteraciones en el metabolismo de los carbohidratos, lípidos y proteínas que tienen en común la existencia de hiperglucemia debido a un fallo en la función de la insulina como hormona reguladora. Este situación puede estar suscitada primariamente por un déficit real de insulina por insuficiencia de las células beta pancreáticas que normalmente la producen o bien por una disminución de la sensibilidad a la misma de los tejidos diana de esta hormona (Tuomi et al., 2014)., Máster en Investigación Biomédica

El término Diabetes Mellitus (DM) engloba a un conjunto de entidades nosológicas que cursan con alteraciones en el metabolismo de los carbohidratos, lípidos y proteínas que tienen en común la existencia de hiperglucemia debido a un fallo en la función de la insulina como hormona reguladora. Este situación puede estar suscitada primariamente por un déficit real de insulina por insuficiencia de las células beta pancreáticas que normalmente la producen o bien por una disminución de la sensibilidad a la misma de los tejidos diana de esta hormona (Tuomi et al., 2014)., Máster en Investigación Biomédica

Producción Científica, The dynamic properties of the actin cytoskeleton in smooth muscle cells play an important role in a number of cardiovascular disease states. The state of actin does not only mediate mechanical stability and contractile function but can also regulate gene expression via myocardin related transcription factors (MRTFs). These transcriptional co-activators regulate genes encoding contractile and cytoskeletal proteins in smooth muscle. Regulation of small non-coding microRNAs (miRNAs) by actin polymerization may mediate some of these effects. MiRNAs are short non-coding RNAs that modulate gene expression by post-transcriptional regulation of target messenger RNA. In this study we aimed to determine a profile of miRNAs that were 1) regulated by actin/MRTF-A, 2) associated with the contractile smooth muscle phenotype and 3) enriched in muscle cells., The Swedish Research Council (grant 2012-2197), The Crafoord Foundation (grant 20150629), Instituto de Salud Carlos III (grant RD12/0042/0006), Ministerio de Economía, Industria y Competitividad (grant BFU2013-45867-R)

Producción Científica, K+ channels encoded by the ether-a-go-go related gene (ERG1 or KCNH2) are important determinants of the cardiac action potential. Expression of both cardiac isoforms (ERG1a and ERG1b) were identified in murine portal vein and distinctive voltage-gated K+ currents were recorded from single myocytes. The aim of the present study was to ascertain the expression and functional impact of ERG channels in murine arteries., British Heart Foundation (projects PG/06/057/20864 and PG/11/93/21943), Medical Research Council (project MR/K019074/1), Ministerio de Economía, Industria y Competitividad (grants BFU2013-45867-R and BFU2016-75360)

Producción Científica, The dynamic properties of the actin cytoskeleton in smooth muscle cells play an important role in a number of cardiovascular disease states. The state of actin does not only mediate mechanical stability and contractile function but can also regulate gene expression via myocardin related transcription factors (MRTFs). These transcriptional co-activators regulate genes encoding contractile and cytoskeletal proteins in smooth muscle. Regulation of small non-coding microRNAs (miRNAs) by actin polymerization may mediate some of these effects. MiRNAs are short non-coding RNAs that modulate gene expression by post-transcriptional regulation of target messenger RNA. In this study we aimed to determine a profile of miRNAs that were 1) regulated by actin/MRTF-A, 2) associated with the contractile smooth muscle phenotype and 3) enriched in muscle cells., The Swedish Research Council (grant 2012-2197), The Crafoord Foundation (grant 20150629), Instituto de Salud Carlos III (grant RD12/0042/0006), Ministerio de Economía, Industria y Competitividad (grant BFU2013-45867-R)

Producción Científica, K+ channels encoded by the ether-a-go-go related gene (ERG1 or KCNH2) are important determinants of the cardiac action potential. Expression of both cardiac isoforms (ERG1a and ERG1b) were identified in murine portal vein and distinctive voltage-gated K+ currents were recorded from single myocytes. The aim of the present study was to ascertain the expression and functional impact of ERG channels in murine arteries., British Heart Foundation (projects PG/06/057/20864 and PG/11/93/21943), Medical Research Council (project MR/K019074/1), Ministerio de Economía, Industria y Competitividad (grants BFU2013-45867-R and BFU2016-75360)

El término Diabetes Mellitus (DM) engloba a un conjunto de entidades nosológicas que cursan con alteraciones en el metabolismo de los carbohidratos, lípidos y proteínas que tienen en común la existencia de hiperglucemia debido a un fallo en la función de la insulina como hormona reguladora. Este situación puede estar suscitada primariamente por un déficit real de insulina por insuficiencia de las células beta pancreáticas que normalmente la producen o bien por una disminución de la sensibilidad a la misma de los tejidos diana de esta hormona (Tuomi et al., 2014)., Máster en Investigación Biomédica

Producción Científica, The dynamic properties of the actin cytoskeleton in smooth muscle cells play an important role in a number of cardiovascular disease states. The state of actin does not only mediate mechanical stability and contractile function but can also regulate gene expression via myocardin related transcription factors (MRTFs). These transcriptional co-activators regulate genes encoding contractile and cytoskeletal proteins in smooth muscle. Regulation of small non-coding microRNAs (miRNAs) by actin polymerization may mediate some of these effects. MiRNAs are short non-coding RNAs that modulate gene expression by post-transcriptional regulation of target messenger RNA. In this study we aimed to determine a profile of miRNAs that were 1) regulated by actin/MRTF-A, 2) associated with the contractile smooth muscle phenotype and 3) enriched in muscle cells., The Swedish Research Council (grant 2012-2197), The Crafoord Foundation (grant 20150629), Instituto de Salud Carlos III (grant RD12/0042/0006), Ministerio de Economía, Industria y Competitividad (grant BFU2013-45867-R)

Producción Científica, K+ channels encoded by the ether-a-go-go related gene (ERG1 or KCNH2) are important determinants of the cardiac action potential. Expression of both cardiac isoforms (ERG1a and ERG1b) were identified in murine portal vein and distinctive voltage-gated K+ currents were recorded from single myocytes. The aim of the present study was to ascertain the expression and functional impact of ERG channels in murine arteries., British Heart Foundation (projects PG/06/057/20864 and PG/11/93/21943), Medical Research Council (project MR/K019074/1), Ministerio de Economía, Industria y Competitividad (grants BFU2013-45867-R and BFU2016-75360)

El término Diabetes Mellitus (DM) engloba a un conjunto de entidades nosológicas que cursan con alteraciones en el metabolismo de los carbohidratos, lípidos y proteínas que tienen en común la existencia de hiperglucemia debido a un fallo en la función de la insulina como hormona reguladora. Este situación puede estar suscitada primariamente por un déficit real de insulina por insuficiencia de las células beta pancreáticas que normalmente la producen o bien por una disminución de la sensibilidad a la misma de los tejidos diana de esta hormona (Tuomi et al., 2014)., Máster en Investigación Biomédica

Producción Científica, Increased vascular tone in essential hypertension involves a sustained rise in total peripheral resistance. A model has been proposed in which the combination of membrane depolarization and higher L‐type Ca2+ channel activity generates augmented Ca2+ influx into vascular smooth muscle cells (VSMCs), contraction and vasoconstriction. The search for culprit ion channels responsible for membrane depolarization has provided several candidates, including members of the canonical transient receptor potential (TRPC) family. TRPC3 and TRPC6 are diacylglycerol‐activated, non‐selective cationic channels contributing to stretch‐ or agonist‐induced depolarization. Conflicting information exists regarding changes in TRPC3/TRPC6 functional expression in hypertension. However, although TRPC3‐TRPC6 channels can heteromultimerize, the possibility that differences in their association pattern may change their functional contribution to vascular tone is largely unexplored. We probe this hypothesis using a model of essential hypertension (BPH mice; blood pressure high) and its normotensive control (BPN mice; blood pressure normal). First, non‐selective cationic currents through homo‐ and heterotetramers recorded from transfected Chinese hamster ovary cells indicated that TRPC currents were sensitive to the selective antagonist Pyr10 only when TRPC6 was present, whereas intracellular anti‐TRPC3 antibody selectively blocked TRPC3‐mediated currents. In mesenteric VSMCs, basal and agonist‐induced currents were more sensitive to Pyr3 and Pyr10 in BPN cells. Consistently, myography studies showed a larger Pyr3/10‐induced vasodilatation in BPN mesenteric arteries. mRNA and protein expression data supported changes in TRPC3 and TRPC6 proportions and assembly, with a higher TRPC3 channel contribution in BPH VSMCs that could favour cell depolarization. These differences in functional and pharmacological properties of TRPC3 and TRPC6 channels, depending on their assembly, could represent novel th, Instituto de Salud Carlos III (project RD12/0042/0006), Programa Estatal de Investigación (project BFU2013-45867-R )

Producción Científica, Increased vascular tone in essential hypertension involves a sustained rise in total peripheral resistance. A model has been proposed in which the combination of membrane depolarization and higher L‐type Ca2+ channel activity generates augmented Ca2+ influx into vascular smooth muscle cells (VSMCs), contraction and vasoconstriction. The search for culprit ion channels responsible for membrane depolarization has provided several candidates, including members of the canonical transient receptor potential (TRPC) family. TRPC3 and TRPC6 are diacylglycerol‐activated, non‐selective cationic channels contributing to stretch‐ or agonist‐induced depolarization. Conflicting information exists regarding changes in TRPC3/TRPC6 functional expression in hypertension. However, although TRPC3‐TRPC6 channels can heteromultimerize, the possibility that differences in their association pattern may change their functional contribution to vascular tone is largely unexplored. We probe this hypothesis using a model of essential hypertension (BPH mice; blood pressure high) and its normotensive control (BPN mice; blood pressure normal). First, non‐selective cationic currents through homo‐ and heterotetramers recorded from transfected Chinese hamster ovary cells indicated that TRPC currents were sensitive to the selective antagonist Pyr10 only when TRPC6 was present, whereas intracellular anti‐TRPC3 antibody selectively blocked TRPC3‐mediated currents. In mesenteric VSMCs, basal and agonist‐induced currents were more sensitive to Pyr3 and Pyr10 in BPN cells. Consistently, myography studies showed a larger Pyr3/10‐induced vasodilatation in BPN mesenteric arteries. mRNA and protein expression data supported changes in TRPC3 and TRPC6 proportions and assembly, with a higher TRPC3 channel contribution in BPH VSMCs that could favour cell depolarization. These differences in functional and pharmacological properties of TRPC3 and TRPC6 channels, depending on their assembly, could represent novel th, Instituto de Salud Carlos III (project RD12/0042/0006), Programa Estatal de Investigación (project BFU2013-45867-R )

Producción Científica, Increased vascular tone in essential hypertension involves a sustained rise in total peripheral resistance. A model has been proposed in which the combination of membrane depolarization and higher L‐type Ca2+ channel activity generates augmented Ca2+ influx into vascular smooth muscle cells (VSMCs), contraction and vasoconstriction. The search for culprit ion channels responsible for membrane depolarization has provided several candidates, including members of the canonical transient receptor potential (TRPC) family. TRPC3 and TRPC6 are diacylglycerol‐activated, non‐selective cationic channels contributing to stretch‐ or agonist‐induced depolarization. Conflicting information exists regarding changes in TRPC3/TRPC6 functional expression in hypertension. However, although TRPC3‐TRPC6 channels can heteromultimerize, the possibility that differences in their association pattern may change their functional contribution to vascular tone is largely unexplored. We probe this hypothesis using a model of essential hypertension (BPH mice; blood pressure high) and its normotensive control (BPN mice; blood pressure normal). First, non‐selective cationic currents through homo‐ and heterotetramers recorded from transfected Chinese hamster ovary cells indicated that TRPC currents were sensitive to the selective antagonist Pyr10 only when TRPC6 was present, whereas intracellular anti‐TRPC3 antibody selectively blocked TRPC3‐mediated currents. In mesenteric VSMCs, basal and agonist‐induced currents were more sensitive to Pyr3 and Pyr10 in BPN cells. Consistently, myography studies showed a larger Pyr3/10‐induced vasodilatation in BPN mesenteric arteries. mRNA and protein expression data supported changes in TRPC3 and TRPC6 proportions and assembly, with a higher TRPC3 channel contribution in BPH VSMCs that could favour cell depolarization. These differences in functional and pharmacological properties of TRPC3 and TRPC6 channels, depending on their assembly, could represent novel th, Instituto de Salud Carlos III (project RD12/0042/0006), Programa Estatal de Investigación (project BFU2013-45867-R )

Producción Científica, Increased vascular tone in essential hypertension involves a sustained rise in total peripheral resistance. A model has been proposed in which the combination of membrane depolarization and higher L‐type Ca2+ channel activity generates augmented Ca2+ influx into vascular smooth muscle cells (VSMCs), contraction and vasoconstriction. The search for culprit ion channels responsible for membrane depolarization has provided several candidates, including members of the canonical transient receptor potential (TRPC) family. TRPC3 and TRPC6 are diacylglycerol‐activated, non‐selective cationic channels contributing to stretch‐ or agonist‐induced depolarization. Conflicting information exists regarding changes in TRPC3/TRPC6 functional expression in hypertension. However, although TRPC3‐TRPC6 channels can heteromultimerize, the possibility that differences in their association pattern may change their functional contribution to vascular tone is largely unexplored. We probe this hypothesis using a model of essential hypertension (BPH mice; blood pressure high) and its normotensive control (BPN mice; blood pressure normal). First, non‐selective cationic currents through homo‐ and heterotetramers recorded from transfected Chinese hamster ovary cells indicated that TRPC currents were sensitive to the selective antagonist Pyr10 only when TRPC6 was present, whereas intracellular anti‐TRPC3 antibody selectively blocked TRPC3‐mediated currents. In mesenteric VSMCs, basal and agonist‐induced currents were more sensitive to Pyr3 and Pyr10 in BPN cells. Consistently, myography studies showed a larger Pyr3/10‐induced vasodilatation in BPN mesenteric arteries. mRNA and protein expression data supported changes in TRPC3 and TRPC6 proportions and assembly, with a higher TRPC3 channel contribution in BPH VSMCs that could favour cell depolarization. These differences in functional and pharmacological properties of TRPC3 and TRPC6 channels, depending on their assembly, could represent novel th, Instituto de Salud Carlos III (project RD12/0042/0006), Programa Estatal de Investigación (project BFU2013-45867-R )

La endotelina es un péptido vasoconstrictor aislado inicialmente del cerdo de donde procede su denominación. Es considerado como el más potente vasoconstrictor conocido por el hombre, es incluso 10 veces más potente que la angiotensina II. Existen tres isoformas y están distribuidas en diversas células y tejidos interviniendo en la modulación del tono vascular, proliferación celular, producción hormonal, balance del sodio y como neurotransmisor. Para la regulación del tono vascular debe de haber un equilibrio de fuerzas vasoconstrictoras y vasodilatadoras generados por el endotelio o que ejercen influencia sobre el mismo, existen varios candidatos en la categoría de los vasoconstrictores, pero el más importante es la endotelina-1 por su potencia y su localización, ya que su acción es ejercida sobre el músculo liso vascular.

Producción Científica, The increased vascular tone that defines essential hypertension is associated with depolarization of vascular smooth muscle cells (VSMCs) and involves a change in the expression profile of ion channels promoting arterial contraction. As a major regulator of VSMC resting membrane potential (VM), K+channel activity is an important determinant of vascular tone and vessel diameter. However, hypertension-associated changes in the expression and/or modulation of K+channels are poorly defined, due to their large molecular diversity and their bed-specific pattern of expression. Moreover, the impact of these changes on the integrated vessel functionand their contribution to the development of altered vascular tone under physiological conditions need to be confirmed. Hypertensive (BPH) and normotensive (BPN) mice strains obtained by phenotypic selection were used to explore whether changes in the functional expression of VSMC inward rectifier K+channels contribute to the more depolarized resting VM and the increased vascular reactivity of hypertensive arteries. We determined the expression levels of inward rectifierK+channel mRNA in several vascular beds from BPN and BPH animals, and their functional contribution to VSMC excitability and vascular tone in mesenteric arteries. We found a decrease in the expression of Kir2.1, Kir4.1, Kir6.x and SUR2 mRNA in BPH VSMCs, and a decreased functional contribution of both KIRand KATP channels in isolated BPH VSMCs. However, only the effect of KATP channel modulators was impaired when exploring vascular tone, suggesting that decreased functional expression of KATP channels may be an important element in the remodelling of VSMCs in essential hypertension., Ministerio de Sanidad, Consumo y Bienestar Social - Instituto de Salud Carlos III (grant R006/009), Ministerio de Ciencia, Innovación y Universidades (grant BFU2010-15898), Junta de Castilla y León (grant VA094A11-2)

Producción Científica, Objective: Phenotypic modulation of vascular smooth muscle cells has been associated with a decreased expression of all voltage-dependent potassium channel (Kv)1 channel encoding genes but Kcna3 (which encodes Kv1.3 channels). In fact, upregulation of Kv1.3 currents seems to be important to modulate proliferation of mice femoral vascular smooth muscle cells in culture. This study was designed to explore if these changes in Kv1 expression pattern constituted a landmark of phenotypic modulation across vascular beds and to investigate the mechanisms involved in the proproliferative function of Kv1.3 channels. Methods and Results: Changes in Kv1.3 and Kv1.5 channel expression were reproduced in mesenteric and aortic vascular smooth muscle cells, and their correlate with protein expression was electrophysiologicaly confirmed using selective blockers. Heterologous expression of Kv1.3 and Kv1.5 channels in HEK cells has opposite effects on the proliferation rate. The proproliferative effect of Kv1.3 channels was reproduced by “poreless” mutants but disappeared when voltagedependence of gating was suppressed. Conclusion: These findings suggest that the signaling cascade linking Kv1.3 functional expression to cell proliferation is activated by the voltage-dependent conformational change of the channels without needing ion conduction. Additionally, the conserved upregulation of Kv1.3 on phenotypic modulation in several vascular beds makes this channel a good target to control unwanted vascular remodeling., Instituto de Salud Carlos III (grant R006/009), Ministerio de Ciencia, Innovación y Universidades (grant BFU2010-15898), Junta de Castilla y León (grant VA094A11-2)

Producción Científica, Objective: Phenotypic modulation of vascular smooth muscle cells has been associated with a decreased expression of all voltage-dependent potassium channel (Kv)1 channel encoding genes but Kcna3 (which encodes Kv1.3 channels). In fact, upregulation of Kv1.3 currents seems to be important to modulate proliferation of mice femoral vascular smooth muscle cells in culture. This study was designed to explore if these changes in Kv1 expression pattern constituted a landmark of phenotypic modulation across vascular beds and to investigate the mechanisms involved in the proproliferative function of Kv1.3 channels. Methods and Results: Changes in Kv1.3 and Kv1.5 channel expression were reproduced in mesenteric and aortic vascular smooth muscle cells, and their correlate with protein expression was electrophysiologicaly confirmed using selective blockers. Heterologous expression of Kv1.3 and Kv1.5 channels in HEK cells has opposite effects on the proliferation rate. The proproliferative effect of Kv1.3 channels was reproduced by “poreless” mutants but disappeared when voltagedependence of gating was suppressed. Conclusion: These findings suggest that the signaling cascade linking Kv1.3 functional expression to cell proliferation is activated by the voltage-dependent conformational change of the channels without needing ion conduction. Additionally, the conserved upregulation of Kv1.3 on phenotypic modulation in several vascular beds makes this channel a good target to control unwanted vascular remodeling., Instituto de Salud Carlos III (grant R006/009), Ministerio de Ciencia, Innovación y Universidades (grant BFU2010-15898), Junta de Castilla y León (grant VA094A11-2)

Producción Científica, The increased vascular tone that defines essential hypertension is associated with depolarization of vascular smooth muscle cells (VSMCs) and involves a change in the expression profile of ion channels promoting arterial contraction. As a major regulator of VSMC resting membrane potential (VM), K+channel activity is an important determinant of vascular tone and vessel diameter. However, hypertension-associated changes in the expression and/or modulation of K+channels are poorly defined, due to their large molecular diversity and their bed-specific pattern of expression. Moreover, the impact of these changes on the integrated vessel functionand their contribution to the development of altered vascular tone under physiological conditions need to be confirmed. Hypertensive (BPH) and normotensive (BPN) mice strains obtained by phenotypic selection were used to explore whether changes in the functional expression of VSMC inward rectifier K+channels contribute to the more depolarized resting VM and the increased vascular reactivity of hypertensive arteries. We determined the expression levels of inward rectifierK+channel mRNA in several vascular beds from BPN and BPH animals, and their functional contribution to VSMC excitability and vascular tone in mesenteric arteries. We found a decrease in the expression of Kir2.1, Kir4.1, Kir6.x and SUR2 mRNA in BPH VSMCs, and a decreased functional contribution of both KIRand KATP channels in isolated BPH VSMCs. However, only the effect of KATP channel modulators was impaired when exploring vascular tone, suggesting that decreased functional expression of KATP channels may be an important element in the remodelling of VSMCs in essential hypertension., Ministerio de Sanidad, Consumo y Bienestar Social - Instituto de Salud Carlos III (grant R006/009), Ministerio de Ciencia, Innovación y Universidades (grant BFU2010-15898), Junta de Castilla y León (grant VA094A11-2)

Producción Científica, The increased vascular tone that defines essential hypertension is associated with depolarization of vascular smooth muscle cells (VSMCs) and involves a change in the expression profile of ion channels promoting arterial contraction. As a major regulator of VSMC resting membrane potential (VM), K+channel activity is an important determinant of vascular tone and vessel diameter. However, hypertension-associated changes in the expression and/or modulation of K+channels are poorly defined, due to their large molecular diversity and their bed-specific pattern of expression. Moreover, the impact of these changes on the integrated vessel functionand their contribution to the development of altered vascular tone under physiological conditions need to be confirmed. Hypertensive (BPH) and normotensive (BPN) mice strains obtained by phenotypic selection were used to explore whether changes in the functional expression of VSMC inward rectifier K+channels contribute to the more depolarized resting VM and the increased vascular reactivity of hypertensive arteries. We determined the expression levels of inward rectifierK+channel mRNA in several vascular beds from BPN and BPH animals, and their functional contribution to VSMC excitability and vascular tone in mesenteric arteries. We found a decrease in the expression of Kir2.1, Kir4.1, Kir6.x and SUR2 mRNA in BPH VSMCs, and a decreased functional contribution of both KIRand KATP channels in isolated BPH VSMCs. However, only the effect of KATP channel modulators was impaired when exploring vascular tone, suggesting that decreased functional expression of KATP channels may be an important element in the remodelling of VSMCs in essential hypertension., Ministerio de Sanidad, Consumo y Bienestar Social - Instituto de Salud Carlos III (grant R006/009), Ministerio de Ciencia, Innovación y Universidades (grant BFU2010-15898), Junta de Castilla y León (grant VA094A11-2)

Producción Científica, Objective: Phenotypic modulation of vascular smooth muscle cells has been associated with a decreased expression of all voltage-dependent potassium channel (Kv)1 channel encoding genes but Kcna3 (which encodes Kv1.3 channels). In fact, upregulation of Kv1.3 currents seems to be important to modulate proliferation of mice femoral vascular smooth muscle cells in culture. This study was designed to explore if these changes in Kv1 expression pattern constituted a landmark of phenotypic modulation across vascular beds and to investigate the mechanisms involved in the proproliferative function of Kv1.3 channels. Methods and Results: Changes in Kv1.3 and Kv1.5 channel expression were reproduced in mesenteric and aortic vascular smooth muscle cells, and their correlate with protein expression was electrophysiologicaly confirmed using selective blockers. Heterologous expression of Kv1.3 and Kv1.5 channels in HEK cells has opposite effects on the proliferation rate. The proproliferative effect of Kv1.3 channels was reproduced by “poreless” mutants but disappeared when voltagedependence of gating was suppressed. Conclusion: These findings suggest that the signaling cascade linking Kv1.3 functional expression to cell proliferation is activated by the voltage-dependent conformational change of the channels without needing ion conduction. Additionally, the conserved upregulation of Kv1.3 on phenotypic modulation in several vascular beds makes this channel a good target to control unwanted vascular remodeling., Instituto de Salud Carlos III (grant R006/009), Ministerio de Ciencia, Innovación y Universidades (grant BFU2010-15898), Junta de Castilla y León (grant VA094A11-2)

Producción Científica, The increased vascular tone that defines essential hypertension is associated with depolarization of vascular smooth muscle cells (VSMCs) and involves a change in the expression profile of ion channels promoting arterial contraction. As a major regulator of VSMC resting membrane potential (VM), K+channel activity is an important determinant of vascular tone and vessel diameter. However, hypertension-associated changes in the expression and/or modulation of K+channels are poorly defined, due to their large molecular diversity and their bed-specific pattern of expression. Moreover, the impact of these changes on the integrated vessel functionand their contribution to the development of altered vascular tone under physiological conditions need to be confirmed. Hypertensive (BPH) and normotensive (BPN) mice strains obtained by phenotypic selection were used to explore whether changes in the functional expression of VSMC inward rectifier K+channels contribute to the more depolarized resting VM and the increased vascular reactivity of hypertensive arteries. We determined the expression levels of inward rectifierK+channel mRNA in several vascular beds from BPN and BPH animals, and their functional contribution to VSMC excitability and vascular tone in mesenteric arteries. We found a decrease in the expression of Kir2.1, Kir4.1, Kir6.x and SUR2 mRNA in BPH VSMCs, and a decreased functional contribution of both KIRand KATP channels in isolated BPH VSMCs. However, only the effect of KATP channel modulators was impaired when exploring vascular tone, suggesting that decreased functional expression of KATP channels may be an important element in the remodelling of VSMCs in essential hypertension., Ministerio de Sanidad, Consumo y Bienestar Social - Instituto de Salud Carlos III (grant R006/009), Ministerio de Ciencia, Innovación y Universidades (grant BFU2010-15898), Junta de Castilla y León (grant VA094A11-2)

Producción Científica, Objective: Phenotypic modulation of vascular smooth muscle cells has been associated with a decreased expression of all voltage-dependent potassium channel (Kv)1 channel encoding genes but Kcna3 (which encodes Kv1.3 channels). In fact, upregulation of Kv1.3 currents seems to be important to modulate proliferation of mice femoral vascular smooth muscle cells in culture. This study was designed to explore if these changes in Kv1 expression pattern constituted a landmark of phenotypic modulation across vascular beds and to investigate the mechanisms involved in the proproliferative function of Kv1.3 channels. Methods and Results: Changes in Kv1.3 and Kv1.5 channel expression were reproduced in mesenteric and aortic vascular smooth muscle cells, and their correlate with protein expression was electrophysiologicaly confirmed using selective blockers. Heterologous expression of Kv1.3 and Kv1.5 channels in HEK cells has opposite effects on the proliferation rate. The proproliferative effect of Kv1.3 channels was reproduced by “poreless” mutants but disappeared when voltagedependence of gating was suppressed. Conclusion: These findings suggest that the signaling cascade linking Kv1.3 functional expression to cell proliferation is activated by the voltage-dependent conformational change of the channels without needing ion conduction. Additionally, the conserved upregulation of Kv1.3 on phenotypic modulation in several vascular beds makes this channel a good target to control unwanted vascular remodeling., Instituto de Salud Carlos III (grant R006/009), Ministerio de Ciencia, Innovación y Universidades (grant BFU2010-15898), Junta de Castilla y León (grant VA094A11-2)

Producción Científica, Abnormal vascular smooth muscle cell (VSMC) proliferation contributes to occlusive and proliferative disorders of the vessel wall. Salicylate and other nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit VSMC proliferation by an unknown mechanism unrelated to anti-inflammatory activity. In search for this mechanism, we have studied the effects of salicylate and other NSAIDs on subcellular Ca2+ homeostasis and Ca2+-dependent cell proliferation in rat aortic A10 cells, a model of neointimal VSMCs. We found that A10 cells displayed both store-operated Ca2+ entry (SOCE) and voltage-operated Ca2+ entry (VOCE), the former being more important quantitatively than the latter. Inhibition of SOCE by specific Ca2+ released-activated Ca2+ (CRAC/Orai) channels antagonists prevented A10 cell proliferation. Salicylate and other NSAIDs, including ibuprofen, indomethacin, and sulindac, inhibited SOCE and thereby Ca2+-dependent, A10 cell proliferation. SOCE, but not VOCE, induced mitochondrial Ca2+ uptake in A10 cells, and mitochondrial depolarization prevented SOCE, thus suggesting that mitochondrial Ca2+ uptake controls SOCE (but not VOCE) in A10 cells. NSAIDs depolarized mitochondria and prevented mitochondrial Ca2+ uptake, suggesting that they favor the Ca2+-dependent inactivation of CRAC/Orai channels. NSAIDs also inhibited SOCE in rat basophilic leukemia cells where mitochondrial control of CRAC/Orai is well established. NSAIDs accelerate slow inactivation of CRAC currents in rat basophilic leukemia cells under weak Ca2+ buffering conditions but not in strong Ca2+ buffer, thus excluding that NSAIDs inhibit SOCE directly. Taken together, our results indicate that NSAIDs inhibit VSMC proliferation by facilitating the Ca2+-dependent inactivation of CRAC/Orai channels which normally is prevented by mitochondria clearing of entering Ca2+., Junta de Castilla y León (grants SAN191/VA1806 and CSI12A08), Ministerio de Ciencia, Innovación y Universidades (grant BFU2009-0867), Deutsche Forschungsgemeinschaft (grant A3)

Producción Científica, Abnormal vascular smooth muscle cell (VSMC) proliferation contributes to occlusive and proliferative disorders of the vessel wall. Salicylate and other nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit VSMC proliferation by an unknown mechanism unrelated to anti-inflammatory activity. In search for this mechanism, we have studied the effects of salicylate and other NSAIDs on subcellular Ca2+ homeostasis and Ca2+-dependent cell proliferation in rat aortic A10 cells, a model of neointimal VSMCs. We found that A10 cells displayed both store-operated Ca2+ entry (SOCE) and voltage-operated Ca2+ entry (VOCE), the former being more important quantitatively than the latter. Inhibition of SOCE by specific Ca2+ released-activated Ca2+ (CRAC/Orai) channels antagonists prevented A10 cell proliferation. Salicylate and other NSAIDs, including ibuprofen, indomethacin, and sulindac, inhibited SOCE and thereby Ca2+-dependent, A10 cell proliferation. SOCE, but not VOCE, induced mitochondrial Ca2+ uptake in A10 cells, and mitochondrial depolarization prevented SOCE, thus suggesting that mitochondrial Ca2+ uptake controls SOCE (but not VOCE) in A10 cells. NSAIDs depolarized mitochondria and prevented mitochondrial Ca2+ uptake, suggesting that they favor the Ca2+-dependent inactivation of CRAC/Orai channels. NSAIDs also inhibited SOCE in rat basophilic leukemia cells where mitochondrial control of CRAC/Orai is well established. NSAIDs accelerate slow inactivation of CRAC currents in rat basophilic leukemia cells under weak Ca2+ buffering conditions but not in strong Ca2+ buffer, thus excluding that NSAIDs inhibit SOCE directly. Taken together, our results indicate that NSAIDs inhibit VSMC proliferation by facilitating the Ca2+-dependent inactivation of CRAC/Orai channels which normally is prevented by mitochondria clearing of entering Ca2+., Junta de Castilla y León (grants SAN191/VA1806 and CSI12A08), Ministerio de Ciencia, Innovación y Universidades (grant BFU2009-0867), Deutsche Forschungsgemeinschaft (grant A3)

Producción Científica, Abnormal vascular smooth muscle cell (VSMC) proliferation contributes to occlusive and proliferative disorders of the vessel wall. Salicylate and other nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit VSMC proliferation by an unknown mechanism unrelated to anti-inflammatory activity. In search for this mechanism, we have studied the effects of salicylate and other NSAIDs on subcellular Ca2+ homeostasis and Ca2+-dependent cell proliferation in rat aortic A10 cells, a model of neointimal VSMCs. We found that A10 cells displayed both store-operated Ca2+ entry (SOCE) and voltage-operated Ca2+ entry (VOCE), the former being more important quantitatively than the latter. Inhibition of SOCE by specific Ca2+ released-activated Ca2+ (CRAC/Orai) channels antagonists prevented A10 cell proliferation. Salicylate and other NSAIDs, including ibuprofen, indomethacin, and sulindac, inhibited SOCE and thereby Ca2+-dependent, A10 cell proliferation. SOCE, but not VOCE, induced mitochondrial Ca2+ uptake in A10 cells, and mitochondrial depolarization prevented SOCE, thus suggesting that mitochondrial Ca2+ uptake controls SOCE (but not VOCE) in A10 cells. NSAIDs depolarized mitochondria and prevented mitochondrial Ca2+ uptake, suggesting that they favor the Ca2+-dependent inactivation of CRAC/Orai channels. NSAIDs also inhibited SOCE in rat basophilic leukemia cells where mitochondrial control of CRAC/Orai is well established. NSAIDs accelerate slow inactivation of CRAC currents in rat basophilic leukemia cells under weak Ca2+ buffering conditions but not in strong Ca2+ buffer, thus excluding that NSAIDs inhibit SOCE directly. Taken together, our results indicate that NSAIDs inhibit VSMC proliferation by facilitating the Ca2+-dependent inactivation of CRAC/Orai channels which normally is prevented by mitochondria clearing of entering Ca2+., Junta de Castilla y León (grants SAN191/VA1806 and CSI12A08), Ministerio de Ciencia, Innovación y Universidades (grant BFU2009-0867), Deutsche Forschungsgemeinschaft (grant A3)

Producción Científica, Abnormal vascular smooth muscle cell (VSMC) proliferation contributes to occlusive and proliferative disorders of the vessel wall. Salicylate and other nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit VSMC proliferation by an unknown mechanism unrelated to anti-inflammatory activity. In search for this mechanism, we have studied the effects of salicylate and other NSAIDs on subcellular Ca2+ homeostasis and Ca2+-dependent cell proliferation in rat aortic A10 cells, a model of neointimal VSMCs. We found that A10 cells displayed both store-operated Ca2+ entry (SOCE) and voltage-operated Ca2+ entry (VOCE), the former being more important quantitatively than the latter. Inhibition of SOCE by specific Ca2+ released-activated Ca2+ (CRAC/Orai) channels antagonists prevented A10 cell proliferation. Salicylate and other NSAIDs, including ibuprofen, indomethacin, and sulindac, inhibited SOCE and thereby Ca2+-dependent, A10 cell proliferation. SOCE, but not VOCE, induced mitochondrial Ca2+ uptake in A10 cells, and mitochondrial depolarization prevented SOCE, thus suggesting that mitochondrial Ca2+ uptake controls SOCE (but not VOCE) in A10 cells. NSAIDs depolarized mitochondria and prevented mitochondrial Ca2+ uptake, suggesting that they favor the Ca2+-dependent inactivation of CRAC/Orai channels. NSAIDs also inhibited SOCE in rat basophilic leukemia cells where mitochondrial control of CRAC/Orai is well established. NSAIDs accelerate slow inactivation of CRAC currents in rat basophilic leukemia cells under weak Ca2+ buffering conditions but not in strong Ca2+ buffer, thus excluding that NSAIDs inhibit SOCE directly. Taken together, our results indicate that NSAIDs inhibit VSMC proliferation by facilitating the Ca2+-dependent inactivation of CRAC/Orai channels which normally is prevented by mitochondria clearing of entering Ca2+., Junta de Castilla y León (grants SAN191/VA1806 and CSI12A08), Ministerio de Ciencia, Innovación y Universidades (grant BFU2009-0867), Deutsche Forschungsgemeinschaft (grant A3)

Producción Científica, Aims: Vascular smooth muscle cell (VSMC) proliferation is involved in cardiovascular pathologies associated with unwanted arterial wall remodelling. Coordinated changes in the expression of several K+ channels have been found to be important elements in the phenotypic switch of VSMCs towards proliferation. We have previously demonstrated the association of functional expression of Kv3.4 channels with proliferation of human uterine VSMCs. Here, we sought to gain deeper insight on the relationship between Kv3.4 channels and cell cycle progression in this preparation. Methods and results: Expression and function of Kv3.4 channels along the cell cycle was explored in uterine VSMCs synchronized at different checkpoints, combining real-time PCR, western blotting, and electrophysiological techniques. Flow cytometry, Ki67 expression and BrdU incorporation techniques allowed us to explore the effects of Kv3.4 channels blockade on cell cycle distribution. We found cyclic changes in Kv3.4 and MiRP2 mRNA and protein expression along the cell cycle. Functional studies showed that Kv3.4 current amplitude and Kv3.4 channels contribution to cell excitability increased in proliferating cells. Finally, both Kv3.4 blockers and Kv3.4 knockdown with siRNA reduced the proportion of proliferating VSMCs. Conclusion: Our data indicate that Kv3.4 channels exert a permissive role in the cell cycle progression of proliferating uterine VSMCs, as their blockade induces cell cycle arrest after G2/M phase completion. The modulation of resting membrane potential (VM) by Kv3.4 channels in proliferating VSMCs suggests that their role in cell cycle progression could be at least in part mediated by their contribution to the hyperpolarizing signal needed to progress through the G1 phase., Ministerio de Sanidad, Consumo y Bienestar Social - Instituto de Salud Carlos III (grants R006/009 and PI041044), Ministerio de Ciencia, Innovación y Universidades (grants BFU2004-05551 and BFU2007-61524), Junta de Castilla y León (grant GR242)

Producción Científica, Objective: Vascular smooth muscle cells (VSMCs) contribute significantly to occlusive vascular diseases by virtue of their ability to switch to a noncontractile, migratory, and proliferating phenotype. Although the participation of ion channels in this phenotypic modulation (PM) has been described previously, changes in their expression are poorly defined because of their large molecular diversity. We obtained a global portrait of ion channel expression in contractile versus proliferating mouse femoral artery VSMCs, and explored the functional contribution to the PM of the most relevant changes that we observed. Methods and Results: High-throughput real-time polymerase chain reaction of 87 ion channel genes was performed in 2 experimental paradigms: an in vivo model of endoluminal lesion and an in vitro model of cultured VSMCs obtained from explants. mRNA expression changes showed a good correlation between the 2 proliferative models, with only 2 genes, Kv1.3 and Kvβ2, increasing their expression on proliferation. The functional characterization demonstrates that Kv1.3 currents increased in proliferating VSMC and that their selective blockade inhibits migration and proliferation. Conclusion: These findings establish the involvement of Kv1.3 channels in the PM of VSMCs, providing a new therapeutical target for the treatment of intimal hyperplasia., Ministerio de Sanidad, Consumo y Bienestar Social - Instituto de Salud Carlos III (grants R006/009, FS041139-0 and PI041044), Ministerio de Ciencia, Innovación y Universidades (grants BFU2004-05551 and BFU2007-61524), Junta de Castilla y León (grant GR242)

Producción Científica, Aims: Vascular smooth muscle cell (VSMC) proliferation is involved in cardiovascular pathologies associated with unwanted arterial wall remodelling. Coordinated changes in the expression of several K+ channels have been found to be important elements in the phenotypic switch of VSMCs towards proliferation. We have previously demonstrated the association of functional expression of Kv3.4 channels with proliferation of human uterine VSMCs. Here, we sought to gain deeper insight on the relationship between Kv3.4 channels and cell cycle progression in this preparation. Methods and results: Expression and function of Kv3.4 channels along the cell cycle was explored in uterine VSMCs synchronized at different checkpoints, combining real-time PCR, western blotting, and electrophysiological techniques. Flow cytometry, Ki67 expression and BrdU incorporation techniques allowed us to explore the effects of Kv3.4 channels blockade on cell cycle distribution. We found cyclic changes in Kv3.4 and MiRP2 mRNA and protein expression along the cell cycle. Functional studies showed that Kv3.4 current amplitude and Kv3.4 channels contribution to cell excitability increased in proliferating cells. Finally, both Kv3.4 blockers and Kv3.4 knockdown with siRNA reduced the proportion of proliferating VSMCs. Conclusion: Our data indicate that Kv3.4 channels exert a permissive role in the cell cycle progression of proliferating uterine VSMCs, as their blockade induces cell cycle arrest after G2/M phase completion. The modulation of resting membrane potential (VM) by Kv3.4 channels in proliferating VSMCs suggests that their role in cell cycle progression could be at least in part mediated by their contribution to the hyperpolarizing signal needed to progress through the G1 phase., Ministerio de Sanidad, Consumo y Bienestar Social - Instituto de Salud Carlos III (grants R006/009 and PI041044), Ministerio de Ciencia, Innovación y Universidades (grants BFU2004-05551 and BFU2007-61524), Junta de Castilla y León (grant GR242)

Producción Científica, Aims: Vascular smooth muscle cell (VSMC) proliferation is involved in cardiovascular pathologies associated with unwanted arterial wall remodelling. Coordinated changes in the expression of several K+ channels have been found to be important elements in the phenotypic switch of VSMCs towards proliferation. We have previously demonstrated the association of functional expression of Kv3.4 channels with proliferation of human uterine VSMCs. Here, we sought to gain deeper insight on the relationship between Kv3.4 channels and cell cycle progression in this preparation. Methods and results: Expression and function of Kv3.4 channels along the cell cycle was explored in uterine VSMCs synchronized at different checkpoints, combining real-time PCR, western blotting, and electrophysiological techniques. Flow cytometry, Ki67 expression and BrdU incorporation techniques allowed us to explore the effects of Kv3.4 channels blockade on cell cycle distribution. We found cyclic changes in Kv3.4 and MiRP2 mRNA and protein expression along the cell cycle. Functional studies showed that Kv3.4 current amplitude and Kv3.4 channels contribution to cell excitability increased in proliferating cells. Finally, both Kv3.4 blockers and Kv3.4 knockdown with siRNA reduced the proportion of proliferating VSMCs. Conclusion: Our data indicate that Kv3.4 channels exert a permissive role in the cell cycle progression of proliferating uterine VSMCs, as their blockade induces cell cycle arrest after G2/M phase completion. The modulation of resting membrane potential (VM) by Kv3.4 channels in proliferating VSMCs suggests that their role in cell cycle progression could be at least in part mediated by their contribution to the hyperpolarizing signal needed to progress through the G1 phase., Ministerio de Sanidad, Consumo y Bienestar Social - Instituto de Salud Carlos III (grants R006/009 and PI041044), Ministerio de Ciencia, Innovación y Universidades (grants BFU2004-05551 and BFU2007-61524), Junta de Castilla y León (grant GR242)

Producción Científica, Aims: Vascular smooth muscle cell (VSMC) proliferation is involved in cardiovascular pathologies associated with unwanted arterial wall remodelling. Coordinated changes in the expression of several K+ channels have been found to be important elements in the phenotypic switch of VSMCs towards proliferation. We have previously demonstrated the association of functional expression of Kv3.4 channels with proliferation of human uterine VSMCs. Here, we sought to gain deeper insight on the relationship between Kv3.4 channels and cell cycle progression in this preparation. Methods and results: Expression and function of Kv3.4 channels along the cell cycle was explored in uterine VSMCs synchronized at different checkpoints, combining real-time PCR, western blotting, and electrophysiological techniques. Flow cytometry, Ki67 expression and BrdU incorporation techniques allowed us to explore the effects of Kv3.4 channels blockade on cell cycle distribution. We found cyclic changes in Kv3.4 and MiRP2 mRNA and protein expression along the cell cycle. Functional studies showed that Kv3.4 current amplitude and Kv3.4 channels contribution to cell excitability increased in proliferating cells. Finally, both Kv3.4 blockers and Kv3.4 knockdown with siRNA reduced the proportion of proliferating VSMCs. Conclusion: Our data indicate that Kv3.4 channels exert a permissive role in the cell cycle progression of proliferating uterine VSMCs, as their blockade induces cell cycle arrest after G2/M phase completion. The modulation of resting membrane potential (VM) by Kv3.4 channels in proliferating VSMCs suggests that their role in cell cycle progression could be at least in part mediated by their contribution to the hyperpolarizing signal needed to progress through the G1 phase., Ministerio de Sanidad, Consumo y Bienestar Social - Instituto de Salud Carlos III (grants R006/009 and PI041044), Ministerio de Ciencia, Innovación y Universidades (grants BFU2004-05551 and BFU2007-61524), Junta de Castilla y León (grant GR242)

Producción Científica, Vascular smooth muscle cells (VSMCs) perform diverse functions that can be classified into contractile and synthetic (or proliferating). All of these functions can be fulfilled by the same cell because of its capacity of phenotypic modulation in response to environmental changes. The resting membrane potential is a key determinant for both contractile and proliferating functions. Here, we have explored the expression of voltage-dependent K+ (Kv) channels in contractile (freshly dissociated) and proliferating (cultured) VSMCs obtained from human uterine arteries to establish their contribution to the functional properties of the cells and their possible participation in the phenotypic switch. We have studied the expression pattern (both at the mRNA and at the protein level) of Kvα subunits in both preparations as well as their functional contribution to the K+ currents of VSMCs. Our results indicate that phenotypic remodeling associates with a change in the expression and distribution of Kv channels. Whereas Kv currents in contractile VSMCs are mainly performed by Kv1 channels, Kv3.4 is the principal contributor to K+ currents in cultured VSMCs. Furthermore, selective blockade of Kv3.4 channels resulted in a reduced proliferation rate, suggesting a link between Kv channels expression and phenotypic remodeling., Ministerio de Sanidad, Consumo y Bienestar Social - Instituto de Salud Carlos III (grants R006/009. FS041139-0 and PI041044), Ministerio de Ciencia, Innovación y Universidades (grants BFU2004-05551 and BFU2007-61524), Junta de Castilla y León (grant GR242)