Contribution of Kv channels to phenotypic remodeling of human uterine artery smooth muscle cells

Producción CientíficaVascular 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

Cell cycle-dependent expression of Kv3.4 channels modulates proliferation of human uterine artery smooth muscle cells

Producción CientíficaAims: 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

Kv1.3 channels can modulate cell proliferation during phenotypic switch by an ion-flux independent mechanism

Producción CientíficaObjective: 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

Seminarios multimedia de implanto-prótesis y patología oclusal

Memoria ID-0116. Ayudas de la Universidad de Salamanca para la innovación docente, curso 2014-2015

Creación de una web del Grupo de Investigación: avances en Salud Oral

Memoria ID-0135. Ayudas de la Universidad de Salamanca para la innovación docente, curso 2015-2016

Cell cycle-dependent expression of Kv3.4 channels modulates proliferation of human uterine artery smooth muscle cells

9 páginas, 6 figuras.[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.Work supported by Ministerio de Sanidad y Consumo, Instituto de Salud Carlos III [R006/009-Heracles network, PI041044 to J.R.L.L.], Ministerio de Educación y Ciencia [BFU2004-05551 to M.T.P.G.; BFU2007-61524 to J.R.L.L.], predoctoral fellowship to OC], and Junta de Castilla y León [GR242].Peer reviewe

Role of KV1.3 channels in intimal hyperlasia

Resumen del póster presentado al American College of Cardiology - 61st Annual Scientific Seesion & Expo celebrado en Chicago (US) del 24 al 27 de marzo de 2012.[Background]: Vascular smooth muscle cells (VSMCs) are able to switch from a contractile to a proliferative phenotype, and this process is central to intimal hyperplasia formation. Phenotypic modulation requires a change in gene expression profile, including a switch in ion transport mechanisms. Potassium (K+) channels have been suggested to have a role in the processes of cell proliferation. Changes in K+ channels expression are associated with functional changes in the electrophysiological properties of VSMCs, which are linked to cell growth. We have previously obtained a global portrait of ion channel expression in contractile versus proliferating VSMCs in different vascular beds, in vitro and in vivo, and have identified a marked increase in Kv1.3 mRNA expression during the switch to a proliferative phenotype. The aim of our study is to investigate the effect of the selective blockade of Kv1.3 channels in VSMC proliferation. For this purpose, we have selected PAP-1 (phenoxyalkoxypsoralen-1), a potent suppressor of T cells proliferation in vitro, which inhibits Kv1.3 with a high selectivity over other K+ channels. [Methods]: Porcine coronary SMCs were isolated and cell proliferation analysis was measured with a BrdU incorporation assay. Using an arterial injury model previously validated by our group, an endothelial denudation injury was induced to murine femoral arteries. A constant infusion of PAP-1 (50 microg/Kg) was administered after injury, through the subcutaneous implant of Alzet osmotic mini-pumps, during 28 days, time-point at which animals were euthanized and arterial segments collected for morphometric and immunohistochemical analysis. [Results]: PAP-1 (10 nM) significantly reduced cell proliferation versus control cells (13,2±1% vs. 18±1%, p<0,05). Intimal proliferation was reduced in PAP-1-treated animals, compared with control, vehicle-treated, mice (Intima-to-media ratios of 0,18±0,11 vs. 1,06±0,40; p=0,01). [Conclusion]: The selective blockade of Kv1.3 channels decreases in vitro VSMCs proliferation and in vivo intimal hyperplasia formation. Our results point to Kv1.3 channels as a new promising therapeutical target to avoid restenosis.Peer reviewe

Kv1.3 channels can modulate cell proliferation during phenotypic switch by an ion-flux independent mechanism

[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 voltage-dependence 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. © 2012 American Heart Association, Inc.Supported by Ministerio de Sanidad, ISCIII grant R006/009 (Red Heracles), Ministerio de Ciencia e Innovación grant BFU2010-15898 (M.T.P.G.) and Junta de Castilla y León grant VA094A11-2 (J.R.L.L.). L.J.M. and S.T. are fellows of the Spanish MICINN and C.R.M. is a fellow of CSIC (JAE-predoc program).Peer Reviewe

Kv1.3 channels modulate human vascular smooth muscle cells proliferation independently of mTOR signaling pathway

Phenotypic modulation (PM) of vascular smooth muscle cells (VSMCs) is central to the process of intimal hyperplasia which constitutes a common pathological lesion in occlusive vascular diseases. Changes in the functional expression of Kv1.5 and Kv1.3 currents upon PM in mice VSMCs have been found to contribute to cell migration and proliferation. Using human VSMCs from vessels in which unwanted remodeling is a relevant clinical complication, we explored the contribution of the Kv1.5 to Kv1.3 switch to PM. Changes in the expression and the functional contribution of Kv1.3 and Kv1.5 channels were studied in contractile and proliferating VSMCs obtained from human donors. Both a Kv1.5 to Kv1.3 switch upon PM and an anti-proliferative effect of Kv1.3 blockers on PDGF-induced proliferation were observed in all vascular beds studied. When investigating the signaling pathways modulated by the blockade of Kv1.3 channels, we found that anti-proliferative effects of Kv1.3 blockers on human coronary artery VSMCs were occluded by selective inhibition of MEK/ERK and PLCγ signaling pathways, but were unaffected upon blockade of PI3K/mTOR pathway. The temporal course of the anti-proliferative effects of Kv1.3 blockers indicates that they have a role in the late signaling events essential for the mitogenic response to growth factors. These findings establish the involvement of Kv1.3 channels in the PM of human VSMCs. Moreover, as current therapies to prevent restenosis rely on mTOR blockers, our results provide the basis for the development of novel, more specific therapies.Supported by grants from the Ministerio de Economía y Competitividad, Instituto de Salud Carlos III (RIC RD12/0042/0006, Red Heracles), Ministerio de Ciencia e Innovación (BFU2010-15898 to MTPG), Junta de Castilla y León (VA094A11-2 to, JRLL), and Fondo de Investigaciones Sanitarias (FIS PI11/00225 to MR)Peer Reviewe