Characterization of Ion Channels Involved in the Proliferative Response of Femoral Artery Smooth Muscle Cells

31 páginas, 6 figuras, adicionales 6 figuras y 3 tablas.[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.This work was supported by Ministerio de Sanidad, Instituto de Salud Carlos III grants R006/009 (Red Heracles), FS041139-0 (M.R.), and PI041044 (J.R.L.-L.); Ministerio de Educacio´n y Ciencia grants BFU2004-05551 (M.T.P.-G.) and BFU2007-61524 (J.R.L.-L.); and Junta de Castilla y Leon grant GR242. Dr Moreno-Domínguez is a fellow of the Spanish Ministerio de Educacion y Ciencia.Peer reviewe

Characterization of ion channels involved in the proliferative response of femoral artery smooth muscle cells

Producción CientíficaObjective: 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

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

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

et al.[Introduction]: K+ 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.This work was funded by the Ministerio de Economia y Competitividad, Instituto de Salud Carlos III, (RIC RD12/0042/0006, Red Heracles) to MR, MH, MTP and JRL; FIS-Instituto Carlos III, PI11/00225 to MR, VALTEC 09-1-0042 to MR, a grant from the Spanish Heart Association to MR., Ministerio de Ciencia e Innovación grant BFU2010-15898 to MTPG and, Junta de Castilla y León grant VA094A11-2 to, JRLLPeer Reviewe

Transcription factor KLF6 upregulates expression of metalloprotease MMP14 and subsequent release of soluble endoglin during vascular injury

17 p.-8 fig.After endothelial injury, the transcription factor Kru¨ppel-like factor 6 (KLF6) translocates into the cell nucleus to regulate a variety of target genes involved in angiogenesis, vascular repair and remodeling, including components of the membrane transforming growth factor beta (TGF-b) receptor complex such as endoglin and activin receptor-like kinase 1. The membrane metalloproteinase 14 (MMP14 or MT1-MMP) targets endoglin to release soluble endoglin and is involved in vascular inflammation and endothelial tubulogenesis. However, little is known about the regulation of MMP14 expression during vascular wounding. In vitro denudation of monolayers of human endothelial cell monolayers leads to an increase in the KLF6 gene transcriptional rate, followed by an upregulation of MMP14 and release of soluble endoglin. Concomitant with this process, MMP14 co-localizes with endoglin in the sprouting endothelial cells surrounding the wound border. MMP14 expression at mRNA and protein levels is increased by ectopic KLF6 and downregulated by KLF6 suppression in cultured endothelial cells. Moreover, after wire-induced endothelial denudation, Klf6?/- mice show lower levels of MMP14 in their vasculature compared with their wild-type siblings. Ectopic cellular expression of KLF6 results in an increased transcription rate of MMP14, and chromatin immunoprecipitation assays show that KLF6 interacts with MMP14 promoter in ECs, this interaction being enhanced during wound healing. Furthermore, KLF6 markedly increases the transcriptional activity of different reporter constructs of MMP14 gene promoter. These results suggest that KLF6 regulates MMP14 transcription and is a critical player of the gene expression network triggered during endothelial repair.This study was funded by grants from Ministerio de Economia y Competitividad of Spain (SAF2013-43421-R to CB and SAF2011-23475 to LMB), Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER; ISCIII-CB06/07/0038 and ER16PIAC707 to CB), Red de Investigación Cooperativa en Salud (RD12/0042/0006 to MR), National Institutes of Health (NIH; DK37340 and DK56621 to SLF) and FEDER funds.MINECO/ICTI2013-2016/SAF2013-43421-RPeer 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

Vascular injury triggers Krüppel-like factor 6 mobilization and cooperation with specificity protein 1 to promote endothelial activation through upregulation of the activin receptor-like kinase 1 gene

29 p.-8 figRATIONALE: Activin receptor-like kinase-1 (ALK1) is an endothelial transforming growth factor β receptor involved in angiogenesis. ALK1 expression is high in the embryo vasculature, becoming less detectable in the quiescent endothelium of adult stages. However, ALK1 expression becomes rapidly increased after angiogenic stimuli such as vascular injury. OBJECTIVE: To characterize the molecular mechanisms underlying the regulation of ALK1 on vascular injury. METHODS AND RESULTS: Alk1 becomes strongly upregulated in endothelial (EC) and vascular smooth muscle cells of mouse femoral arteries after wire-induced endothelial denudation. In vitro denudation of monolayers of human umbilical vein ECs also leads to an increase in ALK1. Interestingly, a key factor in tissue remodeling, Krüppel-like factor 6 (KLF6) translocates to the cell nucleus during wound healing, concomitantly with an increase in the ALK1 gene transcriptional rate. KLF6 knock down in human umbilical vein ECs promotes ALK1 mRNA downregulation. Moreover, Klf6(+/-) mice have lower levels of Alk1 in their vasculature compared with their wild-type siblings. Chromatin immunoprecipitation assays show that KLF6 interacts with ALK1 promoter in ECs, and this interaction is enhanced during wound healing. We demonstrate that KLF6 is transactivating ALK1 gene, and this transactivation occurs by a synergistic cooperative mechanism with specificity protein 1. Finally, Alk1 levels in vascular smooth muscle cells are not directly upregulated in response to damage, but in response to soluble factors, such as interleukin 6, released from ECs after injury. CONCLUSIONS: ALK1 is upregulated in ECs during vascular injury by a synergistic cooperative mechanism between KLF6 and specificity protein 1, and in vascular smooth muscle cells by an EC-vascular smooth muscle cell paracrine communication during vascular remodeling.This work was supported by the Ministerio de Ciencia e Innovación of Spain (MICINN grants SAF2007-61827 and SAF2010-19222 to C.B. and SAF2008-01218 to L.M.B., and predoctoral fellowship BES-2005–7974 to E.M.G-M), Genoma España (MEICA), Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Red HERACLES (RD6/009/0008 to M.R.), and National Institutes of Health & National Center for Research Resources (DK56621 and DK37340 to S.L.F.).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

Kv1.3 blockade inhibits proliferation of vascular smooth muscle cells in vitro and intimal hyperplasia in vivo

© 2020 Published by Elsevier IncThe modulation of voltage-gated K+ (Kv) channels, involved in cell proliferation, arises as a potential therapeutic approach for the prevention of intimal hyperplasia present in in-stent restenosis (ISR) and allograft vasculopathy (AV). We studied the effect of PAP-1, a selective blocker of Kv1.3 channels, on development of intimal hyperplasia in vitro and in vivo in 2 porcine models of vascular injury. In vitro phenotypic modulation of VSMCs was associated to an increased functional expression of Kv1.3 channels, and only selective Kv1.3 channel blockers were able to inhibit porcine VSMC proliferation. The therapeutic potential of PAP-1 was then evaluated in vivo in swine models of ISR and AV. At 15-days follow-up, morphometric analysis demonstrated a substantial reduction of luminal stenosis in the allografts treated with PAP-1 (autograft 2.72 ± 1.79 vs allograft 10.32 ± 1.92 vs allograft + polymer 13.54 ± 8.59 vs allograft + polymer + PAP-1 3.06 ± 1.08 % of luminal stenosis; P = 0.006) in the swine model of femoral artery transplant. In the pig model of coronary ISR, using a prototype of PAP-1-eluting stent, no differences were observed regarding % of stenosis compared to control stents (31 ± 13 % vs 37 ± 18%, respectively; P = 0.372) at 28-days follow-up. PAP-1 treatment was safe and did not impair vascular healing in terms of delayed endothelialization, inflammation or thrombosis. However, an incomplete release of PAP-1 from stents was documented. We conclude that the use of selective Kv1.3 blockers represents a promising therapeutic approach for the prevention of intimal hyperplasia in AV, although further studies to improve their delivery method are needed to elucidate its potential in ISR.This work was supported by a Translational Research Project on Cardiology (2015) of the Sociedad Española de Cardiología and Fondo de Investigación Sanitario Instituto de Salud Carlos III (PI11/00225) to M. Roqué; BFU2016-75360-R from the Ministerio de Economía y Competitividad (MINECO) to M.T. Pérez-García and J.R. López-López; Junta de Castilla y León Grant VA114P17 to M.T. Pérez-García; and the CERCA Programme of the Generalitat de Catalunya.Peer reviewe

Benefit of primary percutaneous coronary interventions in the elderly with ST segment elevation myocardial infarction

Objective: Primary percutaneous coronary intervention (P-PCI) has demonstrated its efficacy in patients with ST segment elevation myocardial infarction (STEMI). However, patients with STEMI ≥75 years receive less P-PCI than younger patients despite their higher in-hospital morbimortality. The objective of this analysis was to determine the effectiveness of P-PCI in patients with STEMI ≥75 years. Methods: We included 979 patients with STEMI ≥75 years, from the ATención HOspitalaria del Síndrome coronario study, a registry of 8142 consecutive patients with acute coronary syndrome admitted at 31 Spanish hospitals in 2014-2016. We calculated a propensity score (PS) for the indication of P-PCI. Patients that received or not P-PCI were matched by PS. Using logistic regression, we compared the effectiveness of performing P-PCI versus non-performance for the composite primary event, which included death, reinfarction, acute pulmonary oedema or cardiogenic shock during hospitalisation. Results: Of the included patients, 81.5 % received P-PCI. The matching provided two groups of 169 patients with and without P-PCI. Compared with its non-performance, P-PCI presented a composite event OR adjusted by PS of 0.55 (95% CI 0.34 to 0.89). Conclusions: Receiving a P-PCI was significantly associated with a reduced risk of major intrahospital complications in patients with STEMI aged 75 years or older