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

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