Characterization of endogenous Kv1.3 channel isoforms in T cells.

Voltage-dependent potassium channel Kv1.3 plays a key role on T-cell activation; however, lack of reliable antibodies has prevented its accurate detection under endogenous circumstances. To overcome this limitation, we created a Jurkat T-cell line with endogenous Kv1.3 channel tagged, to determine the expression, location, and changes upon activation of the native Kv1.3 channels. CRISPR-Cas9 technique was used to insert a Flag-Myc peptide at the C terminus of the KCNA3 gene. Basal or activated channel expression was studied using western blot analysis and imaging techniques. We identified two isoforms of Kv1.3 other than the canonical channel (54 KDa) differing on their N terminus: a longer isoform (70 KDa) and a truncated isoform (43 KDa). All three isoforms were upregulated after T-cell activation. We focused on the functional characterization of the truncated isoform (short form, SF), because it has not been previously described and could be present in the available Kv1.3-/- mice models. Overexpression of SF in HEK cells elicited small amplitude Kv1.3-like currents, which, contrary to canonical Kv1.3, did not induce HEK proliferation. To explore the role of endogenous SF isoform in a native system, we generated both a knockout Jurkat clone and a clone expressing only the SF isoform. Although the canonical isoform (long form) localizes mainly at the plasma membrane, SF remains intracellular, accumulating perinuclearly. Accordingly, SF Jurkat cells did not show Kv1.3 currents and exhibited depolarized resting membrane potential (VM ), decreased Ca2+ influx, and a reduction in the [Ca2+ ]i increase upon stimulation. Functional characterization of these Kv1.3 channel isoforms showed their differential contribution to signaling pathways involved in formation of the immunological synapse. We conclude that alternative translation initiation generates at least three endogenous Kv1.3 channel isoforms in T cells that exhibit different functional roles. For some of these functions, Kv1.3 proteins do not need to form functional plasma membrane channels.

Vascular smooth muscle ion channels in essential hypertension.

Hypertension is a highly prevalent chronic disease and the major risk factor for cardiovascular diseases, the leading cause of death worldwide. Hypertension is characterized by an increased vascular tone determined by the contractile state of vascular smooth muscle cells that depends on intracellular calcium levels. The interplay of ion channels determine VSMCs membrane potential and thus intracellular calcium that controls the degree of contraction, vascular tone and blood pressure. Changes in ion channels expression and function have been linked to hypertension, but the mechanisms and molecular entities involved are not completely clear. Furthermore, the literature shows discrepancies regarding the contribution of different ion channels to hypertension probably due to differences both in the vascular preparation and in the model of hypertension employed. Animal models are essential to study this multifactorial disease but it is also critical to know their characteristics to interpret properly the results obtained. In this review we summarize previous studies, using the hypertensive mouse (BPH) and its normotensive control (BPN), focused on the identified changes in the expression and function of different families of ion channels. We will focus on L-type voltage-dependent Ca2+ channels (Cav1.2), canonical transient receptor potential channels and four different classes of K+ channels: voltage-activated (Kv), large conductance Ca2+-activated (BK), inward rectifiers (Kir) and ATP-sensitive (KATP) K+ channels. We will describe the role of these channels in hypertension and we will discuss the importance of integrating individual changes in a global context to understand the complex interplay of ion channels in hypertension.

miR-126 contributes to the epigenetic signature of diabetic vascular smooth muscle and enhances antirestenosis effects of Kv1.3 blockers.

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. RESUKTS: 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.

Kv1.3 Channel Inhibition Limits Uremia-Induced Calcification in Mouse and Human Vascular Smooth Muscle.

Chronic kidney disease (CKD) significantly increases cardiovascular risk. In advanced CKD stages, accumulation of toxic circulating metabolites and mineral metabolism alterations triggers vascular calcification, characterized by vascular smooth muscle cell (VSMC) transdifferentiation and loss of the contractile phenotype. Phenotypic modulation of VSMC occurs with significant changes in gene expression. Even though ion channels are an integral component of VSMC function, the effects of uremia on ion channel remodeling has not been explored. We used an in vitro model of uremia-induced calcification of human aorta smooth muscle cells (HASMCs) to study the expression of 92 ion channel subunit genes. Uremic serum-induced extensive remodeling of ion channel expression consistent with loss of excitability but different from the one previously associated with transition from contractile to proliferative phenotypes. Among the ion channels tested, we found increased abundance and activity of voltage-dependent K+ channel Kv1.3. Enhanced Kv1.3 expression was also detected in aorta from a mouse model of CKD. Pharmacological inhibition or genetic ablation of Kv1.3 decreased the amount of calcium phosphate deposition induced by uremia, supporting an important role for this channel on uremia-induced VSMC calcification.

Voltage-dependent conformational changes of Kv1.3 channels activate cell proliferation.

The voltage-dependent potassium channel Kv1.3 has been implicated in proliferation in many cell types, based on the observation that Kv1.3 blockers inhibited proliferation. By modulating membrane potential, cell volume, and/or Ca2+ influx, K+  channels can influence cell cycle progression. Also, noncanonical channel functions could contribute to modulate cell proliferation independent of K+ efflux. The specificity of the requirement of Kv1.3 channels for proliferation suggests the involvement of molecule-specific interactions, but the underlying mechanisms are poorly identified. Heterologous expression of Kv1.3 channels in HEK cells has been shown to increase proliferation independently of K+ fluxes. Likewise, some of the molecular determinants of Kv1.3-induced proliferation have been located in the C-terminus region, where individual point mutations of putative phosphorylation sites (Y447A and S459A) abolished Kv1.3-induced proliferation. Here, we investigated the mechanisms linking Kv1.3 channels to proliferation exploring the correlation between Kv1.3 voltage-dependent molecular dynamics and cell cycle progression. Using transfected HEK cells, we analyzed both the effect of changes in resting membrane potential on Kv1.3-induced proliferation and the effect of mutated Kv1.3 channels with altered voltage dependence of gating. We conclude that voltage-dependent transitions of Kv1.3 channels enable the activation of proliferative pathways. We also found that Kv1.3 associated with IQGAP3, a scaffold protein involved in proliferation, and that membrane depolarization facilitates their interaction. The functional contribution of Kv1.3-IQGAP3 interplay to cell proliferation was demonstrated both in HEK cells and in vascular smooth muscle cells. Our data indicate that voltage-dependent conformational changes of Kv1.3 are an essential element in Kv1.3-induced proliferation.

Elastin-like recombinamer-based devices releasing Kv1.3 blockers for the prevention of intimal hyperplasia: An in vitro and in vivo study.

Coronary artery disease (CAD) is the most common cardiovascular disorder. Vascular surgery strategies for coronary revascularization (either percutaneous or open) show a high rate of failure because of restenosis of the vessel, due to phenotypic switch of vascular smooth muscle cells (VSMCs) leading to proliferation and migration. We have previously reported that the inhibition of Kv1.3 channel function with selective blockers represents an effective strategy for the prevention of restenosis in human vessels used for coronary angioplasty procedures. However, delivery systems for controlled release of these drugs have not been investigated. Here we tested the efficacy of several formulations of elastin like recombinamers (ELRs) hydrogels to deliver the Kv1.3 blocker PAP-1 in various restenosis models. The dose and time course of PAP-1 release from ELRs click hydrogels was able to inhibit human VSMC proliferation in vitro as well as remodeling of human vessels in organ culture and restenosis in in vivo models. We conclude that this combination of active compound and advanced delivery method could improve the outcomes of vascular surgery in patients. STATEMENT OF SIGNIFICANCE: Vascular surgery strategies for coronary revascularization show a high rate of failure, because of occlusion (restenosis) of the vessel, due to vascular smooth muscle cells proliferation and migration. We have previously reported that blockers of Kv1.3 channels represent an effective anti-restenosis therapy, but delivery systems for their controlled release have not being explored. Here we tested the efficacy of several formulations of elastin like recombinamers (ELRs) hydrogels to deliver the Kv1.3 blocker PAP-1 in various restenosis models, both in vivo and in vitro, and also in human vessels. We demonstrated that combination of active compound and advanced delivery method could improve the outcomes of vascular surgery in patients.

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

The 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.

Association of Circulating microRNAs with Coronary Artery Disease and Usefulness for Reclassification of Healthy Individuals: The REGICOR Study.

Risk prediction tools cannot identify most individuals at high coronary artery disease (CAD) risk. Oxidized low-density lipoproteins (oxLDLs) and microRNAs are actively involved in atherosclerosis. Our aim was to examine the association of CAD and oxLDLs-induced microRNAs, and to assess the microRNAs predictive capacity of future CAD events. Human endothelial and vascular smooth muscle cells were treated with oxidized/native low-density lipoproteins, and microRNA expression was analyzed. Differentially expressed and CAD-related miRNAs were examined in serum samples from (1) a case-control study with 476 myocardial infarction (MI) patients and 487 controls, and (2) a case-cohort study with 105 incident CAD cases and 455 randomly-selected cohort participants. MicroRNA expression was analyzed with custom OpenArray plates, log rank tests and Cox regression models. Twenty-one microRNAs, two previously undescribed (hsa-miR-193b-5p and hsa-miR-1229-5p), were up- or down-regulated upon cell treatment with oxLDLs. One of the 21, hsa-miR-122-5p, was also upregulated in MI cases (fold change = 4.85). Of the 28 CAD-related microRNAs tested, 11 were upregulated in MI cases -1 previously undescribed (hsa-miR-16-5p)-, and 1/11 was also associated with CAD incidence (adjusted hazard ratio = 0.55 (0.35-0.88)) and improved CAD risk reclassification, hsa-miR-143-3p. We identified 2 novel microRNAs modulated by oxLDLs in endothelial cells, 1 novel microRNA upregulated in AMI cases compared to controls, and one circulating microRNA that improved CAD risk classification.

Myocardin-Dependent Kv1.5 Channel Expression Prevents Phenotypic Modulation of Human Vessels in Organ Culture.

OBJECTIVE: We have previously described that changes in the expression of Kv channels associate to phenotypic modulation (PM), so that Kv1.3/Kv1.5 ratio is a landmark of vascular smooth muscle cells phenotype. Moreover, we demonstrated that the Kv1.3 functional expression is relevant for PM in several types of vascular lesions. Here, we explore the efficacy of Kv1.3 inhibition for the prevention of remodeling in human vessels, and the mechanisms linking the switch in Kv1.3 /Kv1.5 ratio to PM. Approach and Results: Vascular remodeling was explored using organ culture and primary cultures of vascular smooth muscle cells obtained from human vessels. We studied the effects of Kv1.3 inhibition on serum-induced remodeling, as well as the impact of viral vector-mediated overexpression of Kv channels or myocardin knock-down. Kv1.3 blockade prevented remodeling by inhibiting proliferation, migration, and extracellular matrix secretion. PM activated Kv1.3 via downregulation of Kv1.5. Hence, both Kv1.3 blockers and Kv1.5 overexpression inhibited remodeling in a nonadditive fashion. Finally, myocardin knock-down induced vessel remodeling and Kv1.5 downregulation and myocardin overexpression increased Kv1.5, while Kv1.5 overexpression inhibited PM without changing myocardin expression. CONCLUSIONS: We demonstrate that Kv1.5 channel gene is a myocardin-regulated, vascular smooth muscle cells contractile marker. Kv1.5 downregulation upon PM leaves Kv1.3 as the dominant Kv1 channel expressed in dedifferentiated cells. We demonstrated that the inhibition of Kv1.3 channel function with selective blockers or by preventing Kv1.5 downregulation can represent an effective, novel strategy for the prevention of intimal hyperplasia and restenosis of the human vessels used for coronary angioplasty procedures.

Pancreatic ß-cell-specific deletion of insulin-degrading enzyme leads to dysregulated insulin secretion and ß-cell functional immaturity.

Inhibition of insulin-degrading enzyme (IDE) has been proposed as a possible therapeutic target for type 2 diabetes treatment. However, many aspects of IDE's role in glucose homeostasis need to be clarified. In light of this, new preclinical models are required to elucidate the specific role of this protease in the main tissues related to insulin handling. To address this, here we generated a novel line of mice with selective deletion of the Ide gene within pancreatic beta-cells, B-IDE-KO mice, which have been characterized in terms of multiple metabolic end points, including blood glucose, plasma C-peptide, and intraperitoneal glucose tolerance tests. In addition, glucose-stimulated insulin secretion was quantified in isolated pancreatic islets and beta-cell differentiation markers and insulin secretion machinery were characterized by RT-PCR. Additionally, IDE was genetically and pharmacologically inhibited in INS-1E cells and rodent and human islets, and insulin secretion was assessed. Our results show that, in vivo, life-long deletion of IDE from beta-cells results in increased plasma C-peptide levels. Corroborating these findings, isolated islets from B-IDE-KO mice showed constitutive insulin secretion, a hallmark of beta-cell functional immaturity. Unexpectedly, we found 60% increase in Glut1 (a high-affinity/low-Km glucose transporter), suggesting increased glucose transport into the beta-cell at low glucose levels, which may be related to constitutive insulin secretion. In parallel, IDE inhibition in INS-1E and islet cells resulted in impaired insulin secretion after glucose challenge. We conclude that IDE is required for glucose-stimulated insulin secretion. When IDE is inhibited, insulin secretion machinery is perturbed, causing either inhibition of insulin release at high glucose concentrations or constitutive secretion.

Dynamic changes of the extracellular matrix during corneal wound healing.

The extracellular matrix (ECM) confers transparency to the cornea because of the precise organization of collagen fibrils and a wide variety of proteoglycans. We monitored the corneal wound healing process after alkali burns in rabbits. We analyzed the location and expression of collagens and proteoglycans, the clinical impact, and the recovery of optical transparency. After the animals received both general and ocular topical anesthesia, the central cornea of the left eye was burned by placing an 8-mm diameter filter paper soaked in 0.5 N NaOH for 60 s. The eyes were evaluated under a surgical microscope at 1, 3, and 6 months after burning. At each time point, the clinical conditions of the burned and control corneas were observed. The arrangement of collagen fibers in the corneal stroma was visualized by Picrosirius-red staining, Gomori's silver impregnation and transmission electronic microscopy. Corneal light transmittance was also measured. Myofibroblasts presence was analyzed by immunohistochemistry. mRNA expression levels of collagen types I and III, lumican, decorin, keratocan and alpha-smooth muscle actin were determined by quantitative real-time polymerase chain reaction. One month after alkali burn, the ECM was disorganized and filled with lacunae containing different types of cells and collagen type III fibers in the wound area. Corneal opacities were present with attendant loss of light transmittance. Collagen and proteoglycan mRNA expression levels were up-regulated. After three months, wound healing progress was indicated by reduced corneal opacity, increased light transmittance, reorganization of collagen fibers and only collagen type I expression levels were at control levels. After six months, the wound area ECM morphology was similar to controls, but transmittance values remained low, denoting incomplete restoration of the stromal architecture. This multidisciplinary study of the stromal wound healing process revealed changes in corneal transmittance, collagen organization, myofibroblasts presence and ECM composition at 1, 3, and 6 months after alkali burning. Documenting wound resolution during the six-month period provided reliable information that can be used to test new therapies.

Liver-specific ablation of insulin-degrading enzyme causes hepatic insulin resistance and glucose intolerance, without affecting insulin clearance in mice.

The role of insulin-degrading enzyme (IDE), a metalloprotease with high affinity for insulin, in insulin clearance remains poorly understood. OBJECTIVE: This study aimed to clarify whether IDE is a major mediator of insulin clearance, and to define its role in the etiology of hepatic insulin resistance. METHODS: We generated mice with liver-specific deletion of Ide (L-IDE-KO) and assessed insulin clearance and action. RESULTS: L-IDE-KO mice exhibited higher (~20%) fasting and non-fasting plasma glucose levels, glucose intolerance and insulin resistance. This phenotype was associated with ~30% lower plasma membrane insulin receptor levels in liver, as well as ~55% reduction in insulin-stimulated phosphorylation of the insulin receptor, and its downstream signaling molecules, AKT1 and AKT2 (reduced by ~40%). In addition, FoxO1 was aberrantly distributed in cellular nuclei, in parallel with up-regulation of the gluconeogenic genes Pck1 and G6pc. Surprisingly, L-IDE-KO mice showed similar plasma insulin levels and hepatic insulin clearance as control mice, despite reduced phosphorylation of the carcinoembryonic antigen-related cell adhesion molecule 1, which upon its insulin-stimulated phosphorylation, promotes receptor-mediated insulin uptake to be degraded. CONCLUSION: IDE is not a rate-limiting regulator of plasma insulin levels in vivo.

Phenotypic Modulation of Cultured Primary Human Aortic Vascular Smooth Muscle Cells by Uremic Serum.

Patients with chronic kidney disease (CKD) have a markedly increased incidence of cardiovascular disease (CVD). The high concentration of circulating uremic toxins and alterations in mineral metabolism and hormone levels produce vascular wall remodeling and significant vascular damage. Medial calcification is an early vascular event in CKD patients and is associated to apoptosis or necrosis and trans-differentiation of vascular smooth muscle cells (VSMC) to an osteogenic phenotype. VSMC obtained from bovine or rat aorta and cultured in the presence of increased inorganic phosphate (Pi) have been extensively used to study these processes. In this study we used human aortic VSMC primary cultures to compare the effects of increased Pi to treatment with serum obtained from uremic patients. Uremic serum induced calcification, trans-differentiation and phenotypic remodeling even with normal Pi levels. In spite of similar calcification kinetics, there were fundamental differences in osteochondrogenic marker expression and alkaline phosphatase induction between Pi and uremic serum-treated cells. Moreover, high Pi induced a dramatic decrease in cell viability, while uremic serum preserved it. In summary, our data suggests that primary cultures of human VSMC treated with serum from uremic patients provides a more informative model for the study of vascular calcification secondary to CKD.

The secret life of ion channels: Kv1.3 potassium channels and proliferation.

Kv1.3 channels are involved in the switch to proliferation of normally quiescent cells, being implicated in the control of cell cycle in many different cell types and in many different ways. They modulate membrane potential controlling K+ fluxes, sense changes in potential, and interact with many signaling molecules through their intracellular domains. From a mechanistic point of view, we can describe the role of Kv1.3 channels in proliferation with at least three different models. In the "membrane potential model," membrane hyperpolarization resulting from Kv1.3 activation provides the driving force for Ca2+ influx required to activate Ca2+-dependent transcription. This model explains most of the data obtained from several cells from the immune system. In the "voltage sensor model," Kv1.3 channels serve mainly as sensors that transduce electrical signals into biochemical cascades, independently of their effect on membrane potential. Kv1.3-dependent proliferation of vascular smooth muscle cells (VSMCs) could fit this model. Finally, in the "channelosome balance model," the master switch determining proliferation may be related to the control of the Kv1.3 to Kv1.5 ratio, as described in glial cells and also in VSMCs. Since the three mechanisms cannot function independently, these models are obviously not exclusive. Nevertheless, they could be exploited differentially in different cells and tissues. This large functional flexibility of Kv1.3 channels surely gives a new perspective on their functions beyond their elementary role as ion channels, although a conclusive picture of the mechanisms involved in Kv1.3 signaling to proliferation is yet to be reached.

Kv channels and vascular smooth muscle cell proliferation.

Kv channels are present in virtually all VSMCs and strongly influence contractile responses. However, they are also instrumental in the proliferative, migratory, and secretory functions of synthetic, dedifferentiated VSMCs upon PM. In fact, Kv channels not only contribute to all these processes but also are active players in the phenotypic switch itself. This review is focused on the role(s) of Kv channels in VSMC proliferation, which is one of the best characterized functions of dedifferentiated VSMCs. VSMC proliferation is a complex process requiring specific Kv channels at specific time and locations. Their identification is further complicated by their large diversity and the differences in expression across vascular beds. Of interest, both conserved changes in some Kv channels and vascular bed-specific regulation of others seem to coexist and participate in VSMC proliferation through complementary mechanisms. Such a system will add flexibility to the process while providing the required robustness to preserve this fundamental cellular response.

Proliferative Role of Kv11 Channels in Murine Arteries.

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. Methods: Quantitative RT-PCR was undertaken on RNA extracted from a number of murine arteries. Immunofluorescence was performed on single vascular smooth muscle cells using antibodies against the ERG1 expression product (Kv11.1). Single cell electrophysiology was performed on myocytes from portal vein and several different arteries, complimented by isometric tension recordings. Proliferation assays were undertaken on smooth muscle cells isolated from femoral arteries. Results: ERG1 transcripts were detected in all murine blood vessels, and Kv11.1 immunofluorescence was observed in all smooth muscle cells. However, K+ currents with properties consistent with ERG channels were only recorded in portal vein myocytes. Moreover, ERG channel blockers (E4031 or dofetilide, 1 µM) failed to depolarize carotid arteries or produce contraction. Proliferation of arterial smooth muscle cells was associated with a marked increase in ERG1 expression and ERG blockers suppressed proliferation significantly. Conclusions: These data reveal that arterial blood vessels express ERG channels that appear to be functional silent in contractile smooth muscle but contribute to proliferative response.

Regulation of microRNA expression in vascular smooth muscle by MRTF-A and actin polymerization.

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. This analysis was performed using cardiovascular disease-focused miRNA arrays in both mouse and human cells. The potential clinical importance of actin polymerization in aortic aneurysm was evaluated using biopsies from mildly dilated human thoracic aorta in patients with stenotic tricuspid or bicuspid aortic valve. By integrating information from multiple qPCR based miRNA arrays we identified a group of five miRNAs (miR-1, miR-22, miR-143, miR-145 and miR-378a) that were sensitive to actin polymerization and MRTF-A overexpression in both mouse and human vascular smooth muscle. With the exception of miR-22, these miRNAs were also relatively enriched in striated and/or smooth muscle containing tissues. Actin polymerization was found to be dramatically reduced in the aorta from patients with mild aortic dilations. This was associated with a decrease in actin/MRTF-regulated miRNAs. In conclusion, the transcriptional co-activator MRTF-A and actin polymerization regulated a subset of miRNAs in vascular smooth muscle. Identification of novel miRNAs regulated by actin/MRTF-A may provide further insight into the mechanisms underlying vascular disease states, such as aortic aneurysm, as well as novel ideas regarding therapeutic strategies. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.

Differences in TRPC3 and TRPC6 channels assembly in mesenteric vascular smooth muscle cells in essential hypertension.

KEY POINTS: Canonical transient receptor potential (TRPC)3 and TRPC6 channels of vascular smooth muscle cells (VSMCs) mediate stretch- or agonist-induced cationic fluxes, contributing to membrane potential and vascular tone. Native TRPC3/C6 channels can form homo- or heterotetrameric complexes, which can hinder individual TRPC channel properties. The possibility that the differences in their association pattern may change their contribution to vascular tone in hypertension is unexplored. Functional characterization of heterologously expressed channels showed that TRPC6-containing complexes exhibited Pyr3/Pyr10-sensitive currents, whereas TRPC3-mediated currents were blocked by anti-TRPC3 antibodies. VSMCs from hypertensive (blood pressure high; BPH) mice have larger cationic basal currents insensitive to Pyr10 and sensitive to anti-TRPC3 antibodies. Consistently, myography studies showed a larger Pyr3/10-induced vasodilatation in BPN (blood pressure normal) mesenteric arteries. We conclude that the increased TRPC3 channel expression in BPH VSMCs leads to changes in TRPC3/C6 heteromultimeric assembly, with a higher TRPC3 channel contribution favouring depolarization of hypertensive VSMCs. ABSTRACT: 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 therapeutical opportunities.

Molecular Determinants of Kv1.3 Potassium Channels-induced Proliferation.

Changes in voltage-dependent potassium channels (Kv channels) associate to proliferation in many cell types, including transfected HEK293 cells. In this system Kv1.5 overexpression decreases proliferation, whereas Kv1.3 expression increases it independently of K(+) fluxes. To identify Kv1.3 domains involved in a proliferation-associated signaling mechanism(s), we constructed chimeric Kv1.3-Kv1.5 channels and point-mutant Kv1.3 channels, which were expressed as GFP- or cherry-fusion proteins. We studied their trafficking and functional expression, combining immunocytochemical and electrophysiological methods, and their impact on cell proliferation. We found that the C terminus is necessary for Kv1.3-induced proliferation. We distinguished two residues (Tyr-447 and Ser-459) whose mutation to alanine abolished proliferation. The insertion into Kv1.5 of a sequence comprising these two residues increased proliferation rate. Moreover, Kv1.3 voltage-dependent transitions from closed to open conformation induced MEK-ERK1/2-dependent Tyr-447 phosphorylation. We conclude that the mechanisms for Kv1.3-induced proliferation involve the accessibility of key docking sites at the C terminus. For one of these sites (Tyr-447) we demonstrated the contribution of MEK/ERK-dependent phosphorylation, which is regulated by voltage-induced conformational changes.

Regulation of smooth muscle dystrophin and synaptopodin 2 expression by actin polymerization and vascular injury.

OBJECTIVE: Actin dynamics in vascular smooth muscle is known to regulate contractile differentiation and may play a role in the pathogenesis of vascular disease. However, the list of genes regulated by actin polymerization in smooth muscle remains incomprehensive. Thus, the objective of this study was to identify actin-regulated genes in smooth muscle and to demonstrate the role of these genes in the regulation of vascular smooth muscle phenotype. APPROACH AND RESULTS: Mouse aortic smooth muscle cells were treated with an actin-stabilizing agent, jasplakinolide, and analyzed by microarrays. Several transcripts were upregulated including both known and previously unknown actin-regulated genes. Dystrophin and synaptopodin 2 were selected for further analysis in models of phenotypic modulation and vascular disease. These genes were highly expressed in differentiated versus synthetic smooth muscle and their expression was promoted by the transcription factors myocardin and myocardin-related transcription factor A. Furthermore, the expression of both synaptopodin 2 and dystrophin was significantly reduced in balloon-injured human arteries. Finally, using a dystrophin mutant mdx mouse and synaptopodin 2 knockdown, we demonstrate that these genes are involved in the regulation of smooth muscle differentiation and function. CONCLUSIONS: This study demonstrates novel genes that are promoted by actin polymerization, that regulate smooth muscle function, and that are deregulated in models of vascular disease. Thus, targeting actin polymerization or the genes controlled in this manner can lead to novel therapeutic options against vascular pathologies that involve phenotypic modulation of smooth muscle cells.