Alex Cheong

Upregulated TRPC1 Channel in Vascular Injury In Vivo and Its Role in Human Neointimal Hyperplasia

Occlusive vascular disease is a widespread abnormality leading to lethal or debilitating outcomes such as myocardial infarction and stroke. It is part of atherosclerosis and is evoked by clinical procedures including angioplasty and grafting of saphenous vein in bypass surgery. A causative factor is the switch in smooth muscle cells to an invasive and proliferative mode, leading to neointimal hyperplasia. Here we reveal the importance to this process of TRPC1, a homolog of Drosophila transient receptor potential. Using 2 different in vivo models of vascular injury in rodents we show hyperplasic smooth muscle cells have upregulated TRPC1 associated with enhanced calcium entry and cell cycle activity. Neointimal smooth muscle cells after balloon angioplasty of pig coronary artery also express TRPC1. Furthermore, human vein samples obtained during coronary artery bypass graft surgery commonly exhibit an intimal structure containing smooth muscle cells that expressed more TRPC1 than the medial layer cells. Veins were organ cultured to allow growth of neointimal smooth muscle cells over a 2-week period. To explore the functional relevance of TRPC1, we used a specific E3-targeted antibody to TRPC1 and chemical blocker 2-aminoethoxydiphenyl borate. Both agents significantly reduced neointimal growth in human vein, as well as calcium entry and proliferation of smooth muscle cells in culture. The data suggest upregulated TRPC1 is a general feature of smooth muscle cells in occlusive vascular disease and that TRPC1 inhibitors have potential as protective agents against human vascular failure.

MicroRNA-155 promotes resolution of hypoxia-inducible factor 1α activity during prolonged hypoxia

The hypoxia-inducible factor (HIF) is a key regulator of the transcriptional response to hypoxia. While the mechanism underpinning HIF activation is well understood, little is known about its resolution. Both the protein and the mRNA levels of HIF-1α (but not HIF-2α) were decreased in intestinal epithelial cells exposed to prolonged hypoxia. Coincident with this, microRNA (miRNA) array analysis revealed multiple hypoxiainducible miRNAs. Among these was miRNA-155 (miR-155), which is predicted to target HIF-1α mRNA. We confirmed the hypoxic upregulation of miR-155 in cultured cells and intestinal tissue from mice exposed to hypoxia. Furthermore, a role for HIF-1α in the induction of miR-155 in hypoxia was suggested by the identification of hypoxia response elements in the miR-155 promoter and confirmed experimentally. Application of miR-155 decreased the HIF-1α mRNA, protein, and transcriptional activity in hypoxia, and neutralization of endogenous miR-155 reversed the resolution of HIF-1α stabilization and activity. Based on these data and a mathematical model of HIF-1α suppression by miR-155, we propose that miR-155 induction contributes to an isoform-specific negative-feedback loop for the resolution of HIF-1α activity in cells exposed to prolonged hypoxia, leading to oscillatory behavior of HIF-1α-dependent transcription.ABSTRACT The hypoxia-inducible factor (HIF) is a key regulator of the transcriptional response to hypoxia. While the mechanism underpinning HIF activation is well understood, little is known about its resolution. Both the protein and the mRNA levels of HIF-1α (but not HIF-2α) were decreased in intestinal epithelial cells exposed to prolonged hypoxia. Coincident with this, microRNA (miRNA) array analysis revealed multiple hypoxia-inducible miRNAs. Among these was miRNA-155 (miR-155), which is predicted to target HIF-1α mRNA. We confirmed the hypoxic upregulation of miR-155 in cultured cells and intestinal tissue from mice exposed to hypoxia. Furthermore, a role for HIF-1α in the induction of miR-155 in hypoxia was suggested by the identification of hypoxia response elements in the miR-155 promoter and confirmed experimentally. Application of miR-155 decreased the HIF-1α mRNA, protein, and transcriptional activity in hypoxia, and neutralization of endogenous miR-155 reversed the resolution of HIF-1α stabilization and activity. Based on these data and a mathematical model of HIF-1α suppression by miR-155, we propose that miR-155 induction contributes to an isoform-specific negative-feedback loop for the resolution of HIF-1α activity in cells exposed to prolonged hypoxia, leading to oscillatory behavior of HIF-1α-dependent transcription.

A Sphingosine-1–Phosphate-Activated Calcium Channel Controlling Vascular Smooth Muscle Cell Motility

In a screen of potential lipid regulators of transient receptor potential (TRP) channels, we identified sphingosine-1–phosphate (S1P) as an activator of TRPC5. We explored the relevance to vascular biology because S1P is a key cardiovascular signaling molecule. TRPC5 is expressed in smooth muscle cells of human vein along with TRPC1, which forms a complex with TRPC5. Importantly, S1P also activates the TRPC5–TRPC1 heteromultimeric channel. Because TRPC channels are linked to neuronal growth cone extension, we considered a related concept for smooth muscle. We find S1P stimulates smooth muscle cell motility, and that this is inhibited by E3-targeted anti-TRPC5 antibody. Ion permeation involving TRPC5 is crucial because S1P-evoked motility is also suppressed by the channel blocker 2-aminoethoxydiphenyl borate or a TRPC5 ion-pore mutant. S1P acts on TRPC5 via two mechanisms, one extracellular and one intracellular, consistent with its bipolar signaling functions. The extracellular effect appears to have a primary role in S1P-evoked cell motility. The data suggest S1P sensing by TRPC5 calcium channel is a mechanism contributing to vascular smooth muscle adaptation.

Local Delivery of the KCa3.1 Blocker, TRAM-34, Prevents Acute Angioplasty-Induced Coronary Smooth Muscle Phenotypic Modulation and Limits Stenosis

Objective—We previously demonstrated that upregulation of intermediate-conductance Ca2+-activated K+ channels (KCa3.1) is necessary for mitogen-induced phenotypic modulation in isolated porcine coronary smooth muscle cells (SMCs). The objective of the present study was to determine the role of KCa3.1 in the regulation of coronary SMC phenotypic modulation in vivo using a swine model of postangioplasty restenosis. Methods and Results—Balloon angioplasty was performed on coronary arteries of swine using either noncoated or balloons coated with the specific KCa3.1 blocker TRAM-34. Expression of KCa3.1, c-jun, c-fos, repressor element-1 silencing transcription factor (REST), smooth muscle myosin heavy chain (SMMHC), and myocardin was measured using qRT-PCR in isolated medial cells 2 hours and 2 days postangioplasty. KCa3.1, c-jun, and c-fos mRNA levels were increased 2 hours postangioplasty, whereas REST expression decreased. SMMHC expression was unchanged at 2 hours, but decreased 2 days postangioplasty. Use of TRAM-34 coated balloons prevented KCa3.1 upregulation and REST downregulation at 2 hours, SMMHC and myocardin downregulation at 2 days, and attenuated subsequent restenosis 14 and 28 days postangioplasty. Immunohistochemical analysis demonstrated corresponding changes at the protein level. Conclusion—Blockade of KCa3.1 by delivery of TRAM-34 via balloon catheter prevented smooth muscle phenotypic modulation and limited subsequent restenosis.

Robotic multiwell planar patch-clamp for native and primary mammalian cells

Robotic multiwell planar patch-clamp has become common in drug development and safety programs because it enables efficient and systematic testing of compounds against ion channels during voltage-clamp. It has not, however, been adopted significantly in other important areas of ion channel research, where conventional patch-clamp remains the favored method. Here, we show the wider potential of the multiwell approach with the ability for efficient intracellular solution exchange, describing protocols and success rates for recording from a range of native and primary mammalian cells derived from blood vessels, arthritic joints and the immune and central nervous systems. The protocol involves preparing a suspension of single cells to be dispensed robotically into 4–8 microfluidic chambers each containing a glass chip with a small aperture. Under automated control, giga-seals and whole-cell access are achieved followed by preprogrammed routines of voltage paradigms and fast extracellular or intracellular solution exchange. Recording from 48 chambers usually takes 1–6 h depending on the experimental design and yields 16–33 cell recordings.

Regulation of IL-1β–induced NF-κB by hydroxylases links key hypoxic and inflammatory signaling pathways

Significance Oxygen-sensing hydroxylases are a family of enzymes that control the cellular adaptive response to hypoxia. Hydroxylase inhibitors reduce inflammation in vivo; however, the anti-inflammatory mechanism of action remains unclear. IL-1β is a cytokine that potently promotes inflammation through activation of the transcription factor NF-κB. Here, we demonstrate that hydroxylase inhibition leads to a suppression of IL-1β–induced NF-κB activity and provide insight into the underlying mechanism involved. This work develops our understanding of how hydroxylase inhibition regulates IL-1β–induced inflammation and sheds light on our understanding of the association between hypoxic and inflammatory signaling pathways, underscoring the potential use of hydroxylase inhibitors for the treatment of inflammatory disease. Hypoxia is a prominent feature of chronically inflamed tissues. Oxygen-sensing hydroxylases control transcriptional adaptation to hypoxia through the regulation of hypoxia-inducible factor (HIF) and nuclear factor κB (NF-κB), both of which can regulate the inflammatory response. Furthermore, pharmacologic hydroxylase inhibitors reduce inflammation in multiple animal models. However, the underlying mechanism(s) linking hydroxylase activity to inflammatory signaling remains unclear. IL-1β, a major proinflammatory cytokine that regulates NF-κB, is associated with multiple inflammatory pathologies. We demonstrate that a combination of prolyl hydroxylase 1 and factor inhibiting HIF hydroxylase isoforms regulates IL-1β–induced NF-κB at the level of (or downstream of) the tumor necrosis factor receptor-associated factor 6 complex. Multiple proteins of the distal IL-1β–signaling pathway are subject to hydroxylation and form complexes with either prolyl hydroxylase 1 or factor inhibiting HIF. Thus, we hypothesize that hydroxylases regulate IL-1β signaling and subsequent inflammatory gene expression. Furthermore, hydroxylase inhibition represents a unique approach to the inhibition of IL-1β–dependent inflammatory signaling.

Discrete store‐operated calcium influx into an intracellular compartment in rabbit arteriolar smooth muscle

This study tested the hypothesis that store‐operated channels (SOCs) exist as a discrete population of Ca2+ channels activated by depletion of intracellular Ca2+ stores in cerebral arteriolar smooth muscle cells and explored their direct contractile function. Using the Ca2+ indicator fura‐PE3 it was observed that depletion of sarcoplasmic reticulum (SR) Ca2+ by inhibition of SR Ca2+‐ATPase (SERCA) led to sustained elevation of [Ca2+]i that depended on extracellular Ca2+ and slightly enhanced Mn2+ entry. Enhanced background Ca2+ influx did not explain the raised [Ca2+]i in response to SERCA inhibitors because it had marked gadolinium (Gd3+) sensitivity, which background pathways did not. Effects were not secondary to changes in membrane potential. Thus SR Ca2+ depletion activated SOCs. Strikingly, SOC‐mediated Ca2+ influx did not evoke constriction of the arterioles, which were in a resting state. This was despite the fura‐PE3‐indicated [Ca2+]i rise being greater than that evoked by 20 mm[K+]o (which did cause constriction). Release of endothelial vasodilators did not explain the absence of SOC‐mediated constriction, nor did a change in Ca2+ sensitivity of the contractile proteins. We suggest SOCs are a discrete subset of Ca2+ channels allowing Ca2+ influx into a ‘non‐contractile’ compartment in cerebral arteriolar smooth muscle cells.

A dynamic model of the hypoxia-inducible factor 1α (HIF-1α) network

Summary Activation of the hypoxia-inducible factor (HIF) pathway is a critical step in the transcriptional response to hypoxia. Although many of the key proteins involved have been characterised, the dynamics of their interactions in generating this response remain unclear. In the present study, we have generated a comprehensive mathematical model of the HIF-1&agr; pathway based on core validated components and dynamic experimental data, and confirm the previously described connections within the predicted network topology. Our model confirms previous work demonstrating that the steps leading to optimal HIF-1&agr; transcriptional activity require sequential inhibition of both prolyl- and asparaginyl-hydroxylases. We predict from our model (and confirm experimentally) that there is residual activity of the asparaginyl-hydroxylase FIH (factor inhibiting HIF) at low oxygen tension. Furthermore, silencing FIH under conditions where prolyl-hydroxylases are inhibited results in increased HIF-1&agr; transcriptional activity, but paradoxically decreases HIF-1&agr; stability. Using a core module of the HIF network and mathematical proof supported by experimental data, we propose that asparaginyl hydroxylation confers a degree of resistance upon HIF-1&agr; to proteosomal degradation. Thus, through in vitro experimental data and in silico predictions, we provide a comprehensive model of the dynamic regulation of HIF-1&agr; transcriptional activity by hydroxylases and use its predictive and adaptive properties to explain counter-intuitive biological observations.

Expression and function of native potassium channel (KVα1) subunits in terminal arterioles of rabbit

1 In this study we investigated the expression and function of the KVα1 subfamily of voltage‐gated K+ channels in terminal arterioles from rabbit cerebral circulation. 2 K+ current was measured from smooth muscle cells within intact freshly isolated arteriolar fragments. Current activated on depolarisation positive of about –45 mV and a large fraction of this current was blocked by 3,4‐diaminopyridine (3,4‐DAP) or 4‐aminopyridine (4‐AP), inhibitors of KV channels. Expression of cRNA encoding KV1.6 in Xenopus oocytes also generated a 4‐AP‐sensitive K+ current with a threshold for activation near –45 mV. 3 Immunofluorescence labelling revealed KV1.2 to be specifically localised to endothelial cells, and KV1.5 and KV1.6 to plasma membranes of smooth muscle cells. 4 KV channel current in arteriolar fragments was blocked by correolide (which is specific for the KVα1 family of KV channels) but was resistant to recombinant agitoxin‐2 (rAgTX2; which inhibits KV1.6 but not KV1.5). Heterologously expressed KV2.1 was resistant to correolide, and KV1.6 was blocked by rAgTX2. 5 Arterioles that were mildly preconstricted and depolarised by 0.1–0.3 nm endothelin‐1 constricted further in response to 3,4‐DAP, 4‐AP or correolide, but not to rAgTX2. 6 We suggest that KVα1 channels are expressed in smooth muscle cells of terminal arterioles, underlie a major part of the voltage‐dependent K+ current, and have a physiological function to oppose vasoconstriction. KVα1 complexes without KV1.5 appear to be uncommon.

Functional up-regulation of KCNA gene family expression in murine mesenteric resistance artery smooth muscle

This study focused on the hypothesis that KCNA genes (which encode KVα1 voltage‐gated K+ channels) have enhanced functional expression in smooth muscle cells of a primary determinant of peripheral resistance – the small mesenteric artery. Real‐time PCR methodology was developed to measure cell type‐specific in situ gene expression. Profiles were determined for arterial myocyte expression of RNA species encoding KVα1 subunits as well as KVβ1, KVα2.1, KVγ9.3, BKCaα1 and BKCaβ1. The seven major KCNA genes were expressed and more readily detected in endothelium‐denuded mesenteric resistance artery compared with thoracic aorta; quantification revealed dramatic differential expression of one to two orders of magnitude. There was also four times more RNA encoding KVα2.1 but less or similar amounts encoding KVβ1, KVγ9.3, BKCaα1 and BKCaβ1. Patch‐clamp recordings from freshly isolated smooth muscle cells revealed dominant KVα1 K+ current and current density twice as large in mesenteric cells. Therefore, we suggest the increased RNA production of the resistance artery impacts on physiological function, although there is quantitatively less K+ current than might be expected. The mechanism conferring up‐regulated expression of KCNA genes may be common to all the gene family and play a functional role in the physiological control of blood pressure.