Carmelle V. Remillard

A Functional Single-Nucleotide Polymorphism in the TRPC6 Gene Promoter Associated With Idiopathic Pulmonary Arterial Hypertension

Background— Excessive proliferation of pulmonary artery smooth muscle cells (PASMCs) plays an important role in the development of idiopathic pulmonary arterial hypertension (IPAH), whereas a rise in cytosolic Ca2+ concentration triggers PASMC contraction and stimulates PASMC proliferation. Recently, we demonstrated that upregulation of the TRPC6 channel contributes to proliferation of PASMCs isolated from IPAH patients. This study sought to identify single-nucleotide polymorphisms (SNPs) in the TRPC6 gene promoter that are associated with IPAH and have functional significance in regulating TRPC6 activity in PASMCs. Methods and Results— Genomic DNA was isolated from blood samples of 237 normal subjects and 268 IPAH patients. Three biallelic SNPs, −361 (A/T), −254(C/G), and −218 (C/T), were identified in the 2000-bp sequence upstream of the transcriptional start site of TRPC6. Although the allele frequencies of the −361 and −218 SNPs were not different between the groups, the allele frequency of the −254(C→G) SNP in IPAH patients (12%) was significantly higher than in normal subjects (6%; P<0.01). Genotype data showed that the percentage of −254G/G homozygotes in IPAH patients was 2.85 times that of normal subjects. Moreover, the −254(C→G) SNP creates a binding sequence for nuclear factor-&kgr;B. Functional analyses revealed that the −254(C→G) SNP enhanced nuclear factor-&kgr;B–mediated promoter activity and stimulated TRPC6 expression in PASMCs. Inhibition of nuclear factor-&kgr;B activity attenuated TRPC6 expression and decreased agonist-activated Ca2+ influx in PASMCs of IPAH patients harboring the −254G allele. Conclusions— These results suggest that the −254(C→G) SNP may predispose individuals to an increased risk of IPAH by linking abnormal TRPC6 transcription to nuclear factor-&kgr;B, an inflammatory transcription factor.

K + Channels in Apoptosis

A proper rate of programmed cell death or apoptosis is required to maintain normal tissue homeostasis. In disease states such as cancer and some forms of hypertension, apoptosis is blocked, resulting in hyperplasia. In neurodegenerative diseases, uncontrolled apoptosis leads to loss of brain tissue. The flow of ions in and out of the cell and its intracellular organelles is becoming increasingly linked to the generation of many of these diseased states. This review focuses on the transport of K+ across the cell membrane and that of the mitochondria via integral K+-permeable channels. We describe the different types of K+ channels that have been identified, and investigate the roles they play in controlling the different phases of apoptosis: early cell shrinkage, cytochrome c release, caspase activation, and DNA fragmentation. Attention is also given to K+ channels on the inner mitochondrial membrane, whose activity may underlie anti- or pro-apoptotic mechanisms in neurons and cardiomyocytes.

Potassium channels in the regulation of pulmonary artery smooth muscle cell proliferation and apoptosis: Pharmacotherapeutic implications

Maintaining the proper balance between cell apoptosis and proliferation is required for normal tissue homeostasis; when this balance is disrupted, disease such as pulmonary arterial hypertension (PAH) can result. Activity of K+ channels plays a major role in regulating the pulmonary artery smooth muscle cell (PASMC) population in the pulmonary vasculature, as they are involved in cell apoptosis, survival and proliferation. PASMCs from PAH patients demonstrate many cellular abnormalities linked to K+ channels, including decreased K+ current, downregulated expression of various K+ channels, and inhibited apoptosis. K+ is the major intracellular cation, and the K+ current is a major determinant of cell volume. Apoptotic volume decrease (AVD), an early hallmark and prerequisite of programmed cell death, is characterized by K+ and Cl− efflux. In addition to its role in AVD, cytosolic K+ can be inhibitory toward endogenous caspases and nucleases and can suppress mitochondrial cytochrome c release. In PASMC, K+ channel activation accelerates AVD and enhances apoptosis, while K+ channel inhibition decelerates AVD and inhibits apoptosis. Finally, inhibition of K+ channels, by increasing cytosolic [Ca2+] as a result of membrane depolarization‐mediated opening of voltage‐dependent Ca2+ channels, leads to PASMC contraction and proliferation. The goals of this review are twofold: (1) to elucidate the role of K+ ions and K+ channels in the proliferation and apoptosis of PASMC, with an emphasis on abnormal cell growth in human and animal models of PAH, and (2) to elaborate upon the targeting of K+ flux pathways for pharmacological treatment of pulmonary vascular disease.

TRP channels, CCE, and the pulmonary vascular smooth muscle.

Transient receptor potential (TRP) genes represent a novel class of genes that are generally believed to encode for nonselective cation channels. A subfamily of TRP channels, canonical TRP (TRPC), which are highly permeable to Ca2+ (and Na+), co‐assembles with each other to form functional store‐ and receptor‐operated Ca2+ channels. TRPC mRNA and protein have been identified in pulmonary arterial smooth muscle and endothelial cells. The currents generated by Ca2+ influx through store‐ and receptor‐operated Ca2+ channels have also been extensively characterized in these cells. More recently, the attention has shifted to identify the TRP subunits that underlie the function of native channels in the pulmonary vasculature, with the understanding that TRP channels assemble as either homo‐or heterotetramers in vivo. This work in progress has yielded exciting information regarding the involvement of TRP channels in the control of smooth muscle contraction, and cell proliferation and migration. In this review, the authors focus on describing the function and transcriptional regulation of TRP proteins, and the store‐ and receptor‐operated Ca2+ channels for which they are responsible, in pulmonary artery smooth muscle cells (PASMC). They also identify some key TRP proteins whose role in the pulmonary vasculature has been established, as well as some more novel subunits whose role, although intriguing, can only be inferred from other vascular studies. Finally, they describe the involvement of TRP channels in regulating pulmonary vasoconstriction, PASMC proliferation, and pulmonary endothelial barrier function.

Functional ion channels in human pulmonary artery smooth muscle cells: Voltage-dependent cation channels

The activity of voltage-gated ion channels is critical for the maintenance of cellular membrane potential and generation of action potentials. In turn, membrane potential regulates cellular ion homeostasis, triggering the opening and closing of ion channels in the plasma membrane and, thus, enabling ion transport across the membrane. Such transmembrane ion fluxes are important for excitation-contraction coupling in pulmonary artery smooth muscle cells (PASMC). Families of voltage-dependent cation channels known to be present in PASMC include voltage-gated K+ (Kv) channels, voltage-dependent Ca2+-activated K+ (Kca) channels, L- and T-type voltage-dependent Ca2+ channels, voltage-gated Na+ channels and voltage-gated proton channels. When cells are dialyzed with Ca2+-free K+-solutions, depolarization elicits four components of 4-aminopyridine (4-AP)-sensitive Kvcurrents based on the kinetics of current activation and inactivation. In cell-attached membrane patches, depolarization elicits a wide range of single-channel K+ currents, with conductances ranging between 6 and 290 pS. Macroscopic 4-AP-sensitive Kv currents and iberiotoxin-sensitive Kca currents are also observed. Transcripts of (a) two Na+ channel α-subunit genes (SCN5A and SCN6A), (b) six Ca2+ channel α-subunit genes (α1A, α1B, α1x, α1D, α1E and α1G) and many regulatory subunits (α2δ1, β1-4, and γ6), (c) 22 Kv channel α-subunit genes (Kv1.1 – Kv1.7, Kv1.10, Kv2.1, Kv3.1, Kv3.3, Kv3.4, Kv4.1, Kv4.2, Kv5.1, Kv 6.1-Kv6.3, Kv9.1, Kv9.3, Kv10.1 and Kv11.1) and three Kv channel β-subunit genes (Kvβ1-3) and (d) four Kca channel α-subunit genes (S/oα1 and SK2-SK4) and four Kca channel β-subunit genes (Kcaβ1-4) have been detected in PASMC. Tetrodotoxin-sensitive and rapidly inactivating Na+ currents have been recorded with properties similar to those in cardiac myocytes. In the presence of 20 mM external Ca2+, membrane depolarization from a holding potential of −100 mV elicits a rapidly inactivating T-type Ca2+ current, while depolarization from a holding potential of −70 mV elicits a slowly inactivating dihydropyridine-sensitive L-type Ca2+ current. This review will focus on describing the electrophysiological properties and molecular identities of these voltage-dependent cation channels in PASMC and their contribution to the regulation of pulmonary vascular function and its potential role in the pathogenesis of pulmonary vascular disease.

Divergent effects of BMP-2 on gene expression in pulmonary artery smooth muscle cells from normal subjects and patients with idiopathic pulmonary arterial hypertension

Bone morphogenetic proteins (BMPs) inhibit proliferation and induce apoptosis in pulmonary artery smooth muscle cells (PASMCs) from normal subjects. Dysfunction of BMP signaling due to mutations in and/or down-regulation of BMP receptors has been implicated in idiopathic pulmonary arterial hypertension (IPAH). The authors examined whether BMP differentially regulates gene expression in PASMCs from normal subjects and IPAH patients using the Affymetrix microarray analysis. BMP-2 treatment (200 nM for 24 hours) altered expression levels of 6206 genes in normal and IPAH PASMCs. Of these genes, 1063 were regulated oppositely by BMP-2: 523 genes were down-regulated by BMP-2 in normal PASMCs but up-regulated in IPAH PASMCs, whereas 540 genes were up-regulated by BMP-2 in normal PASMCs but down-regulated in IPAH PASMCs. The divergent effects of BMP-2 on gene expression profiles indicate that PASMCs may undergo significant phenotypic changes in IPAH patients during development of the disease. The transition of the antiproliferative effect of BMP-2 in normal PASMCs to its proliferative effect in IPAH patients is attributed potentially to its differential effect on expression patterns of various genes that are involved in cell proliferation and apoptosis. Among the 6206 BMP-2–sensitive genes, there are more than 1800 genes whose expression levels were negatively (correlation coefficient, r, <−0.9) or positively (with r >+ 0.9) correlated with the pulmonary arterial pressure. These results suggest that BMP-mediated gene regulation is significantly altered in PASMCs from IPAH patients and mRNA expression changes in BMP-regulated genes may be involved in the development of IPAH.

Transient Receptor Potential Channels and Caveolin-1: Good Friends in Tight Spaces

Caveolae formation has raised the concept of energy efficiency to new heights. The ultimate purpose of caveolae formation is to colocalize signaling proteins with membrane microdomains in order to facilitate their interaction and improve signal transduction efficiency. Although we know that the main structural protein of caveolae is caveolin, how caveolin interacts with membrane proteins to facilitate their integration into lipid raft domains is unclear. A caveolin-scaffolding domain (CSD) on caveolin itself can associate with membrane proteins such as G proteins and endothelial nitric oxide synthase. In this issue, Kwiatek et al. (p. 1174) report that the TRPC1 channel protein contains a C-terminal CSD-consensus binding sequence that allows for its physical and functional interaction with caveolin-1 in the caveolae of human pulmonary artery endothelial cells (PAEC). Competitive interaction with a CSD-conjugated peptide attenuates thrombin- and thapsigargin-induced Ca2+ influx via store-operated TRPC1 channels. Their data suggest that caveolin-1 can directly regulate TRPC1 function, extending its already ascribed role as a structural protein.

Identification of functional voltage-gated Na+ channels in cultured human pulmonary artery smooth muscle cells

Electrical excitability, which plays an important role in excitation–contraction coupling in the pulmonary vasculature, is regulated by transmembrane ion flux in pulmonary artery smooth muscle cells (PASMC). This study aimed to characterize the electrophysiological properties and molecular identities of voltage-gated Na+ channels in cultured human PASMC. We recorded tetrodotoxin (TTX) sensitive and rapidly inactivating Na+ currents with properties similar to those described in cardiac myocytes. Using RT-PCR, we detected transcripts of seven Na+ channel α genes (SCN2A, 3A, 4A, 7A, 8A, 9A, and 11A), and two β subunit genes (SCN1B and 2B). Our results demonstrate that human PASMC express TTX-sensitive voltage-gated Na+ channels. Their physiological functions remain unresolved, although our data suggest that Na+ channel activity does not directly influence membrane potential, intracellular Ca2+ release, or proliferation in normal human PASMC. Whether their expression and/or activity are heightened in the pathological state is discussed.

Upregulation of Oct-4 isoforms in pulmonary artery smooth muscle cells from patients with pulmonary arterial hypertension.

Oct-4 is a transcription factor considered to be one of the defining pluripotency markers in embryonic stem cells. Its expression has also been demonstrated in adult stem cells, tumorigenic cells, and, most recently and controversially, in somatic cells. Oct-4 pseudogenes also contribute to carcinogenesis. Oct-4 may be involved in the excessive proliferation of pulmonary arterial smooth muscle cells (PASMC) in patients with idiopathic pulmonary arterial hypertension (IPAH), contributing to the pathogenesis of IPAH. In this study, we show that Oct-4 isoforms are upregulated in IPAH-PASMC. Human embryonic stem cells (H9 line) and human PASMC from normotensive subjects were used throughout the investigation as positive and negative controls. In addition to significant upregulation of Oct-4 in a population of IPAH-PASMC, HIF-2alpha, a hypoxia-inducible transcription factor that has been shown to bind to the Oct-4 promoter and induces its expression and transcriptional activity, was also increased. Interestingly, a substantial upregulation of Oct-4 isoforms and HIF-2alpha was also observed in normal PASMC exposed to chronic hypoxia. In conclusion, the data suggest that both Oct-4 isoforms are upregulated and potentially have a significant role in the development of vascular abnormalities associated with the pathogenesis of IPAH and in pulmonary hypertension triggered by chronic hypoxia.

p75 neurotrophin receptor regulates agonist-induced pulmonary vasoconstriction

A member of the TNF receptor family, the p75 neurotrophin receptor (p75(NTR)) has been previously shown to play a role in the regulation of fibrin deposition in the lung. However, the role of p75(NTR) in the regulation of pulmonary vascular tone in the lung is unknown. In the present study, we evaluated the expression of p75(NTR) in mouse pulmonary arteries and the putative role of p75(NTR) in modulating pulmonary vascular tone and agonist responsiveness using wild-type (WT) and p75(NTR) knockout (p75(-/-)) mice. Our data indicated that p75(NTR) is expressed in both smooth muscle and endothelial cells within the pulmonary vascular wall in WT mice. Pulmonary artery rings from p75(-/-) mice exhibited significantly elevated active tension due to endothelin-1-mediated Ca(2+) influx. Furthermore, the contraction due to capacitative Ca(2+) entry (CCE) in response to phenylephrine-mediated active depletion of intracellular Ca(2+) stores was significantly enhanced compared with WT rings. The contraction due to CCE induced by passive store depletion, however, was comparable between WT and p75(-/-) rings. Active tension induced by serotonin, U-46619 (a thromboxane A(2) analog), thrombin, 4-aminopyridine (a K(+) channel blocker), and high extracellular K(+) in p75(-/-) rings was similar to that in WT rings. Deletion of p75(NTR) did not alter pulmonary vasodilation to sodium nitroprusside (a nitric oxide donor). These data suggest that intact p75(NTR) signaling may play a role in modulating pulmonary vasoconstriction induced by endothelin-1 and by active store depletion.