Pulmonary vascular medial hypertrophy caused by excessive pulmonary artery smooth muscle cell (PASMC) proliferation is a major cause for the elevated pulmonary vascular resistance in patients with idiopathic pulmonary arterial hypertension (IPAH). Increased Ca 2؉ influx is an important stimulus for PASMC proliferation. Transient receptor potential (TRP) channel genes encode Ca 2؉ channels that are responsible for Ca 2؉ entry during cell proliferation. Normal human PASMC expressed multiple canonical TRP (TRPC) isoforms; TRPC6 was highly expressed and TRPC3 was minimally expressed. The protein expression of TRPC6 in normal PASMC closely correlated with the expression of Ki67, suggesting that TRPC6 expression is involved in the transition of PASMC from quiescent phase to mitosis. In lung tissues and PASMC from IPAH patients, the mRNA and protein expression of TRPC3 and -6 were much higher than in those from normotensive or secondary pulmonary hypertension patients. Inhibition of TRPC6 expression with TRPC6 small interfering RNA markedly attenuated IPAH-PASMC proliferation. These results demonstrate that expression of TRPC channels correlates with the progression of the cell cycle in PASMC. TRPC channel overexpression may be partially responsible for the increased PASMC proliferation and pulmonary vascular medial hypertrophy in IPAH patients.I diopathic pulmonary arterial hypertension (IPAH) is a fatal disease that causes right heart failure and death. The elevated pulmonary vascular resistance (PVR) and arterial pressure in IPAH patients result mainly from pulmonary vasoconstriction, vascular remodeling, and in situ thrombosis (1). A central aspect of pulmonary vascular remodeling is medial hypertrophy caused by sustained pulmonary vasoconstriction (2-4), excessive pulmonary artery smooth muscle cell (PASMC) proliferation (5), and inhibited PASMC apoptosis (6, 7), resulting in a narrowed vascular lumen and increased PVR. Although its etiology remains unclear, elevated levels of circulating mitogens, dysfunction or down-regulation of receptors and ion channels, upregulation of transporters, and heightened activity of elastases and glycoproteins have been implicated in IPAH (5,6,(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20) Transient receptor potential (TRP) channel genes may encode subunits that form receptor-(ROC) and store-(SOC) operated Ca 2ϩ channels in many cell types, including PASMC and pulmonary artery endothelial cells (PAEC) (28,(30)(31)(32)(33)(34). Ca 2ϩ entry through ROC and SOC increases [Ca 2ϩ ] cyt , allowing for phosphorylation of signal transduction proteins and transcription factors (23,24,(35)(36)(37)(38), that are essential for the progression of the cell cycle (21). High levels of [Ca 2ϩ ] cyt and sufficient levels of Ca 2ϩ in the SR are required for vascular smooth muscle cell proliferation (22,25,39). Because they regulate SR and cytoplasmic Ca 2ϩ , CCE and SOC may play significant roles in regulating cell proliferation (28,29). This study tested the hypothesis that canonical TRP (TRPC...

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Cell apoptosis and proliferation are two counterparts in sharing the responsibility for maintaining normal tissue homeostasis. In recent years, the process of the programmed cell death has gained much interest because of its influence on malignant cell growth and other pathological states. Apoptosis is characterized by a distinct series of morphological and biochemical changes that result in cell shrinkage, DNA breakdown, and, ultimately, phagocytic death. Diverse external and internal stimuli trigger apoptosis, and enhanced K+efflux has been shown to be an essential mediator of not only early apoptotic cell shrinkage, but also of downstream caspase activation and DNA fragmentation. The goal of this review is to discuss the role(s) played by K+transport or flux across the plasma membrane in the regulation of the apoptotic volume decrease and apoptosis. Attention has also been paid to the role of inner mitochondrial membrane ion transport in the regulation of mitochondrial permeability and apoptosis. We provide specific examples of how deregulation of the apoptotic process contributes to pulmonary arterial medial hypertrophy, a major pathological feature in patients with pulmonary arterial hypertension. Finally, we discuss the targeting of K+channels as a potential therapeutic tool in modulating apoptosis to maintain the balance between cell proliferation and cell death that is essential to the normal development and function of an organism.

[Ca 2ϩ ]cyt in pulmonary artery endothelial cells (PAEC). Using combined molecular biological, fluorescence microscopy, and biophysical approaches, we examined the effect of chronic hypoxia (3% O 2, 72 h) on AP-1 DNA binding activity, CCE, and transient receptor potential (TRP) gene expression in human (h) PAEC. EMSA showed that AP-1 binding to hPAEC nuclear protein extracts was significantly enhanced by hypoxia, the increase being dependent on storeoperated Ca 2ϩ influx and sensitive to La 3ϩ , an SOC inhibitor. Hypoxia also increased basal [Ca 2ϩ ]cyt, the amount of CCE produced by store depletion with cyclopiazonic acid, and the amplitude of SOC-mediated currents (I SOC). The increases of CCE amplitude and I SOC current density by hypoxia were paralleled by enhanced TRPC4 mRNA and protein expression. Hypoxia-enhanced CCE and TRPC4 expression were also attenuated by La 3ϩ . These data suggest that hypoxia increases AP-1 binding activity by enhancing Ca 2ϩ influx via La 3ϩ -sensitive TRP-encoded SOC channels in hPAEC. The Ca 2ϩ -mediated increase in AP-1 binding may play an important role in upregulating AP-1-responsive gene expression, in stimulating pulmonary vascular cell proliferation and, ultimately, in pulmonary vascular remodeling in patients with hypoxia-mediated pulmonary hypertension. activating protein-1; store-operated channels; transient receptor potential genes PULMONARY VASCULAR REMODELING in patients and animals with hypoxia-mediated pulmonary hypertension is characterized by pulmonary arterial medial hyper-

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 Ca 2ϩ 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, Ϫ361 (A/T), Ϫ254(C/G), and Ϫ218 (C/T), were identified in the 2000-bp sequence upstream of the transcriptional start site of TRPC6. Although the allele frequencies of the Ϫ361 and Ϫ218 SNPs were not different between the groups, the allele frequency of the Ϫ254(C3 G) SNP in IPAH patients (12%) was significantly higher than in normal subjects (6%; PϽ0.01). Genotype data showed that the percentage of Ϫ254G/G homozygotes in IPAH patients was 2.85 times that of normal subjects. Moreover, the Ϫ254(C3 G) SNP creates a binding sequence for nuclear factor-B. Functional analyses revealed that the Ϫ254(C3 G) SNP enhanced nuclear factor-B-mediated promoter activity and stimulated TRPC6 expression in PASMCs. Inhibition of nuclear factor-B activity attenuated TRPC6 expression and decreased agonist-activated Ca 2ϩ influx in PASMCs of IPAH patients harboring the Ϫ254G allele. Conclusions-These results suggest that the Ϫ254(C3 G) SNP may predispose individuals to an increased risk of IPAH by linking abnormal TRPC6 transcription to nuclear factor-B, an inflammatory transcription factor. (Circulation. 2009;119:2313-2322.)Key Words: calcium Ⅲ hypertension, pulmonary Ⅲ ion channels Ⅲ muscle, smooth Ⅲ NF-kappa B P ulmonary arterial hypertension (PAH) is a fatal and progressive disease characterized by elevated pulmonary vascular resistance resulting from severe pulmonary vascular remodeling. [1][2][3] Approximately 6% of PAH patients have a family history of the condition and are referred to as having familial PAH; the rest are considered to have idiopathic PAH (IPAH). Although the cause of PAH remains unclear, elevated levels of mitogenic, angiogenic, and proinflammatory factors such as platelet-derived growth factor, endothelin-1, interleukin-1␤/-6, soluble CD40 ligand, angiopoietin-1, and serotonin have been reported to correlate with the onset of IPAH. [2][3][4] Other factors associated with IPAH include the downregulation and dysfunction of voltage-gated K ϩ channels 5 and upregulation of the serotonin receptors and transporter. 6 Moreover, mutations in the bone morphogenetic protein receptor-type II gene (BMPR2) have been demonstrated to associate with the development of familial PAH and IPAH. 7,8 However, because BMPR2 mutations are present in Received March 25, 2008; accepted Februar...