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Chronic thromboembolic pulmonary hypertension (CTEPH) is characterized by a fibrotic thrombus persisting and obliterating the lumen of pulmonary arteries; its pathogenesis remains poorly defined. This study investigates a potential contribution for progenitor cell types in the development of vascular obliteration and remodeling in CTEPH patients. Endarterectomized tissue from patients undergoing pulmonary thromboendarterectomy was collected and examined for the structure and cellular composition. Our data show an organized fibrin network structure in unresolved thromboemboli and intimal remodeling in vascular wall tissues, characterized by smooth muscle [alpha]-actin (SM-[alpha]A)-positive cell proliferation in proximal regions (adjacent to thromboemboli) and neoangiogenesis/recanalization in distal regions (downstream from thromboemboli). Cells that are positively stained with CD34 and fetal liver kinase-1 (Flk-1) ([CD34.sup.+][Flk-1.sup.+]) were identified in both the proximal and distal vascular tissues; a subpopulation of [CD34.sup.+][Flk-1.sup.+][CD133.sup.+] cells were further identified by immunostaining. Triple-positive cells are indicative of a population of putative endothelial progenitor cells or potential colony-forming units of endothelial cells. In addition, inflammatory cells ([CD45.sup.+]) and collagen-secreting cells ([procollagen-1.sup.+]) were detected in the proximal vascular wall. Some of the [CD34.sup.+] cells in CTEPH cells isolated from proximal regions were also positive for SM-otA. Our data indicate that putative progenitor cell types are present in the neointima of occluded vessels of CTEPH patients. It is possible that the microenvironment provided by thromboemboli may promote these putative progenitor cells to differentiate and enhance intimal remodeling. pulmonary embolism; endarterectomy; vascular remodeling

Pulmonary vascular remodeling, a major cause for the elevated pulmonary vascular resistance in patients with pulmonary arterial hypertension (PAH), is partially due to increased proliferation of pulmonary arterial smooth muscle cells (PASMC) in the media, resulting in vascular wall thickening. Platelet-derived growth factor (PDGF) is a potent mitogen that may be involved in the progression of PAH. Blockade of PDGF receptors has been demonstrated to have therapeutic potential for patients with severe pulmonary hypertension. Prednisolone is an immunosuppressant shown to have anti-inflammatory and antiproliferative effects on PASMC. This study was designed to investigate whether PDGF and prednisolone affect human PASMC proliferation by regulating the nuclear translocation of NF-[kappa]B (a transcription factor composed of 2 subunits, p50 and p65). Treatment of human PASMC with PDGF (10 ng/ml) significantly increased nuclear translocation of p50 and p65 subunits. Inhibition of NF-[kappa]B activation or nuclear translocation of p50/p65 significantly attenuated PDGF-induced PASMC proliferation (determined by [[sup.3]H]thymidine incorporation). In the presence of prednisolone (200 [micro]M), the PDGF-induced nuclear translocation of p50 and p65 subunits was markedly inhibited (P < 0.05 vs. the cells treated with PDGF alone). These results indicate that PDGF-induced nuclear translocation of NF-[kappa]B may play an important role in stimulating PASMC proliferation (and/or enhancing PASMC survival), whereas prednisolone may exert anti-inflammatory and antiproliferative effects on PASMC by inhibiting NF-[kappa]B nuclear translocation. proliferation; pulmonary arterial hypertension; immunosuppressant; therapy

A rise in cytosolic [Ca.sup.2+] concentration ([[[Ca.sup.2+]].sub.cyt]) m pulmonary artery smooth muscle cells (PASMC) is a trigger for pulmonary vasoconstriction and a stimulus for PASMC proliferation and migration. Multiple mechanisms are involved in regulating [[[Ca.sup.2+]].sub.cyt] in human PASMC. The resting [[[Ca.sup.2+]].sub.cyt] and [Ca.sup.2+] entry are both increased in PASMC from patients with idiopathic pulmonary arterial hypertension (IPAH), which is believed to be a critical mechanism for sustained pulmonary vasoconstriction and excessive pulmonary vascular remodeling in these patients. Here we report that protein expression of NCX1, an NCX family member of [Na.sub.+]/[Ca.sup.2+] exchanger proteins is upregulated in PASMC from IPAH patients compared with PASMC from normal subjects and patients with other cardiopulmonary diseases. The [Na.sup.+]/[Ca.sup.2+] exchanger operates in a forward ([Ca.sup.2+] exit) and reverse ([Ca.sup.2+] entry) mode. By activating the reverse mode of [Na.sup.+]/[Ca.sup.2+] exchange, removal of extracellular [Na.sup.+] caused a rapid increase in [[[Ca.sup.2+]].sub.cyt], which was significantly enhanced in IPAH PASMC compared with normal PASMC. Furthermore, passive depletion of intracellular [Ca.sup.2+] stores using cyclopiazonic acid (10 [micro]M) not only caused a rise in [[[Ca.sup.2+]].sub.cyt] due to [Ca.sup.2+] influx through store-operated [Ca.sup.2+] channels but also mediated a rise in [[[Ca.sup.2+]].sub.cyt] via the reverse mode of [Na.sub.+]/[Ca.sup.2+] exchange. The upregulated NCX1 in IPAH PASMC led to an enhanced [Ca.sup.2+] entry via the reverse mode of [Na.sup.+]/[Ca.sup.2+] exchange, but did not accelerate [Ca.sup.2+] extrusion via the forward mode of [Na.sup.+]/[Ca.sup.2+] exchange. These observations indicate that the upregulated NCX1 and enhanced [Ca.sup.2+] entry via the reverse mode of [Na.sup.+]/[Ca.sup.2+] exchange are an additional mechanism responsible for the elevated [[[Ca.sup.2+]].sub.cyt] in PASMC from IPAH patients. transient receptor potential channel; reverse and forward mode; proliferation doi:10.1152/ajpcell.00383.2006

The pore-forming [alpha]--subunit, Kv1.5, forms functional voltage-gated [K.sup.+] (Kv) channels in human pulmonary artery smooth muscle cells (PASMC) and plays an important role in regulating membrane potential, vascular tone, and PASMC proliferation and apoptosis. Inhibited [K.sup.+] channel expression and function have been implicated in PASMC from patients with idiopathic pulmonary arterial hypertension (IPAH). Here, we report that overexpression of the Kv1.5 channel gene (KCNA5) in human PASMC and other cell lines produced a 15-pS single channel current and a large whole cell current that was sensitive to 4-aminopyridine. Extracellular application of nicotine, bepridil, correolide, and endothelin-1 (ET-1) all significantly and reversibly reduced the Kv1.5 currents, while nicotine and bepridil also accelerated the inactivation kinetics of the currents. Furthermore, we sequenced KCNA5 from IPAH patients and identified 17 single-nucleotide polymorphisms (SNPs); 7 are novel SNPs. There are 12 SNPs in the upstream 5' region, 2 of which may alter transcription factor binding sites in the promoter, 2 nonsynonymous SNPs in the coding region, 2 SNPs in the 3'-untranslated region, and 1 SNP in the 3'-flanking region. Two SNPs may correlate with the nitric oxide-mediated decrease in pulmonary arterial pressure. Allele frequency of two other SNPs in patients with a history of fenfluramine and phentermine use was significantly different from patients who have never taken the anorexigens. These results suggest that 1) Kv1.5 channels are modulated by various agonists (e.g., nicotine and ET-1); 2) novel SNPs in KCNA5 are present in IPAH patients; and 3) SNPs in the promoter and translated regions of KCNA5 may underlie the altered expression and/or function of Kv1.5 channels in PASMC from IPAH patients. voltage-gated [K.sup.+] channel; single-nucleotide polymorphism; drug response; etiology doi:10.1152/ajpcell.00405.2006

Mechanical forces regulate lung maturation in the fetus by promoting type II epithelial differentiation. However, the cell surface receptors that transduce these mechanical cues into cellular responses remain largely unknown. When distal lung type II epithelial cells isolated from embryonic day 19 rat fetuses were cultured on flexible plates coated with laminin, fibronectin, vitronectin, collagen, or elastin and exposed to a level of mechanical strain (5%) similar to that observed in utero, transmembrane signaling responses were induced under all conditions, as measured by ERK activation. However, mechanical stress maximally increased expression of the type II cell differentiation marker surfactant protein C when cells were cultured on laminin substrates. Strain-induced alveolar epithelial differentiation was inhibited by interfering with cell binding to laminin using soluble laminin peptides (IKVIV or YIGSR) or blocking antibodies against integrin [[beta].sub.1], [[alpha].sub3], or [[alpha].sub.6]. Additional studies were carried out with substrates coated directly with different nonactivating anti-integrin antibodies. Blocking integrin [[beta].sub.1] and [[alpha].sub.6] binding sites inhibited both cell adhesion and differentiation, whereas inhibition of [[alpha].sub.3] prevented differentiation without altering cell attachment. These data demonstrate that various integrins contribute to mechanical control of type II lung epithelial cell differentiation on laminin substrates. However, they may act via distinct mechanisms, including some that are independent of their cell anchoring role. laminin; surfactant protein C

The efficacy and safety of sildenafil in the treatment of patients with symptomatic pulmonary arterial hypertension is demonstrated. Sildenafil tends to improve exercise capacity, WHO functional class, and hemodynamics in patients with symptomatic pulmonary arterial hypertension.

Alveolar epithelial apoptosis is an important feature of hyperoxia-induced lung injury in vivo and has been described in the early stages of bronchopulmonary dysplasia (chronic lung disease of preterm newborn). Molecular regulation of hyperoxia-induced alveolar epithelial cell death remains incompletely understood. In view of functional involvement of Fas/FasL system in physiological postcanalicular type II cell apoptosis, we speculated this system may also be a critical regulator of hyperoxia-induced apoptosis. The aim of this study was to investigate the effects of hyperoxia on apoptosis and apoptotic gene expression in alveolar epithelial cells. Apoptosis was studied by TUNEL, electron microscopy, DNA size analysis, and caspase assays. Fas/FasL expression was determined by Western blot analysis and RPA. We determined that in MLE-12 cells exposed to hyperoxia, caspase-mediated apoptosis was the first morphologically and biochemically recognizable mode of cell death, followed by necrosis of residual adherent cells. The apoptotic stage was associated with a threefold upregulation of Fas mRNA and protein expression and increased susceptibility to direct Fas receptor activation, concomitant with a threefold increase of FasL protein levels. Fas gene silencing by siRNAs significantly reduced hyperoxia-induced apoptosis. In murine fetal type II cells, hyperoxia similarly induced markedly increased Fas/FasL protein expression, confirming validity of results obtained in transformed MLE-12 cells. Our findings implicate the Fas/FasL system as an important regulator of hyperoxia-induced type II cell apoptosis. Elucidation of regulation of hyperoxia-induced lung apoptosis may lead to alternative therapeutic strategies for perinatal or adult pulmonary diseases characterized by dysregulated type II cell apoptosis. programmed cell death; bronchopulmonary dysplasia; chronic lung disease; oxygen; alveolar epithelial type II cell