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Factors contributing to the development of a fibrotic vascular scar and pulmonary vascular remodeling leading to chronic thromboembolic pulmonary hypertension (CTEPH) are still unknown. This study investigates the potential contribution of multipotent progenitor cells and myofibroblasts to the development and progression of CTEPH. Histological examination of endarterectomized tissues from patients with CTEPH identified significant neointimal formation. Morphological heterogeneity was observed in cells isolated from these tissues, including a network-like growth pattern and the formation of colony-forming unit-fibroblast-like colonies (CFU-F). Cells typically coexpressed intermediate filaments vimentin and smooth muscle [alpha]-actin. Cells were characterized by immunofluorescence and quantitated by fluorescent-activated cell sorting (FACS) for the presence of cell surface markers typical of mesenchymal progenitor cells; cells were >99% [CD44.sup.+] [CD73.sup.+], [CD90.sup.+], [CD166.sup.+]; >80% [CD29.sup.+]; 45-99% [CD105.sup.+]; [CD34.sup.-] and [CD45.sup.-]. Cells were capable of adipogenic and osteogenic differentiation, determined by Oil Red O and Alizarin Red staining, respectively. Additionally, a population of [Stro-1.sup.+] cells, a marker of bone marrow-derived stromal cells (4.2%), was sorted by FACS and also capable of adipogenic and osteogenic differentiation. In conclusion, this study is the first to identify a myofibroblast cell phenotype to be predominant within endarterectomized tissues, contributing extensively to the vascular lesion/clot. This cell may arise from transdifferentiation of adventitial fibroblasts or differentiation of mesenchymal progenitor cells. The unique microenvironment created by the stabilized clot is likely a factor in stimulating such cellular changes. These findings will be critical in establishing future studies in the development of novel and much needed therapeutic approaches for pulmonary hypertension. progenitor cells; chronic thromboembolic pulmonary hypertension; occlusion; remodeling doi: 10.1152/ajpcell.00416.2009.

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-2[alpha], 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-2[alpha] 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. doi: 10.1152/ajplung.00314.2009 Oct-4 gene; pulmonary arterial smooth muscle cells; hypoxia

The activity of voltage-gated [K.sup.+] ([K.sub.v]) channels plays an important role in regulating pulmonary artery smooth muscle cell (PASMC) contraction, proliferation, and apoptosis. The highly conserved [NH.sub.2]-terminal tetramerization domain (TI) of [K.sub.v] channels is important for proper channel assembly, association with regulatory [K.sub.v] [beta]-subunits, and localization of the channel to the plasma membrane. We recently reported two nonsynonymous mutations (GI82R and E211D) in the KCNA5 gene of patients with idiopathic pulmonary arterial hypertension, which localize to the TI domain of KCNA5. To study the electrophysiological properties and expression patterns of the mutants compared with the wild-type (WT) channel in vitro, we transfected HEK-293 cells with WT KCNA5, G182R, E211D, or the double mutant G182R/E211D channel. The mutants form functional channels; however, whole cell current kinetic differences between WT and mutant channels exist. Steady-state inactivation curves of the G182R and G182R/E211D channels reveal accelerated inactivation; the mutant channels inactivated at more hyperpolarized potentials compared with the WT channel. Channel protein expression was also decreased by the mutations. Compared with the WT channel, which was present in its mature glycosylated form, the mutant channels are present in greater proportion in their immature form in HEK-293 cells. Furthermore, GI82R protein level is greatly reduced in COS-1 cells compared with WT. Immunostaining data support the hypothesis that, while WT protein localizes to the plasma membrane, mutant protein is mainly retained in intracellular packets. Overall, these data support a role for the T1 domain in channel kinetics as well as in KCNA5 channel subcellular localization. voltage-gated [K.sup.+] channels; T1 domain: pulmonary arterial hypertension; [K.sub.v] 1.5 doi:10.1152/ajpcell.00464.2009

Changes in the structure, function and integrity of blood vessels are necessary to the pathogenesis of many diseases, including pulmonary arterial hypertension (PAH), atherosclerosis and neointimal repair. Adult vascular smooth [...], Notch receptor signaling is implicated in controlling smooth muscle cell proliferation and in maintaining smooth muscle cells in an undifferentiated state. Pulmonary arterial hypertension is characterized by excessive vascular resistance, smooth muscle cell proliferation in small pulmonary arteries, leading to elevation of pulmonary vascular resistance, right ventricular failure and death. Here we show that human pulmonary hypertension is characterized by overexpression of NOTCH3 in small pulmonary artery smooth muscle cells and that the severity of disease in humans and rodents correlates with the amount of NOTCH3 protein in the lung. We further show that mice with homozygous deletion of Notch3 do not develop pulmonary hypertension in response to hypoxic stimulation and that pulmonary hypertension can be successfully treated in mice by administration of N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT), a γy-secretase inhibitor that blocks activation of Notch3 in smooth muscle cells. We show a mechanistic link from NOTCH3 receptor signaling through the Hairy and enhancer of Split-5 (HES-5) protein to smooth muscle cell proliferation and a shift to an undifferentiated smooth muscle cell phenotype. These results suggest that the NOTCH3--HES-5 signaling pathway is crucial for the development of pulmonary arterial hypertension and provide a target pathway for therapeutic intervention.

Pulmonary vascular remodeling occurs in patients with chronic thromboembolic pulmonary hypertension (CTEPH). One factor contributing to this vascular wall thickening is the proliferation of pulmonary artery smooth muscle cells (PASMC). Store-operated [Ca.sup.2+] entry (SOCE) and cytosolic free [Ca.sup.2+] concentration ([[[Ca.sup.2+]].sub.cyt]) in PASMC are known to be important in cell proliferation and vascular remodeling in pulmonary hypertension. Rapamycin is widely known for its antiproliferative effects in injured coronary arteries. Although several reports have suggested favorable effects of rapamycin in animal models of pulmonary hypertension, no reports have been published to date in human tissues. Here we report that rapamycin has an inhibitory effect on SOCE and an antiproliferative effect on PASMC derived from endarterectomized tissues of CTEPH patients. Cells were isolated from endarterectomized tissues obtained from patients undergoing pulmonary thromboendarterectomy (PTE). Immunohistochemical analysis indicated high deposition of platelet-derived growth factor (PDGF) in tissue sections from PTE tissues and increased PDGF receptor expression. PDGF transiently phosphorylated Akt, mammalian target of rapamycin (mTOR), and p70S6 kinase in CTEPH cells from CTEPH patients. Acute treatment (30 min) with rapamycin (10 nM) slightly increased cyclopiazonic acid (10 [micro]M)-induced [Ca.sup.2+] mobilization and significantly reduced SOCE. Chronic treatment (24 h) with rapamycin reduced [Ca.sup.2+] mobilization and markedly inhibited SOCE. The inhibitory effects of rapamycin on SOCE were less prominent in control cells. Rapamycin also significantly reduced PDGF-stimulated cell proliferation. In conclusion, the data from this study indicate the importance of the mTOR pathway in the development of pulmonary vascular remodeling in CTEPH and suggest a potential therapeutic benefit of rapamycin (or inhibition of mTOR) in these patients. proliferation; rapamycin; therapy doi: 10.1152/ajplung.90548.2008

Introduction Pulmonary hypertension (PH) is characterized by a progressive increase in pulmonary vascular resistance (PVR) and vascular remodeling, which without treatment leads to right-heart failure and death often within 2-3 [...], Pulmonary hypertension (PH) is an unremitting disease defined by a progressive increase in pulmonary vascular resistance leading to right-sided heart failure. Using mice with genetic deletions of caveolin 1 (Cav1) and eNOS (Nos3), we demonstrate here that chronic eNOS activation secondary to loss of caveolin-1 can lead to PH. Consistent with a role for eNOS in the pathogenesis of PH, the pulmonary vascular remodeling and PH phenotype of Cav1-/- mice were absent in Cav1-/-Nos3-/- mice. Further, treatment of Cav1-/- mice with either MnTMPyP (a superoxide scavenger) or L-NAME (a NOS inhibitor) reversed their pulmonary vascular pathology and PH phenotype. Activation of eNOS in Cav1-/- lungs led to the impairment of PKG activity through tyrosine nitration. Moreover, the PH phenotype in Cav1-/- lungs could be rescued by overexpression of PKG-1. The clinical relevance of the data was indicated by the observation that lung tissue from patients with idiopathic pulmonary arterial hypertension demonstrated increased eNOS activation and PKG nitration and reduced caveolin-1 expression. Together, these data show that loss of caveolin-1 leads to hyperactive eNOS and subsequent tyrosine nitration--dependent impairment of PKG activity, which results in PH. Thus, targeting of PKG nitration represents a potential novel therapeutic strategy for the treatment of PH.

Acute pulmonary embolism occurs in more than half a million people a year in the United States. Chronic thromboembolic pulmonary hypertension (CTEPH) develops in ~4% of these patients due to unresolved thromboemboli. CTEPH is thus a relatively common, progressive, and potentially fatal disease. One currently proposed theory for the poor resolution advocates that modification of fibrinogen in CTEPH patients causes resistance of emboli to fibrinolysis. The current study investigated the regulation of cytosofic [Ca.sup.2+] [([[Ca.sup.2+]].sub.cyt]), central to the control of cell migration, proliferation, and contraction, by chronic exposure of pulmonary artery smooth muscle (PASMC) and endothelial (PAEC) cells to fibrinogen and fibrin. Basal [[[Ca.sup.2+]].sub.cyt] was substantially elevated in PAEC after culture on fibrinogen, fibrin, and thrombin and in PASMC on fibrinogen and fibrin. In PAEC, fibrinogen significantly decreased the peak [[[Ca.sup.2+]].sub.cyt] transient (P thrombin; pulmonary vascular remodeling; calcium regulation

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

Mechanosensitive cation channels and Ca2+ influx through these channels play an important role in the regulation of endothelial cell functions. Transient receptor potential canonical channel 6 (TRPC6) is a diacylglycerol-sensitive nonselective cation channel that forms receptor-operated Ca2+ channels in a variety of cell types. Piezo1 is a mechanosensitive cation channel activated by membrane stretch and shear stress in lung endothelial cells. In this study, we report that TRPC6 and Piezo1 channels both contribute to membrane stretch-mediated cation currents and Ca2+ influx or increase in cytosolic-free Ca2+ concentration ([Ca2+]cyt) in human pulmonary arterial endothelial cells (PAECs). The membrane stretch-mediated cation currents and increase in [Ca2+]cyt in human PAECs were significantly decreased by GsMTX4, a blocker of Piezo1 channels, and by BI-749327, a selective blocker of TRPC6 channels. Extracellular application of 1-oleoyl-2-acetyl-sn-glycerol (OAG), a membrane permeable analog of diacylglycerol, rapidly induced whole cell cation currents and increased [Ca2+]cyt in human PAECs and human embryonic kidney (HEK)-cells transiently transfected with the human TRPC6 gene. Furthermore, membrane stretch with hypo-osmotic or hypotonic solution enhances the cation currents in TRPC6-transfected HEK cells. In HEK cells transfected with the Piezo1 gene, however, OAG had little effect on the cation currents, but membrane stretch significantly enhanced the cation currents. These data indicate that, while both TRPC6 and Piezo1 are involved in generating mechanosensitive cation currents and increases in [Ca2+]cyt in human PAECs undergoing mechanical stimulation, only TRPC6 (but not Piezo1) is sensitive to the second messenger diacylglycerol. Selective blockers of these channels may help develop novel therapies for mechanotransduction-associated pulmonary vascular remodeling in patients with pulmonary arterial hypertension. [ABSTRACT FROM AUTHOR]

Thrombin is a procoagulant inflammatory agonist that can disrupt the endothelium-lumen barrier in the lung by causing contraction of endothelial cells and promote pulmonary cell proliferation. Both contraction and proliferation require increases in cytosolic [Ca.sup.2+] concentration ([[[Ca.sup.2+]].sub.cyt]). In this study, we compared the effect of thrombin on [Ca.sup.2+] signaling in human pulmonary artery smooth muscle (PASMC) and endothelial (PAEC) cells. Thrombin increased the [[[Ca.sup.2+]].sub.cyt] in both cell types; however, the transient response was significantly higher and recovered quicker in the PASMC, suggesting different mechanisms may contribute to thrombin-mediated increases in [[[Ca.sup.2+]].sub.cyt] in these cell types. Depletion of intracellular stores with cyclopiazonic acid (CPA) in the absence of extracellular [Ca.sup.2+] induced calcium transients representative of those observed in response to thrombin in both cell types. Interestingly, CPA pretreatment significantly attenuated thrombin-induced [Ca.sup.2+] release in PASMC; this attenuation was not apparent in PAEC, indicating that a PAEC-specific mechanism was targeted by thrombin. Treatment with a combination of CPA, caffeine, and ryanodine also failed to abolish the thrombin-induced [Ca.sup.2+] transient in PAEC. Notably, thrombin-induced receptor-mediated calcium influx was still observed in PASMC after CPA pretreatment in the presence of extracellular [Ca.sup.2+]. [Ca.sup.2+] oscillations were triggered by thrombin in PASMC resulting from a balance of extracellular [Ca.sup.2+] influx and [Ca.sup.2+] reuptake by the sarcoplasmic reticulum. The data show that thrombin induces increases in intracellular calcium in PASMC and PAEC with a distinct CPA-, caffeine-, and ryanodine-insensitive release existing only in PAEC. Furthermore, a dynamic balance between [Ca.sup.2+] influx, intracellular [Ca.sup.2+] release, and reuptake underlie the [Ca.sup.2+] transients evoked by thrombin in some PASMC. Understanding of such mechanisms will provide an important insight into thrombin-mediated vascular injury during hypertension. sarcoplasmic and endoplasmic reticulum; [Ca.sup.2+] store

Intestinal mucosal restitution occurs as a consequence of epithelial cell migration and reseals superficial wounds after injury. This rapid reepithelialization is mediated in part by a phospholipase C-[gamma]1 (PLC-[gamma]1)-induced [Ca.sup.2+] signaling, but the exact mechanism underlying such signaling and its regulation remains elusive. The small GTP-binding protein Rac1 functions as a pivotal regulator of several signaling networks and plays an important role in regulating cell motility. The current study tests the hypothesis that Rac1 modulates intestinal epithelial cell migration after wounding by altering PLC-[gamma]1-induced [Ca.sup.2+] signaling. Inhibition of Rac1 activity by treatment with its inhibitor NSC-23766 or Rac1 silencing with small interfering RNA decreased store depletion-induced [Ca.sup.2+] influx and suppressed cell migration during restitution, whereas ectopic overexpression of Rac1 increased [Ca.sup.2+] influx and promoted cell migration. Rac1 physically interacted with PLC-[gamma]1 and formed Rac1/PLC-[gamma]1 complex in intestinal epithelial cells. PLC-[gamma]1 silencing in cells overexpressing Rac1 prevented stimulation of store depletion-induced [Ca.sup.2+] influx and cell migration after wounding. Polyamine depletion inhibited expression of both Rac1 and PLC-[gamma]1, decreased Rac1/PLC-[gamma]1 complex levels, reduced [Ca.sup.2+] influx, and repressed cell migration. Overexpression of Rac1 alone failed to rescue [Ca.sup.2+] influx after store depletion and cell migration in polyamine-deficient cells, because it did not alter PLC-[gamma]1 levels. These results indicate that Rac1 promotes intestinal epithelial cell migration after wounding by increasing [Ca.sup.2+] influx as a result of its interaction with PLC-[gamma]1. mucosal injury; early rapid mucosal repair; polyamines; cell migration; capacitative [Ca.sup.2+] entry; cdx2 gene; small guanosine 5'-triphosphate-binding proteins

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