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Repair and regeneration of a diseased lung using stem cells or bioengineered tissues is an exciting therapeutic approach for a variety of lung diseases and critical illnesses. Over the past decade, increasing evidence from preclinical models suggests that mesenchymal stromal cells, which are not normally resident in the lung, can be used to modulate immune responses after injury, but there have been challenges in translating these promising findings to the clinic. In parallel, there has been a surge in bioengineering studies investigating the use of artificial and acellular lung matrices as scaffolds for three-dimensional lung or airway regeneration, with some recent attempts of transplantation in large animal models. The combination of these studies with those involving stem cells, induced pluripotent stem cell derivatives, and/or cell therapies is a promising and rapidly developing research area. These studies have been further paralleled by significant increases in our understanding of the molecular and cellular events by which endogenous lung stem and/or progenitor cells arise during lung development and participate in normal and pathological remodeling after lung injury. For the 2023 Stem Cells, Cell Therapies, and Bioengineering in Lung Biology and Diseases Conference, scientific symposia were chosen to reflect the most cutting-edge advances in these fields. Sessions focused on the integration of "omics" technologies with function, the influence of immune cells on regeneration, and the role of the extracellular matrix in regeneration. The necessity for basic science studies to enhance fundamental understanding of lung regeneration and to design innovative translational studies was reinforced throughout the conference. [ABSTRACT FROM AUTHOR]

A significant number of pregnancies occur at advanced maternal age (>35 yr), which is a risk factor for pregnancy complications. Healthy pregnancies require massive hemodynamic adaptations, including an increased blood volume and cardiac output. There is growing evidence that these cardiovascular adaptations are impaired with age, however, little is known about maternal cardiac function with advanced age. We hypothesized that cardiac adaptations to pregnancy are impaired with advanced maternal age. Younger (4 mo; ~early reproductive maturity in humans) and aged (9 mo; ~35 yr in humans) pregnant Sprague-Dawley rats were assessed and compared with age-matched nonpregnant controls. Two-dimensional echocardiographic images were obtained (ultrasound biomicroscopy; under anesthesia) on gestational day 19 (term = 22 days) and compared with age-matched nonpregnant rats (n = 7-9/group). Left ventricular structure and function were assessed using short-axis images and transmitral Doppler signals. During systole, left ventricular anterior wall thickness increased with age in the nonpregnant rats, but there was no age-related difference between the pregnant groups. There were no significant pregnancy-associated differences in left ventricular wall thickness. Calculated left ventricular mass increased with age in nonpregnant rats and increased with pregnancy only in young rats. Compared with young pregnant rats, the aortic ejection time of aged pregnant rats was greater and Tei index was lower. Overall, the greater aortic ejection time and lower Tei index with age in pregnant rats suggest mildly altered cardiac adaptations to pregnancy with advanced maternal age, which may contribute to adverse outcomes in advanced maternal age pregnancies. NEW & NOTEWORTHY We demonstrated that even before the age of reproductive senescence, rats show signs of age-related alterations in cardiac structure that suggests increased cardiac work. Our data also demonstrate, using an in vivo echocardiographic approach, that advanced maternal age in a rat model is associated with altered cardiac function and structure relative to younger pregnant controls. [ABSTRACT FROM AUTHOR]

Changes in skeletal muscle are an important aspect of overall health. The collection of human muscle to study cellular and molecular processes for research requires a needle biopsy procedure which, in itself, can induce changes in the tissue. To investigate the effect of repeat tissue sampling, we collected skeletal muscle biopsy samples from vastus lateralis separated by 7 days. Cellular infiltrate, central nucleation, enlarged extracellular matrix, and rounding of muscle fibers were used as indices to define muscle damage, and we found that 16/26 samples (61.5%) revealed at least two of these symptoms in the secondary biopsy. The presence of damage influenced outcome measures usually obtained in human biopsies. Damaged muscle showed an increase in the number of small fibers even though average fiber and fiber type-specific cross-sectional area (CSA) were not different. This included higher numbers of embryonic myosin heavy chain-positive fibers (P = 0.001) as well as elevated satellite cell number (P = 0.02) in the damaged areas and higher variability in satellite cell count in the total area (P = 0.04). Collagen content was higher in damaged (P = 0.0003) as well as nondamaged areas (P = 0.05) of the muscle sections of the damaged compared with the nondamaged group. Myofibrillar protein and ribonucleic acid (RNA) fractional synthesis rates were not significantly different between the damaged compared with the nondamaged group. Results indicate that common outcomes as well as outcome variability in human muscle tissue are affected by previous biopsies. Therefore, the extent of potential damage should be assessed when performing repeated biopsies. [ABSTRACT FROM AUTHOR]

The 9th biennial conference titled "Stem Cells, Cell Therapies, and Bioengineering in Lung Biology and Diseases" was hosted virtually, due to the ongoing COVID-19 pandemic, in collaboration with the University of Vermont Larner College of Medicine, the National Heart, Lung, and Blood Institute, the Alpha-1 Foundation, the Cystic Fibrosis Foundation, and the International Society for Cell & Gene Therapy. The event was held from July 12th through 15th, 2021 with a pre-conference workshop held on July 9th. As in previous years, the objectives remained to review and discuss the status of active research areas involving stem cells (SCs), cellular therapeutics, and bioengineering as they relate to the human lung. Topics included 1) technological advancements in the in situ analysis of lung tissues, 2) new insights into stem cell signaling and plasticity in lung remodeling and regeneration, 3) the impact of extracellular matrix in stem cell regulation and airway engineering in lung regeneration, 4) differentiating and delivering stem cell therapeutics to the lung, 5) regeneration in response to viral infection, and 6) ethical development of cellbased treatments for lung diseases. This selection of topics represents some of the most dynamic and current research areas in lung biology. The virtual workshop included active discussion on state-of-the-art methods relating to the core features of the 2021 conference, including in situ proteomics, lung-on-chip, induced pluripotent stem cell (iPSC)-airway differentiation, and light sheet microscopy. The conference concluded with an open discussion to suggest funding priorities and recommendations for future research directions in basic and translational lung biology. [ABSTRACT FROM AUTHOR]

The diaphragm is the main skeletal muscle responsible for inspiration and is susceptible to age-associated decline in function and morphology. Satellite cells in diaphragm fuse into unperturbed muscle fibers throughout life, yet their role in adaptations to hypoxia in diaphragm is unknown. Given their continual fusion, we hypothesize that satellite cell depletion will negatively impact adaptations to hypoxia in the diaphragm, particularly with aging. We used the Pax7CreER/CreER:R26RDTA/DTA genetic mouse model of inducible satellite cell depletion to investigate diaphragm responses to hypoxia in adult (6 mo) and aged (22 mo) male mice. The mice were subjected to normobaric hypoxia at 10% FIO2 or normoxia for 4 wk. We showed that satellite cell depletion had no effect on diaphragm muscle fiber cross-sectional area, fiber-type distribution, myonuclear density, or regulation of extracellular matrix in either adult or aged mice. Furthermore, we showed lower muscle fiber cross-sectional area with hypoxia and age (main effects), while extracellular matrix content was higher and satellite cell abundance was lower with age (main effect) in diaphragm. Lastly, a greater number of Pax3-mRNA+ cells was observed in diaphragm muscle of satellite cell-depleted mice independent of hypoxia (main effect), potentially as a compensatory mechanism for the loss of satellite cells. We conclude that satellite cells are not required for diaphragm muscle adaptations to hypoxia in either adult or aged mice. NEW & NOTEWORTHY Satellite cells show consistent fusion into diaphragm muscle fibers throughout life, suggesting a critical role in maintaining homeostasis. Here, we report identical diaphragm adaptations to hypoxia with and without satellite cells in adult and aged mice. In addition, we propose that the higher number of Pax3-positive cells in satellite cell-depleted diaphragm muscle acts as a compensatory mechanism. [ABSTRACT FROM AUTHOR]

Ethanol metabolism in the liver induces oxidative stress and altered cytokine production preceding myofibroblast activation and fibrogenic responses. The purpose of this study was to determine how ethanol affects the fibrogenic response in precision-cut liver slices (PCLS). PCLS were obtained from chow-fed male Wistar rats (200-300 g) and were cultured up to 96 h in medium, 25 mM ethanol, or 25 mM ethanol and 0.5 mM 4-methylpyrazole (4-MP), an inhibitor of ethanol metabolism. Slices from every time point (24, 48, 72, and 96 h) were examined for glntathione (GSH) levels, lipid peroxidation [thiobarbituric acid-reactive substance (TBARS) assay], cytokine production (ELISA and RT-PCR), and myofibroblast activation [immunoblotting and immunohistochemistry for smooth muscle actin (SMA) and collagen]. Treatment of PCLS with 25 mM ethanol induced significant oxidative stress within 24 h, including depletion of cellular GSH and increased lipid peroxidation compared with controls (P < 0.05). Ethanol treatment also elicited a significant and sustained increase in interleukin-6 (IL-6) production (P < 0.05). Importantly, ethanol treatment accelerates a fibrogenic response after 48 h, represented by significant increases in SMA and collagen 1[alpha](I) production (P < 0.05). These ethanol-induced effects were prevented by the addition of 4-MP. Ethanol metabolism induces oxidative stress (GSH depletion and increased lipid peroxidation) and sustained IL-6 expression in rat PCLS. These phenomena precede and coincide with myofibroblast activation, which occurs within 48 h of treatment. These results indicate the PCLS can be used as in vitro model for studying multicellular interactions during the early stages of ethanol-induced liver injury and fibrogenesis. smooth muscle actin; collagen; IL-6; oxidative stress doi: 10.1152/ajpgi.00287.2009.

Monochloramine (N[H.sub.2]Cl) is a potent, thiol-directed oxidant capable of oxidizing thiol (S-H) residues in a wide variety of proteins. Generated in the stomach by the interaction of bacterial and host products, monochloramine has been shown to dysregulate [Ca.sup.2+] homeostasis and disrupt mucosal integrity. In this report, we show that monochloramine also leads to disturbances in intracellular free zinc concentration ([[[Zn.sup.2+]].sub.i]) in the gastric gland of the rabbit and that the increased [Zn.sup.2+] within the cell causes an independent decrease in cell viability. Changes in [[[Zn.sup.2+]].sub.i] were measured by using the fluorescent reporter FluoZin-3, whereas cell viability was assessed by measuring the conversion of calcein-AM to fluorescent calcein, an assay that is not affected by intracellular oxidation state. Cell death was confirmed using propidium iodide and YO-PRO-1 dye uptake measurements. Our experiments demonstrate that [[[Zn.sup.2+]].sub.i] is increased in gastric glands exposed to N[H.sub.2]Cl and that elevated [[[Zn.sup.2+]].sub.i] decreases cell viability. Chelation of [Zn.sup.2+] with tetrakis-(2-pyridylmethyl) ethylenediamine decreases the toxicity of N[H.sub.2]Cl, but only when administered concurrently. These findings suggest that the toxic effect of thiol oxidants present during chronic gastritis is partially due to dysregulation of [[[Zn.sup.2+]].sub.i] early in the process and that zinc chelation can protect, but not rescue, gastric glands exposed to toxic doses of N[H.sub.2]Cl. gastritis; thiol oxidation; zinc doi: 10.1152/ajpgi.00355.2009.

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

Phosphorylation at serine 68 of phospholemman (PLM) in response to [beta]-adrenergic stimulation results in simultaneous inhibition of cardiac [Na.sup.+]/[Ca.sup.2+] exchanger NCX1 and relief of inhibition of [Na.sup.+]-[K.sup.+]-ATPase. The role of PLM in mediating [beta]-adrenergic effects on in vivo cardiac function was investigated with congenic PLM-knockout (KO) mice. Echocardiography showed similar ejection fraction between wild-type (WT) and PLM-KO hearts. Cardiac catheterization demonstrated higher baseline contractility (+dP/dt) but similar relaxation (-dP/dt) in PLM-KO mice. In response to isoproterenol (Iso), maximal +dP/dt was similar but maximal -dP/dt was reduced in PLM-KO mice. Dose-response curves to Iso (0.5-25 ng) for WT and PLM-KO hearts were super-imposable. Maximal +dP/dt was reached 1-2 min after Iso addition and declined with time in WT but not PLM-KO hearts. In isolated myocytes paced at 2 Hz. contraction and intracellular [Ca.sup.2+] concentration ([[Ca.sup.2+].sub.i]) transient amplitudes and [[[Na.sup.+]].sub.i] reached maximum 2-4 min after Iso addition, followed by decline in WT but not PLM-KO myocytes. Reducing pacing frequency to 0.5 Hz resulted in much smaller increases in [[[Na.sup.+]].sub.i] and no decline in contraction and [[[Ca.sup.2+]].sub.i] transient amplitudes with time in Iso-stimulated WT and PLM-KO myocytes. Although baseline [Na.sup.+]-[K.sup.+]-ATPase current was 41% higher in PLM-KO myocytes because of increased [[alpha].sub.1]- but not [[alpha].sub.2]-subunit activity, resting [[[Na.sup.+]].sub.i] was similar between quiescent WT and PLM-KO myocytes. Iso increased [[alpha].sub.1]-subunit current ([I.sub.[alpha]1]) by 73% in WT but had no effect in PLM-KO myocytes. Iso did not affect [[alpha].sub.2]-subunit current ([I.sub.[alpha]2]) in WT and PLM-KO myocytes. In both WT and NCX1-KO hearts, PLM coimmunoprecipitated with [Na.sup.+]-[K.sup.+]ATPase [[alpha].sub.1]-and [[alpha].sub.2]-subunits, indicating that association of PLM with [Na.sup.+]-[K.sup.+]-ATPase did not require NCX1. We conclude that under stressful conditions in which [[[Na.sup.+]].sub.i] was high, [beta]-adrenergic agonist-mediated phosphorylation of PLM resulted in time-dependent reduction in inotropy due to relief of inhibition of [Na.sup.+]-[K.sup.+]-ATPase. FXYD1; excitation-contraction coupling; contractile function; intracellular [Na.sup.+] and [Ca.sup.2+] regulation; fura-2; sodium-binding benzofuran isophthalate doi:10.1152/ajpheart.00877.2009

Am J Physiol Lung Cell Mol Physiol 298: L57-L66, 2010. First published November 6, 2009; doi: 10.1152/ajplung.00037.2009.--NF-[kappa]B activation in the airway epithelium has been established as a critical pathway in ovalbumin (Ova)-induced airway inflammation in BALB/c mice (Poynter ME, Cloots R, van Woerkom T, Butnor KJ, Vacek P, Taatjes DJ, Irvin CG, Janssen-Heininger YM. J Immunol 173: 7003-7009, 2004). BALB/c mice are susceptible to the development of allergic airway disease, whereas other strains of mice, such as C57BL/6, are considered more resistant. The goal of the present study was to determine the proximal signals required for NF-[kappa]B activation in the airway epithelium in allergic airway disease and to unravel whether these signals are strain-dependent. Our previous studies, conducted in the BALB/c mouse background, demonstrated that transgenic mice expressing a dominant-negative version of I[kappa]B[alpha] in the airway epithelium (CC10-I[kappa]B[[alpha].sub.SR]) were protected from Ova-induced inflammation. In contrast to these earlier observations, we demonstrate here that CC10-I[kappa]B[[alpha].sub.SR] transgenic mice on the C57BL/6 background were not protected from Ova-induced allergic airway inflammation. Consistent with this finding, Ova-induced nuclear localization of the RelA subunit of NF-[kappa]B was not observed in C57BL/6 mice, in contrast to the marked nuclear presence of RelA in BALB/c mice. Evaluation of cytokine profiles in bronchoalveolar lavage demonstrated elevated expression of TNF-[alpha] in BALB/c mice compared with C57BL/6 mice after an acute challenge with Ova. Finally, neutralization of TNF-[alpha] by a blocking antibody prevented nuclear localization of RelA in BALB/c mice after Ova challenge. These data suggest that the mechanism of response of the airway epithelium of immunized C57BL/6 mice to antigen challenge is fundamentally different from that of immunized BALB/c mice and highlight the potential importance of TNF-[alpha] in regulating epithelial NF-[kappa]B activation in allergic airway disease. asthma; BALB/c; ovalbumin; tumor necrosis factor-[alpha]

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

Cardiac [Na.sup.+]-[K.sup.+]-ATPase (NKA) regulates intracellular [Na.sup.+], which in turn affects intracellular [Ca.sup.2+] and contractility via the [Na.sup.+]/[Ca.sup.2+] exchanger. Extracellular [K.sup.+] concentration ([[K.sup.+]]) is a central regulator of NKA activity. Phospholemman (PLM) has recently been recognized as a critical regulator of NKA in the heart. PLM reduces the intracellular [Na.sup.+] affinity of NKA, an effect relieved by PLM phosphorylation. Here we tested whether the NKA [[alpha].sub.1]-vs. [[alpha].sub.2]-isoforms have different external [K.sup.+] sensitivity and whether PLM and PKA activation affects the NKA affinity for [K.sup.+] in mouse cardiac myocytes. We measured the external [[K.sup.+]] dependence of the pump current generated by the ouabain resistant NKA isoform in myocytes from wild-type (WT) mice (i.e., current due to NKA-[[alpha].sub.1]) and mice in which the NKA isoforms have swapped ouabain affinities ([[alpha].sub.1] is ouabain sensitive and [[alpha].sub.2] is ouabain resistant) to assess current due to NKA-[[alpha].sub.2]. We found that NKA-[[alpha].sub.1] has a higher affinity for external [K.sup.+] than NKA-[[alpha].sub.2] [half-maximal pump activation ([K.sub.0.5]) = 1.5 [+ or -] 0.1 vs. 2.9 [+ or -] 0.3 mM]. The apparent external [K.sup.+] affinity of NKA was significantly lower in myocytes from WT vs. PLM-knockout mice ([K.sub.0.5] = 2.0 [+ or -] 0.2 vs. 1.05 [+ or -] 0.08 mM). However, PKA activation by isoproterenol (1 [micro]M) did not alter the [K.sub.0.5] of NKA for external [K.sup.+] in WT myocytes. We conclude that 1) NKA-[[alpha].sub.1] has higher affinity for [K.sup.+] than NKA-[[alpha].sub.2] in cardiac myocytes, 2) PLM decreases the apparent external [K.sup.+] affinity of NKA, and 3) phosphorylation of PLM at the cytosolic domain does not alter apparent extracellular [K.sup.+] affinity of NKA. voltage-clamp; phosphorylation

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

Mitochondria are proposed to be a major oxygen sensor in hypoxic pulmonary vasoconstriction (HPV), a unique response of the pulmonary circulation to low oxygen tension. Mitochondrial factors including reactive oxygen species, cytochrome c, ATP, and magnesium are potent modulators of voltage-gated [K.sup.+] ([K.sub.v]) channels in the plasmalemmal membrane of pulmonary arterial (PA) smooth muscle cells (PASMCs). Mitochondria have also been found close to the plasmalemmal membrane in rabbit main PA smooth muscle sections. Therefore, we hypothesized that differences in mitochondria localization in rat PASMCs and systemic mesenteric arterial smooth muscle cells (MASMCs) may contribute to the divergent oxygen sensitivity in the two different circulations. Cellular localization of mitochondria was compared with immunofluorescent labeling, and differences in functional coupling between mitochondria and [K.sub.v] channels was evaluated with the patch-clamp technique and specific mitochondrial inhibitors antimycin A (acting at complex III of the mitochondrial electron transport chain) and oligomycin A (which inhibits the ATP synthase). It was found that mitochondria were located significantly closer to the plasmalemmal membrane in PASMCs compared with MASMCs. Consistent with these findings, the effects of the mitochondrial inhibitors on [K.sub.v] current ([I.sub.Kv]) were significantly more potent in PASMCs than in MASMCs. The cytoskeletal disruptor cytochalasin B (10 [micro]M) also altered mitochondrial distribution in PASMCs and significantly attenuated the effect of antimycin A on the voltage-dependent parameters of [I.sub.Kv]. These findings suggest a greater structural and functional coupling between mitochondria and [K.sub.v] channels specifically in PASMCs, which could contribute to the regulation of PA excitability in HPV. pulmonary artery; vascular smooth muscle cells; mesenteric artery; [K.sup.+] channel activation; [K.sup.+] channel inactivation; confocal imaging; patch-clamp technique

Optical clarity of larvae makes the zebrafish ideal for real-time analyses of vertebrate organ function through the use of fluorescent reporters of enzymatic activities. A key function of digestive organs is to couple the generation of enzymes with mechanical processes that enable nutrient availability and absorption. However, it has been extremely difficult, and in many cases not possible, to directly observe digestive processes in a live vertebrate. Here we describe a new method to visualize intestinal protein and lipid processing simultaneously in live zebrafish larvae using a quenched fluorescent protein (EnzChek) and phospholipid (PED6). By employing these reagents, we found that wild-type larvae exhibit significant variation in intestinal phospholipase and protease activities within a group but display a strong correlation between the activities within individuals. Furthermore, we found that pancreas function is essential for larval digestive protease activity but not for larval intestinal phospholipase activity. Although fat-free (ffr) mutant larvae were previously described to exhibit impaired lipid processes, we found they also had significantly reduced protease activity. Finally, we selected and evaluated compounds that were previously suggested to have altered phospholipase activity and are known or suspected to have inflammatory effects in the intestinal tract including nonsteroidal anti-inflammatory drugs, and identified a compound that significantly increases intestinal phospholipid processing. Thus the multiple fluorescent reporter-based methodology facilitates the rapid analysis of digestive organ function in live zebrafish larvae. zebrafish; PED6; phospholipase; protease; exocrine pancreas; intestine; lipid

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

Phospholemman (PLM) regulates cardiac [Na.sup.+]/[Ca.sup.2+] exchanger (NCX1) and [Na.sup.+]-[K.sup.+]-ATPase in cardiac myocytes. PLM, when phosphorylated at [Ser.sup.68], disinhibits [Na.sup.+]-[K.sup.+]-ATPase but inhibits NCX1. PLM regulates cardiac contractility by modulating [Na.sup.+]-[K.sup.+]-ATPase and/or NCX1. In this study, we first demonstrated that adult mouse cardiac myocytes cultured for 48 h had normal surface membrane areas, t-tubules, and NCX1 and sarco(endo)plasmic reticulum [Ca.sup.2+]-ATPase levels, and retained near normal contractility, but et [[alpha].sub.1]-subunit of [Na.sup.+]-[K.sup.+]-ATPase was slightly decreased. Differences in contractility between myocytes isolated from wild-type (WT) and PLM knockout (KO) hearts were preserved after 48 h of culture. Infection with adenovirus expressing green fluorescent protein (GFP) did not affect contractility at 48 h. When WT PLM was overexpressed in PLM KO myocytes, contractility and cytosolic [Ca.sup.2+] concentration ([[[Ca.sup.2+]].sub.i]) transients reverted back to those observed in cultured WT myocytes. Both [Na.sup.+]-[K.sup.+]-ATPase current ([I.sub.pump]) and [Na.sup.+]/[Ca.sup.2+] exchange current ([I.sub.NaCa]) in PLM KO myocytes rescued with WT PLM were depressed compared with PLM KO myocytes. Overexpressing the PLMS68E mutant (phosphomimetic) in PLM KO myocytes resulted in the suppression of [I.sub.NaCa] but had no effect on [I.sub.pump]. Contractility, [[[Ca.sup.2+]].sub.i] transient amplitudes, and sarcoplasmic reticulum [Ca.sup.2+] contents in PLM KO myocytes overexpressing the PLMS68E mutant were depressed compared with PLM KO myocytes overexpressing GFP. Overexpressing the PLMS68A mutant (mimicking unphosphorylated PLM) in PLM KO myocytes had no effect on [I.sub.NaCa] but decreased [I.sub.pump]. Contractility, [[[Ca.sup.2+]].sub.i] transient amplitudes, and sarcoplasmic reticulum [Ca.sup.2+] contents in PLM KO myocytes overexpressing the S68A mutant were similar to PLM KO myocytes overexpressing GFP. We conclude that at the single-myocyte level, PLM affects cardiac contractility and [[[Ca.sup.2+]].sub.i] homeostasis primarily by its direct inhibitory effects on [Na.sup.+]/[Ca.sup.2+] exchange. adult mouse cardiac myocyte culture; excitation-contraction coupling; FXYD1; [beta]-adrenergic stimulation; phosphorylation

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

Apoptosis is a complex process essential for normal tissue development and cellular homeostasis. While biochemical events that occur late in the apoptotic process are better characterized, early physiological changes that initiate the progression of cell death remain poorly understood. Previously, we observed that lymphocytes, undergoing apoptosis in response to growth factor withdrawal, experienced a rapid and transient rise in cytosolic pH. We found that the protein responsible was the pH-regulating, plasma membrane protein [Na.sup.+]/[H.sup.+] exchanger isoform 1 (NHE1), and that its activity was impeded by inhibition of the stress-activated kinase, p38 MAP kinase. In the current study, we examined how NHE1 is activated during apoptosis. We identified the phosphorylation sites on NHE1 that regulate its alkalinizing activity in response to a cell death stimulus. Performing targeted mutagenesis, we observed that substitution of Ser726 and Ser729 for alanines produced a mutant form of NHE1 that did not alkalinize in response to an apoptotic stimulus, and expression of which protected cells from serum withdrawal- induced death. In contrast, substitution of Ser726 and Ser729 for glutamic acids raised the basal pH and induced susceptibility to death. Analysis of serine phosphorylation showed that phosphorylation of NHE1 during apoptosis decreased upon mutation of Ser726 and Ser729. Our findings thus confirm a necessary function for NHE1 during apoptosis and reveal the critical regulatory sites that when phosphorylated mediate the alkalinizing activity of NHE1 in the early stages of a cell death response. pH; sodium hydrogen exchanger; mitogen-activated protein kinase

Voltagegated [K.sup.+] (Kv) channels are important in the regulation of pulmonary vascular function having both physiological and pathophysiological implications. The pulmonary vasculature is essential for reoxygenation of the blood, supplying oxygen for cellular respiration. Mitochondria have been proposed as the major oxygen-sensing organelles in the pulmonary vasculature. Using electrophysiological techniques and immunofluorescence, an interaction of the mitochondria with Kv channels was investigated. Inhibitors, blocking the mitochondrial electron transport chain at different complexes, were shown to have a dual effect on Kv currents in freshly isolated rat pulmonary arterial smooth muscle cells (PASMCs). These dual effects comprised an enhancement of Kv current in a negative potential range (manifested as a 5- to 14-mV shift in the Kv activation to more negative membrane voltages) with a decrease in current amplitude at positive potentials. Such effects were most prominent as a result of inhibition of Complex III by antimycin A. Investigation of the mechanism of antimycin A-mediated effects on Kv channel currents ([I.sub.Kv]) revealed the presence of a mitochondria-mediated [Mg.sup.2+] and ATP-dependent regulation of Kv channels in PASMCs, which exists in addition to that currently proposed to be caused by changes in intracellular reactive oxygen species. rat; Kv channel currents; antimycin A; magnesium ions; ATP; Kv channel activation

Phospholemman (PLM, FXYD1), abundantly expressed in the heart, is the primary cardiac sarcolemmal substrate for PKA and PKC. Evidence supports the hypothesis that PLM is part of the cardiac Na-K pump complex and provides the link between kinase activity and pump modulation. PLM has also been proposed to modulate Na/Ca exchanger activity and may be involved in cell volume regulation. This study characterized the phenotype of the PLM knockout (KO) mouse heart to further our understanding of PLM function in the heart. PLM KO mice were bred on a congenic C57/BL6 background. In vivo conductance catheter measurements exhibited a mildly depressed cardiac contractile function in PLM KO mice, which was exacerbated when hearts were isolated and Langendorff perfused. There were no significant differences in action potential morphology in paced Langendorff-perfused hearts. Depressed contractile function was associated with a mild cardiac hypertrophy in PLM KO mice. Biochemical analysis of crude ventricular homogenates showed a significant increase in Na-K-ATPase activity in PLM KO hearts compared with wild-type controls. SDS-PAGE and Western blot analysis of ventricular homogenates revealed small, nonsignificant changes in Na-K-ATPase subunit expression, with two-dimensional gel (isoelectric focusing, SDS-PAGE) analysis revealing minimal changes in ventricular protein expression, indicating that deletion of PLM was the primary reason for the observed PLM KO phenotype. These studies demonstrate that PLM plays an important role in the contractile function of the normoxic mouse heart. Data are consistent with the hypothesis that PLM modulates Na-K-ATPase activity, indirectly affecting intracellular Ca and hence contractile function. FXYD1; contractile function; intracellular sodium regulation

The study's objective was to investigate how estrogen deficiency and run training affect the tibial bone-soleus muscle functional relationship in mice. Female mice were assigned into one of two surgical conditions, ovariectomy (OVX) or sham ovariectomy (sham), and one of two activity conditions, voluntary wheel running (Run) or sedentary (Sed). To determine whether differences observed between OVX and sham conditions could be attributed to estradiol ([E.sub.2]), additional OVX mice were supplemented with [E.sub.2]. Tibial bones were analyzed for their functional capacities, ultimate load, and stiffness. Soleus muscles were analyzed for their functional capacities, maximal isometric tetanic force ([P.sub.o]), and peak eccentric force. The ratios of bone functional capacities to those of muscle were calculated. The bone functional capacities were affected by both surgical condition and activity but more strongly by surgical condition. Ultimate load and stiffness for the sham group were 7-12% greater than those for OVX animals (P = 0.002), whereas only stiffness was greater for Run than for Sed animals (9%; P = 0.015). The muscle functional capacities were affected by both surgical condition and activity; however, in contrast to the bone, the muscle was more affected by activity. [P.sub.o] and peak eccentric force were 10-21% greater for Run than for Sed animals (P [less than or equal to] 0.016), whereas only Po was greater in sham than in OVX animals (9%; P = 0.011). The bone-to-muscle ratios of functional capacities were affected by activity but not by surgical condition or [E.sub.2] supplementation. Thus a mismatch of bone-muscle function occurred in mice that voluntarily ran on wheels, irrespective of estrogen status. biomechanics; functional capacity; exercise; estrogen

Acute hypoxia causes pulmonary vasoconstriction and coronary vasodilation. The divergent effects of hypoxia on pulmonary and coronary vascular smooth muscle cells suggest that the mechanisms involved in oxygen sensing and downstream effectors are different in these two types of cells. Since production of reactive oxygen species (ROS) is regulated by oxygen tension, ROS have been hypothesized to be a signaling mechanism in hypoxia-induced pulmonary vasoconstriction and vascular remodeling. Furthermore, an increased ROS production is also implicated in arteriosclerosis. In this study, we determined and compared the effects of hypoxia on ROS levels in human pulmonary arterial smooth muscle cells (PASMC) and coronary arterial smooth muscle cells (CASMC). Our results indicated that acute exposure to hypoxia ([Po.sub.2] = 25-30 mmHg for 5-10 min) significantly and rapidly decreased ROS levels in both PASMC and CASMC. However, chronic exposure to hypoxia ([Po.sub.2] = 30 mmHg for 48 h) markedly increased ROS levels in PASMC, but decreased ROS production in CASMC. Furthermore, chronic treatment with endothelin-1, a potent vasoconstrictor and mitogen, caused a significant increase in ROS production in both PASMC and CASMC. The inhibitory effect of acute hypoxia on ROS production in PASMC was also accelerated in cells chronically treated with endothelin-1. While the decreased ROS in PASMC and CASMC after acute exposure to hypoxia may reflect the lower level of oxygen substrate available for ROS production, the increased ROS production in PASMC during chronic hypoxia may reflect a pathophysiological response unique to the pulmonary vasculature that contributes to the development of pulmonary vascular remodeling in patients with hypoxia-associated pulmonary hypertension. membrane potential; vascular smooth muscle cells

Mice are useful animal models to study pathogenic mechanisms involved in pulmonary vascular disease. Altered expression and function of voltage-gated [K.sup.+] (Kv) channels in pulmonary artery smooth muscle cells (PASMCs) have been implicated in the development of pulmonary arterial hypertension. Kv currents ([I.sub.K(v)]) in mouse PASMCs have not been comprehensively characterized. The main focus of this study was to determine the biophysical and pharmacological properties of [I.sub.K(v)] in freshly dissociated mouse PASMCs with the patch-clamp technique. Three distinct whole cell [I.sub. K(v)] were identified based on the kinetics of activation and inactivation: rapidly activating and noninactivating currents (in 58% of the cells tested), rapidly activating and slowly inactivating currents (23%), and slowly activating and noninactivating currents (17%). Of the cells that demonstrated the rapidly activating noninactivating current, 69% showed [I.sub. K(v)] inhibition with 4-aminopyridine (4-AP), while 31% were unaffected. Whole cell [I.sub. K(V)] were very sensitive to tetraethylammonium (TEA), as 1 mM TEA decreased the current amplitude by 32% while it took 10 mM 4-AP to decrease [I.sub. K(V)] by a similar amount (37%). Contribution of [Ca.sup.2+]-activated [K.sup.+] ([K.sub.ca]) channels to whole cell [I.sub. K(V)] was minimal, as neither pharmacological inhibition with charybdotoxin or iberiotoxin nor perfusion with [Ca.sup.2+]-free solution had an effect on the whole cell [I.sub. K(v)]. Steady-state activation and inactivation curves revealed a window [K.sup.+] current between -40 and -10 mV with a peak at -31.5 inV. Single-channel recordings revealed large-, intermediate-, and smallamplitude currents, with an averaged slope conductance of 119.4 [+ or -] 2.7, 79.8 [+ or -] 2.8, 46.0 [+ or -] 2.2, and 23.6 [+ or -] 0.6 pS, respectively. These studies provide detailed electrophysiological and pharmacological profiles of the native [K.sub.v] currents in mouse PASMCs. Kv channels

In the intestinal lumen, protein hydrolysate increases the transcription and release of cholecystokinin (CCK) from enteroendocrine cells of the duodenal-jejunal mucosa. Our recent discovery that a G protein-coupled receptor, GPR93, is activated by dietary protein hydrolysate causing induced intracellular calcium-mediated signaling events in intestinal epithelial cells raises a possibility that GPR93 might be involved in the protein hydrolysate induction of CCK expression and/or secretion. Using the enteroendocrine STC-1 cells as a model, the present study demonstrates that increasing expression of GPR93 amplifies the peptone induction of endogenous CCK mRNA levels. A similar increase in CCK transcription, indicated by the luciferase reporter activity driven by an 820-bp CCK promoter, is also observed in response to peptone at a dose as little as 6.25 mg/ml, but not to lysophosphatidic acid (LPA), an agonist of GPR93. We discovered that the upregulation of CCK transcription involves ERK1/2, PKA, and calmodulin-dependent protein kinase-mediated pathways. Additionally, GPR93 activation by peptone induces a response in CCK release at 15 min, which continues over a 2-h period. The cAMP level in STC-1 cells overexpressing GPR93 is induced at a greater extent by peptone than by LPA, suggesting a possible explanation of the different effects of peptone and LPA on CCK transcription and secretion. Our data indicate that GPR93 can contribute to the observed induction of CCK expression and secretion by peptone and provide evidence that G protein-coupled receptors can transduce dietary luminal signals. GPR92; lysophosphatidic acid; peptone; calcium; enteroendocrine doi: 10.1152/ajpgi.00516.2006.

G protein-coupled receptors (GPCRs) have the potential to play a role as molecular sensors responsive to luminal dietary contents. Although such a role for GPCRs has been implicated in the intestinal response to protein hydrolysate, no GPCR directly involved in this process has been previously identified. In the present study, for the first time, we identified GPR93 expression in enterocytes and demonstrated its activation in these cells by protein hydrolysate with [EC.sub.50] of 10.6 mg/ml as determined by the induction of intracellular free [Ca.sup.2+]. In enterocytes, GPR93 was synergistically activated by protein hydrolysate in combination with an agonist, oleoyl-L-[alpha]-lysophosphatidic acid (LPA), which activated the receptor in these enterocytes with [EC.sub.50] of 7.9 nM. The increased intracellular [Ca.sup.2+] by GPR93 activation was observed without the addition of a promiscuous G[alpha] protein and was pertussis toxin sensitive, which suggests G[[alpha].sub.q]- and G[[alpha].sub.i]-mediated pathways. Activated GPR93 also induced pertussis toxinsensitive ERK1/2 phosphorylation. Both nuclear factor of activated T cells and 12-O-tetradecanoylphorbol 13-acetate responsive elements reporter activities were induced by protein hydrolysate in cells exogenously expressing GPR93. The peptidomimetic cefaclor by itself did not activate GPR93 but potentiated the protein hydrolysate response and further amplified the synergistic enhancement of GPR93 activation by protein hydrolysate and LPA. These data suggest that, physiologically, the composition of stimuli might determine GPR93 activity or its sensitivity toward a given activator and suggest a new mechanism of the regulation of mucosal cell proliferation and differentiation and hormonal secretion by dietary products in the lumen. extracellular signal-regulated protein kinase 1/2; intestine; GPR92; GPR93; lysophosphatidic acid

Increased conversion of glucose to lactic acid associated with decreased mitochondrial respiration is a unique feature of tumors first described by Otto Warburg in the 1920s. Recent evidence suggests that the Warburg effect is caused by oncogenes and is an underlying mechanism of malignant transformation. Using a novel approach to measure cellular metabolic rates in vitro, the bioenergetic basis of this increased glycolysis and reduced mitochondrial respiration was investigated in two human cancer cell lines, H460 and A549. The bioenergetic phenotype was analyzed by measuring cellular respiration, glycolysis rate, and ATP turnover of the cells in response to various pharmacological modulators. H460 and A549 cells displayed a dependency on glycolysis and an ability to significantly upregulate this pathway when their respiration was inhibited. The converse, however, was not true. The cell lines were attenuated in oxidative phosphorylation (OXPHOS) capacity and were unable to sufficiently upregulate mitochondrial OXPHOS when glycolysis was disabled. This observed mitochondrial impairment was intimately linked to the increased dependency on glycolysis. Furthermore, it was demonstrated that H460 cells were more glycolytic, having a greater impairment of mitochondrial respiration, compared with A549 cells. Finally, the upregulation of glycolysis in response to mitochondrial ATP synthesis inhibition was dependent on AMP-activated protein kinase activity. In summary, our results demonstrate a bioenergetic phenotype of these two cancer cell lines characterized by increased rate of glycolysis and a linked attenuation in their OXPHOS capacity. These metabolic alterations provide a mechanistic explanation for the growth advantage and apoptotic resistance of tumor cells. oxygen consumption; oxidative phosphorylation; Warburg effect; real time

Phospholemman (PLM) regulates contractility and [Ca.sup.2+] homeostasis in cardiac myocytes. We characterized excitation-contraction coupling in myocytes isolated from PLM-deficient mice backbred to a pure congenic C57BL/6 background. Cell length, cell width, and whole cell capacitance were not different between wild-type and PLM-null myocytes. Compared with wild-type myocytes, Western blots indicated total absence of PLM but no changes in [Na.sup.+]/[Ca.sup.2+] exchanger, sarcoplasmic reticulum (SR) [Ca.sup.2+]-ATPase, [[alpha].sub.1]-subunit of [Na.sup.+]-[K.sup.+]-ATPase, and calsequestrin levels in PLM-null myocytes. At 5 mM extracellular [Ca.sup.2+] concentration ([[[Ca.sup.2+]].sub.o]), contraction and cytosolic [[Ca.sup.2+]] ([[[Ca.sup.2+]].sub.i]) transient amplitudes and SR [Ca.sup.2+] contents in PLM-null myocytes were significantly (P < 0.0004) higher than wild-type myocytes, whereas the converse was true at 0.6 mM [[Ca.sup.2+]].sub.o]. This pattern of contractile and [[[Ca.sup.2+]].sub.i] transient abnormalities in PLM-null myocytes mimics that observed in adult rat myocytes overexpressing the cardiac [Na.sup.+]/[Ca.sup.2+] exchanger. Indeed, we have previously reported that [Na.sup.+]/[Ca.sup.2+] exchange currents were higher in PLM-null myocytes. Activation of protein kinase A resulted in increased inotropy such that there were no longer any contractility differences between the stimulated wild-type and PLM-null myocytes. Protein kinase C stimulation resulted in decreased contractility in both wild-type and PLM-null myocytes. Resting membrane potential and action potential amplitudes were similar, but action potential duration was much prolonged (P < 0.04) in PLM-null myocytes. Whole cell [Ca.sup.2+] current densities were similar between wild-type and PLM-null myocytes, as were the fast- and slow-inactivation time constants. We conclude that a major function of PLM is regulation of cardiac contractility and [Ca.sup.2+] fluxes, likely by modulating [Na.sup.+]/[Ca.sup.2+] exchange activity. heart; mouse; knockout; fura-2; patch-clamp; cytosolic calcium concentration

Overexpression of phospholemman (PLM) in normal adult rat cardiac myocytes altered contractile function and cytosolic [Ca.sup.2+] concentration ([[[Ca.sup.2+]].sub.i]) homeostasis and inhibited [Na.sup.+]/[Ca.sup.2+] exchanger (NCX1). In addition. PLM coimmunoprecipitated and colocalized with NCX1 in cardiac myocyte lysates. In this study, we evaluated whether the cytoplasmic domain of PLM is crucial in mediating its effects on contractility, [[[Ca.sup.2+]].sub.i] transients, and NCX1 activity. Canine PLM or its derived mutants were overexpressed in adult rat myocytes by adenovirus-mediated gene transfer. Confocal immunofluorescence images using canine-specific PLM antibodies demonstrated that the exogenous PLM or its mutants were correctly targeted to sarcolemma, t-tubules, and intercalated discs, with little to none detected in intracellular compartments. Overexpression of canine PLM or its mutants did not affect expression of NCX1, sarco(endo)plasmic reticulum [Ca.sup.2+]-ATPase, [Na.sup.+]-[K.sup.+]-ATPase, and calsequestrin in adult rat myocytes. A COOH-terminal deletion mutant in which all four potential phosphorylation sites ([Ser.sub.62], [Ser.sub.63], [Ser.sub.68], and [Thr.sup.69]) were deleted, a partial COOH-terminal deletion mutant in which [Ser.sub.68] and [Thr.sup.69] were deleted, and a mutant in which all four potential phosphorylation sites were changed to alanine all lost wild-type PLM's ability to modulate cardiac myocyte contractility. These observations suggest the importance of [Ser.sub.68] or [Thr.sup.69] in mediating PLM's effect on cardiac contractility. Focusing on [Ser.sub.68], the [Ser.sub.68] to Glu mutant was fully effective, the [Ser.sub.63] to Ala (leaving [Ser.sub.68] intact) mutant was partially effective, and the [Ser.sub.68] to Ala mutant was completely ineffective in modulating cardiac contractility, [[[Ca.sup.2+]].sub.i] transients, and NCX1 currents. Both the [Ser.sub.63] to Ala and [Ser.sub.68] to Ala mutants, as well as PLM, were able to coimmunoprecipitate NCX1. It is known that [Ser.sub.68] in PLM is phosphorylated by both protein kinases A and C. We conclude that regulation of cardiac contractility, [[[Ca.sup.2+].sub.i]], transients, and NCX1 activity by PLM is critically dependent on [Ser.sub.68]. We suggest that PLM phosphorylation at [Ser.sub.68] may be involved in cAMP- and/or protein kinase C-dependent regulation of cardiac contractility. adult rat myocyte culture; patch-clamp; fura-2; edge detection; excitation-contraction coupling

Phospholemman (FXYD1), a 72-amino acid transmembrane protein abundantly expressed in the heart and skeletal muscle, is a major substrate for phosphorylation in the cardiomyocyte sarcolemma. Biochemical, cellular, and electrophysiological studies have suggested a number of possible roles for this protein, including ion channel modulator, taurine-release channel, [Na.sup.+]/[Ca.sup.2+] exchanger modulator, and Na- KATPase-associated subunit. We have generated a phospholemmandeficient mouse. The adult null mice exhibited increased cardiac mass, larger cardiomyocytes, and ejection fractions that were 9% higher by magnetic resonance imaging compared with wild-type animals. Notably, this occurred in the absence of hypertension. Total Na-K-ATPase activity was 50% lower in the phospholemman-deficient hearts. Expression (per unit of membrane protein) of total Na-K-ATPase was only slightly diminished, but expression of the minor [[alpha].sub.2]-isoform, which has been specifically implicated in the control of contractility, was reduced by 60%. The absence of phospholemman thus results in a complex response, including a surprisingly large reduction in intrinsic Na-K-ATPase activity, changes in Na-KATPase isoform expression, increase in ejection fraction, and increase in cardiac mass. We hypothesize that a primary effect of phospholemman is to modulate the Na-K-ATPase and that its reduced activity initiates compensatory responses. FXYD protein family; heart; mouse; knockout

Diagnostic testing in patients with congenital heart disease is usually performed supine and at rest, conditions not representative of their typical hemodynamics. Upright exercise measurements of blood flow may prove valuable in the assessment of these patients, but data in normal subjects are first required. With the use of a 0.5-T open magnet, a magnetic resonance-compatible exercise cycle, and cine phase-contrast techniques, time-dependent blood flow velocities were measured in the right (RPA), left (LPA), and main (MPA) pulmonary arteries and superior (SVC) and inferior (IVC) vena cavae of 10 healthy 10- to 14-yr-old subjects. Measurements were made at seated rest and during upright cycling exercise (150% resting heart rate). Mean blood flow (l/min) and reverse flow index were computed from the velocity data. With exercise, RPA and LPA mean flow increased 2.0 [+ or -] 0.5 to 3.7 [+ or -] 0.7 (P < 0.05) and 1.6 [+ or -] 0.4 to 2.9 [+ or -] 0.8 (P < 0.05), respectively. Pulmonary reverse flow index (rest vs. exercise) decreased with exercise as follows: MPA: 0.014 [+ or -] 0.012 vs. 0.006 [+ or -] 0.006 [P = not significant (NS)], RPA: 0.005 [+ or -] 0.004 vs. 0.000 [+ or -] 0.000 (P < 0.05), and LPA: 0.041 [+ or -] 0.019 vs. 0.014 [+ or -] 0.016 (P < 0.05). SVC and IVC flow increased from 1.5 [+ or -] 0.2 to 1.9 [+ or -] 0.6 (P = NS) and 1.6 [+ or -] 0.4 to 4.9 [+ or -] 1.3 (P < 0.05), respectively. A 56/44% RPA/LPA flow distribution at both rest and during exercise suggests blood flow distribution is dominated by distal pulmonary resistance. Reverse flow in the MPA appears to originate solely from the LPA while the RPA is in relative isolation. During seated rest, the SVC-to-IVC venous return ratio is 50/50%. With light/moderate cycling exercise, IVC flow increases by threefold, whereas SVC remains essentially constant. congenital heart defects; exercise imaging; flow distribution; retrograde flow

The composition and organization of the extracellular matrix (ECM) contribute to the mechanical properties of tissues. The polymerization of fibronectin into the ECM increases actin organization and regulates the composition of the ECM. In this study, we examined the ability of cell-dependent fibronectin matrix polymerization to affect the tensile properties of an established tissue model. Our data indicate that fibronectin polymerization increases the ultimate strength and toughness, but not the stiffness, of collagen biogels. A fragment of fibronectin that stimulates mechanical tension generation by cells, but is not incorporated into ECM fibrils, did not increase the tensile properties, suggesting that changes in actin organization in the absence of fibronectin fibril formation are not sufficient to increase tensile strength. The actin cytoskeleton was needed to initiate the fibronectin-induced increases in the mechanical properties. However, once fibronectin-treated collagen biogels were fully contracted, the actin cytoskeleton no longer contributed to the tensile strength. These data indicate that fibronectin polymerization plays a significant role in determining the mechanical strength of collagen biogels and suggest a novel mechanism by which fibronectin can be used to enhance the mechanical performance of artificial tissue constructs. tissue engineering; extracellular matrix: mechanical properties

Mirza, M. Ayoub, Xue-Qian Zhang, Belinda A. Ahlers, Anwer Qureshi, Lois L. Carl, Jianliang Song, Amy L. Tucker, J. Paul Mounsey, J. Randall Moorman, Lawrence I. Rothblum, Thomas S. Zhang, and Joseph Y. Cheung. Effects of phospholemman downregulation on contractility and [[Ca.sup.2+]]i transients in adult rat cardiac myocytes. Am J Physiol Heart Circ Physiol 286: H1322-H1330, 2004. First published December 18, 2003; 10.1152/ajpheart.00997. 2003.--Phospholemman (PLM) expression was increased in rat hearts after myocardial infarction (MI). Overexpression of PLM in normal adult rat cardiac myocytes altered contractile function and cytosolic [Ca.sup.2+] concentration ([[Ca.sup.2+]]i) homeostasis in a manner similar to that observed in post-MI myocytes. In this study, we tested whether PLM downregulation in normal adult rat myocytes resulted in contractility and [[Ca.sup.2+]]i transient changes opposite to those observed in post-MI myocytes. Compared with control myocytes infected with adenovirus (Adv) expressing green fluorescent protein (GFP) alone, myocytes infected with Adv expressing both GFP and rat antisense PLM (rASPLM) had 23% less PLM protein (P < 0.012) at 3 days, but no differences were found in sarcoplasmic reticulum (SR) [Ca.sup.2+]-ATPase, [Na.sup.+]/[Ca.sup.2+] exchanger (NCX1), [Na.sup.+]-[K.sup.+]-ATPase, and calsequestrin levels. SR [Ca.sup.2+] uptake and whole cell capacitance were not affected by rASPLM treatment. Relaxation from caffeine-induced contracture was taster, and NCX1 current amplitudes were higher in rASPLM myocytes, indicating that PLM downregulation enhanced NCXI activity. In native rat cardiac myocytes, coimmunoprecipitation experiments indicated an association of PLM with NCXI. At 0.6 mM [[Ca.sup.2+]]o, rASPLM myocytes bad significantly (P < 0.003) lower contraction and [[Ca.sup.+]i transient amplitudes than control GFP myocytes. At 5 mM [[Ca.sup.2+]]o, both contraction and [[Ca.sup.2+]]i transient amplitudes were higher in rASPLM myocytes. This pattern of contractile and [[Ca.sup.2+]]i transient behavior in rASPLM myocytes was opposite to that observed in post-MI rat myocytes. We conclude that downregulation of PLM in normal rat cardiac myocytes enhanced NCX1 function and affected [[Ca.sup.2+]]i transient and contraction amplitudes. We suggest that PLM downregulation offers a potential therapeutic strategy for ameliorating contractile abnormalities in MI myocytes. adult rat myocyte culture; patch clamp; fura-2; edge detection; excitation-contraction coupling

Many patients with anterior cruciate ligament (ACL) injuries have persistent quadriceps muscle atrophy, even after considerable time in rehabilitation. Understanding the factors that regulate muscle mass, and the time course of atrophic events, is important for identifying therapeutic interventions. With a noninvasive animal model of ACL injury, a longitudinal study was performed to elucidate key parameters underlying quadriceps muscle atrophy. Male Long-Evans rats were euthanized at 6, 12, 24, or 48 h or 1, 2, or 4 wk after ACL injury that was induced via tibial compression overload; controls were not injured. Vastus lateralis muscle size was determined by wet weight and fiber cross-sectional area (CSA). Evidence of disrupted neuromuscular communication was assessed via the expression of neural cell adhesion molecule (NCAM) and genes associated with denervation and neuromuscular junction instability. Abundance of muscle RING-finger protein-1 (MuRF-1), muscle atrophy F-box (MAFbx), and 45 s pre-rRNA along with 20S proteasome activity were determined to investigate mechanisms related to muscle atrophy. Finally, muscle damage-related parameters were assessed by measuring IgG permeability, centronucleation, CD68 mRNA, and satellite cell abundance. When compared with controls, we observed a greater percentage of NCAM-positive fibers at 6 h postinjury, followed by higher MAFbx abundance 48 h postinjury, and higher 20S proteasome activity at 1 wk postinjury. A loss of muscle wet weight, smaller fiber CSA, and the elevated expression of run-related transcription factor 1 (Runx1) were also observed at the 1 wk postinjury timepoint relative to controls. There also were no differences observed in any damage markers. These results indicate that alterations in neuromuscular communication precede the upregulation of atrophic factors that regulate quadriceps muscle mass early after noninvasive ACL injury. [ABSTRACT FROM AUTHOR]

The ability to replace defective cells in an airway with cells that can engraft, integrate, and restore a functional epithelium could potentially cure a number of lung diseases. Progress toward the development of strategies to regenerate the adult lung by either in vivo or ex vivo targeting of endogenous stem cells or pluripotent stem cell derivatives is limited by our fundamental lack of understanding of the mechanisms controlling human lung development, the precise identity and function of human lung stem and progenitor cell types, and the genetic and epigenetic control of human lung fate. In this review, we intend to discuss the known stem/progenitor cell populations, their relative differences between rodents and humans, their roles in chronic lung disease, and their therapeutic prospects. Additionally, we highlight the recent breakthroughs that have increased our understanding of these cell types. These advancements include novel lineage-traced animal models and single-cell RNA sequencing of human airway cells, which have provided critical information on the stem cell subtypes, transition states, identifying cell markers, and intricate pathways that commit a stem cell to differentiate or to maintain plasticity. As our capacity to model the human lung evolves, so will our understanding of lung regeneration and our ability to target endogenous stem cells as a therapeutic approach for lung disease. [ABSTRACT FROM AUTHOR]

Ferrets are an attractive mammalian model for several diseases, especially those affecting the lungs, liver, brain, and kidneys. Many chronic human diseases have been difficult to model in rodents due to differences in size and cellular anatomy. This is particularly the case for the lung, where ferrets provide an attractive mammalian model of both acute and chronic lung diseases, such as influenza, cystic fibrosis, A1A emphysema, and obliterative bronchiolitis, closely recapitulating disease pathogenesis, as it occurs in humans. As such, ferrets have the potential to be a valuable preclinical model for the evaluation of cell-based therapies for lung regeneration and, likely, for other tissues. Induced pluripotent stem cells (iPSCs) provide a great option for provision of enough autologous cells to make patient-specific cell therapies a reality. Unfortunately, they have not been successfully created from ferrets. In this study, we demonstrate the generation of ferret iPSCs that reflect the primed pluripotent state of human iPSCs. Ferret fetal fibroblasts were reprogrammed and acquired core features of pluripotency, having the capacity for self-renewal, multilineage differentiation, and a high-level expression of the core pluripotency genes and pathways at both the transcriptional and protein level. In conclusion, we have generated ferret pluripotent stem cells that provide an opportunity for advancing our capacity to evaluate autologous cell engraftment in ferrets. [ABSTRACT FROM AUTHOR]

Cyclic compressive loading (CCL) is a massage mimetic that improves muscle regrowth from atrophy in adult rats. Therefore, we tested if a single bout of CCL increases anabolic signaling and protein synthesis in muscle during normal, weight-bearing conditions in gastrocnemius muscle from adult and aged rats. Male Brown Norway/F344 rats at 10 (adult) and 30 (aged) months of age were assigned control or CCL (receiving a single bout of CCL). Twenty-four hours following a single bout of CCL there was no change in protein synthesis, Akt, or GSK3β signaling at either age, despite adult rats having higher abundance and activation of mechanosensitive pathways (integrins and integrin-linked kinase). Murf1 was elevated in response to CCL in both age groups, potentially indicating muscle remodeling. Muscle from aged rats exhibited an increase in heat shock protein (HSP) 25 and HSP70 and in the cold shock protein RNA-binding motif 3 (RBM3), demonstrating a unique stress response to CCL in aged muscle only. Finally, muscle from aged rats exhibited higher basal protein synthesis that was corroborated by elevated eIF2Bε and rpS6 signaling, without an additional effect of CCL. In summary, a single bout of CCL does not have anabolic effects on skeletal muscle during normal, weight-bearing conditions, even though it has previously been shown to improve regrowth from atrophy. These data demonstrate that interventions that may help recover from atrophy do not necessarily induce muscle hypertrophy in unperturbed conditions. [ABSTRACT FROM AUTHOR]

There is a growing consensus that patients with chronic kidney disease (CKD) should engage in regular exercise, but there is a lack of formal guidelines. In this report, we determined whether combined aerobic and resistance exercise would elicit superior physiological gains, in particular muscular strength, compared with aerobic training alone in nondialysis CKD. Nondialysis patients with CKD stages 3b-5 were randomly allocated to aerobic exercise {AE, n - 21; 9 men; median age 63 [interquartile range (IQR) 58--71] yr; median estimated glomerular filtration rate (eGFR) 24 (IQR 20-30) ml·min-1·1.73 m-2} or combined exercise [CE, n = 20, 9 men, median age 63 (IQR 51-69) yr, median eGFR 27 (IQR 22-32) ml·min-1·1.73 m-2], preceded by a 6-wk run-in control period. Patients then underwent 12 wk of supervised AE (treadmill, rowing, or cycling exercise) or CE training (as AE plus leg extension and leg press exercise) performed three times per week. Outcome assessments of knee extensor muscle strength, quadriceps muscle volume, exercise capacity, and central hemodynamics were performed at baseline, following the 6-wk control period, and at the end of the intervention. AE and CE resulted in significant increases in knee extensor strength of 16 ± 19% (mean ± SD; P = 0.001) and 48 ± 37% (P = 0.001), respectively, which were greater after CE (P = 0.02). AE and CE resulted in 5 ± 7% (P = 0.04) and 9 ± 7% (P = 0.001) increases in quadriceps volume, respectively (P = 0.001), which were greater after CE (P = 0.01). Both AE and CE increased distance walked in the incremental shuttle walk test [28 ± 44 m (P = 0.01) and 32 ± 45 m (P = 0.01), respectively]. In nondialysis CKD, the addition of resistance exercise to aerobic exercise confers greater increases in muscle mass and strength than aerobic exercise alone. [ABSTRACT FROM AUTHOR]

Perinatal exposures are associated with altered risks of childhood allergy. Human studies and our previous work suggest that restricted growth in utero (IUGR) is protective against allergic disease. The mechanisms are not clearly defined, but reduced fetal abundance and altered metabolism of methyl donors are hypothesized as possible underlying mechanisms. Therefore, we examined whether late-gestation maternal dietary methyl donor and cofactor supplementation of the placentally restricted (PR) sheep pregnancy would reverse allergic protection in progeny. Allergic outcomes were compared between progeny from control pregnancies (CON; n = 49), from PR pregnancies without intervention (PR; n = 28), and from PR pregnancies where the dam was fed a methyl donor plus cofactor supplement from day 120 of pregnancy until delivery (PR = Methyl; n = 25). Both PR and PR + Methyl progeny were smaller than CON; supplementation did not alter birth size. PR was protective against cutaneous hypersensitivity responses to ovalbumin (OVA; P = 0.01 in singletons). Cutaneous hypersensitivity responses to OVA in PR = Methyl progeny were intermediate to and not different from the responses of CON and PR sheep. Cutaneous hypersensitivity responses to house dust mites did not differ between treatments. In singleton progeny, upper dermal mast cell density was greater in PR + Methyl than in PR or CON (each P = 0.05). The differences in the cutaneous allergic response were not explained by treatment effects on circulating immune cells or antibodies. Our results suggest that mechanisms underlying in utero programming of allergic susceptibility by IUGR and methyl donor availability may differ and imply that late-gestation methyl donor supplementation may increase allergy risk. [ABSTRACT FROM AUTHOR]

Genetic testing has multiple clinical applications including disease risk assessment, diagnosis, and pharmacogenomics. Pharmacogenomics can be utilized to predict whether a pharmacologic therapy will be effective or to identify patients at risk for treatment-related toxicity. Although genetic tests are typically ordered for a distinct clinical purpose, the genetic variants that are found may have additional implications for either disease or pharmacology. This review will address multiple examples of germline genetic variants that are informative for both disease and pharmacogenomics. The discussed relationships are diverse. Some of the agents are targeted for the disease-causing genetic variant, while others, although not targeted therapies, have implications for the disease they are used to treat. It is also possible that the disease implications of a genetic variant are unrelated to the pharmacogenomic implications. Some of these examples are considered clinically actionable pharmacogenes, with evidence-based, pharmacologic treatment recommendations, while others are still investigative as areas for additional research. It is important that clinicians are aware of both the disease and pharmacogenomic associations of these germline genetic variants to ensure patients are receiving comprehensive personalized care. [ABSTRACT FROM AUTHOR]