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The emergence and global spread of SARS-CoV-2 has resulted in the urgent need for an in-depth understanding of molecular functions of viral proteins and their interactions with the host proteome. Several individual omics studies have extended our knowledge of COVID-19 pathophysiology.sup.1-10. Integration of such datasets to obtain a holistic view of virus-host interactions and to define the pathogenic properties of SARS-CoV-2 is limited by the heterogeneity of the experimental systems. Here we report a concurrent multi-omics study of SARS-CoV-2 and SARS-CoV. Using state-of-the-art proteomics, we profiled the interactomes of both viruses, as well as their influence on the transcriptome, proteome, ubiquitinome and phosphoproteome of a lung-derived human cell line. Projecting these data onto the global network of cellular interactions revealed crosstalk between the perturbations taking place upon infection with SARS-CoV-2 and SARS-CoV at different levels and enabled identification of distinct and common molecular mechanisms of these closely related coronaviruses. The TGF-[beta] pathway, known for its involvement in tissue fibrosis, was specifically dysregulated by SARS-CoV-2 ORF8 and autophagy was specifically dysregulated by SARS-CoV-2 ORF3. The extensive dataset (available at https://covinet.innatelab.org) highlights many hotspots that could be targeted by existing drugs and may be used to guide rational design of virus- and host-directed therapies, which we exemplify by identifying inhibitors of kinases and matrix metalloproteases with potent antiviral effects against SARS-CoV-2. Multi-omics profiling of effects of SARS-CoV-2 and SARS-CoV on A549, a lung-derived human cell line, produces a dataset enabling identification of common and virus-specific mechanisms of infection., Author(s): Alexey Stukalov [sup.1] , Virginie Girault [sup.1] , Vincent Grass [sup.1] , Ozge Karayel [sup.2] , Valter Bergant [sup.1] , Christian Urban [sup.1] , Darya A. Haas [sup.1] , [...]

The distal lung contains terminal bronchioles and alveoli that facilitate gas exchange. Three-dimensional in vitro human distal lung culture systems would strongly facilitate the investigation of pathologies such as interstitial lung disease, cancer and coronavirus disease 2019 (COVID-19) pneumonia caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here we describe the development of a long-term feeder-free, chemically defined culture system for distal lung progenitors as organoids derived from single adult human alveolar epithelial type II (AT2) or KRT5.sup.+ basal cells. AT2 organoids were able to differentiate into AT1 cells, and basal cell organoids developed lumens lined with differentiated club and ciliated cells. Single-cell analysis of KRT5.sup.+ cells in basal organoids revealed a distinct population of ITGA6.sup.+ITGB4.sup.+ mitotic cells, whose offspring further segregated into a TNFRSF12A.sup.hi subfraction that comprised about ten per cent of KRT5.sup.+ basal cells. This subpopulation formed clusters within terminal bronchioles and exhibited enriched clonogenic organoid growth activity. We created distal lung organoids with apical-out polarity to present ACE2 on the exposed external surface, facilitating infection of AT2 and basal cultures with SARS-CoV-2 and identifying club cells as a target population. This long-term, feeder-free culture of human distal lung organoids, coupled with single-cell analysis, identifies functional heterogeneity among basal cells and establishes a facile in vitro organoid model of human distal lung infections, including COVID-19-associated pneumonia. A long-term culture method for organoids derived from single adult human lung cells is used to identify progenitor cells and study SARS-CoV-2 infection., Author(s): Ameen A. Salahudeen [sup.1] [sup.19] , Shannon S. Choi [sup.1] , Arjun Rustagi [sup.2] , Junjie Zhu [sup.3] , Vincent van Unen [sup.4] [sup.5] , Sean M. de la [...]

Although single-cell RNA sequencing studies have begun to provide compendia of cell expression profiles.sup.1-9, it has been difficult to systematically identify and localize all molecular cell types in individual organs to create a full molecular cell atlas. Here, using droplet- and plate-based single-cell RNA sequencing of approximately 75,000 human cells across all lung tissue compartments and circulating blood, combined with a multi-pronged cell annotation approach, we create an extensive cell atlas of the human lung. We define the gene expression profiles and anatomical locations of 58 cell populations in the human lung, including 41 out of 45 previously known cell types and 14 previously unknown ones. This comprehensive molecular atlas identifies the biochemical functions of lung cells and the transcription factors and markers for making and monitoring them; defines the cell targets of circulating hormones and predicts local signalling interactions and immune cell homing; and identifies cell types that are directly affected by lung disease genes and respiratory viruses. By comparing human and mouse data, we identified 17 molecular cell types that have been gained or lost during lung evolution and others with substantially altered expression profiles, revealing extensive plasticity of cell types and cell-type-specific gene expression during organ evolution including expression switches between cell types. This atlas provides the molecular foundation for investigating how lung cell identities, functions and interactions are achieved in development and tissue engineering and altered in disease and evolution. Expression profiling on 75,000 single cells creates a comprehensive cell atlas of the human lung that includes 41 out of 45 previously known cell types and 14 new ones., Author(s): Kyle J. Travaglini [sup.1] [sup.2] , Ahmad N. Nabhan [sup.1] [sup.2] [sup.12] , Lolita Penland [sup.3] [sup.13] , Rahul Sinha [sup.4] [sup.5] , Astrid Gillich [sup.1] [sup.2] , Rene [...]

Bone morphogenetic proteins (BMPs) were once considered only to have a role in bone formation. It is now known that they have pivotal roles in other organ diseases, including heritable pulmonary arterial hypertension (PAH), where genetic mutations in the type II BMP receptor (BMPR2) are the commonest cause of receptor dysfunction. However, it has also recently been demonstrated that aquaporin 1 (Aqp1) dysfunction may contribute to PAH, highlighting that PAH development may involve more than one pathogenic pathway. Whether reduction in BMPR2 affects Aqp1 is unknown. We therefore studied Aqp1 in BMPR2-silenced human pulmonary microvascular endothelial cells (HPMECs). We demonstrated reduced Aqp1 mRNA protein, and function in the BMPR2-silenced cells. Additionally, BMPR2-silenced cells exhibited lower expression of BMP-signaling molecules. In conclusion, decreased BMPR2 appears to affect Aqp1 at the mRNA, protein, and functional levels. This observation may identify a contributory mechanism for PAH. Key words: Aqp1, BMPR2, PAH, HPMEC. On a deja considere que les proteines morphogeniques osseuses (BMP pour << bone morphogenetic proteins >>) jouent uniquement un role dans la formation d'os. On sait maintenant qu'elles jouent un role central dans d'autres maladies, y compris l'hypertension arterielle pulmonaire hereditaire (HAP), ou des mutations genetiques dans les recepteurs aux BMP de type 2 (BMPR2) sont la cause la plus frequente de dysfonctionnement des recepteurs. Toutefois, on a aussi montre recemment qu'un dysfonctionnement de l'aquaporine 1 (Aqp1) pourrait contribuer a l'HAP, ce qui met en relief que l'apparition de l'HAP pourrait mettre enjeu plus d'une voie de signalisation de pathogenie. On ne sait pas si l'abaissement des BMPR2 affecte l'Aqp1. Nous avons donc etudie l'Aqp1 dans des cellules endotheliales de microvaisseaux pulmonaires humains (CEMPH) dont les BMPR2 etaient reprimes. Nous avons montre un abaissement de l'Aqp1 en ARNm, en proteines et quant au fonctionnement dans des cellules dont les BMPR2 etaient reprimes. De plus, les cellules dont les BMPR2 etaient reprimes presentaient une plus faible expression de molecules de signalisation des BMP. En conclusion, l'abaissement des BMPR2 semble affecter l'Aqp1 au niveau de l'ARNm, des proteines et du fonctionnement. Cette observation pourrait permettre de distinguer un mode d'action qui contribuerait a l'HAP. [Traduit par la Redaction] Mots-cles: Aqp1, BMPR2, hypertension arterielle pulmonaire hereditaire, cellules endotheliales de microvaisseaux pulmonaires humains., Introduction Bone morphogenetic proteins (BMPs) are a group of signaling molecules that belong to the transforming growth factor-p (TGF-[beta]) superfamily of proteins. BMPs have multiple roles in the development and [...]

Hyperspectral imaging techniques have recently been applied to many biological applications to improve isolation of individual fluorophores in multi-label samples and identify fluorophores in the presence of highly autofluorescent tissue. Hyperspectral imaging is traditionally performed by collecting fluorescence emission over a broad wavelength range (emission scanning). However, significant light loss and long acquisition times can result from filtering the emission light. Excitation scanning is a novel method of hyperspectral imaging that may provide higher sensitivity for detecting fluorophores than traditional emission-scanning techniques. Excitation scanning is performed by filtering the excitation light over many wavelengths, and subsequently collecting the emission at each excitation wavelength. This results in higher available signal, and shorter acquisition times. Here, we report implementation of an excitation-scanning hyperspectral imaging microscope and preliminary results comparing excitation scanning to emission scanning. A comparative study was conducted using a model of lung injury featuring GFP-expressing pulmonary microvascular endothelial cells (PMVECs) in highly autofluorescent lung tissue. Our results indicate 1-2 orders of magnitude increased signal detection using excitationscanning techniques compared to emission-scanning, and improved sensitivity for detection of GFP in autofluorescent lung tissue. Our future work will further test the efficacy of excitation scanning compared to emission scanning for applications in FRET detection, multi-label studies, and detection of changes in autofluorescence due to lung cancer. Keywords: hyperspectral imaging, microscopy, fluorescence spectroscopy, tunable filters, thin films, INTRODUCTION Fluorescence microscopy has traditionally been performed using bandpass filters to isolate peak fluorescence excitation and emission wavelengths for fluorescent labels. However, the emission spectra of many labels share similar [...]

Memory T lymphocytes are commonly viewed as a major barrier for long-term survival of organ allografts and are thought to accelerate rejection responses due to their rapid infiltration into allografts, low threshold for activation, and ability to produce inflammatory mediators. Because memory T cells are usually associated with rejection, preclinical protocols have been developed to target this population in transplant recipients. Here, using a murine model, we found that costimulatory blockade--mediated lung allograft acceptance depended on the rapid infiltration of the graft by central memory [CD8.sup.+] T cells ([CD44.sup.hi][CD62L.sup.hi][CCR7.sup.+]). Chemokine receptor signaling and alloantigen recognition were required for trafficking of these memory T cells to lung allografts. Intravital 2-photon imaging revealed that CCR7 expression on [CD8.sup.+] T cells was critical for formation of stable synapses with antigen-presenting cells, resulting in IFN-γ production, which induced NO and downregulated alloimmune responses. Thus, we describe a critical role for [CD8.sup.+] central memory T cells in lung allograft acceptance and highlight the need for tailored approaches for tolerance induction in the lung., Introduction While transplantation has become an accepted form of therapy for end-stage organ failure, formidable immunologic barriers limit long-term allograft survival. The currently accepted clinical immunosuppression protocols, consisting of life-long [...]

There is a growing appreciation for the role that acute kidney injury (AKI) plays in the propagation of critical illness. In children, AKI is not only an independent predictor of morbidity and mortality, but is also associated with especially negative outcomes when concurrent with acute lung injury (ALI). Experimental data provide evidence that kidney-lung crosstalk occurs and can be bidirectionally deleterious, although details of the precise molecular mechanisms involved in the AKI-ALI interaction remain incomplete. Clinically, ALI, and the subsequent clinical interventions used to stabilize gas exchange, carry consequences for the homeostasis of kidney function. Meanwhile, AKI negatively affects lung physiology significantly by altering the homeostasis of fluid balance, acid-base balance, and vascular tone. Experimental AKI research supports an 'endocrine' role for the kidney, triggering a cascade of extra-renal inflammatory responses affecting lung homeostasis. In this review, we will discuss the pathophysiologyof kidney-lung crosstalk, the multiple pathways by which AKI affects kidney-lung homeostasis, and discuss how these phenomena may be unique in critically ill children. Understanding how AKI may affect a 'balance of communication' that exists between the kidneys and the lungs is requisite when managing critically ill children, in whom imbalance is the norm. Keywords kidney-lung crosstalk * AKI * ALI * Fluid overload * Renal angina, Introduction Organ cross-talk is of particular importance to practitioners in the intensive care unit (ICU); many disease processes that affect ICU patients carry the potential to affect a number of [...]

Although several clinical studies show a gender dimorphism of immune and organ responsiveness in the susceptibility to and morbidity from shock, trauma, and sepsis, there are conflicting reports on the role of gender in outcomes. In contrast, results obtained from experimental studies clearly support the suggestion that gender plays a significant role in post-injury pathogenesis. Studies performed in a rodent model of trauma-hemorrhage have confirmed that alterations in immune and organ functions after trauma-hemorrhage are more markedly depressed in adult males and in ovariectomized and aged females; however, both are maintained in castrated males and in proestrus females. Moreover, the survival rate of proestrus females subjected to sepsis after trauma-hemorrhage is significantly higher than in age-matched males or ovariectomized females. In this respect, organ functions and immune responses are depressed in males with sepsis or trauma, whereas they are unchanged or are enhanced in females. This article reviews studies delineating the mechanism by which estrogen regulates cerebral nervous, lung, and heart systems in an experimental model of sepsis, trauma, or reperfusion injury. Keywords Shock * Trauma * Males * Females * Sepsis * Reperfusion * Estrogen, Introduction Studies indicate that traumatic injury induces immune dysfunction, which is associated with an increased susceptibility to sepsis, organ failure, and mortality [1-5]. Increased susceptibility to infection and a higher [...]

Surfactant protein A (SP-A) plays an important role in the maintenance of lung lipid homeostasis. Previously, an SP-A receptor, P63 (CKAP4), on type II pneumocyte plasma membranes (PM) was identified by chemical cross-linking techniques. An antibody to P63 blocked the specific binding of SP-A to pneumocytes and the ability of SP-A to regulate surfactant secretion. The current report shows that another biological activity of SP-A, the stimulation of surfactant uptake by pneumocytes, is inhibited by P63 antibody, cAMP exposure resulted in enrichment of P63 on the cell surface as shown by stimulation of SP-A binding, enhanced association of labeled P63 antibody with type II cells, and promotion of SP-A-mediated liposome uptake, all of which were inhibited by competing P63 antibody. Incubation of A549 and type II cells with SP-A also increased P63 localization on the PM. The phosphatidylinositol 3-kinase (PI3-kinase) signaling pathway was explored as a mechanism for the transport of this endoplasmic reticulum (ER)-resident protein to the PM. Treatment with LY-294002, an inhibitor of the PI3-kinase pathway, prevented the SP-A-induced PM enrichment of P63. Exposure of pneumocytes to SP-A or cAMP activated Akt (PKB). Blocking either PI3-kinase or Akt altered SP-A-mediated lipid turnover. The data demonstrate an important role for the PI3-kinase-Akt pathway in intracellular transport of P63. The results add to the growing body of evidence that P63 is critical for SP-A receptor-mediated interactions with type II pneumocytes and the resultant regulation of surfactant turnover. lung; phospholipid; plasma membrane; lipid turnover doi: 10.1152/ajplung.00372.2009.

Activated arginase has been implicated in many diseases including cancer, immune cell dysfunction, infections, and vascular disease. Enhanced arginase activity has been reported in lungs of patients with pulmonary artery hypertension. We used hypoxia as a model for pulmonary hypertension and studied the effect of exposure to hypoxia on arginase activity in human lung microvascular endothelial cells (HMVEC). Hypoxia induces upregulation of arginase activity as well as mRNA and protein levels of arginase II (Arg II), the only arginase isoform we were able to identify in HMVEC. In endothelial cells, arginase shares and competes for the substrate L-arginine with nitric oxide (NO) synthase (NOS). Through regulation of substrate availability for NOS, arginase is able to modulate NO production. To evaluate the role of Arg II in regulation of NO production under hypoxia, we compared NO output (RFL-6 reporter assay) in cells with normal and silenced Arg II. Exposure to hypoxia led to an increase in NO levels produced by HMVEC. Inhibition of Arg II by specific small interfering RNA or by the pharmacological inhibitor BEC additionally enhanced the levels of NO. Another possible role for activated arginase is involvement in regulation of cell proliferation. However, we showed that hypoxia decreased cell proliferation and upregulated Arg II did not have an effect on cell proliferation. Since hypoxia-inducible factors (HIF) are a family of transcriptional factors activated by hypoxia, we tested the possibility of involvement of HIF-1 and HIF-2 in regulation of Arg II under hypoxia. The silencing of HIF-2 but not HIF-1 prevented the activation of Arg II by hypoxia. nitric oxide; hypoxia-inducible factor-2[alpha]; cell proliferation doi: 10.1152/ajpcell.00068.2010.

Integration of cell adhesive, survival, and proliferative processes is essential for capillary morphogenesis of endothelial cells (EC) in vitro and vascular development and function in vivo. Unfortunately, the molecular and cellular mechanisms that impact these processes are poorly defined. Here we examined how lack of bim and/or bcl-2 expression impact lung EC function. The absence of bcl-2 or bim had a significant impact on EC adhesion and migration. Lack of bcl-2 expression decreased lung EC migration, whereas lack of bim expression increased migration compared with their wild-type counterparts. Decreased adhesion to fibronectin and vitronectin was observed in both [bcl-2.sup.-/-] and [bim.sup.-/-] lung EC, with [bcl.sup.-2-/-] EC having very little adhesion to either matrix protein. Capillary morphogenesis was greatly diminished in [bcl.sup.-2-/-] EC, which correlated with decreased lung alveolarization in vivo, an angiogenesis-dependent process. We also observed aberrant production of extracellular matrix proteins, eNOS expression, and nitric oxide production in [bcl-2.sup.-/-] lung EC, which could contribute to inability to undergo capillary morphogenesis. The changes in cell adhesion and migration noted in the absence of bim or bcl-2 were independent of their impact on apoptosis. We observed no significant affect on the steady-state rate of apoptosis of lung EC in the absence of bim or bcl-2. Thus, bcl-2 family members, bim and bcl-2, play a central role in modulation of EC proangiogenic properties, which goes beyond their role as simple mediators of mitochondrial homeostasis and apoptosis. angiogenesis; apoptosis; capillary morphogenesis; extracellular matrix proteins doi: 10.1152/ajplung.00390.2009.

Pulmonary microvascular endothelial cells possess both highly proliferative and angiogenic capacities, yet it is unclear how these cells sustain the metabolic requirements essential for such growth. Rapidly proliferating cells rely on aerobic glycolysis to sustain growth, which is characterized by glucose consumption, glucose fermentation to lactate, and lactic acidosis, all in the presence of sufficient oxygen concentrations. Lactate dehydrogenase A converts pyruvate to lactate necessary to sustain rapid flux through glycolysis. We therefore tested the hypothesis that pulmonary microvascular endothelial cells express lactate dehydrogenase A necessary to utilize aerobic glycolysis and support their growth. Pulmonary microvascular endothelial cell (PMVEC) growth curves were conducted over a 7-day period. PMVECs consumed glucose, converted glucose into lactate, and acidified the media. Restricting extracellular glucose abolished the lactic acidosis and reduced PMVEC growth, as did replacing glucose with galactose. In contrast, slow-growing pulmonary artery endothelial cells (PAECs) minimally consumed glucose and did not develop a lactic acidosis throughout the growth curve. Oxygen consumption was twofold higher in PAECs than in PMVECs, yet total cellular ATP concentrations were twofold higher in PMVECs. Glucose transporter 1, hexokinase-2, and lactate dehydrogenase A were all upregulated in PMVECs compared with their macrovascular counterparts. Inhibiting lactate dehydrogenase A activity and expression prevented lactic acidosis and reduced PMVEC growth. Thus PMVECs utilize aerobic glycolysis to sustain their rapid growth rates, which is dependent on lactate dehydrogenase A. angiogenesis; progenitor cells; repair doi: 10.1152/ajplung.00274.2009.

In this paper, we develop structured tree outflow boundary conditions for modeling the airflow in patient specific human lungs. The utilized structured tree is used to represent the nonimageable vessels beyond the 3D domain. The coupling of the two different scales (1D and 3D) employs a Dirichlet-Neumann approach. The simulations are performed under a variety of conditions such as light breathing and constant flow ventilation (which is characterized by very rapid acceleration and deceleration). All results show that the peripheral vessels significantly impact the pressure, however, the flow is relatively unaffected, reinforcing the fact that the majority of the lung impedance is due to the lower generations rather than the peripheral vessels. Furthermore, simulations of a hypothetical diseased lung (restricted flow in the superior left lobe) under mechanical ventilation show that the mean pressure at the outlets of the 3D domain is about 28% higher. This hypothetical model illustrates potential causes of volutrauma in the human lung and furthermore demonstrates how different clinical scenarios can be studied without the need to assume the unknown flow distribution into the downstream region. [DOI: 10.1115/1.4001679]

Many phytochemicals possess cancer-preventive properties, some putatively through phase II metabolism-mediated mutagen/oxidant quenching. We applied human lung cells in vitro to investigate the effects of several candidate phytopreventive agents, including green tea extracts (GTE), broccoli sprout extracts (BSE), epigallocatechin gallate (EGCG), sulforaphane (SFN), phenethyl isothiocyanate (PEITC), and benzyl isothiocyanate (BITC), on inducing phase II enzymes glutathione S-transferase P1 (GSTP1) and NAD(P)H:quinone oxidoreductase 1 (NQ01) at mRNA and protein levels. Primary normal human bronchial epithelial cells (NHBE), immortalized human bronchial epithelial cells (HBEC), and lung adenocarcinoma cells (A549) were exposed to diet-achievable levels of GTE and BSE (0.5, 1.0, 2.0 mg/L), or individual index components EGCG, SFN, PEITC, BITC (0.5, 1.0, 2.0 [micro]mol/L) for 24 h, 48 h, and 6 d, respectively, mRNA assays employed RNA-specific quantitative RT-PCR and protein assays employed Western blotting. We found that in NHBE cells, while GSTP1 mRNA levels were slightly but significantly increased after exposure to GTE or BSE, NQ01 mRNA increased to 2- to 4-fold that of control when exposed to GTE, BSE, or SFN. Effects on NQ01 mRNA expression in HBEC cells were similar. NQ01 protein expression increased up to 11.8-fold in SFN-treated NHBE cells. Both GSTP1 and NO01 protein expression in A549 cells were constitutively high but not induced under any condition. Our results suggest that NQ01 is more responsive to the studied chemopreventive agents than GSTP1 in human lung cells and there is discordance between single agent and complex mixture effects. We conclude that modulation of lung cell phase II metabolism by chemopreventive agents requires cell- and agent-specific discovery and testing. doi: 10.3945/jn.110.121905.

Mechanical stress is an important modulator of lung morphogenesis, postnatal lung development, and compensatory lung regrowth. The effect of mechanical stress on stem or progenitor cells is unclear. We examined whether proliferative responses of epithelial progenitor cells, including dually immunoreactive (CCSP and proSP-C) progenitor cells (CCSP+/SP-C+) and type II alveolar epithelial cells (ATII), are affected by physical factors found in the lung of emphysematics, including loss of elastic recoil, reduced elastin content, and alveolar destruction. Mice underwent single lung pneumonectomy (PNY) to modulate transpulmonary pressure (mechanical stress) and to stimulate lung regeneration. Control mice underwent sham thoracotomy. Plombage of different levels was employed to partially or completely abolish this mechanical stress. Responses to graded changes in transpulmonary pressure were assessed in elastin-insufficient mice (elastin +/-, ELN+/-) and elastase-treated mice with elastase-induced emphysema. Physiological regrowth, morphometry (linear mean intercept; Lmi), and the proliferative responses of CCSP+/SP-C+, Clara cells, and ATII were evaluated. Plombage following PNY significantly reduced transpulmonary pressure, regrowth, and CCSP+/SP-C+, Clara cell, and ATII proliferation following PNY. In the ELN+/- group, CCSP+/SP-C+ and ATII proliferation responses were completely abolished, although compensatory lung regrowth was not significantly altered. In contrast, in elastase-injured mice, compensatory lung regrowth was significantly reduced, and ATII but not CCSP+/SP-C+ proliferation responses were impaired. Elastase injury also reduced the baseline abundance of CCSP+/SP-C+, and CCSP+/SP-C+ were found to be displaced from the bronchioalveolar duct junction. These data suggest that qualities of the extracellular matrix including elastin content, mechanical stress, and alveolar integrity strongly influence the regenerative capacity of the lung, and the patterns of cell proliferation in the lungs of adult mice. pneumocyte; bronchioalveolar stem cell; elastase; elastin; pneumonectomy; lung regeneration doi: 10.1152/ajplung.90594.2008.

Lung arteriovenous (AV) shunts or malformations cause significant morbidity and mortality in several distinct clinical syndromes. For most patients with lung AV shunts, there is still no optimal treatment. The underlying molecular and cellular etiology for lung AV shunts remains elusive, and currently described animal models have insufficiently addressed this problem. Using a tetracycline-repressible system, we expressed constitutively active Notch4 ([Notch4.sup.*]) specifically in the endothelium of adult mice. More than 90% of mice developed lung hemorrhages and respiratory insufficiency and died by 6-7 wk after gene expression began. Vascular casting and fluorescent microsphere analysis showed evidence of lung AV shunts in affected mice. Cessation of [Notch4.sup.*] expression reversed these pathophysiological effects. Assessment of the vascular morphology revealed enlarged, tortuous vessels in the lungs that resembled arteriovenous malformations. By using whole lung organ culture, we demonstrated the effects of constitutively active Notch4 on the lung vasculature to be a primary lung phenomenon. Together, our results indicate the importance of Notch signaling in maintaining the lung vasculature and offer a new, reliable model with which to study the pathobiology of lung arteriovenous shunts and malformations. vascular; pulmonary; endothelium; Notch doi: 10.1152/ajplung.00188.2009

Saouti N, Westerhof N, Heiderman F, Marcus JT, Stergiopulos N, Westerhof BE, Boonstra A, Postmus PE, Vonk-Noordegraaf A. RC time constant of single lung equals that of both lungs together: a study in chronic thromboembolic pulmonary hypertension. Am J Physiol Heart Circ Physiol 297:H2154-H2160, 2009. First published October 2, 2009; doi: 10.1152/ajpheart.00694.2009.--The product of resistance, R, and compliance, C(RC time), of the entire pulmonary circulation is constant. It is unknown if this constancy holds for individual lungs. We determined R and C in individual lungs in chronic thromboembolic pulmonary hypertension(CTEPH) patients where resistances differ between both lungs. Also, the contribution of the proximal pulmonary arteries(PA) to total lung compliance was assessed. Patients(n = 23) were referred for the evaluation of CTEPH. Pressure was measured by right heart catheterization and flows in the main, left, and right PA by magnetic resonance imaging. Total, left, and right lung resistances were calculated as mean pressure divided by mean flow. Total, left, and right lung compliances were assessed by the pulse pressure method. Proximal compliances were derived from cross-sectional area change [DELTA]A and systolic-diastolic pressure difference [DELTA]P([DELTA]A/[DELTA]P) in main, left, and right PA, multiplied by vessel length. The lung with the lowest blood flow was defined 'low flow'(LF), the contralateral lung 'high flow'(HF). Total resistance was 0.57 [+ or -] 0.28 mmHg x [s.sup.-1] x [ml.sup.-1], and resistances of LF and HF lungs were 1.57 [+ or -] 0.2 vs. 1.00 [+ or -] 0.1 mmHg x [s.sup.-1] x [ml.sup.-1], respectively, P < 0.0001. Total compliance was 1.22 [+ or -] 1.1 ml/ mmHg, and compliances of LF and HF lung were 0.47 [+ or -] 0.11 and 0.62 [+ or -] 0.12 ml/mmHg, respectively, P = 0.01. Total RC time was 0.49 [+ or -] 0.2 s, and RC times for the LF and HF lung were 0.45 [+ or -] 0.2 and 0.45 [+ or -] 0.1 s, respectively, not different. Proximal arterial compliance, given by the sum of main, right, and left PA compliances, was only 19% of total lung compliance. The RC time of a single lung equals that of both lungs together, and pulmonary arterial compliance comes largely from the distal vasculature. compliance; pulmonary hypertension; resistance and capacitance time; resistance doi: 10.1152/ajpheart.00694.2009

West JB. Comparative physiology of the pulmonary blood-gas barrier: the unique avian solution. Am J Physiol Regul Integr Comp Physiol 297: R1625-R1634, 2009. First published September 30, 2009; doi: 10.1152/ajpregu.00459.2009.--Two opposing selective pressures have shaped the evolution of the structure of the blood-gas barrier in air breathing vertebrates. The first pressure, which has been recognized for 100 years, is to facilitate diffusive gas exchange. This requires the barrier to be extremely thin and have a large area. The second pressure, which has only recently been appreciated, is to maintain the mechanical integrity of the barrier in the face of its extreme thinness. The most important tensile stress comes from the pressure within the pulmonary capillaries, which results in a hoop stress. The strength of the barrier can be attributed to the type IV collagen in the extracellular matrix. In addition, the stress is minimized in mammals and birds by complete separation of the pulmonary and systemic circulations. Remarkably, the avian barrier is about 2.5 times thinner than that in mammals and also is much more uniform in thickness. These advantages for gas exchange come about because the avian pulmonary capillaries are unique among air breathers in being mechanically supported externally in addition to the strength that comes from the structure of their walls. This external support comes from epithelial plates that are part of the air capillaries, and the support is available because the terminal air spaces in the avian lung are extremely small due to the flow-through nature of ventilation in contrast to the reciprocating pattern in mammals. evolution; pulmonary circulation; gas exchange; type IV collagen; wall stress; stress failure; respiratory physiology doi: 10.1152/ajpregu.00459.2009.

Bastarache JA, Fremont RD, Kropski JA, Bossert FR, Ware LB. Procoagulant alveolar microparticles in the lungs of patients with acute respiratory distress syndrome. Am J Physiol Lung Cell Mol Physiol 297: L1035-L1041, 2009. First published August 21, 2009; doi: 10.1152/ajplung.00214.2009.--Coagulation and fibrinolysis abnormalities are observed in acute lung injury (ALI) in both human disease and animal models and may contribute to ongoing inflammation in the lung. Tissue factor (TF), the main initiator of the coagulation cascade, is upregulated in the lungs of patients with ALI/acute respiratory distress syndrome (ARDS) and likely contributes to fibrin deposition in the air space. The mechanisms that govern TF upregulation and activation in the lung are not well understood. In the vascular space, TF-bearing microparticles (MPs) are central to clot formation and propagation. We hypothesized that TF-bearing MPs in the lungs of patients with ARDS contribute to the procoagulant phenotype in the air space during acute injury and that the alveolar epithelium is one potential source of TF MPs. We studied pulmonary edema fluid collected from patients with ARDS compared with a control group of patients with hydrostatic pulmonary edema. Patients with ARDS have higher concentrations of MPs in the lung compared with patients with hydrostatic edema (25.5 IQR 21.3-46.9 vs. 7.8 IQR 2.3-27.5 [micro]mol/1, P = 0.009 by Mann-Whitney U-test). These MPs are enriched for TF, have procoagulant activity, and likely originate from the alveolar epithelium [as measured by elevated levels of RAGE (receptor for advanced glycation end products) in ARDS MPs compared with hydrostatic MPs]. Furthermore, alveolar epithelial cells in culture release procoagulant TF MPs in response to a proinflammatory stimulus. These findings suggest that alveolar epithelial-derived MPs are one potential source of TF procoagulant activity in the air space in ARDS and that epithelial MP formation and release may represent a unique therapeutic target in ARDS. coagulation; fibrin deposition; tissue factor; receptor for advanced glycation end products; hydrostatic pulmonary edema doi: 10.1152/ajplung.00214.2009

Tourneux P, Markham N, Seedorf G, Balasubramaniam V, Abman SH. Inhaled nitric oxide improves lung structure and pulmonary hypertension in a model of Neomycin-induced bronchopulmonary dysplasia in neonatal rats. Am J Physiol Lung Cell Mol Physiol 297:L1103-L1111, 2009. First published October 16, 2009; doi: 10.1152/ajplung.00293.2009.--Whether inhaled nitric oxide (iNO) prevents the development of bronchopulmonary dysplasia (BPD) in premature infants is controversial. In adult rats, Neomycin (Bleo) induces lung fibrosis and pulmonary hypertension, but the effects of Bleo on the developing lung and iNO treatment on Bleo-induced neonatal lung injury are uncertain. Therefore, we sought to determine whether early and prolonged iNO therapy attenuates changes of pulmonary vascular and alveolar structure in a model of BPD induced by Bleo treatment of neonatal rats. Sprague-Dawley rat pups were treated with Bleo (1 mg/kg ip daily) or vehicle (controls) from day 2 to 10, followed by recovery from day 11 to 19. Treatment groups received early (days 2-10), late (days 11-19), or prolonged iNO therapy (10 ppm; days 2-19). We found that compared with controls, Bleo increased fight ventricular hypertrophy (RVH), and pulmonary arterial wall thickness, and reduced vessel density alveolarization. In each iNO treatment group, iNO decreased RVH (P < 0.01) and wall thickness (P < 0.01) and restored vessel density after Bleo (P < 0.05). iNO therapy improved alveolarization for each treatment group after Bleo; however, the values remained abnormal compared with controls. Prolonged iNO treatment had greater effects on lung structure after bleomycin than late treatment alone. We conclude that Bleo induces lung structural changes that mimic BPD in neonatal rats, and that early and prolonged iNO therapy prevents right ventricle hypertrophy and pulmonary vascular remodeling and partially improves lung structure. lung development; nitric oxide; alveolarization doi: 10.1152/ajplung.00293.2009

Markovic N, McCaig LA, Stephen J, Mizuguchi S, Veldhuizen RA, Lewis JF, Cepinskas G. Mediators released from LPS-challenged lungs induce inflammatory responses in liver vascular endothelial cells and neutrophilic leukocytes. Am J Physiol Gastrointest Liver Physiol 297: G1066-G1076, 2009. First published October 8, 2009; doi: 10.1152/ajpgi.00278.2009.--The systemic inflammatory response plays an important role in the progression of acute lung injury (ALI) to multiple organ dysfunction syndrome (MODS). However, the role of lung-derived inflammatory mediators in induction of the inflammatory response in remote organs is poorly understood. To address the above, we investigated the effects of lung inflammation on induction of inflammatory response(s) in the liver in vitro. Inflammation in mouse lungs was induced by intranasal administration of lipopolysaccharide (LPS; 1 mg/ml) followed by mechanical ventilation using the isolated perfused mouse lung method to obtain and characterize lung perfusate from the pulmonary circulation. LPS administration to mouse lungs resulted in an increased release of inflammation-relevant cytokines and chemokines into the perfusate (Luminex assay) compared with the saline-controls. Subsequently, primary mouse liver vascular endothelial cells (LVEC) or mouse polymorphonuclear leukocytes (PMN) in vitro were stimulated with the perfusate obtained from saline- or LPS-challenged lungs and assessed for various inflammation-relevant end points. The obtained results indicate that stimulation of LVEC with perfusate obtained from LPS-challenged lungs results in 1) reactive oxygen species (ROS) production; 2) activation of NF-[kappa]B; and 3) expression of E-selectin, ICAM-1, and VCAM-1 and a subsequent increase in PMN rolling and adhesion to LVEC. In addition, perfusate from LPS-challenged lung induced activation of PMN with respect to increased ROS production and upregulation of cell surface levels of adhesion molecules MAC-1 and VLA-4. Heat-inactivation of the perfusate obtained from LPS-challenged lungs was very effective in suppressing increased proadhesive phenotype (i.e., E-selectin and ICAM-1 expression) in LVEC, whereas targeted inhibition (immunoneutralization) of TNF-[alpha] and/or IL-6 in LPS-lung perfusate had no effect. Taken together, these findings indicate that multiple proinflammatory mediators (proteinaceous in nature) released from inflamed lungs act synergistically to induce systemic activation of circulating PMN and promote inflammatory responses in liver vascular endothelial cells. acute lung injury; pulmonary inflammation; systemic inflammation; isolated perfused mouse lung doi: 10.1152/ajpgi.00278.2009

Extravascular lung water includes all of the fluid within the lung but outside of the vasculature. Lung water increases as a result of increased hydrostatic vascular pressure or from an increase in lung endothelial and epithelial permeability or both. Experimentally, extravascular lung water has been measured gravimetrically. Clinically, the chest radiograph is used to determine whether extravascular lung water is present but is an insensitive instrument for determining the quantity of lung water. Bedside measurement of extravascular lung water in patients is now possible using a single indicator thermodilution method. This review critically evaluates the experimental and clinical evidence supporting the potential value of measuring extravascular lung water in patients using the single indicator method. acute lung injury; acute respiratory distress syndrome; pulmonary edema doi: 10.1152/ajplung.00127.2009

We attempted to determine whether intrapulmonary sensory receptors are nourished by the pulmonary or the systemic circulation. Single-unit activity from the cervical vagus nerve was recorded in anesthetized, open chest, mechanically ventilated rabbits, comparing responses to right or left ventricular injection of 2% lidocaine (at 4 mg/kg). Airway mechanosenors [slowly adapting receptor (SARs) and rapidly adapting receptors] were inhibited by lidocaine, whereas chemosensors (C-fiber receptors and high-threshold A[delta]-receptors) were stimulated. Furthermore, all types of airway sensors were perfused preferentially by the pulmonary circulation. For example, 14 of the 15 tested SARs ceased discharge at 4.1 [+ or -] 0.6 s after lidocaine injection into the right ventricle. The blocking effect lasted 35 [+ or -] 6.2 s. In contrast, none of the 15 SARs ceased their activity after lidocaine injection into the left ventricle. Our data show that intrapulmonary sensors are mainly nourished by the pulmonary circulation. Their very short latency indicates that these sensors receive ample blood supply. Thus, intrapulmonary sensors rely on the pulmonary circulation to detect bioactive agents in the blood. lung; vagus nerve; sensory receptor; local anesthetics; airway

Trachealess (Trh) is a PAS domain transcription factor regulating Drosophila tracheogenesis. No other Trh homolog has been associated with a respiratory phenotype. Seeking homolog(s) regulating lung development, we screened murine genomic DNA using trh oligonucleotides, identifying only Npas3. Npas3 mRNA peaks in lung from E10.5 to E13.5, verified by sequencing, with immuno-staining in airway epithelial cells. Npas3-null mice have reduced lung branching morphogenesis but are viable prenatally. Npas3-null newborns die in respiratory distress, with diminished alveolarization, decreased Shh, Fgf9, Fgf10, and Bmp4 mRNAs, and increased Spry2, consistent with reduced FGF signaling. Exogenous FGF10 rescues branching morphogenesis in Npas3-null lungs. In promoter reporter assays, NPAS3 directly upregulates Shh and represses Spry2. [Npas3.sup.+1-] mice have a milder lung phenotype, surviving postnatally, but develop emphysema and airways hyperreactivity. Therefore, absence of a developmentally expressed transcription factor can alter downstream gene expression and multiple signaling pathways in organogenesis. NPAS3 haplo-insufficiency may also lead to emphysema and asthma. branching morphogenesis | emphysema | fibroblast growth factors | sonic hedgehog | transcription factors

Transforming growth factor (TGF)-[beta] mediates hypoxia-induced inhibition of alveolar development in the newborn lung. TGF-[beta] is regulated primarily at the level of activation of latent TGF-[beta]. Fibroblasts expressing Thy-1 (CD90) inhibit TGF-[beta] activation. We hypothesized that loss of Thy-1 due to hypoxia may be a mechanism by which hypoxia increases TGF-[beta] activation and that animals deficient in Thy-1 will simulate the effects of hypoxia on lung development. To determine if loss of Thy-1 occurred during hypoxia, non-transgenic (C57BL/6) wild-type (WT) mice exposed to hypoxia were evaluated for Thy-1 mRNA and protein. To determine if Thy-1 deficiency simulated hypoxia, WT and Thy-1 null (Thy-[1.sup.-/-]) mice were exposed to air or hypoxia from birth to 2 wk, the critical period of lung development, and lung histology, function, parameters related to TGF-[beta] signaling, and extracellular matrix protein content were measured. To test if the phenotype in Thy-[1.sup.-/-] mice was due to excessive TGF-[beta] signaling, measurements were also performed in Thy-[1.sup.-/-] mice administered TGF-[beta] neutralizing antibody (1D11). We observed that hypoxia reduced Thy-1 mRNA and Thy-1 staining in WT mice. Thy-[1.sup.-/-] mice had impaired alveolarization, increased TGF-[beta] signaling, reduced lung epithelial and endothelial cell proliferation but increased fibroblast proliferation, and increased collagen and elastin. Lung compliance was lower, and tissue but not airway resistance was higher in Thy-[1.sup.-/-] mice at 2 wk. Thy-[1.sup.-/-] mice given 1D11 had improved alveolar development and lung function. These data support the hypothesis that hypoxia, by reducing Thy-1, increases TGF-[beta] activation, and thereby inhibits normal alveolar development. transforming growth factor-[beta]; infant; CD90

BACKGROUND: High levels of cadmium exposure are known to cause emphysema in occupationally exposed workers, but little has been reported to date on the association between chronic environmental cadmium exposure [...]

The steady-state turnover of epithelial cells in the lung and trachea is highly relevant to investigators who are studying endogenous stem cells, manipulating gene expression in vivo, or using viral vectors for gene therapy. However, the average lifetime of different airway epithelial cell types has not previously been assessed using currently available genetic techniques. Here, we use Cre/loxP genetic technology to indelibly label a random fraction of ciliated cells throughout the airways of a cohort of mice and follow them in vivo for up to 18 mo. We demonstrate that ciliated airway epithelial cells are a terminally differentiated population. Moreover, their average half-life of 6 mo in the trachea and 17 mo in the lung is much longer than previously available estimates, with significant numbers of labeled cells still present after 18 mo. Cre recombinase; Rosa26R-eYFP; stem cell

Lung side population (SP) cells are resident lung precursor cells with both epithelial and mesenchymal potential that are believed to play a role in normal lung development and repair. Neonatal hyperoxic exposure impairs lung development leading to a long-term decrease in gas exchange surfaces. The hypothesis that lung SP cells are altered during impaired lung development has not been studied. To address this issue, we characterized the endothelial potential of neonatal lung SP and subsets of lung SP from neonatal mice following hyperoxic exposure during room air recovery. Lung SP cells were isolated and sorted on the basis of their capacity to efflux Hoechst 33342. The lung SP was further sorted based on expression of Flk-1 and CD45. In vitro, both [CD45.sup.pos]/[Flk-1.sup.pos] and [CD45.sup.neg]/[Flk-1.sup.pos] bind isolectin B4 and incorporate LDL and form networks in matrigel, indicating that these populations have endothelial cell characteristics. Hyperoxic exposure of neonatal mice resulted in subtle changes in vascular and alveolar density on P13, which persisted with room air recovery to P41. During room air recovery, a decrease in lung SP cells was detected in the hyperoxic-exposed group on postnatal day 13 followed by an increase on day 41. Within this group, the lung SP subpopulation of cells expressing CD45 increased on day 21, 41, and 55. Here, we show that lung SP cells demonstrate endothelial potential and that the population distribution changes in number as well as composition following hyperoxic exposure. The hyperoxia-induced changes in lung SP cells may limit their ability to effectively contribute to tissue morphogenesis during room air recovery. bronchopulmonary dysplasia; microvessel density; alveolarization

Bronchial airway epithelial cells (BAEpC) are among the first cells to encounter M. tuberculosis following airborne infection. However, the response of BAEpC to M. tuberculosis infection has been little studied. This study investigates the response of a human BAEpC cell line (BEAS-2B) to infection with Mycobacterium bovis Bacille Calmette Guerin (BCG). Cultured human BEAS-2B cells were experimentally infected with BCG. Uninfected BEAS-2B cultures were included as controls. Following infection, BEAS-2B cells were evaluated by various methods at various time points up to 3 days. Cell proliferation was evaluated by cellular bioreduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Distribution of cells along the cell cycle was evaluated by FACS analysis of cellular DNA. Apoptotic cells were identified by cell death ELISA and the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling method. Eighty-four apoptosis-relevant genes were screened by PCR gene microarray. Translation of Fas, Fas ligand (Fas-L), and Fas-associated death domain (FADD) were evaluated quantitatively by real-time PCR. Expression of Fas and FADD proteins was evaluated by immunofluorescence and Western blot. Activity of caspase-3 and caspase-8 was evaluated by colorimetric assay of their enzymatic activity. BCG infection of BEAS-2B cells inhibits proliferation, induces cell cycle arrest at the [G.sub.o]/[G.sub.1] phase, causes apoptosis, modulates transcription of multiple apoptosis-relevant genes, promotes translation of Fas, Fas-L, and FADD, upregulates expression of Fas and FADD proteins, and increases activity of caspase-3 and caspase-8. Infection with BCG does not cause any significant change in the secretion of TGF-[beta]. The roles of Fas and FADD as mediators of BCG-induced apoptosis in BEAS-2B cells were tested by partial blockade of Fas and FADD expression with silencing RNA. Partial blockade of Fas or FADD expression results in a decreased apoptotic response to BCG infection. In conclusion, BCG induces cell cycle arrest and apoptosis in BEAS-2B cells. BCG induced apoptosis of BEAS-2B cells via the Fas death receptor pathway. Bacille Calmette Guerin; Fas; Fas ligand; Fas-associated death domain protein; caspase-3; caspase-8

Increasing evidence supports the contribution of genetic influences on susceptibility/severity in acute lung injury (ALI), a devastating syndrome requiring mechanical ventilation with subsequent risk for ventilator-associated lung injury (VALI). To identify VALI candidate genes, we determined that Brown Norway (BN) and Dahl salt-sensitive (SS) rat strains were differentially sensitive to VALI (tidal volume of 20 ml/kg, 85 breaths/rain, 2 h) defined by bronchoalveolar lavage (BAL) protein and leukocytes. We next exploited differential sensitivities and phenotyped both the VALI-sensitive BN and the VALI-resistant SS rat strains by expression profiling coupled to a bioinformatic-intense candidate gene approach (Significance Analysis of Microarrays, i.e., SAM). We identified 106 differentially expressed VALI genes representing gene ontologies such as 'transcription' and 'chemotaxis/cell motility.' We mapped the chromosomal location of the differentially expressed probe sets and selected consomic SS rats with single BN introgressions of chromosomes 2, 13, and 16 (based on the highest density of probe sets) while also choosing chromosome 20 (low probe sets density). VALI exposure of consomic rats with introgressions of BN chromosomes 13 and 16 resulted in significant increases in both BAL cells and protein (compared to parental SS strain), whereas introgression of BN chromosome 2 displayed a large increase only in BAL protein. Introgression of BN chromosome 20 had a minimal effect. These results suggest that genes residing on BN chromosomes 2, 13, and 16 confer increased sensitivity to high tidal volume ventilation. We speculate that the consomic-microarray-SAM approach is a time- and resource-efficient tool for the genetic dissection of complex diseases including VALI. rodent mechanical ventilation; consomics; bioinformatics; microarrays; candidate gene approach

Glycosylation of cell surface proteins can regulate multiple cellular functions. We hypothesized that glycosylation and expression of glycoproteins after epithelial injury is important in mediating repair. We report the use of an in vitro culture model of human airway epithelial cells ([1HAEo.sup.-]) to identify mediators of epithelial repair. We characterized carbohydrate moieties associated with repair by their interaction with the lectin from Cicer arietinum, chickpea agglutinin (CPA). Using CPA, we identified changes in cell surface glycosylation during wound repair. Following mechanical wounding of confluent monolayers of [1HAEo.sup.-] cells, CPA staining increases on the cell surface of groups of cells in proximity to the wound edge. Blocking the CPA carbohydrate ligand inhibited wound repair highlighting the role of the CPA carbohydrate ligand in epithelial repair. Annexin II (AII), a calcium-dependent, membrane-associated protein, was identified as a protein associated with the CPA ligand. By membrane protein biotinylation and immuno-detection, we have shown that following mechanical wounding, the presentation of AII on the cell surface increases coordinate with repair. Cell surface AII accumulates in proximity to the wound. Furthermore, translocation of AII to the cell surface is N-glycosylation dependent. We are the first to demonstrate that following injury, N-glycosylation events and AII presentation on the cell surface of airway epithelial cells are important mediators in repair. airway epithelial repair; Cicer arietinum agglutinin

Transforming growth factor-[alpha] (TGF-[alpha]) and its receptor, the epithelial growth factor receptor (EGFR), have been associated with lung remodeling in premature infants with bronchopulmonary dysplasia (BPD). The goal of this study was to target TGF-[alpha] overexpression to the saccular phase of lung morphogenesis and determine early alterations in gene expression. Conditional lung-specific TGF-[alpha] bitransgenic mice and single-transgene control mice were generated. TGF-[alpha] overexpression was induced by doxycycline (Dox) treatment from embryonic day 16.5 (E16.5) to E18.5. After birth, all bitransgenic pups died by postnatal day 7 (P7). Lung histology at E18.5 and P1 showed abnormal lung morphogenesis in bitransgenic mice, characterized by mesenchymal thickening, vascular remodeling, and poor apposition of capillaries to distal air spaces. Surfactant levels (saturated phosphatidylcholine) were not reduced in bitransgenic mice. Microarray analysis was performed after 1 or 2 days of Dox treatment during the saccular (E17.5, E18.5) and alveolar phases (P4, P5) to identify genes induced by EGFR signaling that were shared or unique to each phase. We found 196 genes to be altered (> 1.5-fold change; P < 0.01 for at least 2 time points), with only 32% similarly altered in both saccular and alveolar phases. Western blot analysis and immunostaining showed that five genes selected from the microarrays (egr-1, SP-B, SP-D, S100A4, and pleiotrophin) were also increased at the protein level. Pathological changes in TGF-[alpha]-overexpressing mice bore similarities to premature infants born in the saccular phase who develop BPD, including remodeling of the distal lung septae and arteries. bronchopulmonary dysplasia; epidermal growth factor receptor; lung remodeling; lung development; transforming growth factor-[alpha]

Respiratory syncytial virus (RSV) is the most common cause of bronchiolitis in infants and children worldwide. We wished to determine whether intratracheal administration of [beta]-agonists improved alveolar fluid clearance (AFC) across the distal respiratory epithelium of RSV-infected mice. Following intranasal infection with RSV strain A2, AFC was measured in anesthetized, ventilated BALB/c mice by instillation of 5% BSA into the dependent lung. We found that direct activation of protein kinase A by forskolin or 8-bromo-cAMP increased AFC at day 2 after infection with RSV. In contrast, short- and long-acting [beta]-agonists had no effect at either day 2 or day 4. Insensitivity to [beta]-agonists was not a result of elevated plasma catecholamines or lung epithelial cell [beta]-adrenergic receptor degradation. Instead, RSV-infected mice had significantly higher levels of phosphorylated PKC[zeta] in the membrane fractions of their lung epithelial cells. In addition, insensitivity to [beta]-agonists was mediated in a paracrine fashion by KC (the murine homolog of CXCL8) and reversed by inhibition of either PKC[zeta] or G protein-coupled receptor kinase 2 (GRK2). These results indicate that insufficient response to [beta]-agonists in RSV may be caused, at least in part, by impaired [beta]-adrenergic receptor signaling, as a consequence of GRK2-mediated uncoupling of [beta]-adrenergic receptors from adenylyl cyclase. paramyxovirus; protein kinase C; G protein-coupled receptor kinase 2; CXCL8

Numerous animal studies have revealed significant effects of sex and sex hormones on normal lung development, lung physiology, and various lung diseases. The primary goal of this review is to summarize knowledge to date on the effects of sex and sex hormones on lung development, physiology, and disease in animals. Specific emphasis will be placed on fibrosis, allergic airway disease, acute lung injury models, respiratory infection, and lung toxicology studies. estrogen; androgen; pulmonary

Elastic fibers play a key role in the structure and function of numerous organs that require elasticity. Elastogenesis is a complex process in which cells first produce a microfibrillar scaffold, composed of numerous structural proteins, upon which tropoelastin assembles to be cross-linked into polymeric elastin. Recently, it was demonstrated that low concentrations of free iron upregulate elastin gene expression in cultured fibroblasts. The present studies were conducted to assess whether low-iron diets would affect the deposition of elastic fibers in an in vivo model. One-day-old chicks were fed semipurified diets containing 1.3 (low), 12 (moderate), and 24 (control) mg/kg of iron. After 3 wk, chicks in the low-iron group were underweight and anemic. Their aortas were smaller with significantly thinner walls than control chicks, yet elastin or collagen content did not decrease relative to total protein. They also demonstrated a significantly lower stress-strain resistance than the controls. Electron microscopy demonstrated that aortic and lung smooth muscle cells were vacuolated and surrounded by loose extracellular matrix and disorganized elastic lamellae with diffuse and fragmented networks of elastic fibers and microfibrils. Immunohistology demonstrated that fibrillin-3 (FBN3) was disorganized and markedly reduced in amount in aortas of the low-iron chicks. Elastin messenger RNA levels were not downregulated in the tissues from the low-iron-fed chicks; however, there was a significant reduction in expression of the FBN1 and FBN3 genes compared with control chicks. The studies indicate that iron deficiency had a pronounced negative effect on elastic fiber development and suggests that fibrillin may have an important role in this pathology.

Abstract: Bleomycin-induced lung fibrosis in mice reproduces some key features of pulmonary fibrosis in humans including alveolar inflammation, myofibroblast proliferation, and collagen deposition. Glucocorticoids have been used as first-line therapy [...]

Homotypic cell-cell adhesion is essential for tissue and organ development, remodeling, regeneration, and physiological function. Whereas a significant number of homotypic cell-cell adhesion molecules have been identified, much more is known about those concentrated in epithelia than in endothelia. Among the endothelial cell-cell adhesion molecules, very little is known that is specific to endothelium in the pulmonary and bronchial circulations. This review focuses primarily on homotypic cell-cell adhesion molecules that are or are likely to be important in lung endothelium. cadherin; immunoglobulin-like

Apoptosis plays a causative role in acute lung injury in part due to epithelial cell loss. We recently reported that zinc protects the lung epithelium during inflammatory stress whereas depletion of intracellular zinc enhances extrinsic apoptosis. In this investigation, we evaluated the relationship between zinc, caspase-3, and cell-to-cell contact via proteins that form the adherens junction complex. Cell adhesion proteins are directly responsible for formation of the mechanical barrier of the lung epithelium. We hypothesized that exposure to inflammatory cytokines, in conjunction with zinc deprivation, would induce caspase-3, leading to degradation of junction proteins, loss of cell-to-cell contact, and compromised barrier function. Primary human upper airway and type I/II alveolar epithelial cultures were obtained from multiple donors and exposed to inflammatory stimuli that provoke extrinsic apoptosis in addition to depletion of intracellular zinc. We observed that zinc deprivation combined with tumor necrosis factor-[alpha], interferon-[gamma] and Fas receptor ligation accelerates caspase-3 activation, proteolysis of E-cadherin and [beta]-catenin, and cellular apoptosis, leading to increased paracellular leak across monolayers of both upper airway and alveolar lung epithelial cultures. Zinc supplementation inhibited apoptosis and paracellular leak, whereas caspase inhibition was less effective. We conclude that zinc is a vital factor in the lung epithelium that protects against death receptor-mediated apoptosis and barrier dysfunction. Furthermore, our findings suggest that although caspase-3 inhibition reduces lung epithelial apoptosis it does not prevent mechanical dysfunction. These findings facilitate future studies aimed at developing therapeutic strategies to prevent acute lung injury. programmed cell death; lung epithelium; caspase; barrier dysfunction; adherens junction

In mice, alveolarization occurs during postnatal days 4 through 12, when secondary alveolar septae create thin-walled alveoli in the distal lung. We hypothesized that genes predominantly expressed in newly forming secondary alveolar septae influence the process of alveolarization. To address this hypothesis, tips of secondary alveolar septae were isolated from sections of postnatal day 6 mouse lung tissue using laser capture microdissection. Total RNA was isolated and amplified from the dissected alveolar septal tips and from intact postnatal day 6 lung tissue. Gene expression in the samples was characterized using Affymetrix mouse U74AN2 GeneChips. Galectin-1 was an abundantly expressed transcript that was enriched in the alveolar septal tips compared with levels in the whole lung tissue. Galectins are [beta]-galactoside-binding proteins involved in the regulation of cell proliferation, differentiation, and apoptosis in fibroblasts, muscle cells and endothelial cells, cell types that are present in the alveolar wall. Immunostaining in postnatal day 6 lung tissue confirmed that galectin-1 protein is concentrated in the tips of secondary alveolar septae, predominantly in myofibroblasts. Fibroblasts isolated from day 6 neonatal mouse lung tissue contained galectin-1 protein. Real-time PCR demonstrated that galectin-1 mRNA levels in mouse lung tissue peak at postnatal day 6. Immunoblot analysis confirmed that peak levels of lung galectin-1 protein are found at postnatal days 6 to 12. The increased expression of galectin-1 at the site and time of ongoing alveolarization in the newborn mouse is suggestive that galectin-1 may play an important role in this critical aspect of lung development. alveolarization; lung development; myofibroblasts; microarray; laser capture microscopy

Fibulin-5 (FBLN5), an extracellular matrix glycoprotein required for normal elastogenesis, is coordinately expressed with elastin during lung injury and repair. We found that treatment with transforming growth factor-[beta] (TGF-[beta]) induced a rapid but transient increase in FBLN5 heterogeneous nuclear RNA (hnRNA) followed by a sustained increased in the steadystate level of FBLN5 mRNA. The transcription start site of the human FBLN5 gene was localized at 221 nucleotides upstream of the translation start site by using primer extension, Northern blots, and functional analysis of transcriptional activity in reporter plasmids containing 5'-flanking regions. TGF-[beta] markedly increased FBLN5 promoter activity in transient transfection assays. Two putative Smad-binding sites were identified within the proximal promoter and are required for this TGF-[beta] induction. Electrophoretic gel mobility shift assay revealed that TGF-[beta] strongly increased binding of Smad2 and Smad3 nuclear complexes to the proximal FBLN5 promoter and induced a Smad2/3-dependent binding of slow migrating nuclear protein complex. FBLN5 mRNA induction by TGF-[beta] was blocked by pretreatment with TGF-[beta] receptor inhibitor SB-431542, the phosphatidylinositol 3-kinase (PI3-kinase) inhibitor LY294002, and actinomycin D. Basal and TGF-[beta]-induced FBLN5 hnRNA and mRNA were strongly and proportionally decreased by LY-294002, as was TGF-[beta]-induced phosphorylation of Akt, but not Smad3, as measured by Western blot analysis. In addition, LY-294002 markedly and proportionally decreased FBLN5 promoter activity in transient transfection analyses with TGF-[beta]-treated or untreated lung fibroblasts. These studies demonstrate that induction of FBLN5 gene expression in lung fibroblasts is mediated via canonical TGF-[beta]/Smad signaling and requires the PI3-kinase/Akt pathway. elastogenesis; emphysema; transcription; Smads; Akt