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High tidal volume ventilation is detrimental to alveolar fluid clearance (AFC), but effects of ventilation pressure (P) on AFC are unknown. In anesthetized BALB/c mice ventilated at constant tidal volume (8 ml/kg), mean AFC rate was 12.8% at 6 cm[H.sub.2]O P, but increased to 37.3% at 18 cm[H.sub.2]O P. AFC rate declined at 22 cm[H.sub.2]O P, which also induced lung damage. Increased AFC at 18 cm[H.sub.2]O P did not result from elevated plasma catecholamines, hypercapnia, or hypocapnia, but was due to augmented [Na.sup.+] and [Cl.sup.-] absorption. PKA agonists and [beta]-agonists stimulated AFC at 10 cm[H.sub.2]O P by upregulating amiloride-sensitive Na+ transport. However, at 18 cm[H.sub.2]O P, PKA agonists and [beta]-agonists reduced AFC. At 15 cm[H.sub.2]O P, the AFC rate was intermediate (mean 26.6%), and forskolin and [beta]-agonists had no effect. Comparable P dependency of AFC and 13-agonist responsiveness was found in C57BL/6 mice. The effect on AFC of increasing P to 18 cm[H.sub.2]O was blocked by adenosine deaminase or an Azb-adenosine receptor antagonist, and could be mimicked by adenosine in mice ventilated at 10 cm[H.sub.2]O P. Modulation of adenosine signaling also resulted in altered responsiveness to [beta]-agonists. These findings indicate that, in the normal mouse lung, basal AFC rates and responses to [beta]-agonists are impacted by ventilation pressure in an adenosine-dependent manner. tidal volume; epithelial sodium channel; adenosine doi: 10.1152/ajplung.00096.2009

Compared with the Toll-like receptor 4 (TLR4) ligand LPS restricted to Gram-negative bacteria, few studies have addressed induction of lung inflammation and concomitant leukocyte recruitment in response to TLR2 ligands. This study is the first report showing that selective TLR2 stimulation by its ligand [Pam.sub.3]-Cys-SerLys-Lys-Lys-Lys-OH ([Pam.sub.3][CSK.sub.4]) within the alveolar compartment promoted lung inflammation in mice and induced the migration of circulatory immune cells including mononuclear phagocytes into the inflamed alveolar space. By using the transgenic [CX.sub.3][CR1.sup.+/GYP] mouse strain for high-purity sorting of circulating and alveolar recruited mononuclear phagocytes together with SMART preamplification and whole genome oligonucleotide microarray techniques, we found that alveolar trafficking of mononuclear phagocytes was associated with profound changes of their gene expression profiles (-900 differentially regulated genes postrecruitment). In particular, alveolar recruited mononuclear phagocytes showed upregulated transcripts of genes encoding cytokines/chemokines and pattern recognition receptor (PRR)-associated molecules. Notably, we observed a dynamic change of the genetic program of recruited mononuclear phagocytes obtained from bronchoalveolar lavage fluid at different time points (24 vs. 48 h) post-[Pam.sub.3][CSK.sub.4] challenge. In early alveolar recruited mononuclear phagocytes, mRNA levels of both proinflammatory (e.g., TNF-[alpha], CCL2, and IL-6) and cenval anti-inflammatory/proresolution [e.g., IL- l-receptor antagonist (IL-1RN), CD200 receptor (CD200R), IL-1 receptor-associated kinase (IRAK-M), IL-10, and Bcl-2-associated X protein (Bax)] mediators were found to be highly upregulated simultaneously. In corresponding cells recruited until later time points, transcript levels of anti-inflammatory/proresolution molecules persisted at the same level, whereas mRNA levels of proinflammatory mediators were found to decline. Collectively, our in vivo study identifies genetic programs by which alveolar recruited mononuclear phagocytes may contribute to the development and termination of pneumonia caused by Gram-positive bacteria. Toll-like receptor doi: 10.1152/ajplung.90433.2008.

The alveolar epithelium plays a critical role in resolving pulmonary edema. We thus hypothesized that its function might be upregulated in rats with heart failure, a condition that severely challenges the lung's ability to maintain fluid balance. Heart failure was induced by left coronary artery ligation. Echocardiographic and cardiovascular hemodynamics confirmed its development at 16 wk postligation. At that time, alveolar fluid clearance was measured by an increase in protein concentration over 1 h of a 5% albumin solution instilled into the lungs. Baseline alveolar fluid clearance was similar in heart failure and age-matched control rats. Terbutaline was added to the instillate to determine whether heart failure rats responded to [beta]-adrenoceptor stimulation. Alveolar fluid clearance in heart failure rats was increased by 194% after terbutaline stimulation compared with a 153% increase by terbutaline in control rats. To determine the mechanisms responsible for this accelerated alveolar fluid clearance, we measured ion transporter expression (ENaC, Na-KATPase, CFTR). No significant upregulation was observed for these ion transporters in the heart failure rats. Lung morphology showed significant alveolar epithelial type II cell hyperplasia in heart failure rats. Thus, alveolar epithelial type II cell hyperplasia is the likely explanation for the increased terbutaline-stimulated alveolar fluid clearance in heart failure rats. These data provide evidence for previously unrecognized mechanisms that can protect against or hasten resolution of alveolar edema in heart failure. albumin; terbutaline

Although alveolar wall thinning has been attributed to apoptosis of interstitial lung lipofibroblasts (LFs), the underlying molecular mechanism(s) remains unknown. Although the physiological vitamin D steroid hormone l[alpha],25(OH)2D3 (1,25D) has been suggested as a local paracrine/ autocrine effector of fetal lung maturation and is known to affect fibroblast apoptosis, its effects on LF apoptosis are unknown. We determined the role of 1,25D and its metabolite, C-3-epimer (3-epi-1,25D), on LF and alveolar type H (ATII) cell differentiation, proliferation, and apoptosis. Embryonic day 19 Sprague-Dawley fetal rat lung LFs and ATII cells were treated with 1,25D or 3-epi-1,25D (1 x [10.sup.-10] to 1 x [10.sup.-8] M) for 24 h, and cell proliferation, apoptosis, and differentiation were assessed. Both 1,25D and 3-epi-1,25D exhibited dose-dependent increases in expression of the key homeostatic epithelial-mesenchymal differentiation markers, increased LF and ATII cell proliferation, and decreased apoptosis. Furthermore, rat pups administered 1,25D from postnatal days 0 to 14 showed increased expressions of key LF and ATII cell differentiation markers, increased Bcl-2-to-Bax ratio as an index of decreased spontaneous alveolar LF and ATII cell apoptosis, increased alveolar count, and a paradoxical increase in septal thickness. We conclude that spatial- and temporal-specific actions of vitamin D play a critical role in perinatal lung maturation by stimulating key alveolar epithelial-mesenchymal interactions and by modulating LF proliferation/ apoptosis. These data not only provide the biological rationale for the presence of an alveolar vitamin D paracrine system, but also provide the first integrated molecular mechanism for increased surfactant synthesis and alveolar septal thinning during perinatal lung maturation. lung development

The adenosine [A.sub.2B] receptor ([A.sub.2B]R) has a wide tissue distribution that includes fibroblasts and endothelial and epithelial cells. The recent generation of an [A.sub.2B][R.sup.-/-] mouse constructed with a [beta]-galactosidase ([beta]-gal) reporter gene under control of the endogenous promoter has provided a valuable tool to quantify [A.sub.2B]R promoter activity (29). To determine the sites of expression of the [A.sub.2B] receptor in the mouse lung, histological and flow cytometric analysis of [beta]-gal reporter gene expression in various lung cell populations was performed. The major site of [A.sub.2B]R promoter activity was found to be the type II alveolar epithelial cells (AECs), identified by coexpression of prosurfactant protein C, with relatively less expression in alveolar macrophages, bronchial epithelial cells, and cells of the vasculature. Highly purified type II AECs were prepared by fluorescence-activated sorting of enhanced green fluorescent protein (eGFP)-positive cells from transgenic mice expressing eGFP under control of the surfactant protein C promoter (21). The type II cells expressed 89-fold higher [A.sub.2B]R mRNA than pulmonary leukocytes, and the [A.sub.2B]R was shown to be functional, as treatment of purified type II AECs with the nonspecific adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA) induced an increase in intracellular cAMP greater that the [beta]-adrenergic agonist isoproterenol that was inhibited completely following treatment by ATL-802, a novel, highly potent ([K.sub.i] = 8.6 nM), and selective (>900 fold over other adenosine receptor subtypes) antagonist of the mouse [A.sub.2B]R. lung; [beta]-galactosidase; G protein-coupled receptor; cAMP

UTP is known to regulate alveolar fluid clearance. However, the relative contribution of alveolar type I cells and type II cells to this process is unknown. In this study, we investigated the effects of UTP on ion transport in type I-like cell (AEC I) and type II-like cell (AEC II) monolayers. Luminal treatment of cell monolayers with UTP increased short-circuit current ([I.sub.sc]) of AEC II but decreased [I.sub.sc] of AEC I. The CI channel blockers NPPB and DIDS inhibited the UTP-induced changes in [I.sub.sc] ([DELTA]Isc) in both types of cells. Amiloride, an inhibitor of epithelial [Na.sup.+] channels (ENaC), abolished the UTP-induced [DELTA][I.sub.sc]. in AEC I, but not in AEC II. The general blocker of [K.sup.+] channels, Ba[Cl.sub.2], eliminated the UTP-induced [DELTA][I.sub.sc] in AEC II, but not in AEC I. The intermediate conductance ([IK.sub.Ca]) blocker, clofilium, also blocked the UTP effect in AEC II. The signal transduction pathways mediated by UTP were the same in AEC I and AEC II. Furthermore, UTP increased CI- secretion in AEC II and [Cl.sup.-] absorption in AEC I. Our results suggest that UTP induces opposite changes in [I.sub.sc] in AEC I and AEC II, likely due to the reversed [Cl.sup.-] flux and different contributions of ENaC and [IK.sub.Ca]. Our results further imply a new concept that type II cells contribute to UTP-induced fluid secretion and type I cells contribute to UTP-induced fluid absorption in alveoli. short-circuit current; fluid transport

Normal alveolarization has been studied in rodents using detailed morphometric techniques and loss of function approaches for growth factors and their receptors. However, it remains unclear how these growth factors direct the formation of secondary septae. We have previously developed a transgenic mouse model in which expression of a soluble dominant-negative FGF receptor (dnFGFR) in the prenatal period results in reduced alveolar septae formation and subsequent alveolar simplification. Retinoic acid (RA), a biologically active derivative of vitamin A, can induce regeneration of alveoli in adult rodents. In this study, we demonstrate that RA induces alveolar reseptation in this transgenic mouse model and that realveolarization in adult mice is FGF dependent. Proliferation in the lung parenchyma, an essential prerequisite for lung regrowth was enhanced after 14 days of RA treatment and was not influenced by dnFGFR expression. During normal lung development, formation of secondary septae is associated with the transient presence of [alpha]-smooth muscle actin ([alpha]SMA)-positive interstitial myofibroblasts. One week after completion of RA treatment, [alpha]SMA expression was detected in interstitial fibroblasts, supporting the concept that RA-initiated realveolarization recapitulates aspects of septation that occur during normal lung development. Expression of dnFGFR blocked realveolarizafion with increased PDGF receptor-[alpha] (PDGFR[alpha])-positive cells and decreased aSMA-positive cells. Taken together, our data demonstrate that FGF signaling is required for the induction of aSMA in the PDGFR[alpha]-positive myofibroblast progenitor and the progression of alveolar regeneration. transgenic mouse model; interstitial myofibroblast; emphysema; platelet derived growth factor receptor

Several respiratory diseases are associated with extensive damage of lung epithelia, and the regulatory mechanisms involved in their regeneration are not clearly defined. Growth factors released by epithelial cells or fibroblasts from injured lungs are important regulators of alveolar repair by stimulating cell motility, proliferation, and differentiation. In addition, [K.sup.+] channels regulate cell proliferation/migration and are coupled with growth factor signaling in several tissues. We decided to explore the hypothesis, never investigated before, that [K.sup.+] could play a prominent role in alveolar repair. We employed a model of mechanical wounding of rat alveolar type II epithelia, in primary culture, to study their response to injury. Wound healing was suppressed by one-half upon epidermal growth factor (EGF) titration with EGF-antibody (Ab) or erbB1/erbB2 tyrosine-kinase inhibition with AG-1478/AG-825. The addition of exogenous EGF slightly stimulated the alveolar wound healing and enhanced, by up to five times, alveolar cell migration measured in a Boyden-type chamber. Conditioned medium collected from injured alveolar monolayers also stimulated cell migration; this effect was abolished in the presence of EGF-Ab. The impact of [K.sup.+] channel modulators was examined in basal and EGF-stimulated conditions. Wound healing was stimulated by pinacidil, an ATP-dependent [K.sup.+] channel ([K.sub.ATP]) activator, which also increased cell migration, by twofold, in basal conditions and potentiated the stimulatory effect of EGF. [K.sub.ATP] or KvLQT1 inhibitors (glibenclamide, clofilium) reduced EGF-stimulated wound healing, cell migration, and proliferation. Finally, EGF stimulated [K.sub.ATP] and KvLQT 1 currents and channel expression. In summary, stimulation of [K.sup.+] channels through autocrine activation of EGF receptors could play a crucial role in lung epithelia repair processes. lung; adenosine 5'-triphosphate-stimulated potassium channel; KvLQT1 [K.sup.+] channels; epidermal growth factor; alveolar cell repair; migration; proliferation

The current study was conducted to elucidate the mechanism through which TNF-[alpha] stimulates expression of MUC 1, a membrane-tethered mucin. A549 human lung alveolar cells treated with TNF-[alpha] exhibited significantly higher MUC 1 protein levels in detergent lysates compared with cells treated with vehicle alone. Increased MUC1 protein levels were correlated with significantly higher levels of MUC1 mRNA in TNF-[alpha]-treated cells compared with controls. However, TNF-[alpha] did not alter MUC1 transcript stability, implying increased de novo transcription induced by the cytokine. TNF-[alpha] increased MUC1 gene promoter activity in A549 cells transfected with a promoter-luciferase reporter plasmid. Both U0126, an inhibitor of MEK1/2, and dominant negative ERK1 prevented TNF-[alpha]-induced MUC1 promoter activation, and antiTNFR1 antibody blocked TNF-[alpha]-stimulated ERK1/2 activation. MUC1 promoter activation by TNF-[alpha] also was blocked by mithramycin A, an inhibitor of Sp1, as well as either deletion or mutation of a putative Spl binding site in the MUC1 promoter located between nucleotides -99 and -90. TNF-[alpha]-stimulated binding of Sp1 to the MUC1 promoter in intact cells was demonstrated by chromatin immunoprecipitation assay. We conclude that TNF-[alpha] induces MUC1 gene transcription through a TNFR1 [right arrow] MEK1/2 [right arrow] ERK1 [right arrow] Sp1 pathway. tumor necrosis factor-[alpha]; Spl; mitogen-activated protein kinase

We have previously shown that cardiogenic pulmonary edema fluid (EF) increases [Na.sup.+] and fluid transport by fetal distal lung epithelia (FDLE) (Rafii B, Gillie DJ, Sulowski C, Hannam V, Cheung T, Otulakowski G, Barker PM and O'Brodovich H. J Physiol 544: 537-548, 2002). We now report the effect of EF on [Na.sup.+] and fluid transport by the adult lung. We first studied primary cultures of adult type II (ATII) epithelium and found that overnight exposure to EF increased [N.sup.+]. transport, and this effect was mainly due to factors other than catecholamines. Plasma did not stimulate [N.sup.+]. transport in ATII. Purification of EF demonstrated that at least some agent(s) responsible for the amiloride-insensitive component resided within the globulin fraction. ATII exposed to globulins demonstrated a conversion of amiloride-sensitive short-circuit current ([I.sub.SC]) to amiloride-insensitive L~ with no increase in total [I.sub.SC]. Patch-clamp studies showed that ATII exposed to EF for 18 h had increased the number of highly selective Nan channels in their apical membrane. In situ acute exposure to EF increased the open probability of [N.sup.+].-permeant ion channels in ATII within rat lung slices. EF did increase, by amiloride-sensitive pathways, the alveolar fluid clearance from the lungs of adult rats. We conclude that cardiogenic EF increases [N.sup.+]. transport by adult lung epithelia in primary cell culture, in situ and in vivo. pulmonary edema fluid: alveolar type ii epithelium; sodium channels; lung fluid clearance

Postnatal developmental stages of lung parenchyma in rhesus monkeys is about one-third that of humans. Alveoli in humans are reported to be formed up to 8 yr of age. We used design-based stereological methods to estimate the number of alveoli ([N.sub.alv]) in male and female rhesus monkeys over the first 7 yr of life. Twenty-six rhesus monkeys (13 males ranging in age from 4 to 1,920 days and lung volumes from 41.7 to 602 [cm.sup.3], 13 females ranging in age from 22 to 2,675 days and lung volumes from 43.5 to 380 [cm.sup.3]) were necropsied and lungs fixed, isotropically oriented, fractionated, sampled, embedded, and sectioned for alveolar counting. Parenchymal, alveolar, alveolar duct core air, and interalveolar septal tissue volumes increased rapidly during the first 2 yr with slowed growth from 2 to 7 yr. The rate of change was greater in males than females. [N.sub.lv] also showed consistent growth throughout the study, with increases in [N.sub.lv] best predicted by increases in lung volume. However, mean alveolar volume showed little relationship with age, lung volume, or body weight but was larger in females and showed a greater size distribution than in males. Alveoli increase in number but not volume throughout postnatal development in rhesus monkeys. stereology; parenchyma; alveolar ducts; postnatal development

Epithelial-mesenchymal transition (EMT), a process whereby fully differentiated epithelial cells undergo transition to a mesenchymal phenotype giving rise to fibroblasts and myofibroblasts, is increasingly recognized as playing an important role in repair and scar formation following epithelial injury. The extent to which this process contributes to fibrosis following injury in the lung is a subject of active investigation. Recently, it was demonstrated that transforming growth factor (TGF)-[beta] induces EMT in alveolar epithelial cells (AEC) in vitro and in vivo, and epithelial and mesenchymal markers have been colocalized to hyperplastic type II (AT2) cells in lung tissue from patients with idiopathic pulmonary fibrosis (IPF), suggesting that AEC may exhibit extreme plasticity and serve as a source of fibroblasts and/or myofibroblasts in lung fibrosis. In this review, we describe the characteristic features of EMT and its mechanistic underpinnings. We further describe the contribution of EMT to fibrosis in adult tissues following injury, focusing especially on the critical role of TGF-[beta] and its downstream mediators in this process. Finally, we highlight recent descriptions of EMT in the lung and the potential implications of this process for the treatment of fibrotic lung disease. Treatment for fibrosis of the lung in diseases such as IPF has heretofore focused largely on amelioration of potential inciting processes such as inflammation. It is hoped that this review will stimulate further consideration of the cellular mechanisms of fibrogenesis in the lung and especially the role of the epithelium in this process, potentially leading to innovative avenues of investigation and treatment. epithelial-mesenchymal transition; alveolar epithelium; pulmonary fibrosis; transforming growth factor-[beta]

Chronic exposure to particulate air pollution is associated with lung function impairment. To determine the molecular mechanism(s) of this phenomenon, we investigated, in an alveolar human epithelial cell line (A549), whether diesel exhaust particles (DEPs), a main component of particulate air pollution, modulates the expression and activity of the matrix metalloprotease (MMP)-1, a collagenase involved in alveolar wall degradation. Interaction of DEPs with cigarette smoke, which also produces structural and functional lung alterations, was also investigated. A noncytotoxic concentration of DEPs induced an increase in MMP-1 mRNA and protein expression and activity in A549 cells without modifying the expression of the MMP inhibitors TIMP-1 and -2. This effect was not potentiated when cells were coexposed to noncytotoxic concentrations of cigarette smoke condensate. DEP-induced MMP-1 was associated with increased ERK 1/2 phosphorylation and upregulation of expression and activity of the NADPH oxidase analog NOX4. Cell transfection with a NOX4 small interfering RNA prevented these phenomena, showing the critical role of a NOX4 ERK 1/2 pathway in DEP-induced MMP-1 expression and activity. Similar results to those observed in A549 cells were obtained in another human lung epithelial cell line, NCI-H292. Furthermore, experiments in mice intratracheally instilled with DEPs confirmed the in vitro findings, showing the induction of NOX4 and MMP-1 protein expression in alveolar epithelial cells. We conclude that alveolar alterations secondary to MMP-1 induction could explain lung function impairment associated with exposure to particulate pollution. chronic obstructive pulmonary disease; environment; pollution; nanoparticles doi:10.1152/ajplung.00445.2006

Patients with interstitial lung diseases, such as idiopathic pulmonary fibrosis (IPF) and bronchopulmonary dysplasia (BPD), suffer from lung fibrosis secondary to myofibroblast-mediated excessive ECM deposition and destruction of lung architecture. Transforming growth factor (TGF)-[beta]1 induces epithelial-mesenchymal transition (EMT) of alveolar epithelial cells (AEC) to myofibroblasts both in vitro and in vivo. Inhaled nitric oxide (NO) attenuates ECM accumulation, enhances lung growth, and decreases alveolar myofibroblast number in experimental models. We therefore hypothesized that NO attenuates TGF-[beta]1-induced EMT in cultured AEC. Studies of the capacity for endogenous NO production in AEC revealed that endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS) are expressed and active in AEC. Total NOS activity was 1.3 pmol x mg [protein.sup.-1] x [min.sup.-1] with 67% derived from eNOS. TGF-[beta]1 (50 pM) suppressed eNOS expression by more than 60% and activity by 83% but did not affect iNOS expression or activity. Inhibition of endogenous NOS with L-NAME led to spontaneous EMT, manifested by increased [alpha]-smooth muscle actin ([alpha]-SMA) expression and a fibroblast-like morphology. Provision of exogenous NO to TGF-[beta]-treated AEC decreased stress fiber-associated [alpha]-SMA expression and decreased collagen I expression by 80%. NO-treated AEC also retained an epithelial morphology and expressed increased lamellar protein, E-cadherin, and pro-surfactant protein B compared with those treated with TGF-[beta] alone. These findings indicate that NO serves a critical role in preserving an epithelial phenotype and in attenuating EMT in AEC. NO-mediated regulation of AEC fate may have important implications in the pathophysiology and treatment of diseases such as IPF and BPD. alveolar epithelium; lung injury; nitric oxide synthases; pulmonary fibrosis; transforming growth factor-[beta] doi:10.1152/ajplung.00475.2006

Pulmonary alveoli, especially in females, are estrogen-responsive structures: ovariectomy in wild-type (WT) adult mice results in alveolar loss, and estradiol replacement induces alveolar regeneration. Furthermore, estrogen receptor (ER)-[alpha] and ER-[beta] are required for the developmental formation of a full complement of alveoli in female mice. We now show ovariectomy resulted in alveolar loss in adult ER-[[beta].sup.-/-] mice but not in adult ER-[[alpha].sup.-/-] mice. Estradiol treatment of ovariectomized ER-[[beta].sup.-/-] mice induced alveolar regeneration. In ovariectomized WT mice, estradiol treatment resulted, within 1 h, in RNA-level gene expression supportive of processes needed to form an alveolar septum, e.g., cell replication, angiogenesis, extracellular matrix remodeling, and guided cell motion. Among these processes, protein expression supporting angiogenesis and cell replication was elevated 1 and 3 h, respectively, after estradiol treatment; similar findings were not present in either mutant. We conclude: 1) loss of signaling via ER-[beta] is not required for postovariectomy-induced alveolar loss or estradiol-induced regeneration; this indicates ER-[alpha] is key for estrogen-related alveolar loss and regeneration in adult female mice; 2) taken together with prior work showing that developmental formation of a full complement of alveoli requires ER-[alpha] and ER-[beta], the present findings indicate the developmental and regenerative formation of alveoli are regulated differently, i.e., signaling for alveolar regeneration is not merely a recapitulation of signaling for developmental alveologenesis; and 3) the timing of estradiol-induced gene expression in lung supportive of processes required to form a septum differs between ovariectomized WT and [ER-[beta].sup.-/-] mice. mutant mice; ovariectomy; hormone replacement therapy; estrogen signaling doi:10.1152/ajplung.00384.2006

Pulmonary injury is associated with the disruption of alveologenesis in the developing lung and causes bronchopulmonary dysplasia (BPD) in prematurely born infants. Transforming growth factor (TGF)-[beta] is an important regulator of cellular differentiation and early lung development, and its levels are increased in newborn lung injury. Although overexpression of TGF-[beta] in the lungs of newborn animals causes pathological features that are consistent with BPD, the role of endogenous TGF-[beta] in the inhibition of the terminal stage of lung development is incompletely understood. In this investigation, the hypothesis that [O.sub.2]-induced injury of the maturing lung is associated with TGF-[beta]-mediated disruption of alveologenesis and microvascular development was tested using a murine model of BPD. Here we report that treatment of developing mouse lungs with TGF-[beta]-neutralizing antibodies attenuates the increase in pulmonary cell phospho-Smad2 nuclear localization, which is indicative of augmented TGF-[beta] signaling, associated with pulmonary injury induced by chronic inhalation of 85% oxygen. Importantly, the neutralization of the abnormal TGF-[beta] activity improves quantitative morphometric indicators of alveologenesis, extracellular matrix assembly, and microvascular development in the injured developing lung. Furthermore, exposure to anti-TGF-[beta] antibodies is associated with improved somatic growth in hyperoxic mouse pups and not with an increase in pulmonary inflammation. These studies indicate that excessive pulmonary TGF-[beta] signaling in the injured newborn lung has an important role in the disruption of the terminal stage of lung development. In addition, they suggest that anti-TGF-[beta] antibodies may be an effective therapy for preventing some important developmental diseases of the newborn lung. bronchopulmonary dysplasia; transforming growth factor-[beta] doi:10.1152/ajplung.00389.2006

Pulmonary ischemia-reperfusion (IR) injury entails acute activation of alveolar macrophages followed by neutrophil sequestration. Although proinflammatory cytokines and chemokines such as TNF-[alpha] and monocyte chemoattractant protein-1 (MCP-1) from macrophages are known to modulate acute IR injury, the contribution of alveolar epithelial cells to IR injury and their intercellular interactions with other cell types such as alveolar macrophages and neutrophils remain unclear. In this study, we tested the hypothesis that following IR, alveolar macrophage-produced TNF-[alpha] further induces alveolar epithelial cells to produce key chemokines that could then contribute to subsequent lung injury through the recruitment of neutrophils. Cultured RAW264.7 macrophages and MLE-12 alveolar epithelial cells were subjected to acute hypoxiareoxygenation (H/R) as an in vitro model of pulmonary IR. H/R (3 h/1 h) significantly induced KC, MCP-1, macrophage inflammatory protein-2 (MIP-2), RANTES, and IL-6 (but not TNF-[alpha]) by MLE- 12 cells, whereas H/R induced TNF-[alpha], MCP-1, RANTES, MIP-1[alpha], and MIP-2 (but not KC) by RAW264.7 cells. These results were confirmed using primary routine alveolar macrophages and primary alveolar type II cells. Importantly, using macrophage and epithelial coculture methods, the specific production of TNF-[alpha] by H/R-exposed RAW264.7 cells significantly induced proinflammatory cytokine/chemokine expression (KC, MCP-1, MIP-2, RANTES, and IL-6) by MLE-12 cells. Collectively, these results demonstrate that alveolar type II cells, in conjunction with alveolar macrophage-produced TNF-[alpha], contribute to the initiation of acute pulmonary IR injury via a proinflammatory cascade. The release of key chemokines, such as KC and MIP-2, by activated type II cells may thus significantly contribute to neutrophil sequestration during IR injury. cytokines; chemokines doi:10.1152/ajplung.00470.2006

To study air space fluid clearance (AFC) under conditions that resemble the clinical setting of pulmonary edema in patients, we developed a new perfused human lung preparation. We measured AFC in 20 human lungs rejected for transplantation and determined the contribution of AFC to lung fluid balance. AFC was then compared with air space and perfusate levels of a biological marker of epithelial injury. The majority of human lungs rejected for transplant had intact basal (75%) and [[beta].sub.2]- adrenergic agonist-stimulated (70%) AFC. For lungs with both basal and stimulated AFC, the basal AFC rate was 19 [+ or -] 10%/h, and the [[beta].sub.2-] adrenergic-stimulated AFC race was 43 [+ or -] 13%/h. Higher rates of AFC were associated with less lung weight gain (Pearson coefficient -0.90, P < 0.0001). Air space and perfusate levels of the type I pneumocyte marker receptor for advanced glycation end products (RAGE) were threefold and sixfold higher, respectively, in lungs without basal AFC compared with lungs with AFC (P < 0.05). These data show that preserved AFC is a critical determinant of favorable lung fluid balance in the perfused human lung, raising the possibility that [[beta].sub.2-]agonist therapy to increase edema fluid clearance may be of value for patients with acute lung injury and pulmonary edema. Also, although additional studies are needed, a biological marker of alveolar epithelial injury may be useful clinically in predicting preserved AFC. alveolar epithelial fluid transport; acute lung injury; acute respiratory distress syndrome; lung transplantation; biological markers; pulmonary edema; primary graft failure; air space fluid clearance doi:10.1152/ajplung.00256.2006

Pneumocystis carinii is an opportunistic fungal pathogen that causes pneumonia (PCP) in immunocompromised individuals. Recent studies have demonstrated that the host's immune response is clearly responsible for the majority of the pathophysiological changes associated with PCP. P. carinii interacts closely with alveolar epithelial cells (AECs); however, the nature and pathological consequences of the epithelial response remain poorly defined. Monocyte chemotactic protein-1 (MCP-1) is involved in lung inflammation, immunity, and epithelial repair and is upregulated during PCP. To determine whether AECs are an important source of MCP-1 in the P. carinii-infected lung, in vivo and in vitro studies were performed. In situ hybridization showed that MCP-1 mRNA was localized to cells with morphological characteristics of AECs in the lungs of infected mice. In vitro studies demonstrated that P. carinii stimulated a time- and dose-dependent MCP-1 response in primary murine type II cells that was preceded by JNK activation. Pharmacological inhibition of JNK nearly abolished P. carinii-stimulated MCP-1 production, while ERK, p38 MAPK, and TNF receptor signaling were not required. Furthermore, delivery of a JNK inhibitory peptide specifically to pulmonary epithelial cells using a recombinant adenovirus vector blocked the early lung MCP-1 response following intratracheal instillation of infectious P. carinii. JNK inhibition did not affect P. carinii-stimulated production of macrophage inflammatory protein-2 in vitro or in vivo, indicating that multiple signaling pathways are activated in P. carinii-stimulated AECs. These data demonstrate that AECs respond to P. carinii in a proinflammatory manner that may contribute to the generation of immune-mediated lung injury. inflammation; epithelial; AIDS doi: 10.1152/ajplung.00452.2006

Pseudomonas aeruginosa is a gram-negative bacilli frequently encountered in human pathology. This pathogen is involved in a large number of nosocomial infections and chronic diseases. Herein we investigated the effects of polyunsaturated fatty acids (PUFA) in chronic Pseudomonas aeruginosa lung infection. C57BL/6 mice were fed for 5 wk with specifically designed diets with high contents in either omega-3 ([omega]-3) or [omega]-6 PUFA and compared to a control diet. P. aeruginosa included in agarose beads was then instilled intratracheally, and the animals were studied for 7 days. On the 4th day, the mice fed with the [omega]-3 diet had a higher lean body mass gain and a lower [omega]-6:[omega]-3 ratio of fatty acids extracted from the lung tissue compared with the other groups (P < 0.05). The [omega]-3 group had the lowest mortality. Distal alveolar fluid clearance (DAFC) as well as the inflammatory response and the cellular recruitment were higher in the [omega]-3 group on the 4th day. The effect on DAFC was independent of [alpha]-epithelial [Na.sup.+] channels ([alpha]-ENaC), [beta]-ENaC, and [[alpha].sub.1]-Na-K-ATPase mRNA expressions, which were not altered by the different diets. In conclusion, a diet enriched in [omega]-3 PUFA can change lung membrane composition and improve survival in chronic pneumonia. This effect on survival is probably multifactorial involving the increased DAFC capacity as well as the optimization of the initial inflammatory response. This work suggests that a better control of the [omega]-6/[omega]-3 PUFA balance may represent an interesting target in the prevention and/or control of P. aeruginosa infection in patients. alveolar liquid clearance doi:10.1152/ajplung.00337.2006.

Alveolar regenerative gene expression is unidentified partly because its onset, after a regenerative stimulus, is unknown. Toward addressing this void, we used a mouse model in which calorie restriction produces alveolar loss, and ad libitum access to food after calorie restriction induces alveolar regeneration. We selected four processes (cell replication, angiogenesis, extracellular matrix remodeling, and guided cell motion) that would be required to convert a flat segment of alveolar wall into a septum that increases gas-exchange surface area. Global gene expression supportive of processes required to form a septum was present within 3 h of allowing calorie-restricted mice food ad libitum. One hour after providing calorie-restricted mice food ad libitum, RNA-level expression supportive of cell replication was present with little evidence of expression supportive of angiogenesis, extracellular matrix remodeling, or guided cell motion. Cell replication was more directly assayed by measuring DNA synthesis in lung. This measurement was made 3 h after allowing calorie-restricted mice food ad libitum because translation may be delayed. Ad libitum food intake, following calorie restriction, elevated DNA synthesis. Thus RNA expression 1 h after allowing calorie-restricted mice food ad libitum supported increased cell replication; measurements at 3 h revealed increased DNA synthesis and RNA expression, supportive of the three other processes required to form a septum. These findings identify the first hour after providing calorie-restricted mice ad libitum access to food as the onset of gene expression in this model that supports processes needed for alveolar regeneration. microarray; cell replication; angiogenesis; extracellular matrix; regeneration; guided cell motion doi:10.1152/ajplung.00146.2006

In patients requiring mechanical ventilation for acute lung injury or acute respiratory distress syndrome (ARDS), tidal volume reduction decreases mortality, but the mechanisms of the protective effect have not been tully explored. To test the hypothesis that alveolar macrophage activation is an early and critical event in the initiation of ventilator-induced lung injury (VILI), rats were ventilated with high tidal volume (H[V.sup.T]) for 10 min to 4 h. Alveolar macrophage counts in bronchoalveolar lavage (BAL) fluid decreased 45% by 20 min of H[V.sub.T] (P < 0.05) consistent with activation-associated adhesion. Depletion of alveolar macrophages in vivo with liposomal clodronate significantly decreased permeability and pulmonary edema following 4 h of H[V.sub.t] (P < 0.05). BAL fluid from rats exposed to 20 min of H[V.sub.t] increased nitric oxide synthase activity nearly threefold in naive primary alveolar macrophages (P < 0.05) indicating that soluble factors present in the air spaces contribute to macrophage activation in VILI. Media from cocultures of alveolar epithelial cell monolayers and alveolar macrophages exposed to 30 rain of stretch in vitro also significantly increased nitrite production in naive macrophages (P < 0.05), but media from stretched alveolar epithelial cells or primary alveolar macrophages alone did not, suggesting alveolar epithelial cell-macrophage interaction was required for the subsequent macrophage activation observed. These data demonstrate that injurious mechanical ventilation rapidly activates alveolar macrophages and that alveolar macrophages play an important role in the initial pathogenesis of VILI. alveolar epithelial barrier function; ventilator-associated lung injury; acute lung injury; acute respiratory distress syndrome

In a recent study (Leroy C, Dagenais A, Berthiaume Y, and Brochiero E. Am J Physiol Lung Cell Mol Physiol 286: L1027-L1037, 2004), we identified an ATP-sensitive [K.sup.+] ([K.sub.ATP]) channel in alveolar epithelial cells, formed by inwardly rectifying [K.sup.+] channel Kir6.1/sulfonylurea receptor (SUR)2B subunits. We found that short applications of [K.sub.ATP], voltage-dependent [K.sup.+] channel KvLQT1, and calcium-activated [K.sup.+] ([K.sub.ca]) channel modulators modified [Na.sup.+] and [C1.sup-] currents in alveolar monolayers. In addition, it was shown previously that a [K.sub.ATP] opener increased alveolar liquid clearance in human lungs by a mechanism possibly related to epithelial sodium channels (ENaC). We therefore hypothesized that prolonged treatment with [K.sup.+] channel modulators could induce a sustained regulation of ENaC activity and/or expression. Alveolar monolayers were treated for 24 h with inhibitors of [K.sub.ATP], KvLQT1, and Kca channels identified by PCR. Glibenclamide and clofilium ([K.sub.ATP] and KvLQT1 inhibitors) strongly reduced basal transepithelial current, amiloride-sensitive [Na.sup.+] current, and forskolin-activated [Cl.sup.-] currents, whereas pinacidil, a [K.sub.ATP] activator, increased them. Interestingly, [K.sup.+] inhibitors or membrane depolarization (induced by valinomycin in high-[K.sup.+] medium) decreased [alpha]-, [beta]-, and [gamma]-ENaC and CFTR mRNA. [alpha]-ENaC and CFTR proteins also declined after glibenclamide or clofilium treatment. Conversely, pinacidil augmented ENaC and CFTR mRNAs and proteins. Since alveolar fluid transport was found to be driven, at least in part, by [Na.sup.+] transport through ENaC, we tested the impact of [K.sup.+] channel modulators on fluid absorption across alveolar monolayers. We found that glibenclamide and clofilium reduced fluid absorption to a level similar to that seen in the presence of amiloride, whereas pinacidil slightly enhanced it. Long-term regulation of ENaC and CFTR expression by [K.sup.+] channel activity could benefit patients with pulmonary diseases affecting ion transport and fluid clearance. lung; ATP-sensitive [K.sup.+] channel; KVLQT1; calcium-activated [K.sup.+] channel; [Na.sup.+] and [Cl.sup.-] transepithelial transport; alveolar clearance

At birth, the lung environment changes from low to relatively high [O.sub.2] tension. Pulmonary blood flow increases and pulmonary artery pressure decreases. Recent data suggest that pulmonary vascular calcium-sensitive [K.sup.+] channel (B[K.sub.Ca] activation mediates perinatal pulmonary vasodilation. Although B[K.sub.Ca] channel expression is developmentally regulated, the molecular mechanisms responsible for B[K.sub.Ca] expression remain unknown. We tested the hypothesis that the low-[O.sup.2] tension environment of the normal fetus modulates B[K.sub.Ca], channel expression. We analyzed B[K.sub.Ca] expression under conditions of hypoxia and normoxia both in vitro and in vivo. B[K.sub.Ca] [alpha]-subunit mRNA expression increased twofold in ovine pulmonary artery smooth muscle cell (PASMC) primary cultures maintained in hypoxia. In vivo, B[K.sub.Ca] expression was similarly affected by hypoxia. When adult Sprague-Dawley rats were placed in hypobaric hypoxic chambers for 3 wk, hypoxic animals showed an increase of threefold in the expression of B[K.sub.Ca] [alpha]- and more than twofold in the expression of B[K.sub.Ca] [[beta].sub.1]-subunit mRNA. Immunochemical staining was consistent with the genetic data. To assess transcriptional activation of the [beta]-subunit of the B[K.sub.Ca] both B[K.sub.Ca] [[beta].sub.]- and [[beta].sub.2]-subunit luciferase ([K.sub.Ca], [beta]:[luc.sup.+]) reporter genes were constructed. Hypoxia increased PASMC [K.sub.Ca] [beta]:[luc.sup.+] reporter expression by threefold and [K.sub.Ca] [beta]:[luc.sup.+] expression by 35%. Fetal PASMC treated with the hypoxia-inducible factor-1 mimetic deferoxamine showed a 63 and 41% increase in B[K.sub.Ca] channel [alpha]- and [[beta].sub.1]-subunit expression, respectively. Together, these results suggest that oxygen tension modulates B[K.sub.Ca] channel subunit mRNA expression, and the regulation is, at least in part, at the transcriptional level. hypoxia; oxygen sensing; hypoxia-inducible factor-1

Recently it has been described that dopamine (DA), via dopaminergic type 2 receptors ([D.sub.2]R), activates the mitogen-activated protein kinase extracellular signal-regulated kinase (MAPK/ERK) proteins in alveolar epithelial cells (AEC), which results in the upregulation of [Na.sup.+]-[K.sup.+]-ATPase. In the present report, we used AEC to investigate the signaling pathway that links DA with ERK activation. Incubation of AEC with DA resulted in rapid and transient stimulation of ERK activity, which was mediated by Ras proteins and the serine/threonine kinase Raf-1. Pretreatment of AEC with Src homology 3 binding peptide, which blocks the interaction between Grb2 and Sos, did not prevent DA activation of ERK. Diacylglycerol (DAG)-dependent protein kinase C (PKC) isoenzymes, involved in the DA-mediated activation of ERK proteins as pretreatment with either bisindolylmaleimide or Ro-31-8220, prevented the phosphorylation of Elk-1, and quinpirole, a [D.sub.2]R activator, stimulates the translocation of PKC[member of]. Together, the data suggest that DA activated MAPK/ERK via Ras, Raf-1 kinase, and DAG-dependent PKC isoenzymes, but, importantly and contrary to the classical model, this pathway did not involve the Grb2-Sos complex formation. alveolar epithelial cell; mitogen-activated protein kinase/extracellular signal-regulated kinase; protein kinase C

Diesel exhaust particles (DEPs) have been shown in vivo as well as in vitro to affect the respiratory function and in particular the immune response to infection and allergens. In the current study, we investigated the effect of DEPs on the production of phosphatidylcholine (PC), a major constituent of surfactant, by rat alveolar type II (ATII) primary cells in vitro. Our results demonstrate that incubation of ATII cells with DEPs lead to a time- and dose-dependent increase in labeled PC release. This effect was mimicked by nitric oxide (NO) donors and cGMP and was abolished by inhibitors of NO synthase (NOS). In addition, a NOS inhibitor inhibits by itself the basal secretion of PC. We next examined the effects of DEPs on NOS gene expression and showed that DEPs increase NO production and upregulate both protein content and mRNA levels of the inducible NOS (NOS II). Together our data demonstrate that DEPs alter the production of surfactant by ATII cells through a NO-dependent signaling pathway. surfactant; nitric oxide

Antisense oligonucleotides were used to manipulate the expression of the respective protein and the patch-clamp technique to study the changes resulting from these manipulations in single-channel characteristics. Findings have indicated that nonselective cation channels belong to the amiloride-sensitive epithelial Na+ (ENaC) family of channels and that the alpha-subunit of ENaC alone or in combination with some protein other than the beta- or gamma-subunit protein is the major component of lung alveolar epithelial cation channels.

Research on the apoptosis of type II alveolar epithelial cells (AEC) shows that it may be induced by either angiotensin (ANG) II or angiotensinogen through pathways involving angiotensin converting enzyme (ACE) and the ANG II receptor. An investigation of the mechanisms underlying the regulation of type II apoptosis shows a definite link to the angiotensin-converting enzyme captopril, a finding that suggests that the ANG II-dependent pathway exists in both human and rat AECs.

The hypothesis that net alveolar fluid clearance would increase in association with hyperplasia of alveolar type II epithelial cells during subacute lung injury was tested. Alveolar fluid clearance was increased in bleomycin-injured rats. This increase was partially inhibited by amiloride of net alveolar fluid transport. The measured increases occurred in the presence of a significant increase in alveolar epithelial permeability to protein as well as a decrease in mRNA for the alpha-subunit of the epithelial sodium channel.

Research was conducted to examine the apoptosis and DNA damage in type 2 alveolar epithelial cells cultured from hyperoxic rats. Adult male rats were treated with >90% humidified oxygen for 48 h and isolated AEC2 from the lungs of oxygen-treated and control rats. Results indicate that keratinocyte growth factor plays an important role as a survival factor in AEC2 by limiting apoptosis in the lung after acute hyperoxic injury.

The influence of mechanical distention on the expression of particular marker proteins in type I and type II pulmonary alveolar epithelial cells is investigated using a culture of type II cells. Type II cells in culture were subjected to tonic mechanical distention and the marker proteins were assayed. Results indicate that these cells were able to express a type I cell marker after distention. The expression of the marker appears to be regulated at the posttranscriptional level.

Research indicates that nitric oxide has a single breath diffusing capacity in alveolar-capillary gas conductance that is unrelated to inspiratory nitric oxide concentrations. Experiments using six mechanically ventilated Chincilla cross-breed rabbits are described. A range of inspiratory nitric oxide concentrations were administered with varying breath-holding times.

Research shows that acute hyperoxia in rats modifies sodium pump activity, affecting alveolar sodium resorption and active sodium transport. Experiments designed to assess the effects of hypoxia on lung sodium-potassium-adenosine triphosphatase activity using healthy male Sprague-Dawley rats are described. Isolated, perfused lung preparations are prepared.

A surface fluorescence method to quantify transalveolar transport of water, protons and solutes in lungs gives sensitive, time resolved biophysical data from intact organs. The lungs are removed and perfused via the pulmonary artery. The airspace is filled with a solution containing a membrane impermeant fluorescent indicator. Fluorescence signals from the lung surfaces originate from the indicator and provide data on intraalveolar fluid composition. The application of this method shows that the mouse lung has high water permeability which may involve water channels.

Exposure of lung type II cells in rat to arsenite and diethylmaleic acid leads to an increase in the intracellular level of glutathione, but does not induce cystine transport. This indicates a different mechanism for cystine transport from the proposed sodium-dependent x(super -)(sub c) system. The cystine transport in the type II cells is regulated by sodium and glutamate and homocysteate can inhibit this transport.

Surfactant protein A (SP-A) induces the release of reactive oxygen species upon binding to alveolar macrophages (AM) through a specific surface receptor with high affinity. A multivalent interaction of SP-A with macrophages elicits this functional response from AM, which can be detected using enhanced chemiluminescence. The release of oxygen radicals from AM is possible only when SP-A is attached to zymosan or the surface of the reaction vial.

Injury of the alveolar epithelium by cigarette smoke is presumed to be an important process in the pathogenesis of smoking-related pulmonary diseases. We investigated the cytotoxic effects of cigarette smoke extract (CSE) on an alveolar type II cell-derived cell line (A549). CSE caused apoptosis at concentrations of 5% or less and necrosis at 10% or more. When CSE was exposed to air before application to A549 cells, the cytotoxic effects were attenuated. CSE caused cell death without direct contact with the cells. Acrolein and hydrogen peroxide, two major volatile factors in cigarette smoke, caused cell death in a similar manner. Aldehyde dehydrogenase, a scavenger of aldehydes, and N-acetylcysteine, a scavenger of oxidants and aldehydes, completely inhibited CSE-induced apoptosis. CSE and acrolein increased intracellular oxidant activity. In conclusion, apoptosis of alveolar epithelial cells may be one of the mechanisms of lung injury induced by cigarette smoking. This cytotoxic effect might be due to an interaction between aldehydes and oxidants present in CSE or formed in CSE-exposed cells. apoptosis; oxidant; aldehyde

Kalinichenko, Vladimir V., Lorena Lim, Brian Shin, and Robert H. Costa. Differential expression of forkhead box transcription factors following butylated hydroxytoluene lung injury. Am J Physiol Lung Cell Mol Physiol 280: L695-L704, 2001.--The forkhead box (Fox) proteins are a growing family of transcription factors that have important roles in cellular proliferation and differentiation and in organ morphogenesis. The Fox family members hepatocyte nuclear factor (HNF)-3[Beta] (Foxa2) and HNF-3/forkhead homolog (HFH)-8 (FREAC-1, Foxf1) are expressed in adult pulmonary epithelial and mesenchymal cells, respectively, but these cells display only low expression levels of the proliferation-specific HFH-11B gene (Trident, Foxm1b). The regulation of these Fox transcription factors in response to acute lung injury, however, has yet to be determined. We report here on the use of butylated hydroxytoluene (BHT)-mediated lung injury to demonstrate that HFH-11 protein and RNA levels were markedly increased throughout the period of lung repair. The maximum levels of HFH-11 were observed by day 2 following BHT injury when both bronchiolar and alveolar epithelial cells were undergoing extensive proliferation. Although BHT lung injury did not alter epithelial cell expression of HNF-3[Beta], a 65% reduction in HFH-8 mRNA levels was observed during the period of mesenchymal cell proliferation. HFH-8-expressing cells were colocalized with platelet endothelial cell adhesion molecule-1-positive alveolar endothelial cells and with [Alpha]-smooth muscle actin-positive peribronchiolar smooth muscle cells. winged helix/forkhead box DNA binding domain; hepatocyte nuclear factor 3/forkhead homolog; alveolar endothelial cell; alveolar type II cell; bronchiolar epithelial cells.

Matrix metalloproteinases and tissue inhibitor of metalloproteinase-2 in fetal rabbit lung. Am J Physiol Lung Cell Mol Physiol 279: L555-L561, 2000.--Cell-extracellular matrix interaction and extracellular matrix remodeling are known to be important in fetal lung development. We investigated the localization of matrix metalloproteinases (MMPs) in fetal rabbit lungs. Immunohistochemistry for type IV collagen, MMP-1, MMP-2, MMP-9, membrane type (MT) 1 MMP, and tissue inhibitor of metalloproteinase (TIMP)-2 and in situ hybridization for MMP-9 mRNA were performed. Gelatin zymography and Western blotting for MTI-MMP in lung tissue homogenates were also studied. MMP-1 and MT1-MMP were detected in epithelial cells, and MMP-2 and TIMP-2 were detected in epithelial cells and some mesenchymal cells in each stage. MMP-9 was found in epithelial cells mainly in the late stage. Gelatin zymography revealed that the ratio of active MMP-2 to latent MMP-2 increased dramatically during the course of development. MT1-MMP was detected in tissue homogenates, especially predominant in the late stage. These findings suggest that MMPs and their inhibitors may contribute to the formation of airways and alveoli in fetal lung development and that activated MMP-2 of alveolar epithelial cells may function to provide an extremely wide alveolar surface. formation of alveoli; type IV collagen; membrane type 1 matrix metalloproteinase; gelatin zymography

Mathew, Rajamma, Newton Y.-T. Fan, Ning Yuan, Praveen N. Chander, Michael H. Gewitz, and Charles T. Stier, Jr. Inhibition of NOS enhances pulmonary vascular changes in stroke-prone spontaneously hypertensive rats. Am. J. Physiol. Lung Cell. Mol. Physiol. 278: L81-L89, 2000.inTo determine the effects of chronic nitric oxide (NO) blockade on the pulmonary vasculature, 58-day-old spontaneously hypertensive rats of the stroke-prone substrain (SHRSP) and Wistar-Kyoto rats (WKY) received [N.sup.[Omega]-nitro-L-arginine (L-NNA; 15 mg [multiplied by] [kg.sup.-1] [multiplied by] [day.sup.-1] orally for 8 days). Relaxation to acetylcholine (ACh) in hilar pulmonary arteries (PAs), the ratio of right ventricular (RV) to body weight (RV/BW) to assess RV hypertrophy (RVH), and the percent medial wall thickness (WT) of resistance PAs were examined. L-NNA did not alter the PA relaxation, RV/BW, or WT in WKY. Although the PA relaxation and RV/BW in control SHRSP were comparable to those in WKY, the WT was increased (31 [+ or -] 2 vs. 19 [+ or -] 1%). L-NNA-treated SHRSP showed two patterns: in one group, the relaxation, RV/BW, and WT were comparable to those in the control SHRSP; in the other, impaired relaxation (36 [+ or -] 7 vs. 88 [+ or -] 4% for WKY) was associated with an increase in WT (37 [+ or -] 1%) and RV/BW (0.76 [+ or -] 0.05). Thus the abnormal pulmonary vasculature in SHRSP at [is less than] 10 wk of age is not accompanied by impaired relaxation in PAs or RVH; however, impaired relaxation is associated with increased WT and RVH. nitric oxide synthase; acetylcholine; [N.sup.[Omega]]-nitro-L-arginine; pulmonary circulation; pulmonary hypertension

Harrod, Kevin S., Bruce C. Trapnell, Kazuhisa Otake, Thomas R. Korfhagen, and Jeffrey A. Whitsett. SP-A enhances viral clearance and inhibits inflammation after pulmonary adenoviral infection. Am. J. Physiol. 277 (Lung Cell. Mol. Physiol. 21): L580-L588, 1999.--Surfactant protein A (SP-A) is a member of the collectin family of host defense molecules expressed primarily in the epithelial cells of the lung. To determine the role of SP-A in pulmonary adenoviral infection, SP-A-deficient (SP-A -/-) mice were intratracheally infected with a replication-deficient recombinant adenovirus, Av1Luc1. Lung inflammation was markedly increased in SP-A -/- compared with SP-A +/+ mice and was associated with increased hemorrhage and epithelial cell injury. Polymorphonuclear cells in bronchoalveolar lavage fluid (BALF) were increased in SP-A -/- mice after administration of adenovirus. Coadministration of adenovirus and purified human SP-A ameliorated adenoviral-induced lung inflammation in SP-A -/- mice. Concentrations of tumor necrosis factor-[Alpha] (TNF-[Alpha]), interleukin (IL)-6, and IL-1[Beta] were increased in BALF of SP-A -/- mice. Likewise, TNF-[Alpha], IL-6, macrophage inflammatory protein (MIP)-1[Alpha], monocyte chemotactic protein-i, and MIP-2 mRNAs were increased in lung homogenates from SP-A -/- mice 6 and 24 h after viral administration. Clearance of adenoviral DNA from the lung and uptake of fluorescent-labeled adenovirus by alveolar macrophages were decreased in SP-A -/- mice. SP-A enhances viral clearance and inhibits lung inflammation during pulmonary adenoviral infection, providing support for the importance of SP-A in antiviral host defense. surfactant protein A; lung epithelium; collectins; alveolar macrophages

Murine and rat models of pneumonia due to Klebsiella pneumoniae were used to investigate the role of intercellular adhesion molecule-1 (ICAM-1) on the type I alveolar epithelial cells in host defense through the enhancement of inflammatory cell microbial activity. Findings have indicated that, in vitro, antimicrobial activity of alveolar macrophages and neutrophils against K. Pneumoniae was significantly enhanced in the presence of rat alveolar epithelial cells that express abundant ICAM-1 in the primary culture.

Vascular endothelial growth factor (VEGF) is a homodimeric glycoprotein that plays a critical role in angiogenesis. Since VEGF is a mitogen for epithelial cells, alternative splicing of mRNA for VEGF can lead to the creation of isoforms of varying mitogenicity and solubility. This developmental process is investigated using adult, recovering newborn, oxygen-injured and control rabbit lungs. Results show that alternative splicing plays a significant role in the regulation of VEGF activity in both developing and injured lungs.

Catecholamine release can avert alveolar flooding after severe hemorrhagic shock. To clarify the role of catecholamine in this upregulation mechanism, rats were hemorrhaged to a mean arterial pressure of 30-35 mmHg for 60 minutes before being resuscitated with a solution of 4% albumin, after which alveolar liquid clearance was measured. The results provide direct in vivo evidence that neutrophil-dependent oxidant injury to alveolar cells helps to blocks the upregulation of fluid clearance by catecholamines if no alteration of paracellular permeability to the protein takes place.