The alveolar compartment, the fundamental gas exchange unit in the lung, is critical for tissue oxygenation and viability. We explored hepatocyte growth factor (HGF), a pleiotrophic cytokine that promotes epithelial proliferation, morphogenesis, migration, and resistance to apoptosis, as a candidate mediator of alveolar formation and regeneration. Mice deficient in the expression of the HGF receptor Met in lung epithelial cells demonstrated impaired airspace formation marked by a reduction in alveolar epithelial cell abundance and survival, truncation of the pulmonary vascular bed, and enhanced oxidative stress. Administration of recombinant HGF to tight-skin mice, an established genetic emphysema model, attenuated airspace enlargement and reduced oxidative stress. Repair in the TSK/+ mouse was punctuated by enhanced akt and stat3 activation. HGF treatment of an alveolar epithelial cell line not only induced proliferation and scattering of the cells but also conferred protection against staurosporine-induced apoptosis, properties critical for alveolar septation. HGF promoted cell survival was attenuated by akt inhibition. Primary alveolar epithelial cells treated with HGF showed improved survival and enhanced antioxidant production. In conclusion, using both loss-of-function and gain-of-function maneuvers, we show that HGF signaling is necessary for alveolar homeostasis in the developing lung and that augmentation of HGF signaling can improve airspace morphology in murine emphysema. Our studies converge on prosurvival signaling and antioxidant protection as critical pathways in HGF–mediated airspace maintenance or repair. These findings support the exploration of HGF signaling enhancement for diseases of the airspace.
The airspace compartment of the mammalian lung, comprised of spherical sacs termed alveoli, harbors the architecture, cellular composition, and molecular armamentarium to perform the critical function of gas exchange or oxygen uptake. Despite the necessity of this alveolar compartment for organismal viability, the mechanism by which alveoli are formed and maintained is obscure. Furthermore, no treatments are currently available that can regenerate the airspace once damaged. In this manuscript, we sought to determine whether hepatocyte growth factor, a cytokine with a functional armamentarium that subserves the critical events of alveolar formation (epithelial proliferation, migration, resistance from apoptosis and angiogenesis), could be an important mediator of alveolar formation and airspace maintenance. Our simple paradigm was that critical homeostatic pathways for the lung should operate both in lung formation and in lung maintenance/regeneration. Using an informative battery of mouse models and cell lines, we show that hepatocyte growth factor is a determinant of alveolar formation and that the enhancement of hepatocyte growth factor signaling can both protect and repair the airspace from pathologic airspace enlargement or emphysema.
The Kit receptor tyrosine kinase functions in hematopoiesis, melanogenesis, and gametogenesis and in interstitial cells of Cajal. We previously identified two upstream hypersensitive site (HS) clusters in mast cells and melanocytes. Here we investigated the roles of these 5′ HS sequences in Kit expression using transgenic mice carrying Kit-GFP reporter constructs. In these mice there is close correspondence between Kit-GFP reporter and endogenous Kit gene expression in most tissues analyzed. Deletion analysis defined the 5′ upstream HS cluster region as critical for Kit expression in mast cells. Furthermore, chromatin immunoprecipitation analysis in mast cells showed that H3 and H4 histone hyperacetylation and RNA polymerase II recruitment within the Kit promoter and in the 5′ HS region were associated with Kit expression. Therefore, the 5′ upstream hypersensitivity sites appear to be critical components of locus control region-mediated Kit gene activation in mast cells.
Rationale: Matrix metalloprotease (MMP)-9 is an elastolytic endopeptidase produced by activated macrophages that may be involved in the development of human pulmonary emphysema and could be inhibited with existing compounds. Mouse models have demonstrated that excess MMP-9 production can result in permanent alveolar destruction.
Objectives: To determine if MMP-9 causes cigarette smoke–induced emphysema using MMP-9 knockout mice and human samples.
Methods: Mouse lungs were analyzed for inflammation and airspace enlargement using a mainstream smoke-exposure model. Human macrophage mRNA was isolated from subjects with emphysema by laser capture microdissection. Human blood monocyte mRNA was isolated from subjects with greater than 30 pack-year smoking history. Human gene expression was determined by quantitative polymerase chain reaction and compared with emphysema severity determined by automated computed tomography analysis. Plasma Clara cell secretory protein and surfactant protein-D were quantified to measure ongoing lung injury.
Measurements and Main Results: Mice deficient in MMP-9 develop the same degree of cigarette smoke–induced inflammation and airspace enlargement as strain-matched controls. Macrophages are the predominant source of MMP-9 production in human emphysema specimens and similar quantities of macrophage MMP-9 mRNA is present in areas of lung with and without emphysema. Circulating monocytes produce more MMP-9 in individuals with advanced emphysema severity despite no correlation of MMP-9 with markers of ongoing lung damage.
Conclusions: These results suggest that MMP-9 in humans who smoke is similar to smoke-exposed mice, where MMP-9 is present in emphysematous lung but not correlated with the emphysema. To the degree that the mechanisms of emphysema in humans who smoke resemble the mouse model, these data suggest specific inhibition of MMP-9 is unlikely to be an effective therapy for cigarette smoke–induced emphysema.
Clinical trial registered with www.clinicaltrials.gov (NCT 00757120).
pulmonary disease, chronic obstructive; laser capture microdissection; mice, knockout
Lipopolysaccharide (LPS) is ubiquitous in the environment. Recent epidemiologic data suggest that occupational exposure to inhaled LPS can contribute to the progression of chronic obstructive pulmonary disease. To address the hypothesis that inhaled LPS can cause emphysema-like changes in mouse pulmonary parenchyma, we exposed C57BL/6 mice to aerosolized LPS daily for 4 weeks. By 3 days after the end of the 4-week exposure, LPS-exposed mice developed enlarged airspaces that persisted in the 4-week recovered mice. These architectural alterations in the lung are associated with enhanced type I, III, and IV procollagen mRNA as well as elevated levels of matrix metalloproteinase (MMP)-9 mRNA, all of which have been previously associated with human emphysema. Interestingly, MMP-9–deficient mice were not protected from the development of LPS-induced emphysema. However, we demonstrate that LPS-induced airspace enlargement was associated with apoptosis within the lung parenchyma, as shown by prominent TUNEL staining and elevated cleaved caspase 3 immunoreactivity. Antineutrophil antiserum-treated mice were partially protected from the lung destruction caused by chronic inhalation of LPS. Taken together, these findings demonstrate that inhaled LPS can cause neutrophil-dependent emphysematous changes in lung architecture that are associated with apoptosis and that these changes may be occurring through mechanisms different than those induced by cigarette smoke.
Rationale: There is growing evidence that alveolar cell apoptosis plays an important role in emphysema pathogenesis, a chronic inflammatory lung disease characterized by alveolar destruction. The association of α1-antitrypsin deficiency with the development of emphysema has supported the concept that protease/antiprotease imbalance mediates cigarette smoke–induced emphysema.
Objectives: We propose that, in addition to its antielastolytic effects, α1-antitrypsin may have broader biological effects in the lung, preventing emphysema through inhibition of alveolar cells apoptosis.
Methods, Measurements, and Main Results: Transduction of human α1-antitrypsin via replication-deficient adeno-associated virus attenuated airspace enlargement and emphysema caused by inhibition of vascular endothelial growth factor (VEGF) receptors with SU5416 in mice, a model of apoptosis-dependent emphysema lacking neutrophilic inflammation. The overexpressed human serine protease inhibitor accumulated in lung cells and suppressed caspase-3 activation and oxidative stress in lungs treated with the VEGF blocker or with VEGF receptor-1 and -2 antibodies. Similar results were obtained in SU5416-treated rats given human α1-antitrypsin intravenously.
Conclusions: Our findings suggest that inhibition of structural alveolar cell apoptosis by α1-antitrypsin represents a novel protective mechanism of the serpin against emphysema. Further elucidation of this mechanism may extend the therapeutic options for emphysema caused by reduced level or loss of function of α1-antitrypsin.
antiprotease; caspase; chronic obstructive pulmonary disease; oxidative stress; serpin
To identify the physiological role of Hck, a functionally redundant member of the Src family of tyrosine kinases expressed in myelomonocytic cells, we generated HckF/F “knock-in” mice which carry a targeted tyrosine (Y) to phenylalanine (F) substitution of the COOH-terminal, negative regulatory Y499-residue in the Hck protein. Unlike their Hck−/− “loss-of-function” counterparts, HckF/F “gain-of-function” mice spontaneously acquired a lung pathology characterized by extensive eosinophilic and mononuclear cell infiltration within the lung parenchyma, alveolar airspaces, and around blood vessels, as well as marked epithelial mucus metaplasia in conducting airways. Lungs from HckF/F mice showed areas of mild emphysema and pulmonary fibrosis, which together with inflammation resulted in altered lung function and respiratory distress in aging mice. When challenged transnasally with lipopolysaccharide (LPS), HckF/F mice displayed an exaggerated pulmonary innate immune response, characterized by excessive release of matrix metalloproteinases and tumor necrosis factor (TNF)α. Similarly, HckF/F mice were highly sensitive to endotoxemia after systemic administration of LPS, and macrophages and neutrophils derived from HckF/F mice exhibited enhanced effector functions in vitro (e.g., nitric oxide and TNFα production, chemotaxis, and degranulation). Based on the demonstrated functional association of Hck with leukocyte integrins, we propose that constitutive activation of Hck may mimic adhesion-dependent priming of leukocytes. Thus, our observations collectively suggest an enhanced innate immune response in HckF/F mice thereby skewing innate immunity from a reversible physiological host defense response to one causing irreversible tissue damage.
knock-in mutation; LPS; endotoxemia; macrophage; neutrophil
Alveolar development comprises the transition of lung architecture from saccules to gas-exchange units during late gestation and early postnatal development. Exposure to hyperoxia disrupts developmental signaling pathways and causes alveolar hypoplasia as seen in bronchopulmonary dysplasia affecting preterm human newborns. Expanding literature suggests that epigenetic changes due to environmental triggers during development may lead to genetically heritable changes in gene expression. Given recent data on altered histone deacetylase (HDAC) activity in lungs of humans and animal models with airspace enlargement/emphysema, we hypothesized that alveolar hypoplasia from hyperoxia exposure in neonatal mice is a consequence of cell cycle arrest and reduced HDAC activity and up-regulation of the cyclin-dependent kinase inhibitor, p21. We exposed newborn mice to hyperoxia and compared lung morphologic and epigenetic changes to room air controls. Further, we pretreated a subgroup of animals with the macrolide antibiotic azithromycin (AZM), known to possess anti-inflammatory properties. Our results showed that hyperoxia exposure resulted in alveolar hypoplasia and was associated with decreased HDAC1 and HDAC2 and increased p53 and p21 expression. Further, AZM did not confer protection against hyperoxia-induced alveolar changes. These findings suggest that alveolar hypoplasia due to hyperoxia is mediated by epigenetic changes affecting cell cycle regulation/senescence during lung development.
Pulmonary lymphangioleiomyomatosis (LAM) is a rare genetic disease characterized by neoplastic growth of atypical smooth muscle–like LAM cells, destruction of lung parenchyma, obstruction of lymphatics, and formation of lung cysts, leading to spontaneous pneumothoraces (lung rupture and collapse) and progressive loss of pulmonary function. The disease is caused by mutational inactivation of the tumor suppressor gene tuberous sclerosis complex 1 (TSC1) or TSC2. By injecting TSC2-null cells into nude mice, we have developed a mouse model of LAM that is characterized by multiple random TSC2-null lung lesions, vascular endothelial growth factor–D expression, lymphangiogenesis, destruction of lung parenchyma, and decreased survival, similar to human LAM. The mice show enlargement of alveolar airspaces that is associated with progressive growth of TSC2-null lesions in the lung, up-regulation of proinflammatory cytokines and matrix metalloproteinases (MMPs) that degrade extracellular matrix, and destruction of elastic fibers. TSC2-null lesions and alveolar destruction were differentially inhibited by the macrolide antibiotic rapamycin (which inhibits TSC2-null lesion growth by a cytostatic mechanism) and a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, simvastatin (which inhibits growth of TSC2-null lesions by a predominantly proapoptotic mechanism). Treatment with simvastatin markedly inhibited MMP-2, MMP-3, and MMP-9 levels in lung and prevented alveolar destruction. The combination of rapamycin and simvastatin prevented both growth of TSC2-null lesions and lung destruction by inhibiting MMP-2, MMP-3, and MMP-9. Our findings demonstrate a mechanistic link between loss of TSC2 and alveolar destruction and suggest that treatment with rapamycin and simvastatin together could benefit patients with LAM by targeting cells with TSC2 dysfunction and preventing airspace enlargement.
Peroxisome proliferator-activated receptor (PPAR)-γ is a nuclear hormone receptor that regulates gene expression, cell proliferation and differentiation. We previously described airway epithelial cell PPARγ deficient mice that develop airspace enlargement with decreased tissue resistance and increased lung volumes. We sought to understand the impact of airspace enlargement in conditionally targeted mice upon the physio-mechanical properties of the lung.
We measured elastic recoil and its determinants, including tissue structure and surface forces. We measured alveolar number using radial alveolar counts, and airspace sizes and their distribution using computer-assisted morphometry.
Air vs. saline-filled pressure volume profiles demonstrated loss of lung elastic recoil in targeted mice that was contributed by both tissue components and surface tension, but was proportional to lung volume. There were no significant differences in surfactant quantity/function nor in elastin and collagen content between targeted animals and littermate controls. Importantly, radial alveolar counts were significantly reduced in the targeted animals and at 8 weeks of age there were 18% fewer alveoli with 32% more alveolar ducts. Additionally, the alveolar ducts were 19% larger in the targeted animals.
Our data suggest that the functional abnormalities, including loss of recoil are secondary to altered force transmission due to differences in the structure of alveolar ducts, rather than changes in surfactant function or elastin or collagen content. These data further define the nature of abnormal lung maturation in the absence of airway epithelial cell PPARγ and identify a putative genetic determinant of dysanapsis, which may serve as a precursor to chronic lung disease.
BACKGROUND: Increased epithelial permeability of the airspaces occurs commonly in the lungs of cigarette smokers. It is likely to be important in augmenting the inflammatory response in the airspaces and hence may have a role in the pathogenesis of emphysema. It has previously been shown that intratracheal instillation of cigarette smoke condensate induces increased epithelial permeability in vivo in rats and in vitro in epithelial cell monolayers, associated with a disturbance in the lung antioxidant, glutathione (GSH). The aim of this study was to assess the role of neutrophils, GSH, and tumour necrosis factor (TNF) in the increased epithelial permeability following intratracheal instillation of cigarette smoke condensate. METHODS: Epithelial permeability of the airspaces was measured in rat lungs as the passage of intratracheally instilled 125-iodine labelled bovine serum albumin (BSA) into the blood. The permeability of a monolayer of human type II alveolar epithelial cells to 125I-BSA was also measured. RESULTS: Cigarette smoke condensate produced a 59.7% increase in epithelial permeability over control values peaking six hours after instillation and returning to control values by 24 hours. Depletion of neutrophils and, to a lesser extent, macrophages by an intraperitoneal injection of antineutrophil antibody did not influence the increased epithelial permeability induced by cigarette smoke condensate. Although instillation of human recombinant TNF alpha produced an increase in epithelial permeability in the rat lung from 0.62 (0.61)% to 1.27 (0.08)%, only a trivial amount of TNF alpha was detected in bronchoalveolar lavage (BAL) fluid in vivo or in culture medium from BAL leucocytes obtained from animals treated with cigarette smoke condensate (94.9 (28.8) units/ml). Furthermore, antiTNF antibody did not abolish the increased epithelial permeability produced by cigarette smoke condensate. The role of GSH was assessed by measuring the changes in both the reduced (GSH) and oxidised form (GSSG) in lung tissue and in BAL fluid. One hour after instillation of cigarette smoke condensate there was a marked fall in the GSH content in the lung (from 809.8 (31.8) to 501.7 (40.5) nmol/g) in association with increased GSSG levels (from 89.8 (2.7) to 148.7 (48.8) nmol/g). This was followed by a return of GSH levels to control values, with a concomitant decrease in GSSG levels six hours after instillation. GSH levels in BAL fluid fell dramatically following cigarette smoke condensate (from 2.56 (0.30) to 0.31 (0.21) nmol/ml) and this fall was sustained up to six hours after instillation of cigarette smoke condensate. CONCLUSIONS: These studies suggest that neutrophils and TNF do not have a major role in the increased epithelial permeability induced by cigarette smoke condensate. However, the data support a role for the depletion of the antioxidant glutathione in the increased epithelial permeability caused by cigarette smoke condensate.
BACKGROUND--Emphysema is currently defined as "a condition of the lung characterised by abnormal, permanent enlargement of the airspaces distal to the terminal bronchiole, accompanied by destruction of their walls, and without obvious fibrosis." The functional and morphological changes that occur in emphysema have largely been attributed to changes in alveolar elastin rather than in collagen. A study was performed to determine whether the amount of collagen in the alveolar wall changes with age in the lungs of non-smokers and of smokers with different types of macroscopically defined emphysema in relation to a microscopic measurement of lung structure. METHODS--Total alveolar wall collagen was measured (as hydroxyproline) in known volumes of distended lung tissue (by reverse phase high pressure liquid chromatography) in the lungs of non-smokers (n = 23) and in regions sampled away from emphysematous lesions in the lungs of 36 smokers (four with no emphysema, 13 with centriacinar emphysema (CAE), nine with panacinar emphysema (PAE), and 10 with a mixture (MIX) of both PAE and CAE). Mean lung airspace wall surface area per unit volume (AWUV) was calculated from at least six random blocks per lung and on histological sections immediately adjacent to those prepared for collagen measurement with a rapid scanning device (fast interval processor). RESULTS--In non-smokers there was no significant correlation between the amount of collagen in the alveolar wall tissue and either mean lung AWUV or increasing patient age when amounts of collagen were expressed either per unit volume of distended lung (40 mm3 sample) or per unit surface area of airspace wall tissue. Smokers without emphysema had similar amounts of collagen to non-smokers. Lungs with PAE and MIX, but not CAE alone, contained significantly more collagen than normal when expressed per unit volume of airspace wall tissue whereas all groups, including CAE, contained significantly raised amounts of collagen when expressed per unit surface area. CONCLUSIONS--There is no significant age related change in the collagen content of the lungs of non-smokers which suggests that, as AWUV is lost with age, the main collagenous framework is maintained. However, in smokers with emphysema there is a loss of airspace wall tissue in regions remote from the macroscopic lesions that is accompanied by a net increase in collagen mass. The greater accumulation of collagen in MIX lungs than in CAE lungs suggests a greater degree of structural damage, indicative of an alternative pathogenetic mechanism operating between the different types of emphysema. Our results suggest an active alveolar wall fibrosis in emphysema as a consequence of cigarette smoking. It is suggested that the definition of emphysema may require further revision to include such change.
Theories of emphysema traditionally revolved around proteolytic destruction of extracellular matrix. Models have recently been developed that show airspace enlargement with the induction of pulmonary cell apoptosis. The purpose of this study was to determine the mechanism by which a model of epithelial cell apoptosis caused airspace enlargement. Mice were treated with either intratracheal microcystin (MC) to induce apoptosis, intratracheal porcine pancreatic elastase (PPE), or their respective vehicles. Mice from all groups were inflated and morphometry was measured at various time points. Physiology measurements were performed for airway resistance, tissue elastance, and lung volumes. The groups were further analyzed by air–saline quasistatic measurements, surfactant staining, and surfactant functional studies. Mice treated with MC showed evidence of reversible airspace enlargement. In contrast, PPE-treated mice showed irreversible airspace enlargement. The airspace enlargement in MC-treated mice was associated with an increase in elastic recoil due to an increase in alveolar surface tension. PPE-treated mice showed a loss of lung elastic recoil and normal alveolar surface tension, a pattern more consistent with human emphysema. Airspace enlargement that occurs with the MC model of pulmonary epithelial cell apoptosis displays physiology distinct from human emphysema. Reversibility, restrictive physiology due to changes in surface tension, and alveolar enlargement associated with heterogeneous alveolar collapse are most consistent with a mild acute lung injury. Inflation near total lung capacity gives the appearance of enlarged alveoli as neighboring collapsed alveoli exert tethering forces.
apoptosis; emphysema; physiology; murine model
Rationale: Adipose-derived stem cells express multiple growth factors that inhibit endothelial cell apoptosis, and demonstrate substantial pulmonary trapping after intravascular delivery.
Objectives: We hypothesized that adipose stem cells would ameliorate chronic lung injury associated with endothelial cell apoptosis, such as that occurring in emphysema.
Methods: Therapeutic effects of systemically delivered human or mouse adult adipose stem cells were evaluated in murine models of emphysema induced by chronic exposure to cigarette smoke or by inhibition of vascular endothelial growth factor receptors.
Measurements and Main Results: Adipose stem cells were detectable in the parenchyma and large airways of lungs up to 21 days after injection. Adipose stem cell treatment was associated with reduced inflammatory infiltration in response to cigarette smoke exposure, and markedly decreased lung cell death and airspace enlargement in both models of emphysema. Remarkably, therapeutic results of adipose stem cells extended beyond lung protection by rescuing the suppressive effects of cigarette smoke on bone marrow hematopoietic progenitor cell function, and by restoring weight loss sustained by mice during cigarette smoke exposure. Pulmonary vascular protective effects of adipose stem cells were recapitulated by application of cell-free conditioned medium, which improved lung endothelial cell repair and recovery in a wound injury repair model and antagonized effects of cigarette smoke in vitro.
Conclusions: These results suggest a useful therapeutic effect of adipose stem cells on both lung and systemic injury induced by cigarette smoke, and implicate a lung vascular protective function of adipose stem cell derived paracrine factors.
pulmonary disease, chronic obstructive; endothelium; cell death; regenerative medicine; human
The Kit gene encodes a receptor tyrosine kinase involved in various biological processes including melanogenesis, hematopoiesis and gametogenesis in mice and human. A large number of Kit mutants has been described so far showing the pleiotropic phenotypes associated with partial loss-of-function of the gene. Hypomorphic mutations can induce a light coat color phenotype while complete lack of KIT function interferes with embryogenesis. Interestingly several intermediate hypomorphic mutations induced in addition growth retardation and post-natal mortality.
In this report we investigated the post-natal role of Kit by using a panel of chemically-induced hypomorphic mutations recently isolated in the mouse. We found that, in addition to the classical phenotypes, mutations of Kit induced juvenile steatosis, associated with the downregulation of the three genes, VldlR, Lpin1 and Lpl, controlling lipid metabolism in the post-natal liver. Hence, Kit loss-of-functions mimicked the inactivation of genes controlling the hepatic metabolism of triglycerides, the major source of energy from maternal milk, leading to growth and viability defects during neonatal development.
This is a first report involving KIT in the control of lipid metabolism in neonates and opening new perspectives for understanding juvenile steatosis. Moreover, it reinforces the role of Kit during development of the liver and underscores the caution that should be exerted in using KIT inhibitors during anti-cancer treatment.
Chronic obstructive pulmonary disease (COPD) is characterized by the presence of airflow obstruction and lung destruction with airspace enlargement. In addition to cigarette smoking, respiratory pathogens play a role in pathogenesis, but specific organisms are not always identified. Recent reports demonstrate associations between the detection of Pneumocystis jirovecii DNA in lung specimens or respiratory secretions and the presence of emphysema in COPD patients. Additionally, human immunodeficiency virus-infected individuals who smoke cigarettes develop early emphysema, but a role for P. jirovecii in pathogenesis remains speculative. We developed a new experimental model using immunocompetent mice to test the interaction of cigarette smoke exposure and environmentally acquired Pneumocystis murina infection in vivo. We hypothesized that cigarette smoke and P. murina would interact to cause increases in total lung capacity, airspace enlargement, and pulmonary inflammation. We found that exposure to cigarette smoke significantly increases the lung organism burden of P. murina. Pulmonary infection with P. murina, combined with cigarette smoke exposure, results in changes in pulmonary function and airspace enlargement characteristic of pulmonary emphysema. P. murina and cigarette smoke exposure interact to cause increased lung inflammatory cell accumulation. These findings establish a novel animal model system to explore the role of Pneumocystis species in the pathogenesis of COPD.
The W/c-kit and Steel loci respectively encode a receptor tyrosine kinase (Kit) and its extracellular ligand, Steel factor, which are essential for the development of hematopoietic, melanocyte, and germ cell lineages in the mouse. To determine the biochemical basis of the Steel/W developmental pathway, we have investigated the response of the Kit tyrosine kinase and several potential cytoplasmic targets to stimulation with Steel in mast cells derived from normal and mutant W mice. In normal mast cells, Steel induces Kit to autophosphorylate on tyrosine and bind to phosphatidylinositol 3'-kinase (PI3K) and phospholipase C-gamma 1 but not detectably to Ras GTPase-activating protein. Additionally, we present evidence that Kit tyrosine phosphorylation acts as a switch to promote complex formation with PI3K. In mast cells from mice homozygous for the W42 mutant allele, Kit is not tyrosine phosphorylated and fails to bind PI3K following Steel stimulation. In contrast, in the transformed mast cell line P815, Kit is constitutively phosphorylated and binds to PI3K in the absence of ligand. These results suggest that Kit autophosphorylation and its physical association with a unique subset of cytoplasmic signaling proteins are critical for mammalian development.
Emphysema is one manifestation of a group of chronic, obstructive, and frequently progressive destructive lung diseases. Cigarette smoking and air pollution are the main causes of emphysema in humans, and cigarette smoking causes emphysema in rodents. This review examines the concept of a homeostatically active lung structure maintenance program that, when attacked by proteases and oxidants, leads to the loss of alveolar septal cells and airspace enlargement. Inflammatory and noninflammatory mechanisms of disease pathogenesis, as well as the role of the innate and adaptive immune systems, are being explored in genetically altered animals and in exposure models of this disease. These recent scientific advances support a model whereby alveolar destruction resulting from a coalescence of mechanical forces, such as hyperinflation, and more recently recognized cellular and molecular events, including apoptosis, cellular senescence, and failed lung tissue repair, produces the clinically recognized syndrome of emphysema.
The receptor tyrosine kinase, c-kit, and its ligand, stem cell factor (SCF), function in a diverse range of biological functions. The role of c-kit in the maintenance and survival of hematopoietic stem cells and of mast cells is well recognized. c-kit also plays an important role in melanogenesis, erythropoiesis and spermatogenesis. Recent work from our laboratory highlights an important role of c-kit in the regulation of expression of two molecules in dendritic cells (DCs), interleukin-6 (IL-6) and Jagged-2 (a ligand of Notch), which are known to regulate T helper cell differentiation. Our study shows that induction of c-kit expression and its signaling in DCs promotes Th2 and Th17 responses but not Th1 response. c-kit inhibition by imatinib mesylate (Gleevec) in DCs was previously shown to promote natural killer cell activation which may be due to dampening of IL-6 production by the DCs. Since dysregulation of c-kit function has been associated with various disease states including cancer, in this perspective we have focused on known and novel functions of c-kit to include molecules such as IL-6 and Notch that were not previously recognized to be within the purview of c-kit biology. We have also reviewed the differential expression pattern of SCF and c-kit on various cell types and its variation during development or pathology. The recognition of previously unappreciated roles for c-kit will provide better insights into its function within and beyond the immune system and pave the way for developing better therapeutic strategies.
c-kit; SCF; interleukin-6; jagged-2; dendritic cells; T cells; differentiation
Chronic obstructive pulmonary disease/emphysema (COPD/emphysema) is characterized by chronic inflammation and premature lung aging. Anti-aging sirtuin 1 (SIRT1), a NAD+-dependent protein/histone deacetylase, is reduced in lungs of patients with COPD. However, the molecular signals underlying the premature aging in lungs, and whether SIRT1 protects against cellular senescence and various pathophysiological alterations in emphysema, remain unknown. Here, we showed increased cellular senescence in lungs of COPD patients. SIRT1 activation by both genetic overexpression and a selective pharmacological activator, SRT1720, attenuated stress-induced premature cellular senescence and protected against emphysema induced by cigarette smoke and elastase in mice. Ablation of Sirt1 in airway epithelium, but not in myeloid cells, aggravated airspace enlargement, impaired lung function, and reduced exercise tolerance. These effects were due to the ability of SIRT1 to deacetylate the FOXO3 transcription factor, since Foxo3 deficiency diminished the protective effect of SRT1720 on cellular senescence and emphysematous changes. Inhibition of lung inflammation by an NF-κB/IKK2 inhibitor did not have any beneficial effect on emphysema. Thus, SIRT1 protects against emphysema through FOXO3-mediated reduction of cellular senescence, independently of inflammation. Activation of SIRT1 may be an attractive therapeutic strategy in COPD/emphysema.
Rationale: Tumor necrosis factor α (TNF-α) has been implicated as a key cytokine in many inflammatory lung diseases. These effects are currently unclear, because a transgenic mouse overexpressing TNF-α in the lung has been shown in separate studies to produce elements of both emphysema and pulmonary fibrosis. Objectives: We sought to elucidate the phenotypic effects of TNF-α overexpression in a mouse model. Measurements: We established the phenotype by measuring lung impedance and thoracic gas volume, and using micro–computed tomography and histology. Main Results: We found that airways resistance in this mouse was not different to control mice, but that lung tissue dampening, elastance, and hysteresivity were significantly elevated. Major heterogeneous abnormalities of the parenchyma were also apparent in histologic sections and in micro–computed tomography images of the lung. These changes included airspace enlargement, loss of small airspaces, increased collagen, and thickened pleural septa. We also found significant increases in lung and chest cavity volumes in the TNF-α–overexpressing mice. Conclusions: We conclude that TNF-α overexpression causes pathologic changes consistent with both emphysema and pulmonary fibrosis combined with a general lung inflammation, and consequently does not model any single human disease. Our study thus confirms the pleiotropic effects of TNF-α, which has been implicated in multiple inflammatory disorders, and underscores the necessity of using a wide range of investigative techniques to link gene expression and phenotype in animal models of disease.
emphysema; micro–computed tomography; plethysmography; pulmonary fibrosis
Background and aims: Interstitial cells of Cajal (ICC) are pacemakers and mediators of motor neurotransmission in gastrointestinal smooth muscles. ICC require cellular signalling via Kit, a receptor tyrosine kinase, for development and maintenance of phenotype. Much of the evidence demonstrating the functions of ICC comes from studies of W/WV mice, which have reduced Kit function and reductions in specific populations of ICC. The aim of the present study was to differentially examine gene expression in the small intestines of wild-type and W/WV mutant mice.
Methods and results: RNA from the jejunums of wild-type and W/WV mutants was analysed using a differential gene display method. Eighteen queries were identified as novel genes that were differentially displayed in wild-type and W/WV mice. One candidate gene, encoding a novel acid phosphatase-like protein, was significantly suppressed in fed and starved W/WV mice. The full length clone of the murine gene and its human counterpart were designated acid phosphatase-like protein 1 (ACPL1). Human ACPL1 cDNA encodes a protein of 428 amino acids with homology to human prostatic acid phosphatase protein. This gene is located at 1q21. ACPL1 was abundantly expressed in the human small intestine and colon. Gene products were found to be cytoplasmic in transfected COS-7 cells. Reverse transcription-polymerase chain reaction analysis revealed expression of ACPL1 mRNA within single isolated ICCs.
Conclusions: Gene analysis showed that ACPL1 was differentially expressed in the small intestines of normal and W/WV mice. ICC within the small intestine expressed mRNA for ACPL1. Specific downregulation of ACPL1 in the jejunums of W/WV mice and high expression in human intestinal tissue suggest that the ACPL1 gene could be associated with ICC function in mice and humans.
gastrointestinal motility; interstitial cells of Cajal; pacemaker deficient mice; acid phosphatase family; acid phosphatase-like protein 1; chromosome 1q21 differential gene display method
Spontaneous premature ovarian failure presents most commonly with secondary amenorrhea. Young women with the disorder are infertile and experience the symptoms and sequelae of estrogen deficiency. The mechanisms that give rise to spontaneous premature ovarian failure are largely unknown, but many reports suggest a genetic mechanism in some cases. The small family size associated with infertility makes genetic linkage analysis studies extremely difficult. Another approach that has proven successful has been to examine candidate genes based on known genetic phenotypes in other species. Studies in mice have demonstrated that c-kit, a transmembrane tyrosine kinase receptor, plays a critical role in gametogenesis. Here we test the hypothesis that human KIT mutations might be a cause of spontaneous premature ovarian failure.
Methods and Results
We examined 42 women with spontaneous premature ovarian failure and found partial X monosomy in two of them. In the remaining 40 women with known 46,XX spontaneous premature ovarian failure we evaluated the entire coding region of the KIT gene. We did this using polymerase chain reaction based single-stranded conformational polymorphism analysis and DNA sequencing. We did not identify a single mutation that would alter the amino acid sequence of the c-KIT protein in any of 40 patients (upper 95% confidence limit is 7.2%). We found one silent mutation at codon 798 and two intronic polymorphisms.
Mutations in the coding regions of the KIT gene appear not to be a common cause of 46,XX spontaneous premature ovarian failure in North American women.
The presence of anti-endothelial cell antibodies and pathogenic T cells may reflect an autoimmune component in the pathogenesis of emphysema. Whether immune modulatory strategies can protect against the development of emphysema is not known.
Sprague Dawley rats were immunized with human umbilical vein endothelial cells (HUVEC) to induce autoimmune emphysema and treated with intrathymic HUVEC-injection and pristane. Measurements of alveolar airspace enlargement, cytokine levels, immuno histochemical, western blot analysis, and T cell repertoire of the lung tissue were performed.
The immunomodulatory strategies protected lungs against cell death as demonstrated by reduced numbers of TUNEL and active caspase-3 positive cells and reduced levels of active caspase-3, when compared with lungs from HUVEC-immunized rats. Immunomodulatory strategies also suppressed anti-endothelial antibody production and preserved CNTF, IL-1alpha and VEGF levels. The immune deviation effects of the intrathymic HUVEC-injection were associated with an expansion of CD4+CD25+Foxp3+ regulatory T cells. Pristane treatment decreased the proportion of T cells expressing receptor beta-chain, Vβ16.1 in the lung tissue.
Our data demonstrate that interventions classically employed to induce central T cell tolerance (thymic inoculation of antigen) or to activate innate immune responses (pristane treatment) can prevent the development of autoimmune emphysema.
Emphysema consists of a unique pattern of alveolar destruction, resulting in marked airspace enlargement with reduction of alveolar capillary exchange area. Classical concepts of the pathogenesis of emphysema have relied on the paradigm set by the inflammation and protease/antiprotease imbalance. We propose herein that cigarette smoke constitutes an environmental hazard that causes alveolar destruction by the interaction of apoptosis, oxidative stress, and protease/antiprotease imbalance. We draw a parallel between organismal aging, organ structural maintenance, and the damage resulting from chronic cigarette smoke inhalation. The stochastic interaction between environmental hazards and the effort of an organism or a particular organ to fend off these hazards results in the accumulation of cellular damage and features characteristic of aging. Inflammation follows as the result of the multiplication of injuries. We highlight the importance of understanding the biology of the interaction of alveolar cells in homeostasis and in alveolar destruction, and the potential role of novel processes related to senescence and stress response. An evolutionary perspective of emphysema that incorporates mechanisms related to aging may lead to important advances in the understanding and therapeutic targeting of chronic obstructive pulmonary disease.
aging; apoptosis; emphysema; inflammation; oxidative stress
Heterozygous elastin gene mutations cause autosomal dominant cutis laxa associated with emphysema and aortic aneurysms. To investigate the molecular mechanisms leading to cutis laxa in vivo, we generated transgenic mice by pronuclear injection of minigenes encoding normal human tropoelastin (WT) or tropoelastin with a cutis laxa mutation (CL). Three independent founder lines of CL mice showed emphysematous pulmonary airspace enlargement. No consistent dermatological or cardiovascular pathologies were observed. One CL and one WT line were selected for detailed studies. Both mutant and control transgenic animals showed elastin deposition into pulmonary elastic fibers, indicated by increased desmosine levels in the lung and by colocalization of transgenic and endogenous elastin by immunostaining. CL mice showed increased static lung compliance and decreased stiffness of lung tissue. In addition, markers of transforming growth factor-β (TGFβ) signaling and the unfolded protein response (UPR) were elevated together with increased apoptosis in the lungs of CL animals. We conclude that the synthesis of mutant elastin in CL activates multiple downstream disease pathways by triggering a UPR, altered mechanical signaling, increased release of TGFβ and apoptosis. We propose that the combined effects of these processes lead to the development of an emphysematous pulmonary phenotype in CL.
chronic obstructive pulmonary disease; mutation; elastin; growth factor; unfolded protein response; transgenic mouse