Pulmonary ischemia induces lung remodeling and angiogenesis.

Cellular remodeling during angiogenesis in the lung is poorly described. Furthermore, it is the systemic vasculature of the lung and surrounding the lung that is proangiogenic when the pulmonary circulation becomes impaired. In a mouse model of chronic pulmonary thromboembolism, after left pulmonary artery ligation (LPAL), the intercostal vasculature, in proximity to the ischemic lung, proliferates and invades the lung (12). In the present study, we performed a detailed investigation of the kinetics of remodeling using histological sections of the left lung of C57Bl/6J mice after LPAL (4 h to 20 days) or after sham surgery. New vessels were seen within the thickened visceral pleura 4 days after LPAL predominantly in the upper portion of the left lung. Connections between new vessels within the pleura and pulmonary capillaries were clearly discerned by 7 days after LPAL. The visceral pleura and the lung parenchyma showed intense tissue remodeling, as evidenced by markedly elevated levels of both proliferating cell nuclear antigen and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling positive cells. Rapidly dividing cells were predominantly macrophages and type II pneumocytes. The increased apoptotic activity was further quantified by caspase-3 activity, which showed a sixfold increase relative to naive lungs, by 24 h after LPAL. Because sham surgeries had little effect on measured parameters, we conclude that both thoracic wound healing and pulmonary ischemia are required for systemic neovascularization.     

Comparison of amounts of collagen and elastin in pleura and parenchyma of dog lung

Pressure-volume characteristics of the lung have been thought to be due primarily to the properties of the network of alveolar septa. However, Hajji et al. (J. Appl. Physiol.: Respirat . Environ. Exercise Physiol. 47: 175-181, 1979) attributed a substantial role to the visceral pleura. Seeking a structural explanation for this result, we compared the relative amounts of collagen fibrils and elastin fibers in the visceral pleura and alveolar parenchyma using stereological measurements in five canine lobes. We found about one-fifth as much collagen and one-tenth as much elastin in the pleura as in the alveolar parenchyma. This structural result confirms the functional conclusions of Hajji et al. We argue that such a substantial structure is not needed for protection against overinflation but may have to do with stabilization of lobe shape or handling of frictional forces.     

Lung hypoplasia and neonatal death in Fgf9-null mice identify this gene as an essential regulator of lung mesenchyme

Mammalian lung develops as an evagination of ventral gut endoderm into the underlying mesenchyme. Iterative epithelial branching, regulated by the surrounding mesenchyme, generates an elaborate network of airways from the initial lung bud. Fibroblast growth factors (FGFs) often mediate epithelial-mesenchymal interactions and mesenchymal Fgf10 is essential for epithelial branching in the developing lung. However, no FGF has been shown to regulate lung mesenchyme. In embryonic lung, Fgf9 is detected in airway epithelium and visceral pleura at E10.5, but is restricted to the pleura by E12.5. We report that mice homozygous for a targeted disruption of Fgf9 exhibit lung hypoplasia and early postnatal death. Fgf9(-/-) lungs exhibit reduced mesenchyme and decreased branching of airways, but show significant distal airspace formation and pneumocyte differentiation. Our results suggest that Fgf9 affects lung size by stimulating mesenchymal proliferation. The reduction in the amount of mesenchyme in Fgf9(-/-) lungs limits expression of mesenchymal Fgf10. We suggest a model whereby FGF9 signaling from the epithelium and reciprocal FGF10 signaling from the mesenchyme coordinately regulate epithelial airway branching and organ size during lung embryogenesis.     

Changes in lung permeability after chronic pulmonary artery obstruction.

We have shown that left pulmonary artery ligation (LPAL) in mice causes a prompt angiogenic response, with new systemic vessels from intercostal arteries penetrating the pleura within 6 days. Because angiogenic vessels in other organs have been shown to exhibit increased permeability, we studied vascular permeability (Evans blue dye extravasation, lung wet weight-to-dry weight ratio, and lavaged protein) in naive C57BL/6 mice and 4 h, and 14 and 21 days after LPAL (4-6 mice/time point). We also measured radiolabel clearance as an index of functional perfusion after LPAL. Tracer clearance from the left lung was maximal by 6 days after LPAL and not different from right lungs. Thus a functional vasculature is established before 6 days of LPAL that results in normal tracer clearance. By 21 days after LPAL, Evans blue-albumin was significantly increased in the left lung relative to both 4 h (no vasculature) and 14 days after LPAL. Only after 21 days of LPAL was left lung wet weight-to-dry weight ratio significantly different from naive lungs. Additionally, lavaged protein was significantly increased both 4 h and 21 days after LPAL relative to control mice. Thus, using three different methods, results consistently demonstrated increased permeability to protein and water 21 days after LPAL. Although changes in surface area of perfusion might affect the interpretation of these results, blood flow measured with labeled microspheres indicated no change in left lung perfusion between 14 and 21 days of LPAL. Thus the lung vasculature, remodeled as a consequence of chronic pulmonary artery obstruction, demonstrates increased water and protein permeability.     

Effect of low-frequency ultrasound on the pleura and adjoining lung tissue

Morphological changes in the pleura and adjacent pulmonary tissue were studied in 50 rabbits exposed to various doses of low-frequency ultrasound. When the exposure did not exceed 20 sec/cm2, no changes in pleural structure were noted. A longer period of exposure was followed by serous effusion. The contact of a wave-guide with the lung tissue caused the necrosis of the pleura and adjacent tissue.     

Laminin alpha 5 is required for lobar septation and visceral pleural basement membrane formation in the developing mouse lung

Laminin alpha/beta/gamma heterotrimers are the major noncollagenous components of all basement membranes. To date, five alpha, three beta, and three gamma chains have been identified. Laminin alpha 5 is expressed early in lung development and colocalizes with laminin alpha1. While laminin alpha1 expression in the lung is restricted to the embryonic period, laminin alpha 5 expression persists throughout embryogenesis and adulthood. Targeted mutation of the mouse laminin alpha 5 gene Lama5 causes embryonic lethality at E14-E17 associated with exencephaly, syndactyly, placentopathy, and kidney defects, all attributable to abnormal basement membranes. In this investigation, lung development in Lama5(-/-) mice up to E16.5 was examined. We observed normal lung branching morphogenesis and vasculogenesis, but incomplete lobar septation and absence of the visceral pleura basement membrane. Preservation of branching morphogenesis was associated with ectopic deposition of laminin alpha 4 in the airway basement membrane. Perturbation of pleural basement membrane formation and right lung septation correlated with absence of laminin alpha 5, which was found to be the only laminin alpha chain present in the normal visceral pleura basement membrane. Our finding of normal lung branching morphogenesis with abnormal lobar septation demonstrates that these processes are not obligatorily linked.     

Epithelial laminin alpha5 is necessary for distal epithelial cell maturation, VEGF production, and alveolization in the developing murine lung

Laminin alpha5 is prominent in the basement membrane of alveolar walls, airways, and pleura in developing and adult lung. Targeted deletion of laminin alpha5 in mice causes developmental defects in multiple organs, but embryonic lethality has precluded examination of the latter stages of lung development. To identify roles for laminin alpha5 in lung development, we have generated an inducible lung epithelial cell-specific Lama5 null (SP-CLama5(fl/-)) mouse through use of the Cre/loxP system, the human surfactant protein C promoter, and the reverse tetracycline transactivator. SP-CLama5(fl/-) embryos exposed to doxycycline from E6.5 died a few hours after birth. Compared to control littermates, SP-CLama5(fl/-) lungs had dilated, enlarged distal airspaces, but basement membrane ultrastructure was preserved. Distal epithelial cell differentiation was perturbed, with a marked reduction of alveolar type II cells and a virtual absence of type I cells. Cell proliferation was reduced and apoptosis was increased. Capillary density was diminished, and this was associated with a decrease in total lung VEGF production. Overall, these findings indicate that epithelial laminin alpha5, independent of its structural function, is necessary for murine lung development, and suggest a role for laminin alpha5 in signaling pathways that promote alveolar epithelial cell differentiation and VEGF expression.     

A possible role of the pleura in lung mechanics

The pressure-volume behavior of excised visceral pleura is studied. The pleura is modeled as a thin incompressible membrane, and the requisite membrane tension is determined from a pseudostrain-energy function. Results are compared to pressure-volume behavior of saline-filled and air-filled parenchyma and to experimental pleural data from the literature. Results suggest that the pleura may play a role as a volume limiter of lung expansion although the need for more detailed analysis is discussed.     

A method for microinjection into subpleural alveoli of rat lung in situ

A new technique is described for the micropuncture of rat lung in the intact thorax. Under pentobarbital sodium anesthesia a glass micropipette is passed through a small area of the parietal pleura, which has been cleared of overlying intercostal muscle. The micropipette is passed into the lung parenchyma and withdrawn again without collapsing the lung. The current application of this technique is in the microinjection of fluid into subpleural alveoli, which is illustrated using a suspension of colloidal gold. The gold particles are immediately dispersed over the surface of many alveoli, a small proportion spreading laterally as far as 4-6 mm. There is no evidence of alveolar flooding.     

Determinants of restrictive lung function in asbestos-induced pleural fibrosis

We evaluated whether restrictive lung function among asbestos-exposed individuals with pleural fibrosis was caused by radiographically inapparent parenchymal inflammation and/or parenchymal fibrosis. All 24 study participants were sheet metal workers who were nonsmokers with normal parenchyma on posteroanterior chest radiograph. These subjects had either normal pleura (n = 7), circumscribed plaques (n = 9), or diffuse pleural thickening (n = 8). After controlling for age, years in the trade, and pack-years of smoking, we found that sheet metal workers with diffuse pleural thickening had a lower forced vital capacity (P less than 0.001), total lung capacity (P less than 0.01), and CO-diffusing capacity of the lung (P less than 0.05) than those with normal pleura. Similarly, sheet metal workers with circumscribed plaques were found to have a reduced forced vital capacity; however, because of the small number of study subjects, this difference (regression coefficient = -11.0) was only marginally significant (P = 0.06). Although circumscribed plaque and diffuse pleural thickening were both associated with a lymphocytic alveolitis and a higher prevalence of parenchymal fibrosis on high-resolution computerized tomography (HRCT) scan, neither a lymphocytic alveolitis nor the finding of parenchymal fibrosis on HRCT scan influenced the relationship between pleural fibrosis and restrictive lung function. We conclude that pleural fibrosis is associated with restrictive lung function and abnormally low diffusion that appears to be independent of our measures of parenchymal injury (chest X-ray, bronchoalveolar lavage, and HRCT scan).(ABSTRACT TRUNCATED AT 250 WORDS)     

The relation between nerve fibres and dopamine cells of the ruminant lung

Synopsis A study of the distribution and ultrastructure of the special chromaffin or dopamine cells of the ruminant lung has been made. The results with fluorescence microscopy show that the adult sheep has larger numbers of such cells than has the lamb, calf or adult goat. Contrary to earlier findings, their numbers have been found to be as great in the intralobular tissue as in the pleura, septa and other structures. Electron microscopic examination of the cells shows that they resemble true mast cells; some contain two types of granule, the appearance of which suggests that part of the amine has been discharged. Although in many places the cells are closely associated with adrenergic nerve fibres no evidence was found to suggest that they had any functional relation with one another.     

In vivo tracheal occlusion in fetal mice induces rapid lung development without affecting surfactant protein C expression

Fetal tracheal occlusion (TO) reverses lung hypoplasia by inducing rapid lung growth. Although increases in lung size accompanied by increased numbers of alveoli and capillaries have been reported, effects of TO on lung development have not been formally assessed. In the present study, the objective was to verify our prediction that the main effect of TO would be to accelerate fetal lung development. We have developed and characterized a new fetal mouse model of TO to best realize this goal. At embryonic day 16.5, pregnant CD1 mice were operated under general anesthesia. One fetus per dam was selected to undergo surgical TO with a surgical clip or a sham operation. The fetuses were delivered 24 or 36 h postsurgery. The maturation of lung parenchyma, evaluated by counting the generations of alveolar saccules from the terminal bronchiole to the pleura, was significantly accelerated in the TO group with a complexity of the gas exchange region comparable with postnatal days 1 and 3 after 24 or 36 h of TO. Cellular proliferation and apoptosis peaks, assessed by immunohistochemistry directed against PCNA and the active form of caspase-3, were significantly increased 24 h after surgery in the TO group compared with the sham group. However, in situ hybridization showed no significant difference in the density of type II pneumocytes expressing surfactant protein C mRNA. Our results show that brief TO during late gestation in fetal mice induces accelerated lung development with minimal effects on surfactant protein C mRNA expression.     

T-zone lymphoma--clinical symptoms, therapy, and prognosis (author's transl)]

The clinical symptoms, response to therapy, and prognosis of T-zone lymphoma were analyzed in 32 cases. This recently defined lymphoma entity developed relatively quickly with generalized lymphadenopathy and general malaise. Hepatomegaly and/or splenomegaly and skin efflorescence were frequent presenting symptoms. A few patients showed hyperimmune reactions and occasionally severe autoimmune hemolytic anemia. The erythrocyte sedimentation rate was usually markedly elevated. There was sometimes a polyclonal increase in serum immunoglobulin, sometimes a reduction. Blood and bone marrow smears from a few patients showed occasional atypical lymphocytes. A remarkable finding was the frequent involvement of lung or pleura (40.5% of the patients). The prognosis is unfavorable. Most of the patients were in stages III or IV at the time of diagnosis. Massive infiltration of organs, resistance to routine therapy, and decreasing resistance to infection resulted in death soon after diagnosis. The probability of survival was 0.48 in the first year after diagnosis. The prognosis for patients in stages I and II was clearly better than that for patients in stages III and IV.     

The effect of long-term treatment with erythromycin on Th1 and Th2 cytokines in diffuse panbronchiolitis

Diffuse panbronchiolitis (DPB) is a chronic progressive disease of the respiratory bronchioles, and has been improved by low-dose, long-term erythromycin (EMC) treatment. The therapeutic benefits may be derived from its anti-inflammatory and immunomodulatory properties rather than antimicrobial effect. However, there are few studies about the mechanism of immunomodulation by EMC treatment for patient with DPB. In this study, we quantified the changes of Th1 and Th2 cytokines in the bronchoalveolar lavage (BAL) fluid from patients with DPB after long term treatment with EMC. After the EMC treatment, a significant reduction in the number of lymphocytes was observed, and the CD4/CD8 ratio was elevated as well. The IL-2 and IFN-gamma levels in the BAL fluid were significantly decreased and the IL-4, IL-5, and IL-13 levels were significantly increased after EMC treatment. Our results suggest that the therapeutic benefits of long-term EMC treatment may be partially due to the immune system's shift from Th1 to Th2 cytokine production.     

Deficiency in the divalent metal transporter 1 increases bleomycin-induced lung injury

Exposure to bleomycin can result in an inflammatory lung injury. The biological effect of this anti-neoplastic agent is dependent on its coordination of iron with subsequent oxidant generation. In lung cells, divalent metal transporter 1 (DMT1) can participate in metal transport resulting in control of an oxidative stress and tissue damage. We tested the postulate that metal import by DMT1 would participate in preventing lung injury after exposure to bleomycin. Microcytic anemia (mk/mk) mice defective in DMT1 and wild-type mice were exposed to either bleomycin or saline via intratracheal instillation and the resultant lung injury was compared. Twenty-four h after instillation, the number of neutrophils and protein concentrations after bleomycin exposure were significantly elevated in the mk/mk mice relative to the wild-type mice. Similarly, levels of a pro-inflammatory mediator were significantly increased in the mk/mk mice relative to wild-type mice following bleomycin instillation. Relative to wild-type mice, mk/mk mice demonstrated lower non-heme iron concentrations in the lung, liver, spleen, and splenic, peritoneal, and liver macrophages. In contrast, levels of this metal were elevated in alveolar macrophages from mk/mk mice. We conclude that DMT1 participates in the inflammatory lung injury after bleomycin with mk/mk mice having increased inflammation and damage following exposure. This finding supports the hypothesis that DMT1 takes part in iron detoxification and homeostasis in the lung.     

Apparent Reversion to Trophism of a Free Omental Graft: A Preliminary Report

In a previous paper we reported evidence which suggested that a free omental graft has trophic characteristics similar to those of the chorionic epithelium of the early embryo, through which it establishes arteriolar or larger connections with aorta, heart and pericardium within 10 days after it has been placed in contact with the denuded surfaces of each structure. The theory of omental trophism was tested by dropping pieces of omentum completely detached into the pleural and peritoneal cavities of white rats. When examined 25 or more days later a percentage of omental grafts appeared healthy and were attached to pleura, lung, heart and to various structures in the peritoneal cavity. In the pleural grafts, histological sections revealed a continuity between the omental grafts and the sub-pleural tissues. A blood-hungry free omental graft appears to seek out its own blood supply. Neither pleura nor serosa was able to repel its primitive probing for a new blood supply.     

Inflammation and ischemia-induced lung angiogenesis

A role for inflammation in modulating the extent of angiogenesis has been shown for a number of organs. The present study was undertaken to evaluate the importance of leukocyte subpopulations for systemic angiogenesis of the lung after left pulmonary artery ligation (LPAL) in a mouse model of chronic pulmonary thromboembolism. Since we (24) previously showed that depletion of neutrophils did not alter the angiogenic outcome, we focused on the effects of dexamethasone pretreatment (general anti-inflammatory) and gadolinium chloride treatment (macrophage inactivator) and studied Rag-1(-/-) mice (T/B lymphocyte deficient). We measured inflammatory cells in bronchoalveolar lavage fluid and lung homogenate macrophage inflammatory protein-2 (MIP-2) and IL-6 protein levels within 24 h after LPAL and systemic blood flow to the lung 14 days after LPAL with labeled microspheres as a measure of angiogenesis. Blood flow to the left lung was significantly reduced after dexamethasone treatment compared with untreated control LPAL mice (66% decrease; P < 0.05) and significantly increased in T/B lymphocyte-deficient mice (88% increase; P < 0.05). Adoptive transfer of splenocytes (T/B lymphocytes) significantly reversed the degree of angiogenesis observed in the Rag-1(-/-) mice back to the level of control LPAL. Average number of lavaged macrophages for each group significantly correlated with average blood flow in the study groups (r(2) = 0.9181; P = 0.01 different from 0). Despite differences in angiogenesis, left lung homogenate MIP-2 and IL-6 did not differ among study groups. We conclude that inflammatory cells modulate the degree of angiogenesis in this lung model where lymphocytes appear to limit the degree of neovascularization, whereas monocytes/macrophages likely promote angiogenesis.     

Erythropoietic protein supportive treatment in small cell lung cancer

Anemia is common among patients with malignant tumors, due to the disease and chemotherapy. Anemia decreases patient's quality of life, and worsens the dose intensity of chemotherapy. The aim of this retrospective data-analysis was to determine the rate of transfusions and the maintenance of chemotherapeutic dose intensity in 9 small cell lung cancer patients receiving beta-erythropoietin, due to anemia observed after the first cycle of chemotherapy. The mean pre-treatment hemoglobin concentration of the patients was 116.67+/-8.17 g/L (mean+/-SD). The mean pre-erythropoietin hemoglobin concentration at baseline was 103.11+/-7.52 g/L. Six cycles of platinum compounds and etoposide were used. The post-treatment hemoglobin concentration of patients was 110.11+/-5.37 g/L (p = 0,028 vs. baseline). During these 54 chemotherapeutic cycles, only 2 patients needed transfusion, each of them once. According to our experience, the use of beta-erythropoietin in 9 anemic small cell lung cancer patients resulted in a low rate of transfusions and maintenance of cytotoxic treatment dose intensity. The adequate use of beta-erythropoietin is of great help to the physician in the management of small cell lung cancer patients.     

Flow limitation and wheezes in a constant flow and volume lung preparation

To facilitate the study of respiratory wheezes in an animal lung model, an isovolume, constant-flow excised dog lung preparation was developed. Dog lungs were inflated to 26 +/- 4 cmH2O and coated with layers of epoxy glue and polyester compound. A rigid shell 2 mm thick was obtained around the entire pleural surface and the extra-pulmonary airways. The adhesive forces between the pleura and the shell were strong enough to hold the lung distended after the inflation pressure was removed. Holes 2 mm diam were drilled through the shell over one of the lung lobes in an array, 4 cm across. The holes penetrated the pleural surface, so that constant flow could be maintained in the expiratory direction by activating a suction pump connected to the trachea. Downstream suction pressure and flow rate were measured with a mercury manometer and a rotameter, respectively. Sounds were recorded by a small (0.6 cm OD) microphone inserted into the trachea. When suction pressure was increased, flow initially increased to 31 +/- 3 l/min. Further increase of suction pressure caused only very slight additional increase in flow (i.e., flow limitation). During this plateau of flow, a pure tone was generated with acoustic properties similar to respiratory wheezes. Both the flow plateau and the wheezing sounds could be eliminated by freezing the lungs. It is concluded that wheezing sounds were associated with flow limitation in this preparation. It is suggested that the stable acoustic properties obtained by this preparation may become useful in the analysis of mechanisms of wheezing lung sounds generation.     

Homogeneity of lung tubulin isoforms during lung maturation

The rat and rabbit lung isoform pattern before (fetal) and after (adult) lung maturation has been analyzed by isoelectric focusing. This pattern has been compared to that of brain tubulin at the same developmental stages. No changes in the pattern of fetal and adult lung tubulin were found. However an increase in brain tubulin heterogeneity was detected from fetal to adult stage.