Interleukin-17A and vascular remodelling in severe asthma; lack of evidence for a direct role.

BACKGROUND: Bronchial vascular remodelling may contribute to the severity of airway narrowing through mucosal congestion. Interleukin (IL)-17A is associated with the most severe asthmatic phenotype but whether it might contribute to vascular remodelling is uncertain. OBJECTIVE: To assess vascular remodelling in severe asthma and whether IL-17A directly or indirectly may cause endothelial cell activation and angiogenesis. METHODS: Bronchial vascularization was quantified in asthmatic subjects, COPD and healthy subjects together with the number of IL-17A+ cells as well as the concentration of angiogenic factors in the sputum. The effect of IL-17A on in vitro angiogenesis, cell migration and endothelial permeability was assessed directly on primary human lung microvascular endothelial cells (HMVEC-L) or indirectly with conditioned medium derived from normal bronchial epithelial cells (NHBEC), fibroblasts (NHBF) and airway smooth muscle cells (ASMC) after IL-17A stimulation. RESULTS: Severe asthmatics have increased vascularity compared to the other groups, which correlates positively with the concentrations of angiogenic factors in sputum. Interestingly, we demonstrated that increased bronchial vascularity correlates positively with the number of subepithelial IL-17A+ cells. However IL-17A had no direct effect on HMVEC-L function but it enhanced endothelial tube formation and cell migration through the production of angiogenic factors by NHBE and ASMC. CONCLUSIONS & CLINICAL RELEVANCE: Our results shed light on the role of IL-17A in vascular remodelling, most likely through stimulating the synthesis of other angiogenic factors. Knowledge of these pathways may aid in the identification of new therapeutic targets.

Human bronchial and parenchymal fibroblasts display differences in basal inflammatory phenotype and response to IL-17A.

BACKGROUND: Chronic inflammation, typified by increased expression of IL-17A, together with airway and parenchymal remodelling are features of chronic lung diseases. Emerging evidence suggests that phenotypic heterogeneity of repair and inflammatory capacities of fibroblasts may contribute to the differential structural changes observed in different regions of the lung. OBJECTIVE: To investigate phenotypic differences in parenchymal and bronchial fibroblasts, either in terms of inflammation and remodelling or the ability of these fibroblasts to respond to IL-17A. METHODS: Four groups of primary fibroblasts were used: normal human bronchial fibroblast (NHBF), normal human parenchymal fibroblast (NHPF), COPD human bronchial fibroblast (CHBF) and COPD human parenchymal fibroblast (CHPF). Cytokine and extracellular matrix (ECM) expression were measured at baseline and after stimulation with IL-17A. Actinomycin D was used to measure cytokine mRNA stability. RESULTS: At baseline, we observed higher protein production of IL-6 in NHPF than NHBF, but higher levels of IL-8 and GRO-α in NHBF. IL-17A induced a higher expression of GRO-α (CXCL1) and IL-6 in NHPF than in NHBF, and a higher level of IL-8 expression in NHBF. IL-17A treatment decreased the mRNA stability of IL-6 in NHBF when compared with NHPF. CHPF expressed higher protein levels of fibronectin, collagen-I and collagen-III than CHBF, NHBF and NHPF. IL-17A increased fibronectin and collagen-III protein only in NHPF and collagen-III protein production in CHBF and CHPF. CONCLUSIONS AND CLINICAL RELEVANCE: These findings provide insight into the inflammatory and remodelling processes that may be related to the phenotypic heterogeneity of fibroblasts from airway and parenchymal regions and in their response to IL-17A.

Monocyte-derived fibrocytes induce an inflammatory phenotype in airway smooth muscle cells.

BACKGROUND: Infiltration of fibrocytes (FC) in the airway smooth muscle is a feature of asthma, but the pathological significance is unknown. OBJECTIVE: We sought to explore whether FC modulate the phenotype of airway smooth muscle cells (ASMC) in asthmatic vs. control subjects. METHODS: Fibrocytes were isolated from CD14+ monocytes from asthmatic and normal subjects. Proliferation of ASMC of asthmatic or normal subjects was analysed by (3) H-thymidine incorporation, cell number counting and Ki-67 expression after treatment of ASMC with FC-conditioned medium (FCCM) or co-culture with FC. ASMC-associated cytokines/chemokines implicated in asthma (TGF-ß1, eotaxin, IL-6 and IL-8) were measured in co-culture or transwell culture of ASMC + FC by ELISA. Immunofluorescence staining was performed to localize these cytokines in ASMC. Cytokine secretion was measured in the transwell culture of ASMC + FC, where NF-κB-p65 or ERK1/2 in ASMC was silenced by siRNA. Contractile phenotype of ASMC in transwell culture was assessed by immunoblotting of α-smooth muscle actin (α-SMA) and myosin light chain kinase (MLCK). RESULTS: Fibrocytes did not affect ASMC proliferation and expression of TGF-ß1, eotaxin, α-SMA and MLCK; however, ASMC production of IL-8 and IL-6 was increased in the co-culture and transwell culture by FC. ASMC treated with FCCM were immunopositive for IL-8/IL-6 and produced more IL-8/IL-6. Furthermore, siRNA silencing of NF-κB-p65 or ERK1/2 in transwell cultures of asthmatic ASMC with normal subject FC decreased IL-8 and IL-6 production. CONCLUSIONS AND CLINICAL RELEVANCE: Fibrocytes promoted IL-8 and IL-6 production by ASMC, demonstrating a proinflammatory role for FC and a possible mechanism of the inflammatory phenotype in asthma.

Pathogenesis of severe asthma.

Patients with severe asthma have asthma symptoms which are difficult to control, require high dosages of medication, and continue to experience persistent symptoms, asthma exacerbations or airflow obstruction. Epidemiological and clinical evidences point to the fact that severe asthma is not a single phenotype. Cluster analyses have identified subclasses of severe asthma using parameters such as patient characteristics, and cytokine profiles have also been useful in classifying moderate and severe asthma. The IL-4/IL-13 signalling pathway accounts for the symptoms experienced by a subset of severe asthmatics with allergen-associated symptoms and high serum immunoglobulin E (IgE) levels, and these patients are generally responsive to anti-IgE treatment. The IL-5/IL-33 signalling pathway is likely to play a key role in the disease pathogenesis of those who are resistant to high doses of inhaled corticosteroid but responsive to systemic corticosteroids and anti-IL5 therapy. The IL-17 signalling pathway is thought to contribute to 'neutrophilic asthma'. Although traditionally viewed as players in the defence mechanism against viral and intracellular bacterial infection, mounting evidence supports a role for Th1 cytokines such as IL-18 and IFN-γ in severe asthma pathogenesis. Furthermore, these cytokine signalling pathways interact to contribute to the spectrum of clinical pathological outcomes in severe asthma. To date, glucocorticoids are the most effective anti-asthma drugs available, yet severe asthma patients are typically resistant to the effects of glucocorticoids. Glucocorticoid receptor dysfunction and histone deacetylase activity reduction are likely to contribute to glucocorticoid resistance in severe asthma patients. This review discusses recent development in different cytokine signalling pathways, their interactions and steroid resistance, in the context of severe asthma pathogenesis.

Induction of glucocorticoid receptor-beta expression in epithelial cells of asthmatic airways by T-helper type 17 cytokines.

BACKGROUND: Corticosteroid insensitivity in asthmatics is associated with an increased expression of glucocorticoid receptor-beta (GR-beta) in many cell types. T-helper type 17 (Th17) cytokine (IL-17A and F) expressions increase in mild and in difficult-to-treat asthma. We hypothesize that IL-17A and F cytokines alone or in combination, induce the expression of GR-beta in bronchial epithelial cells. OBJECTIVES: To confirm the expression of the GR-beta and IL-17 cytokines in the airways of normal subjects and mild asthmatics and to examine the effect of cytokines IL-17A and F on the expression of GR-beta in bronchial epithelial cells obtained from normal subjects and asthmatic patients. METHODS: The expression of IL-17A and F, GR-alpha and GR-beta was analysed in bronchial biopsies from mild asthmatics and normal subjects by Q-RT-PCR. Immunohistochemistry for IL-17 and GR-beta was performed in bronchial biopsies from normal and asthmatic subjects. The expression of IL-6 in response to IL-17A and F and dexamethasone was determined by Q-RT-PCR using primary airway epithelial cells from normal and asthmatic subjects. RESULTS: We detected significantly higher levels of IL-17A mRNA expression in the bronchial biopsies from mild asthmatics, compared with normal. GR-alpha expression was significantly lower in the biopsies from asthmatics compared with controls. The expression of IL-17F and GR-beta in biopsies from asthmatics was not significantly different from that of controls. Using primary epithelial cells isolated from normal subjects and asthmatics, we found an increased expression of GR-beta in response to IL-17A and F in the cells from asthmatics (P< or =0.05). This effect was only partially significant in the normal cells. Dexamethasone significantly decreased the IL-17-induced IL-6 expression in cells from normal individuals but not in those from asthmatics (P< or =0.05). CONCLUSION: Evidence of an increased GR-beta expression in epithelial cells following IL-17 stimulation suggests a possible role for Th17-associated cytokines in the mechanism of steroid hypo-responsiveness in asthmatic subjects.

Coenzyme Q10 with multiple vitamins is generally ineffective in treatment of mitochondrial disease.

We followed 16 patients with a variety of mitochondrial diseases over one to four periods of treatment (2 months each) with coenzyme Q10 plus vitamins K3 and C, riboflavin, thiamine, and niacin, using independent measures of oxidative metabolism to assess efficacy. There were large (> threefold) increases in serum coenzyme Q10 concentrations with treatment, but no measure of oxidative metabolism showed significant improvement with treatment for the group, nor did any individual patient show significant, reproducible, objective clinical improvement. The results suggest that coenzyme Q10 plus vitamin therapy does not significantly improve mitochondrial oxidative metabolism in patients with mitochondrial disease in general. Any clinical benefit that may follow from short-term administration appears slight.

Characterization of the anatomical structures involved in the contractile response of the rat lung periphery.

1. When lung parenchymal strips are challenged with different smooth muscle agonists, the tensile and viscoelastic properties change. It is not clear, however, which of the different anatomical elements present in the parenchymal strip, i.e., small vessel, small airway or alveolar wall, contribute to the response. 2. Parenchymal lung strips from Sprague Dawley rats were suspended in an organ bath filled with Krebs solution (37 degrees C, pH = 7.4) bubbled with 95%O2/5%CO2. Resting tension (T) was set at 1.1 g and sinusoidal oscillations of 2.5% resting length (L0) at a frequency of 1 Hz were applied. Following 1 h of stress adaptation, measurements of length (L) and T were recorded under baseline conditions and after challenge with a variety of pharmacological agents, i.e., acetylcholine (ACh), noradrenaline (NA) and angiotensin II (AII). Elastance (E) and resistance (R) were calculated by fitting changes in T, L and delta L/ delta t to the equation of motion. Hysteresivity (eta, the ratio of the energy dissipated to that conserved) was obtained from the equation eta = (R/E)2 pi f. 3. In order to determine whether small airways or small vessels accounted for the responses to the different pharmacologic agents, further studies were carried out in lung explants. Excised lungs from Sprague Dawley rats were inflated with agarose. Transverse slices of lung (0.5-1.0 mm thick) were cultured overnight. By use of an inverted microscope and video camera, airway and vascular lumen area were measured with an image analysis system. 4. NA, ACh and AII constricted the parenchymal strips. Airways constricted after all agonists, vessels constricted only after All. Atropine (Atr) pre-incubation decreased the explanted airway and vessel response to AII, but no difference was found in the parenchymal strip response. 5. Preincubation with the arginine analogue N omega-nitro-L-arginine (L-NOARG) did not modify the response to ACh but mildly increased the oscillatory response to NA after co-preincubation with propranolol (Prop). 6. These results suggest that during ACh and NA challenge, small vessels do not contribute substantially to the parenchymal strip response. The discrepancy between results in airways, vessels and strips when Atr was administered prior to AII implicates a direct contractile response in the parenchymal strip.

Differential responses of pulmonary arteries and veins to histamine and 5-HT in lung explants of guinea-pigs.

1. The mechanisms by which histamine and 5-HT differentially contract pulmonary arteries and veins are unclear. In lung explants from 26 guinea-pigs, we compared responses of pulmonary arteries and vein to histamine, 5-HT and KCI, and examined potential determinants for the differential responses. Lungs were filled with agarose, sectioned into approximately 1 mm thick slices, and vascular luminal areas measured by image analysis. 2. Histamine and 5-HT produced a concentration-dependent constriction in arteries and veins, greater in the latter. KCl constricted arteries and veins equally. 3. The histamine H1 antagonist chlorpheniramine (10(-4) M) abolished contractions to histamine; the H2 antagonist cimetidine enhanced maximal responses and sensitivity of arteries and veins to histamine, and diminished the differences between their maximal responses; the NO synthase inhibitor Nomega-nitro-L-arginine (L-NOARG) increased the maximal responses of arteries and veins, and the differences between their responses; indomethacin had no effect. 4. Contractions to 5-HT were abolished in arteries and markedly reduced in veins by the 5-HT2 antagonist ketanserin (10(-4) M); L-NOARG potentiated the maximal responses of arteries but not of veins; indomethacin increased the maximal responses of arteries but reduced them in veins. 5. By morphometry, arteries had a greater medial thickness and luminal diameter than veins. 6. The data suggest that in guinea-pigs, H2 receptors are responsible for the differential contractile responses of pulmonary arteries and veins to histamine, whereas endothelium-derived vasoactive substances are responsible for their differential contractile responses to 5-HT.

Combination of theophylline and salbutamol for arrhythmias in severe COPD.

We conducted a single-bind placebo controlled study using 24-hour continuous ambulatory electrocardiographic recordings. The arrhythmogenic potential of the combination of salbutamol and theophylline was investigated in 25 ambulatory subjects with severe chronic airflow obstruction (mean age 65 +/- 8 SD, mean FEV1 31 percent +/- 13 SD predicted). Asymptomatic arrhythmias were very prevalent in the study population: 76 percent of the patients had runs of supraventricular tachycardia while 24 percent had runs of ventricular tachycardia. Individual arrhythmia frequency showed greater between-test variability than previously described in non-COPD subjects. The mode of administration of salbutamol may have affected arrhythmia frequency in that subjects using aerosol nebulizers had more ventricular extrasystoles than those using metered dose inhalers. Although the addition of theophylline to salbutamol significantly increased heart rate and supraventricular extrasystoles, there was no statistically significant increase in ventricular arrhythmias.

Mitochondrial disease. Pulmonary function, exercise performance, and blood lactate levels.

Mitochondrial diseases are a heterogeneous group of disorders in which it has been suggested that genetic defects in oxidative phosphorylation lead to specific alterations in exercise performance and lactate metabolism during exercise. To investigate this possibility, we evaluated pulmonary function tests, incremental exercise testing, and serial blood lactate levels in a group of subjects with mitochondrial disease (M) and compared them with a group of patients with nonmitochondrial (N) myopathies and healthy subjects (H). The two groups were demographically comparable and had no significant differences in pulmonary function. Both groups showed similar degrees of reduced exercise tolerance compared with a group of healthy subjects (M: 61.08% predicted VO2max +/- 19.58 SD, n = 13; N: 62.14 +/- 28.89, n = 7; H: 115.17 +/- 19.35, n = 12; p < 0.001). The mitochondrial disease group more frequently showed abnormalities in cardiac response to exercise than did the nonmitochondrial myopathy subjects (M: 12/13, N: 3/7, H: 3/12, p = 0.002). Minute ventilation greater than predicted occurred with similar frequency in both groups. Although resting lactate level was increased in some subjects with mitochondrial myopathy compared with disease controls, there were no differences between groups for peak venous lactate level normalized for oxygen uptake or the rate of lactate clearance. These findings, while confirming the presence of some specific abnormalities in mitochondrial disease, are against the notion that exercise limitation in this condition directly results from specific abnormalities in oxidative metabolism.

Morphometry of methacholine-induced bronchoconstriction in the rat.

It has been suggested that parenchymal viscoelasticity accounts for a large proportion of resistive pressure losses induced by smooth muscle agonists. We used morphometric methods to evaluate the relative contributions of parenchyma and airways to mechanical changes during methacholine- (MCh) induced bronchoconstriction. We measured tracheal pressure and tidal volume in 17 open-chest mechanically ventilated Sprague-Dawley rats (frequency = 1.5 Hz, tidal volume = 2 ml, positive end-expiratory pressure = 5 cmH2O) after saline (n = 7, C), 16 mg/ml MCh (n = 5, L), and 256 mg/ml MCh (n = 5, H) aerosols. We calculated pulmonary resistance (RL) and elastance (EL) by fitting the equation PL = VRL + VEL + K, where PL is transpulmonary pressure, V is flow, and V is volume. V was obtained numerically from V. Lungs were rapidly frozen in liquid N2 30 s after aerosolization and processed with freeze substitution. Using midsagittal slices of left lung, we measured airway narrowing as the ratio of lumen area (aBM) subtended by the basement membrane perimeter (LBM) to predicted maximal lumen area (ABM = LBM2/4 pi). Air space size was estimated as mean linear intercept (Lm) and parenchymal distortion as standard deviation of linear intercepts (SDi). Lm was increased in L and H animals, reflecting hyperinflation; aBM/ABM in 0.17- to 0.43-mm-diam airways decreased progressively from C to L to H animals, reflecting bronchoconstriction. By use of stepwise multiple linear regression, the contribution of Lm to variance in mechanics was found to consistently exceed or equal that of aBM/ABM. SDi contributed little once Lm was taken into account.(ABSTRACT TRUNCATED AT 250 WORDS)

Exponential fitting of pressure-volume curves: confidence limits and sensitivity to noise.

It has recently become common to model the static pressure-volume curve of the lung as a monoexponential function of the form V = A - Be-KP, where V is volume and P is transpulmonary pressure. The parameters A, B, and particularly K have been employed as descriptors of the intrinsic mechanical properties of the lung. The purpose of the present study was to investigate the sensitivities of A, B, and K to noise in measurements of P and V and to incompleteness of data. Using Monte-Carlo simulation, we found that the presence of typical levels of noise in P led to biased estimates of K and that the 95% confidence intervals about A and K were large compared with the parameter values themselves. These effects were increased as points were systematically removed from either end of the data set. These findings show that values of K estimated from PV data are difficult to interpret without accompanying confidence intervals.

Frequency dependence of pulmonary and chest wall mechanics in young and adult cats.

The frequency (f) dependence of pulmonary and chest wall mechanics was assessed in nine kittens and four cats. Kittens and cats were anesthetized, paralyzed, and mechanically ventilated at various f between 0.13 and 1.6 Hz and 0.09 and 0.79 Hz, respectively. Resistance and dynamic compliance pertaining to the respiratory system (Rrs and Cdyn,rs), lungs (RL and Cdyn,L), and chest wall (RW and Cdyn,W) were estimated by fitting a single-compartment model to data obtained from regular ventilation. Static lung and chest wall compliances (Cst,L and Cst,W) were computed from quasi-static pressure-volume data. Lung tissue resistance (Rti) was estimated with alveolar capsules in open-chest animals. The f dependence of the two-compartment viscoelastic model of the respiratory system was assessed by computing the effective resistance [Rmod,rs(omega)] and compliance [Cmod,rs(omega)] from data obtained at the lowest experimental f. Both Cdyn,L and Cdyn,W decreased with increasing f in all animals. Cdyn,L/Cst,L and Cdyn,W/Cst,W were lower in kittens than in cats. RL and RW decreased markedly with f in all animals. Rti/RL showed a marked f dependence, its values being similar in both young and adult cats at their respective resting f. CstW/Cst,L ratio was higher in kittens than in cats. A better agreement was found between Cmod,rs(omega) and Cdyn,rs than between Rmod,rs(omega) and Rrs.

Differential relaxant responses of pulmonary arteries and veins in lung explants of guinea pigs.

The endothelium regulates vascular tone through release of relaxing or contracting factors, with nitric oxide (NO) being a major endothelium-derived relaxing factor. In the present study, we used a lung explant technique to determine the differential abilities and mechanisms of pulmonary arteries and veins of normal guinea pigs to relax after precontraction. Excised lungs of 15 guinea pigs were filled through the airways with 1% agarose, cut into 1-mm-thick slices, and cultured overnight. Luminal areas of vascular cross sections were measured with an image-analysis system. Vessels were precontracted with U-46619, and responses to histamine, acetylcholine (ACh), sodium nitroprusside, and papaverine were examined. We also determined the effects of N omega-nitro-L-arginine and of indomethacin on ACh-induced responses. We found that histamine relaxed arteries more than veins and that ACh relaxed only arteries. N omega-nitro-L-arginine pretreatment abolished ACh-induced relaxation of arteries and caused ACh-induced contraction of veins, whereas indomethacin markedly augmented ACh-induced relaxation of arteries (maximal relaxation: 48.5 +/- 4.7 vs. 19.2 +/- 5.1% without it) and induced a dose-dependent relaxation of veins (maximal relaxation: 17.0 +/- 4.1%). Sodium nitroprusside induced a significantly greater relaxation of arteries than veins, whereas papaverine relaxed them equally. We conclude that in guinea pigs endothelial NO-mediated relaxation is greater in pulmonary arteries than in veins and that ACh-induced NO-mediated relaxation is reduced by the simultaneous production of cyclooxygenase-derived vasoconstrictors.

Transepithelial prostacyclin gradient in isolated canine trachea.

Cyclooxygenase products of arachidonic acid, potential modulators of airway smooth muscle, have recently been described in bronchoalveolar lavage from canine lungs. To evaluate the possibility that airway epithelium represents a barrier to movement of prostacyclin (PGI2), an important bronchodilator synthesized by isolated airway, we measured the concentrations of 6-oxoprostaglandin F1 alpha (6-oxo-PGF1 alpha), the stable degradation product of PGI2, on the mucosal and serosal sides of isolated canine tracheal segments (CTS) mounted in Ussing chambers. 6-oxo-PGF1 alpha was measured by radioimmunoassay after purification by high-performance liquid chromatography. The concentration of 6-oxo-PGF1 alpha was significantly higher on the serosal than the mucosal side of CTS (1,262 +/- 252 vs. 390 +/- 168 pg.min-1.g-1, n = 8, P less than 0.05). A significant correlation was present between 6-oxo-PGF1 alpha measured on both sides of each CTS (r = 0.778, n = 26, P less than 0.01). 6-oxo-PGF1 alpha production from CTS stripped of mucosa was significantly greater than from isolated mucosa. Radiochromatograms obtained after incubation with [3H]arachidonic acid and calcium ionophore A23187 confirmed PGI2 as the predominant cyclooxygenase product of the submucosa, whereas the mucosa produced only small amounts of PGI2 in proportion to other cyclooxygenase products. PGI2 (10(-8) to 10(-6) M) applied to the mucosal surface of closed tracheal segments precontracted with histamine resulted in no significant relaxation, whereas serosal application showed a concentration-dependent effect. Radiolabeled 6-oxo-PGF1 alpha did not cross the isolated epithelium.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of elastase-induced emphysema on airway responsiveness to methacholine in rats.

We examined the effects of elastase-induced emphysema on lung volumes, pulmonary mechanics, and airway responses to inhaled methacholine (MCh) of nine male Brown Norway rats. Measurements were made before and weekly for 4 wk after elastase in five rats. In four rats measurements were made before and at 3 wk after elastase; in these same animals the effects of changes in end-expiratory lung volume on the airway responses to MCh were evaluated before and after elastase. Airway responses were determined from peak pulmonary resistance (RL) calculated after 30-s aerosolizations of saline and doubling concentrations of MCh from 1 to 64 mg/ml. Porcine pancreatic elastase (1 IU/g) was administered intratracheally. Before elastase RL rose from 0.20 +/- 0.02 cmH2O.ml-1.s (mean +/- SE; n = 9) to 0.57 +/- 0.06 after MCh (64 mg/ml). A plateau was observed in the concentration-response curve. Static compliance and the maximum increase in RL (delta RL64) were significantly correlated (r = 0.799, P less than 0.01). Three weeks after elastase the maximal airway response to MCh was enhanced and no plateau was observed; delta RL64 was 0.78 +/- 0.07 cmH2O.ml-1.s, significantly higher than control delta RL64 (0.36 +/- 0.7, P less than 0.05). Before elastase, increase of end-expiratory lung volume to functional residual capacity + 1.56 ml (+/- 0.08 ml) significantly reduced RL at 64 mg MCh/ml from 0.62 +/- 0.05 cmH2O.ml-1.s to 0.50 +/- 0.03, P less than 0.05.(ABSTRACT TRUNCATED AT 250 WORDS)

Respiratory mechanics and lung development in the rat from early age to adulthood.

The purpose of the present study was to establish how the dependence of respiratory mechanics on lung inflation changes during development. We studied seven groups of rats from 10 days to 3 mo of age at five levels of positive end-expiratory pressure (PEEP) from 0 to 7 hPa (1 hPa = 0.1 kPa approximately 1 cmH(2)O). At each PEEP level, we measured respiratory system resistance and elastance at both 0.9 and 4.8 Hz to partition the mechanical properties into its airway and tissue components. Elastance increased more rapidly with PEEP in the younger animals, which we interpret as reflecting a more pronounced strain stiffening of the younger parenchyma. However, the decrease in airway resistance with PEEP was more pronounced in the older animals. Morphometric analysis showed that mean tissue density decreased and total alveolar surface area increased with age. Our data suggest that the mechanical interdependence between airways and parenchyma is weaker in very young animals compared with mature animals. This may play a role in the hyperresponsiveness of immaturity.

Temporal dynamics of acute isovolume bronchoconstriction in the rat.

The time course of lung impedance changes after intravenous injection of bronchial agonist have produced significant insights into the mechanisms of bronchoconstriction in the dog (J. H. T. Bates, A.-M. Lauzon, G. S. Dechman, G. N. Maksym, and T. F. Shuessler. J. Appl. Physiol. 76: 616-626, 1994). We studied the time course of acute induced bronchoconstriction in five anesthetized paralyzed open-chest rats injected intravenously with a bolus of methacholine. For the 16 s immediately after injection, we held the lung volume constant while applying small-amplitude flow oscillations at 1.48, 5.45, and 19.69 Hz simultaneously, which provided us with continuous estimates of lung resistance (RL) and elastance (EL) at each frequency. This procedure was repeated at initial lung inflation pressures of 0.2, 0.4, and 0.6 kPa. Both RL and EL increased progressively after methacholine administration; however, the rate of change of EL increased dramatically as frequency was increased, whereas RL remained relatively independent of frequency. We interpret these findings in terms of a three-compartment model of the rat lung, featuring two parallel alveolar compartments feeding into a central airway compartment. Model simulations support the notions that both central airway shunting and regional ventilation inhomogeneity developed to a significant degree in our constricted rats. We also found that the rates of increase in both RL and EL were greatly enhanced as the initial lung inflation pressure was reduced, in accord with the notion that parenchymal tethering is an important mechanism limiting the extent to which airways can narrow when their smooth muscle is stimulated to contract.

Airway smooth muscle orientation in intraparenchymal airways.

Airway smooth muscle (ASM) shortening is the central event leading to bronchoconstriction. The degree to which airway narrowing occurs as a consequence of shortening is a function of both the mechanical properties of the airway wall as well as the orientation of the muscle fibers. Although the latter is theoretically important, it has not been systematically measured to date. The purpose of this study was to determine the angle of orientation of ASM (theta) in normal lungs by using a morphometric approach. We analyzed the airway tree of the left lower lobes of four cats and one human. All material was fixed with 10% buffered Formalin at a pressure of 25 cmH2O for 48 h. The fixed material was dissected along the airway tree to permit isolation of generations 4-18 in the cats and generations 5-22 in the human specimen. Each airway generation was individually embedded in paraffin. Five-micrometer-thick serial sections were cut parallel to the airway long axis and stained with hematoxylin-phloxine-saffron. Each block yielded three to five sections containing ASM. To determine theta, we measured the orientation of ASM nuclei relative to the transverse axis of the airway by using a digitizing tablet and a light microscope (x250) equipped with a drawing tube attachment. Inspection of the sections revealed extensive ASM crisscrossing without a homogeneous orientation. The theta was clustered between -20 degrees and 20 degrees in all airway generations and did not vary much between generations in any of the cats or in the human specimen. When theta was expressed without regard to sign, the mean values were 13.2 degrees in the cats and 13.1 degrees in the human. This magnitude of obliquity is not likely to result in physiologically important changes in airway length during bronchoconstriction.

Airway-parenchymal interdependence after airway contraction in rat lung explants.

The constriction of pulmonary airways is limited by the tethering effect exerted by parenchymal attachments. To characterize this tethering effect at the scale of intraparenchymal airways, we studied the pattern of parenchymal distortion due to bronchoconstriction in a rat lung explant system. First, we measured the elastic modulus under tension for 2% (wt/vol) agarose alone (37.6 +/- 1.5 kPa) and for agarose-filled lung (5.7 +/- 1.3 kPa). The latter is similar to the elastic modulus of air-filled lung at total lung capacity (4.5-6 kPa) (S. J. Lai-Fook, T. A. Wilson, R. E. Hyatt, and J. R. Rodarte. J. Appl. Physiol. 40: 508-513, 1976), suggesting that explants can be used as a model of lung tissue distortion. Subsequently, confocal microscopic images of fluorescently labeled 0.5-mm-thick explants prepared from agarose-filled rat lungs inflated to total lung capacity (48 ml/kg) were acquired. Images were taken before and after airway constriction was induced by direct application of 10 mM methacholine, and the pattern of parenchymal distortion was measured from the displacement of tissue landmarks identified in each image for 14 explants. The magnitude of the radial component of tissue displacement was calculated as a function of distance from the airway wall and characterized by a parameter, b, describing the rate at which tissue movement decreased with radial distance. The parameter b was 0.994 +/- 0.19 (SE), which is close to the prediction of b = 1 of micromechanical modeling (T. A. Wilson. J. Appl. Physiol. 33: 472-478, 1972). There was significant variability in b, however, which was correlated with the fractional reduction in airway diameter (r = 0.496). Additionally, parenchymal distortion showed significant torsion with respect to the radial direction. This torsion was similar in concentric zones around the airway, suggesting that it originates from inhomogeneity in the parenchyma rather than inhomogeneous airway constriction. Our results demonstrate the significance of the nonlinear mechanical properties of alveolar walls and the anisotropy of the parenchyma in determining the nature of airway-parenchymal interdependence.