Ultrastructure of lung elastin and collagen in mouse models of spontaneous emphysema.

The tight-skin (Tsk) and beige (bg) mutants of the C57B1/6J strain of mouse spontaneously develop air-space enlargement reminiscent of human emphysema. To determine if this enlargement is accompanied by matrix destruction, as in the human disease, we examined the elastin and collagen matrices of the lungs of both mutants. The ultrastructure of these matrix components was separately visualized by scanning electron microscopy following controlled alkali digestion, which preserves collagen, and formic acid digestion, which enables visualization of elastin. Significant elastin destruction suggestive of an elastolytic process was observed in the lungs of Tsk mice. Thickening of elastin lamellae was observed in the lungs of bg mice, suggesting that congenital matrix remodeling may underlie air-space enlargement in this strain.     

Elastin dehydration through the liquid and the vapor phase: A comparison of osmotic stress models

The swelling and viscoelastic behaviors of samples of purified arterial elastin were investigated to develop a model for studying the viscoelastic behavior of elastin. Two osmotic stress models were used: the vapor phase model (VPM), in which the stress on the elastin sample was applied through the vapor phase by equilibrating the sample over a saline solution, and the liquid phase model (LPM), in which the stress was applied through the liquid phase by equilibrating the sample in aqueous solutions of large molecular weight polymers. The elastin in the VPM showed a highly varied viscoelastic response, and was slightly stiffer and had a slightly higher damping coefficient than the elastin in the LPM at equivalent nominal relative humidities. We believe the difference in behavior of the elastin in the two models was due to geometric distortions of the elastin that occur during dehydration in the VPM. In the LPM, the spaces between the elastin fibrils are filled with water, and in the VPM these spaces collapse when the water is removed. Removal of only the interfibrillar water deswelled the tissue and increased its stiffness and damping coefficient. Viscoelastic spectra obtained at different levels of osmotic stress in the LPM were reducible to one master curve, indicating that the dominant effect of dehydration is a nonspecific reduction of molecular mobility. We conclude that the LPM is a better model than the VPM for studying the effects of dehydration on the mechanical behavior of elastin. © 1996 John Wiley & Sons, Inc.     

Impact of microvascular circulation on peripheral lung stability

The involvement of pulmonary circulation in the mechanical properties was studied in isolated rat lungs. Pulmonary input impedance (ZL) was measured at a mean transpulmonary pressure (Ptpmean) of 2 cmH2O before and after physiological perfusion with either blood or albumin. In these lungs and in a group of unperfused lungs, ZL was also measured at Ptpmean values between 1 and 8 cmH2O. Airway resistance (Raw) and parenchymal damping (G) and elastance (H) were estimated from ZL. End-expiratory lung volume (EELV) was measured by immersion before and after blood perfusion. The orientation of the elastin fibers relative to the basal membrane was assessed in additional unperfused and blood-perfused lungs. Pressurization of the pulmonary capillaries significantly decreased H by 31.5 +/- 3.7% and 18.7 +/- 2.7% for blood and albumin, respectively. Perfusion had no effect on Raw but markedly altered the Ptpmean dependences of G and H < 4 cmH2O, with significantly lower values than in the unperfused lungs. At a Ptpmean of 2 cmH2O, EELV increased by 31 +/- 11% (P = 0.01) following pressurization of the capillaries, and the elastin fibers became more parallel to the basal membrane. Because the organization of elastin fibers results in smaller H values of the individual alveolus, the higher H in the unperfused lungs is probably due to a partial alveolar collapse leading to a loss in lung volume. We conclude that the physiological pressure in the pulmonary capillaries is an important mechanical factor in the maintenance of the stability of the alveolar architecture.     

Aerosol-derived lung morphometry: comparisons with a lung model and lung function indexes.

This study evaluated the ability of aerosol-derived lung morphometry to noninvasively probe airway and acinar dimensions. Effective air-space diameters (EAD) were calculated from the time-dependent gravitational losses of 1-microns particles from inhaled aerosol boluses during breath holding. In 17 males [33 +/- 7 (SD) yr] the relationship between EAD and volumetric penetration of the bolus into the lungs (Vp) could be expressed by the linear power-law function, log (EAD) alpha beta log (Vp). Our EAD values were consistent with Weibel's symmetric lung model A for small airways and more distal air spaces. As lung volume increased from 57 to 87% of total lung capacity (TLC), EAD at Vp of 160 and 550 cm3 increased 70 and 41%, respectively. At 57% TLC, log (EAD) at 160 cm3 was significantly correlated with airway resistance (r = -0.57, P less than 0.0204) but not with forced expired flow between 25 and 75% of vital capacity. Log (EAD) at 400 cm3 was correlated with deposition of 1-micron particles (r = -0.73, P less than 0.0009). We conclude that aerosol-derived lung morphometry is a responsive noninvasive probe of peripheral air-space diameters.     

The distribution of water in arterial elastin: Effects of mechanical stress,osmotic pressure,and temperature

Using gravimetric and radiotracer techniques, we investigated the effects of mechanical stress, osmotic pressure, and temperature on the volumes of the intra- and extrafibrillar water spaces in arterial elastin. We also investigated the effects of temperature on water flow through elastin membranes and on dynamic mechanical properties of elastin rings. Compression by mechanical or osmotic loading reduced the hydration of the elastin in an identical manner. Two distinct stages were evident; at low loads there was extensive water removal from the extrafibrillar space while high loads were required to remove water from the intrafibrillar space. Conversely, dehydration caused by mechanical extension of the matrix was associated with a much smaller loss from the extrafibrillar compartment and a large fractional decrease in the intrafibrillar space. Contraction of the matrix as a result of increased temperature had similar effects on hydration to those produced by extension. Water flux across elastin membranes, corrected for changes in viscosity, and specific hydraulic conductivity both increased as a result of temperature-induced contraction. This effect was attributed to increases in both the fractional volume of the extrafibrillar space and the fiber radius. The elastic modulus decreased with increasing temperature, but there was an increase in viscoelasticity. Previous studies have determined that viscoelasticity depends on the rate of redistribution of intrafibrillar water, so this finding provides additional evidence that heating affects primarily the volume of the intrafibrillar space. © 1995 John Wiley & Sons, Inc.     

Mechanical ventilation uncouples synthesis and assembly of elastin and increases apoptosis in lungs of newborn mice. Prelude to defective alveolar septation during lung development?

Prolonged mechanical ventilation (MV) with O2-rich gas inhibits lung growth and causes excess, disordered accumulation of lung elastin in preterm infants, often resulting in chronic lung disease (CLD). Using newborn mice, in which alveolarization occurs postnatally, we designed studies to determine how MV with either 40% O2 or air might lead to dysregulated elastin production and impaired lung septation. MV of newborn mice for 8 h with either 40% O2 or air increased lung mRNA for tropoelastin and lysyl oxidase, relative to unventilated controls, without increasing lung expression of genes that regulate elastic fiber assembly (lysyl oxidase-like-1, fibrillin-1, fibrillin-2, fibulin-5, emilin-1). Serine elastase activity in lung increased fourfold after MV with 40% O2, but not with air. We then extended MV with 40% O2 to 24 h and found that lung content of tropoelastin protein doubled, whereas lung content of elastin assembly proteins did not change (lysyl oxidases, fibrillins) or decreased (fibulin-5, emilin-1). Quantitative image analysis of lung sections showed that elastic fiber density increased by 50% after MV for 24 h, with elastin distributed throughout the walls of air spaces, rather than at septal tips, as in control lungs. Dysregulation of elastin was associated with a threefold increase in lung cell apoptosis (TUNEL and caspase-3 assays), which might account for the increased air space size previously reported in this model. Our findings of increased elastin synthesis, coupled with increased elastase activity and reduced lung abundance of proteins that regulate elastic fiber assembly, could explain altered lung elastin deposition, increased apoptosis, and defective septation, as observed in CLD.     

Shrinkage of lung after chemical fixation for analysis of pulmonary structure-function relations

Glutaraldehyde is widely used to chemically fix lungs for analysis of pulmonary structure-function relations. Accurate interpretation of observations on fixed tissue requires a clear definition of any artifacts, such as tissue shrinkage, resulting from fixation with glutaraldehyde. Two experimental procedures were used in this study to examine possible shrinkage artifacts resulting from fixation of lung by glutaraldehyde. In the first, isolated perfused dog lungs were rapidly frozen at different transpulmonary pressures. Samples were then freeze substituted at -50 degrees C using 70% ethylene glycol with and without fixatives present. In the second series of experiments, the left lungs of mongrel dogs were fixed by vascular perfusion with glutaraldehyde at different transpulmonary pressures. In both series of experiments any changes in linear dimensions resulting from the fixation procedure were measured. Also, the presence of aldehyde was demonstrated by a positive reaction with Schiff reagent. The results demonstrate that lung tissue fixed either by vascular perfusion or freeze substitution tends to shrink to about the same extent. This shrinkage is reasonably constant at about 9% for transpulmonary pressures of 5 and 15 cmH2O and increases to about 15% when the transpulmonary pressure reaches 25 cmH20.     

Dysregulation of pulmonary elastin synthesis and assembly in preterm lambs with chronic lung disease

Failed alveolar formation and excess, disordered elastin are key features of neonatal chronic lung disease (CLD). We previously found fewer alveoli and more elastin in lungs of preterm compared with term lambs that had mechanical ventilation (MV) with O(2)-rich gas for 3 wk (MV-3 wk). We hypothesized that, in preterm more than in term lambs, MV-3 wk would reduce lung expression of growth factors that regulate alveolarization (VEGF, PDGF-A) and increase lung expression of growth factors [transforming growth factor (TGF)-alpha, TGF-beta(1)] and matrix molecules (tropoelastin, fibrillin-1, fibulin-5, lysyl oxidases) that regulate elastin synthesis and assembly. We measured lung expression of these genes in preterm and term lambs after MV for 1 day, 3 days, or 3 wk, and in fetal controls. Lung mRNA for VEGF, PDGF-A, and their receptors (VEGF-R2, PDGF-Ralpha) decreased in preterm and term lambs after MV-3 wk, with reduced lung content of the relevant proteins in preterm lambs with CLD. TGF-alpha and TGF-beta(1) expression increased only in lungs of preterm lambs. Tropoelastin mRNA increased more with MV of preterm than term lambs, and expression levels remained high in lambs with CLD. In contrast, fibrillin-1 and lysyl oxidase-like-1 mRNA increased transiently, and lung abundance of other elastin-assembly genes/proteins was unchanged (fibulin-5) or reduced (lysyl oxidase) in preterm lambs with CLD. Thus MV-3 wk reduces lung expression of growth factors that regulate alveolarization and differentially alters expression of growth factors and matrix proteins that regulate elastin assembly. These changes, coupled with increased lung elastase activity measured in preterm lambs after MV for 1-3 days, likely contribute to CLD.     

Analysis of stress distribution in the alveolar septa of normal and simulated emphysematic lungs.

The alveolar septum consists of a skeleton of fine collagen and elastin fibers, which are interlaced with a capillary network. Its mechanical characteristics play an important role in the overall performance of the lung. An alveolar sac model was developed for numerical analysis of the internal stress distribution and septal displacements within the alveoli of both normal and emphysematic saline-filled lungs. A scanning electron micrograph of the parenchyma was digitized to yield a geometric replica of a typical two-dimensional alveolar sac. The stress-strain relationship of the alveolar tissue was adopted from experimental data. The model was solved by using commercial finite-element software for quasi-static loading of alveolar pressure. Investigation of the state of stresses and displacements in a healthy lung simulation yielded values that compared well with experimentally reported data. Alteration of the mechanical characteristics of the alveolar septa to simulate elastin destruction in the emphysematic model induced significant stress concentrations (e.g., at a lung volume of 60% total capacity, tensions at certain parts in an emphysematic lung were up to 6 times higher than those in a normal lung). The combination of highly elevated stress sites together with the cyclic loading of breathing may explain the observed progressive damage to elastin fibers in emphysematic patients.     

The Effect of Static Stretch on Elastin Degradation in Arteries

Previously we have shown that gradual changes in the structure of elastin during an elastase treatment can lead to important transition stages in the mechanical behavior of arteries [1]. However, in vivo arteries are constantly being loaded due to systolic and diastolic pressures and so understanding the effects of loading on the enzymatic degradation of elastin in arteries is important. With biaxial tensile testing, we measured the mechanical behavior of porcine thoracic aortas digested with a mild solution of purified elastase (5 U/mL) in the presence of a static stretch. Arterial mechanical properties and biochemical composition were analyzed to assess the effects of mechanical stretch on elastin degradation. As elastin is being removed, the dimensions of the artery increase by more than 20% in both the longitude and circumference directions. Elastin assays indicate a faster rate of degradation when stretch was present during the digestion. A simple exponential decay fitting confirms the time constant for digestion with stretch (0.11±0.04 h⁻¹) is almost twice that of digestion without stretch (0.069±0.028 h⁻¹). The transition from J-shaped to S-shaped stress vs. strain behavior in the longitudinal direction generally occurs when elastin content is reduced by about 60%. Multiphoton image analysis confirms the removal/fragmentation of elastin and also shows that the collagen fibers are closely intertwined with the elastin lamellae in the medial layer. After removal of elastin, the collagen fibers are no longer constrained and become disordered. Release of amorphous elastin during the fragmentation of the lamellae layers is observed and provides insights into the process of elastin degradation. Overall this study reveals several interesting microstructural changes in the extracellular matrix that could explain the resulting mechanical behavior of arteries with elastin degradation.     

Induction of the myofibroblast phenotype following elastolytic injury to mouse lung

The repair of alveolar structures following endotracheal administration of porcine pancreatic elastase (PPE) to mice involves the coordinated deposition of new matrix elements. We determined the induction of the myofibroblast phenotype following elastolytic injury to mouse lung by examining the expression of α-smooth muscle actin (α-SMA) by immunohistochemistry. We also examined elastin and α1(I) collagen mRNA expression by in situ hybridization. Changes in airspace dimensions were assessed by determining mean linear intercept. In untreated mice, α-SMA was localized to vascular structures and large airways, with no detectable expression in alveolar units. PPE induced α-SMA expression in damaged areas surrounding large vessels, in septal remnants, and in the opening ring of alveolar ducts. Elastin and α1(I) collagen mRNA expression were up-regulated in residual alveolar structures and septal walls. PPE dose-response studies indicated that α1(I) collagen and elastin mRNA expression were not induced in areas of normal lung adjacent to damaged lung. The administration of low dose PPE resulted in increased α-SMA protein and elastin mRNA expression in the cells comprising the opening ring of alveolar ducts. Our data suggest that repair mechanisms following elastolytic injury are confined to overtly damaged alveolar structures and involve the induction of the myofibroblast phenotype.     

Effect of temperature on the biaxial mechanics of excised lung parenchyma of the dog.

The influence of temperature on the mechanical properties of excised saline-filled lung parenchyma of the dog was studied at low lung volume. The motivation of this study was to determine whether lung tissue material without the influence of surface tension undergoes a phase transition in the 20-40 degrees C range, as does synthetic elastin studied by Urry in 1984-1986. Dynamic biaxial and uniaxial tensile tests were done, and strain vs. Lagrangian stress curves were recorded during slow cooling and heating between 40 and 10 degrees C. To emphasize the effects of elastin, strains (defined as stretch ratio minus one) were kept below 30%. A slight decrease in compliance occurred with cooling over the entire temperature range. This effect may be attributed to collagen. It was accompanied by a gradual increase in length as the tissue cooled, an effect that may be attributed to elastin. This process was partially reversible with reheating. However, this effect is in contrast with the sudden drastic change in mechanical properties of synthetic elastin described by Urry. Hysteresis, creep, and stress relaxation were small at these low strains. Possible causes of these effects are discussed.     

Connecting incremental shear modulus and Poisson's ratio of lung tissue with morphology and rheology of microstructure

The width and curvature of the collagen and elastin fiber bundles in the human pulmonary interalveolar septa and alveolar mouths are measured. The data, together with the known mechanical properties of collagen and elastin fibers, are used to derive the incremental elastic moduli of the lung tissue. The constitutive equation for small incremental stress and strain superposed on a homeostatic inflated lung is linear and isotropic, and characterized by two material constants.     

Distribution of elastin and collagen in canine lung alveolar parenchyma

We have quantified the fibrous collagen (predominantly type I) and elastin in four locations of perceived mechanical importance: one quasi-planar feature, the alveolar septum or wall (W), and three linear features, the junction (J) of three septa, the free edges (E) of septa, and the line along which two septa join at a distinct angle or bend (B). The frequencies of these four features on light micrographs and the areas of transections through collagen and elastin seen on electron micrographs were combined to give the volumes of collagen and elastin within each feature. We find that E and B have similar compositions and contain most (4/5) of the parenchymal elastin in their relatively heavy cables. The E and B are interconnected and similar in location and composition, and they may constitute a functional entity in which elastin provides tension over a range of lung volumes, opposing septal tensions. In J and W, elastin is typically sparse and fine. Calculations, however, suggest it contributes the dominant portion of septal tension at lower lung volumes. Elastin may be essential to stabilizing septal configuration. Collagen, on the other hand, is distributed relatively evenly throughout E, B, J, and W, consistent with the role of protecting all components against rupture.     

Mechanical interactions between collagen and proteoglycans: implications for the stability of lung tissue.

Collagen and elastin are thought to dominate the elasticity of the connective tissue including lung parenchyma. The glycosaminoglycans on the proteoglycans may also play a role because osmolarity of interstitial fluid can alter the repulsive forces on the negatively charged glycosaminoglycans, allowing them to collapse or inflate, which can affect the stretching and folding pattern of the fibers. Hence, we hypothesized that the elasticity of lung tissue arises primarily from 1) the topology of the collagen-elastin network and 2) the mechanical interaction between proteoglycans and fibers. We measured the quasi-static, uniaxial stress-strain curves of lung tissue sheets in hypotonic, normal, and hypertonic solutions. We found that the stress-strain curve was sensitive to osmolarity, but this sensitivity decreased after proteoglycan digestion. Images of immunofluorescently labeled collagen networks showed that the fibers follow the alveolar walls that form a hexagonal-like structure. Despite the large heterogeneity, the aspect ratio of the hexagons at 30% uniaxial strain increased linearly with osmolarity. We developed a two-dimensional hexagonal network model of the alveolar structure incorporating the mechanical properties of the collagen-elastin fibers and their interaction with proteoglycans. The model accounted for the stress-strain curves observed under all experimental conditions. The model also predicted how aspect ratio changed with osmolarity and strain, which allowed us to estimate the Young's modulus of a single alveolar wall and a collagen fiber. We therefore identify a novel and important role for the proteoglycans: they stabilize the collagen-elastin network of connective tissues and contribute to lung elasticity and alveolar stability at low to medium lung volumes.     

A comparison of fixation methods on lung morphology in a murine model of emphysema

Emphysema is characterized by enlargement of the alveoli, which is the most important parameter to assess the presence and severity of this disease. Alveolar enlargement is primarily defined on morphological criteria; therefore, characterization of this disease with morphological parameters is a prerequisite to study the pathogenesis. For this purpose, different methods of lung fixation were evaluated in a murine model of LPS-induced lung emphysema. Five different methods of lung fixation were evaluated: intratracheal instillation of fixatives, in situ fixation, fixed-volume fixation, vascular whole body perfusion, and vacuum inflation. In addition, the effects of three different fixatives (10% formalin, Carnoy's, and agarose/10% formalin solution) and two embedding methods (paraffin and plastic) were investigated on the murine lung morphology. Mice received intranasal administration of LPS to induce alveolar wall destruction. Quantification of air space enlargement was determined by mean linear intercept analysis, and the histological sections were analyzed for the most optimal fixation method. Additionally, routine immunohistological staining was performed on lung tissue of PBS-treated mice. Intratracheal instillation of formalin or agarose/formalin solution, in situ fixation, and fixed-volume fixation provided a normal lung architecture, in contrast to the lungs fixed via whole body perfusion and vacuum inflation. Formalin-fixed lungs resulted in the most optimal lung morphology for lung emphysema analysis when embedded in paraffin, while for Carnoy's fixed lungs, plastic embedding was preferred. The histological findings, the mean linear intercept measurement, and the immunohistochemistry data demonstrated that fixation by intratracheal instillation of 10% formalin or in situ fixation with 10% formalin are the two most optimal methods to fix lungs for alveolar enlargement analysis to study lung emphysema.     

Elastin protein levels are a vital modifier affecting normal lung development and susceptibility to emphysema

Cigarette smoking is the strongest risk factor for emphysema. However, sensitivity to cigarette smoke-induced emphysema is highly variable, and numerous genetic and environmental factors are thought to mitigate lung response to injury. We report that the quantity of functional elastin in the lung is an important modifier of both lung development and response to injury. In mice with low levels of elastin, lung development is adversely affected, and mice manifest with congenital emphysema. Animals with intermediate elastin levels exhibit normal alveolar structure but develop worse emphysema than normal mice following cigarette smoke exposure. Mechanical testing demonstrates that lungs with low levels of elastin experience greater tissue strains for any given tissue stress compared with wild-type lungs, implying that force-mediated propagation of lung injury through alveolar wall failure may worsen the emphysema after an initial enzymatic insult. Our findings suggest that quantitative deficiencies in elastin predispose to smoke-induce emphysema in animal models and suggest that humans with altered levels of functional elastin could have relatively normal lung function while being more susceptible to smoke-induced lung injury.     

Recombinant human elafin promotes alveologenesis in newborn mice exposed to chronic hyperoxia

Background/aimsElastase inhibitors reverse elastin degradation and abnormal alveologenesis and attenuate the lung structural abnormalities induced by mechanical ventilation with O₂-rich gas. The potential of these molecules to improve endothelial function and to ameliorate severe bronchopulmonary dysplasia (BPD) during lung development is not yet understood. We sought to determine whether the intratracheal treatment of newborn mice with the elastase inhibitor elafin would prevent hyperoxia-induced lung elastin degradation and the cascade of events that cause abnormal alveologenesis.MethodsNewborn mice were exposed to 85% O₂ for 3, 7, 14 or 21 days. Recombinant human elafin was administered by intratracheal instillation from the first day every two days for 20 days. We next used morphometric analyses, quantitative RT-PCR, immunostaining, Western blotting, and ELISA methods to assess the key variables involved in elastogenesis disruption and the potential signaling pathways noted below in recombinant human elafin-treated mouse pups that had been exposed to 85% O₂.ResultsWe found that impaired alveolar development and aberrant elastin production were associated with elevations in whole lung elastase levels in 85% O₂-exposed lungs. Elafin attenuated the structural disintegration that developed in the hyperoxia-damaged lungs. Furthermore, elafin prevented the elastin degradation, neutrophil influx, activation of TGF-β1 and apoptosis caused by 85% O₂ exposure.ConclusionsPulmonary elastase plays an important role in disrupting elastogenesis during O₂-induced damage, which is the result of a pulmonary inflammatory response. Elafin prevents these changes by inhibiting elastase and the TGF-β1 signalling cascade and may be a new therapeutic target for preventing O₂-induced lung injury in neonates.     

Human variation in the peripheral air-space deposition of inhaled particles

Intersubject variability in both peripheral air-space dimensions and breathing pattern [tidal volume (VT) and respiratory frequency (f)] may play a role in determining intersubject variation in the fractional deposition of inhaled particles that primarily deposit in the lung periphery (i.e., distal to conducting airways). In healthy subjects breathing spontaneously at rest, we measured the deposition fraction (DF) of a 2.6-microns monodisperse aerosol by Tyndallometry while simultaneous measurement of VT and f were made. Under these conditions particle deposition occurs primarily in the peripheral air spaces of the lung. As an index of peripheral air-space size, we used measurements of aerosol recovery (RC) as a function of breath-hold time (t) (Gebhart et al. J. Appl. Physiol. 51: 465-476, 1981). In each subject, we measured RC (aerosol expired/aerosol inspired) of a 1.0-micron monodisperse aerosol as a function of breath-hold time for inspiratory capacity breaths of aerosol. The half time (t1/2) (the breath-hold time to reach 50% RC with no breath hold) is proportional to a mean diameter (D) of air spaces filled with aerosol. In the 10 subjects studied, we found a variable DF, range 0.04-0.44 [0.25 +/- 0.12 (SD)]. DF correlated most closely with 1/f, or the period of breathing (r = 0.96, P less than 0.01). There was no significant correlation between DF and t1/2 as an index of peripheral air-space size. In fact there was little deviation in t1/2 in these normal subjects [coefficient of variation (CV) = 0.12].(ABSTRACT TRUNCATED AT 250 WORDS)     

Extracellular matrix proteins: A positive feedback loop in lung fibrosis?

Lung fibrosis is characterized by excessive deposition of extracellular matrix. This not only affects tissue architecture and function, but it also influences fibroblast behavior and thus disease progression. Here we describe the expression of elastin, type V collagen and tenascin C during the development of bleomycin-induced lung fibrosis. We further report in vitro experiments clarifying both the effect of myofibroblast differentiation on this expression and the effect of extracellular elastin on myofibroblast differentiation.Lung fibrosis was induced in female C57Bl/6 mice by bleomycin instillation. Animals were sacrificed at zero to five weeks after fibrosis induction. Collagen synthesized during the week prior to sacrifice was labeled with deuterium. After sacrifice, lung tissue was collected for determination of new collagen formation, microarray analysis, and histology. Human lung fibroblasts were grown on tissue culture plastic or BioFlex culture plates coated with type I collagen or elastin, and stimulated to undergo myofibroblast differentiation by 0–10 ng/ml transforming growth factor (TGF)β₁. mRNA expression was analyzed by quantitative real-time PCR.New collagen formation during bleomycin-induced fibrosis was highly correlated to gene expression of elastin, type V collagen and tenascin C. At the protein level, elastin, type V collagen and tenascin C were highly expressed in fibrotic areas as seen in histological sections of the lung. Type V collagen and tenascin C were transiently increased. Human lung fibroblasts stimulated with TGFβ₁ strongly increased gene expression of elastin, type V collagen and tenascin C. The extracellular presence of elastin increased gene expression of the myofibroblastic markers α smooth muscle actin and type I collagen.The extracellular matrix composition changes dramatically during the development of lung fibrosis. The increased levels of elastin, type V collagen and tenascin C are probably the result of increased expression by fibroblastic cells; reversely, elastin influences myofibroblast differentiation. This suggests a reciprocal interaction between fibroblasts and the extracellular matrix composition that could enhance the development of lung fibrosis.