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.     

Cell-specific expression of human HGF by alveolar type II cells induces remodeling of septal wall tissue in the lung: a morphometric study

Hepatocyte growth factor (HGF) is involved in development and regeneration of the lungs. Human HGF, which was expressed specifically by alveolar epithelial type II cells after gene transfer, attenuated the bleomycin-induced pulmonary fibrosis in an animal model. As there are also regions that appear morphologically unaffected in fibrosis, the effects of this gene transfer to normal lungs is of interest. In vitro studies showed that HGF inhibits the formation of the basal lamina by cultured alveolar epithelial cells. Thus we hypothesized that, in the healthy lung, cell-specific expression of HGF induces a remodeling within septal walls. Electroporation of a plasmid of human HGF gene controlled by the surfactant protein C promoter was applied for targeted gene transfer. Using design-based stereology at light and electron microscopic level, structural alterations were analyzed and compared with a control group. HGF gene transfer increased the volume of distal air spaces, as well as the surface area of the alveolar epithelium. The volume of septal walls, as well as the number of alveoli, was unchanged. Volumes per lung of collagen and elastic fibers were unaltered, but a marked reduction of the volume of residual extracellular matrix (all components other than collagen and elastic fibers) and interstitial cells was found. A correlation between the volumes of residual extracellular matrix and distal air spaces, as well as total surface area of alveolar epithelium, could be established. Cell-specific expression of HGF leads to a remodeling of the connective tissue within the septal walls in the healthy lung, which is associated with more pronounced stretching of distal air spaces at a given hydrostatic pressure during instillation fixation.     

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.     

Comparison of rat and mouse pulmonary tissue mechanical properties and histology.

The present study compares the dynamic mechanical properties and the contents of collagen and elastic fibers (oxytalan + elaunin + fully developed elastic fibers) of mice and rat lung strips. Resistance, elastance (E), and hysteresivity (eta) were obtained during sinusoidal oscillations. The relative amounts of blood vessel, bronchial, and alveolar walls, as well as the mean alveolar diameter were determined. In both species, resistance had a negative and E a positive dependence on frequency, whereas eta remained unchanged. Mice showed higher E and lower eta than rats. Although collagen and elastic fiber contents were similar in both groups, mice had more oxytalan and less elaunin and fully developed elastic fibers than rats. Rats showed less alveolar and more blood vessel walls and higher mean alveolar diameter than mice. In conclusion, mice and rats present distinct tissue mechanical properties, which are accompanied by specific extracellular fiber composition.     

Collagen and elastin fibers in human pulmonary alveolar walls

The morphology and morphometric data of collagen and elastin fibers in the pulmonary alveolar walls are presented. Specimens were obtained from postmortem lungs quick-frozen at specified transpulmonary pressures. Collagen was stained by silver, and elastin was stained by orcein. Photomicrographs were composed by computer. Young lungs typically show small collagen fibers that radiate from the "posts," whereas larger fiber bundles traverse the septum irrespective of capillary blood vessels. In older lungs, rings of collagen around the posts appear enlarged. Elastin bundles do not show obvious variation in pattern with age and inflation pressure. Statistical frequency distributions of the fiber width and curvature are both skewed, but the square root of the width and the cube root of the curvature have approximate normal distributions. Typically, for young lungs at transpulmonary pressure of 4 cmH2O, the mean of (width)1/2 (in micron1/2) for collagen fibers is 0.952 +/- 0.242 (SD), that of (curvature)1/3 (in micron-1/3) is 0.349 +/- 0.094. The corresponding values for elastin are 0.986 +/- 0.255 and 0.395 +/- 0.094.     

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.     

Electron microscopic observations of elastic fibres in the lung and aorta of tight-skin and beta-aminopropionitrile-fed mice.

The lung of the tight-skin (TSK) mouse was characterized by enlargement of the air spaces. Elastin in the alveolar walls of the TSK mouse exhibited fragmentation. The aorta of the TSK mouse was characterized by marked hyperplasia of loose connective tissue in the adventitia. Collagen fibres and ruthenium red-positive materials were markedly increased. Microfibrils surrounding elastin in the adventitia of the aorta were not clear in the TSK mouse. In the lung of the beta-aminopropionitrile (BAPN)-fed mouse, enlargement of the alveolar air spaces was not prominent compared with the TSK mouse. Elastic fibres in the alveolar walls did not show the fragmentation observed in the TSK mouse, and microfibrils surrounding elastin were clearly observed. However, elastic laminae in the media of the BAPN-fed mouse aorta were swollen and fragmented. Elastic fibres in the adventitia exhibited a normal appearance and microfibrils surrounding elastin in the adventitia were clearly observed. The results suggest that the mechanism of the connective tissue abnormality in the TSK mouse is different from that of BAPN, which inhibits the activity of lysyl oxidase. The abnormality of elastin and microfibrils surrounding elastin in the TSK mouse probably plays a role in the deformity or degradation of elastic fibres and the structural changes of the lung.     

Cytochemical visualization of anions in collagenous and elastic fiber-associated connective tissue matrix in neonatal and adult rat lungs using iron-containing stains

The cytochemical reactivity of pulmonary connective tissue matrix component in neonatal and adult rat was evaluated using high iron diamine (HID) to detect sulfate ester end groups and dialyzed iron (DI) to detect sulfated and carboxylated end groups of complex carbohydrates, including glycoproteins and glycosaminoglycans at the ultrastructural level. The HID reaction product, in the form of discrete 5-12 nm silver particles following appropriate intensification with thiocarbohydrazide-silver proteinate, was found associated with cell surfaces, the elastin component of elastic fibers, and at regular intervals along the length of collagen fibers in large airways and deep lung interstitium. Staining was similar in adult and neonatal rats, except in areas where connective tissues were presumably still rapidly developing in the neonatal animals. Here large gaps or spaces containing filamentous structures were observed between collagen and elastic fibers. The distribution of DI-reactive sites was similar to that seen with HID with the exception of elastic fibers in which only the microfibrillar portion stained. The collagen-associated reaction was not regularly disposed like that stained with HID, but rather it formed a tight continuous density around the fiber. These results indicated the presence and location of glycoproteins and glycosaminoglycans in connective tissue ground substance regions prior to the full development of elastic and collagenous elements in neonatal pulmonary airways and parenchyma. They also demonstrate cytochemically the presence of a sulfate ester-containing complex sugar found associated with the elastin component of elastic fibers in the lung.     

Cytochemical visualization of anions in collagenous and elastic fiber-associated connective tissue matrix in neonatal and adult rat lungs using iron-containing stains

Summary The cytochemical reactivity of pulmonary connective tissue matrix components in neonatal and adult rat was evaluated using high iron diamine (HID) to detect sulfate ester end groups and dialyzed iron (DI) to detect sulfated and carboxylated end groups of complex carbohydrates, including glycoproteins and glycosaminoglycans at the ultrastructural level. The HID reaction product, in the form of discrete 5–12 nm silver particles following appropriate intensification with thiocarbohydrazide-silver proteinate, was found associated with cell surfaces, the elastin component of elastic fibers, and at regular intervals along the length of collagen fibers in large airways and deep lung interstitium. Staining was similar in adult and neonatal rats, except in areas where connective tissues were presumably still rapidly developing in the neonatal animals. Here large gaps or spaces cointaining reactive filamentous structures were observed between collagen and elastic fibers. The distribution of DI-reactive sites was similar to that seen with HID with the exception of elastic fibers in which only the microfibrillar portion stained. The collagen-associated reaction was not regularly disposed like that stained with HID, but rather it formed a tight continuous density around the fiber. These results indicated the presence and location of glycoproteins and glycosaminoglycans in connective tissue ground substance regions prior to the full development of elastic and collagenous elements in neonatal pulmonary airways and parenchyma. They also demonstrate cytochemically the presence of a sulfate ester-containing complex sugar found associated with the elastin component of elastic fibers in the lung.Supported by Public Health Servece Grant HL 24748     

Immunolocalization of 67 kDa elastin-binding protein in perinatal rat lungs

Summary 67 kDa elastin-binding protein (RL-67EBP) has been isolated from neonatal rat lungs by the use of an elastin-coupled affinity column, followed by elution with either lactose or synthetic elastin hexapeptide (VGVAPG), and immunohistochemistry has been used on perinatal rat lungs to determine the tissue localization of this protein. No immunoreactive structures occur in fetal lungs, or in the lungs of day-1 and-4 neonates. On day-7 after birth, immunoreactive cells appear in the subepithelial connective tissue of the intrapulmonary airways, from day-10 on, these cells become evenly distributed in the alveolar parenchyma. Occasionally, some cells occur in the alveolar air space, being free from the surface of the alveolar septum. Unpermeabilized cells obtained by bronchoalveolar lavage, show cell surface immunoreactivity, indicating that RL-67EBP is expressed on the surface membrane of the cells. From these findings, it is suggested that the immunoreactive cells are blood-borne monocytes, and that RL-67EBP may function as an elastin peptide receptor by which monocytes mobilize through interstitial connective tissue during their migration from blood to alveolar air space, where they eventually differentiate into alveolar macrophages.     

Age-related changes in the content of fibrous proteins in the rat tissues.

An attempt was made to establish a relationship between the content of elastin and collagen in the rat tissues during the process of aging. The content of collagen fractions and elastin in the rat liver, lung and skin, as well as the elastolytic activity of blood serum were determined. It was found that the concentration of elastin as well as the elastolytic activity of blood serum are increasing during the maturation of rats and the total collagen content is increasing too. After the animals reached the age from twelve to twenty four months--above mentioned values began to decrease. It is concluded that the changes in the content of the two fibrous proteins of the connective tissue depend on age.     

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.     

Use of enzymolysis techniques in studying the mechanical properties of connective tissue components.

The optimum conditions for the selective removal of elastin from connective tissues are described. The process, elastolysis, consists of incubating small samples of connective tissue in buffered saline at ph=8.6 containing 300 microgram/me of a 50-50% mixture of elastase with trypsin inhibitor, for 5-6 hours at room temperature. This process, complimented with other processes for selective removal of lipids, or mucopolysaccharides, or collagen, enables one to examine the contribution of the various components of the connective tissue to its mechanical function. The elastolysis was tested with aortic, valvular and tendon tissues from human, bovine and canine species and it was found that in tensile stress experiments, collagen was unaffected while the low-stress contribution of elastin disappeared.     

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.     

Immunohistochemical evidence for loss of ICAM-1 by alveolar epithelial cells in pulmonary fibrosis

ICAM-1 is an intercellular adhesion molecule of the immunoglobulin supergene family involved in adherence of leukocytes to the endothelium and in leukocytic accumulation in pulmonary injury. In the current study, the antigen retrieval technique was used to detect ICAM-1 immunohistochemically in paraffin sections of lungs from human, mouse and rat as well as in bleomycin- or radiation-induced fibrotic lungs from rat and human. In normal lung tissue, the expression of ICAM-1 on alveolar type I epithelial cells is stronger than on alveolar macrophages and on endothelial cells. Preembedding immuno-electron microscopy of normal rat, mouse and human lung samples revealed sclective ICAM-1 expression on the surface of type I alveolar epithelial cells and, to a lesser extent, on the pulmonary capillary endothelium and on alveolar macrophages. In fibrotic specimens, both focal lack and strengthening of immunostaining on the surface of type I cells was found. Alveolar macrophages were found focally lacking ICAM-1 immunoreactivity. In some cases, rat type II pneumocytes exhibited positive immunoreactions for ICAM-1. Immunoelectron microscopy with preembedded rat lungs (bleomycin-exposed cases) confirmed the altered ICAM-1 distribution at the alveolar epithelial surface. In the alveolar fluid of fibrotic rat lungs, in contrast to that from untreated controls, soluble ICAM-1 was detected by western blot analysis.     

Detection of antileukoprotease in connective tissue of the lung

An indirect immunofluorescence technique was applied to frozen sections of central and peripheral human lung tissue to search for extracellular localizations of antileukoprotease (ALP). Two monoclonal anti-ALP antibodies recognizing different epitopes and polyclonal anti-ALP antibodies were used. ALP was found to be localized along elastic fibers in alveolar septa, and also along elastic fibers in the walls of bronchi, bronchioles and blood vessels. Serous cells of bronchial submucosal glands showed labelling as well. In frozen sections of liver and spleen no label was found. Cells and elastic fibers were not labelled when lung tissue sections were processed with polyclonal or monoclonal anti-ALP antibodies, that were blocked with purified ALP before the immunostaining. The association of ALP with elastic fibers of human pulmonary connective tissue is of importance in understanding the role of the inhibitor in the defense of the lung parenchyma against the action of proteolytic enzymes, which is thought to result in emphysema.     

Detection of antileukoprotease in connective tissue of the lung

Summary An indirect immunofluorescence technique was applied to frozen sections of central and peripheral human lung tissue to search for extracellular localizations of antileukoprotease (ALP). Two monoclonal anti-ALP antibodies recognizing different epitopes and polyclonal anti-ALP antibodies were used. ALP was found to be localized along elastic fibers in alveolar septa, and also along elastic fibers in the walls of bronchi, bronchioles and blood vessels. Serous cells of bronchial submucosal glands showed labelling as well. In frozen sections of liver and spleen no label was found. Cells and elastic fibers were not labelled when lung tissue sections were processed with polyclonal or monoclonal anti-ALP antibodies, that were blocked with purified ALP before the immunostaining. The association of ALP with elastic fibers of human pulmonary connective tissue is of importance in understanding the role of the inhibitor in the defense of the lung parenchyma against the action of proteolytic enzymes, which is thought to result in emphysema.     

Ultrastructural immunolocalization of lysyl oxidase in vascular connective tissue

The localization of lysyl oxidase was examined in calf and rat aortic connective tissue at the ultrastructural level using polyclonal chicken anti-lysyl oxidase and gold conjugated rabbit anti-chicken immunoglobulin G to identify immunoreactive sites. Electron microscopy of calf aortic specimens revealed discrete gold deposits at the interface between extracellular bundles of amorphous elastin and the microfibrils circumferentially surrounding these bundles. The antibody did not react with microfibrils which were distant from the interface with elastin. There was negligible deposition of gold within the bundles of amorphous elastin and those few deposits seen at these sites appeared to be associated with strands of microfibrils. Lysyl oxidase was similarly localized in newborn rat aorta at the interface between microfibrils and nascent elastin fibers. Gold deposits were not seen in association with extracellular collagen fibers even after collagen-associated proteoglycans had been degraded by chondroitinase ABC. However, the antibody did recognize collagen-bound lysyl oxidase in collagen fibers prepared from purified collagen to which the enzyme had been added in vitro. No reaction product was seen if the anti-lysyl oxidase was preadsorbed with purified lysyl oxidase illustrating the specificity of the antibody probe. The present results are consistent with a model of elastogenesis predicting the radial growth of the elastin fiber by the deposition and crosslinking of tropoelastin units at the fiber-microfibril interface.     

Lung and alveolar wall elastic and hysteretic behavior in rats: effects of in vivo elastase treatment.

We investigated the relationship between the microscopic elastic and hysteretic behavior of the alveolar walls and the macroscopic mechanical properties of the whole lung in an in vivo elastase-treated rat model of emphysema. We measured the input impedance of isolated lungs at three levels of transpulmonary pressure (Ptp) and used a linear model to estimate the dynamic elastance and hysteresivity of the lungs. The elastance of the normal lungs increased steeply with Ptp, whereas this dependence diminished in the treated lungs. Hysteresivity decreased significantly with Ptp in the normal lungs, but this dependence disappeared in the treated lungs. To investigate the microscopic origins of these changes, the alveolar walls were immunofluorescently labeled in small tissue strips. By using a fluorescent microscope, the lengths and angular orientations of individual alveolar walls were followed during cyclic uniaxial stretching of the tissue strips. The microstrains (relative change in segment length) and changes in angle of the alveolar walls showed considerable heterogeneity, which was interpreted in terms of a network model. In the normal strips, the alveolar walls showed larger angular changes compared with the treated tissue, whereas the alveolar walls of the treated tissue tended to be more extensible. Hysteresis in the average angle change was also larger in the treated tissue than in the normal tissue. We conclude that the decreased Ptp dependence of elastance and the constant hysteresivity in the treated lungs are related to microstructural remodeling and network phenomena at the level of the alveolar walls.