Arnab Majumdar

Three-dimensional measurement of alveolar airspace volumes in normal and emphysematous lungs using micro-CT

In pulmonary emphysema, the alveolar structure progressively breaks down via a three-dimensional (3D) process that leads to airspace enlargement. The characterization of such structural changes has, however, been based on measurements from two-dimensional (2D) tissue sections or estimates of 3D structure from 2D measurements. In this study, we developed a novel silver staining method for visualizing tissue structure in 3D using micro-computed tomographic (CT) imaging, which showed that at 30 cmH20 fixing pressure, the mean alveolar airspace volume increased from 0.12 nl in normal mice to 0.44 nl and 2.14 nl in emphysematous mice, respectively, at 7 and 14 days following elastase-induced injury. We also assessed tissue structure in 2D using laser scanning confocal microscopy. The mean of the equivalent diameters of the alveolar airspaces was lower in 2D compared with 3D, while its variance was higher in 2D than in 3D in all groups. However, statistical comparisons of alveolar airspace size from normal and emphysematous mice yielded similar results in 2D and 3D: compared with control, both the mean and variance of the equivalent diameters increased by 7 days after treatment. These indexes further increased from day 7 to day 14 following treatment. During the first 7 days following treatment, the relative change in SD increased at a much faster rate compared with the relative change in mean equivalent diameter. We conclude that quantifying heterogeneity in structure can provide new insight into the pathogenesis or progression of emphysema that is enhanced by improved sensitivity using 3D measurements.

Structure–Function Relations in an Elastase-Induced Mouse Model of Emphysema

Emphysema is a progressive disease characterized by the destruction of peripheral airspaces and subsequent decline in lung function. However, the relation between structure and function during disease progression is not well understood. The objective of this study was to assess the time course of the structural, mechanical, and remodeling properties of the lung in mice after elastolytic injury. At 2, 7, and 21 days after treatment with porcine pancreatic elastase, respiratory impedance, the constituents of lung extracellular matrix, and histological sections of the lung were evaluated. In the control group, no changes were observed in the structural or functional properties, whereas, in the treatment group, the respiratory compliance and its variability significantly increased by Day 21 (P < 0.001), and the difference in parameters decreased with increasing positive end-expiratory pressure. The heterogeneity of airspace structure gradually increased over time. Conversely, the relative amounts of elastin and type I collagen exhibited a peak (P < 0.01) at Day 2, but returned to baseline levels by Day 21. Structure-function relations manifested themselves in strong correlations between compliance parameters and both mean size and heterogeneity of airspace structure (r(2) > 0.9). Similar relations were also obtained in a network model of the parenchyma in which destruction was based on the notion that mechanical forces contribute to alveolar wall rupture. We conclude that, in a mouse model of emphysema, progressive decline in lung function is sensitive to the development of airspace heterogeneity governed by local, mechanical, force-induced failure of remodeled collagen.

Design of a new variable-ventilation method optimized for lung recruitment in mice

Variable ventilation (VV), characterized by breath-to-breath variation of tidal volume (Vt) and breathing rate (f), has been shown to improve lung mechanics and blood oxygenation during acute lung injury in many species compared with conventional ventilation (CV), characterized by constant Vt and f. During CV as well as VV, the lungs of mice tend to collapse over time; therefore, the goal of this study was to develop a new VV mode (VV(N)) with an optimized distribution of Vt to maximize recruitment. Groups of normal and HCl-injured mice were subjected to 1 h of CV, original VV (VV(O)), CV with periodic large breaths (CV(LB)), and VV(N), and the effects of ventilation modes on respiratory mechanics, airway pressure, blood oxygenation, and IL-1beta were assessed. During CV and VV(O), normal and injured mice showed regional lung collapse with increased airway pressures and poor oxygenation. CV(LB) and VV(N) resulted in a stable dynamic equilibrium with significantly improved respiratory mechanics and oxygenation. Nevertheless, VV(N) provided a consistently better physiological response. In injured mice, VV(O) and VV(N), but not CV(LB), were able to reduce the IL-1beta-related inflammatory response compared with CV. In conclusion, our results suggest that application of higher Vt values than the single Vt currently used in clinical situations helps stabilize lung function. In addition, variable stretch patterns delivered to the lung by VV can reduce the progression of lung injury due to ventilation in injured mice.

Mechanical Forces Regulate Elastase Activity and Binding Site Availability in Lung Elastin

Many fundamental cellular and extracellular processes in the body are mediated by enzymes. At the single molecule level, enzyme activity is influenced by mechanical forces. However, the effects of mechanical forces on the kinetics of enzymatic reactions in complex tissues with intact extracellular matrix (ECM) have not been identified. Here we report that physiologically relevant macroscopic mechanical forces modify enzyme activity at the molecular level in the ECM of the lung parenchyma. Porcine pancreatic elastase (PPE), which binds to and digests elastin, was fluorescently conjugated (f-PPE) and fluorescent recovery after photobleach was used to evaluate the binding kinetics of f-PPE in the alveolar walls of normal mouse lungs. Fluorescent recovery after photobleach indicated that the dissociation rate constant (k(off)) for f-PPE was significantly larger in stretched than in relaxed alveolar walls with a linear relation between k(off) and macroscopic strain. Using a network model of the parenchyma, a linear relation was also found between k(off) and microscopic strain on elastin fibers. Further, the binding pattern of f-PPE suggested that binding sites on elastin unfold with strain. The increased overall reaction rate also resulted in stronger structural breakdown at the level of alveolar walls, as well as accelerated decay of stiffness and decreased failure stress of the ECM at the macroscopic scale. These results suggest an important role for the coupling between mechanical forces and enzyme activity in ECM breakdown and remodeling in development, and during diseases such as pulmonary emphysema or vascular aneurysm. Our findings may also have broader implications because in vivo, enzyme activity in nearly all cellular and extracellular processes takes place in the presence of mechanical forces.

Mechanical failure, stress redistribution, elastase activity and binding site availability on elastin during the progression of emphysema

Emphysema is a disease of the lung parenchyma with progressive alveolar tissue destruction that leads to peripheral airspace enlargement. In this review, we discuss how mechanical forces can contribute to disease progression at various length scales. Airspace enlargement requires mechanical failure of alveolar walls. Because the lung tissue is under a pre-existing tensile stress, called prestress, the failure of a single wall results in a redistribution of the local prestress. During this process, the prestress increases on neighboring alveolar walls which in turn increases the probability that these walls also undergo mechanical failure. There are several mechanisms that can contribute to this increased probability: exceeding the failure threshold of the ECM, triggering local mechanotransduction to release enzymes, altering enzymatic reactions on ECM molecules. Next, we specifically discuss recent findings that stretching of elastin induces an increase in the binding off rate of elastase to elastin as well as unfolds hidden binding sites along the fiber. We argue that these events can initiate a positive feedback loop which generates slow avalanches of breakdown that eventually give rise to the relentless progression of emphysema. We propose that combining modeling at various length scales with corresponding biological assays, imaging and mechanics data will provide new insight into the progressive nature of emphysema. Such approaches will have the potential to contribute to resolving many of the outstanding issues which in turn may lead to the amelioration or perhaps the treatment of emphysema in the future.

Linking Microscopic Spatial Patterns of Tissue Destruction in Emphysema to Macroscopic Decline in Stiffness Using a 3D Computational Model

Pulmonary emphysema is a connective tissue disease characterized by the progressive destruction of alveolar walls leading to airspace enlargement and decreased elastic recoil of the lung. However, the relationship between microscopic tissue structure and decline in stiffness of the lung is not well understood. In this study, we developed a 3D computational model of lung tissue in which a pre-strained cuboidal block of tissue was represented by a tessellation of space filling polyhedra, with each polyhedral unit-cell representing an alveolus. Destruction of alveolar walls was mimicked by eliminating faces that separate two polyhedral either randomly or in a spatially correlated manner, in which the highest force bearing walls were removed at each step. Simulations were carried out to establish a link between the geometries that emerged and the rate of decline in bulk modulus of the tissue block. The spatially correlated process set up by the force-based destruction lead to a significantly faster rate of decline in bulk modulus accompanied by highly heterogeneous structures than the random destruction pattern. Using the Karhunen-Loève transformation, an estimator of the change in bulk modulus from the first four moments of airspace cell volumes was setup. Simulations were then obtained for tissue destruction with different idealized alveolar geometry, levels of pre-strain, linear and nonlinear elasticity assumptions for alveolar walls and also mixed destruction patterns where both random and force-based destruction occurs simultaneously. In all these cases, the change in bulk modulus from cell volumes was accurately estimated. We conclude that microscopic structural changes in emphysema and the associated decline in tissue stiffness are linked by the spatial pattern of the destruction process.

Modeling the dynamics of airway constriction: effects of agonist transport and binding.

Recent advances have revealed that during exogenous airway challenge, airway diameters cannot be adequately predicted by their initial diameters. Furthermore, airway diameters can also vary greatly in time on scales shorter than a breath. To better understand these phenomena, we developed a multiscale model that allowed us to simulate aerosol challenge in the airways during ventilation. The model incorporates agonist-receptor binding kinetics to govern the temporal response of airway smooth muscle contraction on individual airway segments, which, together with airway wall mechanics, determines local airway caliber. Global agonist transport and deposition are coupled with pressure-driven flow, linking local airway constrictions with global flow dynamics. During the course of challenge, airway constriction alters the flow pattern, redistributing the agonist to less constricted regions. This results in a negative feedback that may be a protective property of the normal lung. As a consequence, repetitive challenge can cause spatial constriction patterns to evolve in time, resulting in a loss of predictability of airway diameters. Additionally, the model offers new insights into several phenomena including the intra- and interbreath dynamics of airway constriction throughout the tree structure.

Proteoglycans maintain lung stability in an elastase-treated mouse model of emphysema.

Extracellular matrix remodeling and tissue rupture contribute to the progression of emphysema. Lung tissue elasticity is governed by the tensile stiffness of fibers and the compressive stiffness of proteoglycans. It is not known how proteoglycan remodeling affects tissue stability and destruction in emphysema. The objective of this study was to characterize the role of remodeled proteoglycans in alveolar stability and tissue destruction in emphysema. At 30 days after treatment with porcine pancreatic elastase, mouse lung tissue stiffness and alveolar deformation were evaluated under varying tonicity conditions that affect the stiffness of proteoglycans. Proteoglycans were stained and measured in the alveolar walls. Computational models of alveolar stability and rupture incorporating the mechanical properties of fibers and proteoglycans were developed. Although absolute tissue stiffness was only 24% of normal, changes in relative stiffness and alveolar shape distortion due to changes in tonicity were increased in emphysema (P < 0.01 and P < 0.001). Glycosaminoglycan amount per unit alveolar wall length, which is responsible for proteoglycan stiffness, was higher in emphysema (P < 0.001). Versican expression increased in the tissue, but decorin decreased. Our network model predicted that the rate of tissue deterioration locally governed by mechanical forces was reduced when proteoglycan stiffness was increased. Consequently, this general network model explains why increasing proteoglycan deposition protects the alveolar walls from rupture in emphysema. Our results suggest that the loss of proteoglycans observed in human emphysema contributes to disease progression, whereas treatments that promote proteoglycan deposition in the extracellular matrix should slow the progression of emphysema.

Particle-hopping models of vehicular traffic: Distributions of distance headways and distance between jams

We calculate the distribution of the distance headways (i.e., the instantaneous gap between successive vehicles) as well as the distribution of instantaneous distance between successive jams in the Nagel-Schreckenberg (NS) model of vehicular traffic. When the maximum allowed speed, Vmax, of the vehicles is larger than unity, over an intermediate range of densities of vehicles, our Monte Carlo (MC) data for the distance headway distribution exhibit two peaks, which indicate the coexistence of “free-flowing” traffic and traffic jams. Our analytical arguments clearly rule out the possibility of occurrence of more than one peak in the distribution of distance headways in the NS model when Vmax=1 as well as in the asymmetric simple exclusion process. Modifying and extending an earlier analytical approach for the NS model with Vmax=1, and introducing a novel transfer matrix technique, we also calculate the exact analytical expression for the distribution of distance between the jams in this model; the corresponding distributions for Vmax > 1 have been computed numerically through MC simulation.

A zipper network model of the failure mechanics of extracellular matrices

Mechanical failure of soft tissues is characteristic of life-threatening diseases, including capillary stress failure, pulmonary emphysema, and vessel wall aneurysms. Failure occurs when mechanical forces are sufficiently high to rupture the enzymatically weakened extracellular matrix (ECM). Elastin, an important structural ECM protein, is known to stretch beyond 200% strain before failing. However, ECM constructs and native vessel walls composed primarily of elastin and proteoglycans (PGs) have been found to fail at much lower strains. In this study, we hypothesized that PGs significantly contribute to tissue failure. To test this, we developed a zipper network model (ZNM), in which springs representing elastin are organized into long wavy fibers in a zipper-like formation and placed within a network of springs mimicking PGs. Elastin and PG springs possessed distinct mechanical and failure properties. Simulations using the ZNM showed that the failure of PGs alone reduces the global failure strain of the ECM well below that of elastin, and hence, digestion of elastin does not influence the failure strain. Network analysis suggested that whereas PGs drive the failure process and define the failure strain, elastin determines the peak and failure stresses. Predictions of the ZNM were experimentally confirmed by measuring the failure properties of engineered elastin-rich ECM constructs before and after digestion with trypsin, which cleaves the core protein of PGs without affecting elastin. This study reveals a role for PGs in the failure properties of engineered and native ECM with implications for the design of engineered tissues.