Maturational changes in extracellular matrix and lung tissue mechanics.

The viscoelastic properties of the pulmonary parenchyma change rapidly postparturition. We compared changes in mechanical properties with changes in tissue composition of rat lung parenchymal strips in three groups of Sprague-Dawley rats: baby (B; 10-14 days), young (Y; approximately 3 wk), and adult (A; approximately 8 wk). Strips were suspended in an organ bath, and resistance (R), elastance (E), and hysteresivity (eta) were calculated during sinusoidal oscillations before and after the addition of acetylcholine (ACh) (10(-3) M). Strips were then fixed in formalin, and sections were stained with hematoxylin and eosin, Verhoff's elastic stain, or Van Gieson's picric acid-fuchsin stain for collagen. The volume proportion of collagen (%Col), the length density of elastic fibers (L(V)/Pr(alv)), and the arithmetic mean thickness of alveolar septae (T(a)) were calculated by morphometry. Tissue was also stained for alpha-smooth muscle actin (ASMA), and the volume proportion of ASMA (%ASMA) was calculated. Hyaluronic acid (HA) was quantitated by radioimmunoassay in separate strips. R and E in B strips were significantly higher, whereas eta was significantly smaller than in Y or A strips. Changes in these parameters with ACh were greater in B strips. T(a), %ASMA, and HA were greatest in B strips, whereas %Col and L(V)/Pr(alv) were least. There were significant positive correlations between R and E vs. T(a) and between percent change in R and eta post-ACh vs. T(a) and vs. %ASMA, and significant negative correlations between R and E vs. %Col and vs. L(V)/Pr(alv) and percent increase in all three mechanical parameters post-ACh vs. %Col. These data suggest that the relatively high stiffness, R, and contractile responsiveness of parenchymal tissues observed in newborns are not directly attributable to the amount of collagen and elastic fibers in the tissue, but rather they are related to the thickened alveolar wall and the relatively greater percent of contractile cells.     

Airway and tissue mechanics in a murine model of asthma: alveolar capsule vs. forced oscillations.

To better address the functional consequences of inflammation on bronchial responsiveness, we studied two groups of BALB/c mice: a nonimmunized control group (n = 8) and a group immunized and challenged with inhaled ovalbumin (n = 8). An alveolar capsule (AC) measured airway resistance (Raw(AC)) and lung elastance (EL). A forced oscillation (FO) technique independently estimated airway resistance (Raw(FO)) and a parameter H(ti) related to tissue elastance. Ovalbumin-immunized and -challenged mice had increased numbers of eosinophils in bronchoalveolar lavage and increased responsiveness to methacholine (MCh). Corresponding parameters from the AC and FO techniques were correlated: Raw(AC) vs. Raw(FO) (r = 0.76) and EL vs. H(ti) (r = 0.88, P < 0.0001 in all cases). AC and FO techniques showed significant increases in tissue elastance in response to MCh but no significant increases in airway resistance. These results demonstrated that the AC and FO techniques yield essentially equivalent results in mice, even when the lung is inhomogeneous, and that the bronchoconstrictive responses to MCh and inflammation in mice are predominantly located in the lung periphery.     

PAI-1 promotes extracellular matrix deposition in the airways of a murine asthma model

Dysregulation of matrix metalloproteinases (MMPs) and ineffective fibrinolysis are associated with the deposition of extracellular matrix (ECM). We hypothesized that elevated plasminogen activator inhibitor (PAI)-1 promotes ECM deposition in the asthmatic airway by inhibiting MMP-9 activity and fibrinolysis. Degree of airway inflammation was similar in PAI-1(-/-) and wild type (WT) mice after ovalbumin (OVA) challenge. PAI-1 production, deposition of collagen and fibrin, and MMP-9 activity in the lung tissue or airways were greater after OVA challenge compared with saline challenge. However, in PAI-1(-/-) mice, collagen deposition was 2-fold less, fibrin deposition was 4-fold less, and MMP-9 activity was 3-fold higher. This is the first direct evidence that the plasmin system regulates ECM deposition in the airways of a murine asthma model, independently of the effect of PAI-1 on inflammatory cells. The results suggest that the PAI-1-dependent inhibition of MMP-9 activity and fibrinolysis is a major mechanism by which ECM deposition occurs.     

Comparative study of cellular and extracellular matrix composition of native and tissue engineered heart valves.

Tissue engineering of heart valves utilizes biodegradable or metabolizable scaffolds for remodeling by seeded autologous cells. The aim of this study was to determine and compare extracellular matrix (ECM) formations, cellular phenotypes and cell location of native and tissue engineered (TE) valve leaflets. Ovine carotid arteries, ovine and porcine hearts were obtained from slaughterhouses. Cells were isolated from carotid arteries and dissected ovine, porcine and TE leaflets. TE constructs were fabricated from decellularized porcine pulmonary valves, seeded ovine arterial cells and subsequent 16 days dynamic in vitro culture using a pulsatile bioreactor. Native and TE valves were studied by histology (hematoxylin-eosin, resorcin-fuchsin, Movat pentachrome), NIR femtosecond multiphoton laser scanning microscopy and scanning electron microscopy (SEM). Cells of native and TE tissues were identified and localized by immunohistochemistry. Arterial, valvular and re-isolated TE-construct cells were processed for immunocytochemistry and Western blotting. ECM analysis and SEM revealed characteristical and comparable structures in native and TE leaflets. Most cells in native leaflets stained strongly positive for vimentin. Cells positive to alpha-smooth muscle actin (alpha-SMA), myosin and calponin were only found at the ventricular (inflow) side of ovine aortic and porcine pulmonary valve leaflets. Cells from TE constructs had a strong expression of vimentin, alpha-SMA, myosin, calponin and h-caldesmon throughout the entire leaflet. Comparable ECM formation and endothelial cell lining of native and TE leaflets could be demonstrated. However, immunostaining revealed significant differences between valvular cell phenotypes of native and TE leaflets. These results may be essential for further cardiovascular tissue engineering efforts.     

Diabetes and arterial extracellular matrix changes in a porcine model of atherosclerosis.

Patients with diabetes are at substantially increased risk for atherosclerosis and clinical cardiovascular events. Because arterial extracellular matrix contains several molecules, including biglycan, versican, hyaluronan, and elastin, that may affect plaque lipid retention and stability, we determined whether diabetes affects plaque content of these molecules in a porcine model of hyperlipidemia and diabetes. Coronary artery sections were studied from non-diabetic normolipidemic (n=11, N-NL), diabetic normolipidemic (n=10, DM-NL), non-diabetic hyperlipidemic (n=16, N-HL), and diabetic hyperlipidemic (n=15, DM-HL) animals. Hyaluronan, biglycan, versican, and apolipoprotein B (apoB) were detected with monospecific peptides or antisera, and elastin with Movat's pentachrome stain, and contents of each were quantified by computer-assisted morphometry. In the hyperlipidemic groups, diabetes was associated with a 4-fold increase in intimal area, with strong correlations between intimal area and immunostained areas for hyaluronan (R(2) = 0.83, p<0.0001), biglycan (R(2) = 0.72, p<0.0001), and apoB (R(2) = 0.23, p=0.0069). In contrast, median (interquartile range) intimal elastin content was significantly lower with diabetes [N-HL: 5.2% (2.4-8.2%) vs DM-HL: 1.5% (0.5-4.2%), p=0.01], and there was a strong negative correlation between intimal total and elastin areas (Spearman r = -0.62, p=0.001). In this porcine model, diabetes was associated with multiple extracellular matrix changes that have been associated with increased lesion instability, greater atherogenic lipoprotein retention, and accelerated atherogenesis.     

The extracellular matrix as a scaffold for tissue reconstruction

The extracellular matrix (ECM) consists of a complex mixture of structural and functional proteins and serves an important role in tissue and organ morphogenesis, maintenance of cell and tissue structure and function, and in the host response to injury. Xenogeneic and allogeneic ECM has been used as a bioscaffold for the reconstruction of many different tissue types in both pre-clinical and human clinical studies. Common features of ECM-associated tissue remodeling include extensive angiogenesis, recruitment of circulating progenitor cells, rapid scaffold degradation and constructive remodeling of damaged or missing tissues. The ECM-induced remodeling response is a distinctly different phenomenon from that of scar tissue formation.     

Elucidation of extracellular matrix mechanics from muscle fibers and fiber bundles

The importance of the extracellular matrix (ECM) in muscle is widely recognized, since ECM plays a central role in proper muscle development (Buck and Horwitz, 1987), tissue structural support (Purslow, 2002), and transmission of mechanical signals between fibers and tendon (Huijing, 1999). Since substrate biomechanical properties have been shown to be critical in the biology of tissue development and remodeling (Engler et al., 2006; Gilbert et al., 2010), it is likely that mechanics are critical for ECM to perform its function. Unfortunately, there are almost no data available regarding skeletal muscle ECM viscoelastic properties. This is primarily due to the impossibility of isolating and testing muscle ECM. Therefore, this note presents a new method to quantify viscoelastic ECM modulus by combining tests of single muscle fibers and fiber bundles. Our results demonstrate that ECM is a highly nonlinearly elastic material, while muscle fibers are linearly elastic.     

Colonial development in Pandorina morum. I. Structure and composition of the extracellular matrix

Every cell in a colony of the freshwater alga Pandorina morum produces a daughter colony in each round of vegetative reproduction. The cells of P. morum structurally resemble those of Chlamydomonas and do not obviously differ from their presumptive unicellular ancestor in ways that would account for their colonial state. It is therefore possible that their colonial association may be the result of altered extracellular matrix, intercellular connections, or modified processes of cell division and matrix formation. The ultrastructural studies and chemical analysis reported here show that the matrix of P. morum is a multilayered structure containing hydroxyproline-rich glycoproteins and sulfated polysaccharide. Significantly, this matrix displays no major differences from the “wall” of Chlamydomonas species. No intercellular connections have been found in mature colonies. The extracellular matrix therefore maintains, but does not initiate, the colonial arrangement of the cells.     

Cell mechanics studied by a reconstituted model tissue

Tissue models reconstituted from cells and extracellular matrix (ECM) simulate natural tissues. Cytoskeletal and matrix proteins govern the force exerted by a tissue and its stiffness. Cells regulate cytoskeletal structure and remodel ECM to produce mechanical changes during tissue development and wound healing. Characterization and control of mechanical properties of reconstituted tissues are essential for tissue engineering applications. We have quantitatively characterized mechanical properties of connective tissue models, fibroblast-populated matrices (FPMs), via uniaxial stretch measurements. FPMs resemble natural tissues in their exponential dependence of stress on strain and linear dependence of stiffness on force at a given strain. Activating cellular contractile forces by calf serum and disrupting F-actin by cytochalasin D yield "active" and "passive" components, which respectively emphasize cellular and matrix mechanical contributions. The strain-dependent stress and elastic modulus of the active component were independent of cell density above a threshold density. The same quantities for the passive component increased with cell number due to compression and reorganization of the matrix by the cells.     

A nonlinear viscoelastic model of lung tissue mechanics.

There have been a number of attempts recently to use linear models to describe the low-frequency (0-2 Hz) dependence of lung tissue resistance (Rti) and elastance (Eti). Only a few attempts, however, have been made to account for the volume dependence of these quantities, all of which require the tissues to be plastoelastic. In this paper we specifically avoid invoking plastoelasticity and develop a nonlinear viscoelastic model that is also capable of accounting for the nonlinear and frequency-dependent features of lung tissue mechanics. The model parameters were identified by fitting the model to data obtained in a previous study from dogs during sinusoidal ventilation. The model was then used to simulate pressure and flow data by use of various types of ventilation patterns similar to those that have been employed experimentally. Rti and Eti were estimated from the simulated data by use of four different estimation techniques commonly applied in respiratory mechanics studies. We found that the estimated volume dependence of Rti and Eti is sensitive to both the ventilation pattern and the estimation technique, being in error by as much as 217 and 22%, respectively.     

Control of cytoskeletal mechanics by extracellular matrix, cell shape, and mechanical tension.

We have investigated how extracellular matrix (ECM) alters the mechanical properties of the cytoskeleton (CSK). Mechanical stresses were applied to integrin receptors on the apical surfaces of adherent endothelial cells using RGD-coated ferromagnetic microbeads (5.5-microns diameter) in conjunction with a magnetic twisting device. Increasing the number of basal cell-ECM contacts by raising the fibronectin (FN) coating density from 10 to 500 ng/cm2 promoted cell spreading by fivefold and increased CSK stiffness, apparent viscosity, and permanent deformation all by more than twofold, as measured in response to maximal stress (40 dyne/cm2). When the applied stress was increased from 7 to 40 dyne/cm2, the stiffness and apparent viscosity of the CSK increased in parallel, although cell shape, ECM contacts, nor permanent deformation was altered. Application of the same stresses over a lower number ECM contacts using smaller beads (1.4-microns diameter) resulted in decreased CSK stiffness and apparent viscosity, confirming that this technique probes into the depth of the CSK and not just the cortical membrane. When magnetic measurements were carried out using cells whose membranes were disrupted and ATP stores depleted using saponin, CSK stiffness and apparent viscosity were found to rise by approximately 20%, whereas permanent deformation decreased by more than half. Addition of ATP (250 microM) under conditions that promote CSK tension generation in membrane-permeabilized cells resulted in decreases in CSK stiffness and apparent viscosity that could be detected within 2 min after ATP addition, before any measurable change in cell size.(ABSTRACT TRUNCATED AT 250 WORDS)     

Glycosaminoglycans contribute to extracellular matrix fiber recruitment and arterial wall mechanics

Elastic and collagen fibers are well known to be the major load-bearing extracellular matrix (ECM) components of the arterial wall. Studies of the structural components and mechanics of arterial ECM generally focus on elastin and collagen fibers, and glycosaminoglycans (GAGs) are often neglected. Although GAGs represent only a small component of the vessel wall ECM, they are considerably important because of their diverse functionality and their role in pathological processes. The goal of this study was to study the mechanical and structural contributions of GAGs to the arterial wall. Biaxial tensile testing was paired with multiphoton microscopic imaging of elastic and collagen fibers in order to establish the structure–function relationships of porcine thoracic aorta before and after enzymatic GAG removal. Removal of GAGs results in an earlier transition point of the nonlinear stress–strain curves \((p<0.05)\). However, stiffness was not significantly different after GAG removal treatment, indicating earlier but not absolute stiffening. Multiphoton microscopy showed that when GAGs are removed, the adventitial collagen fibers are straighter, and both elastin and collagen fibers are recruited at lower levels of strain, in agreement with the mechanical change. The amount of stress relaxation also decreased in GAG-depleted arteries \((p<0.05)\). These findings suggest that the interaction between GAGs and other ECM constituents plays an important role in the mechanics of the arterial wall, and GAGs should be considered in addition to elastic and collagen fibers when studying arterial function.     

The extracellular matrix viscoelasticity as a regulator of cell and tissue dynamics

The extracellular matrix mechanical properties regulate processes in development, cancer, and fibrosis. Among the distinct mechanical properties, the vast majority of research has focused on the extracellular matrix's elasticity as the primary determinant of cell and tissue behavior. However, both cells and the extracellular matrix are not only elastic but also viscous. Despite viscoelasticity being a universal feature of living tissues, our knowledge of the influence of the extracellular matrix's viscoelasticity in cell behavior is limited. This mini-review describes some of the recent findings that have highlighted the role of the extracellular matrix's viscoelasticity in cell and tissue dynamics.     

Mechanical model for a collagen fibril pair in extracellular matrix

In this paper, we model the mechanics of a collagen pair in the connective tissue extracellular matrix that exists in abundance throughout animals, including the human body. This connective tissue comprises repeated units of two main structures, namely collagens as well as axial, parallel and regular anionic glycosaminoglycan between collagens. The collagen fibril can be modeled by Hooke’s law whereas anionic glycosaminoglycan behaves more like a rubber-band rod and as such can be better modeled by the worm-like chain model. While both computer simulations and continuum mechanics models have been investigated for the behavior of this connective tissue typically, authors either assume a simple form of the molecular potential energy or entirely ignore the microscopic structure of the connective tissue. Here, we apply basic physical methodologies and simple applied mathematical modeling techniques to describe the collagen pair quantitatively. We found that the growth of fibrils was intimately related to the maximum length of the anionic glycosaminoglycan and the relative displacement of two adjacent fibrils, which in return was closely related to the effectiveness of anionic glycosaminoglycan in transmitting forces between fibrils. These reveal the importance of the anionic glycosaminoglycan in maintaining the structural shape of the connective tissue extracellular matrix and eventually the shape modulus of human tissues. We also found that some macroscopic properties, like the maximum molecular energy and the breaking fraction of the collagen, were also related to the microscopic characteristics of the anionic glycosaminoglycan.     

Effects of chronic L-NAME treatment lung tissue mechanics, eosinophilic and extracellular matrix responses induced by chronic pulmonary inflammation

The importance of lung tissue in asthma pathophysiology has been recently recognized. Although nitric oxide mediates smooth muscle tonus control in airways, its effects on lung tissue responsiveness have not been investigated previously. We hypothesized that chronic nitric oxide synthase (NOS) inhibition by N(omega)-nitro-L-arginine methyl ester (L-NAME) may modulate lung tissue mechanics and eosinophil and extracellular matrix remodeling in guinea pigs with chronic pulmonary inflammation. Animals were submitted to seven saline or ovalbumin exposures with increasing doses (1 approximately 5 mg/ml for 4 wk) and treated or not with L-NAME in drinking water. After the seventh inhalation (72 h), animals were anesthetized and exsanguinated, and oscillatory mechanics of lung tissue strips were performed in baseline condition and after ovalbumin challenge (0.1%). Using morphometry, we assessed the density of eosinophils, neuronal NOS (nNOS)- and inducible NOS (iNOS)-positive distal lung cells, smooth muscle cells, as well as collagen and elastic fibers in lung tissue. Ovalbumin-exposed animals had an increase in baseline and maximal tissue resistance and elastance, eosinophil density, nNOS- and iNOS-positive cells, the amount of collagen and elastic fibers, and isoprostane-8-PGF(2alpha) expression in the alveolar septa compared with controls (P<0.05). L-NAME treatment in ovalbumin-exposed animals attenuated lung tissue mechanical responses (P<0.01), nNOS- and iNOS-positive cells, elastic fiber content (P<0.001), and isoprostane-8-PGF(2alpha) in the alveolar septa (P<0.001). However, this treatment did not affect the total number of eosinophils and collagen deposition. These data suggest that NO contributes to distal lung parenchyma constriction and to elastic fiber deposition in this model. One possibility may be related to the effects of NO activating the oxidative stress pathway.     

Frequency dependence of respiratory system mechanics during induced constriction in a murine model of asthma.

Airway dysfunction in asthma is characterized by hyperresponsiveness, heterogeneously narrowed airways, and closure of airways. To test the hypothesis that airway constriction in ovalbumin (OVA)-sensitized OVA-intranasally challenged (OVA/OVA) mice produces mechanical responses that are similar to those reported in asthmatic subjects, respiratory system resistance (Rrs) and elastance (Edyn,rs) spectra were obtained in OVA/OVA and control mice during intravenous methacholine (MCh) infusions. In control mice, MCh at 1,700 microg x kg(-1) x min(-1) produced 1) a 495 and 928% increase of Rrs at 0.5 Hz and 19.75 Hz, respectively, 2) a 33% rise in Edyn,rs at 0.5 Hz, and 3) a mild frequency (f)-dependent increase of Edyn,rs. The same MCh dose in OVA/OVA mice produced 1) elevations of Rrs at 0.5 Hz and 19.75 Hz of 1,792 and 774%, respectively, 2) a 390% rise in Edyn,rs at 0.5 Hz, and 3) marked f-dependent increases of Edyn,rs. During constriction, the f dependence of mechanics in control mice was consistent with homogeneous airway narrowing; however, in OVA/OVA mice, f dependence was characteristic of heterogeneously narrowed airways, closure of airways, and airway shunting. These mechanisms amplify the pulmonary mechanical responses to constrictor stimuli at physiological breathing rates and have important roles in the pathophysiology of human asthma.     

Oral tolerance attenuates changes in in vitro lung tissue mechanics and extracellular matrix remodeling induced by chronic allergic inflammation in guinea pigs

Recent studies emphasize the presence of alveolar tissue inflammation in asthma. Immunotherapy has been considered a possible therapeutic strategy for asthma, and its effect on lung tissue had not been previously investigated. Measurements of lung tissue resistance and elastance were obtained before and after both ovalbumin and acetylcholine challenges. Using morphometry, we assessed eosinophil and smooth muscle cell density, as well as collagen and elastic fiber content, in lung tissue from guinea pigs with chronic pulmonary allergic inflammation. Animals received seven inhalations of ovalbumin (1-5 mg/ml; OVA group) or saline (SAL group) during 4 wk. Oral tolerance (OT) was induced by offering ad libitum ovalbumin 2% in sterile drinking water starting with the 1st inhalation (OT1 group) or after the 4th (OT2 group). The ovalbumin-exposed animals presented an increase in baseline and in postchallenge resistance and elastance related to baseline, eosinophil density, and collagen and elastic fiber content in lung tissue compared with controls. Baseline and post-ovalbumin and acetylcholine elastance and resistance, eosinophil density, and collagen and elastic fiber content were attenuated in OT1 and OT2 groups compared with the OVA group. Our results show that inducing oral tolerance attenuates lung tissue mechanics, as well as eosinophilic inflammation and extracellular matrix remodeling induced by chronic inflammation.     

Changes in the composition of the extracellular matrix in patellar tendinopathy

ObjectiveTo compare the chemical levels and mRNA expression of proteoglycan and collagen in normal human patellar tendons and tendons exhibiting chronic overuse tendinopathy.MethodsSulfated glycosaminoglycan and hydroxyproline content were investigated by spectrophotometric measurement using papain-digested samples. Deglycosylated proteoglycan core proteins were analysed by Western blot using specific antibodies. Total mRNA isolated from samples of frozen tendons was assayed by relative quantitative RT-PCR for decorin, biglycan, fibromodulin, versican, aggrecan, and collagens Type I, II and III and normalised to glyceraldehyde-3-phosphate dehydrogenase.ResultsThere was a significant increase in sulfated glycosaminoglycan content in pathologic tendons compared to normal. This was attributed to an increased deposition of the large aggregating proteoglycans versican and aggrecan and the small proteoglycans biglycan and fibromodulin, but not decorin. Aggrecan and versican were extensively degraded in both normal and pathologic tendons, biglycan was more fragmented in the pathologic tendons while predominantly intact fibromodulin and decorin were present in normal and pathologic tendons. There was a greater range in total collagen content but no change in the level of total collagen in pathologic tendons. There were no significant differences between the pathologic and normal tendon for all genes, however p values close to 0.05 indicated a trend in downregulation of Type I collagen and fibromodulin, and upregulation in versican and Type III genes in pathologic tissue.ConclusionThe changes in proteoglycan and collagen levels observed in patellar tendinopathy appear to be primarily due to changes in the metabolic turnover of these macromolecules. Changes in the expression of these macromolecules may not play a major role in this process.