The different roles of myosin IIA and myosin IIB in contraction of 3D collagen matrices by human fibroblasts

Contraction of 3D collagen matrices by fibroblasts frequently is used as an in vitro model of wound closure. Different iterations of the model – all conventionally referred to as “contraction” – involve different morphological patterns. During floating matrix contraction, cells initially are round without stress fibers and subsequently undergo spreading. During stressed matrix contraction, cells initially are spread with stress fibers and subsequently undergo shortening. In the current studies, we used siRNA silencing of myosin IIA (MyoIIA) and myosin IIB (MyoIIB) to test the roles of myosin II isoforms in fibroblast interactions with 3D collagen matrices and collagen matrix contraction. We found that MyoIIA but not MyoIIB was required for cellular global inward contractile force, formation of actin stress fibers, and morphogenic cell clustering. Stressed matrix contraction required MyoIIA but not MyoIIB. Either MyoIIA or MyoIIB was sufficient for floating matrix contraction (FMC) stimulated by platelet-derived growth factor. Neither MyoIIA or MyoIIB was necessary for FMC stimulated by serum. Our findings suggest that myosin II-dependent motor mechanisms for collagen translocation during extracellular matrix remodeling differ depending on cell tension and growth factor stimulation.     

Decreased PDGF Receptor Kinase Activity in Fibroblasts Contracting Stressed Collagen Matrices

Fibroblasts cultured in mechanically stressed collagen matrices proliferate, whereas cells in floating collagen matrices become quiescent. Previous research indicated that one factor contributing to cell quiescence in floating matrices was reduced receptor autophosphorylation in response to PDGF stimulation (i.e., PDGF receptor desensitization). To learn more about the mechanism of PDGF receptor desensitization, we analyzed changes in PDGF receptor autophosphorylation and receptor kinase activity after stressed collagen matrices were switched to floating conditions, which results in rapid cell contraction and dissipation of mechanical stress. PDGF receptor desensitization occurred during contraction stimulated by serum but not in the absence of serum, and desensitization was prevented by inhibitors of contraction but not by inhibitors of the contraction-activated cyclic AMP signaling pathway. Receptor desensitization resulted from decreased receptor kinase activity rather than from elevated protein tyrosine phosphatase activity, and only receptors unoccupied at the time of contraction were affected. After contraction, radiolabeled PDGF binding to the cells was decreased, which suggested that receptor desensitization resulted from a contraction-dependent change in receptor availability or affinity.     

Mutational hotspots in the mitochondrial genome of lung cancer

In lung fibrosis tissue architecture and function is severely hampered by myofibroblasts due to excessive deposition of extracellular matrix and tissue contraction. Myofibroblasts differentiate from fibroblasts under the influence of transforming growth factor (TGF) β₁ but this process is also controlled mechanically by cytoskeletal tension. In healthy lungs, the cytoskeleton of fibroblasts is mechanically strained during breathing. In stiffer fibrotic lung tissue, this mechanical stimulus is reduced, which may influence fibroblast-to-myofibroblast differentiation. Therefore, we investigated the effect of cyclic mechanical stretch on fibroblast-to-myofibroblast differentiation.Primary normal human lung fibroblasts were grown on BioFlex culture plates and stimulated to undergo myofibroblast differentiation by 10 ng/ml TGFβ₁. Cells were either or not subjected to cyclic mechanical stretch (sinusoidal pattern, maximum elongation 10%, 0.2 Hz) for a period of 48 h on a Flexercell apparatus. mRNA expression was analyzed by real-time PCR.Cyclic mechanical loading reduced the mRNA expression of the myofibroblast marker α-smooth muscle actin and the extracellular matrix proteins type-I, type-III, and type-V collagen, and tenascin C. These outcomes indicate that fibroblast-to-myofibroblast differentiation is reduced. Cyclic mechanical loading did not change the expression of the fibronectin ED-A splice variant, but did decrease the paracrine expression of TGFβ₁, thereby suggesting a possible regulation mechanism for the observed effects. The data suggest that cyclic loading experienced by healthy lung cells during breathing may prevent fibroblasts from differentiating towards myofibroblasts.     

LPA-stimulated fibroblast contraction of floating collagen matrices does not require Rho kinase activity or retraction of fibroblast extensions

Fibroblasts synthesize, organize, and maintain connective tissues during development and in response to injury and fibrotic disease. These morphogenetic processes depend on cell-matrix remodeling, which has been investigated using cells cultured in three-dimensional collagen matrices. The current studies were carried out to test the role of Rho kinase activity and retraction of fibroblast extensions on the matrix remodeling process. We found that remodeling (contraction) of floating collagen matrices stimulated by lysophosphatidic acid (LPA) did not require Rho kinase activity or retraction of fibroblast extensions. On the other hand, LPA-stimulated contraction of restrained matrices became Rho kinase dependent after the matrices were allowed to develop mechanical loading for 2-4 h, suggesting that the remodeling process itself was able to feed back to modulate cell behavior in an iterative process. Modulation was specific for LPA since fibroblast-collagen matrix contraction stimulated by platelet-derived growth factor was Rho kinase dependent before or after mechanical loading developed.     

Fibroblast biology in three-dimensional collagen matrices

Research on fibroblast biology in three-dimensional collagen matrices offers new opportunities to understand the reciprocal and adaptive interactions that occur between cells and surrounding matrix in a tissue-like environment. Such interactions are integral to the regulation of connective tissue morphogenesis and dynamics that characterizes tissue homeostasis and wound repair. During fibroblast-collagen matrix remodeling, mechanical signals from the remodeled matrix feed back to modulate cell behavior in an iterative process. As mechanical loading (tension) within the matrix increases, the mechanisms used by cells to remodel the matrix change. Fibroblasts in matrices that are under tension or relaxed respond differently to growth factor stimulation, and switching between mechanically loaded and unloaded conditions influences whether cells acquire proliferative/biosynthetic active or quiescent/resting phenotypes.     

Decreased level of PDGF-stimulated receptor autophosphorylation by fibroblasts in mechanically relaxed collagen matrices

The goal of our studies was to characterize the interrelationship between extracellular matrix organization and fibroblast proliferation in response to growth factors. We compared fibroblasts in monolayer culture with cells in contracted collagen matrices that were mechanically stressed or relaxed. In response to platelet-derived growth factor (PDGF), DNA synthesis by fibroblasts in mechanically relaxed collagen matrices was 80-90% lower than in monolayer culture and 50% lower than in mechanically stressed matrices. Fibroblasts in monolayer and contracted collagen matrix cultures contained similar levels of PDGF receptors, but differed in their autophosphorylation response. Cells in mechanically relaxed matrices showed lowest levels of autophosphorylation, 90% less than cells in monolayer culture. Experiments comparing receptor expression and capacity for PDGF- stimulated autophosphorylation showed that cells in mechanically relaxed collagen matrices never developed normal receptor autophosphorylation. Furthermore, when mechanically stressed collagen matrices were switched to mechanically relaxed conditions, capacity for receptor autophosphorylation decreased within 1-2 h and remained low. Based on immunomicroscopic observations and studies on down-regulation of receptors by PDGF binding, it appeared that most PDGF receptors in monolayer or contracted collagen matrix cultures were localized on the cell surface and accessible to PDGF binding. In related studies, we found that EGF receptors of fibroblasts in mechanically relaxed collagen matrices also showed low levels of autophosphorylation in response to EGF treatment. Based on these results, we suggest that mechanical interactions between cells and their surrounding matrix provide regulatory signals that modulate autophosphorylation of growth factor receptors and cell proliferation.     

Fibroblast-collagen-matrix contraction: growth-factor signalling and mechanical loading

Fibroblast-collagen-matrix contraction provides a unique way to study reciprocal geometric and mechanical interactions between fibroblasts and extracellular matrix. Such interactions are difficult to appreciate or examine in routine cell culture because the culture surface is usually fixed in place. Forces exerted on collagen fibrils by cells cause isometric tension to develop in the cells if the collagen resists deformation; by contrast, the cells remain mechanically unloaded in the absence of matrix resistance. Recent evidence suggests that the state of cellular mechanical loading determines the mechanism that cells use to regulate contraction.     

P21-activated kinase 1: convergence point in PDGF- and LPA-stimulated collagen matrix contraction by human fibroblasts

Fibroblast three-dimensional collagen matrix culture provides a tissue-like model that can be used to analyze cell form and function. The physiological agonists platelet-derived growth factor (PDGF) and lysophosphatidic acid (LPA) both stimulate human fibroblasts to contract floating collagen matrices. In this study, we show that the PDGF and LPA signaling pathways required for matrix contraction converge on p21-activated kinase 1 (PAK1) and its downstream effector cofilin1 and that contraction depends on cellular ruffling activity, rather than on the protrusion and retraction of cellular dendritic extensions. We also show that, depending on the agonist, different Rho effectors cooperate with PAK1 to regulate matrix contraction, Rho kinase in the case of PDGF and mDia1 in the case of LPA. These findings establish a unified framework for understanding the cell signaling pathways involved in fibroblast contraction of floating collagen matrices.     

Masters and servants of the force: the role of matrix adhesions in myofibroblast force perception and transmission

Fibroblast-to-myofibroblast differentiation - a key event in the development of fibrocontractive diseases and in wound granulation tissue contraction - is hallmarked by the formation of stress fibers and the neo-expression of alpha-smooth muscle actin. Incorporation of the smooth muscle actin isoform into stress fibers confers to myofibroblasts a high contractile activity which is transmitted to the extracellular matrix at sites of specialized adhesions, termed 'fibronexus' in tissue and 'supermature focal adhesions' in two-dimensional cell culture. Myofibroblast differentiation requires a mechanically restrained environment in conjunction with the action of growth factors like transforming growth factor beta and specialized matrix molecules like the ED-A splice variant of fibronectin. This mini-review discusses the roles of myofibroblast adhesions in sensing matrix stress, in transmitting contractile force to the extracellular environment and in creating the high intracellular tension that is required for myofibroblast development.     

Myofibroblast contraction activates latent TGF-beta1 from the extracellular matrix

The conjunctive presence of mechanical stress and active transforming growth factor β1 (TGF-β1) is essential to convert fibroblasts into contractile myofibroblasts, which cause tissue contractures in fibrotic diseases. Using cultured myofibroblasts and conditions that permit tension modulation on the extracellular matrix (ECM), we establish that myofibroblast contraction functions as a mechanism to directly activate TGF-β1 from self-generated stores in the ECM. Contraction of myofibroblasts and myofibroblast cytoskeletons prepared with Triton X-100 releases active TGF-β1 from the ECM. This process is inhibited either by antagonizing integrins or reducing ECM compliance and is independent from protease activity. Stretching myofibroblast-derived ECM in the presence of mechanically apposing stress fibers immediately activates latent TGF-β1. In myofibroblast-populated wounds, activation of the downstream targets of TGF-β1 signaling Smad2/3 is higher in stressed compared to relaxed tissues despite similar levels of total TGF-β1 and its receptor. We propose activation of TGF-β1 via integrin-mediated myofibroblast contraction as a potential checkpoint in the progression of fibrosis, restricting autocrine generation of myofibroblasts to a stiffened ECM.     

Hepatic stellate cells require a stiff environment for myofibroblastic differentiation

The myofibroblastic differentiation of hepatic stellate cells (HSC) is a critical event in liver fibrosis and is part of the final common pathway to cirrhosis in chronic liver disease from all causes. The molecular mechanisms driving HSC differentiation are not fully understood. Because macroscopic tissue stiffening is a feature of fibrotic disease, we hypothesized that mechanical properties of the underlying matrix are a principal determinant of HSC activation. Primary rat HSC were cultured on inert polyacrylamide supports of variable but precisely defined shear modulus (stiffness) coated with different extracellular matrix proteins or poly-L-lysine. HSC differentiation was determined by cell morphology, immunofluorescence staining, and gene expression. HSC became progressively myofibroblastic as substrate stiffness increased on all coating matrices, including Matrigel. The degree rather than speed of HSC activation correlated with substrate stiffness, with cells cultured on supports of intermediate stiffness adopting stable intermediate phenotypes. Quiescent cells on soft supports were able to undergo myofibroblastic differentiation with exposure to stiff supports. Stiffness-dependent differentiation required adhesion to matrix proteins and the generation of mechanical tension. Transforming growth factor-β treatment enhanced differentiation on stiff supports, but was not required. HSC differentiate to myofibroblasts in vitro primarily as a function of the physical rather than the chemical properties of the substrate. HSC require a mechanically stiff substrate, with adhesion to matrix proteins and the generation of mechanical tension, to differentiate. These findings suggest that alterations in liver stiffness are a key factor driving the progression of fibrosis.     

Tensional homeostasis in dermal fibroblasts: Mechanical responses to mechanical loading in three-dimensional substrates

Many soft connective tissues are under endogenous tension, and their resident cells generate considerable contractile forces on the extracellular matrix. The present work was aimed to determine quantitatively how fibroblasts, grown within three-dimensional collagen lattices, respond mechanically to precisely defined tensional loads. Forces generated in response to changes in applied load were measured using a tensional culture force monitor. In a number of variant systems, resident cells consistently reacted to modify the endogenous matrix tension in the opposite direction to externally applied loads. That is, increased external loading was followed immediately by a reduction in cell-mediated contraction whilst decreased external loading elicited increased contraction. Responses were cell-mediated and not a result of material properties of the matrices. This is the first detailed characterisation of a tensional homeostatic response in cells. The maintained force, after 8 h in culture, was typically around 40–60 dynes/million cells. Maintenance of an active tensional homeostasis has widespread implications for cells in culture and forwhole tissue function. J. Cell. Physiol. 175:323–332, 1998. © 1998 Wiley-Liss, Inc.     

Dendritic fibroblasts in three-dimensional collagen matrices

Cell motility determines form and function of multicellular organisms. Most studies on fibroblast motility have been carried out using cells on the surfaces of culture dishes. In situ, however, the environment for fibroblasts is the three-dimensional extracellular matrix. In the current research, we studied the morphology and motility of human fibroblasts embedded in floating collagen matrices at a cell density below that required for global matrix remodeling (i.e., contraction). Under these conditions, cells were observed to project and retract a dendritic network of extensions. These extensions contained microtubule cores with actin concentrated at the tips resembling growth cones. Platelet-derived growth factor promoted formation of the network; lysophosphatidic acid stimulated its retraction in a Rho and Rho kinase-dependent manner. The dendritic network also supported metabolic coupling between cells. We suggest that the dendritic network provides a mechanism by which fibroblasts explore and become interconnected to each other in three-dimensional space.     

ATP modulates load-inducible IL-1beta,COX 2, and MMP-3 gene expression in human tendon cells

Tendon cells receive mechanical signals from the load bearing matrices. The response to mechanical stimulation is crucial for tendon function. However, overloading tendon cells may deteriorate extracellular matrix integrity by activating intrinsic factors such as matrix metalloproteinases (MMPs) that trigger matrix destruction. We hypothesized that mechanical loading might induce interleukin-1beta (IL-1beta) in tendon cells, which can induce MMPs, and that extracellular ATP might inhibit the load-inducible gene expression. Human tendon cells isolated from flexor digitorum profundus tendons (FDPs) of four patients were made quiescent and treated with ATP (10 or 100 microM) for 5 min, then stretched equibiaxially (1 Hz, 3.5% elongation) for 2 h followed by an 18-h-rest period. Stretching induced IL-1beta, cyclooxygenase 2 (COX 2), and MMP-3 genes but not MMP-1. ATP reduced the load-inducible gene expression but had no effect alone. A medium change caused tendon cells to secrete ATP into the medium, as did exogenous UTP. The data demonstrate that mechanical loading induces ATP release in tendon cells and stimulates expression of IL-1beta, COX 2, and MMP-3. Load-induced endogenous IL-1beta may trigger matrix remodeling or a more destructive pathway(s) involving IL-1beta, COX 2, and MMP-3. Concomitant autocrine and paracrine release of ATP may serve as a negative feedback mechanism to limit activation of such an injurious pathway. Attenuation or failure of this negative feedback mechanism may result in the progression to tendinosis.     

Local, three-dimensional strain measurements within largely deformed extracellular matrix constructs

The ability to create extracellular matrix (ECM) constructs that are mechanically and biochemically similar to those found in vivo and to understand how their properties affect cellular responses will drive the next generation of tissue engineering strategies. To date, many mechanisms by which cells biochemically communicate with the ECM are known. However the mechanisms by which mechanical information is transmitted between cells and their ECM remain to be elucidated. "Self-assembled" collagen matrices provide an in vitro-model system to study the mechanical behavior of ECM. To begin to understand how the ECM and the cells interact mechanically, the three-dimensional (3D) mechanical properties of the ECM must be quantified at the micro-(local) level in addition to information measured at the macro-(global) level. Here we describe an incremental digital volume correlation (IDVC) algorithm to quantify large (>0.05) 3D mechanical strains in the microstructure of 3D collagen matrices in response to applied mechanical loads. Strain measurements from the IDVC algorithm rely on 3D confocal images acquired from collagen matrices under applied mechanical loads. The accuracy and the precision of the IDVC algorithm was verified by comparing both image volumes collected in succession when no deformation was applied to the ECM (zero strain) and image volumes to which simulated deformations were applied in both ID and 3D (simulated strains). Results indicate that the IDVC algorithm can accurately and precisely determine the 3D strain state inside largely deformed collagen ECMs. Finally, the usefulness of the algorithm was demonstrated by measuring the microlevel 3D strain response of a collagen ECM loaded in tension.     

Role of vitronectin and fibronectin receptors in oral mucosal and dermal myofibroblast differentiation

Background information. The activation of fibroblasts into myofibroblasts is a crucial event in healing that is linked to remodelling and scar formation, therefore we determined whether regulation of myofibroblast differentiation via integrins might affect wound healing responses in populations of patient‐matched HOFs (human oral fibroblasts) compared with HDFs (human dermal fibroblasts). Results. Both the HOF and HDF cell types underwent TGF‐β1 (transforming growth factor‐β1)‐induced myofibroblastic differentiation [upregulation of the expression of α‐sma (α‐smooth muscle actin)], although analysis of unstimulated cells indicated that HOFs contained higher basal levels of α‐sma than HDFs (P<0.05). Functional blocking antibodies against the integrin subunits α5 (fibronectin) or αv (vitronectin) were used to determine whether the effects of TGF‐β1 were regulated via integrin signalling pathways. α‐sma expression in both HOFs and HDFs was down‐regulated by antibodies against both α5 and αv. Functionally, TGF‐β1 inhibited cell migration in an in vitro wound model and increased the contraction of collagen gels. Greater contraction was evident for HOFs compared with HDFs, both with and without stimulation by TGF‐β1 (P<0.05). When TGF‐β1‐stimulated cells were incubated with blocking antibodies against α5 and αv, gel contraction was decreased to that of non‐stimulated cells; however, blocking αv or α5 could not restore cellular migration in both HOFs and HDFs. Conclusions. Despite intrinsic differences in their basal state, the cellular events associated with TGF‐β1‐induced myofibroblastic differentiation are common to both HOFs and HDFs, and appear to require differential integrin usage; up‐regulation of α‐sma expression and increases in collagen gel contraction are vitronectin‐ and fibronectin‐receptor‐dependent processes, whereas wound re‐population is not.     

Nicotine inhibits myofibroblast differentiation in human gingival fibroblasts

Cigarette smoking has been suggested as a risk factor for several periodontal diseases. It has also been found that smokers respond less favorably than non-smokers to periodontal therapy. Previous work in our lab has shown that nicotine inhibits human gingival cell migration. Since myofibroblasts play an important role in wound closure, we asked if nicotine affects gingival wound healing process by regulating myofibroblast differentiation. Human gingival fibroblasts (HGFs) from two patients were cultured in 10% fetal bovine serum cell culture medium. Cells were pretreated with different doses of nicotine (0, 0.01, 0.1, and 1 mM) for 2 h, and then incubated with transforming growth factor beta (TGF-beta1) (0, 0.25, 0.5, and 1 ng/ml) with or without nicotine for 30 h. The expression level of alpha-smooth muscle actin (alpha-SMA), a specific marker for myofibroblasts, was analyzed by Western blots, immunocytochemistry, and real-time polymerase chain reaction (real-time PCR). Phosphorylated p38 mitogen-activated protein kinase (Phospho-p38 MAPK) activity was analyzed by Western blots. TGF-beta1 induced an increase of alpha-SMA protein and mRNA expression, while nicotine (1 mM) inhibited the TGF-beta1-induced expression of alpha-SMA but not beta-actin. Nicotine treatment down-regulated TGF-beta1-induced p38 MAPK phosphorylation. Our results demonstrated for the first time that nicotine inhibits myofibroblast differentiation in human gingival fibroblasts in vitro; supporting the hypothesis that delayed wound healing in smokers may be due to decreased wound contraction by myofibroblasts.     

Multiple roles of alpha-smooth muscle actin in mechanotransduction

Alpha-smooth muscle actin (SMA), an actin isoform that contributes to cell-generated mechanical tension, is normally restricted to cells of vascular smooth muscle, but SMA can also be expressed in certain non-muscle cells, most notably myofibroblasts. These cells are present in healing wounds, scars, and fibrocontractive lesions where they contribute to fibrosis. In myofibroblasts, cell-generated traction forces associated with SMA contribute to matrix remodeling, but exogenous mechanical forces can also increase SMA expression. Force-induced SMA utilizes a feed-forward amplification loop involving a priori SMA in focal adhesions, the binding of the p38 MAP kinase to SMA filaments, activation of Rho and binding of serum response factor to the CArG-B box of the SMA promoter. Thus, in addition to its importance as a structural protein in tissue remodeling and contraction, SMA may serve as a mechanotransducer, based on its ability to physically link mechanosensory elements and to enhance its own, force-induced expression.