Fibroblast biology: Signals targeting the synovial fibroblast in arthritis

Fibroblast-like cells in the synovial lining (type B lining cells), stroma and pannus tissue are targeted by many signals, such as the following: ligands binding to cell surface receptors; lipid soluble, small molecular weight mediators (eg nitric oxide [NO], prostaglandins, carbon monoxide); extracellular matrix (ECM)-cell interactions; and direct cell-cell contacts, including gap junctional intercellular communication. Joints are subjected to cyclic mechanical loading and shear forces. Adherence and mechanical forces affect fibroblasts via the ECM (including the hyaluronan fluid phase matrix) and the pericellular matrix (eg extracellular matrix metalloproteinase inducer [EMMPRIN]) matrices, thus modulating fibroblast migration, adherence, proliferation, programmed cell death (including anoikis), synthesis or degradation of ECM, and production of various cytokines and other mediators [1]. Aggressive, transformed or transfected mesenchymal cells containing proto-oncogenes can act in the absence of lymphocytes, but whether these cells represent regressed fibroblasts, chondrocytes or bone marrow stem cells is unclear.     

The Role of Vinculin in the Regulation of the Mechanical Properties of Cells

Vinculin couples as a focal adhesion protein the extracellular matrix (ECM) through integrins to the actomyosin cytoskeleton. During the last years vinculin has become the focus of cell mechanical measurements and a key protein regulating the transmission of contractile forces. In earlier reports vinculin has been described as an inhibitor of cell migration on planar substrates, because knock-out of vinculin in F9 mouse embryonic carcinoma cells and mouse embryonic fibroblasts showed increased cell motility on 2D substrates. The role of vinculin in cell invasion through a 3D extracellular matrix is still fragmentarily investigated. This review presents vinculin in its role as a regulator of cellular mechanical functions. Contractile force generation is reduced when vinculin is absent, or enhanced when vinculin is present. Moreover, the generation of contractile forces is a prerequisite for cell invasion through a dense 3D ECM, where the pore-size is smaller than the diameter of the cell nucleus (<2 μm). Measurements of cell’s biophysical properties will be presented. In summary, vinculin’s leading role among focal adhesion proteins in regulating the mechanical properties of cells will be discussed.     

Fibroblast to myofibroblast transition is enhanced by increased cell density

Idiopathic pulmonary fibrosis (IPF) is a chronic disease of the lung caused by a rampant inflammatory response that results in the deposition of excessive extracellular matrix (ECM). IPF patient lungs also develop fibroblastic foci that consist of activated fibroblasts and myofibroblasts. In concert with ECM deposition, the increased cell density within fibroblastic foci imposes confining forces on lung fibroblasts. In this work, we observed that increased cell density increases the incidence of the fibroblast-to-myofibroblast transition (FMT), but mechanical confinement imposed by micropillars has no effect on FMT incidence. We found that human lung fibroblasts (HLFs) express more α-SMA and deposit more collagen matrix, which are both characteristics of myofibroblasts, in response to TGF-β1 when cells are seeded at a high density compared with a medium or a low density. These results support the hypothesis that HLFs undergo FMT more readily in response to TGF-β1 when cells are densely packed, and this effect could be dependent on increased OB-cadherin expression. This work demonstrates that cell density is an important factor to consider when modelling IPF in vitro, and it may suggest decreasing cell density within fibroblastic foci as a strategy to reduce IPF burden.     

A phenomenological cohesive model for the macroscopic simulation of cell–matrix adhesions

Cell adhesion is crucial for cells to not only physically interact with each other but also sense their microenvironment and respond accordingly. In fact, adherent cells can generate physical forces that are transmitted to the surrounding matrix, regulating the formation of cell–matrix adhesions. The main purpose of this work is to develop a computational model to simulate the dynamics of cell–matrix adhesions through a cohesive formulation within the framework of the finite element method and based on the principles of continuum damage mechanics. This model enables the simulation of the mechanical adhesion between cell and extracellular matrix (ECM) as regulated by local multidirectional forces and thus predicts the onset and growth of the adhesion. In addition, this numerical approach allows the simulation of the cell as a whole, as it models the complete mechanical interaction between cell and ECM. As a result, we can investigate and quantify how different mechanical conditions in the cell (e.g., contractile forces, actin cytoskeletal properties) or in the ECM (e.g., stiffness, external forces) can regulate the dynamics of cell–matrix adhesions.     

Effects of interleukin-18 on cardiac fibroblast function and gene expression

Fibroblasts are the primary cell type responsible for synthesis and remodeling of the extracellular matrix in the heart. A number of factors including growth factors, hormones and mechanical forces have been identified that modulate the production of extracellular matrix by cardiac fibroblasts. Inflammatory mediators including pro-inflammatory cytokines and chemokines also impact fibrosis of the heart. Recent studies have illustrated that interleukin-18 promotes a pro-fibrotic response in cardiac fibroblasts; however the effects of this cytokine on other aspects of fibroblast function have not been examined. While fibroblasts have long been known for their role in production and remodeling of the extracellular matrix, other functions of these cells are only now beginning to be appreciated. We hypothesize that exposure to interleukin-18 will stimulate other aspects of fibroblast behavior important in myocardial remodeling including proliferation, migration and collagen reorganization. Fibroblasts were isolated from adult male rat hearts and bioassays performed to determine the effects of interleukin-18 on fibroblast function. Treatment of fibroblasts with interleukin-18 (1-100ng/ml) resulted in increased production of extracellular matrix components and remodeling or contraction of three-dimensional collagen scaffolds by these cells. Furthermore, exposure to interleukin-18 stimulated fibroblast migration and proliferation. Treatment of heart fibroblasts with interleukin-18 resulted in the rapid activation of the c-Jun N-terminal kinase (JNK) and phosphoinositide 3-kinase (PI3-kinase) pathways. Studies with pharmacological inhibitors illustrated that activation of these pathways is critical to interleukin-18 mediated alterations in fibroblast function. These studies illustrate that interleukin-18 plays a role in modulation of cardiac fibroblast function and may be an important component of the inflammation-fibrosis cascade during pathological myocardial remodeling.     

Prostaglandins but not leukotrienes alter extracellular matrix protein deposition and cytokine release in primary human airway smooth muscle cells and fibroblasts

Eicosanoids are lipid-signaling mediators released by many cells in response to various stimuli. Increasing evidence suggests that eicosanoids such as leukotrienes and prostaglandins (PGs) may directly mediate remodeling. In this study, we assessed whether these substances could alter extracellular matrix (ECM) proteins and the inflammatory profiles of primary human airway smooth muscle cells (ASM) and fibroblasts. PGE(2) decreased both fibronectin and tenascin C in fibroblasts but only fibronectin in ASM. PGD(2) decreased both fibronectin and tenascin C in both ASM and fibroblasts, whereas PGF(2α) had no effect on ECM deposition. The selective PGI(2) analog, MRE-269, decreased fibronectin but not tenascin C in both cell types. All the PGs increased IL-6 and IL-8 release in a dose-dependent manner in ASM and fibroblasts. Changes in ECM deposition and cytokine release induced by prostaglandins in both ASM and fibroblasts were independent of an effect on cell number. Neither the acute nor repeated stimulation with leukotrienes had an effect on the deposition of ECM proteins or cytokine release from ASM or fibroblasts. We concluded that, collectively, these results provide evidence that PGs may contribute to ECM remodeling to a greater extent than leukotrienes in airway cells.     

Mechanical stress regulates the mechanotransduction and metabolism of cardiac fibroblasts in fibrotic cardiac diseases

Fibrotic cardiac diseases are characterized by myocardial fibrosis that results in maladaptive cardiac remodeling. Cardiac fibroblasts (CFs) are the main cell type responsible for fibrosis. In response to stress or injury, intrinsic CFs develop into myofibroblasts and produce excess extracellular matrix (ECM) proteins. Myofibroblasts are mechanosensitive cells that can detect changes in tissue stiffness and respond accordingly. Previous studies have revealed that some mechanical stimuli control fibroblast behaviors, including ECM formation, cell migration, and other phenotypic traits. Further, metabolic alteration is reported to regulate fibrotic signaling cascades, such as the transforming growth factor-β pathway and ECM deposition. However, the relationship between metabolic changes and mechanical stress during fibroblast-to-myofibroblast transition remains unclear. This review aims to elaborate on the crosstalk between mechanical stress and metabolic changes during the pathological transition of cardiac fibroblasts.     

Extracellular matrix alters the relationship between tritiated thymidine incorporation and proliferation of MC3T3-E1 cells during osteogenesis in vitro

Bone cells in vivo exist in direct contact with extracellular matrix, which regulates their basic biological processes including metabolism, development, growth and differentiation. Thus, the in vitro activity of cells cultured on tissue culture treated plastic could be different from the activity of cells cultured on their natural substrate. We selected MC3T3-E1 pre-osteoblastic cells to study the effect of extracellular matrix on cell proliferation because these cells undergo a progressive developmental sequence of proliferation and differentiation. MC3T3-E1 cells were cultured on plastic or plastic coated with ECM, fibronectin, collagen type I, BSA or poly l-lysine and their ability to proliferate was assessed by incorporation of [3H]dT or by enumeration of cells. Our results show that (1) ECM inhibits incorporation of [3H]dT by MC3T3-E1 cells; (2) collagen type I, but not BSA, poly l-lysine or fibronectin also inhibits incorporation of [3H]dT; (3) the level of ECM inhibition of [3H]dT incorporation is directly related to the number of cells cultured, but unrelated to the cell cycle distribution or endogenous thymidine content; (4) the kinetic profile of [3H]dT uptake suggest that ECM inhibits transport of [3H]dT from the extracellular medium, and (5) cell counts are similar in cultures whether cells are grown on plastic or ECM. These results suggest that decreased incorporation of [3H]dT by cells cultured on ECM is not reflective of bone cell proliferation.     

Mechanical Cell-Matrix Feedback Explains Pairwise and Collective Endothelial Cell Behavior In Vitro

In vitro cultures of endothelial cells are a widely used model system of the collective behavior of endothelial cells during vasculogenesis and angiogenesis. When seeded in an extracellular matrix, endothelial cells can form blood vessel-like structures, including vascular networks and sprouts. Endothelial morphogenesis depends on a large number of chemical and mechanical factors, including the compliancy of the extracellular matrix, the available growth factors, the adhesion of cells to the extracellular matrix, cell-cell signaling, etc. Although various computational models have been proposed to explain the role of each of these biochemical and biomechanical effects, the understanding of the mechanisms underlying in vitro angiogenesis is still incomplete. Most explanations focus on predicting the whole vascular network or sprout from the underlying cell behavior, and do not check if the same model also correctly captures the intermediate scale: the pairwise cell-cell interactions or single cell responses to ECM mechanics. Here we show, using a hybrid cellular Potts and finite element computational model, that a single set of biologically plausible rules describing (a) the contractile forces that endothelial cells exert on the ECM, (b) the resulting strains in the extracellular matrix, and (c) the cellular response to the strains, suffices for reproducing the behavior of individual endothelial cells and the interactions of endothelial cell pairs in compliant matrices. With the same set of rules, the model also reproduces network formation from scattered cells, and sprouting from endothelial spheroids. Combining the present mechanical model with aspects of previously proposed mechanical and chemical models may lead to a more complete understanding of in vitro angiogenesis.     

Dissecting the influence of cellular senescence on cell mechanics and extracellular matrix formation in vitro

Tissue formation and healing both require cell proliferation and migration, but also extracellular matrix production and tensioning. In addition to restricting proliferation of damaged cells, increasing evidence suggests that cellular senescence also has distinct modulatory effects during wound healing and fibrosis. Yet, a direct role of senescent cells during tissue formation beyond paracrine signaling remains unknown. We here report how individual modules of the senescence program differentially influence cell mechanics and ECM expression with relevance for tissue formation. We compared DNA damage-mediated and DNA damage-independent senescence which was achieved through over-expression of either p16^Ink4a or p21^Cip1 cyclin-dependent kinase inhibitors in primary human skin fibroblasts. Cellular senescence modulated focal adhesion size and composition. All senescent cells exhibited increased single cell forces which led to an increase in tissue stiffness and contraction in an in vitro 3D tissue formation model selectively for p16 and p21-overexpressing cells. The mechanical component was complemented by an altered expression profile of ECM-related genes including collagens, lysyl oxidases, and MMPs. We found that particularly the lack of collagen and lysyl oxidase expression in the case of DNA damage-mediated senescence foiled their intrinsic mechanical potential. These observations highlight the active mechanical role of cellular senescence during tissue formation as well as the need to synthesize a functional ECM network capable of transferring and storing cellular forces.     

Cells in 3D matrices under interstitial flow: Effects of extracellular matrix alignment on cell shear stress and drag forces

Interstitial flow is an important regulator of various cell behaviors both in vitro and in vivo, yet the forces that fluid flow imposes on cells embedded in a 3D extracellular matrix (ECM), and the effects of matrix architecture on those forces, are not well understood. Here, we demonstrate how fiber alignment can affect the shear and pressure forces on the cell and ECM. Using computational fluid dynamics simulations, we show that while the solutions of the Brinkman equation accurately estimate the average fluid shear stress and the drag forces on a cell within a 3D fibrous medium, the distribution of shear stress on the cellular surface as well as the peak shear stresses remain intimately related to the pericellular fiber architecture and cannot be estimated using bulk-averaged properties. We demonstrate that perpendicular fiber alignment of the ECM yields lower shear stress and pressure forces on the cells and higher stresses on the ECM, leading to decreased permeability, while parallel fiber alignment leads to higher stresses on cells and increased permeability, as compared to a cubic lattice arrangement. The Spielman–Goren permeability relationships for fibrous media agreed well with CFD simulations of flow with explicitly considered fibers. These results suggest that the experimentally observed active remodeling of ECM fibers by fibroblasts under interstitial flow to a perpendicular alignment could serve to decrease the shear and drag forces on the cell.     

Aligned forces: Origins and mechanisms of cancer dissemination guided by extracellular matrix architecture

Organized extracellular matrix (ECM), in the form of aligned architectures, is a critical mediator of directed cancer cell migration by contact guidance, leading to metastasis in solid tumors. Current models suggest anisotropic force generation through the engagement of key adhesion and cytoskeletal complexes drives contact-guided migration. Likewise, disrupting the balance between cell–cell and cell–ECM forces, driven by ECM engagement for cells at the tumor–stromal interface, initiates and drives local invasion. Furthermore, processes such as traction forces exerted by cancer and stromal cells, spontaneous reorientation of matrix-producing fibroblasts, and direct binding of ECM modifying proteins lead to the emergence of collagen alignment in tumors. Thus, as we obtain a deeper understanding of the origins of ECM alignment and the mechanisms by which it is maintained to direct invasion, we are poised to use the new paradigm of stroma-targeted therapies to disrupt this vital axis of disease progression in solid tumors.     

An Introductory Review of Cell Mechanobiology

Mechanical loads induce changes in the structure, composition, and function of living tissues. Cells in tissues are responsible for these changes, which cause physiological or pathological alterations in the extracellular matrix (ECM). This article provides an introductory review of the mechanobiology of load-sensitive cells in vivo, which include fibroblasts, chondrocytes, osteoblasts, endothelial cells, and smooth muscle cells. Many studies have shown that mechanical loads affect diverse cellular functions, such as cell proliferation, ECM gene and protein expression, and the production of soluble factors. Major cellular components involved in the mechanotransduction mechanisms include the cytoskeleton, integrins, G proteins, receptor tyrosine kinases, mitogen-activated protein kinases, and stretch-activated ion channels. Future research in the area of cell mechanobiology will require novel experimental and theoretical methodologies to determine the type and magnitude of the forces experienced at the cellular and sub-cellular levels and to identify the force sensors/receptors that initiate the cascade of cellular and molecular events     

Integrin β1 is required for maintenance of vascular tone in postnatal mice

Connective tissue is required for maintaining the integrity of tissues. Integrins are the cell surface receptors responsible for cell attachment to extracellular matrix; however, their tissue-specific role in this process is poorly understood. Here, we test whether integrin β1 is required for blood vessel maintenance and integrity in adult mice. We show that adult mice containing a fibroblast/smooth muscle cell-specific deletion of integrin β1 exhibit impaired bleeding time and maintenance of vessel architecture, including progressively reduced levels of extracellular matrix (ECM). Vessels also possessed diminished levels of α-smooth muscle actin (α-SMA), and cells derived from vessels showed reduced production of mRNAs encoding ECM and α-SMA as well as reduced α-SMA protein and stress fibers and ECM contraction. Integrin β1 in adult fibroblasts/smooth muscle cells/pericytes is required for vasoconstriction and vascular maintenance.     

Relationship between cell stiffness and stress fiber amount,assessed by simultaneous atomic force microscopy and live-cell fluorescence imaging

Actomyosin stress fibers, one of the main components of the cell’s cytoskeleton, provide mechanical stability to adherent cells by applying and transmitting tensile forces onto the extracellular matrix (ECM) at the sites of cell–ECM adhesion. While it is widely accepted that changes in spatial and temporal distribution of stress fibers affect the cell’s mechanical properties, there is no quantitative knowledge on how stress fiber amount and organization directly modulate cell stiffness. We address this key open question by combining atomic force microscopy with simultaneous fluorescence imaging of living cells, and combine for the first time reliable quantitative parameters obtained from both techniques. We show that the amount of myosin and (to a lesser extent) actin assembled in stress fibers directly modulates cell stiffness in adherent mouse fibroblasts (NIH3T3). In addition, the spatial distribution of stress fibers has a second-order modulatory effect. In particular, the presence of either fibers located in the cell periphery, aligned fibers or thicker fibers gives rise to reinforced cell stiffness. Our results provide basic and significant information that will help design optimal protocols to regulate the mechanical properties of adherent cells via pharmacological interventions that alter stress fiber assembly or via micropatterning techniques that restrict stress fiber spatial organization.     

Techniques for assessing 3-D cell–matrix mechanical interactions in vitro and in vivo

Cellular interactions with extracellular matrices (ECM) through the application of mechanical forces mediate numerous biological processes including developmental morphogenesis, wound healing and cancer metastasis. They also play a key role in the cellular repopulation and/or remodeling of engineered tissues and organs. While 2-D studies can provide important insights into many aspects of cellular mechanobiology, cells reside within 3-D ECMs in vivo, and matrix structure and dimensionality have been shown to impact cell morphology, protein organization and mechanical behavior. Global measurements of cell-induced compaction of 3-D collagen matrices can provide important insights into the regulation of overall cell contractility by various cytokines and signaling pathways. However, to understand how the mechanics of cell spreading, migration, contraction and matrix remodeling are regulated at the molecular level, these processes must also be studied in individual cells. Here we review the evolution and application of techniques for imaging and assessing local cell–matrix mechanical interactions in 3-D culture models, tissue explants and living animals.     

Paracrine cellular and extracellular matrix interactions with mesenchymal progenitors during pulmonary alveolar septation

Alveolar development in humans primarily occurs postnatally and requires a carefully orchestrated expansion of distal epithelial and mesenchymal progenitor populations and coordinated differentiation, to create a highly segmented gas‐exchange surface. The regulation of alveolarization normally assimilates cues from paracrine cell–cell, cell–extracellular matrix, and mechanical interactions which are superimposed on cells and the extracellular matrix through phasic respiratory movement. In bronchopulmonary dysplasia, the entire process is precociously initiated when cellular and extracellular components are adapted to the saccular stage where movement and circulation are much more limited. This review focuses on mesenchymal cells (fibroblasts, endothelial cells, and pericytes), and epithelial cells are primarily discussed as sources of growth factor ligands or recipients of ligands produced by mesenchymal cells. Some interstitial fibroblasts differentiate to contractile myofibroblasts, containing a smooth muscle‐actin rich cytoskeleton, which connects with tensile and elastic elements in the extracellular matrix, and together comprise a load‐bearing network that diffuses mechanical forces during respiration. Other interstitial fibroblasts assimilate neutral lipid droplets, which regulate the differentiation of distal epithelial progenitors and surfactant production by alveolar type 2 cells. Pericytes organize and reinforce the capillary network as it expands to match the coverage of type 1 epithelial cells. Hyperoxia and the mechanical load imposed by positive pressure mechanical ventilation disrupt these paracrine interactions, leaving thickened alveolar walls, airways and arterioles, thereby diminishing gas‐exchange surface area. Better understanding of these mechanisms of alveolar septation will lead to more effective treatments to preserve and perhaps augment the surface usual sequence of events that drive alveolarization. Birth Defects Research (Part A) 100:227–239, 2014. © 2014 Wiley Periodicals, Inc.     

Integrins and cell signaling in chondrocytes

Integrins are adhesion receptor heterodimers that transmit information from the extracellular matrix (ECM) to the cell through activation of cell signaling pathways. Chondrocytes express several members of the integrin family including alpha5beta1 which is the primary chondrocyte receptor for fibronectin. Cell signaling mediated through integrins regulates several chondrocyte functions including differentiation, matrix remodeling, responses to mechanical stimulation and cell survival. Integrin-mediated activation of members of the mitogen-activated protein kinase family likely plays a key role in transmitting signals regulating chondrocyte gene expression. Upstream mediators of mitogen-activated protein kinase (MAP kinase) activation include focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2 (pyk2) which are both expressed by chondrocytes. A better understanding of chondrocyte integrin signaling is needed to define the mechanisms by which the ECM regulates chondrocyte function.     

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.     

Fibroblast adhesion results in the induction of a matrix remodeling gene expression program

Fibrosis is believed to occur through the failure to terminate the normal tissue remodeling program. Tissue repair intimately involves the ability of fibroblasts to attach to extracellular matrix (ECM), resulting in cell migration and ECM contraction. Elevated, activated adhesive signaling is a key phenotypic hallmark of fibrotic cells. The precise contribution of adhesion to tissue remodeling and repair and fibrotic responses in fibroblasts is unclear, but involves focal adhesion kinase (FAK). FAK signals downstream of integrin-mediates attachment of fibroblasts to extracellular matrix. In this report, we show that FAK is required for the expression of a cohort of mRNAs encoding ECM and matrix remodeling genes including CCN2, alpha-smooth muscle actin (SMA) and type I collagen. Adhesion of fibroblasts to fibronectin, a component of the provisional matrix deposited in the initial phases of tissue repair, also resulted in the induction of CCN2, alpha-SMA and type I collagen mRNAs. Endothelin-1 (ET-1), a key inducer of pro-fibrotic gene expression, was also induced upon fibroblast attachment to ECM, and antagonism of the ET-1 receptors significantly reduced the ability of adhesion to induce expression of CCN2, alpha-SMA and type I collagen mRNAs. These results suggest that adhesion of fibroblasts to matrix during the initial phases of tissue remodeling and repair may actively contribute to the tissue repair program through the induction of pro-fibrotic gene expression.