Mechanosensing and fibrosis (original) (raw)
Cell mechanoresponses relevant to fibrosis. As early sentinels of homeostatic disruption, endothelial and epithelial cells have refined mechanosensing capabilities that allow them to detect fine changes in the physical environment. Multiple endothelial and epithelial mechanotransducers have been identified, including those in cell adhesion protein complexes, primary cilia, and mechanically gated ion channels (refs. 46–52 and Table 1). Mechanical activation of these sensory systems can alter endothelial and epithelial barrier function, inflammatory signaling, migration, invasion, and proliferation. Moreover, these cell types have the capacity to transmit signals to neighboring cells to amplify mechanoresponses. For example, endothelial cells are known to transmit signals to the surrounding microenvironment through angiocrine signals such as growth factors and chemokines (53). Epithelial cells similarly transmit paracrine signals that communicate mechanical signals to nearby mesenchyme (54). One particularly pertinent example is that of liver sinusoidal endothelial cells (LSECs), which actively secrete angiocrine signals that maintain neighboring hepatic stellate cell (HSC) quiescence (55), thereby preventing the “activation” or transdifferentiation of HSCs into fibrogenic myofibroblasts, key mediators of hepatic fibrosis. Studies suggest that LSECs are also capable of reverting HSCs from an activated to a quiescent phenotype through a nitric oxide–dependent pathway downstream of the mechanosensitive transcription factor Krüppel-like factor 2 (KLF2) (56). Similar homeostatic epithelial signals in the lung are thought to underlie maintenance of fibroblast quiescence (57–59), while endothelial-derived angiocrine signals are implicated in lung regeneration and fibrosis (60).
Mechanosensory molecules and pathways potentially active in fibrosis
Aberrant mechanical activation may underlie persistently altered endothelial and epithelial cell states in fibrosis. For example, vascular leakage and vasculopathies are implicated in fibrosis (61, 62), and vascular barrier function responds directly to mechanical forces (63, 64) and to alterations in matrix stiffness (65–67). In the kidney, mechanical forces play a critical role in maintaining endothelial barrier function (68–70), while capillary functional alterations due to disturbed blood flow often lead to reduced vascular density, capillary rarefaction, and inflammation, which together exacerbate the progression of kidney fibrosis (71–73). Together these observations identify multiple potentially important roles for epithelial and endothelial mechanosignaling in acute injury responses and the transition to fibrotic tissue remodeling.
While tissue-resident interstitial mesenchymal cells (including organ-specific fibroblasts, pericytes, and stellate cells) respond to signals from epithelial and endothelial cells, they also directly respond to changes in their mechanical environment. Moreover, as the primary matrix-producing cells, they also have the capacity to remodel their tissue mechanical environment. For example, stretch promotes fibroblast matrix production (74), which reinforces the tissue and ultimately shields resident cells from further stretch in an attempt to restore mechanical homeostasis (4). In cases of pathological fibrotic remodeling, matrix stiffening promotes a nonhomeostatic feedback loop that amplifies matrix deposition in mesenchymal cells in a cell-autonomous fashion (27). Cultured lung fibroblasts, HSCs, and portal fibroblasts all respond to pathophysiological matrix stiffness changes with increased ECM gene expression, protein production, and deposition (27, 75–78). Relative to physiologically compliant matrices, stiff matrices promote characteristics of mesenchymal activation, including proliferation, apoptosis resistance, contractility, and expression of an invasive phenotype (27, 77, 79). Together, these findings implicate fibrotic tissue stiffening in the amplification of mesenchymal cell activation. Intriguingly, dynamically altering matrix stiffness can recapitulate not just HSC activation upon matrix stiffening, but also partial reversion of the myofibroblastic phenotype upon matrix softening (80, 81), reinforcing the critical role of mechanics in determining mesenchymal cell fate. Similarly, pathological fibroblasts isolated from patients with idiopathic pulmonary fibrosis retain the capacity to become largely inactivated by culture on physiologically compliant matrices (82). These studies accentuate the need to identify the mechanisms that orchestrate fibroblast mechanobiological responses.
Mechanisms of cell-matrix mechanosensing. Adherent cells derive critical signals from their interactions with the ECM to regulate survival, morphology, migration, and higher-level cell phenotypes. The mechanical information transmitted from the ECM is largely processed through integrin-based adhesions, which provide a mechanical linkage from the ECM to the intracellular cytoskeleton and cellular signaling pathways (ref. 83 and Figure 2). Integrins, comprising heterodimers of α and β subunits, recognize specific polypeptide sequences in ECM proteins. In mechanosensing, integrins serve as mechanical linkages and scaffolds upon which complex signaling interactions can be organized, as their short intracellular domains have no inherent signaling domains (83). Activated integrins are bound to the actomyosin system through dynamic associations with integrin- or F-actin–binding proteins, such as talin and vinculin (84). Sensing of matrix stiffness by cultured fibroblasts requires the coordinated interactions of integrins with fibronectin outside the cell, and with talin and actin inside the cell. Together these dynamically interacting proteins form a clutch mechanism whereby competing adhesion, contraction, and adhesion-reinforcement kinetics enable mechanosensitive assembly, growth, and maintenance of integrin-based cell-matrix adhesion complexes (85). Specific integrin heterodimers display catch-bond behaviors (86, 87), with decreasing off-rate kinetics under increased loading. This feature permits the mechanical environment to further impact the interactions and behavior of specific integrins. The GPI-anchored glycoprotein Thy-1 has also recently been shown to modify integrin interactions (88), potentially amplifying or attenuating matrix stiffness sensitivity to cell-matrix adhesions. Adhesion complexes themselves are well known to integrate mechanical signals rapidly and thereby assemble on time scales immediately comparable to dynamic changes in internal or externally applied forces (89). Proteomics-based efforts have illuminated the tremendous complexity of cell-matrix adhesion complexes beyond the core integrin adhesion machinery, identifying more than 500 individual proteins in the adhesome as well as dynamic alterations in adhesome composition depending on changes in the mechanical environment (90, 91). Force-induced alterations to the composition, interactions, and activation state of signaling molecules within cell-matrix adhesions are thought to underlie the cellular response to the physical environment. However, the overwhelming complexity of these interactions has prompted a focus on downstream transcriptional points of signal integration (reviewed below), or on integrins themselves as upstream points of signal initiation.
Mechanosensing mechanisms in injury, repair, and fibrosis. Cells receive mechanical cues via mechanosensitive proteins at the cell membrane–cytoskeletal cortex interface (e.g., PIEZO1/2), as well as cell-cell and cell-matrix adhesions, with cadherins and integrins being the most common mechanical signaling interfaces. Mechanical signal processing occurs through adhesion protein clustering, stabilization of protein-protein interactions (e.g., integrin-talin), and activation of biochemical and transcriptional signaling pathways. These signals may initiate at cell-cell or cell-matrix adhesion sites, or as a consequence of cytoskeletal remodeling (actin, myosin, Rho/ROCK) within the cytoplasm. Cytoskeletal remodeling can also transmit forces across the nuclear envelope (nesprins, lamins), potentially directly altering the environment for transcription. The combination of forces directly transmitted to the nucleus and the nuclear localization of mechanoactivated transcriptional regulators combine with a variety of epigenetic mechanisms to transiently or persistently alter cellular programs that drive injury, repair, and fibrosis responses.
Interest in targeting integrins in fibrosis originally gained momentum with the delineation of αvβ6 integrin as a key mechanism for extracellular TGF-β activation, highlighting this integrin’s therapeutic potential as a target in preclinical models of pulmonary and liver fibrosis (92–94). Studies expanded the investigation of integrin targeting to additional organs and introduced small-molecule integrin inhibitors, particularly directed at the αv subunit (95–99). Recent cell biological studies reveal an important role for αv integrins in mechanosensing (100, 101), potentially linking these integrins to mechanobiological responses that occur in fibrotic tissue remodeling. Thus while integrin targeting remains complicated by the relatively limited repertoire and broad tissue distribution of these key cell adhesion proteins, along with the complex intracellular signaling cascades triggered by cell adhesion, the promising results in preclinical models suggest the potential for integrins to emerge as viable therapeutic targets in fibrosis.
Cell-cell mechanosensing. Cells also derive important information, including mechanical signals, from cell-cell adhesions. The most studied of the cell-cell adhesion mechanotransducers are cadherin-based adhesions, which form via homotypic interactions between membrane-spanning cadherins on adjacent cells (ref. 102 and Figure 2). As in cell-matrix adhesions, the physical state of actomyosin tension plays an important role in the formation, organization, and downstream signaling from these cell-cell adhesion complexes, while the junctions themselves provide signals that regulate cell polarity, collective migration, barrier function, and cell state. Forces transmitted across cell-cell adhesions contribute to long-range tissue integrity and morphogenesis (103) and are essential to collective migration (104). A well-described endothelial mechanotransducing complex that mediates responses to luminal shear is localized at cell-cell junctions and contains VE-cadherin, PECAM-1, and two VEGF receptors, VEGFR2 and VEGFR3 (105–110). Flow and mechanical force trigger engagement of PECAM-1 with the vimentin cytoskeleton and activation of an Src family kinase, leading to sequential activation of VEGF receptors, PI3K, and endothelial nitric oxide synthase, and generation of nitric oxide to regulate vascular tone. VEGF receptor tyrosine kinase inhibitors are well-tolerated therapies used to treat various malignancies, and preclinical studies show promise in promoting deactivation of myofibroblasts and treatment of fibrosis (111).
Beyond shear sensing, cell-cell adhesions are critical to barrier function. Internally generated cytoskeletal forces or externally generative physical forces can disrupt cell-cell adhesion, altering both mechanical homeostasis and barrier function (64). Recently, physical cues have been identified as important regulators of cell-cell signaling via the Notch pathway (112–114), with significant therapeutic implications for pulmonary, dermal, and renal fibrosis (60, 115, 116). Thus, cell-cell adhesions provide both mechanical and biochemical signals that are likely relevant to both endothelial and epithelial injury, as well as the transition from injury to healing or fibrotic scarring. While less appreciated, interstitial cells such as fibroblasts also express cadherins that mediate physical cell-cell signaling cues to modulate cell phenotype (117, 118), and recent studies specifically implicate cadherin 11 in fibrotic tissue remodeling (119).