Myofibroblasts and lung fibrosis induced by carbon nanotube exposure

Abstract

Carbon nanotubes (CNTs) are newly developed materials with unique properties and a range of industrial and commercial applications. A rapid expansion in the production of CNT materials may increase the risk of human exposure to CNTs. Studies in rodents have shown that certain forms of CNTs are potent fibrogenic inducers in the lungs to cause interstitial, bronchial, and pleural fibrosis characterized by the excessive deposition of collagen fibers and the scarring of involved tissues. The cellular and molecular basis underlying the fibrotic response to CNT exposure remains poorly understood. Myofibroblasts are a major type of effector cells in organ fibrosis that secrete copious amounts of extracellular matrix proteins and signaling molecules to drive fibrosis. Myofibroblasts also mediate the mechano-regulation of fibrotic matrix remodeling via contraction of their stress fibers. Recent studies reveal that exposure to CNTs induces the differentiation of myofibroblasts from fibroblasts in vitro and stimulates pulmonary accumulation and activation of myofibroblasts in vivo. Moreover, mechanistic analyses provide insights into the molecular underpinnings of myofibroblast differentiation and function induced by CNTs in the lungs.In view of the apparent fibrogenic activity of CNTs and the emerging role of myofibroblasts in the development of organ fibrosis, we discuss recent findings on CNT-induced lung fibrosis with emphasis on the role of myofibroblasts in the pathologic development of lung fibrosis. Particular attention is given to the formation and activation of myofibroblasts upon CNT exposure and the possible mechanisms by which CNTs regulate the function and dynamics of myofibroblasts in the lungs. It is evident that a fundamental understanding of the myofibroblast and its function and regulation in lung fibrosis will have a major influence on the future research on the pulmonary response to nano exposure, particle and fiber-induced pneumoconiosis, and other human lung fibrosing diseases.

Keywords: Animal model; Carbon nanotube; Extracellular matrix; Lung fibrosis; Mechanism; Myofibroblast.

Fig. 1

CNT-induced lung fibrosis. CNTs are respirable fibers with a tendency to deposit, penetrate and accumulate in lung tissues (left box). Exposure to CNTs induces acute phase responses including an inflammatory response, represented by the recruitment of Mac2-positive macrophages, and a fibrotic response, shown by Picro-Sirius Red staining for collagens I and III. Acute phase responses start as early as day 1, reach an apex on day 7, and decline after day 7 to significantly lower levels on day 14 post-exposure. In this scenario, day 7 post-exposure may represent an acute-to-chronic transition of CNT-induced pathology in mouse lungs (middle box). CNT-induced chronic phase responses are characterized by interstitial fibrosis and formation of epithelioid granulomas, shown by Masson’s Trichrome staining for collagen fibers on day 28 post-exposure. CNT-induced lung fibrosis appears to be persistent and irreversible in studies for up to 1 year post-exposure (right box)

Fig. 2

Fibroblasts and myofibroblasts in fibrosis. Fibroblasts and myofibroblasts act as the effector cells in organ fibrosis. Upon fibrogenic stimulation, tissue resident fibroblasts are activated to migrate and proliferate. Activated fibroblasts are the major progenitor cells to differentiate into myofibroblasts, indicated by the de novo expression of α-SMA. Myofibroblasts possess several characteristics, which distinguish them from fibroblasts and render them unique and critical functions in organ fibrosis

Fig. 3

α-SMA expression induced by MWCNTs in mouse lungs. Pulmonary exposure to MWCNTs (XNRI MWNT-7, 40 μg) for 7 days strongly induces α-SMA expression. α-SMA expression in well-formed fibrotic foci is shown by immunohistochemistry (upper panel) and in less well-formed, early stage fibrotic foci by immunofluorescence (lower panel), respectively (scale bar: 20 µm; for immunofluorescence, red: α-SMA staining, blue: nuclear DAPI staining)

Fig. 4

Regulation of myofibroblast formation by TGF-β1. a Schematic presentation of TGF-β1 signaling in myofibroblast formation. Upon stimulation, latent TGF-β1 is activated and active TGF-β1 is released to bind to its receptors on the cell surface to drive the Smad-dependent pathway, which directly up-regulates the transcription of fibrotic genes encoding α-SMA, collagens, and fibronectin. Binding of active TGF-β1 to its receptors also elicits a number of Smad-independent pathways, such as the PI3K-AKT signaling, which may promote myofibroblast differentiation and function. b Role of TGF-β1 in CNT-stimulated myofibroblast differentiation. CNTs induce the production and secretion of TGF-β1 by macrophages and epithelial cells, which serves as a paracrine factor to stimulate fibroblast-to-myofibroblast differentiation. CNTs also directly induce fibroblasts to produce and secrete TGF-β1, which functions as an autocrine factor for fibroblasts to differentiate into myofibroblasts. CNTs may directly promote fibroblast-to-myofibroblast differentiation by mimicking the ECM or intracellular collagen fibers to generate mechanical stress. In addition, CNTs stimulate epithelial cells to produce and secrete TGF-β1, which may induce the trans-differentiation of epithelial cells to myofibroblasts via EMT

Fig. 5

Mediators of CNT-induced myofibroblast differentiation in the lungs. CNTs stimulate multiple mechanisms and mediators capable of promoting myofibroblast formation and function, including a pro-fibrotic growth factors TGF-β1 and PDGF, b Th2 cytokines IL-4 and IL-13, c ROS, d pro-fibrotic cytokines TNF-α, IL-6, IL-1α and IL-1β, e TIMP1, and f tissue stiffness. Activation of these signaling cascades may induce myofibroblast differentiation and activation directly or by boosting fibroblast proliferation to increase the myofibroblast precursor pool, leading to fibrosis in the lungs