0023-6837/03/8312-1689$03.00/0 LABORATORY INVESTIGATION Vol. 83, No. 12, p. 1689, 2003 Copyright © 2003 by The United States and Canadian Academy of Pathology, Inc. Printed in U.S.A. MINIREVIEW Normal and Pathologic Soft Tissue Remodeling: Role of the Myofibroblast, with Special Emphasis on Liver and Kidney Fibrosis Alexis Desmoulière, Ian A. Darby, and Giulio Gabbiani Groupe de Recherches pour l’Etude du Foie (AD), INSERM E0362, Université Victor Segalen Bordeaux 2, Bordeaux, France; Wound Healing and Microvascular Biology Group (IAD), School of Medical Sciences, RMIT University, Victoria, Australia; and Department of Pathology (GG), Centre Médical Universitaire, Geneva, Switzerland he retractile and remodeling phenomena accom- Definition and Cytoskeletal Markers of Myofibroblasts panying and, in pathologic situations, following T Granulation tissue, which allows the replacement of the healing of an open wound have attracted the the injured tissue, is mainly characterized by fibroblast interest of physicians and scientists for many centu- proliferation, angiogenesis, and extracellular matrix ries. It has long been assumed that these phenomena deposition. Fibroblasts acquire smooth muscle (SM) depend on extracellular matrix reorganization; how- features characterizing the myofibroblast (for review, ever, the demonstration that fibroblastic cells acquire see Desmoulière and Gabbiani, 1996; Schürch et al, contractile features during the evolution of granulation 1998; Serini and Gabbiani, 1999; Tomasek et al, 2002) tissue (for review, see Schürch et al, 1998) has that represents the main cellular element present in switched the interest from the extracellular to the granulation tissue (Gabbiani, 1998). Myofibroblasts intracellular compartment. The past 30 years have contain cytoplasmic bundles of microfilaments or seen the accumulation of advances in the field of stress fibers, which with mechanism similar but not contractile fibroblasts or myofibroblasts that presently identical to those taking place in SM cells, play a role appear to be the key cell for the production of granu- in contraction (Grinnell, 1994, 2000). Myofibroblasts lation tissue remodeling. Myofibroblasts are obviously are interconnected by gap junctions and are also also producing extracellular matrix components such connected to the extracellular matrix by a specialized as collagen and fibronectin, and it has become clearer structure called fibronexus, a transmembrane com- that granulation tissue remodeling depends on the plex involving intracellular microfilaments in continuity coordinate activities of intracellular and extracellular with extracellular fibronectin fibers (Eyden, 1993; molecules. The interactions of these molecules are Singer et al, 1984). More recently, the fibronexus has under the control of microenvironmental factors such been assimilated to the mature or supermature focal as transforming growth factor-␤ (TGF-␤) or other cy- contact, ie, three-dimensional transcellular structure tokines and mechanical stress. The purpose of our containing the fibronectin isoform ED-A and ␣-SM review is to summarize recent advances made in this actin; these are organized by intracellularly and extra- field by several laboratories, with special emphasis on cellularly originated forces and play a role in the the evolution of fibrotic phenomena in the liver and the establishment and modulation of the myofibroblastic kidney. phenotype (Dugina et al, 2001). Recently, it has been shown that ␣-SM actin is crucial for focal adhesion maturation in myofibroblasts (Hinz et al, 2003). Fur- thermore, myofibroblasts are the main cellular type DOI: 10.1097/01.LAB.0000101911.53973.90 involved in extracellular matrix deposition during tis- Received July 21, 2001. sue repair (Zhang et al, 1994). To replace the damaged This work was supported in part by the Swiss National Science Foundation tissue, myofibroblasts participate actively in the syn- (grant no. 31–61.336.00) and a visiting fellowship by the University of thesis of extracellular matrix components such as Bordeaux 2 (to IAD). Address reprint requests to: Dr. A. Desmoulière, GREF, INSERM E0362, collagens I and III, tenascin, and fibronectin. Myofibro- Université Victor Segalen Bordeaux 2, 146, rue Léo-Saignat, 33076 blasts are also responsible for the synthesis of en- Bordeaux cedex, France. E-mail: [email protected] zymes involved in matrix degradation. Laboratory Investigation • December 2003 • Volume 83 • Number 12 1689 Desmoulière et al Myofibroblasts express different sets of cytoskeletal related to the contractile activity of myofibroblasts. proteins that represent useful markers of cell differen- Indeed, it has been recently demonstrated both in vitro tiation; the study of their expression allows the char- and in vivo a direct correlation between the level of acterization of fibroblastic phenotypic modifications ␣-SM actin expression and myofibroblast contraction corresponding to functional changes that take place (Hinz et al, 2001a, 2001b). This point, and the role of during physiologic and pathologic repair processes mechanical factors in proto-myofibroblastic and myo- (Sappino et al, 1990; Schmitt-Gräff et al, 1994). The fibroblastic differentiation, will be discussed exten- cytoskeleton of eukaryotic cells comprises three types sively below (see “Force Generation by the Myofibro- of filaments. The intermediate filaments, which are blast: Role and Factors Involved” section). Proto- rigid and permanent, strengthen cells against me- myofibroblasts and myofibroblasts can also express chanical stress; their composition depends on cell other SM cytoskeletal proteins, including desmin, type. In mesenchymal cells, they are homopolymers of caldesmon, and SM-myosin heavy chains (for review, vimentin or desmin in the cytoplasm and of lamins in see Desmoulière and Gabbiani, 1996); however, while the inner surface of the nuclear membrane (Raats and particularly useful in vivo to characterize the SM Bloemendal, 1992). The microtubules are hollow tubes differentiation of the fibroblastic cells in different that play a crucial role in cell organization and drive pathologic situations, the roles of these proteins are intracellular transport via motor proteins; they consist not fully understood. In contrast, differentiated myofi- mainly of tubulin. Finally, the microfilaments are thin broblasts do not express smoothelin, a recently de- and flexible, essential for intracellular movements, and scribed late differentiation marker of both vascular and consisting of actin and myosin. Actin exists as six parenchymal SM cells (Christen et al, 2001; Van der different isoforms: two cytoplasmic or nonmuscle ac- Loop et al, 1997). The expression of smoothelin is at tins (␤ and ␥), and four muscle actins (two striated present the most reliable criterion to distinguish be- muscle actins: ␣-skeletal and ␣-cardiac; and two SM tween the differentiated myofibroblast, as defined actins: ␣ and ␥) (Vandekerckhove and Weber, 1978; here, and the mature SM cell (Van der Loop et al, Buckingham et al, 1984). The actin filament is a 1996). two-stranded helix of actin monomers and can asso- ciate with a myriad of actin binding proteins (capping, Myofibroblasts during Normal Healing in the Skin severing, cross-linking, bundling, nucleating, seques- tering, or motor proteins) that enable the filament to Normal wound healing includes a number of overlap- perform a variety of functions. Myosins are motor ping phases. After injury, there is an early inflamma- proteins that hydrolyze ATP, providing the energy for tory step characterized by hemorrhage and clotting. their movement along actin filaments. A large variety During this phase, platelet degranulation occurs, re- of myosin isoforms is present in muscle (striated or leasing a cocktail of growth factors into the local smooth) and nonmuscle cells. Conventional or class II environment that helps to attract inflammatory cells, myosins are hexamers of two heavy chains and two particularly monocytes and granulocytes, to the site of pairs of light chains (Loukianov et al, 1997; Schiaffino damage. These cells in turn release numerous factors and Reggiani, 1996). Numerous recent studies have that stimulate the repair process. At this time, the illuminated our understanding of dynamics of actin wound has a provisional extracellular matrix consist- cytoskeleton. Assembly and disassembly of actin fila- ing largely of fibrin. This provisional matrix serves to ment bundles result from the association of various seal the wound temporarily and allows the invasion of actin binding proteins and are regulated by small cells that carry out the repair process by fibrogenesis GTPases related to the Ras superfamily. Rearrange- and angiogenesis (Yamada and Clark, 1996). It also ment of actin cytoskeleton, particularly at the surface provides a substrate on which epithelial cells migrate membrane, mediates different cellular processes. Rho to re-epithelialize the wound. In the next phase, allow- GTPases for instance, in response to extracellular ing the development of the granulation tissue, fibro- signals, regulate the interaction between stress fibers, blasts invade the wound and commence replacing the contractile actin-myosin filaments and focal adhesion provisional matrix with a more mature wound matrix complexes (Hall, 1998; Machesky and Hall, 1996; containing collagen, fibronectin, and other matrix mol- Molitoris, 1997). Markers of actin cytoskeleton are ecules. In this phase, angiogenesis