INTRODUCTION

TOP ABSTRACT INTRODUCTION ROLES IN HEALTH AND... DEFINITION OF A MYOFIBROBLAST ORIGIN OF MYOFIBROBLASTS AND... ACTIVATION, PROLIFERATION, AND... ROLE IN WOUND REPAIR ROLE IN FIBROSIS CONCLUSIONS AND SPECULATION REFERENCES

A GROWING BODY OF LITERATURE over the last decade has made it evident that there is phenotypic heterogeneity among fibroblasts and that some express features of smooth muscle differentiation (73, 136, 214, 216). These smooth-muscle-like cells, or myofibroblasts as they were termed by Gabbiani (82, 83) who pioneered this field, take part in the growth, development, and repair of normal tissue as well as the diseases affecting many different organs. These cells belong to a unique class, and, even allowing for specific functions for those cells in a given organ or tissue, there is an amazing similarity in their morphology, function, and biochemical repertoire regardless of their location. Nonetheless, in a given tissue, they may express some specific appearances and functions, i.e., phenotypic and functional heterogeneity. Because of this propensity and their location next to epithelial or parenchymal cells, we have suggested they might be termed "juxtaparenchymal cells" (247).

In this review, we give an overview of myofibroblasts, illustrating similarities and differences in their biochemical/physiological/immunologic properties, and we indicate the role that these cells play in specific disease states. The major soluble factors secreted by these cells are discussed, and important receptors on myofibroblasts are listed. We have slanted the discussion toward the intestinal myofibroblasts (247): the interstitial cells of Cajal (ICC) and the subepithelial intestinal myofibroblast. This review is not meant to be entirely comprehensive of the field of myofibroblasts. It focuses on recently discovered information about the interactions of myofibroblasts with epithelial and parenchymal cells and the molecules that mediate these interactions. Furthermore, we have purposely referenced review articles when possible to amplify the reference base.

	ROLES IN HEALTH AND DISEASE

TOP ABSTRACT INTRODUCTION ROLES IN HEALTH AND... DEFINITION OF A MYOFIBROBLAST ORIGIN OF MYOFIBROBLASTS AND... ACTIVATION, PROLIFERATION, AND... ROLE IN WOUND REPAIR ROLE IN FIBROSIS CONCLUSIONS AND SPECULATION REFERENCES

Table 1 lists various tissue myofibroblasts and what is thought to be their normal function. Some of these functions are well proven, and others can be inferred from the known properties of myofibroblasts in other tissues. In general, there are several common normal activities of myofibroblasts. First, through mesenchymal-epithelial interactions, myofibroblasts are key components of organogenesis or morphogenesis, i.e., the growth and differentiation of the tissue or organ (227). They do so through the secretion of soluble mediators of inflammation and growth factors (Table 2) and expression of their receptors (Table 3) and through secretion and formation of interstitial matrix and/or basement membrane molecules (Table 4) (20, 73, 82, 247). Myofibroblasts also play a fundamental role in many disease states, either through activation and proliferation or through deletion (Table 5) (51, 214, 216). They play a central role in wound healing, presumably as an extension or accentuation of their role in normal growth and differentiation (82, 83, 99, 120, 136). They appear to be involved in the formation and repair of the extracellular matrix (ECM) and proliferation and differentiation of epithelial (or parenchymal), vascular and neurogenic elements (50, 215, 250, 262).

Healing is facilitated by the fact that the myofibroblasts are contractile, which aids in reducing the amount of denuded surface area of wounded tissue (163, 192, 242). An extension of this contractile capability allows these cells to participate in the ejection of fluid from the gastric glands (237) and in the motility of intestinal villi (120). Their relationship to myoepithelial cells, which have this function in the breast lobule (204) and the seminiferous tubules of the testis (106), is unclear. The contractile property of pericytes and specialized pericytes such as the hepatic stellate (Ito) cell, the mesangial cell of the kidney, and the glomus cell of peripheral vessels gives these myofibroblasts the ability to locally autoregulate blood flow (122, 124, 160, 177, 216, 247). The ability to accumulate and extrude calcium causes cyclic increases and decreases in calcium concentration in these cells, permitting actin-myosin contraction (77). This process is probably the same as or similar to the one responsible for the ability of the ICC to perform a pacemaker function in the intestine (212). Because myofibroblasts are interconnected through gap junctions (120, 212, 246), the electrical signals created by cyclic ion movements can be transmitted through the syncytium and thus through the length of the resident organ.

Myofibroblasts play a major role in the inflammatory response. These cells are avid producers of both chemokines and cytokines (105, 178, 179, 227, 235, 267) and are capable of augmenting or downregulating the inflammatory response by the secretion of these soluble mediators of inflammation (Table 2). They also synthesize prostaglandins, expressing both the constitutive cyclooxygenase-1 (COX-1 or PHS-1) gene product and the inducible COX-2 (PHS-2) protein (19, 69, 102, 105, 167, 240, 241, 259). In some tissues they make both nitric oxide and carbon monoxide, gases known or proposed to be important neurotransmitters and regulators of motility and inflammation (1, 14, 39, 69, 162, 167, 202, 240, 241). When activated, myofibroblasts also express adhesion molecules such as intracellular adhesion molecule-1, vascular cell adhesion molecule, and neural cell adhesion molecule (100, 133, 151, 178, 192). Thus lymphocytes, mast cells, and neutrophils may dock on the myofibroblasts and participate in organized immunological and inflammatory reactions (31, 48, 67, 105, 201, 236). Myofibroblasts also express  and  integrins that are part of the adhesion mechanism of myofibroblasts to matrix proteins (192). Through these or other properties, myofibroblasts participate in the formation of tissue granulomas (208, 247). Granulomas themselves are impressive factories of cytokines and other inflammatory mediators (87, 114).

Last, production of matrix molecules such as collagen, glycosaminoglycans, tenascin, and fibronectin in the interstitial space or basement membrane (Table 4) is part of the structure, growth, differentiation, and wound healing function of myofibroblasts (20, 227). These processes, when unchecked, deranged, or repeated, can result in tissue fibrosis (4, 25, 71, 72, 88, 138, 150). Therefore, fibrotic disease is a major pathological end point of activated and proliferating myofibroblasts in most, if not all, tissues.

Disease states not mentioned in Table 3 are inflammatory pseudotumors of the lung, liver, or stomach (64) and neoplastic transformation of the myofibroblasts themselves. Recent evidence indicates that stromal tumors of the gastrointestinal tract are derived from c-kit mutations in gastrointestinal myofibroblasts (103). Another benign neoplasm of myofibroblasts is the desmoid tumor, which can occur in any tissue but develops most commonly in the mesentery of the gut (92). Although desmoids can and do occur sporadically, they are particularly common (increased 850 times) in the familial adenomatous polyposis (FAP) syndrome. FAP is a hereditary malignancy presenting as colonic polyps and colorectal carcinoma (233) that is due to mutations in the adenomatous polyposis coli gene (92). Malignant myofibroblasts are also observed in clearly aggressive tumors such as angiosarcomas, fibrosarcomas, histiocytomas, and mesotheliomas (46, 216). Myofibroblasts are also responsible for the desmoplastic reaction seen in many cancers, i.e., the proliferation of fibrotic tissue within or adjacent to the tumor itself. This is most often observed in breast carcinoma, carcinoid tumors, Hodgkin's disease, and malignant melanomas (70, 153, 204, 218, 264). The role of myofibroblasts in both hereditary and nonhereditary colon cancer is discussed in more detail later in part II of this review [which will appear in the August issue (191)] (see also Ref. 131).

	DEFINITION OF A MYOFIBROBLAST

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Myofibroblasts may be defined morphologically and immunologically through identification of expressed cytoskeletal proteins (214, 216). The simplest definition of a myofibroblast is that they are smooth-muscle-like fibroblasts. Some investigators choose to call them smooth-muscle-like cells or activated smooth muscle cells (159, 163, 237). Others refer to them as lipocytes because of their propensity to store retinoids (vitamin A) or as stellate cells, because of a shape change of transiently differentiated myofibroblasts (see below). Myofibroblasts may well represent an intermediate state between fibroblasts and smooth muscle cells. This is best demonstrated in the prostate (98, 125, 181), in the pericytes surrounding the fetal vessels of the placental stem villus (134), and in the stromal myofibroblasts of the breast (204).

In both cell culture in vitro and native tissues in situ, myofibroblasts possess several distinguishing morphological characteristics, some of which are present in fibroblasts or smooth muscle cells (Fig. 1). They display prominent cytoplasmic actin microfilaments (stress fibers), and they are connected to each other by adherens and gap junctions (51, 239). These cells are also in contact with the ECM by focal contacts once known as the fibronexus, a transmembrane complex made up of intracellular contractile microfilaments and the ECM protein fibronectin (65). Both fibronexus formation and stress fiber assembly are regulated by Rho, a newly described member of the RAS superfamily of small guanosine triphosphatases (GTPases) (94), specifically in mammalian cells by Rho A. These small, monomeric GTP-binding proteins also regulate myofibroblast morphology (191, 265). Often, an incomplete basal lamina surrounds the myofibroblasts. Gap junctions couple some myofibroblasts to the tissue smooth muscle, and the cells are commonly in close apposition to varicosities of nerve fibers (134, 212, 243).

View larger version (144K): [in this window] [in a new window]   Fig. 1.   A: transmission electron micrograph of a cultured lumen intestinal subepithelial myofibroblast (18Co). The cell membrane displays numerous caveolae. Stress fibers (bundles of actin microfilaments) are prominent. The cytoplasm is rich in rough endoplasmic reticulum, Golgi apparatus, and mitochondria. B: nucleus of an activated myofibroblast shows multiple indentations. Adherens (C) and gap junctions (D) are present between myofibroblasts. [From Valentich et al. (246).]

In some tissues, e.g., the liver (Ito cells) (88), intestine [both the ICC (243) and the subepithelial myofibroblasts] (78, 246), the orbital myofibroblast (195, 254), the synoviocyte of the joint space (11), and brain (astrocyte) (15, 166, 193), the myofibroblasts exist in two distinct morphological states (Fig. 2): 1) the "activated" myofibroblast, as described above, and 2) the stellate-transformed myofibroblast, which is considered to be a transiently differentiated myofibroblast. This generalization, correlating appearance and function, has not been verified in every tissue where such morphological heterogeneity has been seen. Agents (e.g., prostaglandins, cholera toxin, vasoactive intestinal polypeptide) that increase the cAMP content of activated myofibroblasts and cell-soluble cAMP analogs themselves induce stellate transformation in vitro within 24 h (78) and stop myofibroblast proliferation (5, 119).

View larger version (181K): [in this window] [in a new window]   Fig. 2.   Phase-contrast micrographs (A and B) and scanning electron micrographs (C and D) of stellate 18Co cells. The stellate myofibroblast displays a highly refractile cell body on phase-contrast microscopy and possesses a highly arborized array of cell processes with several orders of bifurcation. The cell processes are devoid of microvilli, whereas the cell body shows a dense array of long microvilli, giving it a shaggy appearance. [From Valentich et al. (246).]

Immunohistochemical characterization of myofibroblasts is based on antibody reactions to two of the three filament systems of eukaryotic cells (75, 116). These three systems are composed of 1) actin, a component of the microfilaments; 2) vimentin, desmin, lamin, or glial fibrillary acidic protein (GFAP), members of the intermediate filament system; and 3) the tubulins of the microtubules. Myofibroblasts have not been characterized with regard to tubulins. The  and  actins are expressed by all cells, including myofibroblasts, which may also express -smooth muscle (-SM) actin (214, 216). Myofibroblasts stain negatively for -cardiac and -skeletal actin (216). Myofibroblasts are not well characterized with regard to the newly defined myosin isoforms (75, 161, 217). In some tissues, such as the intestine and reticular cells of lymph nodes and spleen, myofibroblasts stain positive for smooth muscle heavy chain myosin or tropomyosin (old terminology) (216, 243).

Vimentin, desmin, and -SM actin are the three filaments most often used to classify myofibroblasts (161). Expression of these proteins may vary with the tissue studied within species and is subject to environmental factors, e.g., whether the cells are studied in situ or in culture and, even within a given tissue, whether the cells are activated by hormonal or cytokine treatment or by disease (191). Based on immunohistochemical staining of these filaments in a given tissue, a classification system has been proposed (134, 216). Myofibroblasts that express only vimentin are termed V-type myofibroblasts, those that express vimentin and desmin are called VD-type, those that express vimentin, -SM actin, and desmin are called VAD-type, those that express vimentin and -SM actin are called VA-type, and those that express vimentin and myosin are called VM-type.

In the intestine, the ICC express immunoreactive vimentin, -SM actin, and smooth muscle tropomyosin, suggesting that they are members of the V or VM (243) class of myofibroblasts. However, we have not been able to find references indicating that ICC have been explored with antibodies to -SM actin. The intestinal subepithelial myofibroblasts (ISEMFs) stain positive for vimentin and -SM actin and negative (or weakly) for desmin (VA-type). They also express smooth muscle myosin (thus may be called VAM-type myofibroblasts), although expression of myosin is less than that seen in corresponding smooth muscle cells in the same tissue (120, 153, 246). It is possible that both intestinal myofibroblasts, the ICC and ISEMF, could be the VA(M)-type.

Specific monoclonal antibodies have been developed to identify myofibroblasts in certain tissues. For example, the monoclonal antibody Gb42 recognizes placental myofibroblasts (134). The 8E1 monoclonal antibody reacts with many of the stellate-shaped myofibroblasts, such as GFAP-positive astrocytes, and both intestinal myofibroblasts, the ICC and ISEMF (79). Anti-GFAP serum stains astrocytes, pancreatic periacinar stellate cells, and hepatic stellate (Ito) cells (30). The PR2D3 antibody stains subepithelial myofibroblasts in the stomach and intestine, lung myofibroblasts, periductular myofibroblasts of the kidney, testes, and breast, Ito cells of the liver, umbilical cord stellate myofibroblasts, and both vascular and tissue smooth muscle of most organs (199). Antibodies against the protooncogene c-kit, the receptor for stem cell factor (SCF or steel factor), react with ICC (109, 132, 244) and possibly with pulmonary alveolar myofibroblasts (61). No systematic studies have been reported concerning the reactivity of all the other myofibroblasts to c-kit antibodies.

In many of the studies quoted above, only a subset of the (myo)fibroblasts stain with -SM actin antibodies in vivo or in vitro (culture). In vivo, not all fibroblastic-appearing cells are myofibroblasts. In culture, treatment with transforming growth factor- (TGF-) may induce uniform -SM actin staining of all the cells, providing the cells are of a single clone (226). Furthermore, activation to an -SM actin-expressing phenotype may require both TGF- and a specific cell-matrix interaction (118, 222) (see ACTIVATION, PROLIFERATION, AND MIGRATION OF MYOFIBROBLASTS).

	ORIGIN OF MYOFIBROBLASTS AND ROLE IN GROWTH AND DEVELOPMENT

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It is unclear whether myofibroblasts originate from progenitor stem cells (possibly neuroepithelial stem cells) (24, 157) from the neural crest (117) or simply transdifferentiate from resident tissue fibroblasts (81) or from tissue (e.g., vascular, intestinal, or uterine) smooth muscle cells (204). The close anatomic relationship of pericytes to vascular smooth muscle and of intestinal myofibroblasts to intestinal smooth muscle suggests a bidirectional route of transdifferentiation. These various possibilities are depicted in Fig. 3. It has recently been suggested that renal tubular cells (a cell of endoderm origin) might differentiate into myofibroblasts (mesenchymal cells) under noxious stimuli (171, 177, 198). However, it is equally likely that the peritubular interstitial myofibroblasts are proliferating under these circumstances and simply replacing apoptotic tubular cells in these disease states.

View larger version (23K): [in this window] [in a new window]   Fig. 3.   Proposed scheme depicting the origin, transdifferentiation, activation, and stellate transformation of myofibroblasts. PDGF, platelet-derived growth factor; TLP, tethered ligand protein; TGF-, transforming growth factor-; IL-1, interleukin-1; EGF, epidermal growth factor; bFGF, basic fibroblast growth factor; IGFI, insulin-like growth factor I; CTGF, connective tissue growth factor.

Two soluble factors have been shown to promote differentiation from embryonic stem cells: PDGF and SCF. PDGF has two chains, A and B, and exists as a homodimer (PDGF-AA or PDGF-BB) or as a heterodimer (PDGF-AB). Each form acts on separate receptors:  receptors that are nondiscriminatory and can bind AA, BB, and AB dimers or  receptors that are specific for the B chain (126). After ligand binding, there are two separate intercellular signaling pathways for the PDGF receptor: a mitogen-activated protein kinase (MAPK) path and one involving phosphatidylinositol 3-kinase (PI3K). Depending on the cell types, one pathway may be required for cell activation and/or proliferation and the other pathway for cell motility (migration) (3, 72, 150). For example, smooth muscle cells proliferate in response to the MAPK cascade and migrate in response to the PI3K path, whereas hepatic stellate cells and endothelial cells respond with both proliferation and migration via the PI3K (72).

Disruption of the PDGF-AA gene in mice is lethal in 50% of affected animals (26). The surviving animals are almost completely devoid of lung alveolar myofibroblasts (also called pulmonary contractile interstitial cells) and develop emphysema due to failure of lung septation. In contrast, animals born with disruption of the PDGF-BB gene have a virtual absence of renal mesangial cells and failure of the formation of the complex structure of the glomerulus (141, 234). The PDGF-BB-deficient animals also lack pericytes and thus develop microaneurysms, reminiscent of those seen in the diabetic retina, and leaky vessels that cause tissue edema and hemorrhage (142). Intestinal subepithelial myofibroblasts (119) and hepatic stellate cells (147) proliferate in response to low concentrations of PDGF-BB, suggesting that this growth factor is important in the growth and development of these cells.

The protooncogene c-kit is the transmembrane glycoprotein tyrosine kinase (III) receptor (160 kDa molecular mass) for SCF, a growth factor secreted by epithelial cells, white blood cells, and (myo)fibroblasts. SCF is also a member of the PDGF family, and the tyrosine kinase type III family also includes receptors for granulocyte/macrophage colony-stimulating factor (GM-CSF). Intestinal ICC (in situ and in culture) express c-kit as detected by rat anti-kit (ACK2) monoclonal antibodies (18, 109, 132, 174, 244). Mutations in the c-kit locus, the W mutants, result in abnormalities in the number, structure, and function of the ICC (18, 52, 174, 212, 249). Furthermore, mutants of the ligand SCF, steel (Sl) mutants, also show morphological and functional abnormalities of the ICC (257). Thus the PDGF family of growth factors seems crucial for the embryological development of myofibroblasts. Unfortunately, no systematic study of the various different tissue myofibroblasts has been reported in PDGF or SCF knockout mice or in mutants of their respective receptors.

TGF-, PDGF, insulin-like growth factor II (IGF-II), and interleukin-4 (IL-4) appear to be the most important growth factors for the transdifferentiation of fibroblasts to myofibroblasts or of stellate-transformed myofibroblasts into activated myofibroblasts (45, 61, 145, 211, 239). When myofibroblasts from the intestine (74), breast (203), skin (17, 111, 263), liver (88), lung (214), prostate (181), nose (256), and joint synovium (214) are treated with TGF- in serum-containing media, they express -SM actin, reduce the number of vitamin A lipid droplets, and expand the rough endoplasmic reticulum, i.e., they take on the morphology of an activated myofibroblast. Conversely, interferon (IFN)- and IFN- (56, 90) decrease the expression of -SM actin in myofibroblasts. It is not clear whether they do so by transdifferentiating myofibroblasts back to the fibroblast state, inducing them to undergo stellate transformation, or simply downregulating the amount of -SM actin in the cell.

	ACTIVATION, PROLIFERATION, AND MIGRATION OF MYOFIBROBLASTS

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Fibroblasts or stellate-transformed myofibroblasts become activated and proliferate when cultured on plastic in serum-containing growth culture media, especially when seeded at low cell density (74, 155). In vivo activation, as signified by the development of -SM actin positivity, may be separable from proliferation. Whereas many fibrogenic cytokines [IL-1, tumor necrosis factor (TNF)-, PDGF, fibroblast growth factor (FGF), and TGF-] have been incriminated in this process (138), TGF- appears to be the most important cytokine causing the development of -SM actin staining and an activated phenotype (25, 45, 88, 97, 155, 165, 223, 238) capable of collagen secretion (25, 45, 165). The source of TGF- in damaged tissue may be from white blood cells, parenchymal or epithelial cells, or from the myofibroblast itself in an autocrine fashion (22, 25, 45, 88). Recently, it has been determined that the activation of the myofibroblast requires the presence of matrix molecules, specifically, the ED-A (EIIIA) domain of fibronectin (118, 222). Tissue injury gives rise to this specific ED-A domain splice variant of fibronectin. ED-A is the binding site for cell membranes and for other matrix molecules. It has been shown in both skin granulation tissue (222) and hepatic (118) models that this fibronectin ED-A domain is necessary for TGF- to trigger -SM actin expression and collagen secretion by myofibroblasts. Following activation of the myofibroblast, PDGF or connective tissue growth factor (CTGF), a member of the PDGF family (29), appears to be the factor primarily responsible for myofibroblast proliferation (71, 72, 89, 119, 147). TGF- was once considered the prime factor (88, 214, 216), but it is now thought that TGF- acts predominantly through the induction of PDGF receptors on or synthesis of CTGF by the myofibroblasts (72, 89, 111, 112, 263). Thus TGF- is predominantly a cytodifferentiating rather than a proliferating growth factor.

The TGF-s are a large superfamily of soluble factors important in growth, development, and fibrogenesis (149). TGF-1, TGF-2, and TGF-3 are encoded from three separate genes. TGF-1 is the isoform usually upregulated in the presence of tissue injury. It is secreted in a latent form after cleavage from a large promolecule and then noncovalently binds to another peptide on the cell membrane called the latency-associated peptide, which, in turn, is formed from the cleavage fragments of the TGF- precursor. This latent TGF- is stored on the surface of the cell or on the extracellular matrix, awaiting conversion by unknown mechanisms to active TGF-. In contrast to their apparent (but probably indirect) proliferative effect on myofibroblasts (see above), TGF-s cause G₁ phase cell cycle arrest of epithelial and smooth muscle cells and may even induce apoptosis (25). TGF- acts through a superfamily of serine-threonine kinase cell surface receptors. All three TGF-s bind first to the type II (RII) receptor that assembles and phosphorylates the type I (RI) receptor, activating this serine-threonine kinase and transducing the signal (25). Microsatellite (genomic) instability due to defects in DNA mismatch repair systems of the TGF- receptors has been incriminated in the unregulated growth of cancer and in vascular atherosclerosis/restenosis (159). This raises the question of a role for myofibroblasts in these two diseases [see part II of this review (191)].

TGF- (138), a member of the epidermal growth factor (EGF) family, as well as EGF itself (138, 214, 216, 226), GM-CSF (25), both acidic and basic FGF (aFGF and bFGF, respectively) (25, 119, 185, 214, 216, 226), and IGF-I and IGF-II (25, 226) are candidate growth factors promoting myofibroblast proliferation (see more details in Growth Factors). Proinflammatory cytokines such as TNF-, IL-1, IL-6, and IL-8 may also cause activation and proliferation (119, 138, 177, 185, 246) as does IL-4, a protein generally thought of as an anti-inflammatory cytokine (61, 156, 211).

ANG II or aldosterone, thrombin, and endothelin are also important soluble factors reported to promote myofibroblast activation (15, 34, 35, 77, 246, 258). Endothelin is capable of rapidly transdifferentiating the stellate morphology of intestinal myofibroblasts to the activated phenotype within 30 min of addition to cell culture media (78, 80, 246). After it activates the myofibroblasts, endothelin may subsequently inhibit their proliferation (147, 148).

Cocultures of fibroblasts and myofibroblasts with cancer cells of several different types induce transdifferentiation of fibroblasts to myofibroblasts and activation and proliferation of myofibroblasts (13, 19, 46, 70, 136, 149, 153, 204, 246, 264). This property of neoplastic cells, perhaps via secretion of growth hormones such as TGF-, may well be responsible for the desmoplastic reaction (excessive fibrosis) seen in many cancers.

	ROLE IN WOUND REPAIR

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The process of wound healing is a highly orchestrated event that entails the release of proinflammatory cytokines, eicosanoids of the cyclooxygenase, lipoxygenase, and cytochrome P-450 family, nitric oxide, and a host of growth factors; the secretion of collagen and other matrix proteins; the elaboration of angiogenic, angiostatic, and nerve growth factors; and, finally, if it is a deep or open wound, the formation of granulation tissue that then becomes a scar (fibrosis) (20, 57, 187, 208, 214, 247, 252). Myofibroblasts appear to be key cells in these various events. They become activated and proliferate in the early stages of wounding. They respond to proinflammatory cytokines with elaboration of matrix proteins and additional growth factors and then disappear by apoptosis following repair or scar formation (51, 54, 55, 113, 164, 266).

Repair Processes

Conversely, if the wound is deep, the subepithelial tissues that contain interstitial substance, blood vessels, nerves, and fibroblasts must be reconstituted. If the basement membrane has been destroyed by the noxious stimulus, epithelial cells and mesenchymal elements form a new basement membrane (252). Epithelial stem cells then undergo mitosis and proliferate and migrate along the newly formed basement membrane. This latter process is a coordinated event involving secretion of matrix proteins and growth factors. Thus myofibroblasts appear to play roles both in the restitution and repair processes.

A key event in the process of wound repair by either restitution or proliferation is contraction of the underlying granulation tissue or gastrointestinal lamina propria to limit the exposed surface area of the wound (51, 120, 163, 192, 208, 242). Myofibroblasts contain smooth muscle myosin isoforms in addition to -SM actin, the requisite machinery for contraction and/or motility. The ability of myofibroblasts to carry out these processes depends on changes in the cellular cytoskeleton as well as in the Rho-regulated fibronexus and on the expression of integrins that allow attachment of the myofibroblasts to the extracellular matrix (192, 242).

The fibronexus, discovered and characterized by Eyden (65) and Singer and colleagues (228-232) connects the myofibroblast to the extracellular matrix through a transmembrane integrin complex that joins the actin stress fiber of the myofibroblast to ECM fibronectin (32, 192). The Rho family of small GTPases includes Rac 1-3, Cdc 42, and Rho A-H, which respond to PDGF, TNF- or bradykinin, or lipopolysaccharide A, respectively. In mammals, Rho A acts on the actin cytoskeleton to cause myofibroblast shape change or motility (6, 265).

ECM

Growth Factors

Individual growth factors may be produced by the epithelial cells alone (trefoil proteins), by mesenchymal cells such as myofibroblasts or inflammatory cells, particularly macrophages and lymphocytes, and some by both cell types (67). Furthermore, the various inflammatory cytokines, eicosanoids, and growth factors released during tissue damage may directly affect the epithelium or parenchymal cell of the injured tissue, or these agents may act more proximally on myofibroblasts to induce these cells to secrete additional cytokines, eicosanoids, or growth factors (67). Thus an in vivo epithelial proliferative response could be the result of a cytodifferentiating effect of mediators on the myofibroblasts, inducing them to express receptors for other factors or to secrete specific epithelial proliferating growth factors. Examples of this are TGF-1, which induces the expression of PDGF receptors on or CTGF secretion by the myofibroblasts, causing them to proliferate in response to PDGF (137), or the secretion of hepatocyte growth factor (HGF) or keratinocyte growth factor (KGF) by myofibroblasts in response to IL-1 (37) or immune stimulation (10, 66).

IL-1 (60), IL-6 (261), IL-15 (196), and TNF- (121, 139) have also been identified as being involved in tissue repair and have been shown to be mitogenic for several mesenchymal and epithelial cell lines. Furthermore, combinations of cytokines and growth factors may have offsetting effects on epithelial proliferation, so the ultimate consequence of these various factors in vivo can be quite complicated.

Factors secreted by myofibroblasts such as EGF and TGF- (12, 188), IGF-I and IGF-II (144, 145), HGF (28, 84, 173), and members of the FGF family, including aFGF, bFGF (also known as FGF-2) (110), KGF (also known as FGF-7) (107, 207), and IL-11 (38, 175, 194), have been demonstrated to be the major paracrine growth factors for epithelial and parenchymal cells. The trefoil peptides, secreted by the epithelial cells themselves, have similar effects through autocrine stimulation (186). These factors may also have nonmitogenic effects on intestinal cells as well, e.g., they may regulate secretory and contractile processes as well as regulate blood flow (245).

The trefoil peptides are so named because of a distinctive pairing of six cysteine residues that results in three interchained loops, thus giving a "three leaf" trefoil shape (38, 186). There are three such trefoil proteins that are small, highly stable molecules secreted principally by the goblet (mucus)-secreting cells of the epithelium and not by myofibroblasts. The stomach secretes peptide pS2 in the fundus and spasmolytic polypeptide (SP) in the antrum, whereas the breast epithelium secretes only pS2 and the pancreas secretes only pancreatic SP (pSP) (129). In contrast, the intestinal epithelium secretes only intestinal trefoil factor (ITF). Targeted gene disruption of ITF causes abnormal epithelial cells and increased susceptibility to various models of injury, resulting in a colitis-like picture (7, 130). Exogenous administration of ITF repairs the damage susceptibility in this knockout model and also protects the gastric mucosa against other injuries such as those induced by ethanol or chronic indomethacin administration (7). PS2 gene knockout mice have a different disease phenotype; they develop extensive neoplastic adenomas in the antrum of the stomach, which then progress to carcinoma in situ (140).

TGF-, EGF, and the EGF human homologue urogastrone (EGF/URO) are members of the same family of polypeptides and act on a common cell membrane receptor (12, 188). The TGF-/EGF receptor appears to be upregulated in the mucosa of injured intestine and other organs. TGF- is expressed in epithelial cells, myofibroblasts, and monocytes/macrophages, whereas EGF seems to be produced primarily by the epithelial cells of the salivary gland and Brunner's glands of the duodenum. TGF- is synthesized as a 160-amino acid precursor molecule that spans the cell membrane. Proteases release the soluble 50-amino acid form. It is unclear whether the membrane-bound form is active as a growth factor for adjacent cells. The soluble factor is trophic (mitogenic) for a number of cell lines in vitro and intestinal epithelial cells in vivo. It may well have differentiating functions as well. Ulceration of the human gastrointestinal mucosa causes the development of a specific cell lineage from epithelial stem cells that bud from the crypts next to an ulcer and then ramify to form a small gland. These budding glands secrete EGF/URO, which stimulates epithelial proliferation and promotes ulcer healing (260).

The FGF family (aFGF and bFGF) are important mitogens for myofibroblasts and have powerful neurotrophic and angiogenic properties that are important for tissue healing (68, 110). Other angiogenic factors secreted by myofibroblasts include the CXC family of cytokines such as IL-8 and epithelial neutrophil-activating peptide (ENA-78) (5, 127, 178). Cell-to-cell contact such as that occurring in restitution or wound healing has its antiapoptotic action via the adhesion molecule N-cadherin. When the adjacent cells touch, there is homophilic binding of N-cadherin molecules, which activate the FGF family of receptors. In this way, cell contact mimics the antiapoptotic effect of bFGF (91).

IGF-I and IGF-II are structurally related polypeptides that have various metabolic, proliferative, and differentiating effects through endocrine, autocrine, and paracrine mechanisms (144, 145). The effects are mediated by IGF-I receptors and insulin receptors. There is an IGF-II receptor, but its role in signal transduction is unclear. IGF is present in the circulation (from liver) and is also secreted in a paracrine fashion by myofibroblasts adjacent to epithelial and parenchymal cells (144). The IGF actions are determined by the availability of free IGF, the form that interacts with its receptors. In turn, the amount of free IGF is modulated by the level of high-affinity IGF-binding proteins (IGFBPs), of which six have been identified (42-44). These IGFBPs not only regulate the bioavailability of IGF but also inhibit or enhance its action on target tissues. Although the IGFs are weakly mitogenic for epithelial and parenchymal cells, they seem to be powerfully mitogenic for myofibroblasts (226) and other smooth muscle cells (255).

A new member of the family of factors stimulating epithelial growth is IL-11, a multifunctional cytokine originally derived from bone marrow stromal cells (175, 194). It regulates the growth of hematopoietic and lymphoid cells by acting on the IL-6 family of cytokine receptors. It stimulates proliferation of small intestinal crypts and accelerates recovery of the intestinal mucosa from models of damage (143). It also has trophic effects on neurons, preadipocytes, and myofibroblasts of the lung (175). TGF- and IL-1 are potent stimulants of IL-11 production. Paradoxically, IL-11 has been shown to inhibit epithelial cell proliferation in the lung by altering phosphorylation of the retinoblastoma protein (194). Thus it is possible that the proliferative effect of IL-11 on epithelia occurs via activation of myofibroblasts, with subsequent secretion of epithelial proliferating factors by these cells, rather than being a direct effect of IL-11 on the epithelium itself.

KGF is a member of the FGF family (FGF-7) (107, 207). This factor is unique because, unlike other members of the FGF family, it does not appear to have activity on fibroblasts, endothelial cells, or other nonepithelial targets. This is a consequence of the epithelial cell expression of the KGF receptor (KGFR), a transmembrane tyrosine kinase that binds KGF and aFGF with high affinity and binds bFGF much more poorly. The KGFR is nearly identical to the FGF receptor type II, except for alterations in a 49-amino acid residue in one of its extracellular loops. FGFR-II does not bind KGF but shows a high affinity for both aFGF and bFGF. KGFR expression is limited to epithelial cells, whereas FGFR-II is present in a variety of tissues including fibroblasts. KGF, initially isolated from lung fibroblasts, appears to be a myofibroblast-secreted epithelial growth factor with specific roles in epithelial growth and differentiation. The KGFR is expressed on the epithelial cells, and KGF has been shown to induce proliferation and differentiation of a host of epithelial and parenchymal cells, including intestinal epithelial cells, type II pneumocytes, hepatocytes, and keratinocytes of the skin. Its expression and secretion are regulated by IL-1 (37). Its synthesis is significantly upregulated in the lamina propria of inflamed intestine (66). Thus KGF represents a prime example of a mediator causing a specific mesenchymal-epithelial interaction.

HGF, also known as scatter factor because it induces cell migration as well as proliferation, is synthesized and secreted by fibroblasts and myofibroblasts (28, 84). HGF is a glycoprotein heparin-binding heterodimer related to plasminogen, consisting of a heavy  chain and a light  chain held together by disulfide bonds (84). It is produced as a single-chain precursor protein and proteolytically cleaved to form HGF. The HGF receptor, prominently expressed by epithelial cells, is encoded by the protooncogene c-met. This receptor is a heterodimeric glycoprotein of 190 kDa linked with two disulfide bonds; the  chain is extracellular, while the membrane-spanning  chain has the cytoplasmic domain of a tyrosine kinase. C-met also is regulated by proteases that cleave both chains from a 178-kDa common precursor. Thus HGF is only active if the correct proteases are present. Like TGF-, HGF has effects on cell division, motility, and apoptosis and appears to have angiogenic activity (28). Its synthesis is stimulated by IL-1 (28). Not only does it cause proliferation of epithelial cells but it also affects parenchymal cells such as liver and bone (28). Thus HGF, like KGF, is a major mediator of epithelial-mesenchymal interactions and epithelial morphogenesis (207).

The process of repair is completed by the terminal differentiation of epithelial and parenchymal cells and by apoptosis of the -SM actin myofibroblasts (51, 81, 216). The factors that terminate the repair process are poorly understood. The role of IL-10, INF-, and INF- in either the downregulation (90, 197) of myofibroblasts or induction of their apoptosis (95, 128) needs further investigation.

	ROLE IN FIBROSIS

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With repeated cycles of injury and repair or if, for unclear reasons, there is loss of the signals that discontinue the healing process, organ fibrosis occurs. The important functions of the myofibroblasts in the fibrosis of tissues such as the skin, lung, pancreas, and kidney are well described (see references in Table 4). The effects of PDGF, TGF-, and other growth factors in the fibrotic process have been studied in detail (72, 93, 167, 214, 239) and are beyond the scope of this review (see Refs. 20 and 96 for detailed reviews of fibrosis).

Factors that act on myofibroblasts are important in tissue fibrosis. Recently, the key role of TGF- in fibrosis has been accentuated by the finding of fibrosis of multiple organs, including the liver, kidney (both renal interstitium and glomerulus), and adipose tissue in a transgenic mouse overexpressing TGF- (45). PDGF-BB causes fibrosis in the kidney (239), and, given the propensity of TGF- to upregulate PDGF receptors, an equally important role for PDGF cannot be ruled out. IGF-I has been shown also to induce collagen mRNA and IGF binding protein-5 mRNA in rat intestinal smooth muscle (269), raising the question of an important role for this growth factor in organ fibrogenesis (268). IL-1, IL-6, INF-, TNF-, and bFGF have also been incriminated as fibrogenic cytokines (101, 205). Potential abnormalities in matrix secretion, degradation of matrix by MMPs, and inhibition of MMPs by TIMPs (see above) that might result in fibrosis are under investigation (21, 96, 154). An understanding of these processes and the development of effective pharmacological or biological inhibitors would be important advances in the treatment of disease.

	CONCLUSIONS AND SPECULATION

TOP ABSTRACT INTRODUCTION ROLES IN HEALTH AND... DEFINITION OF A MYOFIBROBLAST ORIGIN OF MYOFIBROBLASTS AND... ACTIVATION, PROLIFERATION, AND... ROLE IN WOUND REPAIR ROLE IN FIBROSIS CONCLUSIONS AND SPECULATION REFERENCES

Myofibroblasts are ubiquitous cells with similar properties and functions that play important roles in growth and development, wound repair, and disease. Either their absence or their activation and proliferation in a given tissue or organ can lead to specific diseases as outlined in Table 3. However, because they are present in virtually every tissue, it is possible that they may play a role in multisystem diseases as well. For example, do abnormalities in pericytes account for some of the multifocal effects of chronic hypertension (208)? Are the multiple abnormalities of diabetes mellitus due to stimulation of or damage to vascular, renal, intestinal, and skin myofibroblasts? It is intriguing that high glucose concentrations induce TGF-1 production by the glomerular mesangial cell (135), and PDGF and bFGF improve wound healing in genetically diabetic animals (87). What is the role of myofibroblasts in aging, a condition in which myofibroblasts are reported to be morphologically abnormal (169)? These are but a few of the intriguing questions raised by this unique family of pleiotropic cells.