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Address reprint requests to Boris Hinz, Ph.D., Laboratory of Tissue Repair and Regeneration, Matrix Dynamics Group, University of Toronto, Room 241, Fitzgerald Bldg, 150 College St, Toronto, Ontario, Canada M5S 3E2
Laboratory of Experimental Cancer Research, Department of Radiation Oncology and Experimental Cancer Research, Ghent University Hospital, Ghent, Belgium
Laboratory of Experimental Cancer Research, Department of Radiation Oncology and Experimental Cancer Research, Ghent University Hospital, Ghent, Belgium
The discovery of the myofibroblast has opened new perspectives for the comprehension of the biological mechanisms involved in wound healing and fibrotic diseases. In recent years, many advances have been made in understanding important aspects of myofibroblast basic biological characteristics. This review summarizes such advances in several fields, such as the following: i) force production by the myofibroblast and mechanisms of connective tissue remodeling; ii) factors controlling the expression of α-smooth muscle actin, the most used marker of myofibroblastic phenotype and, more important, involved in force generation by the myofibroblast; and iii) factors affecting genesis of the myofibroblast and its differentiation from precursor cells, in particular epigenetic factors, such as DNA methylation, microRNAs, and histone modification. We also review the origin and the specific features of the myofibroblast in diverse fibrotic lesions, such as systemic sclerosis; kidney, liver, and lung fibrosis; and the stromal reaction to certain epithelial tumors. Finally, we summarize the emerging strategies for influencing myofibroblast behavior in vitro and in vivo, with the ultimate goal of an effective therapeutic approach for myofibroblast-dependent diseases.
Myofibroblasts regulate connective tissue remodeling by combining the extracellular matrix (ECM)–synthesizing features of fibroblasts with cytoskeletal characteristics of contractile smooth muscle cells. Since their first description in granulation tissue 40 years ago, remarkable progress has been made in understanding myofibroblast biological characteristics and their participation in physiological and pathological situations.
It is well established that myofibroblasts have multiple origins, contribute importantly to connective tissue remodeling by exerting traction forces and synthesizing ECM components, regress and disappear by apoptosis on wound epithelialization, and may persist in fibrotic situations and cause organ dysfunction.
many new findings have emerged to advance our understanding of myofibroblast biological features, but many questions remain unanswered. Unresolved questions include the following: i) what is the progenitor or precursor cell for the myofibroblast, ii) is there a specific myofibroblast marker, iii) what regulates myofibroblast contractile activity, and iv) what is the basis for myofibroblast persistence in chronic or progressive fibrosis? Recent rapid progress in microRNA (miRNA) and epigenetics research
has furnished new tools and concepts for assessing molecular regulation of myofibroblast differentiation and perpetuation of the myofibroblast phenotype, as potentially mediated by epigenetic mechanisms. The ultimate aim is to suggest therapeutic strategies for influencing the widespread pathological situations that depend on this enigmatic cell.
Myofibroblast Basics
During normal tissue repair, such as skin wound healing, controlled and transient activation of myofibroblasts contributes to restoration of tissue integrity by forming a mechanically sound scar.
However, when myofibroblast activities become excessive and persist, beneficial tissue repair turns into the detrimental tissue deformities characteristic of organ fibrosis. In this review, we will discuss in more detail fibrosis of the skin, lungs, liver, and kidney. In addition to these organs, myofibroblasts play a substantial role promoting heart fibrosis and vascular remodeling, but because of space limitation, these topics are not covered herein, and we refer the reader to recent reviews of this rapidly growing body of literature.
Another fibrotic condition is the desmoplastic or stromal reaction to epithelial tumors, during which myofibroblasts contribute to the mediator and mechanical environment that promotes tumor progression.
We will discuss the similarities of the tumor-associated fibroblasts and myofibroblasts with those present in other fibrotic lesions.
Specific Aspects of Myofibroblast Contraction
High contractile activity of myofibroblasts is necessary for generating tissue contractures. In addition to the expression of α-smooth muscle actin (α-SMA; gene ACTA) in stress fibers, which promotes stronger force generation compared with other actin isoforms in fibroblastic cells, myofibroblasts appear to use specific modes of contraction.
In contrast to the reversible and comparably short-lived contraction of striated and smooth muscles, myofibroblast contractile activity, together with ECM synthesis and degradation, leads to connective tissue remodeling, followed by irreversible and long contractures in a process that can span weeks, months, or even years.
It is still unknown how myofibroblasts stabilize contractions that occur at the cellular or subcellular level to counteract the stress present in a tissue undergoing remodeling. Recent in vitro studies
indicate that myofibroblasts use a lockstep or ratchet mechanism of cyclic and incremental contractile events. This mechanism consists of strong (micronewtons) and far-ranging (tens of micrometers) contractions mediated by RhoA/Rho-associated kinase and weak (approximately 100 pN) and short-ranging (approximately 0.4 μm) cyclic contractions promoted by changes in intracellular calcium concentrations.
The model proposes that strong isometric contraction generates slack in the myofibroblast-associated fibrous and stressed collagen. Such tension-released fibrils are then straightened by the weak, but repeated, subcellular contractile events. By local ECM remodeling and/or deposition of new ECM, the shortened and repeatedly stressed collagen fibrils stabilize the status quo of the ECM, and a new myofibroblast contraction cycle can begin. It is intriguing that the level of stress (ie, the resistance of collagen fibers to pulling) may determine which mechanism of contraction will be engaged.
Characteristic Features of the Myofibroblast
Although there is considerable evidence to indicate the fibroblastic origin of myofibroblasts, other cell types, mostly from mesenchymal lineages, have been suggested as alternative or additional precursors.
Because different subsets of myofibroblast precursors are recruited in different organs, we will further address the question of the myofibroblast origin later. To target the fibrotic activity of myofibroblasts, irrespective of their provenance, it is important to define common denominators, a need that has stimulated the search for specific molecular markers. The most widely used molecular marker of the differentiated myofibroblast in research and clinical diagnostics is the de novo expression of α- SMA.
The convenience of a unique molecular marker has fostered the misconception that a myofibroblast must express α-SMA to be a myofibroblast. However, the most important defining feature of myofibroblasts is the de novo development of in vivo stress fibers and contractile force.
Vascular smooth muscle alpha-actin gene transcription during myofibroblast differentiation requires Sp1/3 protein binding proximal to the MCAT enhancer.
YB-1 coordinates vascular smooth muscle alpha-actin gene activation by transforming growth factor beta1 and thrombin during differentiation of human pulmonary myofibroblasts.
NOX4/NADPH oxidase expression is increased in pulmonary fibroblasts from patients with idiopathic pulmonary fibrosis and mediates TGFbeta1-induced fibroblast differentiation into myofibroblasts.
Oxidative stress in scleroderma: maintenance of scleroderma fibroblast phenotype by the constitutive up-regulation of reactive oxygen species generation through the NADPH oxidase complex pathway.
). It remains to be confirmed whether these markers are tissue and/or condition specific or whether they are useful in identifying myofibroblasts in more general terms. To our knowledge, none of the other markers described over the years is unique to the myofibroblast.
Vascular smooth muscle alpha-actin gene transcription during myofibroblast differentiation requires Sp1/3 protein binding proximal to the MCAT enhancer.
YB-1 coordinates vascular smooth muscle alpha-actin gene activation by transforming growth factor beta1 and thrombin during differentiation of human pulmonary myofibroblasts.
Vascular smooth muscle alpha-actin gene transcription during myofibroblast differentiation requires Sp1/3 protein binding proximal to the MCAT enhancer.
NOX4/NADPH oxidase expression is increased in pulmonary fibroblasts from patients with idiopathic pulmonary fibrosis and mediates TGFbeta1-induced fibroblast differentiation into myofibroblasts.
Oxidative stress in scleroderma: maintenance of scleroderma fibroblast phenotype by the constitutive up-regulation of reactive oxygen species generation through the NADPH oxidase complex pathway.
The table summarizes recently identified proteins and factors that have regulated myofibroblast differentiation either directly or indirectly. For well-established myofibroblast modulators, such as TGFβ1, review articles are referenced.
Mechanical resistance of the ECM, in conjunction with the action of profibrotic transforming growth factor β1 (TGFβ1), is an amply documented primary stimulus for myofibroblast differentiation and persistence.
Contracting cells then generate the conditions that make them even more contractile in a detrimental feed-forward loop. The chicken-and-egg question of how fibroblastic cells are initiated to become contractile in the soft ECM present after the injury may be answered by recent biomechanical studies. In an animal model of liver fibrosis, increased tissue stiffness precedes the activation of fibroblastic cells and the accumulation of collagen, thus suggesting that such early mechanical changes may be sufficient to trigger the contraction cascade.
Collagen cross-linking catalyzed by lysyl oxidase (LOX) enzymes is one possible factor responsible for early structural changes and tissue stiffening in these conditions.
Fibrosis-specific and stable collagen cross-links are further formed by lysyl hydroxylase and pro-collagen-lysine, 2-oxoglutarate 5-dioxygenase, which are responsible for pyridinoline and aldehyde-derived collagen cross-links in the fibrotic skin.
Furthermore, antibody-mediated inhibition of the enzyme LOX-like 2 (LOXL2) suppresses fibrosis and its progression in a variety of organ systems, including tumor desmoplasia.
However, it remains unclear whether this effect is due to reduced ECM stiffness. The potential role of initial tissue stiffening in the onset of fibrosis is particularly interesting given that cultured myofibroblasts can activate latent TGFβ1 from a sufficiently stiffened ECM by integrin-mediated contraction.
These findings establish a direct link between the mechanical and chemical factors regulating myofibroblast differentiation and ultimately causing fibrosis.
Table 1 summarizes myofibroblast-inducing and myofibroblast-inhibiting factors.
Myofibroblast Differentiation and Regulation of ACTA Gene Expression
Because α-SMA expression is a common key element for detection of myofibroblast differentiation and a major player in contractile force production, most of the available data focus on the regulatory mechanisms underlying expression of this gene. Abundant information is available on transcriptional regulation of the ACTA gene, which indicates complex combinatorial mechanisms involving both stimulatory and inhibitory factors (Figure 1A and Table 1).
Figure 1Myofibroblast epigenetics. A: Transcriptional regulation of the ACTA gene in myofibroblast differentiation. Activating or repressing transcription factors are shown directed at the relative locations of their cognate binding elements in the ACTA gene promoter. The interaction of Kruppellike factor (KLF) 4 with Smad3 results in decreased Smad3 binding to the Smad-binding element (SBE). In contrast, MRTF enhances serum response factor binding to its cognate element to activate differentiation. PPARγ and NF-κB are also shown, but the location/nature of their direct interaction with the promoter is uncertain (indicated with a question mark). The activation of Notch signaling activates its downstream factor CSL. CArG, CC(A/T)6GG; C/EBP, CCAAT enhancer binding protein; CSL, from CBF1/RBP-J in mammals, suppressor of hairless [Su(H)] in drosophila and xenopus, and Lag-1 in Caenorhabditis elegans; IM-CAT, binding element with sequence CATCCT; LAP, liver activator protein; LIP, liver inhibitory protein; NKE, Nkx2.5 binding element; SRF, serum response factor; TEF, transcription enhancer factor; THR, TGF-β1 hypersensitivity region; TSS, transcription start site. B: DNA methylation and miRNAs in myofibroblast differentiation. Methylated CpG islands are shown in the ACTA gene promoter in the fibroblast, which are maintained by DNMT. The differentiated myofibroblast shows reduced methylation with activation of ACTA gene expression. The methylated DNA-binding protein MeCP2 promotes myofibroblast differentiation; however, multiple target genes are regulated by this protein. The indicated miRNA species regulate their target mRNAs with downstream consequences on myofibroblast differentiation, as indicated. Although ACTA is used as a hallmark gene of the myofibroblast, other fibrosis-relevant genes have been regulated by hypermethylation as well (see Myofibroblast DNA Methylation). C: Histone deacetylation and myofibroblast differentiation. The indicated HDAC isoforms modulate differentiation via multiple mechanisms. In addition, inhibition/depletion of HDAC4 suppresses TGFβ activation of Akt via 5′-TG-3′-interacting factor 1,2 (TGIF1,2) and protein phosphatase 2A/1 (PP2A/PP1).
Among the activators are serum response factor and the transcription enhancer factor-1 family member R-transcription enhancer factor, which bind to CC(A/T)6GG (CArG) and CATTCCT (MCAT) elements, respectively, in the upstream regulatory sequence of the α-SMA promoter.
another element found to be important in TGFβ1-induced differentiation is a proximal TGFβ control element, to which several factors [eg, Krüppel-like factor 5, specificity protein (Sp) 1, and Sp3] can bind to activate transcription.
Vascular smooth muscle alpha-actin gene transcription during myofibroblast differentiation requires Sp1/3 protein binding proximal to the MCAT enhancer.
Other upstream elements include the TGFβ hypersensitivity region and an Smad-binding element, which are activated by binding to Sp1/Sp3 and Smad3, respectively.
Vascular smooth muscle alpha-actin gene transcription during myofibroblast differentiation requires Sp1/3 protein binding proximal to the MCAT enhancer.
Studies in several different cell types have implicated additional transcription factors, such as CCAAT enhancer–binding protein β, CSL (from CBF1/RBP-J in mammals, Suppressor of Hairless [Su(H)] in Drosophila and Xenopus, and Lag-1 in Caenorhabditis elegans) (a down3stream target of Notch signaling), and c-Myb, in regulation of ACTA gene expression (Table 1).
An important factor linking ACTA gene transcription to the level of mechanical stress and the state of the contractile cytoskeleton is myocardin-related transcription factor A (MRTF-A).
In a mechanism that involves the F-actin–organizing factor mDia1, MRTF-A docks to CArG elements and enhances the transcriptional activity of serum response factor.
Several factors have down-regulated α-SMA expression and, thus, may be responsible for active suppression of myofibroblast differentiation (Table 1). Among these factors is Kruppellike factor 4, which can compete for binding to the TGFβ control element by its activators and interact with the Mad homology 2 (MH2) domain of Smad3 to suppress its binding to the ACTA promoter.
YB-1 coordinates vascular smooth muscle alpha-actin gene activation by transforming growth factor beta1 and thrombin during differentiation of human pulmonary myofibroblasts.
In the case of Nkx2.5, reduction in its expression correlates with myofibroblast differentiation in lung fibroblasts. Thus, differentiation may be mediated by a derepression mechanism dependent on decreased expression of one or more of these repressors.
Smad-Independent Regulation of the α-SMA Promoter
In addition to Smad signaling, the multiple, complex signaling mechanisms implicated in regulation of myofibroblast differentiation may also involve mitogen-activated protein (MAP) kinases.
This is likely mediated by CSL interacting with its binding element identified in the ACTA promoter. Induction of Jagged1, a ligand for Notch1, results in activation of this signaling pathway, with subsequent activation of ACTA gene transcription. Impaired Notch signaling in vivo results in reduced myofibroblast differentiation in an animal model.
Another important pathway in myofibroblast differentiation associated with the desmoplastic response in tumors is Hedgehog signaling, although the downstream target gene(s) have not been identified.
All these differing signaling pathways are likely to work in concert at one level or another in more complex scenarios to ultimately affect ACTA gene expression, both directly and indirectly, via other regulatory genes.
Myofibroblast Epigenetics: A Fibrotic Memory?
Various approaches have demonstrated differences in the protein, gene, and transcriptional profiles between myofibroblast precursors from different origins and their fibrotic counterparts.
Microarray studies have shown up-regulation of many genes, consistent with activation by TGFβ1, Wnt, and connective tissue growth factor (CCN2) signaling pathways, which are abnormally expressed in patients with fibrotic disorders.
Gene profiling of scleroderma skin reveals robust signatures of disease that are imperfectly reflected in the transcript profiles of explanted fibroblasts.
A common observation in profiling studies is that specific signatures of myofibroblasts are retained over several passages in vitro, suggesting a myofibroblast memory that may be mediated by epigenetic modifications, with consequent preservation of the myofibroblast phenotype and its persistence. Epigenetic regulation of gene expression in a variety of fibrotic conditions has involved DNA methylation, modification of histones, and regulation of miRNAs targeting select genes (Figure 1, B and C).
Myofibroblast DNA Methylation
In the case of DNA methylation, modification at CpG islands represents an important mechanism for gene silencing, and this appears to be the case for the ACTA gene
(Figure 1B). Lung alveolar epithelial type II cells, which do not express α-SMA, exhibit high levels of methylation in the three CpG islands identified in the regulatory regions of this gene and in intronic regions. In contrast, lung fibroblasts exhibit significantly lower levels of DNA methylation in this gene, and its inhibition using 5-aza-2′-deoxycytidine activates gene transcription. DNA methylation is not limited to the ACTA gene in fibrotic conditions. In lung fibroblasts, hypermethylation of the promoter region of the TYHY1 gene has been implicated in its silencing, correlating with fibrosis in the lung.
In scleroderma fibroblasts, ECM-inhibitory genes, such as Smad7 or Fli-1 are also hypermethylated and consequently down-regulated, suggesting that DNA methylation could cause derepression of ECM genes involved in fibrosis.
Similarly induced specific deficiency of DNA methyl transferases (DNMTs), using small-interfering RNAs, causes activation of ACTA gene transcription in these fibroblasts
(Figure 1B). In the fibrotic kidney, DNMT1 is associated with persistent fibroblast activation and fibrogenesis via promotion of hypermethylation of RASAL1, a gene that encodes an inhibitor of the Ras oncogene.
The use of these approaches to alter DNMT expression and/or inhibition of DNA methylation is likely to affect other genes as well, which may indirectly affect ACTA gene expression. This is the case in a study
of hepatic stellate cells (HSCs), in which inhibition of DNA methylation results in activation of PPARγ and NF-κB, which then repress ACTA gene expression. Direct and indirect effects of the methylated DNA-binding protein MeCP2 have also been observed. MeCP2 binds directly to the ACTA gene promoter and is essential for ACTA gene expression.
Moreover, MeCP2 deficiency significantly reduces pulmonary fibrosis and myofibroblast differentiation in an animal model. The relative contribution of these direct versus indirect mechanisms may be decisive in determining the ultimate effect on myofibroblast differentiation.
Myofibroblasts, Fibrosis, and miRNAs
In addition to epigenetic modulation through DNA methylation, different miRNAs appear to contribute to the myofibroblast memory
(Figure 1B and Table 1). miR-29 expression is markedly reduced in skin biopsy specimens and explanted skin fibroblasts from patients with scleroderma, suggesting a post-translational epigenetic mechanism for increased ECM gene expression.
In fibrotic lung, low levels of miR-29 correlate with high expression levels of profibrotic genes, including previously unrecognized ECM and ECM remodeling genes. Knockdown or TGFβ1-mediated down-regulation of miR-29 in cultured lung fibroblast derepressed fibrosis-associated genes.
Down-regulation of miR-29 in cardiac fibroblasts in vitro and in vivo induces fibrosis-associated genes, whereas its overexpression has a fibrosis-suppressing effect.
Conversely, in an animal model of kidney fibrosis, up-regulation of miR-192 is reported on TGFβ1 signaling, and overexpression of an miR-192 analogue induces ECM production.
promote expression of fibrotic genes in mouse mesangial cells. Another miRNA involved in fibrosis is miR-132, which mediates inhibition of MeCP2 expression, reduces PPARγ expression, and consequently enhances ACTA gene expression in lung fibroblast.
Such an indirect mechanism has also been reported for additional miRNA species. Among these, Let7 and miR-21 affect lung myofibroblast differentiation via effects on HMGA2 and Smad7, respectively, although additional effects on other target genes have not been excluded.
will hopefully provide a more complete picture of the miRNA species involved in the different aspects of fibrosis.
Myofibroblast Histone Modifications
Histone modification is involved in the perpetuation of fibrosis. The importance of histone modification in regulation of ACTA gene expression is generally indirect.
the direct target genes of these modified histones have not been identified. Histone acetylation is usually associated with activation of target gene expression; thus, the mechanism of this inhibition on ACTA gene expression is likely due to activation of other genes that repress its expression. In addition to histone acetylation, methylation of histones is involved in myofibroblast development and fibrosis.
Considering the rapid progress made in understanding epigenetic processes, it will be years until the first potential targets are identified and tested for their anti-fibrotic potential.
Organ Fibrosis
Systemic Sclerosis
The Role of Fibrosis in Systemic Sclerosis
Systemic sclerosis or scleroderma is a chronic autoimmune and fibrotic disease of unknown etiology with no effective disease-modifying therapies and significant mortality. The disease is highly heterogeneous in its clinical manifestations. Widespread fibrosis affecting virtually every organ distinguishes scleroderma from organ-based fibrotic processes, such as pulmonary fibrosis, keloids, glomerulosclerosis, and hepatic fibrosis. The dermis, lung parenchyma, heart, gastrointestinal tract, tendons, and ligaments are prominently affected, with endocrine organs, such as the thyroid gland, occasionally affected.
Interestingly, neither the liver nor the central nervous system show significant fibrosis in scleroderma. Activated fibroblasts and myofibroblasts are the primary effector cells of fibrosis in scleroderma.
The skin in scleroderma is characterized by replacement of the normal dermal architecture with collagen-rich connective tissue. Progressive dermal thickening and sclerosis obliterate eccrine and sebaceous glands, hair follicles, and small blood vessels. Analysis demonstrates excessive deposition of the main fibrillar collagens (types I and III) and types V and VII collagens, normally restricted to the dermal-epidermal junction.
Shared expression of phenotypic markers in systemic sclerosis indicates a convergence of pericytes and fibroblasts to a myofibroblast lineage in fibrosis.
Fibrillin accumulation is of particular interest, because fibrillin-1 regulates the activation of ECM-bound latent TGFβ1, and mice with a fibrillin-1 mutation (tight skin mice) develop a sclerodermalike phenotype.
Microarray analysis of scleroderma skin reveals up-regulation of genes normally associated with bone and cartilage, including type XI collagen and cartilage oligomeric matrix protein (COMP). Integrins involved in cell-cell and cell-ECM adhesion and latent growth factor activation are also overexpressed in the lesional tissue.
Fibroblasts and myofibroblasts are the principal stromal cells responsible for the excessive ECM deposition and remodeling in scleroderma. Although in situ hybridization studies have identified an increased proportion of biosynthetically active fibroblasts in scleroderma dermis, in vitro clonal analysis of explanted skin fibroblasts shows substantial heterogeneity with respect to collagen synthesis, suggesting the existence of distinct subpopulations of dermal mesenchymal cells with different origins. Cells positive for α-SMA are present in the skin with lesional scleroderma but are not detected in healthy skin.
Shared expression of phenotypic markers in systemic sclerosis indicates a convergence of pericytes and fibroblasts to a myofibroblast lineage in fibrosis.
Accumulation is prominent in areas with significant collagen deposition. Myofibroblastic cells are also observed in the esophagus and lung, even in the liver, despite the absence of fibrosis. Culture of bronchoalveolar lavage fluids from patients with scleroderma shows spontaneous outgrowth of α-SMA–positive cells with high production of collagen and fibronectin.
In contrast, myofibroblasts are not detected in bronchoalveolar lavage fluids from healthy individuals. The number of myofibroblasts in the lesional skin in scleroderma correlates with both the extent of local collagen deposition and the clinical assessment of skin stiffness.
Moreover, the myofibroblast score in the skin shows a decrease over 6 to 12 months in patients treated with cyclophosphamide, suggesting that myofibroblast levels in the skin may be used as biomarkers of changing skin involvement.
The origin of myofibroblasts in the fibrotic lesions has not been conclusively established. They may arise from the in situ activation of normally quiescent resident fibroblasts in response to extracellular triggers, such as TGFβ1, Wnt, Jagged/Notch, CCN2, endothelin-1, lysophophatidic acid, and other signaling molecules, as well as hypoxia and mechanical stress due to increased ECM stiffness.
Each of these stimuli, which induce α-SMA expression and stress fiber formation in explanted healthy skin fibroblasts, has been aberrantly expressed or regulated in scleroderma. Alternatively, myofibroblasts in scleroderma lesional tissue may arise from other cell types, including vascular smooth muscle cells, pericytes,
Shared expression of phenotypic markers in systemic sclerosis indicates a convergence of pericytes and fibroblasts to a myofibroblast lineage in fibrosis.
Because both myofibroblasts and pericytes are associated with vascular remodeling and injury, which are prominent in patients with scleroderma, vascular smooth muscle cells and pericytes might provide the pathogenetic link between vascular injury and fibrosis characteristic of scleroderma. Although the significance of mesenchymal differentiation pathways in the context of scleroderma has not been conclusively demonstrated, and the mechanisms underlying such cellular plasticity in vivo remain mostly unknown, it is tempting to consider scleroderma therapies that are based on targeting these events.
Persistence of Scleroderma Fibroblasts
Historically, the study of scleroderma fibroblasts focuses primarily on the skin, because it is readily accessible for biopsy. Pioneering studies by Leroy
30 years ago are the first to demonstrate the feasibility of studying explanted skin fibroblasts in scleroderma. These observations indicate that scleroderma-derived skin fibroblasts display a biosynthetically activated phenotype in vitro that is independent of extracellular signals. A cell-autonomous activated phenotype is maintained for multiple in vitro passages.
In some studies, most explanted scleroderma fibroblasts are positive for α-SMA in vitro, with increased production of collagen and tissue inhibitor of metalloproteinase.
Myofibroblasts from scleroderma skin synthesize elevated levels of collagen and tissue inhibitor of metalloproteinase (TIMP-1) with two forms of TIMP-1.
Apoptosis resistance may be due to activation of the Akt prosurvival pathway and may account for the persistence of myofibroblasts in scleroderma skin.
Another potential mechanistic explanation accounting for the persistent α-SMA expression in scleroderma is provided by the demonstration that pharmacological inhibition of focal adhesion kinase phosphorylation significantly attenuates the myofibroblast phenotype of these cells.
Scleroderma fibroblasts show an elevated level of focal adhesion kinase phosphorylation, presumably reflecting their stimulation by autocrine TGFβ1. Consistent with the autocrine TGFβ1 hypothesis, explanted scleroderma fibroblasts show constitutive nuclear localization of phosphorylated Smad2/3, even in the absence of added TGFβ1.
These studies suggest that an autocrine TGFβ1-stimulatory loop induces focal adhesion kinase phosphorylation and apoptosis resistance, with consequent persistence of α-SMA–expressing myofibroblasts.
In addition to enhanced ECM production, scleroderma fibroblasts in culture produce chemokines and growth factors and spontaneously generate reactive oxygen radicals, such as H2O2, through the NADPH oxidase (NOX) complex pathway, which is up-regulated in scleroderma skin.
Oxidative stress in scleroderma: maintenance of scleroderma fibroblast phenotype by the constitutive up-regulation of reactive oxygen species generation through the NADPH oxidase complex pathway.
In turn, intracellular production of reactive oxygen species is at least partially responsible for the constitutive up-regulation of collagen synthesis in these cells. The expression of surface receptors for relevant growth factors and chemokines, including TGFβ1, platelet-derived growth factor, and CCR2, are also elevated on scleroderma fibroblasts, suggesting an additional feed-forward amplification mechanism that might underlie the persistently activated phenotype.
Renal interstitial fibrosis is a common pathological feature of chronic kidney disease (CKD), irrespective of etiology. The myofibroblast is a major player in the onset and evolution of renal fibrosis and has been implicated in pathogenesis.
Despite their pivotal role in disease progression, the source of renal myofibroblasts is still a matter of debate. Several progenitors have been proposed in addition to local fibroblasts, including circulating fibrocytes, local pericytes,
using a mouse model of renal interstitial fibrosis and genetic cell lineage tracing. However, under the same conditions of kidney obstruction, but using a different approach, EMT was observed by another group.
Tubuloepithelial-to-Mesenchymal Cross Talk Contributes to Kidney Myofibroblast Generation
It appears increasingly likely that loss of the normal, homeostatic microenvironment, due to alterations in the tubulointerstitium per se, triggers myofibroblast activation and causes progression of fibrosis.
This established concept is based on the histological evidence that proximal tubule cell (PTC) injury precedes interstitial fibrosis and that regions of active interstitial fibrosis predominantly exhibit a peritubular, rather than a perivascular, distribution. This notion is supported by in vitro studies
showing that cortical fibroblast proliferation is stimulated by paracrine signals generated by PTC in co-culture.
Moreover, in proteinuric nephropathies with progressive injury of the glomerular filtering barrier, abnormal uptake of ultrafiltered proteins by PTCs induces release of TGFβ1, which, in turn, promotes interstitial fibrogenesis.
Re-establishment in vitro of epithelial cell polarization and differentiation inhibits proliferation of mesenchymal stem cells (MSCs) from co-cultured adipose tissue fragments,
confirming that uninjured differentiated renal tubular cells in their normal configuration contribute to the maintenance of the homeostatic state of MSCs. A seminal work
evaluating the effects in vitro of aristolochic acid on several PTC lines reveals that damaged epithelial cells markedly up-regulate expression of profibrotic TGFβ1 and CCN2. This up-regulation is associated with a marked increase in the percentage of cells in the G2/M phase of the cell cycle, which is confirmed in vivo using different acute kidney disease mouse models.
The contribution of PTCs is further supported in a tetracycline-inducible transgenic mouse model, in which conditional overexpression of TGFβ1, limited to renal tubules, induces widespread peritubular fibrosis and focal nephron degeneration.
During the course of this process, the remnants of one cell were removed by phagocytosis by neighboring cells with a mechanism akin to that seen in tubular atrophy.
Outside these degenerating tubules, a marked proliferation of resident fibroblasts, without contribution of EMT and only sparse macrophages, is associated with progressive deposition of ECM.
Notch signaling is involved in orchestrating kidney development in tubular epithelial cells and plays a role in tubulointerstitial development.
Interestingly, specific expression of cleaved Notch1 in tubular epithelial cells causes fibrosis in the surrounding interstitium, with a histological appearance that resembles the lesions seen in human CKD. Conversely, tubular-specific deletion of Notch signaling protects against kidney fibrosis.
The importance of an altered tubule-interstitial microenvironment to fibrosis is also supported by the clinical observation that the severity of acute kidney injury (AKI) with massive tubular epithelial cell death is a robust predictor of progression to CKD.
Even if mechanisms for AKI to CKD progression are unknown, it can be hypothesized that AKI progression toward CKD arises from incomplete repair of regenerating tubules. Another explanation is that persistently high profibrotic signaling activity in regenerating tubule epithelium produces paracrine activity that drives fibroblast proliferation and inflammation. Thus, in severe AKI and CKD, epithelial-mesenchymal cross talk alterations possibly lead to tubulointerstitial fibrosis and kidney failure.
It appears that an altered renal microenvironment is the main cause of myofibroblast differentiation in renal fibrosis, and cross talk between epithelial cells and fibroblasts is pivotal for maintenance of the local homeostatic microenvironment, a concept previously suggested for idiopathic pulmonary fibrosis
Therefore, interfering with tubular-mesenchymal cross talk emerges as a promising strategy for the development of new anti-fibrotic drugs, which are not solely directed to block myofibroblast activation.
Pulmonary Fibrosis
Structural Aspects of the Lung Relevant for Fibrosis
The adult human lung is a structurally complex organ system composed of >40 cell types. The upper conducting airway, composed of the trachea and a series of branching bronchi and bronchioles, terminates in the gas-exchanging alveoli of the lower respiratory tract. At each level, the epithelium-lined airways and endothelium-lined vasculature are integrated, both structurally and functionally, by an interconnected reticulum of mesenchymal cells and ECM extending from the upper airway down to the alveoli.
The lung is susceptible to various forms of short-/long-term injuries, both airborne and blood borne, that may culminate in fibrosis. In addition, fibrosis may involve the small conducting airways (eg, asthma and chronic obstructive pulmonary disease), vasculature (eg, pulmonary hypertension), or pleura (eg, pleural fibrosis). Some forms of fibrosis, such as acute lung injury or cryptogenic organizing pneumonia, are at least partially reversible, whereas others, in particular idiopathic pulmonary fibrosis, are progressive and usually fatal.
A central role for the myofibroblast in tissue remodeling and fibrosis involving these different tissue compartments and varied disease processes has been demonstrated.
The concept that lung myofibroblasts may derive from multiple cellular sources, including bone marrow progenitors and the lung epithelium, has been previously reviewed.
Depending on the experimental model and tools used, conclusions on participation of myofibroblast progenitors to lung fibrosis sometimes appear contradictory. For example, recent studies using a surfactant protein C-CreER(T2) knock-in allele to follow the fate of type II alveolar cells in vivo indicated no contribution to myofibroblasts in the fibrotic reaction to an acute lung injury involving intratracheal instillation of bleomycin.
The resolving nature of bleomycin-induced lung fibrosis in mice contrasts with the progressive nature of the most recalcitrant forms of lung fibrosis in humans, in particular idiopathic pulmonary fibrosis. Whether chronicity of the injury may account for processes, such as EMT, remains to be determined. Chronicity and irreversibility of the fibrotic process may also be linked to the epigenetic (dys) regulation
that controls the differentiation and fate of endogenous reparative cells.
Specific Features of Myofibroblasts in the Lung
The fate of myofibroblasts, regardless of their origin(s), in injured tissues may ultimately determine whether normal healing occurs or whether progression to end-stage fibrosis ensues.
There is likely to be significant heterogeneity in lung fibroblasts, including anti-fibrotic subpopulations, such as Thy-1–expressing fibroblasts and lipofibroblasts.
The mechanisms that produce an apoptosis-resistant myofibroblast phenotype have not been fully elucidated, although some of the same factors that mediate myofibroblast differentiation appear to promote myofibroblast survival.
Activation of the pro-survival phosphatidylinositol 3-kinase/AKT pathway by transforming growth factor-beta1 in mesenchymal cells is mediated by p38 MAPK-dependent induction of an autocrine growth factor.
A member of the NOX family of enzymes, NOX4 has been required for TGFβ1-induced myofibroblast differentiation, ECM production, and contractility of lung myofibroblasts.
NOX4/NADPH oxidase expression is increased in pulmonary fibroblasts from patients with idiopathic pulmonary fibrosis and mediates TGFbeta1-induced fibroblast differentiation into myofibroblasts.
NOX4 is up-regulated in lungs of human subjects with idiopathic pulmonary fibrosis, and genetic or pharmacological targeting of NOX4 attenuated lung fibrogenesis in two different murine models of lung injury.
It is unknown if the profibrotic effects of NOX4 are related solely to activation of myofibroblasts or if its expression in alveolar epithelial cells may also contribute to fibrogenesis.
It is also yet to be determined if targeting NOX4 modulates the apoptosis-resistant phenotype of myofibroblasts and whether this strategy may be effective in established fibrosis. However, the proof of concept
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