Focal adhesion size controls tension-dependent recruitment of alpha-smooth muscle actin to stress fibers

Expression of alpha-smooth muscle actin (alpha-SMA) renders fibroblasts highly contractile and hallmarks myofibroblast differentiation. We identify alpha-SMA as a mechanosensitive protein that is recruited to stress fibers under high tension. Generation of this threshold tension requires the anchoring of stress fibers at sites of 8-30-microm-long "supermature" focal adhesions (suFAs), which exert a stress approximately fourfold higher (approximately 12 nN/microm2) on micropatterned deformable substrates than 2-6-microm-long classical FAs. Inhibition of suFA formation by growing myofibroblasts on substrates with a compliance of < or = 11 kPa and on rigid micropatterns of 6-microm-long classical FA islets confines alpha-SMA to the cytosol. Reincorporation of alpha-SMA into stress fibers is established by stretching 6-microm-long classical FAs to 8.1-microm-long suFA islets on extendable membranes; the same stretch producing 5.4-microm-long classical FAs from initially 4-microm-long islets is without effect. We propose that the different molecular composition and higher phosphorylation of FAs on supermature islets, compared with FAs on classical islets, accounts for higher stress resistance.     

Myofibroblast development is characterized by specific cell-cell adherens junctions

Myofibroblasts of wound granulation tissue, in contrast to dermal fibroblasts, join stress fibers at sites of cadherin-type intercellular adherens junctions (AJs). However, the function of myofibroblast AJs, their molecular composition, and the mechanisms of their formation are largely unknown. We demonstrate that fibroblasts change cadherin expression from N-cadherin in early wounds to OB-cadherin in contractile wounds, populated with alpha-smooth muscle actin (alpha-SMA)-positive myofibroblasts. A similar shift occurs during myofibroblast differentiation in culture and seems to be responsible for the homotypic segregation of alpha-SMA-positive and -negative fibroblasts in suspension. AJs of plated myofibroblasts are reinforced by alpha-SMA-mediated contractile activity, resulting in high mechanical resistance as demonstrated by subjecting cell pairs to hydrodynamic forces in a flow chamber. A peptide that inhibits alpha-SMA-mediated contractile force causes the reorganization of large stripe-like AJs to belt-like contacts as shown for enhanced green fluorescent protein-alpha-catenin-transfected cells and is associated with a reduced mechanical resistance. Anti-OB-cadherin but not anti-N-cadherin peptides reduce the contraction of myofibroblast-populated collagen gels, suggesting that AJs are instrumental for myofibroblast contractile activity.     

Transforming growth factor-beta1 promotes the morphological and functional differentiation of the myofibroblast

The myofibroblast is responsible for the generation of contractile force associated with wound contraction and pathological contractures and is characterized by the presence of alpha-smooth muscle (alpha-sm) actin-containing stress fibers, vinculin-containing fibronexus adhesion complexes, and fibronectin fibrils containing the ED-A splice variant. Transforming growth factor-beta1 (TGF-beta1) can promote the expression of alpha-sm actin in myofibroblasts, but the functional significance of this increased expression is unclear. In this study, we demonstrate, using the stress-relaxed collagen lattice contraction assay, that TGF-beta1 promoted a dose-dependent increase in the generation of contractile force in myofibroblasts and a concomitant increase in the expression of alpha-sm actin. We also demonstrate that TGF-beta1 enhanced the formation of the structural elements important in myofibroblast contractile force generation and transmission, including stress fibers, vinculin-containing fibronexus adhesion complexes, and fibronectin fibrils, and that this enhancement occurred prior to, and independent of, alpha-sm actin expression. This differentiated myofibroblast phenotype was not stable. Removal of TGF-beta1 resulted in reduced expression of alpha-sm actin as well as a decreased assembly of stress fibers and vinculin-containing adhesion complexes; however, there was no reduction in fibronectin fibrils. We conclude that TGF-beta1 promotes the morphological and functional differentiation of the myofibroblast by first enhancing the formation of the structural elements characteristic of the myofibroblast followed by increased expression of alpha-sm actin and contractile force generation.     

Effect of actomyosin contractility on focal contacts of myofibroblasts and structure of stress fibers

Myofibroblasts from rat lung were cultivated. These cells in addition to beta- and gamma-cytoplasmic actins, expressed alpha-smooth muscle actin (alpha-SMA) and formed a system of "supermature" focal contacts, which were connected with thick stress-fibers expressing alpha-SMA and myosin II. Reduction of actin-myison contractility by inhibitors BDM and ML-7 lead to stress fiber reorganization, e.g., decrease in their thickness, a selective disappearance of alpha-SMA expression and myosin translocation from bundles to the cytoplasm. Using immunofluorescence, interference-reflection microscopy and morphometry, we have demonstrated that an inhibition of actin-myosin contractility also leads to dispersion of myofibroblastic focal contacts. Phase-contrast and DIC video-enhanced microscopy of live cells showed morphological reorganization at the leading edge after inhibitory treatment. Thus, actin-myosin contractility controls the structure of "supermature" focal contacts of myofibroblasts and alpha-SMA expression in stress fibers.     

Rho-mediated assembly of stress fibers is differentially regulated in corneal fibroblasts and myofibroblasts

Corneal keratocytes (stromal cells) are activated to fibroblasts and myofibroblasts during wound healing. Myofibroblast transdifferentiation is accompanied by the expression of α-smooth muscle actin (α-SMA) and the assembly of a robust stress fiber network and larger focal adhesions (FAs). The regulation of the assembly of stress fibers was evaluated in cultured corneal fibroblast and myofibroblast phenotypes. In both cell types, the inhibition of Rho GTPase activity by microinjecting C3 transferase into the cells resulted in the disassembly of stress fibers and FAs. However, the inhibition of the Rho-associated kinases ROKα and ROKβ with their inhibitor, Y27632, or by overexpression of their mutant kinase-dead forms resulted in only a partial loss of the stress fibers and FAs in myofibroblasts but a total loss in fibroblasts. ROK inhibitor-sensitive and -resistant stress fibers in myofibroblasts contained α-SMA, nonmuscle myosin II, tropomyosin, and calponin. The ROK inhibition-resistant stress fibers and FAs were lost upon the overexpression of the dominant-negative form of mDia1 (a mammalian homolog of Drosophila diaphanous protein). These findings indicated that while the assembly of stress fibers in fibroblasts critically involves both ROK and mDia1, in myofibroblasts, the assembly of α-SMA-containing stress fibers also occurs independently of ROK and involves Rho/mDia1.     

Transforming growth factor-beta 1 specifically induce proteins involved in the myofibroblast contractile apparatus

Transforming growth factor-beta(1) (TGF-beta(1)) induces alpha-smooth muscle actin (alpha-SMA) and collagen synthesis in fibroblast both in vivo and in vitro and plays a significant role in tissue repair and the development of fibrosis. During these processes the fibroblasts differentiate into activated fibroblasts (so called myofibroblasts), characterized by increased alpha-SMA expression. Because TGF-beta(1) is considered the main inducer of the myofibroblast phenotype and cytoskeletal changes accompany this differentiation, the main objective of this investigation was to study how TGF-beta(1) alters protein expression of cytoskeletal-associated proteins. Metabolic labeling of cell cultures by [(35)S]methionine, followed by protein separation on two-dimensional gel electrophoresis, displayed approximately 2500 proteins in the pI interval of 3-10. Treatment of TGF-beta(1) led to specific spot pattern changes that were identified by mass spectrometry and represent specific induction of several members of the contractile apparatus such as calgizzarin, cofilin, and profilin. These proteins have not previously been shown to be regulated by TGF-beta(1), and the functional role of these proteins is to participate in the depolymerization and stabilization of the microfilaments. These results show that TGF-beta(1) induces not only alpha-SMA but a whole set of actin-associated proteins that may contribute to the increased contractile properties of the myofibroblast. These proteins accompany the induced expression of alpha-SMA and may participate in the formation of stress fibers, cell contractility, and cell spreading characterizing the myofibroblasts phenotype.     

Masters and servants of the force: the role of matrix adhesions in myofibroblast force perception and transmission

Fibroblast-to-myofibroblast differentiation - a key event in the development of fibrocontractive diseases and in wound granulation tissue contraction - is hallmarked by the formation of stress fibers and the neo-expression of alpha-smooth muscle actin. Incorporation of the smooth muscle actin isoform into stress fibers confers to myofibroblasts a high contractile activity which is transmitted to the extracellular matrix at sites of specialized adhesions, termed 'fibronexus' in tissue and 'supermature focal adhesions' in two-dimensional cell culture. Myofibroblast differentiation requires a mechanically restrained environment in conjunction with the action of growth factors like transforming growth factor beta and specialized matrix molecules like the ED-A splice variant of fibronectin. This mini-review discusses the roles of myofibroblast adhesions in sensing matrix stress, in transmitting contractile force to the extracellular environment and in creating the high intracellular tension that is required for myofibroblast development.     

The NH2-terminal peptide of alpha-smooth muscle actin inhibits force generation by the myofibroblast in vitro and in vivo

Myofibroblasts are specialized fibroblasts responsible for granulation tissue contraction and the soft tissue retractions occurring during fibrocontractive diseases. The marker of fibroblast-myofibroblast modulation is the neo expression of alpha-smooth muscle actin (alpha-SMA), the actin isoform typical of vascular smooth muscle cells that has been suggested to play an important role in myofibroblast force generation. Actin isoforms differ slightly in their NH2-terminal sequences; these conserved differences suggest different functions. When the NH2-terminal sequence of alpha-SMA Ac-EEED is delivered to cultured myofibroblast in the form of a fusion peptide (FP) with a cell penetrating sequence, it inhibits their contractile activity; moreover, upon topical administration in vivo it inhibits the contraction of rat wound granulation tissue. The NH2-terminal peptide of alpha-skeletal actin has no effect on myofibroblasts, whereas the NH2-terminal peptide of beta-cytoplasmic actin abolishes the immunofluorescence staining for this isoform without influencing alpha-SMA distribution and cell contraction. The FPs represent a new tool to better understand the specific functions of actin isoforms. Our findings support the crucial role of alpha-SMA in wound contraction. The alpha-SMA-FP will be useful for the understanding of the mechanisms of connective tissue remodeling; moreover, it furnishes the basis for a cytoskeleton-dependent preventive and/or therapeutic strategy for fibrocontractive pathological situations.     

Mechanisms of force generation and transmission by myofibroblasts

The myofibroblast has been shown to have a key role in tissue reconstruction after injury and pathological changes characterized by fibrosis. Force generation by the myofibroblast depends on the isometric contraction of stress fibers containing alpha-smooth muscle actin, and is mediated through Rho/Rho-kinase. The force is transmitted by vinculin and tensin-containing "supermature" focal adhesions, which connect stress fibers with the extracellular matrix. Force production and transmission by the myofibroblast are modulated by the coordinated action of cytokines, extracellular matrix components and mechanical tension. Regulation of these phenomena will be important for therapeutic strategies aimed at influencing fibrocontractive diseases.     

Initiation of attachment and generation of mature focal adhesions by integrin-containing filopodia in cell spreading

Integrin receptors, and associated cytoplasmic proteins mediate adhesion, cell signaling and connections to the cytoskeleton. Using fluorescent protein chimeras, we analyzed initial integrin adhesion in spreading fibroblasts with Total Internal Reflection Fluorescence (TIRF) microscopy. Surprisingly, sequential radial projection of integrin and actin containing filopodia formed the initial cell-matrix contacts. These Cdc42-dependent, integrin-containing projections recruited cytoplasmic focal adhesion (FA) proteins in a hierarchical manner; initially talin with integrin and subsequently FAK and paxillin. Radial FA structures then anchored cortical actin bridges between them and subsequently cells reorganized their actin, a process promoted by Src, and characterized by lateral FA reorientation to provide anchor points for actin stress fibers. Finally, the nascent adhesions coalesced until they formed mature FAs.     

Nicotine inhibits myofibroblast differentiation in human gingival fibroblasts

Cigarette smoking has been suggested as a risk factor for several periodontal diseases. It has also been found that smokers respond less favorably than non-smokers to periodontal therapy. Previous work in our lab has shown that nicotine inhibits human gingival cell migration. Since myofibroblasts play an important role in wound closure, we asked if nicotine affects gingival wound healing process by regulating myofibroblast differentiation. Human gingival fibroblasts (HGFs) from two patients were cultured in 10% fetal bovine serum cell culture medium. Cells were pretreated with different doses of nicotine (0, 0.01, 0.1, and 1 mM) for 2 h, and then incubated with transforming growth factor beta (TGF-beta1) (0, 0.25, 0.5, and 1 ng/ml) with or without nicotine for 30 h. The expression level of alpha-smooth muscle actin (alpha-SMA), a specific marker for myofibroblasts, was analyzed by Western blots, immunocytochemistry, and real-time polymerase chain reaction (real-time PCR). Phosphorylated p38 mitogen-activated protein kinase (Phospho-p38 MAPK) activity was analyzed by Western blots. TGF-beta1 induced an increase of alpha-SMA protein and mRNA expression, while nicotine (1 mM) inhibited the TGF-beta1-induced expression of alpha-SMA but not beta-actin. Nicotine treatment down-regulated TGF-beta1-induced p38 MAPK phosphorylation. Our results demonstrated for the first time that nicotine inhibits myofibroblast differentiation in human gingival fibroblasts in vitro; supporting the hypothesis that delayed wound healing in smokers may be due to decreased wound contraction by myofibroblasts.     

Hyaluronan Controls the Deposition of Fibronectin and Collagen and Modulates TGF-β1 Induction of Lung Myofibroblasts

The contribution of hyaluronan-dependent pericellular matrix to TGF-β1-driven induction and maintenance of myofibroblasts is not understood. Hyaluronan is an extracellular matrix (ECM) glycosaminoglycan important in cell adhesion, proliferation and migration, and is implicated in myofibroblast formation and maintenance. Reduced turnover of hyaluronan has been linked to differentiation of myofibroblasts and potentiation of lung fibrosis. Fibronectin is a fibril forming adhesive glycoprotein that is also upregulated following induction with TGF-β1. Although they are known to bind each other, the interplay between hyaluronan and fibronectin in the pericellular matrix during myofibroblast induction and matrix assembly is not clear. This study addresses the role of hyaluronan and its interaction with fibrillar matrix components during myofibroblast formation. Hyaluronan and fibronectin were increased and co-localized in the ECM following myofibroblast induction by TGF-β1. Inhibition of hyaluronan synthesis in TGF-β1-induced lung myofibroblasts over a 4 day period with 4-methyl umbelliferone (4-MU) further enhanced myofibroblast morphology, caused increased deposition of fibronectin and type I collagen in the ECM, and increased expression of alpha-smooth muscle actin and hyaluronan synthase 2 (HAS2) mRNA. Hyaluronan oligosaccharides or hyaluronidase treatment, which more effectively disrupted the pericellular matrix, had similar effects. CD44 and β1 integrins co-localized in the cell membrane and along some stress fibers. However, CD44 and hyaluronan were specifically excluded from focal adhesions, and associated primarily with cortical actin. Time-lapse imaging of the immediate effects of hyaluronidase digestion showed that hyaluronan matrix primarily mediates attachment of membrane and cortical actin between focal contacts, suggesting that surface adhesion through hyaluronan and CD44 is distinct from focal adhesion through β1 integrins and fibronectin. Fluorescein-labeled hyaluronan bound regularly along fibronectin fibers and co-localized more with β1 integrin and less with CD44. Therefore, the hyaluronan matrix can interfere with the assembly of fibrillar ECM components, and this interplay regulates the degree of myofibroblast formation. These data also suggest that adhesion through hyaluronan matrix impacts cytoskeletal organization, and is potentially part of a clutch mechanism that regulates stick and slip of myofibroblasts by affecting the adhesion to and organization of fibronectin and collagen.     

Regulation of alpha-smooth muscle actin gene expression in myofibroblast differentiation from rat lung fibroblasts

Myofibroblasts express alpha-smooth muscle actin and have a phenotype intermediate between fibroblasts and smooth muscle cells. Their emergence can be induced by cytokines such as transforming growth factor beta; but the regulatory mechanism for induction of alpha-smooth muscle actin gene expression in myofibroblast differentiation has not been determined. To examine this mechanism at the level of the alpha-smooth muscle actin promoter, rat lung fibroblasts were transfected with varying lengths of the alpha-smooth muscle actin promoter linked to the chloramphenicol acetyl transferase reporter gene and treated with transforming growth factor beta1. The results show that the shortest inducible promoter was 150 base pairs long, suggesting the presence in this region of cis-elements of potential importance in transforming growth factor beta1 induced myofibroblast differentiation. Transfection of "decoy" oligonucleotides corresponding to sequences for four suspected regulatory factors demonstrated that only the transforming growth factor beta control element is involved in the regulation of transforming growth factor beta1-induced alpha-smooth muscle actin expression in myofibroblast differentiation. Consistent with this conclusion is the finding that a mutation in the transforming growth factor beta control element caused a significant reduction in promoter activity. These observations taken together show that alpha-smooth muscle actin promoter regulation during myofibroblast differentiation is uniquely different from that in smooth muscle cells and other cell lines. Since myofibroblasts play a key role in wound contraction and synthesis of extracellular matrix, clarification of this differentiation mechanism should provide new insight into fibrogenesis and suggest future novel strategies for modulation of wound healing and controlling fibrosis.     

Assembly and mechanosensory function of focal adhesions: experiments and models

Initial integrin-mediated cell-matrix adhesions (focal complexes) appear underneath the lamellipodia, in the regions of the "fast" centripetal flow driven by actin polymerization. Once formed, these adhesions convert the flow behind them into a "slow", myosin II-driven mode. Some focal complexes then turn into elongated focal adhesions (FAs) associated with contractile actomyosin bundles (stress fibers). Myosin II inhibition does not suppress formation of focal complexes but blocks their conversion into mature FAs and further FA growth. Application of external pulling force promotes FA growth even under conditions when myosin II activity is blocked. Thus, individual FAs behave as mechanosensors responding to the application of force by directional assembly. We proposed a thermodynamic model for the mechanosensitivity of FAs, taking into account that an elastic molecular aggregate subject to pulling forces tends to grow in the direction of force application by incorporating additional subunits. This simple model can explain a variety of processes typical of FA behavior. Assembly of FAs is triggered by the small G-protein Rho via activation of two major targets, Rho-associated kinase (ROCK) and the formin homology protein, Dia1. ROCK controls creation of myosin II-driven forces, while Dia1 is involved in the response of FAs to these forces. Expression of the active form of Dia1, allows the external force-induced assembly of mature FAs, even in conditions when Rho is inhibited. Conversely, downregulation of Dia1 by siRNA prevents FA maturation even if Rho is activated. Dia1 and other formins cap barbed (fast growing) ends of actin filaments, allowing insertion of the new actin monomers. We suggested a novel mechanism of such "leaky" capping based on an assumption of elasticity of the formin/barbed end complex. Our model predicts that formin-mediated actin polymerization should be greatly enhanced by application of external pulling force. Thus, the formin-actin complex might represent an elementary mechanosensing device responding to force by enhancement of actin assembly. In addition to its role in actin polymerization, Dia1 seems to be involved in formation of links between actin filaments and microtubules affecting microtubule dynamics. Alpha-tubulin deacetylase HDAC6 cooperates with Dia1 in formation of such links. Since microtubules are known to promote FA disassembly, the Dia1-mediated effect on microtubule dynamics may possibly play a role in the negative feedback loop controlling size and turnover of FAs.     

Transforming growth factor beta stimulates fibroblast-collagen matrix contraction by different mechanisms in mechanically loaded and unloaded matrices

Studies were carried out to test the idea that transforming growth factor beta (TGFbeta) stimulated fibroblast contraction of collagen matrices by different mechanisms depending on mechanical loading on the cells and matrix. Under mechanically unloaded conditions (floating matrices), TGFbeta stimulated contraction directly as an agonist and indirectly by preactivating cells to express the myofibroblast phenotype. Increased contraction of floating matrices by preactivated cells appeared to result in part from an autocrine mechanism. Under mechanically loaded conditions (stressed matrices), TGFbeta had no direct agonist effect on contraction. Fibroblasts preactivated to become myofibroblasts showed increased ability to transfer tension to stressed matrices, and tension persisted even after the cells' actin cytoskeleton was disrupted. Our findings are consistent with the idea that fibroblasts activated to become myofibroblasts in vitro have increased contractile activity and indicate that multiple mechanisms that differ depending on mechanical loading on the cells and matrix are involved.     

Endothelin-1 promotes myofibroblast induction through the ETA receptor via a rac/phosphoinositide 3-kinase/Akt-dependent pathway and is essential for the enhanced contractile phenotype of fibrotic fibroblasts

The endothelins are a family of endothelium-derived peptides that possess a variety of functions, including vasoconstriction. Endothelin-1 (ET-1) is up-regulated during tissue repair and promotes myofibroblast contraction and migration, hence contributing to matrix remodeling during tissue repair. Here, we show that addition of ET-1 to normal lung fibroblasts induces expression of proteins that contribute to a contractile phenotype, including alpha-smooth muscle actin (alpha-SMA), ezrin, moesin, and paxillin. We confirm that ET-1 enhances the ability of lung fibroblasts to contract extracellular matrix, a function essential for tissue repair, through induction of de novo protein synthesis. Blockade of the Akt/phosphoinositide 3-kinase (PI3-kinase) pathway with LY294002 and wortmannin prevents the ability of ET-1 to induce alpha-SMA, ezrin, paxillin, and moesin and to promote matrix contraction. Dominant negative rac and Akt blocked the ability of ET-1 to promote formation of alpha-SMA stress fibers. Using specific ET-1 receptor inhibitors, we show that ET-1 induces collagen matrix contraction through the ETA, but not the ETB, receptor. Relative to normal pulmonary fibroblasts, fibroblasts cultured from scars of patients with the fibrotic disease systemic sclerosis (scleroderma) show enhanced ET-1 expression and binding. Systemic sclerosis lung fibroblasts show increased ability to contract a collagen matrix and elevated expression of the procontractile proteins alpha-SMA, ezrin, paxillin, and moesin, which are greatly reduced by antagonizing endogenous ET-1 signaling. Thus, blocking ET-1 or the PI3-kinase/Akt cascades might be beneficial in reducing scar formation in pulmonary fibrosis.     

Transforming growth factor-beta 1 induces alpha-smooth muscle actin expression in granulation tissue myofibroblasts and in quiescent and growing cultured fibroblasts

Granulation tissue fibroblasts (myofibroblasts) develop several ultrastructural and biochemical features of smooth muscle (SM) cells, including the presence of microfilament bundles and the expression of alpha-SM actin, the actin isoform typical of vascular SM cells. Myofibroblasts have been proposed to play a role in wound contraction and in retractile phenomena observed during fibrotic diseases. We show here that the subcutaneous administration of transforming growth factor- beta 1 (TGF beta 1) to rats results in the formation of a granulation tissue in which alpha-SM actin expressing myofibroblasts are particularly abundant. Other cytokines and growth factors, such as platelet-derived growth factor and tumor necrosis factor-alpha, despite their profibrotic activity, do not induce alpha-SM actin in myofibroblasts. In situ hybridization with an alpha-SM actin probe shows a high level of alpha-SM actin mRNA expression in myofibroblasts of TGF beta 1-induced granulation tissue. Moreover, TGF beta 1 induces alpha-SM actin protein and mRNA expression in growing and quiescent cultured fibroblasts and preincubation of culture medium containing whole blood serum with neutralizing antibodies to TGF beta 1 results in a decrease of alpha-SM actin expression by fibroblasts in replicative and non-replicative conditions. These results suggest that TGF beta 1 plays an important role in myofibroblast differentiation during wound healing and fibrocontractive diseases by regulating the expression of alpha-SM actin in these cells.     

The N-cadherin/catenin complex in colon fibroblasts and myofibroblasts.

Fibroblasts and myofibroblasts were isolated respectively from normal colon mucosa and from colon cancers. Immunostaining with an antibody against alpha-smooth muscle actin (alpha-SMA) of the tissues of origin and of early passage cultures showed equal proportions of alpha-SMA positive myofibroblasts in vivo as in vitro. Immunocytochemistry, immunoprecipitation of metabolically labelled cells followed by Western blotting and RT-PCR of RNA isolates demonstrated the presence of a N-cadherin/catenin complex in both fibroblasts and myofibroblasts. This complex was found preferentially at the cell cell boundaries. Immunocytochemistry and, to a lesser extent, co-immunoprecipitation indicated partial colocalisation of catenins and alpha-SMA. Transforming growth factor beta1 (TGF-beta1) greatly enhanced the expression of alpha-SMA, but left the N-cadherin/catenin complex unaltered. We speculate that the N-cadherin/catenin complex may have different functions in myofibroblasts than in fibroblasts because of its interaction with alpha-SMA.     

Cofilin is a marker of myofibroblast differentiation in cells from porcine aortic cardiac valves

The formation of myofibroblasts in valve interstitial cell (VIC) populations contributes to fibrotic valvular disease. We examined myofibroblast differentiation in VICs from porcine aortic valves. In normal valves, cells immunostained for alpha-smooth muscle actin (alpha-SMA, a myofibroblast marker) were rare (0.69 +/- 0.48%), but in sclerotic valves of animals fed an atherogenic diet, myofibroblasts were spatially clustered and abundant (31.2 +/- 6.3%). In cultured VIC populations from normal valves, SMA-positive myofibroblasts were also spatially clustered, abundant (21% positive cells after 1 passage), and stained for collagen type I and vimentin but not desmin. For an analysis of stem cells, two-color flow cytometry of isolated cells stained with Hoechst 33342 demonstrated that 0.5% of VICs were side population cells; none stained for SMA. Upon culture, sorted side population cells generated approximately 85% SMA-positive cells, indicating that some myofibroblasts originate from a rare population with stem cell characteristics. Plating cells on rigid collagen substrates enabled the formation of myofibroblasts after 5 days in culture, which was completely blocked by culture of cells on compliant collagen substrates. Exogenous tensile force also significantly increased SMA expression in VICs. Isotope-coded affinity tags and mass spectrometry were used to identify differentially expressed proteins in myofibroblast differentiation of VICs. Of the nine proteins that were identified, cofilin expression and phospho-cofilin were strongly increased by conditions favoring myofibroblast differentiation. Knockdown of cofilin with small-interfering RNA inhibited collagen gel contraction and reduced myofibroblast differentiation as assessed by the SMA incorporation into stress fibers. When compared with normal valves, diseased valves showed strong immunostaining for cofilin that colocalized with SMA in clustered cells. We conclude that in VICs, cofilin is a marker for myofibroblasts in vivo and in vitro that arise from a rare population of stem cells and require a rigid matrix for formation.     

NAD(P)H oxidase-derived reactive oxygen species regulate angiotensin-II induced adventitial fibroblast phenotypic differentiation

Phenotypic differentiation of adventitial fibroblasts into myofibroblasts is an essential feature of vascular remodeling. The present study was undertaken to test the hypothesis that reactive oxygen species (ROS) are involved in rat adventitial fibroblast differentiation to myofibroblast. Activation of alpha-smooth muscle actin (alpha-SMA) was used as a marker of myofibroblast. Angiotensin II increased intracellular ROS in adventitial fibroblasts that was completely inhibited by the free radical scavenger NAC, the NAD(P)H oxidase inhibitor DPI, and transfection of antisense gp91phox oligonucleotides. Myofibroblast differentiation was prevented by inhibition of ROS generation with DPI, NAC, and antisense gp91phox as shown by decreased expression of alpha-SMA. Angiotensin II rapidly induced phosphorylation of p38 MAPK and JNK, both of which were inhibited by DPI, NAC, antisense gp91phox, and the selective AT1 receptor antagonist, losartan. Inhibiting p38MAPK with SB202190 or JNK with SP600125 also reduced angiotensin II-induced alpha-SMA expression. These findings demonstrate that angiotensin II induces adventitial fibroblast differentiation to myofibroblast via a pathway that involves NADPH oxidase generation of ROS and activation of p38MAPK and JNK pathways.