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.     

Osteopontin regulates α-smooth muscle actin and calponin in vascular smooth muscle cells

vSMCs (vascular smooth muscle cells) lose differentiation markers and gain uncontrolled proliferative activity during the early stages of atherosclerosis. Previous studies have shown that OPN (osteopontin) mRNA and protein levels increase significantly on induction of proliferative activity by allylamine (an atherogenic amine) and that this response can be inhibited by OPN antibodies. We have investigated the role of OPN in vSMC differentiation. Primary cultures of aortic mouse vSMCs were transfected with an OPN expression plasmid and several vSMC differentiation markers including α-SM actin (α-smooth muscle actin), SM22-α, tropomyosin and calponin were monitored in this cellular model. α-SM actin and calponin protein levels were significantly decreased by OPN overexpression. Down-regulation of α-SM actin and calponin was also observed on extracellular treatment of mouse vSMCs with recombinant OPN. In addition, calponin mRNA was significantly decreased under serum-restricted conditions when OPN mRNA was dramatically increased, while α-SM actin mRNA remained unchanged. These data indicate that OPN down-regulates α-SM actin and calponin expression through an extracellular signalling pathway. Functional connectivity between OPN and vSMC differentiation markers has been established. Since vSMCs lose differentiation features during early atherosclerosis, a mechanistic basis for OPN functions as a critical regulator of proliferative cardiovascular disease has been presented.     

The myofibroblast markers α-SM actin and β-actin are differentially expressed in 2 and 3-D culture models of fibrotic and normal skin

To characterize the differences between fibrotic myofibroblasts and normal fibroblasts, we studied two differentiation markers: -smooth muscle (SM) actin, a specific marker of myofibroblast differentiation, and -actin, which is overexpressed in the fibrotic tissue. Experiments were performed on fibroblasts isolated from normal pig skin and on subcutaneous myofibroblasts isolated from pig radiation-induced fibrosis. Three culture models were used: cells in monolayers, equivalent dermis, consisting of fibroblasts embedded into a matrix composed of type I collagen, and in vitro reconstituted skin, in which the matrix and containing life fibroblasts were overlaid with keratinocytes. Samples were studied using immunofluorescence and western-blotting. In monolayers cultures, both fibrosis and normal cells expressed -SM actin. Furthermore, similar amounts of -actin protein were found. In these conditions, the resulting alterations in the phenotypes of cells made comparison of cultured fibrotic and normal cells irrelevant. Under the two 3-D culture models, normal fibroblasts no longer expressed -SM actin. They expressed -actin at the basal level. Moreover, the fibrotic myofibroblasts in both 3-D models retained their differentiation features, expressing -SM actin and overexpressing -actin. We found that this normalization was mainly related to the genomic programmation acquired by the cells in the tissue. Cellular motility and microenvironment were also involved, whereas cellular proliferation was not a major factor. Consequently, both three-dimensional models allowed the study of radiation-induced fibrosis in vitro, provided good extrapolations to in vivo conditions and avoided certain of culture artefacts.     

GM-CSF-induced granulation tissue formation: relationships between macrophage and myofibroblast accumulation

We have studied the formation of granulation tissue around osmotic minipumps delivering granulocyte macrophage-colony stimulating factor (GM-CSF) chronologically in the rat using electron microscopy and immunohistochemistry at the light and electron microscopic levels, with specific antibodies against α-smooth muscle (SM) actin and rat macrophages. At 2 and 3 days after pump implantation, GM-CSF application produced an extensive inflammatory reaction characterized by edema and the accumulation of polymorphonuclear cells and macrophages. Gradually, polymorphonuclear cells decreased in number and macrophages became arranged in large clusters. The expression of α-SM actin in fibroblastic cells of the granulation tissue started from the 4th day after pump implantation and progressed up to the 7th day. Double immunofluorescence staining showed macrophage clusters in relation to α-SM actinrich fibroblastic cells. Electron microscopic examination confirmed that the fibroblasts containing α-SM actinpositive stress fibers were found initially in close proximity to clustered macrophages. The delivery of plateletderived growth factor (PDGF) and tumor necrosis factor-α (TNF-α) by the osmotic minipump induced an accumulation of macrophages, but in a much smaller number compared with those seen after GM-CSF application; these macrophages were never assembled in clusters and, furthermore, TNF-α and PDGF did not stimulate α-SM actin expression in fibroblastic cells. Our results suggest that after GM-CSF administration, the cluster-like accumulation of macrophages plays an important role in stimulating α-SM actin expression in myofibroblasts. Our results may be relevant to the understanding of the processes leading to granulation tissue formation in this and other experimental models.     

Smooth muscle actin isoforms: A tug of war between contraction and compliance

In higher vertebrates, smooth muscle (SM) contains two tissue-specific actin isoforms: α-SMA and γ-SMA, which predominate in vascular and visceral SM, respectively. Whether α-SMA has been extensively studied and recognized for its contractile activity in SM and SM-like cells such as myofibroblasts, myoepithelial and myoid cells, the distribution and role of γ-SMA remained largely unknown. We developed a new specific monoclonal antibody against γ-SMA and confirmed that γ-SMA predominates in the visceral system and is minor in the vascular system, although more expressed in highly compliant veins than in stiff arteries. Contrary to α-SMA, γ-SMA is absent from myofibroblasts in vitro, and in fibrotic diseases in vivo. We raised the hypothesis that, whereas α-SMA is responsible for the “contractile” activity, γ-SMA would be involved in the “compliance” of SM and SM-like cells. Several models support this hypothesis, namely veins vs. arteries and the physiological modifications occurring in the uterus and mammary glands during pregnancy and lactation. Our results suggest that, in addition to enteric smooth muscles, γ-SMA is expressed in all the tissues submitted to an important dilation including veins, gravid uterus, and lactating mammary glands. The hypothesis of two complementary mechanical roles for the two SMA isoforms is sustained by their different intracellular distributions and by functional assays.     

Transforming Growth Factor-β1 Modulates Myofibroblastic Phenotype of Rat Palatal Fibroblastsin Vitro

The effects of transforming growth factor-β1 (TGF-β1) on normal rat palatal fibroblastsin vitrowere investigated in the present study in order to unravel the precise mechanisms by which the phenotypic modulation of fibroblasts occurs during the scar formation process. TGF-β1 dramatically changed the morphology of normal palatal fibroblasts from polygonal into an elongated shape, which was very similar to that of fibroblasts derived from experimental immature scar tissue in rat palatal mucosa. This morphological transition was concomitant with an increase in the expression of α-smooth muscle (α-SM) actin protein, a marker for myofibroblasts, when determined by immunocytochemistry. An immunoblot study also revealed that α-SM actin expression in palatal fibroblasts became evident after 24 h of TGF-β1 treatment and increased time-dependently up to 72 h. Northern blot analysis showed that TGF-β1 stimulated endogenous TGF-β1 mRNA expression in palatal fibroblasts within 24 h. Neither epidermal growth factor nor basic fibroblast growth factor had any effect on either α-SM actin expression or TGF-β1 mRNA expression. Pretreatment of palatal fibroblasts with TGF-β1 significantly increased the contractile capacity in a three-dimensional collagen gel culture, even when the culture medium was deprived of TGF-β1 for 72 h of the experimental period. Moreover, the contractility of scar fibroblasts, which highly expressed α-SM actin protein and TGF-β1 mRNA, was significantly lowered by a neutralizing antibody to TGF-β1. These data strongly suggest that TGF-β1 is a potential inducer of phenotypic expression of myofibroblasts in palatal fibroblasts and that autoinduction of TGF-β1 mRNA expression may play an important role in the scar formation process in palatal mucosa.     

Remodeling of the vascular tunica media is essential for development of collateral vessels in the canine heart

Previous studies have shown that neointima formation and adventitial remodeling play an important role in the enlargement of collateral vessels (CVs) during coronary arteriogenesis in the dog heart. In this study, we investigated the importance of remodeling of the tunica media in the same model. Basal membrane (BM), contractile and cytoskeletal components of smooth muscle cells (SMCs) were studied in growth of coronary CVs induced by chronic occlusion of the left circumflex (LCX) coronary artery by routine histology, electron microscopy (EM), and immunoconfocal microscopy using antibodies against α-smooth actin (α-SM actin), calponin, desmin, and laminin. In addition, matrix metalloproteinase-2 (MMP-2) and tissue inhibitor-1 of matrix metalloproteinase (TIMP-1) were investigated. The data showed that (1) in normal small arteries (NVs) laminin formed a network in which SMCs were encaged;α-SM actin, calponin and desmin were evenly expressed in SMCs; (2) in early (2 weeks) growing CVs the laminin network was disrupted, desmin was significantly reduced in SMCs, but α-SM actin and calponin still highly expressed; (3) in actively (6 weeks) growing CVs laminin was still weak in the tunica media (TM), but without network-like structure. Desmin was further reduced in SMCs of TM, whereas α-SM actin and calponin showed little changes, although they were significantly decreased in intimal SMCs; (4) in mature CVs, the network-like structure was re-formed, and α-SM actin, calponin, and desmin were all similar to that in normal vessels; (5) histology for BM confirmed laminin staining; (6) EM revealed that in NVs the SMCs contained abundant contractile filaments and were surrounded by a layer of BM whereas in growing CVs, BM structure was not observed, but the SMCs in the media still contained many myofilaments; (7) MMP-2 was highly expressed in the media of early growing vessels, but decreased in TM of actively growing vessels where TIMP-1 expression was high. In conclusion, our data revealed features of TM of growing CVs. Disruption and degradation of BM facilitate SMC proliferation, and together with reduction of desmin and fragmentation of the internal elastic lamina enable the vascular wall to expand and enlarge when blood pressure and shear stress increase. MMP2 may be an important player in regulating SMC phenotype, proliferation, migration and maintaining integrity of the vascular wall through governing proteolysis during arteriogenesis. (Mol Cell Biochem 264: 201–210, 2004)     

Cytoskeletal features of alveolar myofibroblasts and pericytes in normal human and rat lung.

Frozen or paraffin-embedded human and rat lung specimens were stained with antibodies against total actin, alpha-smooth muscle (SM) actin, vimentin, desmin, or gelsolin. Alveolar interstitial myofibroblasts [i.e., contractile interstitial cells (CIC)] were labeled by total actin antibody but not by alpha-SM actin antibody. They stained for vimentin and gelsolin and, in rat lungs, most of them for desmin. Pericytes located around venules at the junction of three alveolar septa were always positive for alpha-SM actin and never for desmin. Tissue samples were also immunostained by an alpha-SM actin antibody and studied by electron microscopy. With this technique we confirmed that cells, identified as pericytes on the basis of their location, were intensely labeled by alpha-SM actin antibodies, whereas alveolar myofibroblasts were not. We conclude that in the lung interstitium pericytes and alveolar myofibroblasts have distinct cytoskeletal features, alpha-SM actin antibody staining being a simple method to distinguish between them. Furthermore, it appears that alveolar myofibroblasts have a peculiar pattern of cytoskeletal protein composition which, in the rat, is similar to that previously described for stromal cells in uterine submucosa, liver sinusoids (Ito cells), or the core of intestinal villi.     

Fetal and postnatal sera differentially modulate human dermal fibroblast phenotypic and functional features in vitro

Fetal wounds heal without scar formation, fibrosis, or contracture. Compared with adult wounds, they are characterized by major differences in the extracellular matrix and the absence of myofibroblastic cells. The reasons for these differences are not well known and determination of factors affecting the absence of scarring in the fetus may lead to strategies for controlling adult pathological scarring. In the present study, we have assessed the effects of serum on the behavior of normal human dermal fibroblasts. Using an in vitro approach, we investigated the effects of fetal and adult serum on cell properties such as growth rate, collagen synthesis, gelatinase activities, and differentiation to myofibroblasts using biochemical, morphological, and ultrastructural parameters. We studied the induction of α-smooth muscle (α-SM) actin in fibroblasts, and its correlation with increased collagen gel contraction by the cells. Our results showed that, compared with FBS (fetal bovine serum), postnatal calf serum (PCS) decreased mitogenic activity and collagenase synthesis but not collagen synthesis. Furthermore, cells cultured with PCS differentiated to myofibroblasts with an increase in cell diameter, number of stress fibers, α-SM actin expression, and collagen gel contraction. To characterize the molecules involved in this differentiation process, the amount of transforming growth factor β (TGFβ) in FBS and PCS was determined and the effect of neutralizing anti-TGFβ antibody was evaluated. It was determined that FBS contained more TGFβ than PCS, but that essentially all the TGFβ was latent in both sera. However, results obtained with anti-TGFβ antibody show that active TGFβ is present when human dermal fibroblasts are cultured with medium containing PCS. These results suggest that, in the presence of PCS but not FBS, the cells either produce active TGFβ or an enzyme that is able to activate latent serum TGFβ. Alternatively, sera may contain two different forms of latent TGFβ, the PCS form being activated by the dermal fibroblast cells. A similar mechanism may be involved, at least in part, in skin wound healing and may underlie the appearance of myofibroblasts in postnatal wounds. J. Cell. Physiol. 171:1–10, 1997. © 1997 Wiley-Liss, Inc.     

LR8 expression in fibroblasts of healthy and fibrotic human tissues

LR8 gene was first reported in a subpopulation of cultured human lung fibroblasts expressing the receptor for C1q-globular domain, and it was not detectable in cultured endothelial cells and smooth muscle cells. LR8 mRNA levels were higher in fibrotic lungs. In this study we assessed LR8 production in human tissues and determined if the distribution of fibroblasts producing LR8 is affected in fibrosis. Normal and fibrotic tissue sections from human liver, lung and kidneys were immunostained with antibodies to LR8 and examined for the presence of fibroblasts staining positively and negatively. The cells were also examined for co-expression of α-smooth muscle actin (SMA), a marker for myofibroblasts. The results showed that LR8 was expressed by fibroblasts, smooth muscle cells, endothelial cells, bile duct cells, pulmonary alveolar cells and distal and proximal kidney tubule cells. Connective tissues of normal and fibrotic tissues contained fibroblasts staining positively and negatively with anti- LR8 antibody. The number of LR8-positive cells was higher in fibrotic tissues, but differences were not statistically significant. Fibroblasts producing both LR8 and SMA were present in higher numbers in fibrotic tissues as compared to normal tissues and the differences were statistically significant (p<0.05). Our results show that fibroblast subtypes differing in LR8 expression are present in human tissues, and that in fibrotic tissues cells co-expressing LR8 and SMA are present. Our results indicate that LR8 expressing cells may participate in the early stages of fibrotic diseases and that fibroblasts expressing LR8, not LR8 negative cells, have potential to become myofibroblasts in fibrotic tissues.     

Modulation of actin mRNAs in cultured vascular cells by matrix components and TGF-β1

Summary Alpha-smooth muscle actin is currently considered a marker of smooth muscle cell differentiation. However, during various physiologic and pathologic conditions, it can be expressed, sometimes only transiently, in a variety of other cell types, such as cardiac and skeletal muscle cells, as well as in nonmuscle cells. In this report, the expression of actin mRNAs in cultured rat capillary endothelial cells (RFCs) and aortic smooth muscle cells (SMCs) has been studied by Northern hybridization in two-dimensional cultures seeded on individual extracellular matrix proteins and in three-dimensional type I collagen gels. In two-dimensional cultures, in addition to cytoplasmic actin mRNAs which are normally found in endothelial cell populations, RFCs expressed α-smooth muscle (SM) actin mRNA at low levels. α-SM actin mRNA expression is dramatically enhanced by TGF-β₁. In addition, double immunofluorescence staining with anti-vWF and anti-α-SM-1 (a monoclonal antibody to α-SM actin) shows that RFCs co-express the two proteins. In three dimensional cultures, RFCs still expressed vWF, but lost staining for α-SM actin, whereas α-SM actin mRNA became barely detectable. In contrast to two-dimensional cultures, the addition of TGF-β₁ to the culture media did not enhance α-SM actin mRNA in three-dimensional cultures, whereas it induced rapid capillary tube formation. Actin mRNA expression was modulated in SMCs by extracellular matrix components and TGF-β₁ with a pattern very different from that of RFCs. Namely, the comparison of RFCs with other cell types such as bovine aortic endothelial cells shows that co-expression of endothelial and smooth muscle cell markers is very unique to RFCs and occurs only in particular culture conditions. This could be related to the capacity of these microvascular endothelial cells to modulate their phenotype in physiologic and pathologic conditions, particularly during angiogenesis, and could reflect different embryologic origins for endothelial cell populations. Supported by a Post-Doctoral Fellowship from the Swiss National Science Foundation (OK) and grant HL-RO1-28373 (JAM) from the Department of Human Services, Public Health Service, Washington, D.C.     

The Fibronectin Domain ED-A Is Crucial for Myofibroblastic Phenotype Induction by Transforming Growth Factor-β1

Transforming growth factor-β1 (TGFβ1), a major promoter of myofibroblast differentiation, induces α-smooth muscle (sn) actin, modulates the expression of adhesive receptors, and enhances the synthesis of extracellular matrix (ECM) molecules including ED-A fibronectin (FN), an isoform de novo expressed during wound healing and fibrotic changes. We report here that ED-A FN deposition precedes α-SM actin expression by fibroblasts during granulation tissue evolution in vivo and after TGFβ1 stimulation in vitro. Moreover, there is a correlation between in vitro expression of α-SM actin and ED-A FN in different fibroblastic populations. Seeding fibroblasts on ED-A FN does not elicit per se α-SM actin expression; however, incubation of fibroblasts with the anti-ED-A monoclonal antibody IST-9 specifically blocks the TGFβ1-triggered enhancement of α-SM actin and collagen type I, but not that of plasminogen activator inhibitor-1 mRNA. Interestingly, the same inhibiting action is exerted by the soluble recombinant domain ED-A, but neither of these inhibitory agents alter FN matrix assembly. Our findings indicate that ED-A–containing polymerized FN is necessary for the induction of the myofibroblastic phenotype by TGFβ1 and identify a hitherto unknown mechanism of cytokine-determined gene stimulation based on the generation of an ECM-derived permissive outside in signaling, under the control of the cytokine itself.     

Connective tissue growth factor is induced in bleomycin-induced skin scleroderma

The origin of fibrotic cells within connective tissue is unclear. For example, the extent to which microvascular pericytes contribute to the number of myofibroblasts present in dermal fibrosis in uncertain. Connective tissue growth factor (CTGF/CCN2) is a marker and mediator of fibrosis. In this report, we use an antibody recognizing CCN2 to assess the cell types in mouse dermis which express CCN2 in the bleomycin model of skin scleroderma. Control (PBS injected) and fibrotic (bleomycin-injected) dermis was examined for CCN2, α-smooth muscle actin (α-SMA) (to detect myofibroblasts), and NG2 (to detect pericytes) expression. Consistent with previously published data, CCN2 expression was largely absent in the dermis of control mice. However, upon exposure to bleomycin, CCN2 was observed in the dermis. Cells that expressed CCN2 were α−SMA-expressing myofibroblasts. Approximately 85% of myofibroblasts were NG2-positive, CCN2-expressing pericytes, indicating that pericytes significantly contributed to the presence of myofibroblasts in sclerotic dermis. Thus CCN2 is induced in fibrotic skin, correlating with the induction of myofibroblast induction. Moreover, CCN2-expressing pericytes significantly contribute to the appearance of myofibroblasts in bleomycin-induced skin scleroderma.     

Contractile proteins in pericytes at the blood-brain and blood-retinal barriers

Evidence from a variety of sources suggests that pericytes have contractile properties and may therefore function in the regulation of capillary blood flow. However, it has been suggested that contractility is not a ubiquitous function of pericytes, and that pericytes surrounding true capillaries apparently lack the machinery for contraction. The present study used a variety of techniques to investigate the expression of contractile proteins in the pericytes of the CNS. The results of immunocytochemistry on cryosections of brain and retina, retinal whole-mounts and immunoblotting of isolated brain capillaries indicate strong expression of the smooth muscle isoform of actin (α-SM actin) in a significant number of mid-capillary pericytes. Immunogold labelling at the ultrastructural level showed that α-SM actin expression in capillaries was exclusive to pericytes, and endothelial cells were negative. Compared to α-SM actin, non-muscle myosin was present in lower concentrations. By contrast, smooth muscle myosin isoforms, were absent. Pericytes were strongly positive for the intermediate filament protein vimentin, but lacked desmin which was consistently found in vascular smooth muscle cells. These results add support for a contractile role in pericytes of the CNS microvasculature, similar to that of vascular smooth muscle cells.     

Glutathione redox regulates TGF-β-induced fibrogenic effects through Smad3 activation

Transforming growth factor-β (TGF-β) plays a pivotal role in the fibrogenic action involved in the induction of connective tissue growth factor (CTGF), extracellular matrix and fibroblast transformation. Smad3 mediates TGF-β signaling related to the fibrotic response. In human lung fibroblasts or bronchial smooth muscle cells, we demonstrated that an increase in the intracellular glutathione level suppressed TGF-β1-induced phosphorylation of Smad3, while inhibiting TGF-β1-induced expressions of CTGF, collagen type1, fibronectin and transformation into myofibroblasts, which are characterized by the expression of α-smooth muscle actin. These data indicate that the intracellular glutathione redox status regulates TGF-β-induced fibrogenic effects through Smad3 activation.     

Expression of collagen VI α5 and α6 chains in human muscle and in Duchenne muscular dystrophy-related muscle fibrosis

Collagen VI is a major extracellular matrix (ECM) protein with a critical role in maintaining skeletal muscle functional integrity. Mutations in COL6A1, COL6A2 and COL6A3 genes cause Ullrich Congenital Muscular Dystrophy (UCMD), Bethlem Myopathy, and Myosclerosis. Moreover, Col6a1(-/-) mice and collagen VI deficient zebrafish display a myopathic phenotype. Recently, two additional collagen VI chains were identified in humans, the α5 and α6 chains, however their distribution patterns and functions in human skeletal muscle have not been thoroughly investigated yet. By means of immunofluorescence analysis, the α6 chain was detected in the endomysium and perimysium, while the α5 chain labeling was restricted to the myotendinous junctions. In normal muscle cultures, the α6 chain was present in traces in the ECM, while the α5 chain was not detected. In the absence of ascorbic acid, the α6 chain was mainly accumulated into the cytoplasm of a sub-set of desmin negative cells, likely of interstitial origin, which can be considered myofibroblasts as they expressed α-smooth muscle actin. TGF-β1 treatment, a pro-fibrotic factor which induces trans-differentiation of fibroblasts into myofibroblasts, increased the α6 chain deposition in the extracellular matrix after addition of ascorbic acid. In order to define the involvement of the α6 chain in muscle fibrosis we studied biopsies of patients affected by Duchenne Muscular Dystrophy (DMD). We found that the α6 chain was dramatically up-regulated in fibrotic areas where, in contrast, the α5 chain was undetectable. Our results show a restricted and differential distribution of the novel α6 and α5 chains in skeletal muscle when compared to the widely distributed, homologous α3 chain, suggesting that these new chains may play specific roles in specialized ECM structures. While the α5 chain may have a specialized function in tissue areas subjected to tensile stress, the α6 chain appears implicated in ECM remodeling during muscle fibrosis.     

Loss of LRP1 promotes acquisition of contractile-myofibroblast phenotype and release of active TGF-β1 from ECM stores

In healing tissue, fibroblasts differentiate to α-smooth muscle actin (SMA)-expressing contractile-myofibroblasts, which pull the wound edges together ensuring proper tissue repair. Uncontrolled expansion of the myofibroblast population may, however, lead to excessive tissue scarring and finally to organ dysfunction. Here, we demonstrate that the loss of low-density lipoprotein receptor-related protein (LRP) 1 overactivates the JNK1/2-c-Jun-Fra-2 signaling pathway leading to the induction of α-SMA and periostin expression in human lung fibroblasts (hLF). These changes are accompanied by increased contractility of the cells and the integrin- and protease-dependent release of active transforming growth factor (TGF)-β1 from the extracellular matrix (ECM) stores. Liberation of active TGF-β1 from the ECM further enhances α-SMA and periostin expression thus accelerating the phenotypic switch of hLF. Global gene expression profiling of LRP1-depleted hLF revealed that the loss of LRP1 affects cytoskeleton reorganization, cell-ECM contacts, and ECM production. In line with these findings, fibrotic changes in the skin and lung of Fra-2 transgenic mice were associated with LRP1 depletion and c-Jun overexpression. Altogether, our results suggest that dysregulation of LRP1 expression in fibroblasts in healing tissue may lead to the unrestrained expansion of contractile myofibroblasts and thereby to fibrosis development. Further studies identifying molecules, which regulate LRP1 expression, may provide new therapeutic options for largely untreatable human fibrotic diseases.     

Phenotypic changes in the regenerating rabbit bladder muscle. Role of interstitial cells and innervation on smooth muscle cell differentiation

 We have studied the phenotypic changes in regenerating smooth muscle (SM) tissue of detrusor muscle after local application of a necrotizing, freeze–thaw injury to the serosal surface of rabbit bladder. Bromo-deoxyuridine (BrdU) incorporation and immunofluorescence studies were performed on bladder cryosections from day 2 up to day 15 after surgery with monoclonal antibodies specific for some cytoskeletal markers [desmin, vimentin, non-muscle (NM) myosin] and for SM-specific proteins (α-actin, myosin, and SM22). Four days after lesion, some clls incorporated in regenerating SM bundles are BrdU positive, but all display a phenotypic pattern identical to that of the interstitial, highly proliferating cells, i.e., expression of vimentin. By days 7–15 the differentiation profile of regenerating SM returns to that of uninjured SM tissue (appearance of desmin, SM-type α-actin, and SM myosin). A chemical denervation achieved by 6-hydroxydopamine treatment for 2 weeks induces the formation of vimentin/SM α-actin/NM myosin/SM22-containing myofibroblasts in the interstitial, fibroblast-like cells of uninjured bladder. In the bladder wall, alteration of reinnervation during the regenerating SM process produces: (1) in the outer region, the activation of vimentin/SM α-actin/desmin myofibroblasts in the de novo SM cell bundles; and (2) in the inner region of bladder, including the muscularis mucosae, the formation of proliferating, fully differentiated SM cells peripherally to newly formed SM cell bundles. These findings suggest that: (1) the de novo SM tissue formation in the bladder can occur via incorporation of interstitial cells into growing SM bundles; and (2) the alteration of reinnervation during the regenerating process induces a spatial-specific differentiation of interstitial myofibroblasts in SM cells before SM cell bundling. Accepted: 14 May 1997     

Keratocyte phenotype mediates proteoglycan structure: a role for fibroblasts in corneal fibrosis

In pathological corneas, accumulation of fibrotic extracellular matrix is characterized by proteoglycans with altered glycosaminoglycans that contribute to the reduced transparency of scarred tissue. During wound healing, keratocytes in the corneal stroma transdifferentiate into fibroblasts and myofibroblasts. In this study, molecular markers were developed to identify keratocyte, fibroblast, and myofibroblast phenotypes in primary cultures of corneal stromal cells and the structure of glycosaminoglycans secreted by these cells was characterized. Quiescent primary keratocytes expressed abundant protein and mRNA for keratocan and aldehyde dehydrogenase class 3 and secreted proteoglycans containing macromolecular keratan sulfate. Expression of these marker compounds was reduced in fibroblasts and also in transforming growth factor-beta-induced myofibroblasts, which expressed high levels of alpha-smooth muscle actin, biglycan, and the extra domain A (EDA or EIIIA) form of cellular fibronectin. Collagen types I and III mRNAs were elevated in both fibroblasts and in myofibroblasts. Expression of these molecular markers clearly distinguishes the phenotypic states of stromal cells in vitro. Glycosaminoglycans secreted by fibroblasts and myofibroblasts were qualitatively similar to and differed from those of keratocytes. Chondroitin/dermatan sulfate abundance, chain length, and sulfation were increased as keratocytes became fibroblasts and myofibroblasts. Fluorophore-assisted carbohydrate electrophoresis analysis demonstrated increased N-acetylgalactosamine sulfation at both 4- and 6-carbons. Hyaluronan, absent in keratocytes, was secreted by fibroblasts and myofibroblasts. Keratan sulfate biosynthesis, chain length, and sulfation were significantly reduced in both fibroblasts and myofibroblasts. The qualitatively similar expression of glycosaminoglycans shared by fibroblasts and myofibroblasts suggests a role for fibroblasts in deposition of non-transparent fibrotic tissue in pathological corneas.