Effects of TRAP-1-Like Protein (TLP) Gene on Collagen Synthesis Induced by TGF-β/Smad Signaling in Human Dermal Fibroblasts

Background

Hypertrophic scars are pathologic proliferations of the dermal skin layer resulting from excessive collagen deposition during the healing process of cutaneous wounds. Current research suggests that the TGF-β/Smad signaling pathway is closely associated with normal scar and hypertrophic scar formation. TRAP-1-like protein (TLP), a cytoplasmic protein, has been reported to efficiently regulate Smad2- and Smad3-dependent signal expression in the TGF-β pathway. The relationship between TLP and Type I/III collagen (Col I/III) synthesis explored in the present study provides an effective target for wound healing and gene therapy of hypertrophic scarring.

Objective

To investigate the effects of TLP on collagen synthesis in human dermal fibroblasts.

Methods

Lentiviral vectors encoding TLP was constructed to transfect fibroblasts derived from normal human skin. The expression of Col I/III and phosphorylation of Smad2 and Smad3 in fibroblasts were examined after TLP treatment. In addition, the comparison of TLP expression in normal skin tissues and in hypertrophic scar tissues was performed, and the effect of TLP on cell viability was analyzed by MTT assay.

Results

TLP expression in hypertrophic scar tissue was markedly higher than in normal skin tissue. The Real Time PCR and Western blot test results both revealed that the synthesis of Col I/III was positively correlated with the expression of TLP. TLP also facilitate Smad2 phosphorylation while, conversely, inhibiting Smad3 phosphorylation. TLP may play a cooperative role, along with the cytokine TGF-β1, in improving the overall cell viability of skin fibroblasts.

Conclusions

TLP likely acts as a molecular modulator capable of altering the balance of Smad3- and Smad2-dependent signaling through regulation of phosphorylation, thus facilitating collagen synthesis in fibroblasts. Based on genetic variation in TLP levels in different tissues, these results suggest that TLP plays a key role in the process of TGF-β1/Smad3 signaling that contributes to wound healing and genesis of pathologic scars.     

TGF-β1 and serum both stimulate contraction but differentially affect apoptosis in 3D collagen gels

Apoptosis of fibroblasts may be key for the removal of cells following repair processes. Contraction of three-dimensional collagen gels is a model of wound healing and remodeling. Here two potent inducers of contraction, TGF-β1 and fetal calf serum (FCS) were evaluated for their effect on fibroblast apoptosis in contracting collagen gels. Human fetal lung fibroblasts were cultured in floating type I collagen gels, exposed to TGF-β1 or FCS, and allowed to contract for 5 days. Apoptosis was evaluated using TUNEL and confirmed by DNA content profiling. Both TGF-β1 and serum significantly augmented collagen gel contraction. TGF-β1 also increased apoptosis assessed by TUNEL positivity and DNA content analysis. In contrast, serum did not affect apoptosis. TGF-β1 induction of apoptosis was associated with augmented expression of Bax, a pro-apoptotic member of the Bax/Bcl-2 family, inhibition of Bcl-2, an anti-apoptotic member of the same family, and inhibition of both cIAP-1 and XIAP, two inhibitors of the caspase cascade. Serum was associated with an increase in cIAP-1 and Bcl-2, anti-apoptotic proteins. Interestingly, serum was also associated with an apparent increase in Bax, a pro-apoptotic protein. Blockade of Smad3 with either siRNA or by using murine fibroblasts deficient in Smad3 resulted in a lack of TGF-β induction of augmented contraction and apoptosis. Contraction induced by different factors, therefore, may be differentially associated with apoptosis, which may be related to the persistence or resolution of the fibroblasts that accumulate following injury.     

The Anti-Scar Effects of Basic Fibroblast Growth Factor on the Wound Repair In Vitro and In Vivo

Hypertrophic scars (HTS) and keloids are challenging problems. Their pathogenesis results from an overproduction of fibroblasts and excessive deposition of collagen. Studies suggest a possible anti-scarring effect of basic fibroblast growth factor (bFGF) during wound healing, but the precise mechanisms of bFGF are still unclear. In view of this, we investigated the therapeutic effects of bFGF on HTS animal model as well as human scar fibroblasts (HSF) model. We show that bFGF promoted wound healing and reduced the area of flattened non-pathological scars in rat skin wounds and HTS in the rabbit ear. We provide evidence of a new therapeutic strategy: bFGF administration for the treatment of HTS. The scar elevation index (SEI) and epidermal thickness index (ETI) was also significantly reduced. Histological reveal that bFGF exhibited significant amelioration of the collagen tissue. bFGF regulated extracellular matrix (ECM) synthesis and degradation via interference in the collagen distribution, the α-smooth muscle actin (α-SMA) and transforming growth factor-1 (TGF-β1) expression. In addition, bFGF reduced scarring and promoted wound healing by inhibiting TGFβ1/SMAD-dependent pathway. The levels of fibronectin (FN), tissue inhibitor of metalloproteinase-1 (TIMP-1) collagen I, and collagen III were evidently decreased, and matrix metalloproteinase-1 (MMP-1) and apoptosis cells were markedly increased. These results suggest that bFGF possesses favorable therapeutic effects on hypertrophic scars in vitro and in vivo, which may be an effective cure for human hypertrophic scars.     

miR-145 Contributes to Hypertrophic Scarring of the Skin by Inducing Myofibroblast Activity

Hyperthrophic scarring of the skin is caused by excessive activity of skin myofibroblasts after wound healing and often leads to functional and/or aesthetic disturbance with significant impairment of patient quality of life. MicroRNA (miRNA) gene therapies have recently been proposed for complex processes such as fibrosis and scarring. In this study, we focused on the role of miR-145 in skin scarring and its influence in myofibroblast function. Our data showed not only a threefold increase of miR-145 levels in skin hypertrophic scar tissue but also in transforming growth factor β1 (TGF-β1)-induced skin myofibroblasts compared with healthy skin or nontreated fibroblasts (p < 0.001). Consistent with the upregulation of miR-145 induced by TGF-β1 stimulation of fibroblasts, the expression of Kruppel-like factor 4 (KLF4) was decreased by 50% and α-smooth muscle actin (α-SMA) protein expression showed a threefold increase. Both could be reversed by miR-145 inhibition (p < 0.05). Restoration of KLF4 levels equally abrogated TGF-β1–induced α-SMA expression. These data demonstrate that TGF-β1 induces miR-145 expression in fibroblasts, which in turn inhibits KLF4, a known inhibitor of α-SMA, hence upregulating α-SMA expression. Furthermore, treatment of myofibroblasts with a miR-145 inhibitor strongly decreased their α-1 type I collagen expression, TGF-β1 secretion, contractile force generation and migration. These data demonstrate that upregulation of miR-145 plays an important role in the differentiation and function of skin myofibroblasts. Additionally, inhibition of miR-145 significantly reduces skin myofibroblast activity. Taken together, these results suggest that miR-145 is a promising therapeutic target to prevent or reduce hypertrophic scarring of the skin.     

RAGE and TGF-β1 Cross-Talk Regulate Extracellular Matrix Turnover and Cytokine Synthesis in AGEs Exposed Fibroblast Cells

AGEs accumulation in the skin affects extracellular matrix (ECM) turnover and triggers diabetes associated skin conditions and accelerated skin aging. The receptor of AGEs (RAGE) has an essential contribution to cellular dysfunction driven by chronic inflammatory responses while TGF-β1 is critical in both dermal homeostasis and inflammation. We investigated the contribution of RAGE and TGF-β1 to the modulation of inflammatory response and ECM turnover in AGEs milieu, using a normal fibroblast cell line. RAGE, TGF-β1, collagen I and III gene and protein expression were upregulated after exposure to AGEs-BSA, and MMP-2 was activated. AGEs-RAGE was pivotal in NF-κB dependent collagen I expression and joined with TGF-β1 to stimulate collagen III expression, probably via ERK1/2 signaling. AGEs-RAGE axis induced upregulation of TGF-β1, TNF-α and IL-8 cytokines. TNF-α and IL-8 were subjected to TGF-β1 negative regulation. RAGE’s proinflammatory signaling also antagonized AGEs-TGF-β1 induced fibroblast contraction, suggesting the existence of an inhibitory cross-talk mechanism between TGF-β1 and RAGE signaling. RAGE and TGF-β1 stimulated anti-inflammatory cytokines IL-2 and IL-4 expression. GM-CSF and IL-6 expression appeared to be dependent only on TGF-β1 signaling. Our data also indicated that IFN-γ upregulated in AGEs-BSA milieu in a RAGE and TGF-β1 independent mechanism. Our findings raise the possibility that RAGE and TGF-β1 are both involved in fibrosis development in a complex cross-talk mechanism, while also acting on their own individual targets. This study contributes to the understanding of impaired wound healing associated with diabetes complications.     

Expression of TGF-β3 in isolated fibroblasts from foreskin

Background:

The multifunctional transforming growth factor beta (TGF-β) is a glycoprotein that exists in three isoforms. TGF-β3 expression increases in fetal wound healing and reduces fibronectin and collagen I and III deposition, and also improves the architecture of the neodermis which is a combination of blood vessels and connective tissue during wound healing. Fibroblasts are key cells in the wound healing process. TGF-β3 plays a critical role in scar-free wound healing and fibroblast actions in the wound healing process. The aim of this study was to express the TGF-β3 gene (tgf-b3) in human foreskin fibroblasts (HFF’s).

Methods:

We obtained HFF’s from a newborn and a primary fibroblast culture was prepared. The cells were transfected with TGF-β3-pCMV6-XL5 plasmid DNA by both lipofection and electroporation. Expression of TGF-β3 was measured by enzyme-linked immunosorbent assay (ELISA).

Results:

The highest TGF-β3 expression (8.3-fold greater than control) was obtained by lipofection after 72 hours using 3 µl of transfection reagent. Expression was 1.4-fold greater than control by electroporation.

Conclusions:

In this study, we successfully increased TGF-β3 expression in primary fibroblast cells. In the future, grafting these transfected fibroblasts onto wounds can help the healing process without scarring.Key Words: Fibroblasts, Gene expression, TGF-β3     

MicroRNA 181b Regulates Decorin Production by Dermal Fibroblasts and May Be a Potential Therapy for Hypertrophic Scar

Hypertrophic scarring is a frequent fibroproliferative complication following deep dermal burns leading to impaired function and lifelong disfigurement. Decorin reduces fibrosis and induces regeneration in many tissues, and is significantly downregulated in hypertrophic scar and normal deep dermal fibroblasts. It was hypothesized that microRNAs in these fibroblasts downregulate decorin and blocking them would increase decorin and may prevent hypertrophic scarring. Lower decorin levels were found in hypertrophic scar as compared to normal skin, and in deep as compared to superficial dermis. A decorin 3’ un-translated region reporter assay demonstrated microRNA decreased decorin in deep dermal fibroblasts, and microRNA screening predicted miR- 24, 181b, 421, 526b, or 543 as candidates. After finding increased levels of mir-181b in deep dermal fibroblasts, it was demonstrated that TGF-β₁ stimulation decreased miR-24 but increased miR-181b and that hypertrophic scar and deep dermis contained increased levels of miR-181b. By blocking miR-181b with an antagomiR, it was possible to increase decorin protein expression in dermal fibroblasts. This suggests miR-181b is involved in the differential expression of decorin in skin and wound healing. Furthermore, blocking miR-181b reversed TGF-β₁ induced decorin downregulation and myofibroblast differentiation in hypertrophic scar fibroblasts, suggesting a potential therapy for hypertrophic scar.     

Asiatic Acid Isolated From Centella Asiatica Inhibits TGF-β1-induced Collagen Expression in Human Keloid Fibroblasts via PPAR-γ Activation

Keloids are fibroproliferative disorders characterized by exuberant extracellular matrix deposition and transforming growth factor (TGF)-β/Smad pathway plays a pivotal role in keloid pathogenesis. Centella asiatica extract has been applied in scar management for ages. As one of its major components, asiatic acid (AA) has been recently reported to inhibit liver fibrosis by blocking TGF-β/Smad pathway. However, its effect on keloid remains unknown. In order to investigate the effects of AA on cell proliferation, invasion and collagen synthesis, normal and keloid fibroblasts were exposed to TGF-β1 with or without AA. Relevant experiments including 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay, 5-ethynyl-2-deoxyuridine (EdU) incorporation assay, Transwell invasion assay, enzyme-linked immunosorbent assay, Western blot, quantitative polymerase chain reaction and RNA interference assay were conducted. As a result, keloid fibroblasts showed higher responsiveness to TGF-β1 stimulation than normal fibroblasts in terms of invasion and collagen synthesis. AA could suppress TGF-β1-induced expression of collagen type I, inhibit Smad 2/3 phosphorylation and plasminogen activator inhibitor-1 (PAI-1) expression, while elevate Smad 7 protein level. Noteworthy, the effects of AA on keloid fibroblasts could be abrogated by PPAR-γ antagonist GW9662 and by silencing of PPAR-γ. The present study demonstrated that AA inhibited TGF-β1-induced collagen and PAI-1 expression in keloid fibroblasts through PPAR-γ activation, which suggested that AA was one of the active constituents of C. asiatica responsible for keloid management, and could be included in the arsenal for combating against keloid.     

Inhibition of Contractile Function in Human Joint Capsule Myofibroblasts by Targeting the TGF-β1 and PDGF Pathways

Background

Contractile myofibroblasts (MFs) accumulate in the joint capsules of patients suffering from posttraumatic joint stiffness. MF activation is controlled by a complex local network of growth factors and cytokines, ending in the increased production of extracellular matrix components followed by soft tissue contracture. Despite the tremendous growth of knowledge in this field, inconsistencies remain in practice and prevention.

Methods and Findings

In this in vitro study, we isolated and cultured alpha-smooth muscle actin (α-SMA) positive human joint capsule MFs from biopsy specimens and investigated the effect of profibrotic and antifibrotic agents on MF function. Both TGF-β1 and PDGF significantly induced proliferation and increased extracellular matrix contraction in an established 3D collagen gel contraction model. Furthermore, both growth factors induced α-SMA and collagen type I gene expression in MFs. TGF-β1 down-regulated TGF-β1 and TGF-β receptor (R) 1 and receptor (R) 2 gene expression, while PDGF selectively down-regulated TGF-β receptor 2 gene expression. These effects were blocked by suramin. Interestingly, the anti-oxidant agent superoxide dismutase (SOD) blocked TGF-β1 induced proliferation and collagen gel contraction without modulating the gene expression of α-SMA, collagen type I, TGF-β1, TGF-β R1 and TGF-β R2.

Conclusions

Our results provide evidence that targeting the TGF-β1 and PDGF pathways in human joint capsule MFs affects their contractile function. TGF-β1 may modulate MF function in the joint capsule not only via the receptor signalling pathway but also by regulating the production of profibrotic reactive oxygen species (ROS). In particular, anti-oxidant agents could offer promising options in developing strategies for the prevention and treatment of posttraumatic joint stiffness in humans.     

Thy-1-Integrin αvβ5 Interactions Inhibit Lung Fibroblast Contraction-induced Latent Transforming Growth Factor-β1 Activation and Myofibroblast Differentiation

Myofibroblasts, key effector cells in tissue fibrosis, are specialized contractile cells. Lung myofibroblast contraction induces integrin α_vβ₅-dependent latent transforming growth factor (TGF)-β1 activation suggests that myofibroblast contractility may be a driving force for the persistent myofibroblast differentiation observed in fibrotic lungs. Understanding the mechanisms that regulate fibroblast contraction and mechanotransduction will add new insights into the pathogenesis of lung fibrosis and may lead to new therapeutic approaches for treating fibrotic lung diseases. We and others previously demonstrated that lung fibroblast expression of Thy-1 prevents lung fibrosis. The mechanisms underlying the anti-fibrotic effect of Thy-1 are not well understood. In this study, we showed that Thy-1 interacts with integrin α_vβ₅, both in a cell-free system and on the cell surface of rat lung fibroblasts. Thy-1-integrin α_vβ₅ interactions are RLD-dependent because mutated Thy-1, in which RLD is replaced by RLE, loses the ability to bind the integrin. Furthermore, Thy-1 expression prevents fibroblast contraction-induced, integrin α_vβ₅-dependent latent TGF-β1 activation and TGF-β1-dependent lung myofibroblast differentiation. In contrast, lack of Thy-1 expression or disruption of Thy-1-α_vβ₅ interactions renders lung fibroblasts susceptible to contraction-induced latent TGF-β1 activation and myofibroblast differentiation. These data suggest that Thy-1-integrin α_vβ₅ interactions inhibit contraction-induced latent TGF-β1 activation, presumably by blocking the binding of extracellular matrix-bound latent TGF-β1 with integrin α_vβ₅. Our studies suggest that targeting key mechanotransducers to inhibit mechanotransduction might be an effective approach to inhibit the deleterious effects of myofibroblast contraction on lung fibrogenesis.     

Regulation of TGF-β1-driven Differentiation of Human Lung Fibroblasts: EMERGING ROLES OF CATHEPSIN B AND CYSTATIN C*

Lung matrix homeostasis partly depends on the fine regulation of proteolytic activities. We examined the expression of human cysteine cathepsins (Cats) and their relative contribution to TGF-β1-induced fibroblast differentiation into myofibroblasts. Assays were conducted using both primary fibroblasts obtained from patients with idiopathic pulmonary fibrosis and human lung CCD-19Lu fibroblasts. Pharmacological inhibition and genetic silencing of Cat B diminished α-smooth muscle actin expression, delayed fibroblast differentiation, and led to an accumulation of intracellular 50-kDa TGF-β1. Moreover, the addition of Cat B generated a 25-kDa mature form of TGF-β1 in Cat B siRNA-pretreated lysates. Inhibition of Cat B decreased Smad 2/3 phosphorylation but had no effect on p38 MAPK and JNK phosphorylation, indicating that Cat B mostly disturbs TGF-β1-driven canonical Smad signaling pathway. Although mRNA expression of cystatin C was stable, its secretion, which was inhibited by brefeldin A, increased during TGF-β1-induced differentiation of idiopathic pulmonary fibrosis and CCD-19Lu fibroblasts. In addition, cystatin C participated in the control of extracellular Cats, because its gene silencing restored their proteolytic activities. These data support the notion that Cat B participates in lung myofibrogenesis as suggested for stellate cells during liver fibrosis. Moreover, we propose that TGF-β1 promotes fibrosis by driving the effective cystatin C-dependent inhibition of extracellular matrix-degrading Cats.     

Macitentan inhibits the transforming growth factor-β profibrotic action,blocking the signaling mediated by the ETR/TβRI complex in systemic sclerosis dermal fibroblasts

IntroductionSystemic sclerosis (SSc) is a complex and not fully understood autoimmune disease associated with fibrosis of multiple organs. The main effector cells, the myofibroblasts, are collagen-producing cells derived from the activation of resting fibroblasts. This process is regulated by a complex repertoire of profibrotic cytokines, and among them transforming growth factor beta (TGF-β) and endothelin-1 (ET-1) play a major role. In this paper we show that TGF-β and ET-1 receptors co-operate in myofibroblast activation, and macitentan, an ET-1 receptor antagonist binding ET-1 receptors, might interfere with both TGF-β and ET-1 pathways, preventing myofibroblast differentiation.MethodsFibroblasts isolated from healthy controls and SSc patients were treated with TGF-β and ET-1 and successively analyzed for alpha smooth muscle actin (α-SMA) and collagen (Col1A1) expression and for the Sma and Mad Related (SMAD) phosphorylation. We further tested the ability of macitentan to interfere with these process. Furthermore, we silenced ET-1 and endothelin-1 receptor A expression and evaluated the formation of an ET-1/TGF-β receptor complex by immunoprecitation assay.ResultsWe showed myofibroblast activation in SSc fibroblasts assessing the expression of α-SMA and Col1A1, after stimulation with TGF-β and ET-1. Macitentan interfered with both ET-1- and TGF-β-induced fibroblast activation. To explain this unexpected inhibitory effect of macitentan on TGF-β activity, we silenced ET-1 expression on SSc fibroblasts and co-immunoprecipitated these two receptors, showing the formation of an ET-1/TGF-β receptor complex.ConclusionsDuring SSc, ET-1 produced by activated endothelia contributes to myofibroblast activation using TGF-β machinery via an ET-1/TGF-β receptor complex. Macitentan interferes with the profibrotic action of TGF-β, blocking the ET-1 receptor portion of the ET-1/TGF-β receptor complex.     

The Effect of p38MAPK on Cyclic Stretch in Human Facial Hypertrophic Scar Fibroblast Differentiation

Hypertrophic scars (HTS), the excessive deposition of scar tissue by fibroblasts, is one of the most common skin disorders. Fibroblasts derived from surgical scar tissue produce high levels of α-smooth muscle actin (α-SMA) and transforming growth factor-β1 (TGF-β1). However, the molecular mechanisms for this phenomenon is poorly understood. Thus, the purpose of this study was to evaluate the molecular mechanisms of HTS and their potential therapeutic implications. Fibroblasts derived from skin HTS were cultured and characterized in vitro. The fibroblasts were synchronized and randomly assigned to two groups: cyclic stretch and cyclic stretch pre-treated with SB203580 (a p38MAPK inhibitor). Cyclic stretch at 10% strain was applied at a loading frequency of 10 cycles per minute (i.e. 5 seconds of tension and 5 seconds of relaxation) for 0 h, 6 h and 12 h. Cyclic stretch on HTS fibroblasts led to an increase in the expression of α-SMA and TGF-β1 mRNA and protein and the phosphorylation of p38MAPK. SB203580 reversed these effects and caused a decrease in matrix contraction. Furthermore, HTS fibroblast growth was partially blocked by p38MAPK inhibition. Therefore, the mechanism of cyclic stretch involves p38 MAPK, and its inhibition is suggested as a novel therapeutic strategy for HTS.     

Amphiregulin,an Epidermal Growth Factor Receptor Ligand,Plays an Essential Role in the Pathogenesis of Transforming Growth Factor-β-induced Pulmonary Fibrosis

Dysregulated amphiregulin (AR) expression and EGR receptor (EGFR) activation have been described in animal models of pulmonary fibrosis and in patients with idiopathic pulmonary fibrosis. However, the exact role of AR in the pathogenesis of pulmonary fibrosis has not been clearly defined. Here, we show that a potent profibrogenic cytokine TGF-β1 significantly induced the expression of AR in lung fibroblasts in vitro and in murine lungs in vivo. AR stimulated NIH3T3 fibroblast cell proliferation in a dose-dependent manner. Silencing of AR expression by siRNA or chemical inhibition of EGFR signaling, utilizing AG1478 and gefitinib, significantly reduced the ability of TGF-β1 to stimulate fibroblast proliferation and expression of α-smooth muscle actin, collagen, and other extracellular matrix-associated genes. TGF-β1-stimulated activation of Akt, ERK, and Smad signaling was also significantly inhibited by these interventions. Consistent with these in vitro findings, AR expression was impressively increased in the lungs of TGF-β1 transgenic mice, and either siRNA silencing of AR or chemical inhibition of EGFR signaling significantly reduced TGF-β1-stimulated collagen accumulation in the lung. These studies showed a novel regulatory role for AR in the pathogenesis of TGF-β1-induced pulmonary fibrosis. In addition, these studies suggest that AR, or AR-activated EGFR signaling, is a potential therapeutic target for idiopathic pulmonary fibrosis associated with TGF-β1 activation.