Collagen Synthesis In Keloid Fibroblasts Research Articles

SIRT6 Inhibits the Proliferation and Collagen Synthesis of Keloid Fibroblasts through MAPK/ERK Pathway.

Keloid, a fibroproliferative disorder, significantly impacts patients' quality of life, yet effective therapies remain elusive. This study explored the role of silent information regulator 6 (SIRT6) in modulating the proliferation, invasion, and collagen synthesis of keloid fibroblasts. Keloid and normal skin specimens were collected, and fibroblasts were isolated from the keloid tissue. SIRT6 recombinant adenovirus (Ad) was constructed to infect keloid fibroblasts to overexpress SIRT6. This study entails three groups: Control group, adenovirus-Negative Control (Ad-NC) group, and Ad-SIRT6 group. SIRT6 protein and mRNA levels were measured via Western blotting and Quantitative reverse transcription polymerase chain reaction (qRT-PCR), respectively. Cell viability was determined using 5-ethynyl-2'-deoxyuridine (EdU) assay. Flow cytometry was exploited to measure cell apoptosis. To investigate cell migration, wound healing assay and Transwell assay were employed. Western blotting was also utilized to study the expression levels of apoptotic proteins, collagen deposition-related proteins, and Mitogen-Activated Protein Kinases (MAPK)/extracellular regulated protein kinases (ERK) pathway-related proteins. Compared to the control and Ad-NC groups, the Ad-SIRT6 group exhibited significantly elevated SIRT6 level; diminished cell proliferation, migration and invasion; reduced protein levels of α-smooth muscle actin (α-SMA), collagen I, collagen III, phospho SMAD Family Member 3 (p-Smad3), transforming growth factor-β 1 (TGF-β1), and MAPK/ERK pathway proteins (phospho extracellular signal-regulated protein kinase 1/2 (p-ERK1/2), phospho MAP kinase-ERK kinase (p-MEK) and phospho-c-Raf (p-c-Raf)). Treatment with epidermal growth factor (EGF), an MAPK/ERK pathway agonists, reversed the inhibitory effect of SIRT6 on cell activity and inhibited apoptosis in keloid fibroblasts. SIRT6 overexpression in keloid fibroblasts attenuates proliferation, invasion, and collagen synthesis, while fostering apoptosis, likely through the suppression of MAPK/ERK pathway activity. This suggests a potential therapeutic target for keloid treatment.

Mediation of Anti-Keloid Effects of mTOR Inhibitors by Autophagy-Independent Machinery

Abstract Objective: Blocking mechanistic target of rapamycin (mTOR) activation with mTOR inhibitors has promising therapeutic potential for keloids. However, the precise mechanism of mTOR inhibitors remains unclear. This study was aimed to investigate the role of autophagy machinery in the anti-keloid effects of mTOR inhibitors. Methods: We first validated the biological effects induced by the mTOR inhibitors rapamycin (100 nmol/L) and KU-0063794 (5 μmol/L) on the proliferation, apoptosis, migration, and collagen synthesis of keloid fibroblasts (KFs) derived from Han Chinese persons through a Cell Counting Kit-8 assay, 5-Bromo-2’-deoxyuridine incorporation, Annexin V/propidium iodide staining, migration, and western blotting. To explore whether autophagy machinery is involved in the anti-keloid effects of mTOR inhibitors, we first blocked the autophagy activation induced by rapamycin and KU-0063794 with a pharmacological autophagy inhibitor (wortmannin) or by silencing the key autophagy gene (ATG5), and we then re-evaluated these biological effects on KFs. Results: Blocking mTOR activation with either rapamycin or KU-0063794 completely inhibited proliferation, migration, and collagen synthesis of primary KFs but did not affect apoptosis. Incubating KFs with the autophagy inhibitor wortmannin or performing ATG5 silencing abrogated the subsequent activation of autophagic activity induced by rapamycin (rapamycin + E-64d + pepstatin vs. rapamycin + wortmannin + E-64d + pepstatin: 1.88 ± 0.38 vs. 1.02 ± 0.35, F = 6.86, P = 0.013), (non-sense control + rapamycin vs. ATG5 small interfering RNA + rapamycin: 1.46 ± 0.18 vs. 0.75 ± 0.20, respectively; F = 7.68, P = 0.01) or KU-0063794 (KU-0063794 + E-64d + pepstatin vs. KU-0063794 + wortmannin + E-64d + pepstatin: 1.65 ± 0.35 vs. 0.76 ± 0.17, F = 10.01, P = 0.004), (NC + KU-0063794 vs. ATG5 small interfering RNA + KU-0063794: 1.59 ± 0.50 vs. 0.77 ± 0.09, F = 5.93, P = 0.02) as evidenced by decreased accumulation of LC3-II. However, blockage of autophagy induction in mTOR inhibitor-treated KFs with both methods did not disturb their anti-keloid effects, such as inhibition of cell viability, cell migration, and collagen synthesis (P > 0.05 each). Conclusion: Blocking mTOR activation with the mTOR inhibitors rapamycin and KU-0063794 showed anti-keloid effects in KFs. Restoration of autophagy inhibition by mTOR inhibitors does not contribute to their anti-keloid effects.

Curcumol inhibits keloid fibroblast proliferation and collagen synthesis through the ERK signaling pathway

To assess the effects of curcumol on the proliferation, apoptosis and collagen synthesis of keloid fibroblasts and explore the underlying mechanism. Keloid fibroblasts were treated with different concentrations of curcumol (10, 20, 40, 80 and 160 mg/L) or with 160 mg/L curcumol and 20 μmol/L ISO (an ERK signaling pathway activator). Western blotting was performed to detect the expression levels of proliferation-associated proteins (cyclin D1 and PCNA), fibrosis marker proteins (Col1A1, Col3A1 and α-SMA), apoptosis proteins (Bcl-2, Bax and cleaved caspase-3) and ERK signaling pathway proteins (p-ERK1/2, p-MEK and p-c-Raf) in the cells. MTT assay and flow cytometry were used to evaluate the proliferation and apoptosis rate of the treated cells, respectively. Curcumol at 10, 20, 40, 80 and 160 mg/L all reduced the protein expressions of cyclin D1, PCNA and Bcl-2, inhibited the expressions of fibrotic marker proteins Col1A1, Col3A1 and α-SMA, decreased the levels of ERK signaling pathway proteins p-ERK1/2, p-MEK and p-c-Raf, and increased the expressions of Bax and cleaved caspase-3 proteins (P < 0.05). Curcumol treatment at 160 mg/L obviously inhibited the proliferation and collagen synthesis, promoted cell apoptosis and inhibited the ERK signaling pathway in the keloid fibroblasts; treatment with ISO significantly reversed the effects of curcumol on the proliferation, apoptosis, collagen synthesis and ERK signal pathway of the cells. Curcumol regulates proliferation, apoptosis and collagen synthesis in keloid fibroblasts through the ERK signaling pathway.

Resveratrol inhibits proliferation and promotes apoptosis of keloid fibroblasts by targeting HIF-1α

A keloid is characterized by red, tickling, hard, and irregular raised tissues, and it tends to outgrow its origin. It frequently occurs in young adults and appears to be refractory to prevailing therapies. Resveratrol is a new drug that has anti-proliferative effect. In this study, keloid-derived fibroblasts were cultured under hypoxia environment and was treated by resveratrol. CCK-8 assay and Annexin V-FITC were used to evaluate cell activity and apoptosis level. Western blot and RT-qPCR were also used to assess the expression of HIF-α, Collagen I and Collagen III. Besides, siRNA was also used to explore the mechanisms of resveratrol’s effect. In this study, hypoxia promotes proliferation and inhibits apoptosis of keloid fibroblasts. These findings highlight the potential obstacle in treating keloids. Furthermore, we demonstrated that resveratrol could reverse the effect of hypoxia on keloids through down-regulation of HIF-1α. Moreover, collagen synthesis in keloid fibroblasts was also inhibited by resveratrol, which corresponded with HIF-1α suppression. These results provide evidence for resveratrol’s treatment effect against keloids through inhibiting cell proliferation and promoting cell apoptosis, while, HIF-1α may play the key role in this process.

Ginsenoside Rg3 inhibits keloid fibroblast proliferation, angiogenesis and collagen synthesis in vitro via the TGF‑β/Smad and ERK signaling pathways.

A wide range of therapeutic options exists for the treatment of keloids, all of which have their own strengths; however, a high risk of side-effects and frequent recurrence remains. Therefore, the present study aimed to identify improved therapeutic approaches or drugs for the treatment of keloids. Ginsenoside Rg3 (Rg3) has been reported to exert numerous antitumor effects, thus indicating that Rg3 may be a potential therapeutic agent that targets keloids. The present study determined the effects of Rg3 on human keloid fibroblasts (KFs) in vitro, and further explored the associated molecular and cellular mechanisms. Keloid scar specimens were obtained from patients, aged between 22 and 35 years, without systemic diseases and primary cells were isolated from keloid tissues. In each assay, KFs were divided into three groups and were cultured in medium with or without various concentrations of Rg3 (50 or 100 μg/ml). Cell viability assay, flow cytometry, quantitative polymerase chain reaction, cell migration assay, immunofluorescence staining, western blot analysis, Transwell cell invasion assay and immunohistochemical analysis were used to analyze the KFs and keloid explant cultures. The results of the present study demonstrated that Rg3 was able to exert an inhibitory effect on the transforming growth factor-β/Smad and extracellular signal-regulated kinase signaling pathways in KFs. The proliferation, migration, invasion, angiogenesis and collagen synthesis of KFs were markedly suppressed following treatment with Rg3. Furthermore, the results of an ex vivo assay indicated that Rg3 inhibited angiogenesis and reduced collagen accumulation in keloids. Significant statistical differences existed between the control and Rg3-treated groups (P<0.05). All of these experimental results suggested that Rg3 may serve as a reliable drug for the treatment of patients with keloids.

DKK3 regulates cell proliferation, apoptosis and collagen synthesis in keloid fibroblasts via TGF-β1/Smad signaling pathway.

It has been reported that Dickkopf-3 (DKK3) down-regulation was examined in keloid fibroblasts, but the biological functions of DKK3 have not yet been investigated. In this study, we examined the expression of DKK3 in human keloid tissues, further evaluated the biological function of DKK3 and explored its potential molecular mechanism in transforming growth factor-β1 (TGF-β1)-induced keloid fibroblasts. Our results showed that DKK3 mRNA expression in human keloid tissues is down-regulated. DKK3 overexpression inhibited cell proliferation in TGF-β1-induced keloid fibroblasts transfected with pcDNA3.1-DKK3. Furthermore, DKK3 overexpression remarkably upregulated the protein expression levels of Bax and caspase-3, but decreased the protein expression of Bcl-2. In addition, DKK3 overexpression dramatically inhibited the protein and mRNA levels of collagen I (Col-I), collagen III (Col-III) and α-smooth muscle actin (α-SMA). Moreover, the protein expression of TGF-β receptor I (TGF-β RI), TGF-β receptor II (TGF-β RII), the phosphorylation of Smad2 (p-Smad2) and Smad3 (p-Smad3) was dramatically inhibited by pcDNA3.1-DKK3. LY2109761, a TGF-β receptor inhibitor, also suppressed cell proliferation, apoptosis and collagen synthesis in TGF-β1-induced keloid fibroblasts. Taken together, DKK3 overexpression could inhibit cell proliferation, induced cell apoptosis, and suppressed collagen synthesis through TGF-β1/Smad signaling in TGF-β1-induced keloid fibroblasts. Our findings suggest that DKK3 is a novel and promising molecular target for keloid treatment.

Resveratrol Inhibits Proliferation and Promotes Apoptosis of Keloid Fibroblasts by Targeting HIF-1α

Keloid is characterized by red, tickling, hard and irregular raised tissues, and tends to outgrow its origin. It occurs frequently in young adults and appears as refractory to prevailing therapies. Although histological studies have revealed striking similarities of keloids with cancer, such as uncontrolled cell growth, and many of the therapies in clinical practice including -fluorouracil and tamoxifen, excision, and radiation therapy were borrowed from cancer management, the etiology of keloid has remained to be explored to provide insights for developing effective therapeutic modalities. Hypoxic stress around tumors was frequently reported as conferring growth momentum to tumor cells. The hypoxia caused by fast growing of keloid fibroblasts may also be a risk factor that antagonizes therapies. Resveratrol has received intense attention in cancer researches since its anti-proliferative effect on human pulmonary artery smooth muscle cells were revealed. In this study, we first revealed that hypoxia promotes proliferation and inhibit apoptosis of keloid fibroblasts, which highlights the potential obstacle in treating keloids. Further, we demonstrated resveratrol could reverse the effect of hypoxia on keloids, which involves down-regulation of HIF-1α. Moreover, the collagen synthesis of keloid fibroblasts was also inhibited by resveratrol, which was corresponding with HIF-1α suppression. These results provide complementary evidence to the multiple molecular signalings controlled by resveratrol and suggest a pathway implicates resveratrol to regulate expression of procollagen expression via HIF-1α.

Role of collagen triple helix repeat containing-1 in tumor and inflammatory diseases.

Initially, collagen triple helix repeat containing-1 (CTHRC1) is expressed mainly in adventitial fibroblasts and neointimal smooth muscle cells of balloon-injured vessels, and increases cell migration, promotes tissue repair in response to injury. A variety of studies demonstrated that over-expression of CTHRC1 in solid tumors results in enhancement of migration and invasion of tumor cells, and is associated with decreased overall survival and disease-free survival. CTHRC1 expression is elevated in hepatitis B virus-infected patients and highly correlated with hepatocellular carcinoma progression as well. Furthermore, CTHRC1 plays a pivotal role in a great many fields, including increases bone mass, prevents myelination, reverses collagen synthesis in keloid fibroblasts, and increases fibroblast-like synoviocytes migration speed and abundant production of arthritic pannus in rheumatoid arthritis. Therefore, it will provide new insight into the pathogenesis of tumor and autoimmune diseases, and will shed new light on the therapy of related clinical diseases.

Heat Shock Protein 90 Inhibitor Decreases Collagen Synthesis of Keloid Fibroblasts and Attenuates the Extracellular Matrix on the Keloid Spheroid Model

Heat Shock Protein 90 Inhibitor Decreases Collagen Synthesis of Keloid Fibroblasts and Attenuates the Extracellular Matrix on the Keloid Spheroid Model

Treg‐enriched CD4+ T cells attenuate collagen synthesis in keloid fibroblasts

Keloid is an inflammatory and fibrotic disease with an unknown pathogenesis. Regulatory T cells (Tregs) of CD4+ lineage can suppress other effector CD4+ T cells and modulate the immune response. A relative decrease in the number of Tregs may be involved in the pathogenesis of inflammatory and fibrotic diseases. We therefore investigated the number of Tregs in keloids using immunohistochemistry and examined the interaction between Tregs and keloid fibroblasts (KFs) using a coculture system. It was found that the ratio of Tregs/CD4+ T cells was lower compared with that in other common inflammatory skin conditions. In addition, Treg-enriched CD4+ T cells reduced collagen synthesis by KFs. Our findings suggest that a local imbalance of Tregs contributes to the development of keloids and that correction of this imbalance might represent a novel therapeutic approach to keloid fibrosis.

Oxymatrine inhibits collagen synthesis in keloid fibroblasts via inhibition of transforming growth factor‐β1/Smad signaling pathway

Keloids are benign dermal tumors characterized by fibroblastic proliferation and excessive accumulation of collagen. Oxymatrine (OMT) is an alkaloid extracted from the Chinese herb Sophora japonica with capacities of anti-fibrosis. To evaluate the effects of OMT on collagen production and to explore its mechanisms. OMT was applied to human keloid fibroblasts in vitro. Collagen, transforming growth factor (TGF)-β1, TGF-β receptor, and Smads were analyzed by Western Blot, reverse transcription polymerase chain reaction, and immunofluorescence. We found that both collagen synthesis and Smad3 production were significantly suppressed in a dose-dependent administration of OMT. However, expression of TGF-β1, TGF-β receptor1, TGF-β receptor2, Smad4, and Smad7 was unchanged. We also found that OMT reversed phosphorylation and nuclear translocation of Smad3 induced by TGF-β1. OMT inhibited collagen synthesis, which might be associated with TGF-β/Smad signaling pathway. These findings suggest that OMT may be a promising candidate to prevent keloid and other fibrotic diseases.

Compound Astragalus and Salvia miltiorrhiza extract inhibits cell proliferation, invasion and collagen synthesis in keloid fibroblasts by mediating transforming growth factor-β / Smad pathway

The transforming growth factor (TGF)-β/Smad pathway plays a key role in keloid development. We have previously demonstrated that compound Astragalus and Salvia miltiorrhiza extract (CASE) inhibits liver fibrosis and reduces invasion capacity of HepG2 cells by mediating the TGF-β/Smad pathway. We therefore hypothesize that CASE may also exert antifibrotic effects in keloids by mediating the TGF-β/Smad pathway. To investigate the effects of CASE on cell proliferation, invasion and collagen synthesis in keloid fibroblasts, and to explore the effects of CASE on the TGF-β/Smad signal pathway in order to elucidate its mechanisms of action. The inhibitory effects of CASE on keloid fibroblasts were evaluated. Cell proliferation was studied by MTT assay; cell invasion was observed utilizing Transwell invasion chambers; and collagen synthesis in keloid fibroblasts was measured by (3) H-proline incorporation assay. Expression of proteins induced by TGF-β1 and their intracellular localization in keloid fibroblasts were investigated by Western blot and immunofluorescence, respectively. Plasminogen activator inhibitor-1 (PAI-1) transcriptional activity was measured by real-time reverse transcription-polymerase chain reaction. CASE significantly inhibited cell proliferation induced by newborn bovine serum as well as collagen synthesis and cell invasion induced by TGF-β1 in keloid fibroblasts, while it showed weak effects on normal fibroblasts. The phosphorylation of Smad2/3 was markedly reduced by CASE treatment, while CASE exhibited stronger inhibitory effects on linker region phosphorylation (pSmad2L and pSmad3L) compared with effects on C-terminal region phosphorylation (pSmad2C and pSmad3C). In addition, CASE blocked formation of Smad2/3/4 complexes and their nuclear translocation, but upregulated Smad7 expression in a dose-dependent manner. PAI-1 mRNA and protein levels were also suppressed by CASE treatment. These results suggest that CASE exhibits inhibitory effects on cell proliferation, invasion and collagen synthesis in keloid fibroblasts, and its mechanisms of action may involve the TGF-β/Smad pathway.

Collagen triple helix repeat containing-1 inhibits transforming growth factor-β1-induced collagen type I expression in keloid

Keloids are benign skin tumours occurring during wound healing in genetically predisposed patients. There is evidence that transforming growth factor (TGF)-b is involved in keloid formation. Collagen triple helix repeat containing- 1 (Cthrc1) was identified as a novel gene expressed in the adventitia and neointima on arterial injury. It is indicated to be a cell type-specific inhibitor of TGF-b, which functionally increases cell migration while reducing collagen type I and III deposition. However, to our knowledge expression and regulatory mechanisms of Cthrc1 and TGF-b1 in keloid and normal skin have not been studied before. Cthrc1 gene regulation and potential role in keloid formation were determined, and its correlation with TGF-b1 involved in keloid pathogenesis was examined in human fibroblasts of keloids and normal skin. The expression of Cthrc1 and TGF-b1 was investigated in fibroblasts of keloid and normal skin. Collagen type I expression and collagen synthesis in keloid fibroblasts induced by TGF-b1 were examined. Then, recombinant Cthrc1 was applied to assess its correlation with TGF-b1. Increased TGF-b1 and Cthrc1 expression was examined in keloid compared with normal skin. Cthrc1 expression increased in a concentration-dependent manner induced by TGF-b1 in keloid fibroblasts. TGF-b1 stimulated collagen type I expression and collagen synthesis in keloid fibroblasts, which can be reversed by recombinant Cthrc1. TGF-b1 was upregulated in keloid fibroblasts and recombinant Cthrc1 inhibited TGF-b1-stimulated collagen type I synthesis, which suggests that Cthrc1 may be a potential therapeutic option for keloids.

Troglitazone suppresses transforming growth factor-β1-induced collagen type I expression in keloid fibroblasts

Peroxisome proliferator-activated receptor (PPAR)-gamma agonists are increasingly used in patients with diabetes and some studies have suggested a beneficial effect on organ fibrosis. However their effects on dermal fibrosis in keloids are unknown. To investigate the effect of the PPAR-gamma agonist troglitazone on transforming growth factor (TGF)-beta1-induced collagen type I expression in keloid fibroblasts. Keloid fibroblasts were cultured and exposed to different concentrations of troglitazone in the presence of TGF-beta1. The mRNA expression of PPAR-gamma was determined by semiquantitative reverse transcriptase-polymerase chain reaction. The protein of PPAR-gamma, Smad2, Smad3, phoshpo-Smad2/3 and collagen type I was determined by Western blotting and collagen synthesis was evaluated by measuring (3)H-proline incorporation. The effect of troglitazone on cell viability was evaluated by the colorimetric conversion of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide. PPAR-gamma was expressed at a moderate level in keloid fibroblasts. Troglitazone depressed TGF-beta1-stimulated collagen type I expression and collagen synthesis in keloid fibroblasts in a concentration-dependent manner. Moreover, troglitazone inhibited expression and phosphorylation of TGF-beta1-induced Smad2/3. Cell viability was unaffected. These inhibitory effects of troglitazone were reversed by the PPAR-gamma-specific antagonist GW9662. Our data suggest that PPAR-gamma is present in keloid fibroblasts and PPAR-gamma activation inhibits TGF-beta1-induced collagen type I expression at least in part by decreasing collagen synthesis. PPAR-gamma may be a promising therapeutic target for keloids.

165 Prostaglandin E2 Regulates Normal and Keloid Fibroblast Migration and Collagen Synthesis: Implications for Wound Healing

Background: Prostaglandin E2 (PGE2) is an important inflammatory mediator, which has been shown to regulate fibroblast chemotaxis and extracellular matrix production through four G-coupled protein receptors (EP1, EP2, EP3, EP4). Previous studies have linked abnormal PGE2 signaling with the development of improper damage repair and tissue fibrosis in the airway. This study begins to address the role of PGE2 in dermal fibrosis, particularly keloid formation. Such an analysis is consistent with data indicating inflammatory/immune abnormalities associated with keloids. Hypothesis: Keloid fibroblasts respond to PGE2 appropriately, but in a quantitatively diminished manner. Methods: Cultured fibroblasts from normal human skin and keloid lesions were used for these studies. Fibroblast migration was assayed using a well established two dimensional motility assay. Collagen production was analyzed using Western blot and commercial ELISA methodology. Results: Administration of PGE2 decreased keloid fibroblast migration in a dose-dependent manner, via a mechanism which appears to involve the EP2/EP4-cAMP-PKA signal transduction pathway. PGE2 reversed the transforming growth factor β-1-induced increase in collagen type I in both normal and keloid fibroblasts. Discussion: Our hypothesis is partially supported by the results, which show that the response of keloid fibroblasts to PGE2 is qualitatively and quantitatively identical to normal fibroblasts. PGE2 decreases the rates of migration and collagen synthesis of keloid fibroblasts, raising the possibility that exogenous PGE2 may have potential therapeutic applications to reduce keloid formation. Acknowledgments: The authors would like to thank Children’s Hospital of Pittsburgh for providing funding for this research.

Modulation of Collagen Synthesis in Keloid Fibroblasts by Silencing Smad2 with siRNA

Modulation of Collagen Synthesis in Keloid Fibroblasts by Silencing Smad2 with siRNA

Green Tea Extract and (−)-Epigallocatechin-3-Gallate Inhibit Mast Cell-Stimulated Type I Collagen Expression in Keloid Fibroblasts via Blocking PI-3K/Akt Signaling Pathways

Keloid, a chronic fibro-proliferative disease, exhibits distinctive histological features characterized by an abundant extracellular matrix stroma, a local infiltration of inflammatory cells including mast cells (MCs), and a milieu of enriched cytokines. Previous studies have demonstrated that co-culture with MCs stimulate type I collagen synthesis in fibroblasts, but the signaling mechanisms remain largely unknown. In this study, we investigated the signaling pathways involved in MC-stimulated type I collagen synthesis and the effects of green tea extract (GTE) and its major catechin, (-)-epigallocatechin-3-gallate (EGCG), on collagen homeostasis in keloid fibroblasts. Our results showed that MCs significantly stimulated type I collagen expression in keloid fibroblasts, and the upregulation of type I collagen was significantly attenuated by blockade of phosphatidylinositol-3-kinase (PI-3K), mammalian target of rapamycin (mTOR), and p38 MAPK signaling pathways, but not by blockade of ERK1/2 pathway. Furthermore, GTE and EGCG dramatically inhibited type I collagen production possibly by interfering with the PI-3K/Akt/mTOR signaling pathway. Our findings suggest that interaction between MCs and keloid fibroblasts may contribute to excessive collagen accumulation in keloids and imply a therapeutic potential of green tea for the intervention and prevention of keloids and other fibrotic diseases.

Modulation of Collagen Synthesis in Keloid Fibroblasts by Silencing Smad2 with siRNA

Keloids represent a dysregulated response to cutaneous wounding that results in an excessive deposition of extracellular matrix, especially types I and III collagen. Transforming growth factor (TGF)-beta plays a central role in the pathogenesis of fibrosis by inducing and sustaining activation of keloid fibroblast. However, the underlying mechanisms are poorly understood. In this study, the authors examined the function of Smad2, a recently characterized intracellular effector of TGF-beta signaling, in keloid fibroblasts using small interfering RNA (siRNA). Three pairs of siRNA duplexes targeting human Smad2 were designed; the most efficient one was selected and used for further research. Keloid fibroblasts were treated with or without Smad2 siRNA, and the expression levels of related genes were examined by reverse-transcriptase polymerase chain reaction and immunofluorescence. The down-regulation of Smad2 by siRNA led to a significant decrease in mRNA levels of Smad2 in both a dose-dependent and time-dependent manner. The knockdown of Smad2 expression in protein level was confirmed using immunofluorescence. The mRNA levels of types I and III procollagen were also significantly and uniquely decreased following the reduction of Smad2 by siRNA. The results indicate that Smad2 plays an important role in TGF-beta-induced fibrosis in keloids. Down-regulation of Smad2 expression in keloid fibroblasts can significantly decrease procollagen gene expression. Also, siRNA targeting Smad2 was an efficient reagent with which to reduce extracellular matrix deposition and attenuate process of fibrosis. It could be a new, promising therapeutic approach for improving skin wound healing and inhibiting progression of fibrotic conditions by interrupting the TGF-beta signaling pathway.

Decreased Prostaglandin E2 Production by Inflammatory Cytokine and Lower Expression of EP2 Receptor Result in Increased Collagen Synthesis in Keloid Fibroblasts

We investigated the metabolism of arachidonic acid in normal skin-derived fibroblasts (NF) as well as in keloid-derived fibroblasts (KF) in response to macrophage migration inhibitory factor (MIF), a pluripotent cytokine. We found that MIF enhanced cyclooxygenase-2 activity in NF more than in KF. Consistent with this finding, prostaglandin E(2) (PGE(2)), an antifibrogenic molecule, was more significantly increased in NF than in KF by MIF treatment. As regarding E prostanoid receptor 2, the level of expression was significantly lower in KF than in NF. On the other hand, Forskolin, a direct activator of adenylcyclase, decreased collagen synthesis in both NF and KF, which indicates that cAMP plays an important role in regulating collagen synthesis. As PGE(2) induces cAMP production, it is conceivable that increased collagen synthesis in KF might be owing to decreased PGE(2) and cAMP production. These findings may aid in the development of a therapeutic strategy for the regulation of collagen synthesis in keloid fibroblasts.

Exposure of fibroblasts derived from keloid patients to low-energy electromagnetic fields: preferential inhibition of cell proliferation, collagen synthesis, and transforming growth factor beta expression in keloid fibroblasts in vitro.

We studied the effects of electromagnetic fields (EMFs) on cell proliferation and collagen synthesis in both normal and keloid fibroblasts in vitro. Treatment of keloid fibroblasts with 60-Hz EMFs for 10 days resulted in the inhibition of cell proliferation, whereas no significant change in cell proliferation of normal fibroblasts was observed. EMFs inhibited collagen synthesis in keloid fibroblasts but not in normal fibroblasts without altering the ratio of type III to type I collagen, indicating that EMFs inhibit type I and type III collagen synthesis to the same extent. EMFs also decreased the expression of transforming growth factor beta (TGF-beta) in keloid fibroblasts. These results suggest that EMFs may have a useful therapeutic potential for the treatment of keloid.