Insights into How Plant-Derived Extracts and Compounds Can Help in the Prevention and Treatment of Keloid Disease: Established and Emerging Therapeutic Targets.

Keloid is a disease in which fibroblasts abnormally proliferate and synthesize excessive amounts of extracellular matrix, including collagen and fibronectin, during the healing process of skin wounds, causing larger scars that exceed the boundaries of the original wound. Currently, surgical excision, cryotherapy, radiation, laser treatment, photodynamic therapy, pressure therapy, silicone gel sheeting, and pharmacotherapy are used alone or in combinations to treat this disease, but the outcomes are usually unsatisfactory. The purpose of this review is to examine whether natural products can help treat keloid disease. I introduce well-established therapeutic targets for this disease and various other emerging therapeutic targets that have been proposed based on the phenotypic difference between keloid-derived fibroblasts (KFs) and normal epidermal fibroblasts (NFs). We then present recent studies on the biological effects of various plant-derived extracts and compounds on KFs and NFs. Associated ex vivo, in vivo, and clinical studies are also presented. Finally, we discuss the mechanisms of action of the plant-derived extracts and compounds, the pros and cons, and the future tasks for natural product-based therapy for keloid disease, as compared with existing other therapies. Extracts of Astragalus membranaceus, Salvia miltiorrhiza, Aneilema keisak, Galla Chinensis, Lycium chinense, Physalis angulate, Allium sepa, and Camellia sinensis appear to modulate cell proliferation, migration, and/or extracellular matrix (ECM) production in KFs, supporting their therapeutic potential. Various phenolic compounds, terpenoids, alkaloids, and other plant-derived compounds could modulate different cell signaling pathways associated with the pathogenesis of keloids. For now, many studies are limited to in vitro experiments; additional research and development are needed to proceed to clinical trials. Many emerging therapeutic targets could accelerate the discovery of plant-derived substances for the prevention and treatment of keloid disease. I hope that this review will bridge past, present, and future research on this subject and provide insight into new therapeutic targets and pharmaceuticals, aiming for effective keloid treatment.

Tripterine Inhibits Proliferation and Promotes Apoptosis of Keloid Fibroblasts by Targeting ROS/JNK Signaling.

Keloids are benign skin tumors characterized by excessive fibroblast proliferation and collagen deposition. The current treatment of keloids with hormone drug injection, surgical excision, radiotherapy, physical compression, laser therapy, cryotherapy often have unsatisfactory outcomes. The phytochemical compounds have shown great potential in treating keloids. Tripterine, a natural triterpene derived from the traditional Chinese medicine Thunder God Vine (Tripterygium wilfordii), was previously reported to exhibit an anti-scarring bioactivity in mouse embryonic fibroblast NIH/3T3 cells. Accordingly, our study was dedicated to explore its role in regulating the pathological phenotypes of keloid fibroblasts. Human keloid fibroblasts were treated with tripterine (0-10 µM) for 24 hours. Cell viability, proliferation, migration, apoptosis, and extracellular matrix (ECM) deposition were determined by CCK-8, EdU, wound healing, Transwell, flow cytometry, western blotting, and RT-qPCR assays. The effects of tripterine treatment on reactive oxygen species (ROS) generation and JNK activation in keloid fibroblasts were assessed by DCFH-DA staining and western blotting analysis. Tripterine at the concentrations higher than 4 µM attenuated the viability of human keloid fibroblasts in a dose-dependent manner. Treatment with tripterine (4, 6, and 8 µM) dose-dependently inhibited cell proliferation and migration, promoted cell apoptosis, reduced α-SMA, Col1, and Fn expression, induced ROS production, and enhanced JNK phosphorylation in keloid fibroblasts. Collectively, tripterine ameliorates the pathological characteristics of keloid fibroblasts that are associated with keloidformation and growth by inducing ROS generation and activating JNK signalingpathway.

Glabridin from Glycyrrhiza glabra Possesses a Therapeutic Role against Keloid via Attenuating PI3K/Akt and Transforming Growth Factor-ß1/SMAD Signaling Pathways.

Glabridin (Gla) is a typical flavonoid isolated from the Glycyrrhiza glabra with various bioactivities and is a common additive in many cosmetics. In our study, we evaluated the antiscarring effect of Gla from G. glabra in a rabbit ear hyperplastic scar model. Hematoxylin and eosin staining and Masson staining were applied to determine the pathological changes and collagen fibers of scar tissue in rabbits. The results suggested that Gla could reduce rabbit ear scar hyperplasia, inhibit inflammation, and decrease collagen production. Furthermore, the in vitro cell experiments were applied to determine the effects of Gla on human keloid fibroblasts (HKFs), and we observed that Gla suppressed the HKF cells' proliferation via inducing apoptosis. Subsequently, we found that Gla reduced collagen production in HKF cells. The further molecular mechanisms investigations suggested that Gla played a therapeutic role against keloid by attenuating PI3K/Akt and TGFß1/SMAD pathways. Our study would be beneficial for extending the applications of the known sweet plant of G. glabra.

Effect of the combination of photobiomodulation therapy and the intralesional administration of corticoid in the preoperative and postoperative periods of keloid surgery: A randomized, controlled, double-blind trial protocol study.

Keloid scars are characterized by the excessive proliferation of fibroblasts and an imbalance between the production and degradation of collagen, leading to its buildup in the dermis. There is no "gold standard" treatment for this condition, and the recurrence is frequent after surgical procedures removal. In vitro studies have demonstrated that photobiomodulation (PBM) using the blue wavelength reduces the proliferation speed and the number of fibroblasts as well as the expression of TGF-ß. There are no protocols studied and established for the treatment of keloids with blue LED. Therefore, the purpose of this study is to determine the effects of the combination of PBM with blue light and the intralesional administration of the corticoid triamcinolone hexacetonide on the quality of the remaining scar by Vancouver Scar Scale in the postoperative period of keloid surgery. A randomized, controlled, double-blind, clinical trial will be conducted involving two groups: 1) Sham (n = 29): intralesional administration of corticoid (IAC) and sham PBM in the preoperative and postoperative periods of keloid removal surgery; and 2) active PBM combined with IAC (n = 29) in the preoperative and postoperative periods of keloid removal surgery. Transcutaneous PBM will be performed on the keloid region in the preoperative period and on the remaining scar in the postoperative period using blue LED (470 nm, 400 mW, 4J per point on 10 linear points). The patients will answer two questionnaires: one for the assessment of quality of life (Qualifibro-UNIFESP) and one for the assessment of satisfaction with the scar (PSAQ). The team of five plastic surgeons will answer the Vancouver Scar Scale (VSS). All questionnaires will be administered one, three, six, and twelve months postoperatively. The keloids will be molded in silicone prior to the onset of treatment and prior to excision to assess pre-treatment and post-treatment size. The same will be performed for the remaining scar at one, three, six, and twelve months postoperatively. The removed keloid will be submitted to histopathological analysis for the determination of the quantity of fibroblasts, the organization and distribution of collagen (picrosirius staining), and the genic expression of TGF-ß (qPCR). All data will be submitted to statistical analysis. Trial registration: This study is registered in ClinicalTrials.gov (ID: NCT04824612).

Wubeizi Ointment Suppresses Keloid Formation through Modulation of the mTOR Pathway.

BACKGROUND: Wubeizi (Rhus chinensis Mill.) ointment has been shown as an effective treatment for keloids. However, the protective mechanisms of Wubeizi ointment are not fully understood. The mammalian target of rapamycin (mTOR) has been demonstrated to be associated with keloid pathogenesis. In the present study, we investigated if Wubeizi ointment suppressed keloid formation through the modulation of key molecules of the rapamycin (mTOR) pathway including phosphatase and tensin homolog (PTEN), phosphatidylinositol 3-kinase (PI3K), and protein kinase B (Akt). METHODS: A keloid mouse model and human keloid-derived fibroblasts were developed and treated with Galla chinensis. Immunohistochemistry, western blot, and reverse transcription-PCR were used to detect PI3K, PTEN, Akt, and mTOR in keloid tissues and keloid fibroblasts. The apoptosis and proliferation rate of keloid fibroblasts was, respectively, analyzed by flow cytometry according to the MTT assay. Statistical analysis was done using SPSS version 20.0. For two variable comparisons, a two independent samples t-test was used. For multiple variable comparisons, data were analyzed by one-way analysis of variance (ANOVA) followed by pairwise q-tests. RESULTS: Our in vivo and in vitro studies showed that Wubeizi ointment suppressed keloid formation through inhibition of fibroblast proliferation and promotion of fibroblast apoptosis. The underlying basis involves downregulation of p-Akt and p-mTOR as well as upregulation of PTEN. CONCLUSION: These findings may contribute to a better understanding of the mechanisms of Wubeizi ointment for treating keloids.

Effects of hyperbaric oxygen therapy on the expression levels of the inflammatory factors interleukin-12p40, macrophage inflammatory protein-1ß, platelet-derived growth factor-BB, and interleukin-1 receptor antagonist in keloids.

BACKGROUND: Our study aimed to screen and explore the expression of inflammatory factors in keloid patients and to investigate how hyperbaric oxygen (HBO) therapy affects the expression levels of interleukin-12p40 (IL-12p40), macrophage inflammatory protein-1ß (MIP-1ß), platelet-derived growth factor-BB (PDGF-BB), and interleukin-1 receptor antagonist (IL-1Ra). OBJECTIVE: 30 patients were randomly selected and divided into the following 3 groups: keloid samples from keloid patients treated with HBO therapy (A), keloid samples from keloid patients treated without HBO therapy (B), and normal control skin samples derived from individuals who had no clear scarring (C). Each group included 10 samples. METHODS: Inflammatory factors in the keloid tissues were measured with the MILLIPLEX multiplexed Luminex system. Hematoxylin and eosin staining, immunohistochemical staining, and Western blotting were used to observe the morphological differences in different tissues and the expression levels. RESULTS: The expression levels of inflammatory mediators, including IL-12p40, MIP-1ß, PDGF-BB, and IL-1Ra, in keloid tissues were significantly different from those in samples of normal skin. Hematoxylin and eosin staining showed significantly greater inflammatory infiltration in keloid tissue. Significantly different expression levels were observed in group A, B, and C. CONCLUSION: Significantly altered levels of inflammatory factors in the samples from keloid patients were observed, suggesting that formation of a keloid is potentially related to inflammatory responses. HBO therapy could significantly affect the expression levels of IL-12p40, MIP-1ß, PDGF-BB, and IL-1Ra, indicating that the effects of HBO therapy are associated with the attenuation of inflammatory responses.

Hyperbaric oxygen therapy can ameliorate the EMT phenomenon in keloid tissue.

BACKGROUND: Hyperbaric oxygen therapy (HBOT) has been widely used in the clinical setting. In this study, HBOT therapy was evaluated for its ability to ameliorate the epithelial-to-mesenchymal transition (EMT) phenomenon in keloid tissue. METHODS: Keloid patients were randomly divided into two groups: keloid patients (K group, 9 patients) and keloid patients receiving HBOT (O group, 9 patients). A third group with normal skin (S group, 9 patients) was established for control. Before HBOT and surgery, a laser Doppler flowmeter was used to measure the keloid blood supply of patients in the O group. Hematoxylin and eosin (H&E) staining was used to observe morphology. E-cadherin, ZO-1, vimentin, fibronectin, vascular endothelial growth factor (VEGF), and hypoxia inducible factor (HIF)-1α were measured by immunofluorescence staining and Western blot analysis. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to evaluate the mRNA expression level of these factors as well. RESULTS: In the O group, keloid blood perfusion was significantly reduced after patients received HBOT. Compared with the K group, lower expression levels of vimentin, vibronectin, VEGF, and HIF-1α were observed in the O group, whereas the expression of E-cadherin and ZO-1 was significantly higher. The mRNA expression of E-cadherin and ZO-1 was also increased after HBOT. CONCLUSIONS: The expression levels of factors related to the EMT phenomenon were significantly reversed in keloid patients after they received HBOT, indicating that HBOT may be an effective therapy against the EMT phenomenon in keloid patients.

Molecular signalings in keloid disease and current therapeutic approaches from natural based compounds.

CONTEXT: Keloid is an excessive dermal scar occurring in response to skin injuries. Several therapeutic strategies have been proposed to ease the aggressiveness of keloid scarring. Even though the principle mechanism underlying the disease propagation still remains unidentified, several signaling pathways were highly focused as plausible pathways involving keloid formation, including transforming growth factor-beta 1 (TGF-ß1), mitogen-activated protein kinase (MAPK), insulin-like growth factor-I (IGF-I), and integrin pathways. Natural compounds containing multiple bioeffective properties such as quercetin, asiaticoside, Astragalus membranaceus Bunge. (Leguminosae), and Salvia miltiorrhiza Bunge. (Lamiaceae) extracts, curcuminoids, oxymatrine, madecassoside, and Aneilema keisak Hassk. (Commelinaceae) are claimed as candidates for therapeutic treatment against keloid disorder. OBJECTIVE: This review investigates current mechanisms regarding keloid formation and provides scientific evidence supporting the therapeutic potential of natural compounds. METHODS: This review obtained and analyzed a number of literature data items from various databases including Pubmed, ScienceDirect, and Elton B. Stephens Company (EBSCO). RESULT: Several phytochemical compounds are able to suppress keloid scar development through manipulating various components in the complex signaling cascades. CONCLUSION: The present review may be helpful to future studies that further examine the molecular mechanism of keloid etiology as well as investigate the anti-keloid property in natural compounds.

Evaluation of genistein ability to modulate CTGF mRNA/protein expression, genes expression of TGFß isoforms and expression of selected genes regulating cell cycle in keloid fibroblasts in vitro.

Keloids are characterized by overgrowth of connective tissue in the skin that arises as a consequence of abnormal wound healing. Normal wound healing is regulated by a complex set of interactions within a network of profibrotic and antifibrotic cytokines that regulate new extracellular matrix (ECM) synthesis and remodeling. These proteins include transforming growth factor ß (TGFß) isoforms and connective tissue growth factor (CTGF). TGFß1 stimulates fibroblasts to synthesize and contract ECM and acts as a central mediator of profibrotic response. CTGF is induced by TGFß1 and is considered a downstream mediator of TGFß1action in fibroblasts. CTGF plays a crucial role in keloid pathogenesis by promoting prolonged collagen synthesis and deposition and as a consequence sustained fibrotic response. During keloids formation, besides imbalanced ECM synthesis and degradation, fibroblast proliferation and it's resistance to apoptosis is observed. Key genes that may play a role in keloid formation and growth involve: suppressor gene p53.,cyclin-depend- ent kinase inhibitor CDKN1A (p21) and BCL2 family genes: antiapoptotic BCL-2 and proapoptotic BAX. Genistein (4',5,7-trihydroxyisoflavone) exhibits multidirectional biological action. The concentration of genistein is relatively high in soybean. Genistein has been shown as effective antioxidant and chemopreventive agent. Genistein can bind to estrogen receptors (ERs) and modulate estrogen action due to its structure similarity to human estrogens. Genistein also inhibits transcription factors NFκB. Akt and AP-l signaling pathways, that are important for cytokines expression and cell proliferation, differentiation, survival and apoptosis. The aim of the study was to investigate genistein as a potential inhibitor of CTGF and TGFß1, ß2 and ß3 isoforms expression and a potential regulator of p53. CDKN1A(p21), BAX and BCL-2 expression in normal fibroblasts and fibroblasts derived from keloids cultured in vitro. Real time RT-QPCR was used to estimate transcription level of selected genes in normal and keloid fibroblasts treated with genistein. Secreted/cell-associated CTGF protein was evaluated in cell growth's medium by ELISA. Total protein quantification was evaluated by fluorimetric assay in cells llsates (Quant-iT TM Protein Assay Kit). It was found that TGFß1, ß2 and ß3 genes expression are decreased by genistein. Genistein suppresses the expression of CTGF mRNA and CTGF protein in a concentration dependent manner, p53 and p21 genes expression are modulated by genistein in concentration dependent manner. The agent also modulates BAX/BCL-2 ratio in examined cells in vitro.

Asiatic acid isolated from Centella asiatica inhibits TGF-ß1-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)-ß/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-ß/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-ß1 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-ß1 stimulation than normal fibroblasts in terms of invasion and collagen synthesis. AA could suppress TGF-ß1-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-ß1-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.

[Effects of oxymatrine on the expressions of pro-collagen and fibronectin of fibroblasts derived from human hyperplastic scars].

OBJECTIVE: To study the effects of oxymatrine (OM) on the expressions of pro-collagen I (PC I), pro-collagen II (PC III), fibronectin (FN), matrix metalloproteinase-1 (MMP-1) mRNA of fibroblasts from keloid (KFb), hyperplastic scar (HFb), and normal skin (NFb), and to compare with hydrocortisone (HC). METHODS: The primary KFb, HFb and NFb were derived from patients and cultured in vitro using tissue block culture method. The fibroblasts were treated with 500 microg/mL OM, 2 microg/mL HC, or without any medicine (as the control). The mRNA expressions of PC I, PC III, FN, MMP-1 of the fibroblasts were detected using RT-PCR. RESULTS: Under the normal condition, when compared with NFb, the mRNA expressions of PC I of KFb and HFb increased by 31.7% and 34.2% (both P < 0.05). Besides, the mRNA expression of PC III of KFb increased by 44.9% (P < 0.01). OM down-regulated the mRNA expressions of FN and PC I of HFb by 18.8% and 23.6% respectively (both P < 0.05). HC decreased the mRNA expressions of FN and PC I of HFb by 26.8% and 43.6% respectively (P < 0.05, P < 0.01). Meantime, OM up-regulated the mRNA expression of MMP-1 of KFb by 21.8% (P < 0.05). CONCLUSIONS: OM suppressed the synthesis of extracellular matrix (ECM) possibly through down-regulating the mRNA expressions of PC I and FN. Compared with HC, OM could promote the degradation of ECM through inducing the MMP-1 mRNA expressions of KFb. Therefore, OM could be potentially used in treatment of hypertrophic scar and keloid.

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

BACKGROUND: The transforming growth factor (TGF)-ß/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-ß/Smad pathway. We therefore hypothesize that CASE may also exert antifibrotic effects in keloids by mediating the TGF-ß/Smad pathway. OBJECTIVES: 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-ß/Smad signal pathway in order to elucidate its mechanisms of action. METHODS: 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-ß1 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. RESULTS: CASE significantly inhibited cell proliferation induced by newborn bovine serum as well as collagen synthesis and cell invasion induced by TGF-ß1 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. CONCLUSIONS: 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-ß/Smad pathway.

Addition of novel degenerate electrical waveform stimulation with photodynamic therapy significantly enhances its cytotoxic effect in keloid fibroblasts: first report of a potential combination therapy.

BACKGROUND: We recently reported use of photodynamic therapy (PDT) for treating keloid disease (KD). However, in view of high recurrence rates post any treatment modality, adjuvant therapies should be considered. Additionally, we previously demonstrated the effect of a novel electrical waveform, the degenerate wave (DW) on differential gene expression in keloid fibroblasts. OBJECTIVE: In this study, we evaluated the in vitro cytotoxic effect of PDT at 5J/cm(2) and 10J/cm(2) of red light (633 ± 3nm) using 5-aminolevulinic acid (ALA) and methyl aminolevulinate (MAL) with and without DW, on keloid fibroblasts compared to normal skin fibroblasts. METHODS: The rate of intracellular photosensitizer (protoporphyrin IX, PPIX) generation and disintegration, reactive oxygen species (ROS) generation, LDH cytotoxicity, WST-1 cytoproliferation, apoptosis by Caspase-3 activation, mitochondrial membrane potential assessment by JC-1 aggregates, qRT-PCR, flow cytometry and In-Cell Western Blotting were performed. RESULTS: PPIX accumulation and disintegration rate was higher in keloid than normal fibroblasts after incubation with MAL compared to ALA. Increased cytotoxicity and decreased cytoproliferation were observed for keloid fibroblasts after PDT+DW treatment compared to PDT alone. ROS generation, mitochondrial membrane depolarization, apoptosis (Caspase-3 activation) and collagens I and III gene down-regulation were higher in keloid compared to normal skin fibroblasts after MAL-PDT+DW treatment. An increase in the number of cells entering apoptosis and necrosis was observed after PDT+DW treatment by flow cytometry analysis. All positive findings were statistically significant (P<0.05). CONCLUSION: The cytotoxic effect of PDT on keloid fibroblasts can be enhanced significantly with addition of DW stimulation, indicating for the first time the utility of this potential combinational therapy.

Vitamin D: a novel therapeutic approach for keloid, an in vitro analysis.

BACKGROUND: Vitamin D and its metabolites play an important role in calcium homeostasis, bone remodelling, hormone secretion, cell proliferation and differentiation. Recent studies also suggest a beneficial role of vitamin D in slowing the progression of tissue fibrosis. However, their effects on dermal fibrosis and keloids are unknown. Objectives To investigate the effect of 1,25-dihydroxyvitamin D3 (1,25D) in the pathogenesis of tissue fibrosis by keloid fibroblasts (KFs). METHODS: KFs were cultured and exposed to different concentrations of 1,25D in the presence or absence of transforming growth factor (TGF)-ß1. KF phenotypes and protein production were analysed by real-time reverse transcriptase-polymerase chain reaction, Western blot, immunofluorescence and multiplex enzyme-linked immunosorbent assay techniques. Collagen synthesis was evaluated by measuring (3) H-proline incorporation. The effect of 1,25D on cell proliferation and viability was evaluated by Formazan assay, proliferating cell nuclear antigen expression and the colorimetric conversion of 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide. RESULTS: We confirmed the presence of vitamin D receptors (VDRs) in cultured keloid fibroblasts. Fibroblasts transfected with a vitamin D response element reporter construct and exposed to the active vitamin D metabolite 1,25D showed increased promoter activity indicating VDR functionality in these cells. Incubation of KFs with 1,25D suppressed TGF-ß1-induced collagen type I, fibronectin and α-smooth muscle actin expression. 1,25D also modulated plasminogen activator inhibitor-1 and matrix metalloproteinase-9 expression induced by TGF-ß1. Interestingly, 1,25D induced hepatocyte growth factor mRNA expression and protein secretion in keloid fibroblasts. CONCLUSIONS: This study highlights key mechanistic pathways through which vitamin D decreases fibrosis, and provides a rationale for studies to test vitamin D supplementation as a preventive and/or early treatment strategy for keloid and related fibrotic disorders.

A novel in vitro assay for electrophysiological research on human skin fibroblasts: degenerate electrical waves downregulate collagen I expression in keloid fibroblasts.

Electrical stimulation (ES) has been used for the treatment of wounds and has been shown to alter gene expression and protein synthesis in skin fibroblasts in vitro. Here, we have developed a new in vitro model system for testing the effects of precisely defined, different types of ES on the collagen expression of normal and keloid human skin fibroblasts. Keloid fibroblasts were studied because they show excessive collagen production. Both types of fibroblasts were electrically stimulated with alternating current (AC), direct current (DC) or degenerate waves (DW). Cells were subjected to 20, 75 and 150mV/mm electric field strengths at 10 and 60Hz frequencies. At lower electric fields, all types of ES upregulated collagen I in both cell types compared to controls. However, at higher electric field strength (150mV/mm) and frequency (60Hz), DW maximally downregulated collagen I in keloid fibroblasts, yet had significantly lower cytotoxic effects on normal fibroblasts than AC and DC. Compared to unstimulated cells, both normal skin and keloid fibroblasts showed a significant decrease in collagen I expression after 12h of DW and AC stimulation. In contrast, increasing amplitude of DC upregulated collagen I and PAI-1 gene transcription in normal and keloid fibroblasts, along with increased cytotoxicity effects. Thus, our new preclinical assay system shows highly differential effects of specific types of ES on human fibroblast collagen expression and cytotoxicity and identifies DW of electrical current (DW) as a promising, novel therapeutic strategy for suppressing excessive collagen I formation in keloid disease.

Overview about the keloid scars and the elaboration of a non-invasive, unconventional treatment.

Although the healing means 2 types of biological mechanisms that seem to be "pathologic", the swell and the granulations are a normal process in the biology of the human being, representing two systemic functions: the adaptation and the morphogenesis. There is a pathological healing in which the fundamental healing phenomenon is deviated from the normal. There are three variable parameters responsible for the pathological evolution of a scar: the cellular population, the fundamental matrix and the fibers. The healing evolution can be deviated to an intense maturation with an "old", atrophic scar, or to an incomplete maturation and the result is a hypertrophic or a keloid scar. For the hypertrophic scars, the excision and the skin graft lead to good results and the relapses are rare; the keloid relapse is always at the border between the graft and the wound edge, or between the two skin grafts. These are the considerations for which the treatments are mixed, combined (surgical, drugs, physiotherapy) both in our country and abroad, but the results are still frustrating. That is why new, modern methods of treatment are used today: criotherapy, laser, ultrasounds. However, even those treatments are not very successful: tissue expander, external press therapy, corticosteroids injections, other pharmacological agents (retinoic acid, colchicines, antineoplasics). We propose a regenerative, alternative, non-invasive treatment starting from the results we obtained in a research work 4 years ago, when we irradiated the fibroblasts in an electromagnetic high frequency millimeter waves field, and we obtained the fibroblasts apoptosis and the reorganization of the collagen fibers by changing the piezoelectric emission.

Suppression of TGF-ß1/SMAD pathway and extracellular matrix production in primary keloid fibroblasts by curcuminoids: its potential therapeutic use in the chemoprevention of keloid.

Keloid is a fibrotic disease characterized by abnormal accumulation of extracellular matrix (ECM) in the dermis. It is a late spreading skin overgrowth and may be considered a plastic surgeon's nightmare. In nature, curcuminoid is composed of curcumin, demethoxycurcumin (DMC) and bisdemethoxycurcumin (bDMC). Curcuminoids have been found to inhibit fibrosis. However, their role in the synthesis of ECM in the keloid fibroblasts (KFs) has remained unclear. In this series of studies, a total of seven primary KFs cultures were used as the KFs model for investigating the inhibitory effect of curcuminoids on the expression of ECM and TGF-ß1. A sensitive and reproducible HPLC method was developed to provide a quantitative analysis on the cellular uptake of curcuminoids onto the KF cells. The level of ECM in the primary KFs was elevated. The elevation of ECM and TGF-ß1/p-SMAD-2 level was substantially blocked by the cellular uptake of curcumin in a dose-dependent manner in all the seven primary KFs. The results have led to the conclusion that the excessive production of ECM in the KF cells could be blocked and/or rapidly decreased by curcumin.

Green tea polyphenol epigallocatechin-3-gallate suppresses collagen production and proliferation in keloid fibroblasts via inhibition of the STAT3-signaling pathway.

Keloids are benign skin tumors characterized by collagen accumulation and hyperproliferation of fibroblasts. To find an effective therapy for keloids, we explored the pharmacological potential of (-)-epigallocatechin-3-gallate (EGCG), a widely investigated tumor-preventive agent. When applied to normal and keloid fibroblasts (KFs) in vitro, proliferation and migration of KFs were more strongly suppressed by EGCG than normal fibroblast proliferation and migration (IC(50): 54.4 microM (keloid fibroblast (KF)) versus 63.0 microM (NF)). The level of Smad2/3, signal transducer and activator of transcription-3 (STAT3), and p38 phosphorylation is more enhanced in KFs, and EGCG inhibited phosphorylation of phosphatidylinositol-3-kinase (PI3K), extracellular signal-regulated protein kinase 1/2 (ERK1/2), and STAT3 (Tyr705 and Ser727). To evaluate the contribution of these pathways to keloid pathology, we treated KFs with specific inhibitors for PI3K, ERK1/2, or STAT3. Although a PI3K inhibitor significantly suppressed proliferation, PI3K and MEK/ERK inhibitors had a minor effect on migration and collagen production. However, a JAK2/STAT3 inhibitor and a STAT3 siRNA strongly suppressed proliferation, migration, and collagen production by KFs. We also found that treatment with EGCG suppressed growth and collagen production in the in vivo keloid model. This study demonstrates that EGCG suppresses the pathological characteristics of keloids through inhibition of the STAT3-signaling pathway. We propose that EGCG has potential in the treatment and prevention of keloids.

Human skin keloid fibroblasts display bioenergetics of cancer cells.

Cultured human skin keloid fibroblasts (KFs) showed bioenergetics similar to cancer cells in generating ATP mainly from glycolysis as demonstrated by increased lactate production. Activities of hexokinase, glyceraldehyde-3-phosphate dehydrogenase, and lactate dehydrogenase were also significantly higher compared with normal fibroblasts (NFs). Inhibitors of glycolysis decreased the rate of ATP biosynthesis more significantly in KFs suggesting their reliance on glycolysis. In contrast, ATP generation in NFs was derived mainly from oxidative phosphorylation (OXPHOS), which was more compromised by mitochondrial/respiratory inhibitors. However, when fortified with excess exogenous respiratory substrates, ATP production was increased to a similar maximal level in both types of fibroblasts. In spite of this seemingly equal total capacity, ATP biosynthesis and intracellular ATP concentration were significantly higher in KFs, which further increased their ATP production when exposed to hypoxia and hypoxia-mimetics: desferrioxamine and cobalt chloride. This upregulation was again significantly compromised by glycolytic inhibitors. The rate of generation of reactive oxygen species was lower in KFs possibly due to their switch to aerobic glycolysis from mitochondrial OXPHOS. Thus, cultured skin KFs could provide a human cell model to study the de-regulation of bioenergetics of proliferative cells and their response to the HIF (hypoxia-inducible factor) signaling.

Prostaglandin E2 inhibition of keloid fibroblast migration, contraction, and transforming growth factor (TGF)-beta1-induced collagen synthesis.

Keloid formation has been linked to aberrant fibroblast activity, exacerbated by growth factors and inflammatory mediators. Prostaglandin E2 (PGE2), synthesized from arachidonic acid by cyclooxygenases (COX) and synthases (PGES), acts as both an inflammatory mediator and fibroblast modulator. Although PGE2 has known antifibrotic effects in the lower airway, its role in dermal fibrosis in general, and keloid formation in particular, remains unclear. This study focused on: (1) the effects of PGE2 on keloid fibroblast migration, contraction, and collagen synthesis and (2) endogenous PGE2 synthesis in response interleukin-1beta. PGE2 decreased keloid fibroblast migration and contraction via an EP2/EP4-cAMP mechanism that disrupted actin cytoskeletal dynamics and reversed transforming growth factor-beta1-induced collagen I and III synthesis. Impaired fibroblast PGE2 production has been linked to lower airway fibrosis and recently to keloid formation. Here, we showed that interleukin-1beta stimulation leads to nuclear factor-kappaB translocation to the nucleus, resulting in up-regulation of COX-2 and microsomal PGE2 synthase 1. Up-regulation of COX-2 in, and secretion of PGE2 by keloid fibroblasts are diminished compared with their normal fibroblast counterparts. We suggest that the antifibrotic effects of PGE2 during keloid formation are potentially diminished due to aberrant paracrine fibroblast signaling. Exogenous PGE2 may supplement decreased endogenous levels and inhibit keloid formation or progression.