Fibroblast-populated Collagen Lattice Contraction Research Articles

Roles of bovine sialoglycoproteins for anti-skin aging and accelerating skin wound healing.

Fibroblasts are the most predominant cell subpopulation in the dermal layer of human skin, they play an important role in maintaining skin architecture and function. The senescence of fibroblasts is one of major causes of skin aging and chronic wound in the elderly, which is accompanied with a reduction of α2,6-sialylation on the cell surface. In this study, we investigated the effects of the bovine sialoglycoproteins on normal human dermal fibroblasts (NHDF). The results showed that bovine sialoglycoproteins could promote the proliferation and migration of NHDF cells, and accelerate the contraction of fibroblast-populated collagen lattice (FPCL). The average doubling time of NHDF cells treated with bovine sialoglycoproteins (0.5 mg/mL) was 31.1 ± 1.0 h whereas that was 37.9 ± 2.7 h for the control (p ˂ 0.05). Moreover, the expression of basic fibroblast growth factor (FGF-2) was upregulated, while that of transforming growth factor-beta 1 (TGF-β1) and human type I collagen (COL-I) were downregulated in treated NHDF cells. Furthermore, bovine sialoglycoproteins treatment significantly enhanced the α2,6-sialylation on the cell surfaces, which was consistent with the upregulation of α2,6-sialyltransferase I (ST6GAL1) expression. These results indicated that the bovine sialoglycoproteins might be developed as a reagent against skin aging in the cosmetic industry, or as a new candidate for accelerating skin wound healing and inhibiting scar formation.

IL-10 alleviates lipopolysaccharide-induced skin scarring via IL-10R/STAT3 axis regulating TLR4/NF-κB pathway in dermal fibroblasts.

Hypertrophic scar (HS) is a severe fibrotic skin disease. It has always been a major problem in clinical treatment, mainly because its pathogenesis has not been well understood. The roles of bacterial contamination and prolonged wound inflammation were considered significant. IL‐10 is a potent anti‐inflammatory cytokine and plays a pivotal role in wound healing and scar formation. Here, we investigate whether IL‐10 alleviates lipopolysaccharide (LPS)‐induced inflammatory response and skin scarring and explore the possible mechanism of scar formation. Our results showed that the expression of TLR4 and pp65 was higher in HS and HS‐derived fibroblasts (HSFs) than their counterpart normal skin (NS) and NS‐derived fibroblasts (NSFs). LPS could up‐regulate the expression of TLR4, pp65, Col I, Col III and α‐SMA in NSFs, but IL‐10 could down‐regulate their expression in both HSFs and LPS‐induced NSFs. Blocking IL‐10 receptor (IL‐10R) or the phosphorylation of STAT3, their expression was up‐regulated. In addition, in vitro and in vivo models results showed that IL‐10 could alleviate LPS‐induced fibroblast‐populated collagen lattice (FPCL) contraction and scar formation. Therefore, IL‐10 alleviates LPS‐induced skin scarring via IL‐10R/STAT3 axis regulating TLR4/NF‐κB pathway in dermal fibroblasts by reducing ECM proteins deposition and the conversion of fibroblasts to myofibroblasts. Our results indicate that IL‐10 can alleviate the LPS‐induced harmful effect on wound healing, reduce scar contracture, scar formation and skin fibrosis. Therefore, the down‐regulation of inflammation may lead to a suitable scar outcome and be a better option for improving scar quality.

IL-10 Alleviates Lipopolysaccharide-Induced Skin Scarring <i>via</i> IL-10R/STAT3 Axis Regulating TLR4/NF-κB Pathway in Dermal Fibroblasts

Background: Hypertrophic scar (HS) is a serious fibrotic skin disease. The roles of bacterial contamination and prolonged inflammation in wound were considered to be significant. IL-10 plays a pivotal role in wound healing and scar formation. Here, we investigate whether IL-10 alleviates lipopolysaccharide (LPS)-induced inflammatory response and skin scarring, explore the possible mechanism in scar formation. Methods: RT-qPCR, Western blotting, histochemistry, immunostaining, ELISA array, electron microscope, fibroblast-populated collagen lattice (FPCL) and a rabbit ear scar model were used to investigate and validate the effect of IL-10 on LPS-stimulated scar formation. Findings: Our results showed that the expression of TLR4 and pp65 was higher in HS and HS-derived fibroblasts (HSFs) than their counterpart normal skin (NS) and NS-derived fibroblasts (NSFs). LPS could upregulate the expression of TLR4, pp65, Col I, Col III and α-SMA in NSFs, but IL-10 could downregulate their expression in both HSFs and LPS-induced NSFs. Blocking IL-10 receptor (IL-10R) or the phosphorylation of STAT3, their expression was upregulated. In addition, in vitro and in vivo models results showed that IL-10 could alleviate LPS-induced fibroblast-populated collagen lattice (FPCL) contraction and scar formation. Interpretation: Our study suggests that IL-10 may improve LPS-induced harmful to wound healing, reduce scar contracture and scar formation via IL-10R/STAT3 axis regulating TLR4/NF-κB pathway in dermal fibroblasts by reducing ECM proteins deposition and the conversion of HSFs to NSFs. Therefore, the downregulation of inflammation may be a better option for improving scar quality, and become potential therapeutic targets for scarring. Funding Statement: This research was supported by grants from the National Natural Science Foundation of China (81571914, and 81741105), and the Natural Science Foundation of Shaanxi Province (2018JQ8048). Declaration of Interests: The authors declare no conflicts of interest. Ethics Approval Statement: Written consent was obtained from all participants before surgery. All protocols used in this study were approved by the Ethics Committee of Xijing Hospital, affiliated to the Fourth Military Medical University of China. Animal experiments were approved by the Experimental Animal Committee of the Fourth Military Medical University (Xi’an, China).

Wnt4 negatively regulates the TGF-β1-induced human dermal fibroblast-to-myofibroblast transition via targeting Smad3 and ERK.

Abnormal activation of Wnt signaling has been demonstrated in the wound healing process and the pathogenesis of fibrotic disorders, with Wnt4 specifically identified as having a key role in the pathogenesis of renal, pulmonary and liver fibrosis. Wnt4 also was found to be upregulated by transforming growth factor-β1 (TGF-β1) in fetal and postnatal murine fibroblasts and bone marrow mesenchymal cells, suggesting an underlying cooperation between Wnt4 and TGF-β1 in fibrosis. However, the specific roles of Wnt4 in TGF-β1-induced skin myofibroblast transition and hypertrophic scar formation remain unclear. In the present study, we first observed reduced Wnt4 expression in hypertrophic scar tissue compared with that in normal skin tissue. Following upregulation by TGF-β1, Wnt4 inhibited the TGF-β1-induced transdifferentiation of fibroblasts into myofibroblasts. Using fibroblast-populated collagen lattice contraction assays, we showed that the increased contractility induced by TGF-β1 was significantly blocked by exogenous Wnt4 and the α-smooth muscle actin (α-SMA) expression was decreased in fibroblasts in the collagen lattices. In addition, knockdown of Wnt4 resulted in further increases in α-SMA and collagen I expressions. Further investigation showed that Wnt4 could inhibit the autocrine effect of TGF-β1 as well as block the phosphorylation of Smad3 and ERK but not of AKT or JNK. Lastly, using hypertrophic scar-derived fibroblasts, we showed that the elevated α-SMA and collagen I levels were markedly reduced after treatment with Wnt4. Taken together, our results suggest that Wnt4 negatively regulates TGF-β1-induced fibroblast activation, which may represent a novel therapeutic strategy for the treatment and prevention of hypertrophic scars.

A novel FPCL model producing directional contraction through induction of fibroblast alignment by biphasic pulse direct current electric field

Although parallel alignment of fibroblasts to the tension lines of scar has been evidenced in vivo, how scar contracture generates directional contraction remains largely unclear due to the lack of effective in vitro model. Fibroblast populated collagen lattice (FPCL), a widely used in vitro model, fails to mimic scar contracture since it produces concentric contraction with the random orientation of fibroblast. We hypothesized that a novel FPCL model with fibroblast alignment might produce directional contraction and then simulate scar contracture better. Here, we showed that although direct current electric fields (DCEFs) enabled fibroblasts aligned perpendicularly to the field vector, it also promoted electrotactic migration of fibroblast in FPCL. By contrast, biphasic pulse direct current electric fields (BPDCEFs), featured by reversal of the EF direction periodically, abolished the electrotactic migration, but induced fibroblast alignment in a pulse frequency dependent manner. Specifically, BPDCEF at a pulse frequency of 0.0002 Hz induced fibroblast alignment comparable to that induced by DCEF under the same field strength (300 mV/mm), leading to an enhanced contraction of FPCL along the direction of cell alignment. FPCL pretreated by BPDCEF showed an elliptical contraction whereas it was concentric in control FPCL. Further study revealed that F-actin redistributions acted as a key mechanism for the induction of fibroblasts alignment by BPDCEF. Cytochalasin D, an inhibitor of actin dynamics, abolished F-actins redistribution, and significantly suppressed the fibroblasts alignment and the directional contraction of FPCL. Importantly, BPDCEF significantly increased RhoA activity in fibroblasts, while this response was attenuated by C3 transferase pre-treatment, a potent inhibitor of RhoA, caused F-actin depolymerization and actin filament bundle randomly distributed. Taken together, our study suggests a crucial role for fibroblast orientation in scar contracture, and provides a novel FPCL model that may be feasible and effective for investigating scar contracture in vitro.

ILK–PI3K/AKT pathway participates in cutaneous wound contraction by regulating fibroblast migration and differentiation to myofibroblast

The interactions between fibroblasts and the extracellular matrix in wound contraction are mainly mediated via integrin signaling. Integrin-linked kinase (ILK) is a key mediator in integrin signal transduction. We investigated the role of ILK in cutaneous wound contraction. We found that ILK was involved in cutaneous wound healing in rats, and ILK and PI3K/AKT inhibitors inhibited wound contraction and re-epithelialization, consequently delaying wound healing in vivo. Further, using in vitro studies, we demonstrated that ILK and PI3K/AKT inhibitors suppressed the contraction of fibroblast-populated collagen lattices, inhibited fibroblast migration, and interrupted the effect of TGF-β1 on promoting alpha smooth muscle actin (α-SMA) expression in fibroblasts. When ILK expression was directly blocked by ILK small interfering RNA transfection, the migration and α-SMA expression of normal dermal fibroblasts were significantly suppressed as well. The data suggest that the ILK–PI3K/AKT signaling pathway mediates cutaneous wound contraction by regulating fibroblast migration and differentiation to myofibroblasts.

Cryptotanshinone downregulates the profibrotic activities of hypertrophic scar fibroblasts and accelerates wound healing: A potential therapy for the reduction of skin scarring.

Hypertrophic scar (HS) is a skin fibrotic disease that causes major clinically problematic symptoms. Cryptotanshinone (CT) is an important ingredient of Danshen (Salvia miltiorrhiza Bunge extract) that has been used to treat cardio-cerebral vascular diseases. Its clinical efficacy in HS remains unclear. To investigate whether CT can inhibit HS fibrosis, HS-derived fibroblastic cells (HSFs) were established and treated with or without CT. Type-collagen-I (Col1), type-collagen-III (Col3) and α-smooth muscle actin (α-SMA) expression were measured by western blot and real-time quantitative polymerase chain reaction. HSFs migration and contraction were assessed with the scratch assay and the fibroblast-populated collagen lattice (FPCL) contraction assay, respectively. Wound healing in CT-treated Balb/c mice was assessed by immunohistochemical analysis of collagen expression and Masson's trichrome staining analysis of collagen deposition. CT treatment of HSFs down-regulated Col1, Col3 and α-SMA mRNA and protein expression, HSFs migration, and HSFs contraction, and improved FPCL architecture. In mice, CT treatment accelerated wound healing: the scar margins were narrow and there was less collagen deposition in the regenerated tissue. Thus, CT promotes wound healing and decreases excessive deposition of extracellular matrix components. CT may help to prevent and reduce scarring.

Downregulation of Scar Fibroblasts by Antineoplastic Drugs: A Potential Treatment for Fibroproliferative Disorders

The various fibroproliferative disorders affecting humans have in common excess fibroblast activity and persistent overexpression or dysregulated activity of transforming growth factor beta (TGF-β). Cancer has many similar characteristics. Antineoplastic drugs can downregulate fibroblast activity and cytokine growth factors. This study evaluates the effect of six antineoplastic drugs on keloid and Dupuytren’s disease fibroblasts. Keloid, normal scar, Dupuytren’s affected palmar fascia, and normal palmar fascia fibroblasts were grown and seeded into Fibroblast Populated Collagen Lattices (FPCLs). The FPCLs were treated with one of six antineoplastic drugs or left untreated as controls. At 7 days, supernatants were extracted from all FPCLs and assayed for expression of Transforming Growth Factor beta (TGF)-β1 and TGF-β2. All six antineoplastic drugs significantly inhibited FPCL contraction in both fibroproliferative conditions compared with the untreated controls (p β1 and TGF-β2 expression was downregulated in the supernatants of all FPCLs by the drug exposure. Cytotoxicity did not occur in these studies and was not the reason for the results. Although antineoplastic drugs can have significant side effects when given systemically, these results may be minimized when given to small areas involved in fibroproliferative scarring or when given topically or intralesionally. These in vitro results suggest that antineoplastic drugs may have a utility for treating various fibroproliferative disorders and warrant further investigation.

Inhibitory effect of TGF-β peptide antagonist on the fibrotic phenotype of human hypertrophic scar fibroblasts

Context: TGF-β plays a central role in hypertrophic scar (HS) formation and development.Objective: This study investigated the role of a TGF-β antagonist peptide in inhibiting fibrotic behavior of human HS-derived fibroblasts (HSFs).Materials and methods: HSFs were seeded at a density of 3.1 × 104/cm2 and were subjected to treatment of peptide antagonist (30 μM) or TGF-β receptor inhibitor LY2109761 (10 μM) or without treatment followed by the analyses of quantitative PCR, Elisa, in vitro wounding and fibroblast-populated collagen lattice (FPCL) assays.Results: qPCR and Elisa analyses showed that the peptide could, respectively, reduce the gene (at 48 h) and protein (at 72 h) expression levels of collagen I (86 ± 4.8%; 56.6 ± 7.3%), collagen III (73 ± 10.7%; 43.7 ± 7.2%), fibronectin (90 ± 8.9%; 21.1 ± 2.8%), and TGF-β1 (85 ± 9.3%; 25.0 ± 9.4%) as opposed to the non-treated group (p < 0.05), as the LY2109761 group similarly did. Cell proliferation was also significantly inhibited at day 5 (CCK-8 assay) by both peptide and LY2109761 treatments compared with the non-treated group (p < 0.05). The peptide also significantly inhibited cell migration as opposed to blank control at 24 h (43 ± 6.7% versus 60 ± 2.1%, p < 0.05) and at 48 h (63.9 ± 3.1% versus 95 ± 4.1%, p < 0.05). Similar to LY2109761, the peptide antagonist significantly reduced HS FPCL contraction compared with the non-treated group with significant differences in surface area at 48 h (0.71 ± 0.06 cm2 versus 0.51 ± 0.06 cm2, p < 0.05) and at 72 h (0.65 ± 0.02 cm2 versus 0.42 ± 0.01 cm2, p < 0.05).Conclusion: The TGF-β antagonist peptide may serve as an important drug for HS prevention and reduction given the obvious benefits of good biosafety, low cost, and easy manufacture and delivery.

Enhanced Contraction of a Normal Breast-Derived Fibroblast-Populated Three-Dimensional Collagen Lattice via Contracted Capsule Fibroblast-Derived Paracrine Factors: Functional Significance in Capsular Contracture Formation.

The authors' aim was to identify morphological, genotypic, and cytokine profiles of normal breast-derived fibroblasts, noncontracted breast implant capsule (Baker grades 1 and 2) fibroblasts, and contracted breast implant capsule (Baker grades 3 and 4) fibroblasts, and to investigate the paracrine effects of contracted breast capsule fibroblast--conditioned media on a breast-derived fibroblast-populated three-dimensional collagen lattice. Primary breast-derived fibroblasts (n = 5), noncontracted breast capsule fibroblasts (n = 5), and contracted breast capsule fibroblasts (n = 5) were cultured, and conditioned media were obtained from passage 1 cells. Cells were immunostained for alpha smooth muscle actin to identify myofibroblasts. A panel of 16 inflammatory, fibrosis, extracellular matrix, and tissue remodeling-related genes were investigated using quantitative reverse transcriptase polymerase chain reaction and cytokine arrays. Fibroblast-populated collagen lattices were fabricated and treated with conditioned media, and lattice contracture was measured over 5 days. Several inflammatory and fibrotic genes were significantly dysregulated in contracted breast capsule fibroblasts compared with noncontracted breast capsule fibroblasts and breast-derived fibroblasts (p < 0.05). Breast-derived fibroblast-populated collagen lattices treated with contracted breast capsule fibroblast-conditioned media demonstrated increased lattice contraction compared with treatment with normal 10% serum media (control), breast-derived fibroblasts, or noncontracted breast capsule fibroblast-conditioned media (p < 0.05). Breast-derived fibroblasts supplemented with contracted breast capsule fibroblast-conditioned media transformed into a contracted breast capsule fibroblast-like cell (p < 0.05). The authors show that contracted breast capsule-derived fibroblasts induce normal breast fibroblast transformation and contraction via paracrine signaling, which may contribute to capsular contracture formation.

Mesenchymal stromal cells reverse hypoxia-mediated suppression of α-smooth muscle actin expression in human dermal fibroblasts

During wound healing, fibroblasts deposit extracellular matrix that guides angiogenesis and supports the migration and proliferation of cells that eventually form the scar. They also promote wound closure via differentiation into α-smooth muscle actin (SMA)-expressing myofibroblasts, which cause wound contraction. Low oxygen tension typical of chronic nonhealing wounds inhibits fibroblast collagen production and differentiation. It has been suggested that hypoxic mesenchymal stromal cells (MSCs) secrete factors that promote wound healing in animal models; however, it is unclear whether these factors are equally effective on the target cells in a hypoxic wound environment. Here we investigated the impact of MSC-derived soluble factors on the function of fibroblasts cultured in hypoxic fibroblast-populated collagen lattices (FPCLs). Hypoxia alone significantly decreased FPCL contraction and α-SMA expression. MSC-conditioned medium restored hypoxic FPCL contraction and α-SMA expression to levels similar to normoxic FPCLs. SB431542, an inhibitor of transforming growth factor-β1 (TGF-β1)-mediated signaling, blocked most of the MSC effect on FPCL contraction, while exogenous TGF-β1 at levels similar to that secreted by MSCs reproduced the MSC effect. These results suggest that TGF-β1 is a major paracrine signal secreted by MSCs that can restore fibroblast functions relevant to the wound healing process and that are impaired in hypoxia.

Comparative Evaluation of Agents on Dupuytrenʼs Contracture and Keloid Fibrosis

INTRODUCTION: Neoplasms are the result of abnormal cell proliferation. Dupuytren's contracture and keloid scar are characterized by fibroblast hyperproliferation and collagen deposition resulting in abnormal scar tissue. This similarity has lead to investigation of antiproliferative/antimetabolite agents used in the treatment of neoplasms to control/alleviate Dupuytren's contracture and keloid scar. While individual drug treatments show some decrease in contraction, there has not been a reported investigation comparing antiproliferative/antimetabolite substances and their proposed mechanism of action. The purpose of this experimental series is to comparatively evaluate respective levels of TGF-β1 and -β2, the rate and extent of contraction of fibroblasts derived from keloid scar and Dupuytren's contracture when treated with antiproliferative/antimetabolite agents. METHODS: Fibroblasts obtained from explants of Dupuytren's contracture and palmar fascia (control) as well as keloids and normal scar (control) were utilized. These fibroblast populated collagen lattices (FPCL) were exposed to 72 hours of non-cytotoxic doses of 5-Fluorouracil, Methotrexate, Paclitaxel, Tamoxifen, Mitomycin-C, and Bleomycin. Standardized photography was utilized to evaluate FPCL contraction. The supernatant of FPCL was analyzed using TGF-β1 and -β2 immunoassays. RESULTS: Dupuytren's and keloid fibroblasts' FPCLs exhibit increased contraction when compared with controls. These abnormal scar types showed a significant decrease of contraction after exposure to particular antiproliferative agents. TGF-β1 secretion did not result in a significant difference between abnormal scars when compared with controls. Various antiproliferative treatment agents on abnormal scar groups decreased the expression of TGF-β1compared to the untreated controls. Abnormal scar types had elevated TGF-β2 when compared with controls. Treatment of fibroblasts with all drugs tested resulted in significant down-regulation of TGF-β2 expression compared to untreated fibroblasts from these abnormal scar types. CONCLUSION: Non-cytotoxic doses of antiproliferative/antimetabolite agents used in this study decrease fibroblast contraction significantly when compared with untreated fibroblasts. These agents also cause a significant decrease in both TGF-β1 and -β2, a likely cause of decreased fibroblast contraction. Antiproliferative and antimetbolite agents, especially Paclitaxel, Methotrexate and Tamoxifen, are effective in vitro to limit fibroblast contraction. Further testing is warranted to provide evidence of their efficacy in vivo. DISCLOSURES: Funded in part with a grant from the Plastic Surgery Foundation. This material is the result of work supported with resources and the use of facilities at the Bay Pines V.A. Healthcare System. The information presented herein do not express the views of the Department of Veterans Affairs or the United States Government. Dr. Robson is a consultant for SteadMed Medical, Inc.

Paracrine Effects of Adipose-Derived Stem Cells on Keratinocytes and Dermal Fibroblasts

BackgroundAdipose-derived stem cells (ASCs) are mesenchymal stem cells that have recently been applied to tissue repair and regeneration. Keratinocytes and dermal fibroblasts play key roles in cutaneous wound healing.ObjectiveWe investigated the paracrine effects of ASCs on HaCaT cells (i.e., immortalized human keratinocytes) and human dermal fibroblasts to explore the mechanism of the effects of ASCs on cutaneous wound healing.MethodsHaCaT cells and primary cultured human dermal fibroblasts were treated with 50% conditioned medium of ASCs (ASC-CM). Viability, in vitro wound healing, and fibroblast-populated collagen lattice contraction assays were conducted, and reverse transcription-polymerase chain reaction (RT-PCR) for the type I procollagen α1 chain gene was performed.ResultsThe proliferation of HaCaT cells and fibroblasts was increased by ASC-CM in the viability assay. ASC-CM promoted in vitro wound healing of HaCaT cells and increased the contraction of the fibroblast-populated collagen lattice. RT-PCR showed that the transcription of the type I procollagen α1 chain gene in fibroblasts was upregulated by ASC-CM.ConclusionThe stimulatory effect of ASC on cutaneous wound healing may be partially mediated by paracrine effects of ASCs on other skin cells. Application of ASCs or ASC-derived molecules could be an innovative therapeutic approach in the treatment of chronic wounds and other conditions.

Effect of integrin-linked kinase on fibroblast populated collagen lattice contraction

Objective To investigate the influences of integrin-linked kinase (ILK) on fibroblast populated collagen lattice (FPCL) contraction and the mechanism.Methods The human skin fibroblasts from plastic patients were used to construct FPCLs.The effects of ILK-AKT inhibitor LY294002 on FPCL contraction were observed.The expressions of ILK and α-SMA in fibroblasts was detected by using Western blotting after ILK siRNA transfection or LY294002 treatment.Results FPCLs in each group had various extents of contraction,but collagen gels without fibroblasts had no contraction.The contraction rate of FPCLs in LY294002 group at 24 and 48 h [ ( 20.23 ± 9.57 ) ％,(22.47 ± 10.93 ) ％ ] was significantly lower than that in control group [ (48.73 ±6.60)％,(54.33 ± 12.88)％ ] (P ＜0.05).It was also significantly lower than that in control group and DMSO group at 72,96 and 120 h (P ＜0.05).The expression of α-SMA in ILK siRNA transfected group (0.992 ±0.255) and LY294002 group ( 1.225 ±0.323)was significantly lower than other groups (2.009 ± 0.820,2.190 ± 0.577,1.758 ± 0.732 ) ( P ＜ 0.05 ).Conclusion The contraction of FPCL and expression of α-SMA in skin fibroblasts can be inhibited via blocking ILK pathway. Key words: Integrin-linked kinase; Wound; Contraction; α-SMA

Rat Mast Cells Enhance Fibroblast Proliferation and Fibroblast-Populated Collagen Lattice Contraction through Gap Junctional Intercellular Communications

Mast cells' association with fibrosis is known, but the mechanics of that association are unclear. The hypothesis is that mast cells promote fibroblast profibrotic activities through heterocellular gap junctional intercellular communications. Casting populated collagen lattices with both human mastocytoma cell line (HMC-1), an established mast cell line, and fibroblasts enhances lattice contraction via gap junctional intercellular communications. Unfortunately, in monolayer culture, HMC-1 cells and fibroblasts do not form heterocellular gap junctional intercellular communications. Freshly isolated rat peritoneal mast cells, however, establish these communications with fibroblasts in monolayer culture. Isolated rat peritoneal mast cells, however, survive only 7 days. Establishing a rat mast cell line that grows in the same medium as fibroblasts advances the study of mast cell-fibroblast interactions. HMC-1 cells thrive without supplements, suggesting that they release the factor(s) necessary for their viability. Spent HMC-1 medium may contain the factor(s) that generate a viable rat mast cell line. Rat peritoneal-isolated mast cells grew in culture medium containing spent HMC-1 medium for 4 weeks. At 4 weeks, rat mast cells (RMC-1) were successfully maintained in Dulbecco's Modified Eagle Medium with 10% serum. RMC-1 cells formed heterocellular gap junctional intercellular communications with fibroblasts, enhancing both fibroblast proliferation and co-cultured RMC-1/fibroblast/populated collagen lattice contraction. Enhanced fibroblast proliferation and lattice contraction failed to occur by including RMC-1 cells unable to establish gap junctional intercellular communications with fibroblasts, but cell proliferation was not affected by including degranulated RMC-1 cells. Heterocellular gap junctional intercellular communications with mast cells increase in fibroblast proliferation and fibroblast PCL contraction, two hypertrophic scar fibroblast activities.

Effect of 405-nm high-intensity narrow-spectrum light on fibroblast-populated collagen lattices: an in vitro model of wound healing

High-intensity narrow-spectrum (HINS) 405-nm light is a novel technology developed to address the significant problem of health-care associated infection. Its potential for wound-decontamination applications is assessed on mammalian cells and bacteria. The fibroblast-populated collagen lattice (FPCL) is used as an in vitro model of wound healing, and the effect of HINS light on contraction is examined. Effects on cell proliferation, morphological changes, and α-smooth muscle actin (α-SMA) expression are investigated. Bactericidal effects are assessed using the bacterium Staphylococcus epidermidis. Low doses of HINS light were found to have no significant inhibitory effects on FPCL contraction, cell proliferation, or α-SMA expression. Doses of up to 18 Jcm(-2) had no significant inhibitory effects on FPCL cell numbers, and this dose was shown to cause almost complete inactivation of bacteria. These results show that HINS light has potential for disinfection applications without adversely influencing wound healing.

Calcium-Dependent Signaling in Dupuytren's Disease

Previous studies suggest that Dupuytren's disease is caused by fibroblast and myofibroblast contractility. Cell contractility in smooth muscle cells is caused by calcium-dependent and calcium-independent signaling mechanisms. In the calcium-dependent pathway, calcium/calmodulin activates myosin light chain kinase (MLCK). In this study, the effects of calcium/calmodulin inhibition with the FDA-approved drug fluphenazine on Dupuytren's fibroblast contractility and MLCK expression were tested. Fibroblast lines from the palmar fascia of patients with Dupuytren's disease were explanted and used for in vitro study. The effect of fluphenazine on Dupuytren's fibroblast migration was determined using a scratch migration assay, and contractility was determined using fibroblast-populated collagen lattice (FPCL) assays. Immunohistochemical staining of MLCK in different samples of Dupuytren's tissue and normal fascia were compared. Fluphenazine demonstrated a dose-dependent inhibition of Dupuytren's fibroblast migration, with the maximum inhibition of migration observed at 20μM (69.8 ± 1.9%). Fluphenazine also inhibited FPCL contraction in a dose-dependent manner. Maximal inhibition was observed at a fluphenazine concentration of 20μM (52.5 ± 6.1%). Immunohistological staining illustrated that MLCK was predominantly expressed throughout the cytoplasm of select fibroblasts within Dupuytren's nodules, yet was absent in the fibroblasts of Dupuytren's cords and normal palmar fascia. Fluphenazine inhibits Dupuytren's fibroblast contractility and migration through inhibition of MLCK in vitro. However, the inconsistent expression of MLCK throughout Dupuytren's tissue suggests that calcium-dependent signaling may not be a primary mode of contracture formation. Fluphenazine inhibition of MLCK is not likely to be a target for the treatment of Dupuytren's disease.

Dupuytren's Fibroblast Contractility by Sphingosine-1-Phosphate Is Mediated Through Non-Muscle Myosin II

Previous studies suggest that Dupuytren's disease is caused by fibroblast and myofibroblast contractility within Dupuytren's nodules; however, the stimulus for cell contractility is unknown. Sphingosine-1-phosphate (S1P) is a serum-derived lysophospholipid mediator that enhances cell contractility by activating the S1P receptor, S1P(2). It is hypothesized that S1P stimulates Dupuytren's fibroblast contractility through S1P(2) activation of non-muscle myosin II (NMMII). This investigation examined the role of S1P and NMMII activation in Dupuytren's disease progression and suggests potential targets for treatment. We enmeshed Dupuytren's fibroblasts into fibroblast-populated collagen lattices (FPCLs) and assayed S1P-stimulated FPCL contraction in the presence of the S1P(2) receptor inhibitor JTE-013, the Rho kinase inhibitor Y-27632, the myosin light chain kinase inhibitor ML-7, and the NMMII inhibitor blebbistatin. Tissues from Dupuytren's fascia (n = 10) and normal palmar fascia (n = 10) were immunostained for NMMIIA and NMMIIB. Sphingosine-1-phosphate stimulated FPCL contraction in a dose-dependent manner. Inhibition of S1P(2) and NMMII prevented S1P-stimulated FPCL contraction. Rho kinase and myosin light chain kinase inhibited both S1P and control FPCL contraction. Dupuytren's nodule fibroblasts robustly expressed NMMIIA and NMMIIB, compared with quiescent-appearing cords and normal palmar fascia. Sphingosine-1-phosphate promotes Dupuytren's fibroblast contractility through S1P(2), which stimulates activation of NMMII. NMMII isoforms are ubiquitously expressed throughout Dupuytren's nodules, which suggests that nodule fibroblasts are primed to respond to S1P stimulation to cause contracture formation. S1P-promoted activation of NMMII may be a target for disease treatment.

Periostin differentially induces proliferation, contraction and apoptosis of primary Dupuytren's disease and adjacent palmar fascia cells

Dupuytren's disease, (DD), is a fibroproliferative condition of the palmar fascia in the hand, typically resulting in permanent contracture of one or more fingers. This fibromatosis is similar to scarring and other fibroses in displaying excess collagen secretion and contractile myofibroblast differentiation. In this report we expand on previous data demonstrating that POSTN mRNA, which encodes the extra-cellular matrix protein periostin, is up-regulated in Dupuytren's disease cord tissue relative to phenotypically normal palmar fascia. We demonstrate that the protein product of POSTN, periostin, is abundant in Dupuytren's disease cord tissue while little or no periostin immunoreactivity is evident in patient-matched control tissues. The relevance of periostin up-regulation in DD was assessed in primary cultures of cells derived from diseased and phenotypically unaffected palmar fascia from the same patients. These cells were grown in type-1 collagen-enriched culture conditions with or without periostin addition to more closely replicate the in vivo environment. Periostin was found to differentially regulate the apoptosis, proliferation, α smooth muscle actin expression and stressed Fibroblast Populated Collagen Lattice contraction of these cell types. We hypothesize that periostin, secreted by disease cord myofibroblasts into the extra-cellular matrix, promotes the transition of resident fibroblasts in the palmar fascia toward a myofibroblast phenotype, thereby promoting disease progression.

Simvastatin impairs smad-3 phosphorylation and modulates transforming growth factor β1-mediated activation of intestinal fibroblasts

Transforming growth factor (TGF) beta1, acting through the smad pathway, is critical to fibroblast-mediated intestinal fibrosis. Simvastatin exhibits antifibrotic properties. This study assessed the effects of simvastatin on TGF-beta1-mediated intestinal fibroblast activation. Human intestinal fibroblasts were activated with TGF-beta1 with or without simvastatin or the cholesterol pathway intermediates farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP). Collagen-Ialpha2 expression was assessed by reverse transcriptase-polymerase chain reaction. Connective tissue growth factor (CTGF) and smad phosphorylation were evaluated by western blot, and plasminogen activator inhibitor (PAI) 1 activity by enzyme-linked immunosorbent assay. Fibroblast filamentous (F)-actin accumulation was assessed by confocal microscopy and contraction by a fibroblast-populated collagen lattice (FPCL) model. TGF-beta1 treatment of fibroblasts induced smad-2/3 phosphorylation, CTGF and collagen-Ialpha2 production, F-actin bundling, FPCL contraction and PAI-1 activation. Pretreatment with simvastatin inhibited the induction of CTGF and collagen-Ialpha2, PAI-1 activation, F-actin bundling and FPCL contraction. The inhibitory effect of simvastatin on PAI-1 activation was reversed by GGPP and FPP. Simvastatin pretreatment inhibited TGF-beta1-mediated phosphorylation of smad-3. Simvastatin abrogates TGF-beta1-mediated intestinal fibroblast activation by inhibition of smad-3 phosphorylation. These findings offer a mechanism for the antifibrotic effects of simvastatin and a therapeutic entry point in the treatment of intestinal fibrosis.