Populated Collagen Lattice Research Articles

Stretching Promotes Wound Contraction Through Enhanced Expression of Endothelin Receptor B and TRPC3 in Fibroblasts.

The conditioned medium from stretched keratinocyte was added to the fibroblast populated collagen lattice. Then, we analyzed the levels of endothelin receptor in the human hypertrophic scar tissue and stretched fibroblasts. To address the function of TRPC3, we have used an overexpression system with the collagen lattice. Finally, the TRPC3 overexpressing fibroblasts were transplanted to mouse dorsal skin, and the rate of skin wound contraction was assessed. Conditioned medium from stretched keratinocytes increased the rate of contraction of fibroblast populated collagen lattice. In human hypertrophic scar and stretched fibroblasts, endothelin receptor type B was increased. Cyclic stretching of TRPC3 overexpressing fibroblasts activated NFATc4, and stretched human fibroblasts showed more activation of NFATc4 in response to ET-1. The wound treated with TRPC3 overexpressing fibroblasts showed more contraction than control wound. These findings suggest that cyclical stretching of wounds have an effect on both keratinocytes and fibroblasts, where keratinocytes secret more ET-1, and fibroblasts develop more sensitivity to ET-1 by expressing more endothelin receptors and TRPC3.

Cysteine-rich domain of type III collagen N-propeptide inhibits fibroblast activation by attenuating TGFβ signaling

TGFβ is a key regulator of the dynamic reciprocity between cells and the extracellular matrix that drives physiologic and pathologic responses in both tissue repair and tumor microenvironments. Our studies define type III Collagen (Col3) as a suppressor of scar formation and desmoplasia through its effects, in part, on myofibroblasts. TGFβ stimulates activation of myofibroblasts, and here, we demonstrate that cultured Col3-deficient fibroblasts have increased TGFβ signaling compared to wild-type fibroblasts. Moreover, kinetic binding studies show that a synthetic peptide containing a Col3 cysteine-rich (CR) domain found within its N-propeptide binds in a dose-dependent manner to TGFβ1, while a CR control peptide with mutated cysteines does not, suggesting that Col3 attenuates TGFβ signaling in part through the N-propeptide CR domain. Consistent with this hypothesis, the CR peptide attenuates TGFβ signaling in fibroblasts and 4T1 breast cancer cells and suppresses fibroblast activation and contraction, as assessed by α-smooth-muscle actin staining, cell wrinkling of deformable silicone, and stressed-fibroblast populated collagen lattice contraction assays. Finally, CR peptide treatment of orthotopically injected breast cancer cells (4T1) suppresses intratumoral fibroblast activation and inhibits primary tumor growth compared to CR control. Treatment with the CR peptide decreases both intratumoral canonical and non-canonical downstream TGFβ signaling targets, consistent with its extracellular binding to TGFβ. Taken together, our results suggest that the Col3 N-propeptide CR domain binds TGFβ1 and attenuates (but importantly does not eliminate) TGFβ signaling in fibroblasts and cancer cells. Expanding on our previous work, this study demonstrates an additional mechanism by which Col3 regulates cell behaviors in post-injury and tumor microenvironments and suggests that novel Col3-targeted strategies could effectively control biologic responses in vivo and improve anti-scarring/fibrosis and oncologic therapies.

<p>The Pro-Fibrotic Behavior of Human Tenon’s Capsule Fibroblasts in Medically Treated Glaucoma Patients</p>

PurposeThe aim of this study was to compare human Tenon’s capsule fibroblasts (HTCFs) obtained from patients who received medical therapy for glaucoma (glaucomatous patients) and patients not treated for glaucoma (non-glaucomatous patients) in terms of wound healing and fibrosis.Patients and MethodsBioartificial tissues (BATs) were generated using primary HTCF-populated collagen lattices. Pro-fibrotic gene expression within HTCFs was compared between glaucomatous patients and non-glaucomatous patients after BAT culture. The BATs were also assessed regarding fibroblast–myofibroblast transition, collagen remodeling and collagen contraction using alpha-smooth muscle actin immunohistochemistry, picrosirius red staining and collagen contraction assays, respectively.ResultsPro-fibrotic gene expression in BAT-cultured HTCFs derived from glaucomatous patients was significantly increased compared to non-glaucomatous patients. BATs imbued with HTCFs collected from glaucomatous patients exhibited a greater proportion of myofibroblasts as well as increased collagen contraction/remodeling compared to HTCFs isolated from non-glaucomatous patients.ConclusionHTCFs from glaucomatous and non-glaucomatous patients differ in the expression of genes involved in fibrosis, proportion of fibroblasts undergoing transdifferentiation into myofibroblasts, contractile properties and collagen remodeling. These results suggest that for any number of reasons, at a cellular level, patients who received medical therapy for glaucoma have eyes primed for fibrosis.

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.

Fibroblast populated collagen lattices exhibit opposite biophysical conditions by fibrin or hyaluronic acid supplementation.

Fibrin and hyaluronic acid are important components of the provisional wound matrix. Through interactions with fibroblasts, they provide biophysical cues that regulate the viscoelastic properties of the extracellular matrix. To understand the roles of fibrin and hyaluronic acid in a collagenous environment, we used fibroblast populated collagen lattices (collagen, collagen-fibrin, and collagen-hyaluronic acid). Compared with collagen and collagen-hyaluronic acid cultures, collagen-fibrin cultures showed less contraction, which is correlated with increased elastic (G') and complex (|G*|) moduli, and reduced proportions of dendritic fibroblasts, despite increased αv integrin expression. Stiffness decreased during culture in collagen-fibrin environment, meanwhile phase shift (δ) values increased, clearly associated with the rise in fibrinolytic and gelatinolytic activities. These processes changed the viscoelastic properties of the system toward G' and |G*| values observed on day 5 in collagen cultures. Although less collagen turnover was observed in collagen-fibrin cultures than in collagen and collagen-hyaluronic acid cultures, collagen neosynthesis was apparently insufficient to contribute to the overall viscoelastic properties of the system. Collagen-hyaluronic acid cultures showed very limited changes during time. Firstly, they exhibited the highest δ values, suggesting an increase in the viscous behavior due to the hygroscopic properties of hyaluronic acid. These results showed that fibrin and hyaluronic acid not only affect differently the viscoelastic properties of the culture, they can tune fibroblastic activity by regulating cell attachment and extracellular matrix remodeling.

English

Povidone-iodine (PI) 10% solution is an effective antiseptic. However, it appears to be toxic to the cells involved in wound healing. The aim of this study is to evaluate the toxicity of PI on cultured human fibroblast using fibroblast populated collagen lattice (FPCL) model. The cultured human fibroblast was divided into 6 groups i.e. 5 groups were exposed by PI 1, 0.1, 0.01, 0.001 and 0.0001%, and 1 group was exposed by phosphate-buffered-saline (PBS). Twenty-four hours later, the media was washed using PBS. The size of the FPCL media on each group was observed over time by serial photographs, which then were measured by Image-J computer program. Exposure of 0.1, 0.01, 0.001 and 0.0001 PI caused an obvious reduction of fibroblast’s contraction capability on FPCL media, which described temporary fibroblast injury, that showing a concentration-dependent recovery phenomenon after 48th hour. Furthermore, 1% PI exposure leads to a permanent fibroblast injury. In conclusion, PI exposure in concentration more than 0.1% has a permanent toxic effect on fibroblast that clearly observed using a simple FPCL model.

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.

Type I collagen extracted from rat-tail and bovine Achilles tendon for dental application: a comparative study

Background: Collagen has attracted great interest as a biomaterial for various dental and medical uses. Objective: Investigate the characteristics and biocompatibility of type I collagen extracted from rat-tail tendon and bovine Achilles tendon for dental application. Materials and methods: Type-I collagen was extracted from rat-tail and bovine Achilles tendon using pepsin. The purity of collagen extracts was examined using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The biocompatibility with human gingival fibroblasts (HGFs) and human oral keratinocytes (HOKs) was examined using an MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Scanning electron microscope (SEM) illustrations of purified collagen alone and collagen with HGFs and HOKs were presented. A three-dimensional wound-healing model of fibroblast populated collagen lattice (FPCL) was used to determine the capability of both sources of collagen to induce wound healing in vitro. Cellular collagen lattices were fabricated to examine the contraction rate of these collagens. Results: The average yield of collagen extracted from rat-tail and bovine Achilles tendon were 21.8±14.9% and 5.4±0.4%, respectively. The SDS-PAGE analysis showed that the extracts were composed of alpha 1, alpha 2 and beta chains with little contamination of other small proteins. The MTT assay showed good proliferation of cells cultured with each collagen extract, indicating that collagen extracts were non-toxic to the cells. SEM and the FPCL analysis showed that both types of collagen were biocompatible with both HGFs and HOKs, inducing good contraction in the in vitro model. Conclusion: Type-I collagen extracted from rat-tail and bovine Achilles tendon appeared to be biocompatible with HGFs and HOKs. Both biomaterials may be of use in dental practice.

Calcium-Based Nanoparticles Accelerate Skin Wound Healing

IntroductionNanoparticles (NPs) are small entities that consist of a hydroxyapatite core, which can bind ions, proteins, and other organic molecules from the surrounding environment. These small conglomerations can influence environmental calcium levels and have the potential to modulate calcium homeostasis in vivo. Nanoparticles have been associated with various calcium-mediated disease processes, such as atherosclerosis and kidney stone formation. We hypothesized that nanoparticles could have an effect on other calcium-regulated processes, such as wound healing. In the present study, we synthesized pH-sensitive calcium-based nanoparticles and investigated their ability to enhance cutaneous wound repair.MethodsDifferent populations of nanoparticles were synthesized on collagen-coated plates under various growth conditions. Bilateral dorsal cutaneous wounds were made on 8-week-old female Balb/c mice. Nanoparticles were then either administered intravenously or applied topically to the wound bed. The rate of wound closure was quantified. Intravenously injected nanoparticles were tracked using a FLAG detection system. The effect of nanoparticles on fibroblast contraction and proliferation was assessed.ResultsA population of pH-sensitive calcium-based nanoparticles was identified. When intravenously administered, these nanoparticles acutely increased the rate of wound healing. Intravenously administered nanoparticles were localized to the wound site, as evidenced by FLAG staining. Nanoparticles increased fibroblast calcium uptake in vitro and caused contracture of a fibroblast populated collagen lattice in a dose-dependent manner. Nanoparticles also increased the rate of fibroblast proliferation.ConclusionIntravenously administered, calcium-based nanoparticles can acutely decrease open wound size via contracture. We hypothesize that their contraction effect is mediated by the release of ionized calcium into the wound bed, which occurs when the pH-sensitive nanoparticles disintegrate in the acidic wound microenvironment. This is the first study to demonstrate that calcium-based nanoparticles can have a therapeutic benefit, which has important implications for the treatment of wounds.

Wnt/β-catenin pathway forms a negative feedback loop during TGF-β1 induced human normal skin fibroblast-to-myofibroblast transition

Fibroblast-to-myofibroblast transition is a key event during wound healing and hypertrophic scar formation. Previous studies suggested Wnt/β-catenin signaling might be involved in the wound healing. However, its specific role in skin fibroblast-to-myofibroblast transition remains unclear. To investigate the specific role of β-catenin during the transforming growth factor-β1 induced normal skin myofibroblasts transition. By real-time quantitative polymerase chain reaction, Western-blot and immunocytochemistry, the activation of Wnt/β-catenin pathway in cultured human normal skin fibroblasts during TGF-β1 induced fibroblast-to-myofibroblast transition was investigated. The effects of β-catenin on myofibroblasts transition were also investigated when SB-216763, over-expression and siRNA of β-catenin were utilized. In addition, fibroblasts populated collagen lattices contraction assays were conducted to examine the effects of β-catenin on the contractility of the fibroblasts induced by TGF-β1. Furthermore, the effects of β-catenin on the expression of α-smooth muscle actin and collagen types I and III in hypertrophic scar derived fibroblasts were studied. The expression of Wnts mRNA and β-catenin protein was up-regulated by TGF-β1 stimulation during the myofibroblasts transition. Both of SB-216763 and β-catenin over-expression was paralleled with decreased expression of α-smooth muscle actin, collagen types I and III, while siRNA targeting β-catenin leads to up-regulation of α-smooth muscle actin, collagen types I and III. The increased contractility and α-smooth muscle actin expression of the fibroblasts in the collagen lattices induced by TGF-β1 was inhibited by SB-216763. In addition, the expression levels of α-smooth muscle actin, collagen types I and III in hypertrophic scar derived fibroblasts were also down-regulated by SB-216763. Specifically in normal skin fibroblasts, β-catenin might be involved in the myofibroblasts transition and negatively regulate the TGF-β1-induced myofibroblast transition.

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

On a class of admissible constitutive behaviors in free-floating engineered tissues

A commonly used assay for studying cell–matrix interactions is the free-floating fibroblast populated collagen lattice, which was introduced in 1979. Briefly, fibroblasts are seeded within an initially thin, amorphous, untethered, circular gel consisting of reconstituted fibrillar collagen. Although the gel remains traction free and circular, the cells typically contract the gel to less than 50% of its original diameter within hours to days. Cellular mechanotransduction mechanisms are fundamental to this contraction, but there has not been a careful study of the associated mechanics. In this paper, we model the initial contraction of a circular gel by assuming a homogeneous, axisymmetric finite deformation while allowing possible radial variations in material properties, including material symmetry. For the class of constitutive relations considered, we show that equilibrium and boundary conditions only admit trivial solutions (i.e., no deformation, no contraction) unless radial variations exist in the material behavior, including cell contraction. Although more complete data are needed to model better this initial-boundary value problem, the present results are consistent with both the salient features of the gel assay and recent observations reported in the literature that cells often introduce regional variations in tissue properties in vivo in an attempt to achieve, maintain, or restore mechanical homeostasis.

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.

Thrombospondin 1 is a key mediator of transforming growth factor β-mediated cell contractility in systemic sclerosis via a mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK)-dependent mechanism

BackgroundThe mechanism underlying the ability of fibroblasts to contract a collagen gel matrix is largely unknown. Fibroblasts from scarred (lesional) areas of patients with the fibrotic disease scleroderma show enhanced ability to contract collagen relative to healthy fibroblasts. Thrombospondin 1 (TSP1), an activator of latent transforming growth factor (TGF)β, is overexpressed by scleroderma fibroblasts. In this report we investigate whether activation of latent TGFβ by TSP1 plays a key role in matrix contraction by normal and scleroderma fibroblasts.MethodsWe use the fibroblast populated collagen lattices (FPCL) model of matrix contraction to show that interfering with TSP1/TGFβ binding and knockdown of TSP1 expression suppressed the contractile ability of normal and scleroderma fibroblasts basally and in response to TGFβ. Previously, we have shown that ras/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) mediates matrix contraction basally and in response to TGFβ.ResultsDuring mechanical stimulation in the FPCL system, using a multistation tensioning-culture force monitor (mst-CFM), TSP1 expression and p-ERK activation in fibroblasts are enhanced. Inhibiting TSP1 activity reduced the elevated activation of MEK/ERK and expression of key fibrogenic proteins. TSP1 also blocked platelet-derived growth factor (PDGF)-induced contractile activity and MEK/ERK activation.ConclusionsTSP1 is a key mediator of matrix contraction of normal and systemic sclerosis fibroblasts, via MEK/ERK.

ShRNA targeting SFRP2 promotes the apoptosis of hypertrophic scar fibroblast

Hypertrophic scars result from a dysregulated process in wound healing. Although the basic mechanism is unclear, increased proliferation and decreased cell apoptosis are noticed in the development of hypertrophic scar. In previous study, we found that secreted frizzled-related protein 2 (SFRP2), which was associated with cell proliferation, apoptosis, and differentiation, was dramatically upregulated in hypertrophic scar (HS) tissue. In this study short hairpin RNA (shRNA) targeting SFRP2 was employed to characterize SFRP2 function in hypertrophic scar-derived fibroblasts (HSFb). Cell proliferation was assessed by MTT, dynamic growth curves, and BRDU assays. Meanwhile, Cell apoptosis was detected using fluorescence-activated cell sorting (FACS). Caspase-3 activity was assayed by spectrophotometry. Fibroblast populated collagen lattice (FPCL) model was employed to evaluate the contractility of HSFb. Further, real-time PCR and western blot were used to measure the mRNA and protein expressions of α-SMA in HSFb. In addition, mRNA levels of type I and III procollagen were assayed by quantitative real-time PCR. The results revealed that shRNA targeting SFRP2 significantly promoted the apoptosis of HSFb, while it had no effect on the cell proliferation. Decreased synthesis of a-smooth muscle actin (α-SMA) in HSFb and reduced contraction of fibroblasts in the FPCL model were observed. Quantitative RT-PCR suggested that the mRNAs of type I and III procollagen were significantly downregulated. In conclusion, as a novel anti-apoptosis gene, SPRP2 was present in hypertrophic scars. Importantly, shRNA targeting SFRP2 may provide a new approach to preventing the formation of HS.

Differences in the mechanism of collagen lattice contraction by myofibroblasts and smooth muscle cells

Both rat derived vascular smooth muscle cells (SMC) and human myofibroblasts contain α smooth muscle actin (SMA), but they utilize different mechanisms to contract populated collagen lattices (PCLs). The difference is in how the cells generate the force that contracts the lattices. Human dermal fibroblasts transform into myofibroblasts, expressing α-SMA within stress fibers, when cultured in lattices that remain attached to the surface of a tissue culture dish. When attached lattices are populated with rat derived vascular SMC, the cells retain their vascular SMC phenotype. Comparing the contraction of attached PCLs when they are released from the culture dish on day 4 shows that lattices populated with rat vascular SMC contract less than those populated with human myofibroblast. PCL contraction was evaluated in the presence of vanadate and genistein, which modify protein tyrosine phosphorylation, and ML-7 and Y-27632, which modify myosin ATPase activity. Genistein and ML-7 had no affect upon either myofibroblast or vascular SMC-PCL contraction, demonstrating that neither protein tyrosine kinase nor myosin light chain kinase was involved. Vanadate inhibited myofibroblast-PCL contraction, consistent with a role for protein tyrosine phosphatase activity with myofibroblast-generated forces. Y-27632 inhibited both SMC and myofibroblast PCL contraction, consistent with a central role of myosin light chain phosphatase.

Investigating the Role of P311 in the Hypertrophic Scar

The mechanisms of hypertrophic scar formation are not fully understood. We previously screened the differentially expressed genes of human hypertrophic scar tissue and identified P311 gene as upregulated. As the activities of P311 in human fibroblast function are unknown, we examined the distribution of it and the effects of forced expression or silencing of expression of P311. P311 expression was detected in fibroblast-like cells from the hypertrophic scar of burn injury patients but not in peripheral blood mononuclear cells, bone marrow mesenchymal stem cells, epidermal cells or normal skin dermal cells. Transfection of fibroblasts with P311 gene stimulated the expression of alpha-smooth muscle actin (α-SMA), TGF-β1 and α1(I) collagen (COL1A1), and enhanced the contraction of fibroblast populated collagen lattices (FPCL). In contrast, interference of fibroblast P311 gene expression decreased the TGF-β1 mRNA expression and reduced the contraction of fibroblasts in FPCL. These results suggest that P311 may be involved in the pathogenesis of hypertrophic scar via induction of a myofibroblastic phenotype and of functions such as TGF-β1 expression. P311 could be a novel target for the control of hypertrophic scar development.

OR9,54 IGFBP-6 and IGF-II: novel mediators of myofibroblast differentiation in Dupuytren's Disease?

Background: Dupuytren’s Disease (DD), a debilitating contracture of the hand, is typical of other fibrotic diseases in displaying increased levels of extra-cellular matrix (ECM)associated Transforming Growth Factor (TGF)-b and contractile (myo)fibroblasts. TGF-b induces myofibroblast differentiation in DD and regulates the deposition of molecules into the collagenenriched ECM. In addition, TGF-b promotes the accumulation and translocation of b-catenin to the nucleus to trans-activate gene transcription. We have shown that TGF-b signaling and b-catenin accumulation correlate with repressed IGFBP6 transcription and IGFBP-6 secretion in primary cultures of DD cells. In parallel, we have shown that cellular b-catenin levels are increased and ECMassociated IGFBP-6 levels are depleted in DD tissues relative to patient-matched, unaffected tissue controls. Data from a related fibrosis, desmoid tumour, indicate that IGFBP6 transcription can be repressed by b-catenin interactions with the TCF/Lef transcription factor complex. We hypothesize that TGF-b-induced b-catenin interactions with the TCF/Lef transcription factor complex represses IGFBP6 transcription in DD, resulting in decreased levels of IGFBP-6 and increased IGF-II bioavailability. As IGF-II has been shown to act in combination with TGF-b to promote myofibroblast differentiation in other systems, we further hypothesize that IGFBP-6 repression and increased IGF-II bioavailability are previously unrecognized mediators of myofibroblast differentiation in DD. Methods: Cell proliferation (WST-1) assays were performed on collagen-coated substrates with or without IGFBP-6, IGF-II or TGF-b treatments. Myofibroblast differentiation was assessed by stressed Fibroblast Populated Collagen Lattice (sFPCL) contraction assays and SDS-PAGE for a-smooth muscle actin levels. Chromatin Immunoprecipitation (ChIP) assays are underway to assess TGF-binduced b-catenin/TCF/Lef transcription complex interactions with the IGFBP6 promoter in DD and control cells. Results: TGF-b (12.5 ng.ml) or IGFBP-6 (200 and 400ng/ml) did not affect the basal proliferation rates of DD or control cells while IGF-II (100ng/ml) inhibited the proliferation of control cells but not DD cells. TGF-b induced contractility and a-smooth muscle actin levels, indicative of myofibroblast differentiation. IGFBP-6 (400ng/ml) inhibited TGF-b induced contractility and asmooth muscle actin levels in DD and control cells. IGF-II induced the contractility of DD and control cells but did not alter asmooth muscle actin levels. ChIP assays to confirm TGF-b-induced b-catenin/TCF/LEF transcription complex binding to the IGFBP6 promoter are underway. Discussion/Conclusion: TGF-b, IGF-II and IGFBP-6 do not induce DD or control cell proliferation. TGF-b induces myofibroblast differentiation and IGFBP-6 at 400ng/ml, incorporated into the collagen-rich ECM, inhibits this process. IGF-II promotes DD and control cell contractility but not a-smooth muscle actin levels, suggesting that it acts through an alternative mechanism. We are currently investigating the Rho kinase-mediated actin/myosin polymerization in IGF-II-induced cell contraction. Our data suggest that IGFBP-6 acts as an inhibitor of myofibroblast differentiation that is repressed by TGF-b signaling, thereby promoting IGF-IIinduced contractility. IGFBP-6 may have potential as a therapeutic target for the future development of non-surgical treatment interventions in DD and, potentially, other disease conditions where changes in IGF-II bioavailability are primary components of the pathophysiology.

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.

Overexpression of manganese superoxide dismutase in human dermal fibroblasts enhances the contraction of free floating collagen lattice: implications for ageing and hyperplastic scar formation

Cell-matrix interactions are of significant importance for tissue homeostasis of the skin and, if disturbed, may lead to ageing and hyperplastic scar formation. We have studied fibroblasts stably overexpressing manganese superoxide dismutase (MnSOD) with a defined capacity for the removal of superoxide anions and concomitant accumulation of hydrogen peroxide to evaluate the role of enhanced MnSOD activity on the dynamics of cell-matrix interactions in the three-dimensional collagen lattice contraction assay. MnSOD overexpressing fibroblast populated collagen lattices revealed a significantly enhanced contraction compared to collagen lattices populated with vector control cells. The enhanced collagen lattice contraction was in part due to an increase in active TGF-beta1 and the accumulation of H2O2 in MnSOD overexpressing fibroblasts populated collagen lattices. Inhibition of TGF-beta1 signalling by the ALK4,5,7 kinases' inhibitor SB431542 at least partly inhibited the enhanced collagen lattice contraction of MnSOD overexpressing fibroblasts populated lattices. In addition, supplementation of vector control fibroblast populated collagen lattices with recombinant TGF-beta1 concentration dependently enhanced the collagen lattice contraction. In the presence of the antioxidant Ebselen, a mimic of H2O2 and other hydroperoxides/peroxynitrite-detoxifying glutathione peroxidase, collagen lattice contraction and the activation of TGF-beta1 were significantly reduced in collagen lattices populated with MnSOD overexpressing fibroblasts. Collectively, these data suggest that H2O2 or other hydroperoxides or peroxynitrite or a combination thereof may function as important second messengers in collagen lattice contraction and act at least in part via TGF-beta1 activation.