Fibroblast-populated Collagen Lattice Research Articles

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.

In vitro study of the effects of plaunotol on oral cell proliferation and wound healing

Plaunotol is an acyclic diterpene alcohol extracted from a medicinal plant called plau-noi, Croton stellatopilosus Ohba, and has been widely used for the treatment of gastric ulcers in Japan. The aim of this study was to examine the effects of plaunotol on human gingival fibroblasts (HGFs) and human oral keratinocytes (HOKs). To assess the cytotoxic effect, HGFs and HOKs were treated with plaunotol. Subsequently, the morphology of cells was recorded and cells were subjected to MTT assay. To investigate cell proliferation effect, cells were treated with plaunotol and counted with a haemocytometer. To determine wound healing effect, the number of cells repopulated into the wounded areas in monolayer culture and in fibroblast-populated collagen lattice (FPCL) was measured. The results showed that 10 and 1 μg/ml (33 and 3.3 μmol/l) plaunotol induced toxicity in HGFs and HOKs, respectively. However, 0.1 μg/ml (0.33 μmol/l) plaunotol promoted HGF proliferation and wound healing in monolayer and FPCL models. In contrast, 0.1 μg/ml plaunotol could not induce HOK proliferation nor in vitro wound healing using monolayer culture, but it induced wound healing in a modified FPCL model. Our data suggested that plaunotol could promote oral cell proliferation and wound healing in vitro and may have an implication on oral wound healing.

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.

Standardized, Defined Serum-free Culture of a Human Skin Equivalent on Fibroblast-populated Collagen Scaffold

The defined, serum-free media used in the cultivation of skin equivalents are liable to inter-laboratory variations, require the preparation of multiple additives, and are potentially difficult to replicate. In this study, we assessed the use of standardized, serum-free and bovine pituitary extract-free keratinocyte culture media in the development of a human skin equivalent. After culture at the air-liquid interface for 3 weeks on a fibroblast-populated collagen lattice, an orthokeratinized, pluristratified epithelium was produced which expressed cytokeratins, cornified cell envelope precursors (involucrin, transglutaminase 1, filaggrin) and desmosomal components (desmoglein and desmocollin 1 and 3, plakophilin 1) in a differentiation-specific manner. There was also evidence of basement membrane reconstitution with collagen IV/VII, laminin 5, α6 and β4 integrin subunit expression at the epithelial-matrix junction. Overall, our findings demonstrate that readily available, defined organotypic culture media can be used to generate a reproducible skin equivalent with hallmarks of epidermal differentiation.

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.

The effect of MMP inhibitor GM6001 on early fibroblast-mediated collagen matrix contraction is correlated to a decrease in cell protrusive activity

Although fibroblasts play an essential part during the wound healing response, the mechanisms by which they mediate tissue remodelling and contraction are still unclear. Using live cell and matrix imaging within 3D free-floating fibroblast-populated collagen lattices as a model for tissue contraction, we compared the behaviour of a range of fibroblasts with low and high contraction abilities and analysed the effect of the broad spectrum MMP-inhibitor GM6001 on cell behaviour and matrix contraction. We identified two mechanisms underlying matrix contraction, one via direct cell-mediated contractile activity, the second through matrix degradation. These appear to be linked to cell morphology and regulated by the collagen concentration within the matrix. Cells with a rounded morphology proliferated in the matrix but did not remodel it efficiently, resulting in a poor ability to contract matrices. Cells with an elongated morphology showed higher levels of protrusive activity, leading to efficient matrix remodelling and contraction. GM6001 inhibited week-long matrix contraction to various extents with the different cell lines. However, quantitative analysis of the cell protrusive activity showed that GM6001 consistently decreased cell dynamics in 3D by about 20%, and this was correlated with a significant reduction in early matrix contraction. Overall our results suggest that although fibroblast-mediated matrix contraction depends on both cell dynamics and MMP-mediated matrix degradation, the efficiency of GM6001 treatment in preventing contraction might be linked to a direct effect on cell dynamics.

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.

Bacterial lipopolysaccharide promotes profibrotic activation of intestinal fibroblasts

Fibroblasts play a critical role in intestinal wound healing. Lipopolysaccharide (LPS) is a cell wall component of commensal gut bacteria. The effects of LPS on intestinal fibroblast activation were characterized. Expression of the LPS receptor, toll-like receptor (TLR) 4, was assessed in cultured primary human intestinal fibroblasts using flow cytometry and confocal microscopy. Fibroblasts were treated with LPS and/or transforming growth factor (TGF) beta1. Nuclear factor kappaB (NFkappaB) pathway activation was assessed by inhibitory kappaBalpha (IkappaBalpha) degradation and NFkappaB promoter activity. Fibroblast contractility was measured using a fibroblast-populated collagen lattice. Smad-7, a negative regulator of TGF-beta1 signalling, and connective tissue growth factor (CTGF) expression were assessed using reverse transcriptase-polymerase chain reaction and western blot. The NFkappaB pathway was inhibited by IkappaBalpha transfection. TLR-4 was present on the surface of intestinal fibroblasts. LPS treatment of fibroblasts induced IkappaBalpha degradation, enhanced NFkappaB promoter activity and increased collagen contraction. Pretreatment with LPS (before TGF-beta1) significantly increased CTGF production relative to treatment with TGF-beta1 alone. LPS reduced whereas TGF-beta1 increased smad-7 expression. Transfection with an IkappaBalpha plasmid enhanced basal smad-7 expression. Intestinal fibroblasts express TLR-4 and respond to LPS by activating NFkappaB and inducing collagen contraction. LPS acts in concert with TGF-beta1 to induce CTGF. LPS reduces the expression of the TGF-beta1 inhibitor, smad-7.

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.

The Modulus of Fibroblast-Populated Collagen Gels is not Determined by Final Collagen and Cell Concentration: Experiments and an Inclusion-Based Model

The fibroblast-populated collagen lattice is an attractive model tissue for in vitro studies of cell behavior and as the basis for bioartificial tissues. In spite of its simplicity-containing only collagen and cells-the system is surprisingly difficult to describe mechanically because of the ability of the cells to remodel the matrix, including compaction at short times and synthesis and/or degradation (and cell proliferation) at longer times. The objectives of this work were to measure the equilibrium modulus of fibroblast-populated gels with different collagen and cell concentrations, and to use that characterization as the basis for a theoretical model that could be used to predict gel mechanics based on conditions. Although many observations were as expected (e.g., the gel compacts more when there are more cells in it, and the gel is stiffer when there is more collagen in it), an unexpected result arose: the final modulus of the gel was not dependent solely on the final composition. Even if it compacted more than a gel that was originally at a high collagen concentration, a gel that started at a low collagen concentration remained less stiff than the higher-concentration gel. In light of these results and experimental studies by others, we propose a model in which the gel compaction is not homogeneous but consists instead of extreme densification near the cells in an otherwise unchanged matrix. By treating the dense regions as spherical inclusions, we used classical composite material theory to develop an expression for the modulus of a compacted gel based on the initial collagen density and the final inclusion (i.e., cell) density. The new model fit the data for moderately compacted gels well but broke down, as expected, for larger volume fractions at which the underlying model assumptions did not apply.

Induction of the CXC Chemokine Interferon-γ-Inducible Protein 10 Regulates the Reparative Response Following Myocardial Infarction

Interferon-gamma-inducible protein (IP)-10/CXCL10, an angiostatic and antifibrotic chemokine with an important role in T-cell trafficking, is markedly induced in myocardial infarcts, and may regulate the reparative response. To study the role of IP-10 in cardiac repair and remodeling. We studied cardiac repair in IP-10-null and wild-type (WT) mice undergoing reperfused infarction protocols and examined the effects of IP-10 on cardiac fibroblast function. IP-10-deficient and WT animals had comparable acute infarct size. However, the absence of IP-10 resulted in a hypercellular early reparative response and delayed contraction of the scar. Infarcted IP-10(-/-) hearts exhibited accentuated early dilation, followed by rapid wall thinning during infarct maturation associated with systolic dysfunction. Although IP-10-null and WT mice had comparable cytokine expression, the absence of IP-10 was associated with marked alterations in the cellular content of the infarct. IP-10(-/-) infarcts had more intense infiltration with CD45(+) leukocytes, Mac-2(+) macrophages, and alpha-smooth muscle actin (alpha-SMA)(+) myofibroblasts than WT infarcts but exhibited reduced recruitment of the subpopulations of leukocytes, T lymphocytes and alpha-SMA(+) cells that expressed CXCR3, the IP-10 receptor. IP-10 did not modulate cardiac fibroblast proliferation and apoptosis but significantly inhibited basic fibroblast growth factor-induced fibroblast migration. In addition, IP-10 enhanced growth factor-mediated wound contraction in fibroblast-populated collagen lattices. Endogenous IP-10 is an essential inhibitory signal that regulates the cellular composition of the healing infarct and promotes wound contraction, attenuating adverse remodeling. IP-10-mediated actions may be due, at least in part, to direct effects on fibroblast migration and function.

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.

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.

Pentoxifylline modifies three-dimensional collagen lattice model contraction and expression of collagen types I and III by human fibroblasts derived from post-burn hypertrophic scars and from normal skin

Fibroblasts are thought to be partially responsible for the persisting contractile forces that result in burn contractures. Using a monolayer cell culture and fibroblast populated collagen lattice (FPCL) three-dimensional model we subjected hypertrophic scar and non-cicatricial fibroblasts to the antifibrogenic agent pentoxifylline (PTF – 1 mg/mL) in order to reduce proliferation, collagen types I and III synthesis and model contraction. Fibroblasts were isolated from post-burn hypertrophic scars (HSHF) and non-scarred skin (NHF). Cells were grown in monolayers or incorporated into FPCL's and exposed to PTF. In monolayer, cell number proliferation was reduced (46.35% in HSHF group and 37.73% in NHF group, p < 0.0001). PTF selectively inhibited collagen III synthesis in the HSHF group while inhibition was more evident to type I collagen synthesis in the NHF group. PTF also reduced contraction in both (HSHF and NHF) FPCL.