Collagen Lattice Research Articles

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.

Mechano‐regulation of contractile phenotype in myofibroblasts

During wound healing, myofibroblast (MF) appearance correlates with increased wound tension leading to the hypothesis that the mechanics of the wound environment regulate MF formation and function. In this study we examined how the mechanics of a high‐tension environment regulate the MF contractile phenotype. To address this we cultured rat dermal fibroblasts in compliant (low‐tension) and stiff (high‐tension) collagen lattices. Stiff‐cultured cells acquired the MF contractile phenotype of increased focal adhesions and stress fiber bundling and highly expressed the contractile cytoskeletal genes smooth muscle (SM) α‐actin, SM γactin and SM 22‐α. In contrast, compliant‐cultured cells had a fibroblast phenotype and low expression of contractile genes. We next asked if this mechano‐regulation was associated with dynamic changes in the actin cytoskeleton. In support of this, we found induction of F‐actin formation increased contractile gene expression in compliant‐cultured cells, and destabilization of the F‐actin cytoskeleton down‐regulated contractile gene expression in stiff‐cultured cells. We next plan to address the role of the actin‐sensitive transcription factor, myocardin‐related transcription factor‐A, in this process. Our goal is to identify the combination of external and internal factors regulating MF formation, maintenance, and function. Supported by NIH grant R01 GM60651.

Antioxidant Effect on Myofibroblast Differentiation

Antioxidants have been shown to decrease various fibrotic cells in culture, however little is known about their effects on dermal fibrotic cells. In this study we will investigate the effects of several different antioxidants on dermal myofibroblast differentiation. This will be accomplished by measuring the percent of myofibroblasts and cell number in differing treatment groups. The antioxidants chosen for our study include: carcinine, epigallocatechin gallate, and lycopene. Immortalized human fibroblasts treated with TGF‐β are plated at a concentration of 0.2 x 105 cells per coverslip. After incubating 72 hours, the coverslips are fixed, stained with immunofluorescence, and photographed. The preliminary results suggest that myofibroblast presence is somewhat reduced by carcinine; however this antioxidant had little effect on the cell number. Experiments with epigallocatechin gallate and lycopene are underway. The antioxidants that affect myofibroblast differentiation in the coverslips will then be tested in stress‐relaxed collagen lattices. These studies may be useful to develop future therapies for Dupuytren's contracture and other fibrotic tissue pathologies. Funded by a UCO student RCSA Award.Grant Funding Source: UCO RCSA Award

4-Methylumbelliferone inhibits tumour cell growth and the activation of stromal hyaluronan synthesis by melanoma cell-derived factors

There is a close correlation between tumour progression and hyaluronan production, either by tumour cells or by stromal cells that are stimulated by tumour-derived factors. Inhibition of tumour stimulation of fibroblast hyaluronan may suppress tumour growth and invasion. To examine the effect of the hyaluronan synthesis inhibitor 4-methylumbelliferone (4-MU) on the growth of and hyaluronan synthesis by fibroblasts and C8161 and MV3 melanoma cell lines, invasion, and inhibition of tumour cell-derived factor activation of fibroblasts. Effects of 4-MU on growth and hyaluronan synthesis by fibroblasts and melanoma cells were examined in monolayer culture and fibroblast-contracted collagen lattices, and their effects on the growth and invasion of tumour cells into collagen lattices were also studied. 4-MU caused a dose-dependent growth inhibition of fibroblast and melanoma cells with maximum inhibition at 0·5 mmol L(-1) 4-MU. At this dose, 4-MU inhibited (3) H-glucosamine incorporation into fibroblast glycosaminoglycans by 52%, and hyaluronan synthesis by 64%. The relative inhibition was more pronounced when fibroblasts were stimulated with C8161 melanoma cell-conditioned medium. 4-MU reduced the level of hyaluronan in fibroblast-contracted collagen lattices, and inhibited both the growth on and invasion into the lattices by melanoma cells. This growth inhibition appears to be predominantly independent of inhibition of hyaluronan synthesis. The effect on growth inhibition was reversible, and 4-MU had no effect on apoptosis. 4-MU is a potent inhibitor of hyaluronan synthesis, induction of stromal hyaluronan accumulation by tumour cells, and fibroblast and melanoma cell proliferation, and results suggest that 4-MU may have potential as a tumour cell anti-invasive and antiproliferative agent.

Systemic vasculopathy with altered vasoreactivity in a transgenic mouse model of scleroderma

IntroductionVasculopathy, including altered vasoreactivity and abnormal large vessel biomechanics, is a hallmark of systemic sclerosis (SSc). However, the pathogenic link with other aspects of the disease is less clear. To assess the potential role of transforming growth factor beta (TGF-β) overactivity in driving these cardiovascular abnormalities, we studied a novel transgenic mouse model characterized by ligand-dependent activation of TGF-β signaling in fibroblasts.MethodsThe transgenic mouse strain Tβ RIIΔk-fib is characterized by balanced ligand-dependent upregulation of TGF-β signaling. Aortic and cardiac tissues were examined with histologic, biochemical, and isolated organ bath studies. Vascular and perivascular architecture was examined by hematoxylin and eosin (H&E) and special stains including immunostaining for TGF-β1 and phospho-Smad2/3 (pSmad2/3). Confirmatory aortic smooth muscle cell proliferation, phenotype, and functional assays, including signaling responses to exogenous TGF-β and endothelin-1, were performed. Aortic ring contractile responses to direct and receptor-mediated stimulation were assessed.ResultsAortic ring contractility and relaxation were diminished compared with wild-type controls, and this was associated with aortic adventitial fibrosis confirmed histologically and with Sircol assay. TGF-β1 and pSmad 2/3 expression was increased in the adventitia and smooth muscle layer of the aorta. Aortic smooth muscle cells from transgenic animals showed significant upregulation of TGF-β- responsive genes important for cytoskeletal function, such as transgelin and smoothelin, which were then resistant to further stimulation with exogenous TGF-β1. These cells promoted significantly more contraction of free floating type I collagen lattices when compared with the wild-type, but were again resistant to exogenous TGF-β1 stimulation. Aortic ring responses to receptor-mediated contraction were reduced in the transgenic animals. Specifically, bosentan reduced endothelin-mediated contraction in wild-type animals, but had no effect in transgenic animals, and endothelin axis gene expression was altered in transgenic animals. Transgenic mice developed cardiac fibrosis.ConclusionsThe histologic, biochemical, and functional phenotype of this transgenic mouse model of scleroderma offers insight into the altered biomechanical properties previously reported for large elastic arteries in human SSc and suggests a role for perturbed TGF-β and endothelin activity in this process.

Intense Pulsed Light Effects on the Expression of Extracellular Matrix Proteins and Transforming Growth Factor Beta-1 in Skin Dermal Fibroblasts Cultured within Contracted Collagen Lattices

Intense Pulsed Light Effects on the Expression of Extracellular Matrix Proteins and Transforming Growth Factor Beta-1 in Skin Dermal Fibroblasts Cultured within Contracted Collagen Lattices

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.

Increased cAMP Levels Modulate Transforming Growth Factor-β/Smad-induced Expression of Extracellular Matrix Components and Other Key Fibroblast Effector Functions

cAMP is a key messenger of many hormones and neuropeptides, some of which modulate the composition of extracellular matrix. Treatment of human dermal fibroblasts with dibutyryl cyclic AMP and forskolin antagonized the inductive effects of transforming growth factor-beta (TGF-beta) on the expression of collagen, connective tissue growth factor, tissue inhibitor of matrix metalloproteinase-1, and plasminogen activator inhibitor type I, four prototypical TGF-beta-responsive genes. Increased intracellular cAMP prevented TGF-beta-induced Smad-specific gene transactivation, although TGF-beta-mediated Smad phosphorylation and nuclear translocation remained unaffected. However, increased cAMP levels abolished TGF-beta-induced interaction of Smad3 with its transcriptional co-activator cAMP-response element-binding protein (CREB)-binding protein (CBP)/p300. Overexpression of the transcriptional co-activator CBP/p300 rescued Smad-specific gene transcription in the presence of cAMP suggesting that sequestration of limited amounts of CBP/p300 by the activated cAMP/CREB pathway is the molecular basis of this inhibitory effect. These findings were extended by two functional assays. Increased intracellular cAMP levels suppressed the inductive activity of TGF-beta to contract mechanically unloaded collagen lattices and resulted in an attenuation of fibroblast migration of mechanically induced cell layer wounds. Of note, cAMP and TGF-beta synergistically induced hyaluronan synthase 2 (HAS2) expression and hyaluronan secretion, presumably via putative CREB-binding sites adjacent to Smad-binding sites within the HAS2 promoter. Our findings identify the cAMP pathway as a potent but differential and promoter-specific regulator of TGF-beta-mediated effects involved in extracellular matrix homeostasis.

Conjunctival Interleukin-13 Expression in Mucous Membrane Pemphigoid and Functional Effects of Interleukin-13 on Conjunctival Fibroblasts in Vitro

Interleukin-13 (IL-13) is the dominant effector cytokine of fibrosis in pulmonary and liver disease. Excessive conjunctival fibrosis in the immunobullous disease ocular mucous membrane pemphigoid (MMP) causes blindness; the pathogenesis of scarring in this disease is incompletely understood. To determine whether IL-13 is involved in conjunctival fibrosis in MMP, we studied the expression of IL-13 in ocular MMP patients before and after systemic immunosuppression and examined the effects of IL-13 on normal human conjunctival fibroblasts. We found high stromal cell expression of IL-13 in active ocular MMP by immunohistochemistry; 80% of these cells were CD3-positive T cells. Following immunosuppression, in clinically uninflamed, treated, ocular MMP patients, the number of IL-13 positive cells was significantly reduced, but this was still fourfold greater than in normal conjunctiva. IL-13 stimulated collagen lattice contraction and migration, and decreased production of mmp-3 and mmp-10 by human conjunctival fibroblasts. The addition of T cell culture supernatant to IL-13 synergistically augmented fibroblast migration. IL-13 also up-regulated surface expression of HLA-DR, CD80, CD40, and CD154 by conjunctival fibroblasts, suggesting a potential mechanism for fibroblast-T cell cross talk, via which fibroblasts may actively engage in perpetuating chronic inflammation and continued fibrosis. Together, these findings suggest that IL-13 is involved in conjunctival fibrosis in MMP, and that IL-13 has both profibrotic and pro-inflammatory effects on human conjunctival fibroblasts.

Incorporation of Hydroxyapatite Sol Into Collagen Gel to Regulate the Contraction Mediated by Human Bone Marrow-Derived Stromal Cells

In this paper, hydroxyapatite (HA) sol composed of nanosized HA particles was incorporated into collagen gel to regulate cell-mediated collagen contraction. Human bone marrow derived stromal cells (hMSCs) were cultured for 37 days in gels with HA to collagen ratios of 0:1, 0.5:1, 1:1, 1.5:1, and 2:1, respectively. The contraction of gels was evaluated by measuring diameter change with incubation time, and the microstructure of gels at the end of culture was visualized by SEM. The cytotoxicity of HA sol was evaluated according to the percentage of maximum lactate dehydrogenase (LDH) release from hMSCs. A combination of Hoechst 33342, propidium iodide, and calcein esters was used for imaging the live/dead cells and the distribution of loaded cells within the constructs. By incorporating HA sol into the collagen gel, the hMSCs medicated contraction was delayed and the extent of contraction was reduced as the proportion of HA relative to the collagen increased. Depending on the ratio of HA sol incorporated within the collagen gel, hMSC cells revealed different morphology and a change in the distribution of loaded cells within the gels. No significant cytotoxicity was noticed as a consequence of incorporating HA sol into the collagen lattice.

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.

Effects of cell concentration and collagen concentration on contraction kinetics and mechanical properties in a bone marrow stromal cell‐collagen construct

A cell-collagen construct is commonly used to investigate the phenomenon of wound healing and to estimate the variables for tissue engineering. The purpose of this study was to assess the effects of cell concentration and collagen concentration on the contraction kinetics and mechanical properties of bone marrow stromal cell (BMSC) seeded collagen lattices. To investigate the effects of both variables on the contraction kinetics, the construct contraction was monitored up to 13 days. Incremental stress- relaxation tests were carried out after a 2-week incubation to obtain the stress-strain profiles, which were subsequently assessed in a quasilinear viscoelastic (QLV) model. During contraction, aligned BMSCs were observed first in the interior portion of the ring, followed by the middle portion and finally in the exterior portion. Constructs seeded with a higher initial cell concentration (higher than 1 x 10(5) cells/mL) or lower initial collagen concentration (lower than 2 mg/mL) exhibited faster contraction, higher ultimate stress, and superior elasticity and reduced relaxation behavior (p < 0.05). The cell-collagen model was successfully used to yield information regarding the initial cell concentration and the initial collagen concentration on contraction kinetics and mechanical behavior, which may have possible application in tissue engineering.

The α11β1 Integrin Has a Mechanistic Role in Control of Interstitial Fluid Pressure and Edema Formation in Inflammation

Collagen-binding integrins may be involved in controlling interstitial fluid pressure (Pif), transcapillary fluid flux, and tissue fluid volume. Our aim was to explore whether the newly discovered collagen binding alpha11beta1 integrin has a mechanistic role in inflammatory edema formation. In collagen matrices seeded with a mixture of mast cells and fibroblasts, fibroblasts lacking the alpha11 integrin subunit (alpha11(-/-)) contracted collagen gels less efficiently than control fibroblasts, suggesting that the alpha11beta1 integrin is able to mediate tensile force in connective tissues. In alpha11(-/-) mice, control Pif in skin did not differ from the pressure found in wild-type mice. Whereas a reduction in Pif was found in control mice after inducing inflammation, thereby contributing to fluid extravasation and edema formation, such a reduction was not seen in alpha11(-/-) mice. That this effect is mediated through the extracellular compartment is suggested by a similar plasma protein extravasation ratio in alpha11(-/-) and wild-type mice. Our data suggest that alpha11beta1 integrins on dermal fibroblasts mediate collagen lattice remodeling and have a mechanistic role in controlling Pif in inflammation and thereby fluid extravasation and edema formation in vivo.

Functional Consequences of Mitochondrial DNA Deletions in Human Skin Fibroblasts: Increased Contractile Strength in Collagen Lattices Is Due to Oxidative Stress-Induced Lysyl Oxidase Activity

Deletions within the mitochondrial DNA (mtDNA) are thought to contribute to extrinsic skin aging. To study the translation of mtDNA deletions into functional and structural changes in the skin, we seeded human skin fibroblasts into collagen gels to generate dermal equivalents. These cells were either derived from Kearns-Sayre syndrome (KSS) patients, who constitutively carry large amounts of the UV-inducible mitochondrial common deletion, or normal human volunteers. We found that KSS fibroblasts, in comparison with normal human fibroblasts, contracted the gels faster and more strongly, an effect that was dependent on reactive oxygen species. Gene expression and Western blot analysis revealed significant upregulation of lysyl oxidase (LOX) in KSS fibroblasts. Treatment with the specific LOX inhibitor beta-aminopropionitrile decreased the contraction difference between KSS and normal human fibroblast equivalents. Also, addition of the antioxidant N-tert-butyl-alpha-phenylnitrone reduced the contraction difference by inhibiting collagen gel contraction in KSS fibroblasts, and both beta-aminopropionitrile and N-tert-butyl-alpha-phenylnitrone diminished LOX activity. These data suggest a causal relationship between mtDNA deletions, reactive oxygen species production, and increased LOX activity that leads to increased contraction of collagen gels. Accordingly, increased LOX expression was also observed in vivo in photoaged human and mouse skin. Therefore, mtDNA deletions in human fibroblasts may lead to functional and structural alterations of the skin.

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.

Tumor Necrosis Factor-α in Ocular Mucous Membrane Pemphigoid and Its Effect on Conjunctival Fibroblasts

First, to determine whether tumor necrosis factor-(TNF)-alpha is expressed in the conjunctiva of ocular mucous membrane pemphigoid (MMP) and the consequences of systemic immunosuppressive treatment on this expression. Second, to investigate the in vitro effects of TNFalpha on human conjunctival fibroblasts. The expression of TNFalpha in conjunctival tissues of patients with actively inflamed ocular MMP (n = 10), patients with clinically noninflamed ocular MMP after systemic immunosuppressive treatment (n = 10), and normal subjects (n = 10) was studied by immunohistochemistry. The effect of TNFalpha on functional assays of human conjunctival fibroblast activity were investigated, including migration, collagen lattice contraction, matrix metalloproteinase (mmp), and tissue inhibitor of matrix metalloproteinase (timp) secretion, proliferation, and surface expression of HLA-DR, ICAM, CD80, CD86, CD40, CD40-ligand. In active ocular MMP, TNFalpha is expressed by a large number of stromal infiltrating cells (234 cells/mm(2)), and although the level of stromal TNFalpha expression is significantly reduced after immunosuppressive treatment (90 cells/mm(2)), these levels are still significantly elevated compared with normal conjunctiva (10 cells/mm(2), P < 0.05). TNFalpha stimulates increased migration by conjunctival fibroblasts (P < 0.001), increased production of mmp-9 (P = 0.01), decreased production of timp-2 (P = 0.01) and timp-4 (P = 0.04), and upregulated expression of CD40 and ICAM (P = 0.04). No significant effects of TNFalpha on fibroblast proliferation or collagen lattice contraction were detected. Increased conjunctival expression of TNFalpha in ocular MMP suggests that systemic TNFalpha antagonists are likely to be effective in controlling severe disease unresponsive to conventional systemic immunosuppression. Residual TNFalpha expression persists in clinically noninflamed disease. TNFalpha appears to have profibrotic and proinflammatory effects on human conjunctival fibroblasts.

β-catenin and transforming growth factor β have distinct roles regulating fibroblast cell motility and the induction of collagen lattice contraction

Backgroundβ-catenin and transforming growth factor β signaling are activated in fibroblasts during wound healing. Both signaling pathways positively regulate fibroblast proliferation during this reparative process, and the effect of transforming growth factor β is partially mediated by β-catenin. Other cellular processes, such as cell motility and the induction of extracellular matrix contraction, also play important roles during wound repair. We examined the function of β-catenin and its interaction with transforming growth factor β in cell motility and the induction of collagen lattice contraction.ResultsFloating three dimensional collagen lattices seeded with cells expressing conditional null and stabilized β-catenin alleles, showed a modest negative relationship between β-catenin level and the degree of lattice contraction. Transforming growth factor β had a more dramatic effect, positively regulating lattice contraction. In contrast to the situation in the regulation of cell proliferation, this effect of transforming growth factor β was not mediated by β-catenin. Treating wild-type cells or primary human fibroblasts with dickkopf-1, which inhibits β-catenin, or lithium, which stimulates β-catenin produced similar results. Scratch wound assays and Boyden chamber motility studies using these same cells found that β-catenin positively regulated cell motility, while transforming growth factor β had little effect.ConclusionThis data demonstrates the complexity of the interaction of various signaling pathways in the regulation of cell behavior during wound repair. Cell motility and the induction of collagen lattice contraction are not always coupled, and are likely regulated by different intracellular mechanisms. There is unlikely to be a single signaling pathway that acts as master regulator of fibroblast behavior in wound repair. β-catenin plays dominant role regulating cell motility, while transforming growth factor β plays a dominant role regulating the induction of collagen lattice contraction.

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.

The effects of replicative senescence and telomerase on contraction and motility of fibroblast cells

Fibroblast cells are important in wound healing as they differentiate into myofibroblasts that can migrate into a wound and create tension and aging skin seems to take longer to repair a wound. This study was designed to look at the migration and contraction ability of early (EP) and late (LP) passage cells as compared to cells with telomerase (hTERT), an enzyme that lengthens telomeres delaying senescence. To assess contraction, all cells were grown on coverslips with and without transforming growth factor beta, a wound healing agent, and stained for alpha smooth muscle actin (α‐sma) a myofibroblast marker. Collagen lattice assays were used to assess contraction by measuring lattice diameter as it contracted over time then stained for the presence of α‐sma. Finally, a scratch assay was used to measure the ability for each type of cell to migrate into a "wounded" area. Contraction by EP cells was greater than LP cells in a five day collagen lattice. Results on migration assays and α‐sma in coverslips and collagen lattice are still being analyzed; however expected results would show that EP and hTERT cells should migrate faster and have more α‐sma than LP cells. If these results are shown, they would suggest that the formation of α‐sma in EP and hTERT cells allows faster migration and better contraction than in LP cells. Funding provided by the University of Central Oklahoma.Grant Funding SourceUCOIRB