CD109, a TGF-�� co-receptor, attenuates extracellular matrix production in scleroderma skin fibroblasts

Introduction

Scleroderma or systemic sclerosis (SSc) is a complex connective tissue disease characterized by fibrosis of skin and internal organs. Transforming growth factor beta (TGF-β) plays a key role in the pathogenesis of SSc fibrosis. We have previously identified CD109 as a novel TGF-β co-receptor that inhibits TGF-β signaling. The aim of the present study was to determine the role of CD109 in regulating extracellular matrix (ECM) production in human SSc skin fibroblasts.

Methods

CD109 expression was determined in skin tissue and cultured skin fibroblasts of SSc patients and normal healthy subjects, using immunofluorescence, western blot and RT-PCR. The effect of CD109 on ECM synthesis was determined by blocking CD109 expression using CD109-specific siRNA or addition of recombinant CD109 protein, and analyzing the expression of ECM components by western blot.

Results

The expression of CD109 proteinis markedly increased in SSc skin tissue in vivo and in SSc skin fibroblasts in vitro as compared to their normal counterparts. Importantly, both SSc and normal skin fibroblasts transfected with CD109-specific siRNA display increased fibronectin, collagen type I and CCN2 protein levels and enhanced Smad2/3 phosphorylation compared with control siRNA transfectants. Furthermore, addition of recombinant CD109 protein decreases TGF-β₁-induced fibronectin, collagen type I and CCN2 levels in SSc and normal fibroblasts.

Conclusion

The upregulation of CD109 protein in SSc may represent an adaptation or consequence of aberrant TGF-β signaling in SSc. Our finding that CD109 is able to decrease excessive ECM production in SSc fibroblasts suggest that this molecule has potential therapeutic value for the treatment of SSc.     

Lipoic acid plays a role in scleroderma: insights obtained from scleroderma dermal fibroblasts

Introduction

Systemic sclerosis (SSc) is a connective tissue disease characterized by fibrosis of the skin and organs. Increase in oxidative stress and platelet-derived growth factor receptor (PDGFR) activation promote type I collagen (Col I) production, leading to fibrosis in SSc. Lipoic acid (LA) and its active metabolite dihydrolipoic acid (DHLA) are naturally occurring thiols that act as cofactors and antioxidants and are produced by lipoic acid synthetase (LIAS). Our goals in this study were to examine whether LA and LIAS were deficient in SSc patients and to determine the effect of DHLA on the phenotype of SSc dermal fibroblasts. N-acetylcysteine (NAC), a commonly used thiol antioxidant, was included as a comparison.

Methods

Dermal fibroblasts were isolated from healthy subjects and patients with diffuse cutaneous SSc. Matrix metalloproteinase (MMPs), tissue inhibitors of MMPs (TIMP), plasminogen activator inhibitor 1 (PAI-1) and LIAS were measured by enzyme-linked immunosorbent assay. The expression of Col I was measured by immunofluorescence, hydroxyproline assay and quantitative PCR. PDGFR phosphorylation and α-smooth muscle actin (αSMA) were measured by Western blotting. Student’s t-tests were performed for statistical analysis, and P-values less than 0.05 with two-tailed analysis were considered statistically significant.

Results

The expression of LA and LIAS in SSc dermal fibroblasts was lower than normal fibroblasts; however, LIAS was significantly higher in SSc plasma and appeared to be released from monocytes. DHLA lowered cellular oxidative stress and decreased PDGFR phosphorylation, Col I, PAI-1 and αSMA expression in SSc dermal fibroblasts. It also restored the activities of phosphatases that inactivated the PDGFR. SSc fibroblasts produced lower levels of MMP-1 and MMP-3, and DHLA increased them. In contrast, TIMP-1 levels were higher in SSc, but DHLA had a minimal effect. Both DHLA and NAC increased MMP-1 activity when SSc cells were stimulated with PDGF. In general, DHLA showed better efficacy than NAC in most cases.

Conclusions

DHLA acts not only as an antioxidant but also as an antifibrotic because it has the ability to reverse the profibrotic phenotype of SSc dermal fibroblasts. Our study suggests that thiol antioxidants, including NAC, LA, or DHLA, could be beneficial for patients with SSc.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-014-0411-6) contains supplementary material, which is available to authorized users.     

Decreased expression of the NF-κB family member RelB in lung fibroblasts from Smokers with and without COPD potentiates cigarette smoke-induced COX-2 expression

Background

Heightened inflammation, including expression of COX-2, is associated with COPD pathogenesis. RelB is an NF-κB family member that attenuates COX-2 in response to cigarette smoke by a mechanism that may involve the miRNA miR-146a. There is no information on the expression of RelB in COPD or if RelB prevents COX-2 expression through miR-146a.

Methods

RelB, Cox-2 and miR-146a levels were evaluated in lung fibroblasts and blood samples derived from non-smokers (Normal) and smokers (At Risk) with and without COPD by qRT-PCR. RelB and COX-2 protein levels were evaluated by western blot. Human lung fibroblasts from Normal subjects and smokers with and without COPD, along with RelB knock-down (siRNA) in Normal cells, were exposed to cigarette smoke extract (CSE) in vitro and COX-2 mRNA/protein and miR-146a levels assessed.

Results

Basal expression of RelB mRNA and protein were significantly lower in lung cells derived from smokers with and without COPD, the latter of which expressed more Cox-2 mRNA and protein in response to CSE. Knock-down of RelB in Normal fibroblasts increased Cox-2 mRNA and protein induction by CSE. Basal miR-146a levels were not different between the three groups, and only Normal fibroblasts increased miR-146a expression in response to smoke. There was a positive correlation between systemic RelB and Cox-2 mRNA levels and circulating miR-146a levels were higher only in GOLD stage I subjects.

Conclusions

Our data indicate that RelB attenuates COX-2 expression in lung structural cells, such that loss of pulmonary RelB may be an important determinant in the aberrant, heightened inflammation associated with COPD pathogenesis.     

Rac Inhibition Reverses the Phenotype of Fibrotic Fibroblasts

Background

Fibrosis, the excessive deposition of scar tissue by fibroblasts, is one of the largest groups of diseases for which there is no therapy. Fibroblasts from lesional areas of scleroderma patients possess elevated abilities to contract matrix and produce α−smooth muscle actin (α-SMA), type I collagen and CCN2 (connective tissue growth factor, CTGF). The basis for this phenomenon is poorly understood, and is a necessary prerequisite for developing novel, rational anti-fibrotic strategies.

Methods and Findings

Compared to healthy skin fibroblasts, dermal fibroblasts cultured from lesional areas of scleroderma (SSc) patients possess elevated Rac activity. NSC23766, a Rac inhibitor, suppressed the persistent fibrotic phenotype of lesional SSc fibroblasts. NSC23766 caused a decrease in migration on and contraction of matrix, and α−SMA, type I collagen and CCN2 mRNA and protein expression. SSc fibroblasts possessed elevated Akt phosphorylation, which was also blocked by NSC23766. Overexpression of rac1 in normal fibroblasts induced matrix contraction and α−SMA, type I collagen and CCN2 mRNA and protein expression. Rac1 activity was blocked by PI3kinase/Akt inhibition. Basal fibroblast activity was not affected by NSC23766.

Conclusion

Rac inhibition may be considered as a novel treatment for the fibrosis observed in SSc.     

MiR-29a Reduces TIMP-1 Production by Dermal Fibroblasts via Targeting TGF-β Activated Kinase 1 Binding Protein 1, Implications for Systemic Sclerosis

Background

Systemic sclerosis (SSc) is an autoimmune connective tissue disease characterised by skin and internal organs fibrosis due to accumulation of extra cellular matrix (ECM) proteins. Tissue inhibitor of metalloproteinases 1 (TIMP-1) plays a key role in ECM deposition.

Aim

To investigate the role of miR-29a in regulation of TAB1-mediated TIMP-1 production in dermal fibroblasts in systemic sclerosis.

Methods

Healthy control (HC) and SSc fibroblasts were cultured from skin biopsies. The expression of TIMP-1, MMP-1 and TGF-β activated kinase 1 binding protein 1 (TAB1) was measured following miR-29a transfection using ELISA, qRT-PCR, and Western Blotting. The functional effect of miR-29a on dermal fibroblasts was assessed in collagen gel assay. In addition, HeLa cells were transfected with 3′UTR of TAB1 plasmid cloned downstream of firefly luciferase gene to assess TAB1 activity. HC fibroblasts and HeLa cells were also transfected with Target protectors in order to block the endogenous miR-29a activity.

Results

We found that TAB1 is a novel target gene of miR-29a, also regulating downstream TIMP-1 production. TAB1 is involved in TGF-β signal transduction, a key cytokine triggering TIMP-1 production. To confirm that TAB1 is a bona fide target gene of miR-29a, we used a TAB1 3′UTR luciferase assay and Target protector system. We showed that miR-29a not only reduced TIMP-1 secretion via TAB1 repression, but also increased functional MMP-1 production resulting in collagen degradation. Blocking TAB1 activity by pharmacological inhibition or TAB1 knockdown resulted in TIMP-1 reduction, confirming TAB1-dependent TIMP-1 regulation. Enhanced expression of miR-29a was able to reverse the profibrotic phenotype of SSc fibroblasts via downregulation of collagen and TIMP-1.

Conclusions

miR-29a repressed TAB1-mediated TIMP-1 production in dermal fibroblasts, demonstrating that miR-29a may be a therapeutic target in SSc.     

Differentiation potential of CD14+ monocytes into myofibroblasts in patients with systemic sclerosis

Background

Circulating monocytes are a highly plastic and functionally heterogeneic cell type with an activated phenotype in patients with systemic sclerosis (SSc). CD14⁺ monocytes have the potential to differentiate into extra-cellular matrix (ECM) producing cells, possibly participating in fibrogenesis.

Aim

To study the effect of GM-CSF, IL-4 and endothelin -1 (ET-1) alone or in combination on monocyte differentiation into myofibroblasts.

Methods

CD14⁺ cells were isolated from peripheral blood from 14 SSc patients and healthy controls by positive selection and incubated with different combinations of GM-CSF, IL-4 and ET-1 for 14 days. Type-1 collagen and α-SMA were detected by Western blot, qPCR and confocal microscopy. HLA-DR, CD11c and CD14 expression was analysed by flow cytometry. A collagen gel contraction assay was performed for functional myofibroblast assessment.

Results

GM-CSF both induced collagen and α-SMA expression after 14 days. ET-1 further increased GM-CSF-induced collagen expression in a dose dependent manner up to 30-fold. IL-4/GM-CSF combination leads to a more DC-like phenotype of monocytes associated with reduced collagen and α-SMA expression compared to GM-CSF alone. Collagen and α-SMA expression was higher in monocytes from SSc patients and monocytes were more prone to obtain a spindle form. In contrast to controls, ET-1 and IL-4 alone were sufficient to induce α-SMA expression in monocytes from SSc patients. Despite the induction of α-SMA expression, monocyte-derived myofibroblasts only had a moderate capability of contraction in functional analyses.

Conclusion

SSc monocytes display increased maturation towards myofibroblasts demonstrated by their phenotype and α-SMA expression when compared to monocytes from healthy controls, however only with minor functional contraction properties.     

B lymphocytes and B-cell activating factor promote collagen and profibrotic markers expression by dermal fibroblasts in systemic sclerosis

Introduction

B lymphocytes might play a pathogenic role in dermal fibrosis in systemic sclerosis (SSc). B-cell activating factor (BAFF), a key cytokine for B-cell activation, is increased in the serum and the skin of patients with SSc. However, the ability of B cells directly to stimulate dermal fibroblasts and the role of BAFF are not fully understood. We therefore investigated the involvement of B cells and BAFF in the expression of collagen and profibrotic markers by dermal fibroblasts.

Methods

Cocultures of blood B cells from healthy blood donors and normal or SSc dermal fibroblasts stimulated with anti-IgM and BAFF were performed. Alpha-SMA, TIMP1, MMP9, COL1A1, COL1A2, and COL3A1 mRNA expression were determined by quantitative RT-PCR. Soluble collagen, BAFF, IL-6, IL-1β, TGF-β1, and CCL2 protein secretion were assessed.

Results

Coculture of blood B cells and dermal fibroblasts isolated from SSc patients induced IL-6, TGF-β1, CCL2, and collagen secretion, as well as Alpha-SMA, TIMP1, and MMP9 expression in dermal fibroblasts. Transwell assays demonstrated that this induction was dependent on cell-cell contact. Addition of anti-IgM and BAFF to the coculture increased IL-6, CCL2, TGF-β1, and collagen secretion. B cell- and BAFF-induced collagen secretion was highly reduced by anti-TGF-β1 antibodies.

Conclusions

Our results showed for the first time a direct role of B cells on the production of collagen by dermal fibroblasts, which is further enhanced by BAFF. Thus, these results demonstrate a new pathogenic role of B cells and BAFF in fibrosis and systemic sclerosis.     

Estradiol promotes the development of a fibrotic phenotype and is increased in the serum of patients with systemic sclerosis

Introduction

Systemic sclerosis (SSc) is more prevalent in women. Our goal is to determine the effects of 17β-estradiol (E2) on the development of fibrosis and to compare circulating levels of estrogens in SSc patients and healthy controls.

Methods

Using primary human dermal fibroblasts, we evaluated the effect of E2 on fibronectin (FN) expression with and without the estrogen receptor (ER) antagonist ICI 182,780, inhibitors of signaling, propyl-pyrazole-triol, an ERα specific ligand, and genistein, an ERβ selective ligand, to identify the signaling pathways mediating E2''s effect. We confirmed the fibrotic effect of E2 in human skin using an ex vivo organ culture model. Lastly, we measured levels of E2 and estrone in serum samples from SSc patients with diffuse cutaneous involvement and healthy controls using mass spectrometry.

Results

E2 increased expression of FN in dermal fibroblasts. ICI 182,780, inositol-1,4,5-triphosphate inhibitor, and p38 mitogen-activated protein kinase inhibitor blocked the effects of E2 on FN. Propyl-pyrazole-triol, but not genistein, significantly increased FN expression. Ex vivo, E2 induced fibrosis of human skin. The effects of E2 were abrogated by ICI 182,780. Circulating levels of E2 and estrone were significantly increased in sera of patients with diffuse cutaneous SSc.

Conclusion

Our findings implicate estrogens in the fibrotic process and may explain the preponderance of SSc in women. ICI 182,780 or other ER signaling antagonists may be effective agents for the treatment of fibrosis.     

RNA Interference against Platelet-Derived Growth Factor Receptor α mRNA Inhibits Fibroblast Transdifferentiation in Skin Lesions of Patients with Systemic Sclerosis

Objective

To down-regulate expression of mRNA for the platelet-derived growth factor receptor (PDGFR)-α, block the signalling pathway of PDGF and its receptor, and study their influence on fibroblast transdifferentiation to myofibroblasts in systemic sclerosis (SSc).

Methods

Fibroblasts from skin lesions of SSc patients and health adult controls were cultured in vitro, and α-smooth muscle actin (α-SMA) expression was determined by immunocytochemistry. Both groups of fibroblasts were stimulated with PDGF-AA, transforming growth factor β₁ (TGF-β₁), and costimulated with PDGF-AA and TGF-β₁, then PDGFR-α and α-SMA mRNA and protein expression were detected with RT-PCR and WB respectively. Three pairs of siRNAs targeting different PDGFR-α mRNA sequences were synthesized for RNAi. SSc and control fibroblasts were transfected with PDGFR-α siRNA; stimulated with PDGF-AA; and assessed for PDGFR-α and α-SMA mRNA and protein expression.

Results

Although the fibroblasts from both groups had similar morphology, the SSc skin lesions had significantly more myofibroblasts than control skin lesions. PDGF-AA stimulation, TGF-β₁ stimulation, and costimulation significantly up-regulated PDGFR-α and α-SMA mRNA and protein expression in SSc fibroblasts compared to control (P<0.05), and costimulation had the strongest effects (P<0.05). All three pairs of siRNAs suppressed PDGFR-α mRNA and protein expression (P<0.05), but siRNA₁₄₉₅ had the highest gene-silencing efficiency (P<0.05). PDGFR-α siRNA attenuated the effects of PDGF-AA through up-regulating PDGFR-α and α-SMA mRNA and protein expression and inhibiting fibroblast transdifferentiation to myofibroblasts in SSc (P<0.05).

Conclusions

PDGFR-α over-expression in SSc fibroblasts bound PDGF-AA more efficiently and promoted fibroblast transdifferentiation, which was enhanced by TGF-β₁. PDGFR-α siRNA down-regulated PDGFR-α expression, blocked binding to PDGF-AA, and inhibited fibroblast transdifferentiation to myofibroblasts.     

Versican is upregulated in circulating monocytes in patients with systemic sclerosis and amplifies a CCL2-mediated pathogenic loop

Introduction

Altered phenotypes of circulating monocytes of patients with systemic sclerosis (SSc) have been reported, but the role of these alterations in the pathogenesis of SSc remains unclear. This study was undertaken to identify molecules that are preferentially expressed by SSc monocytes, and to investigate the roles of these molecules in the pathogenic process of SSc.

Methods

We analyzed circulating CD14⁺ monocytes isolated from 36 patients with SSc and 32 healthy control subjects. The monocytes'' gene expression profiles were assessed by Oligo GEArray^® (SABiosciences, Frederic, MA, USA) and semiquantitative or quantitative PCR; their protein expression was evaluated in culture supernatants of unstimulated monocytes by immunoblotting or ELISA, and by immunocytostaining. Monocyte chemoattractant activity of CCL2 was assessed in a TransWell^® system (Corning Incorporated, Corning, NY, USA) in the presence or absence of chondroitin sulfate (CS).

Results

A step-wise approach to profiling gene expression identified that versican and CCL2 were upregulated in SSc monocytes. Subsequent analysis of proteins expressed in monocyte culture supernatants confirmed enhanced production of versican and CCL2 in SSc monocytes compared with control monocytes. CCL2 bound to CS chains of versican and colocalized with versican in the monocytes'' Golgi apparatus. Finally, CCL2 had a greater ability to mediate monocyte migration when bound to CS chains, because this binding provided efficient formation of CCL2 gradients and protection from protease attack.

Conclusion

Circulating monocytes with elevated versican and CCL2 levels may contribute to the fibrotic process in a subset of SSc patients by amplifying a positive feedback loop consisting of versican, CCL2, and the influx of monocytes.     

Monocyte surface expression of Fcγ receptor RI (CD64), a biomarker reflecting type-I interferon levels in systemic lupus erythematosus

Introduction

More than half of systemic lupus erythematosus (SLE) patients show evidence of excess type I interferon (IFN-I) production, a phenotype associated with renal disease and certain autoantibodies. However, detection of IFN-I proteins in serum is unreliable, and the measurement of interferon-stimulated gene (ISG) expression is expensive and time consuming. The aim of this study was to identify a surrogate marker for IFN-I activity in clinical samples for monitoring disease activity and response to therapy.

Methods

Monocyte surface expression of Fcγ receptors (FcγRs), chemokine receptors, and activation markers were analyzed with flow cytometry in whole blood from patients with SLE and healthy controls. FcγR expression also was measured in peripheral blood mononuclear cells (PBMCs) from healthy controls cultured with Toll-like receptor (TLR) agonists, cytokines, or serum from SLE patients. Expression of ISGs was analyzed with real-time PCR.

Results

Circulating CD14⁺ monocytes from SLE patients showed increased surface expression of FcγRI (CD64). The mean fluorescent intensity of CD64 staining correlated highly with the ISG expression (MX1, IFI44, and Ly6E). In vitro, IFN-I as well as TLR7 and TLR9 agonists, induced CD64 expression on monocytes from healthy controls. Exposure of monocytes from healthy controls to SLE sera also upregulated the expression of CD64 in an IFN-I-dependent manner. Decreased CD64 expression was observed concomitant with the reduction of ISG expression after high-dose corticosteroid therapy.

Conclusions

Expression of CD64 on circulating monocytes is IFN-I inducible and highly correlated with ISG expression. Flow-cytometry analysis of CD64 expression on circulating monocytes is a convenient and rapid approach for estimating IFN-I levels in SLE patients.     

Expression of TGF-β3 in isolated fibroblasts from foreskin

Background:

The multifunctional transforming growth factor beta (TGF-β) is a glycoprotein that exists in three isoforms. TGF-β3 expression increases in fetal wound healing and reduces fibronectin and collagen I and III deposition, and also improves the architecture of the neodermis which is a combination of blood vessels and connective tissue during wound healing. Fibroblasts are key cells in the wound healing process. TGF-β3 plays a critical role in scar-free wound healing and fibroblast actions in the wound healing process. The aim of this study was to express the TGF-β3 gene (tgf-b3) in human foreskin fibroblasts (HFF’s).

Methods:

We obtained HFF’s from a newborn and a primary fibroblast culture was prepared. The cells were transfected with TGF-β3-pCMV6-XL5 plasmid DNA by both lipofection and electroporation. Expression of TGF-β3 was measured by enzyme-linked immunosorbent assay (ELISA).

Results:

The highest TGF-β3 expression (8.3-fold greater than control) was obtained by lipofection after 72 hours using 3 µl of transfection reagent. Expression was 1.4-fold greater than control by electroporation.

Conclusions:

In this study, we successfully increased TGF-β3 expression in primary fibroblast cells. In the future, grafting these transfected fibroblasts onto wounds can help the healing process without scarring.Key Words: Fibroblasts, Gene expression, TGF-β3     

B-cell depletion therapy in patients with diffuse systemic sclerosis associates with a significant decrease in PDGFR expression and activation in spindle-like cells in the skin

Introduction

Recently, several studies assessing the clinical efficacy of rituximab (RTX) in systemic sclerosis (SSc) have reported encouraging results. We aimed at exploring whether RTX exerts its beneficial effects on fibrosis through attenuation of platelet-derived growth factor receptor (PDGFR) pathway activation.

Methods

We immunohistochemically assessed skin biopsies obtained from eight patients with SSc prior to and 6 months following RTX treatment, three control SSc patients (at the same time points) and three healthy subjects. We assessed the expression of platelet-derived growth factor, PDGFR and phosphorylated (activated) PDGFR.

Results

We found a strong correlation of PDGFRα and PDGFRβ expression on spindle-like cells and collagen deposition in SSc biopsies (r = 0.97 and r = 0.96 for PDGFRα and PDGFRβ, respectively; P < 0.0001 for both), indicating a strong link between PDGFR expression and fibrosis. Expression of PDGFRα and PDGFRβ in the papillary dermis significantly decreased following RTX administration (mean ± standard error of the mean at baseline vs. 6 months, respectively: PDGFRα, 42.05 ± 5.03 vs. 26.85 ± 3.00, P = 0.004; and PDGFRβ, 37.14 ± 4.94 vs. 24.01 ± 3.27, P = 0.012). Similarly, expression of phosphorylated PDGFRα and PDGFRβ in the papillary dermis significantly decreased following RTX administration (P = 0.006 and P = 0.013 for phospho-PDGFRα and phospho-PDGFRβ, respectively). No changes in platelet-derived growth factor tissue expression or serum levels were found following RTX treatment.

Conclusion

RTX may favorably affect skin fibrosis through attenuation of PDGFR expression and activation, a finding that supports a disease-modifying role of RTX in SSc. Large-scale, multicenter studies are needed to further explore the efficacy of RTX in SSc.     

Defective alterations in the collagen network to prostacyclin in COPD lung fibroblasts

Background

Prostacyclin analogs are potent vasodilators and possess anti-inflammatory properties. However, the effect of prostacyclin on extracellular matrix (ECM) in COPD is not well known. Collagen fibrils and proteoglycans are essential ECM components in the lung and fibroblasts are key players in regulating the homeostasis of ECM proteins. The aim was to study the synthesis of prostacyclin and its effect on fibroblast activity and ECM production, and in particular collagen I and the collagen-associated proteoglycans biglycan and decorin.

Methods

Parenchymal lung fibroblasts were isolated from lungs from COPD patients (GOLD stage IV) and from lungs and transbronchial biopsies from control subjects. The prostacyclin analog iloprost was used to study the effect of prostacyclin on ECM protein synthesis, migration, proliferation and contractile capacity of fibroblasts.

Results

TGF-β₁ stimulation significantly increased prostacyclin synthesis in fibroblasts from COPD patients (p < 0.01), but showed no effect on fibroblasts from control subjects. Collagen I synthesis was decreased by iloprost in both control and COPD fibroblasts (p < 0.05). Conversely, iloprost significantly altered biglycan and decorin synthesis in control fibroblasts, but iloprost displayed no effect on these proteoglycans in COPD fibroblasts. Proliferation rate was reduced (p < 0.05) and contractile capacity was increased in COPD fibroblasts (p < 0.05) compared to control fibroblasts. Iloprost decreased proliferative rate in control fibroblasts (p < 0.05), whereas iloprost attenuated contraction capacity in both COPD (p < 0.01) and control fibroblasts (p < 0.05).

Conclusions

Iloprost reduced collagen I synthesis and fibroblast contractility but did not affect the collagen-associated proteoglycans or proliferation rate in fibroblasts from COPD patients. Enhanced prostacyclin production could lead to improper collagen network fibrillogenesis and a more emphysematous lung structure in severe COPD patients.     

Crosstalk between Shh and TGF-β Signaling in Cyclosporine-Enhanced Cell Proliferation in Human Gingival Fibroblasts

Background

Immunosuppressant cyclosporine-A induces gingival hyperplasia, which is characterized by increased fibroblast proliferation and overproduction of extracellular matrix components and regulated by transforming growth factor-beta (TGF-β). The TGF-β and Sonic hedgehog (Shh) pathways both mediate cell proliferation. Crosstalk between these pathways in cancer has recently been proposed, but the hierarchical pattern of this crosstalk remains unclear. In normal fibroblasts, a TGF-β-stimulating Shh pattern was observed in induced fibrosis. However, Shh pathway involvement in cyclosporine-enhanced gingival proliferation and the existence of crosstalk with the TGF-β pathway remain unclear.

Methodology/Principal Findings

Cyclosporine enhanced mRNA and protein levels of TGF-β and Shh in human gingival fibroblasts (RT-PCR and western blotting). A TGF-β pathway inhibitor mitigated cyclosporine-enhanced cell proliferation and an Shh pathway inhibitor attenuated cyclosporine-enhanced proliferation in fibroblasts (MTS assay and/or RT-PCR of PCNA). Exogenous TGF-β increased Shh expression; however, exogenous Shh did not alter TGF-β expression. The TGF-β pathway inhibitor mitigated cyclosporine-upregulated Shh expression, but the Shh pathway inhibitor did not alter cyclosporine-upregulated TGF-β expression.

Conclusions/Significance

The TGF-β and Shh pathways mediate cyclosporine-enhanced gingival fibroblast proliferation. Exogenous TGF-β increased Shh expression, and inhibition of TGF-β signaling abrogated the cyclosporine-induced upregulation of Shh expression; however, TGF-β expression appeared unchanged by enhanced or inhibited Shh signaling. This is the first study demonstrating the role of Shh in cyclosporine-enhanced gingival cell proliferation; moreover, it defines a hierarchical crosstalk pattern in which TGF-β regulates Shh in gingival fibroblasts. Understanding the regulation of cyclosporine-related Shh and TGF-β signaling and crosstalk in gingival overgrowth will clarify the mechanism of cyclosporine-induced gingival enlargement and help develop targeted therapeutics for blocking these pathways, which can be applied in pre-clinical and clinical settings.     

Antifibrotic properties of caveolin-1 scaffolding domain in vitro and in vivo

Lung fibrosis involves the overexpression of ECM proteins, primarily collagen, by alpha-smooth muscle actin (ASMA)-positive cells. Caveolin-1 is a master regulator of collagen expression by cultured lung fibroblasts and of lung fibrosis in vivo. A peptide equivalent to the caveolin-1 scaffolding domain (CSD peptide) inhibits collagen and tenascin-C expression by normal lung fibroblasts (NLF) and fibroblasts from the fibrotic lungs of scleroderma patients (SLF). CSD peptide inhibits ASMA expression in SLF but not NLF. Similar inhibition of collagen, tenascin-C, and ASMA expression was also observed when caveolin-1 expression was upregulated using adenovirus. These observations suggest that the low caveolin-1 levels in SLF cause their overexpression of collagen, tenascin-C, and ASMA. In mechanistic studies, MEK, ERK, JNK, and Akt were hyperactivated in SLF, and CSD peptide inhibited their activation and altered their subcellular localization. These studies and experiments using kinase inhibitors suggest many differences between NLF and SLF in signaling cascades. To validate these data, we determined that the alterations in signaling molecule activation observed in SLF also occur in fibrotic lung tissue from scleroderma patients and in mice with bleomycin-induced lung fibrosis. Finally, we demonstrated that systemic administration of CSD peptide to bleomycin-treated mice blocks epithelial cell apoptosis, inflammatory cell infiltration, and changes in tissue morphology as well as signaling molecule activation and collagen, tenascin-C, and ASMA expression associated with lung fibrosis. CSD peptide may be a prototype for novel treatments for human lung fibrosis that act, in part, by inhibiting the expression of ASMA and ECM proteins.