To investigate the role of focal adhesion kinase (FAK) in transforming growth factor (TGF)-β-induced myofibroblast transdifferentiation of human Tenon's fibroblasts.
Primary cultured human Tenon's fibroblasts were exposed to TGF-β1 for up to 48 hours. The mRNA levels of FAK, α smooth muscle actin (αSMA), and β-actin were determined by quantitative real time reverse transcription polymerase chain reaction. The protein levels of collagen type I, FAK, phospho-FAK, αSMA, and β-actin were determined by Western immunoblots. After the small interfering RNA targeting FAK (siRNAFAK) molecules were delivered into the cells, the expressions of αSMA proteins were determined by Western immunoblots.
In human Tenon's fibroblasts, TGF-β1 significantly increased the mRNA and protein expressions of αSMA. However, when the action of FAK was inhibited using siRNAFAK, the TGF-β1-induced expression of αSMA was attenuated.
Our data suggest that FAK may be associated with the TGF-β1-induced transdifferentiation of human Tenon's fibroblasts to myofibroblasts, which is the essential step of subconjunctival fibrosis.
Fibroblast; Focal adhesion protein-tyrosine kinases; Myofibroblast; Transforming growth factors
To gain a better understanding of the roles of interleukins (ILs) in subconjunctival fibrosis, we investigated their expression in transforming growth factor-β1 (TGF-β1)-stimulated Tenon’s fibroblasts and examined their association with the transdifferentiation of fibroblasts to myofibroblasts.
After primary culture, fibroblasts derived from human Tenon’s capsule were exposed to TGF-β1. The expression of α-smooth muscle actin (α-SMA) protein was assessed by western immunoblots and immunofluorescence. The mRNA levels of various ILs were also evaluated by multiplex reverse transcription (RT)-PCR. Using the small interfering RNAs (siRNAs) specific for IL-6 and IL-11 and the promoter deletion assay, the contributions of IL-6 and IL-11 to TGF-β1-induced induction of α-SMA were determined.
In human Tenon’s fibroblasts, TGF-β1 stimulated the expression of α-SMA protein determined by western blot analysis and also increased the mRNA levels of IL-6 and IL-11 determined by multiplex RT-PCR. On the western immunoblots and immunofluorescence, the increased expression of α-SMA was attenuated only by the siRNAs specific for IL-6 but not by the siRNAs specific for IL-11. When the activator protein-1 binding sites of the IL-6 promoter region were deleted, the stimulation effects of TGF-β1 decreased.
Our data show that autocrine IL-6 may participate in the TGF-β1-induced transdifferentiation of human Tenon’s fibroblasts to myofibroblasts, which is known to be an essential step for subconjunctival fibrosis.
To investigate the expression of the matricellular protein SPARC (secreted acidic cysteine-rich glycoprotein) in scarred human Tenon’s capsule and in cultured human Tenon’s fibroblasts (HTF), and to analyze the influence of SPARC on cell proliferation and collagen matrix contraction in vitro.
Human Tenon's capsule scars obtained from surgical revisions after filtration surgery were analyzed for SPARC expression by immunohistochemistry. In cultured HTF cells, SPARC expression was assessed by northern and western blot analyses after incubation with transforming growth factor (TGF)-β1 and TGF-β2. Cell proliferation was determined by bromodeoxyuridine (BrdU)–labeling and HTF cells-mediated collagen matrix contraction by morphometric measurements of three-dimensional collagen lattices after treatment with SPARC and/or TGF-β1.
In scarred human Tenon’s capsule specimens, an increased expression of SPARC was mainly localized to the extracellular matrix and to blood vessel walls as compared to healthy control Tenon’s capsule. In cultured HTF cells, treatment with TGF-β1 more than TGF-β2 induced the expression of SPARC both on the mRNA and protein level. Incubation of HTF cells with SPARC resulted in an increase in collagen matrix contraction and cell proliferation. Moreover, a combined incubation of SPARC and TGF-β1 stimulated HTF cell proliferation significantly over the levels that were observed after single treatment.
Our data provide evidence that SPARC contributes to excessive wound healing and scar formation in human Tenon’s capsules after filtration surgery and may thus represent a novel target for anti-fibrotic strategies.
Transdifferentiation of human Tenon fibroblasts to myofibroblasts and subsequent deposition of extracellular matrix is a key step in the scarring after glaucoma filtration surgery. The p38 signaling pathway plays an important role in cell proliferation and differentiation, and its upstream regulators and downstream molecules are widely distributed in the eye. We aimed to investigate the role of p38 in the activation of Tenon fibroblasts and that of the anti-fibrotic mechanism of rosiglitazone in the modulation of the p38 signaling pathway.
Cultured Tenon fibroblasts were stimulated with transforming growth factor (TGF)-β1. Activation of p38 was examined by western blot analysis. Rosiglitazone and blocking of the p38 signaling pathway by SB203580 were used to antagonize stimulation by TGF-β1. Fibroblast motility was examined by wound closure assay; alpha-smooth muscle actin, connective tissue growth factor, and collagen type I were determined by qPCR and western blot. Expression and localization of alpha-smooth muscle actin were determined by immunofluorescence staining.
Phosphorylated p38 was upregulated in fibroblasts stimulated with TGF-β1, and this effect was substantially inhibited by rosiglitazone. Proliferation and migration of fibroblasts were suppressed by rosiglitazone and SB203580. Expression of alpha-smooth muscle actin, connective tissue growth factor, and collagen type I were decreased at the mRNA and protein levels by rosiglitazone and SB203580. However, the inhibitory effect of SB203580 on transcription and protein expression was weaker than that of rosiglitazone. Similar phenomena were found on immunofluorescence microscopy of alpha-smooth muscle actin.
The p38 signaling pathway mediates the TGF-β1-induced transdifferentiation of human Tenon fibroblasts to myofibroblasts. Rosiglitazone can exert anti-fibrotic activity by interfering with the TGF-β/p38 signaling pathway and might be useful for modulating scar formation after glaucoma filtration surgery.
Platelet-derived growth factor A (PDGF-A) signals solely through PDGF-Rα, and is required for fibroblast proliferation and transdifferentiation (fibroblast to myofibroblast conversion) during alveolar development, because pdgfa-null mice lack both myofibroblasts and alveoli. However, these PDGF-A-mediated mechanisms remain incompletely defined. At postnatal days 4 and 12 (P4 and P12), using mouse lung fibroblasts, we examined (a) how PDGF-Rα correlates with ki67 (proliferation marker) or alpha-smooth muscle actin (αSMA, myofibroblast marker) expression, and (b) whether PDGF-A directly affects αSMA or modifies stimulation by transforming growth factor beta (TGFβ).
Using flow cytometry we examined PDGF-Rα, αSMA and Ki67 in mice which express green fluorescent protein (GFP) as a marker for PDGF-Rα expression. Using real-time RT-PCR we quantified αSMA mRNA in cultured Mlg neonatal mouse lung fibroblasts after treatment with PDGF-A, and/or TGFβ.
The intensity of GFP-fluorescence enabled us to distinguish three groups of fibroblasts which exhibited absent, lower, or higher levels of PDGF-Rα. At P4, more of the higher than lower PDGF-Rα + fibroblasts contained Ki67 (Ki67+), and Ki67+ fibroblasts predominated in the αSMA + but not the αSMA- population. By P12, Ki67+ fibroblasts comprised a minority in both the PDGF-Rα + and αSMA+ populations. At P4, most Ki67+ fibroblasts were PDGF-Rα + and αSMA- whereas at P12, most Ki67+ fibroblasts were PDGF-Rα- and αSMA-. More of the PDGF-Rα + than - fibroblasts contained αSMA at both P4 and P12. In the lung, proximate αSMA was more abundant around nuclei in cells expressing high than low levels of PDGF-Rα at both P4 and P12. Nuclear SMAD 2/3 declined from P4 to P12 in PDGF-Rα-, but not in PDGF-Rα + cells. In Mlg fibroblasts, αSMA mRNA increased after exposure to TGFβ, but declined after treatment with PDGF-A.
During both septal eruption (P4) and elongation (P12), alveolar PDGF-Rα may enhance the propensity of fibroblasts to transdifferentiate rather than directly stimulate αSMA, which preferentially localizes to non-proliferating fibroblasts. In accordance, PDGF-Rα more dominantly influences fibroblast proliferation at P4 than at P12. In the lung, TGFβ may overshadow the antagonistic effects of PDGF-A/PDGF-Rα signaling, enhancing αSMA-abundance in PDGF-Rα-expressing fibroblasts.
These findings clearly demonstrate that substratum nanotopography inhibits TGFβ-induced corneal myofibroblast differentiation and suggests that structural features of this scale help to stabilize the keratocyte and fibroblast phenotypes in vivo.
The transition of corneal fibroblasts to the myofibroblast phenotype is known to be important in wound healing. The purpose of this study was to determine the effect of topographic cues on TGFβ-induced myofibroblast transformation of corneal cells.
Rabbit corneal fibroblasts were cultured on nanopatterned surfaces having topographic features of varying sizes. Cells were cultured in media containing TGFβ at concentrations ranging from 0 to 10 ng/mL. RNA and protein were collected from cells cultured on topographically patterned and planar substrates and analyzed for the myofibroblast marker α-smooth muscle actin (αSMA) and Smad7 expression by quantitative real time PCR. Western blot and immunocytochemistry analysis for αSMA were also performed.
Cells grown on patterned surfaces demonstrated significantly reduced levels of αSMA (P < 0.002) compared with planar surfaces when exposed to TGFβ; the greatest reduction was seen on the 1400 nm surface. Smad7 mRNA expression was significantly greater on all patterned surfaces exposed to TGFβ (P < 0.002), whereas cells grown on planar surfaces showed equal or reduced levels of Smad7. Western blot analysis and αSMA immunocytochemical staining demonstrated reduced transition to the myofibroblast phenotype on the 1400 nm surface when compared with cells on a planar surface.
These data demonstrate that nanoscale topographic features modulate TGFβ-induced myofibroblast differentiation and αSMA expression, possibly through upregulation of Smad7. It is therefore proposed that in the wound environment, native nanotopographic cues assist in stabilizing the keratocyte/fibroblast phenotype while pathologic microenvironmental alterations may be permissive for increased myofibroblast differentiation and the development of fibrosis and corneal haze.
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).
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.
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).
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.
To investigate the interfering effect of Y-27632, a ROCK-I selective inhibitor, on the signal transduction pathway of transforming growth factor-β1 (TGF-β1) in ocular Tenon capsule fibroblasts (OTFS) in vitro.
After OTFS from passages 4 to 6 in vitro were induced by TGF-β1 and then treated by Y-27632, the changes of the OTFS cell cycles were analyzed via flow cytometry, and the proteins expression of the α-smooth muscular actin (α-SMA), connective tissue growth factor (CTGF), collagen I were calculated by Western blot. After OTFS treated by the different concentrations of Y-27632, the expression levels of the α-SMA, CTGF and collagen I mRNA were assayed by RT-PCR.
Y-27632 had no markedly effect on the OTFS cell cycles. After treated by TGF-β1, OTFS in G1 period significantly increased. The cell cycles distribution by both TGF-β1 and Y-27632 had no remarkable difference from that in control group. Y-27632 significantly inhibited the proteins expressions of both α-SMA and CTGF, while to some extent inhibited that of collagen I. TGF-β1 significantly promoted the proteins expressions of α-SMA, CTGF and collagen I. After OTFS treated by both TGF-β1 and Y-27632, of α-SMA, the protein expression was similar with that in control group (P=0.066>0.05), but the protein expression of CTGF or collagen I, respectively, was significantly different from that in control group (P=0.000<0.01). The differences of expressions of the α-SMA, CTGF and collagen I mRNA in 30, 150, 750µmol/L Y-27632 group were statistically significant, compared with those in control group, respectively (α-SMA, P=0.002, 0.000, 0.000; CTGF, P=0.014, 0.002, 0.001; collagen I, P=0.003, 0.002, 0.000).
Blocking the Rho/ROCK signaling pathway by using of Y-27632 could inhibit the cellular proliferation and the expression of both CTGF and α-SMA whatever OTFS induced by TGF-β1 or not. Y-27632 suppressed the expression of collagen I mRNA without induction.
Y-27632; ocular Tenon's capsule fibroblasts; transforming growth factor beta type 1; α-smooth muscular actin; connective tissue growth factor; collagen I
IGF-1 is elevated in pulmonary fibrosis and acute lung injury, where fibroblast activation is a prominent feature. We previously demonstrated that blockade of IGF pathway in murine model of lung fibrosis improved outcome and decreased fibrosis. We now expand that study to examine effects of IGF pathway on lung fibroblast behaviors that could contribute to fibrosis.
We first examined mice that express αSMA promoter upstream of GFP reporter treated with A12, a blocking antibody to IGF-1 receptor, after bleomycin induced lung injury. We then examined the effect of IGF-1 alone, or in combination with the pro-fibrotic cytokine TGFβ on expression of markers of myofibroblast activation in vitro, including αSMA, collagen α1, type 1, collagen α1, type III, and TGFβ expression.
After bleomycin injury, we found decreased number of αSMA-GFP + cells in A12 treated mice, validated by αSMA immunofluorescent staining. We found that IGF-1, alone or in combination with TGF-β, did not affect αSMA RNA expression, promoter activity, or protein levels when fibroblasts were cultured on stiff substrate. IGF-1 stimulated Col1a1 and Col3a1 expression on stiff substrate. In contrast, IGF-1 treatment on soft substrate resulted in upregulation of αSMA gene and protein expression, as well as Col1a1 and Col3a1 transcripts. In conclusion, IGF-1 stimulates differentiation of fibroblasts into a myofibroblast phenotype in a soft matrix environment and has a modest effect on αSMA stress fiber organization in mouse lung fibroblasts.
Fibroblasts; IGF; Fibrosis
To synthesize a ternary cationic copolymer called CS-g-(PEI-b-mPEG) and characterize its features as a non-viral siRNA carrier; in turn, to investigate the influence of small interfering RNA (siRNA) targeting IκB kinase subunit β (IKKβ) on the proliferation of human Tenon’s capsule fibroblasts (HTFs) in vitro.
First, a novel cationic copolymer composed of low molecular weight, linear poly(ethyleneimine) [PEI] blocked with polyethylene glycol (PEG) and grafted onto a chitosan (CS) molecule was synthesized. CS-g-(PEI-b-mPEG) was then compacted with 21nt siRNA at various copolymer/siRNA charge (N/P) ratios, and the resulting complexes were characterized by dynamic light scattering, gel electrophoresis, and serum incubation. Cell Titer 96® AQueous One Solution cell proliferation assay was used to investigate the cytotoxicity of this cationic copolymer. Second, siRNAs targeting IKKβ (IKKΒ-siRNAs) were delivered into the HTFs using CS-g-(PEI-b-mPEG) as the vehicle. Real-time reverse transcription polymerase chain reaction (RT–PCR) subsequently assessed the mRNA level of IKKβ, and western blot assay was used to determine protein expression. After IKKB-siRNA transfection, Cell Titer 96® AQueous One Solution cell proliferation assay was used to evaluate the proliferation of HTFs.
The diameter of the CS-g-(PEI-b-mPEG)/siRNA complexes tended to decrease whereas their zeta potential tended to increase as the N/P ratio increased. The CS-g-(PEI-b-mPEG) copolymer showed good siRNA binding ability and high siRNA protection capacity. Furthermore, the copolymer presented remarkable transfection efficiency and showed much less cytotoxicity than 25 kDa PEI. IKKB-siRNAs were successfully delivered into HTFs using CS-g-(PEI-b-mPEG) as a vector. As a result, the expression of IKKβ was downregulated at both the mRNA and protein levels, and the activation of nuclear factor-κB (NF-κB) in the HTFs was subsequently inhibited. Most impressively, the proliferation of HTFs was also effectively suppressed through the blocking of the NF-κB pathway.
All the results demonstrate that CS-g-(PEI-b-mPEG) is a promising candidate for siRNA delivery, featuring excellent biocompatibility, biodegradability, and transfection efficiency. The RNA interference (RNAi) strategy using cationic copolymers as siRNA carriers will be a safe and efficient anti-scarring method following glaucoma filtration surgery.
Idiopathic pulmonary fibrosis is a common and invariably fatal disease with limited therapeutic options. Ca2+-activated KCa3.1 potassium channels play a key role in promoting TGFβ1 and bFGF-dependent profibrotic responses in human lung myofibroblasts (HLMFs). We hypothesised that KCa3.1 channel-dependent cell processes regulate HLMF αSMA expression via Smad2/3 signalling pathways.
In this study we have compared the phenotype of HLMFs derived from non-fibrotic healthy control lungs (NFC) with cells derived from IPF lungs. HLMFs grown in vitro were examined for αSMA expression by immunofluorescence (IF), RT-PCR and flow cytommetry. Basal Smad2/3 signalling was examined by RT-PCR, western blot and immunofluorescence. Two specific and distinct KCa3.1 blockers (TRAM-34 200 nM and ICA-17043 [Senicapoc] 100 nM) were used to determine their effects on HLMF differentiation and the Smad2/3 signalling pathways.
IPF-derived HLMFs demonstrated increased constitutive expression of both α-smooth muscle actin (αSMA) and actin stress fibres, indicative of greater myofibroblast differentiation. This was associated with increased constitutive Smad2/3 mRNA and protein expression, and increased Smad2/3 nuclear localisation. The increased Smad2/3 nuclear localisation was inhibited by removing extracellular Ca2+ or blocking KCa3.1 ion channels with selective KCa3.1 blockers (TRAM-34, ICA-17043). This was accompanied by de-differentiation of IPF-derived HLMFs towards a quiescent fibroblast phenotype as demonstrated by reduced αSMA expression and reduced actin stress fibre formation.
Taken together, these data suggest that Ca2+- and KCa3.1-dependent processes facilitate “constitutive” Smad2/3 signalling in IPF-derived fibroblasts, and thus promote fibroblast to myofibroblast differentiation. Importantly, inhibiting KCa3.1 channels reverses this process. Targeting KCa3.1 may therefore provide a novel and effective approach for the treatment of IPF and there is the potential for the rapid translation of KCa3.1-directed therapy to the clinic.
Idiopathic pulmonary fibrosis (IPF); Fibrosis; Lung; Myofibroblast; KCa3.1; Ion channel; Differentiation; Smad 2; Smad 3
A critical component of corneal scarring is the TGFβ-induced differentiation of corneal keratocytes into myofibroblasts. Inhibitors of this differentiation are potentially therapeutic for corneal scarring. In this study, we tested the relative effectiveness and mechanisms of action of two electrophilic peroxisome proliferator-activated receptor gamma (PPARγ) ligands: cyano-3,12-dioxolean-1,9-dien-28-oic acid-metheyl ester (CDDO-Me) and 15-deoxy-Δ-12,14-prostaglandin J2 (15d-PGJ2) for inhibiting TGFβ-induced myofibroblast differentiation in vitro. TGFβ was used to induce myofibroblast differentiation in cultured, primary human corneal fibroblasts. CDDO-Me and 15d-PGJ2 were added to cultures to test their ability to inhibit this process. Myofibroblast differentiation was assessed by measuring the expression of myofibroblast-specific proteins (αSMA, collagen I, and fibronectin) and mRNA (αSMA and collagen III). The role of PPARγ in the inhibition of myofibroblast differentiation by these agents was tested in genetically and pharmacologically manipulated cells. Finally, we assayed the importance of electrophilicity in the actions of these agents on TGFβ-induced αSMA expression via Western blotting and immunofluorescence. Both electrophilic PPARγ ligands (CDDO-Me and 15d-PGJ2) potently inhibited TGFβ-induced myofibroblast differentiation, but PPARγ was only partially required for inhibition of myofibroblast differentiation by either agent. Electrophilic PPARγ ligands were able to inhibit myofibroblast differentiation more potently than non-electrophilic PPARγ ligands, suggesting an important role of electrophilicity in this process. CDDO-Me and 15d-PGJ2 are strong inhibitors of TGFβ-induced corneal fibroblast to myofibroblast differentiation in vitro, suggesting this class of agents as potential novel therapies for corneal scarring warranting further study in pre-clinical animal models.
Hepatic fibrosis, the major complication of virtually all types of chronic liver damage, usually begins in portal areas, and its severity has been correlated to liver progenitor cells (LPC) expansion from periportal areas, even if the primary targets of injury are intralobular hepatocytes. The aim of this study was to determine the potential fibrogenic role of LPC, using a new experimental model in which rat liver fibrosis was induced by chronic carbon tetrachloride (CCl4) administration for 6 weeks, in combination with chronic acetylaminofluorene treatment (AAF), which promotes activation of LPC compartment. Treatment with CCl4 alone caused a significant increase in serum transaminase activity as well as liver fibrosis initiating around central veins and leading to formation of incomplete centro-central septa with sparse fibrogenic cells expressing α-smooth muscle actin (αSMA). In AAF/CCl4-treated animals, the fibrogenic response was profoundly worsened, with formation of multiple porto-central bridging septa leading to cirrhosis, whereas hepatocellular necrosis and inflammation were similar to those observed in CCl4-treated animals. Enhanced fibrosis in AAF/CCl4 group was accompanied by ductule forming LPC expanding from portal areas, αSMA-positive cells accumulation in the fibrotic areas and increased expression of hepatic collagen type 1, 3 and 4 mRNA. Moreover, CK19-positive LPC expressed the most potent fibrogenic cytokine transforming growth factor-β (TGFβ) without any expression of αSMA, desmin or fibroblast-specific protein-1, demonstrating that LPC did not undergo an epithelial-mesenchymal transition. In this new experimental model, LPC, by expressing TGFβ contributed to the accumulation of αSMA-positive myofibroblasts in the ductular reaction leading to enhanced fibrosis but also to disease progression and to a fibrotic pattern similar to that observed in human.
2-Acetylaminofluorene; toxicity; Actins; analysis; Animals; Antigens, CD; analysis; Antigens, Differentiation, Myelomonocytic; analysis; Carbon Tetrachloride; toxicity; Epithelial-Mesenchymal Transition; Keratin-19; analysis; Liver; pathology; Liver Cirrhosis, Experimental; etiology; Male; Rats; Rats, Sprague-Dawley; Stem Cells; physiology; Transforming Growth Factor beta; genetics; fibrosis; liver progenitor cells; TGFβ; epithelial-mesenchymal transition
The transformation of fibroblasts to myofibroblasts is critical to corneal wound healing, stromal haze formation, and scarring. It has recently been demonstrated that the provision of biomimetic substratum topographic cues inhibits the progression toward the myofibroblast phenotype under the influence of transforming growth factor β1 (TGF-β1). The objective of this study was to determine the effect of another fundamental biophysical cue, substrate compliance, on TGF-β1–induced myofibroblast transformation of primary corneal cells isolated from human and rabbit corneas.
Human and rabbit corneal fibroblasts were cultured on surfaces of varying substrate compliance (4–71 kPa) and tissue culture plastic (TCP) (>1 gigapascal [GPa]). Cells were cultured in media containing TGF-β1 at concentrations of 0, 1, or 10 ng/mL for 72 hours. RNA and protein were collected from cells cultured on polyacrylamide gels and TCP and were analyzed for the expression of α-smooth muscle actin (α-SMA), a key marker of myofibroblast transformation, using quantitative PCR, immunocytochemistry, and Western blot.
Cells grown on more compliant substrates demonstrated significantly reduced amounts of α-SMA mRNA compared with TCP. Immunocytochemistry and Western blot analysis determining the presence of α-SMA corroborated this finding, thus confirming a reduced transformation to the myofibroblast phenotype on more compliant substrates compared with cells on TCP in the presence of TGF-β1.
These data indicate that substrate compliance modulates TGF-β1–induced expression of α-SMA and thus influences myofibroblast transformation in the corneal stroma. This provides further evidence that biomimetic biophysical cues inhibit myofibroblast transformation and participate in stabilizing the native cellular phenotype.
Substratum compliance modulates the TGF-β1–induced expression of α-SMA in human and rabbit corneal stromal cells in vitro, thus indicating that compliance influences myofibroblast transformation in the corneal stroma.
myofibroblast transformation; biophysical cues; α-smooth muscle actin; corneal wound healing
To identify and determine the function of the proteins associated with failed filtering blebs following trabeculectomy.
Tenon's tissues, obtained during surgery for failed filtering blebs or obtained during cataract surgery on normal eyes, were analyzed by proteomics. The proteins showing significant differences between the two tissues were selected for identification by mass spectrometry. The location and expression pattern of ribosomal S6 kinase 2 (RSK2), one of the altered proteins, were determined. The effect of basic fibroblast growth factor (bFGF) on the expression pattern and function of RSK2 in NIH3T3 fibroblast cells was then investigated by an RNA knockdown technique.
Eight proteins were found differentially expressed in failed filtering blebs; the identified proteins included those associated with intracellular signaling pathways. The expression of RSK2, one of the identified proteins, was found to be decreased compared with that of the control. RSK2 was located in Tenon’s tissue using an immunohistochemical technique. In culture, the bFGF-induced cell proliferation was inhibited by the RNA knockdown of RSK2. The level of mRNA and protein expression of actin was increased by RSK2 RNA knockdown, but bFGF-induced protein expression of actin was not promoted by RSK2 RNA knockdown. Whereas RSK2 RNA knockdown increased the expression and activity of mitogen-activated protein kinase (MAPK), activation of MAPK induced by bFGF was not promoted by RSK2 knockdown.
The expression of eight proteins in the failed filtering blebs was significantly different from that in the Tenon’s capsules used as a control. The effect of RSK2 expression on fibroblast cells suggests that RSK2 may be associated with wound healing in filtering blebs.
Purpose. This study was to determine the effect of CTGF-small interfering RNA (siRNA) on TGF-β2-induced proliferation in human Tenon capsule fibroblasts (HTFs). Methods. HTFs were transfected with four of CTGF-siRNAs separately for screening of gene silencing efficacy that was determined by transcript level measured by quantitative real-time PCR (qRT-PCR). Recombinant TGF-β2 was added into the culture to stimulate the proliferation of HTFs. The gene silencing efficacy of the siRNAs was evaluated by qRT-PCR and immunofluorescence of CTGF transcript and protein levels. The viability of HTFs was determined by cell counting kit-8 (CCK-8). FCM was used to assess cell cycle after CTGF-siRNA transfection. Results. The expression of CTGF and proliferation of HTFs were increased significantly by TGF-β2 stimulation. The transfection of CTGF-siRNA abolished the upregulation of CTGF and cell proliferation induced by TGF-β2. The analysis of cell cycle indicated that CTGF-siRNA treatment stimulated cells from S phase to G0/G1 phase in comparison with the inverse physiologic function of TGF-β2. Conclusion. CTGF targeting siRNA could effectively suppress the expression of CTGF and attenuate the proliferation of HTFs. The siRNA approach may provide a therapeutic option for eliminating filtration bleb scarring after glaucoma filtration surgery (GFS).
Transforming growth factor-β (TGF-β) is considered to be essential to induce epithelial-to-mesenchymal transition (EMT) which plays central roles in wound healing in ocular fibrotic complication. The present study investigates whether small interference RNAs (siRNAs) targeting the type II receptor of TGF-β (TβRII) could be used to minimize the TGF-β action.
TGF-β receptor type II (TβRII) specific siRNAs designed from the Nakamura human gene sequence were used to transfect the cultured lens epithelial cells (LECs). The optimal transfection of scramble siRNA-Cy3 labeled duplexes in cultured LECs were examined by laser scanning confocal microscope and flow cytometry. TβRII protein expression and transcript levels were analyzed by immunofluorescence, western blotting, and real time PCR, respectively. Western blotting was performed to examine protein expression of fibronectin and alpha-smooth muscle actin (α-SMA). Scratch assay was used to determine cell migration. Cell morphology was observed after transfection by inverted microscope.
The optimal transfection rate of scramble siRNA-Cy3 labeled duplexes was efficient in that nearly to 50% in cultured LECs. TβRII specific siRNAs significantly reduced the receptor transcript and protein expression in cultured LECs. The gene knockdown inhibited LECs transdifferentiation, as it abrogated the expression of fibronectin and α-SMA, and retarded cell migration on the scratch assay. In addition, after transfection with TβRII specific siRNA, the cultured LECs did not show fibroblast-like shape which was one of the feature signs of EMT. Wound scratch assays indicated that the number of cultured LECs migrated into the wounded area was significantly lower in TβRII specific siRNA treated group (12.8±3.27/7.85 mm2), compared with normal (57.8±3.06/7.85 mm2) and scrambled RNA transfected group (50.8±3.64/7.85 mm2; p<0.0001).
Our results provided additional evidence to support that TGF-β pathway was involved in the development of EMT of human posterior capsule opacification, while how TβRII was involved should be further investigated.
Glaucoma, a prevalent blinding disease is commonly associated with increased intraocular pressure due to impaired aqueous humor (AH) drainage through the trabecular meshwork (TM). Although increased TM tissue contraction and stiffness in association with accumulation of extracellular matrix (ECM) are believed to be partly responsible for increased resistance to AH outflow, the extracellular cues and intracellular mechanisms regulating TM cell contraction and ECM production are not well defined. This study tested the hypothesis that sustained activation of Rho GTPase signaling induced by lysophosphatidic acid (LPA), TGF-β and connective tissue growth factor (CTGF) influences TM cell plasticity and fibrogenic activity which may eventually impact resistance to AH outflow. Various experiments performed using human TM cells revealed that constitutively active RhoA (RhoAV14), TGF-β2, LPA and CTGF significantly increase the levels and expression of Fibroblast Specific Protein-1 (FSP-1), α-smooth muscle actin (αSMA), collagen-1A1 and secretory total collagen, as determined by q-RT-PCR, immunofluorescence, immunoblot, flow cytometry and the Sircol assay. Significantly, these changes appear to be mediated by Serum Response Factor (SRF), myocardin-related transcription factor (MRTF-A), Slug and Twist-1, which are transcriptional regulators known to control cell plasticity, myofibroblast generation/activation and fibrogenic activity. Additionally, the Rho kinase inhibitor-Y27632 and anti-fibrotic agent-pirfenidone were both found to suppress the TGF-β2-induced expression of αSMA, FSP-1 and collagen-1A1. Taken together, these observations demonstrate the significance of RhoA/Rho kinase signaling in regulation of TM cell plasticity, fibrogenic activity and myofibroblast activation, events with potential implications for the pathobiology of elevated intraocular pressure in glaucoma patients.
Glaucoma; Trabecular meshwork; Aqueous humor; Intraocular pressure; Fibrosis; Plasticity; Serum response factor; Slug; Rho GTPase
Heat shock protein 27 (Hsp27), a member of the small heat shock protein family, is an apoptosis regulator. Our previous proteomic study showed that Hsp27 mainly expressed in human oocyte, and that Hsp27 expression was downregulated in the ovaries derived from women with the polycystic ovary syndrome (PCOS), a well known endocrinal disorder with abnormal apoptotic activity and folliculogenesis. However, the exact effects of Hsp27 downregulation on oocyte development have not yet been clarified.
The expression of Hsp27 gene was downregulated in the mouse oocytes cultured in vitro using siRNA adenovirus infection, while the activity of Hsp27 was decreased by microinjection of polyclonal Hsp27 antibody into the cytoplasm of germinal vesicle (GV) oocytes. Oocyte maturation rate was evaluated by morphological observation. Early stage of apoptosis was determined using Annexin-V staining analysis and some critical apoptotic factors and cytokines were also monitored at both mRNA level by real time RT-PCR and protein expression level by immunofluorescence and western blot.
Hsp27 expressed at high level in maturing oocytes. Infection with AdshHsp27, and microinjection of Hsp27 antibody into GV oocytes, resulted in the improved oocyte development and maturation. Germinal vesicle breakdown (GVBD) rates were significantly increased in two AdshHsp27-treated groups (88.7%, 86.0%) and Hsp27 antibody-injected group (77.0%) when compared with control (76.2% in AdGFP, 64.4% in IgG-injected), respectively. In addition, the rates of metaphase II (MII) development in two AdshHsp27-treated groups (73.8%, 76.4%) and Hsp27 antibody-injected group (67.3%) were higher than that in the controls (59.6% in AdGFP, 55.1% in IgG-injected). We also found that the rates of early stage of apoptosis in Hsp27 downregulated groups (46.5% and 45.6%) were higher than that in control group (34.1%) after 8 h of IVM. Similarly, downregulation of Hsp27 caused a significantly enhanced the expression of apoptotic factors (caspase 8, caspase 3) and cytokines (bmp 15 and gdf 9).
Downregulation of Hsp27 improved the maturation of mouse oocytes, while increased early stage of apoptosis in oocytes by inducing the activation of extrinsic, caspase 8-mediated pathway.
Systemic sclerosis (SSc) is an autoimmune inflammatory disorder of unknown etiology characterized by fibrosis of the skin and internal organs. Ang II (angiotensin II), a vasoconstrictive peptide, is a well-known inducer of kidney, heart, and liver fibrosis. The goal of this study was to investigate the profibrotic potential of Ang II in the mouse skin.
Ang II was administered by subcutaneous osmotic mini pumps to C57BL/6 male mice. Collagen-content measurements were performed with Gomori Trichrome staining and hydroxyproline assay. The mRNA expression level of collagens, TGF-β1, TGF-β2, TGF-β3, CTGF, αSMA, CD3, Emr1, CD45/B220, MCP1, and FSP1 were quantified with real-time polymerase chain reaction (PCR). Immunostaining was performed for markers of inflammation and fibrosis, including, phospho-Smad2, αSMA, CD3, Mac3, CD45/B220, and CD163B. Fibrocytes were identified by double staining with CD45/FSP1 and CD45/PH4. Endothelial cells undergoing endothelial-to-mesenchymal transition (EndoMT) were identified by double staining with VE-cadherin/FSP1.
Ang II-infused mice develop prominent dermal fibrosis in the area proximal to the pump, as shown by increased collagen and CTGF mRNA levels, increased hydroxyproline content, and more tightly packed collagen fibers. In addition, elevated mRNA levels of TGF-β2 and TGF-β3 along with increased expression of pSmad2 were observed in the skin of Ang II-treated mice. Dermal fibrosis was accompanied by an increased number of infiltrating fibrocytes, and an increased number of αSMA-positive cells, as well as CD163B+ macrophages in the upper dermis. This correlated with significantly increased mRNA levels of αSMA, Emr1, and MCP1. Infiltration of CD3-, CD45/B220-, and Mac3-positive cells was observed mainly in the hypodermis. Furthermore, an increased number of double-positive VE-cadherin/FSP1 cells were detected in the hypodermis only.
This work demonstrates that Ang II induces both inflammation and fibrosis in the skin via MCP1 upregulation and accumulation of activated fibroblasts. Additionally, our data suggest that populations of these fibroblasts originate from circulating blood cells. Ang II infusion via osmotic minipumps could serve as a useful mouse model of skin fibrosis to gain new insights into pathogenic mechanisms and to test new antifibrotic therapies.
Statins are the most commonly prescribed drugs for the treatment of hypercholesterolemia. Statins exert not only lipid-lowering but also other cellular effects, including anti-fibrotic properties. The purposes of this study were to determine the effect of simvastatin on Transforming growth factor (TGF)-β1-induced myofibroblast differentiation and collagen production in nasal polyp-derived fibroblasts (NPDFs) and to verify the mechanism of the effect of simvastatin in TGF-β1-induced myofibroblast differentiation in NPDFs.
NPDFs were pre-treated with simvastatin with or without mevalonate or Y-27643 for 2 hours prior to induction by TGF-β1. The expression of α-smooth muscle actin (SMA) and collagen type IV mRNA was determined by a reverse transcription-polymerase chain reaction, and the expression of α-SMA protein was determined by immunofluoescent cytochemical staining. Total soluble collagen production was analyzed by the SirCol collagen dye-binding assay. Phosphorylation of Smad 2/3 was evaluated by Western blot analysis.
In TGF-β1-induced NPDFs, simvastatin significantly inhibited the expressions of α-SMA and collagen type IV mRNA and reduced α-SMA and collagen protein levels. Pre-treatment with mevalonate reversed the effect of simvastatin. The expression of α-SMA mRNA and protein was significantly decreased by pre-treatment with Y-27632. The TGF-β1-induced expression of pSmad 2/3 protein was notably decreased by pre-treatment with simvastatin.
We showed that simvastatin inhibits TGF-β1-induced myofibroblast differentiation (expression of α-SMA) and collagen production in NPDFs and Rho/Rock and TGF-β/Smad signaling is involved as an underlying mechanism. The results of our study suggest that simvastatin is a possible candidate for the suppression of nasal polyp formation.
Idiopathic pulmonary fibrosis (IPF) is a progressive disease of insidious onset, and is responsible for up to 30,000 deaths per year in the U.S. Excessive production of extracellular matrix by myofibroblasts has been shown to be an important pathological feature in IPF. TGF-β1 is expressed in fibrotic lung and promotes fibroblast to myofibroblast differentiation (FMD) as well as matrix deposition.
To identify the mechanism of Arsenic trioxide’s (ATO)’s anti-fibrotic effect in vitro, normal human lung fibroblasts (NHLFs) were treated with ATO for 24 hours and were then exposed to TGF-β1 (1 ng/ml) before harvesting at multiple time points. To investigate whether ATO is able to alleviate lung fibrosis in vivo, C57BL/6 mice were administered bleomycin by oropharyngeal aspiration and ATO was injected intraperitoneally daily for 14 days. Quantitative real-time PCR, western blotting, and immunofluorescent staining were used to assess the expression of fibrotic markers such as α-smooth muscle actin (α-SMA) and α-1 type I collagen.
Treatment of NHLFs with ATO at very low concentrations (10-20nM) inhibits TGF-β1-induced α-smooth muscle actin (α-SMA) and α-1 type I collagen mRNA and protein expression. ATO also diminishes the TGF-β1-mediated contractile response in NHLFs. ATO’s down-regulation of profibrotic molecules is associated with inhibition of Akt, as well as Smad2/Smad3 phosphorylation. TGF-β1-induced H2O2 and NOX-4 mRNA expression are also blocked by ATO. ATO-mediated reduction in Smad3 phosphorylation correlated with a reduction of promyelocytic leukemia (PML) nuclear bodies and PML protein expression. PML-/- mouse embryonic fibroblasts (MEFs) showed decreased fibronectin and PAI-1 expression in response to TGF-β1. Daily intraperitoneal injection of ATO (1 mg/kg) in C57BL/6 mice inhibits bleomycin induced lung α-1 type I collagen mRNA and protein expression.
In summary, these data indicate that low concentrations of ATO inhibit TGF-β1-induced fibroblast to myofibroblast differentiation and decreases bleomycin induced pulmonary fibrosis.
Arsenic trioxide; IPF; TGF-β1; Pulmonary fibrosis; PML; Bleomycin
Fibroblast to myofibroblast transition is believed to contribute to airway remodelling in lung diseases such as asthma and chronic obstructive pulmonary disease. This study examines the role of aclidinium, a new long-acting muscarinic antagonist, on human fibroblast to myofibroblast transition.
Human bronchial fibroblasts were stimulated with carbachol (10−8 to 10−5 M) or transforming growth factor-β1 (TGF-β1; 2 ng/ml) in the presence or absence of aclidinium (10−9 to 10−7 M) or different drug modulators for 48 h. Characterisation of myofibroblasts was performed by analysis of collagen type I and α-smooth muscle actin (α-SMA) mRNA and protein expression as well as α-SMA microfilament immunofluorescence. ERK1/2 phosphorylation, RhoA-GTP and muscarinic receptors (M) 1, 2 and 3 protein expression were determined by western blot analysis and adenosine 3′-5′ cyclic monophosphate levels were determined by ELISA. Proliferation and migration of fibroblasts were also assessed.
Collagen type I and α-SMA mRNA and protein expression, as well as percentage α-SMA microfilament-positive cells, were upregulated in a similar way by carbachol and TGF-β1, and aclidinium reversed these effects. Carbachol-induced myofibroblast transition was mediated by an increase in ERK1/2 phosphorylation, RhoA-GTP activation and cyclic monophosphate downregulation as well as by the autocrine TGF-β1 release, which were effectively reduced by aclidinium. TGF-β1 activated the non-neuronal cholinergic system. Suppression of M1, M2 or M3 partially prevented carbachol- and TGF-β1-induced myofibroblast transition. Aclidinium dose-dependently reduced fibroblast proliferation and migration.
Aclidinium inhibits human lung fibroblast to myofibrobast transition.
Fibroblast; myofibroblast; anticholinergic; asthma; COPD; airway epithelium; interstitial fibrosis
Calcific aortic stenosis, characterized by excessive fibrosis and deposition of bone-like calcified tissue, affects roughly 2–3% of the US population over 65. Recent studies have suggested that statins have a positive effect on the progression of aoritic stenosis, likely due to their ability to affect the resident cell population, known as valvular interstitial cells (VICs). VICs are fibroblastic cells that can differentiate to form activated myofibroblasts, displaying increased alpha smooth muscle actin (αSMA) expression, contractility, and collagen production.
Methods and Results
In culture, VICs spontaneously form multicellular aggregates that subsequently develop into calcified nodules, providing an in vitro model for aortic stenosis. Using real-time microscopic tracking, we observed that confluent VIC monolayers spontaneously contract into rounded nodules, suggesting that myofibroblastic contractility is a critical step in the process of nodule formation. Over-expression of αSMA increased VIC calcific nodule formation and contractility, while knock-down of αSMA with siRNAs reduced these phenotypes, suggesting that the expression and contractile properties of αSMA are essential to the formation of nodules. Statin treatment of VICs reduced αSMA expression, inhibited contractility, and decreased nodule formation. When statins were used to treat preformed nodules, no decrease in the number of calcified nodules was observed, suggesting that statins may play more of a preventative role in aortic stenosis than a cure.
Our studies provide evidence of a causal relationship between VIC myofibroblastic activity and initial VIC calcific nodule formation. Furthermore we demonstrate that pravastatin inhibition of calcific nodule formation is related to inhibition of myofibroblastic activity.
valvular interstitial cells; myofibroblast; calcific nodule; statin; contractility
Aims: To determine the presence and origin of myofibroblasts in pterygia.
Methods: 86 specimens including head, body, and fibrovascular tissue from 52 primary and 34 recurrent pterygia and five exenterated eyes without pterygia were searched for the origin of myofibroblasts. All tissues were subjected to haematoxylin and eosin staining, immunohistochemistry using antibodies against alpha smooth muscle actin (α-SMA), desmin, vimentin, and caldesmon, and transmission electron microscopy (TEM). The phenotype of fibroblasts subcultured in a serum free medium from pterygium fibrovascular tissues was characterised by the above antibodies. Bundles of dense fibrous tissues were noted in 86% of the fibrovascular tissue specimens evaluated. Cells within these bundles were characterised as myofibroblasts based on positive staining to α-SMA, but negative to desmin and caldesmon, markers for smooth muscle cells. Interestingly, positive α-SMA staining was also found in the periorbital fibroadipose tissue posterior to Tenon’s capsule near the nasal conjunctiva in all exenterated specimens. All first passage fibroblasts expressed vimentin, some were positive to α-SMA, but all were negative to desmin or caldesmon. Cells in pterygium fibrovascular tissues showed ultrastructural features of intracytoplasmic bundles of microfilaments, consistent with myofibroblastic differentiation.
Conclusion: These studies collectively demonstrate the presence of contractile myofibroblasts bundle in pterygia and in the periorbital fibroadipose tissue posterior to Tenon’s capsule of exenterated eyes without pterygium.
pterygium; myofibroblasts; recurrence; proliferation