Gel Contraction Research Articles

Basic Study of Heating Response Measurement for Nanosheet Particle/Polymer Composite Gel Actuator with Anisotropic Contraction

A soft actuator is expected to be applied in the next generation of robotics. This study focuses on soft gel actuators hybridized with nanosheet liquid crystals. The resulting soft actuator has a highly hydrophilic property, and is suitable for underwater use. When the gel in water is heated to more than the transition temperature, the gel contracts; conversely, it swells when it is cooled. This gel actuator remarkably has an anisotropic contraction characteristic because the orientation of the nanosheets is uniformly arranged. However, little is known about its dynamic characteristics of the thermal contraction. As a basic analysis, this paper investigates the heating step response through experiments, and reveals the variation of the time constant against the dimensions.

376 Tsc2 disruption in mesenchymal progenitors regulates hair follicles and TGF beta signaling

The genetic disorder tuberous sclerosis complex (TSC) results in benign tumor formation due to biallelic inactivating mutations in TSC1 or TSC2 and increased mechanistic target of rapamycin complex 1 (mTORC1) signaling. TSC skin tumors often display follicular abnormalities including variable enlargement, increased numbers, and abnormal structure. In an earlier abstract we reported that mice with mesenchymal-specific deletion of Tsc2 (Tsc2cKO) displayed increased dermal thickness, increased hairshaft diameter, and earlier hair regrowth at 7 weeks of age in regions where Tsc2 was disrupted. To identify Tsc2-dependent transcriptional programs, Tsc2cKO or WT mice were crossed with mice carrying the RiboTag allele, and RNA sequencing was performed on ribosome-associated transcripts in primary neonatal dermal fibroblasts isolated from these mice (Tsc2KO or WT cells) treated with or without 20nM rapamycin for 24hr, an inhibitor of mTORC1 used in the treatment of TSC. Gene enrichment analysis indicated that genes down-regulated in Tsc2KO cells were enriched for those involved in the response to transforming growth factor beta (TGFβ), a known regulator of both catagen and mesenchymal cell differentiation. Treatment with rapamycin normalized these transcripts. Biological responses to TGFβ1 in Tsc2-deficient fibroblasts were subsequently analyzed. Tsc2KO cells showed reduced TGFβ1-stimulated collagen gel contraction compared to control cells (P=0.026). Using a luciferase reporter, TGFβ1-induced Smad activity was reduced by about half in Tsc2KO cells. In summary, our results suggest defects in hair follicles observed in mice with mesenchymal Tsc2 disruption may be the result of an impaired TGFβ1 response. Due to the critical role of TGFβ in cell fate determination, overcoming reduced TGFβ may also be a novel treatment approach in TSC.

Spontaneous differentiation of periodontal ligament stem cells into myofibroblast during ex vivo expansion.

Periodontitis is characterized by the chronic inflammation and destruction of tooth-supporting tissues. Periodontal ligament stem cell (PDLSC) is the mesenchymal stem cell (MSC) population isolated from periodontal ligament, which is the key tissue for regeneration of periodontal tissues. Although transplantation of PDLSCs is proposed as novel regenerative therapy, limited information is available, regarding the characteristic change of PDLSCs during ex vivo expansion. In this study, we encountered morphological change of PDLSCs during standard cell culture and aimed to investigate the change of PDLSCs in stem cell characteristics and to search for the culture condition to maintain stem cell properties. Characteristics of PDLSCs were examined using in vitro osteoblast and adipocyte differentiation. Myofibroblast differentiation was confirmed using immunohistochemistry and collagen gel contraction assay. Replicative senescence was examined by β-gal staining. PDLSCs changed their morphology from spindle to flat and wide during ex vivo expansion. After the morphological change, PDLSCs showed several features of myofibroblast including extensive stress fiber formation, contraction activity, and myofibroblast marker expression. Upon the morphological change, osteoblastic and adipocyte differentiation capacity were reduced and expression of stem cell-related genes were decreased. β-Gal staining was not always correlated with the morphological change of PDLSCs. Moreover, exogenous addition of bFGF and PDGF-BB served to maintain spindle shape and osteoblastic differentiation potential of PDLSCs. This study demonstrates that spontaneous differentiation of PDLSCs during ex vivo expansion and may provide the important information of cell culture condition of PDLSCs for clinical use.

Specific Features of Fibrotic Lung Fibroblasts Highly Sensitive to Fibrotic Processes Mediated via TGF-β-ERK5 Interaction.

Lung fibrosis is associated with lung tissue contraction due to abnormal accumulation of myofibroblasts, which aggressively promote the fibrotic process. Transforming growth factor (TGF)-β signaling in fibroblasts promotes extracellular matrix (ECM) synthesis and fibroblast migration and differentiation into myofibroblasts. Inhibition of extracellular signal-regulated kinase (ERK)5 blocks lung fibroblast activation by suppressing TGF-β signaling. Here, we examined the effects of an ERK5 inhibitor on TGF-β1-induced fibrosis in lung fibroblasts. The effects of ERK5 inhibition following TGF-β1 exposure were evaluated in lung fibroblasts isolated from fibrotic human lung tissues. Fibroblast-mediated collagen gel contraction and fibroblast migration towards fibronectin were assessed. Phenotypic differences in fibrotic fibroblasts were examined using the cap analysis gene expression method for genome-wide quantification of promoter activity. TGF-β1stimulated contraction of collagen gels, fibroblast migration, and α-smooth muscle actin and fibronectin expression, and Smad3 phosphorylation were increased in fibrotic fibroblasts as compared to normal lung fibroblasts. Treatment with the ERK5 inhibitor blocked these responses to a greater extent in fibroblasts from patients with usual interstitial pneumonia as compared to nonspecific interstitial pneumonia, independent of bone morphogenetic protein/Smad1 regulation. Moreover, 223 genes including fibulin-5 -which is involved in the TGF-β1-ERK5 signaling network- were upregulated in fibrotic fibroblasts, and ECM regulation was found to be enriched in the Reactome analysis. ERK5 inhibition attenuated the high sensitivity of fibrotic fibroblasts to TGF-β1/Smad3 signaling. Thus, the ERK5 pathway components and fibulin-5 are potential therapeutic targets to prevent lung fibrosis progression.

Extracellular matrix components isolated from diabetic mice alters cardiac fibroblast function through the AGE/RAGE signaling cascade

Roughly 30 million Americans suffer from diabetes and these individuals are at an increased risk of developing cardiovascular complications. The most common type of cardiovascular complication is heart failure, where the heart is unable to adequately pump blood throughout the body. Heart failure can be a result of the stiffening of the left ventricle which occurs when cardiac fibroblasts become “active” and begin to remodel the extracellular matrix (ECM). Fibroblast “activation” can be triggered by the AGE/RAGE signaling cascade. Advanced Glycated End products (AGEs) are produced and accumulate in the ECM overtime in a healthy individual, but under hyperglycemic conditions, this process is accelerated. We aim to investigate how the presence of AGEs in the either diabetic or non‐diabetic ECM can affect fibroblast ECM remodeling as well as determine the role of AGE/RAGE signaling during this process. In order to assess this question diabetic and non‐diabetic fibroblasts were embedded in 3D matrices composed of collagen isolated from either diabetic or non‐diabetic mice. Fibroblast function was assessed using gel contraction, migration, proliferation, and protein expression. The role of AGE/RAGE signaling during the 3D matrix experiment was determined by either activating the signaling pathway using AGE‐BSA or inhibiting specific signaling components through drug treatments. Non‐diabetic fibroblasts displayed similar gel contraction to diabetic cells when embedded in diabetic collagen. Thus, suggesting the diabetic ECM can alter fibroblast function from “inactive” to “active” states. In addition, increasing the AGE/RAGE cascade leads to increase gel contraction whereas inhibiting the cascade leads to little or no gel contraction. These results indicate 1) the ECM from diabetic and non‐diabetic mice differ from one another, 2) diabetic ECM can impact fibroblast function and shift them towards an “active” state, and 3) that fibroblasts can modify the ECM through activation of the AGE/RAGE signaling cascade. These results suggest the importance of understanding the impact diabetes has on the ECM and fibroblast function, more specially how these two components are intertwined with one another. Unraveling this issue will provide better insight into the impact of diabetic and cardiovascular complications.Support or Funding InformationUS Army Medical Research Award #81XWH‐16‐1‐0710University of Mississippi School of Pharmacy; Department of BioMolecular SciencesThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Oxygen-dependent contraction and degradation of the extracellular matrix mediated by interaction between tumor and endothelial cells

Understanding the mechanisms of cancer development and metastasis requires comprehensive analyses of interactions between normal and tumor cells and the extracellular matrix (ECM) in hypoxic tumor microenvironments. However, the scope of many tumor microenvironment studies is limited to verifying the development and performance of microenvironment-producing cell culture platforms. This study describes the effects of co-culture and hypoxia on contraction and degradation of the ECM. Collagen type I gel was placed in the gel channel of an oxygen tension–controllable microfluidic device as a tumor tissue substrate. MDA-MB-231 human breast cancer cells and/or human umbilical vein endothelial cells (HUVECs) were cultured inside the gel or on the adjacent media channels, respectively. Changes in the collagen gel were evaluated by generating normoxic (21% O2) or hypoxic (1% O2) conditions via variation of the supply of gas mixture. HUVECs induced collagen gel contraction and degradation more strongly than MDA-MB-231 cells. Although interaction between co-cultured MDA-MB-231 cells and HUVECs promoted gel contraction and degradation, hypoxia attenuated the effect. Immunofluorescence staining indicated decreased expression of secretory matrix metalloproteinase-7 (MMP-7) inside the collagen gel under hypoxic conditions, but no morphologic changes in cells were observed. Separate Western blot analyses using cells cultured on cell culture dishes confirmed reduced endogenous MMP expression in hypoxia-exposed HUVECs. These results demonstrate that hypoxic conditions affect collagen gel contraction and degradation by altering the expression of MMP-7 in co-cultured MDA-MB-231 cells and HUVECs.

ERK3‐MK5 Signaling in Murine Ventricular Fibroblasts and its Role in Fibroblast Migration

Background and objectivesMAP kinase‐activated protein kinase‐5 (MK5), a protein serine/threonine kinase identified as a substrate for p38α/β, ERK3, and ERK4 MAPKs, is expressed in the heart. However, the interacting partners and physiological function of MK5 are just beginning to be understood. Both MK5 and ERK3 haploinsufficiency attenuates the increase in cardiac collagen 1‐α1mRNA induced by constriction of the transverse aorta. Additionally, following myocardial ischemia induced by ligation of the left anterior descending coronary artery, scar size and collagen content were reduced in MK5 haploinsufficient mice compared with wildtype littermates. This study was to examine ERK3‐MK5 signaling in cardiac ventricular fibroblasts.MethodsCardiac fibroblasts were isolated from MK5+/+and MK5−/− mice and male Sprague‐Dawley rats. Subconfluent cultures of fibroblasts from passages 2 and 3 were used. Protein expression, as well as the ratio of filamentous (F) to monomeric (G) actin, was determined by immunoblotting. The presence of ERK3‐MK5 complexes was determined by proximity ligation assay (PLA) and co‐immunoprecipitation assays. Cell motility and myofibroblast contraction were studied by scratch‐wound and collagen gel contraction assays, respectively. The subcellular distribution of ERK3, MK5, smooth muscle α‐actin (αSMA), and F‐actin was determined by immunocytofluorescence and confocal microscopy.ResultsMK5 immunoreactivity was primarily localized in the nucleus whereas that of ERK3 was in the cytoplasm. In actively dividing fibroblasts, phospho‐MK5 immunoreactivity appeared to be associated with the cytoskeleton and pseudopodia. Following serum stimulation, ERK3 immunoreactivity redistributed to membrane ruffles and/or lamellipodia. ERK3, but not ERK4 or p38α, immunoreactivity was detected in MK5 immunoprecipitates from fibroblast lysates. PLA revealed ERK3‐MK5 complexes in the cytoplasm, which were less abundant following siRNA‐mediated knockdown of MK5 (MK5‐kd). MK5‐kd cells showed reduced contraction of collagen gels, cell spreading, and formation of dendritic extensions. The ratio of F‐actin to G‐actin decreased upon MK5 knockdown. Furthermore. confocal fluorescence microscopy revealed a reduction in F‐actin in MK5‐kd fibroblasts compared to fibroblasts transfected with scrambled RNA. MK5+/+ fibroblasts contained prominent, intersecting bundles of αSMA immunoreactivity in the leading edge of the cells, whereas MK5−/− fibroblasts had thin, parallel bundles of αSMA in this region. Cell migration, in response to serum, and/or angiotensin II, was reduced upon siRNA‐mediated knockdown of either MK5 or ERK3.ConclusionCo‐immunoprecipitation and PLA suggest that ERK3 and MK5 form complexes in cardiac fibroblasts and that these complexes are located in the cytosol. Following serum stimulation, phospho‐MK5 and ERK3 immunoreactivity redistributed to the leading edges of the membranes. Suppressing ERK3 or MK5 expression decreased cell motility. Hence, MK5 and ERK3 signaling may play overlapping roles in regulating cardiac fibroblast function.Support or Funding InformationThis study was supported by a grant from the Heart and Stroke Foundation of Canada.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Torula yeast (Candida utilis)-derived glucosylceramide contributes to dermal elasticity in vitro.

Glucosylceramide (GlcCer) is derived from several plants, such as rice, maize, and wheat, and has been reported to retain moisture by functioning as a barrier between the epidermis and the environment. However, there is insufficient research on the effect of GlcCer on dermal elasticity and wrinkles. In this study, we investigated the effects of torula yeast extract and torula yeast-derived GlcCer on dermal elasticity. We measured cell proliferation, collagen production, and collagen gel contraction using human dermal fibroblasts. Torula yeast extract and torula yeast-derived GlcCer increased dermal fibroblast proliferation and collagen production. Collagen gel contraction was promoted by torula yeast extract and torula yeast-derived GlcCer. These results indicate that GlcCer may affect dermal elasticity. Torula yeast extract and torula yeast-derived GlcCer may contribute to the maintenance of dermal elasticity. PRACTICAL APPLICATIONS: In this study, we found that torula yeast-derived glucosylceramide (GlcCer) has an additional function of improving dermal elasticity. With improved elasticity, skin becomes more resilient, thus preventing wrinkles. GlcCer has already been used in cosmetic products to retain skin moisture. Therefore, torula yeast-derived GlcCer can be expected to have several cosmetic applications.

MiR-1246 as a therapeutic target in oral submucosa fibrosis pathogenesis

Oral submucous fibrosis (OSF) is a precancerous condition of oral cancer with a complex etiology. Our previous work has demonstrated that non-coding RNA miR-1246 contributes to the cancer stemness of oral cancer. In the current study, we sought to investigate the effect of the inhibition of miR-1246 on the oral fibrogenesis. The expression levels of miR-1246 in OSF tissues and fibrotic buccal mucosal fibroblasts (fBMFs) were examined by qRT-PCR. Collagen gel contraction and migration assays were conducted to evaluate the myofibroblast activities. The relationship between miR-1246 and type I collagen was assessed and the protein expression of type I collagen was determined by Western blot. MiR-1246 expression was upregulated in both OSF specimen and fBMFs compared to the normal counterparts. Inhibition of miR-1246 successfully suppressed the myofibroblast activities, including collagen gel contractility and migration capacity. Moreover, the expression of miR-1246 was positively correlated with type I collagen and the expression of type I collagen was abrogated by repression of miR-1246. MiR-1246 is not only critical to the maintenance of oral stemness but also important to the activation of myofibroblasts. Our results showed that miR-1246 is positively associated with the type I collagen, which may be a downstream effector of miR-1246 and responsible for the fibrosis effect on fBMFs.

Neuronal PAS Domain 2 (Npas2)-Deficient Fibroblasts Accelerate Skin Wound Healing and Dermal Collagen Reconstruction.

The circadian clock, which consists of endogenous self-sustained and cell-autonomous oscillations in mammalian cells, is known to regulate a wide range of peripheral tissues. The unique upregulation of a clock gene, neuronal PAS domain protein 2 (Npas2), observed along with fibroblast aging prompted us to investigate the role of Npas2 in the homeostasis of dermal structure using in vivo and in vitro wound healing models. Time-course healing of a full-thickness skin punched wound exhibited significantly faster wound closure in Npas2-/- mice than wild-type (WT) C57Bl/6J mice. Dorsal skin fibroblasts isolated from WT, Npas2+/-, and Npas2-/- mice exhibited consistent profiles of core clock gene expression except for Npas2 and Per2. In vitro behavioral characterizations of dermal fibroblasts revealed that Npas2-/- mutation was associated with increased proliferation, migration, and cell contraction measured by floating collagen gel contraction and single-cell force contraction assays. Npas2 knockout fibroblasts carrying sustained the high expression level of type XII and XIV FAICT collagens and synthesized dermis-like thick collagen fibers in vitro. Confocal laser scanning microscopy demonstrated the reconstruction of dermis-like collagen architecture in the wound healing area of Npas2-/- mice. This study indicates that the induced Npas2 expression in fibroblasts may interfere with skin homeostasis, wound healing, and dermal tissue reconstruction, providing a basis for novel therapeutic target and strategy. Anat Rec, 2019. © 2019 Wiley Periodicals, Inc.

Sphingosine 1-phosphate induces epicardial progenitor cell differentiation into smooth muscle-like cells.

Epicardial progenitor cells (EpiCs) which are derived from the proepicardium have the potential to differentiate into coronary vascular smooth muscle cells during development. Whether sphingosine 1-phosphate (S1P), a highly hydrophobic zwitterionic lysophospholipid in signal transduction, induces the differentiation of EpiCs is unknown. In the present study, we demonstrated that S1P significantly induced the expression of smooth muscle cell specific markers α-smooth muscle actin and myosin heavy chain 11 in the EpiCs. And the smooth muscle cells differentiated from the EpiCs stimulated by S1P were further evaluated by gel contraction assay. To further confirm the major subtype of sphingosine 1-phosphate receptors (S1PRs) involved in the differentiation of EpiCs, we used the agonists and antagonists of different S1PRs. The results showed that the S1P1/S1P3 antagonist VPC23019 and the S1P2 antagonist JTE013 significantly attenuated EpiCs differentiation, while the S1P1 agonist SEW2871 and antagonist W146 did not affect EpiCs differentiation. These results collectively suggested that S1P, principally through its receptor S1P3, increases EpiCs differentiation into VSMCs and thus indicated the importance of S1P signaling in the embryonic coronary vasculature, while S1P2 plays a secondary role.

Protease-activated receptor 2 activates CRAC-mediated Ca2+ influx to cause prostate smooth muscle contraction.

Protease‐activated receptor 2 (PAR2) is a G‐protein‐coupled receptor that contributes to prostate fibrosis and lower urinary tract symptoms (LUTS). In addition to fibrosis, aberrant smooth muscle tone in the prostate has been hypothesized to play a role. We therefore examined PAR2 expression in primary human prostate smooth muscle cells (PSMC) and studied the downstream signaling effects of PAR2 activation. Signaling pathways involved in the process were assessed using the PAR2 activating peptide SLIGKV‐NH2. We show that PAR2 is expressed in PSMC and that PAR2 activation mediates a biphasic elevation in intracellular Ca2+ and phosphorylation of myosin light chain 20 (MLC20), causing cellular contraction as assessed in a gel contraction assay. Intracellular Ca2+ flux was inhibited by a phosphoinositide hydrolysis inhibitor, U73122, showing a requirement for phospholipase C β (PLCβ) activation. PSMC expressed mRNA for L‐type voltage dependent Ca2+ channels (VDCC) as well as Ca2+ release activated channels (CRAC), a hitherto unreported finding. Secondary intracellular Ca2+ oscillations were abrogated only by BTP2, the CRAC channel inhibitor, but not by nifedipine, an inhibitor of VDCC. These data suggest that, PAR2 activation and subsequent Ca2+ entry through CRAC channels are important mechanisms in prostate smooth muscle contraction.

An in vitro study of scarring formation mediated by human Tenon fibroblasts: Effect of Y-27632, a Rho kinase inhibitor.

Scar formation is the most common cause for failure of glaucoma filtration surgery because of increased fibroblast proliferation and activation. We have now examined the effect of Y‐27632, a Rho‐associated protein kinase (ROCK) inhibitor, on postsurgical scarring formation in human Tenon fibroblasts (HTFs). Collagen gel contraction assay was used to compare contractility activity of Y‐27632 with several antiglaucoma drugs. Immunofluorescence and western blotting were used to examine expression of scar formation–related factors. We found that Y‐27632 inhibited collagen gel contraction, as well as α‐smooth muscle actin and vimentin expression; these were promoted by treatment with latanoprost, timolol, or transforming growth factor (TGF)–β. To investigate the effect of Y‐27632 in postsurgical scarring, we mimicked TGF‐β secretion by stimulating HTFs with TGF‐β prior to Y‐27632 treatment. HTFs cultured in the presence of TGF‐β significantly increased gel contraction. In contrast, when HTFs were treated with 10μM Y‐27632, contraction was significantly inhibited. Furthermore, Y‐27632 reduced TGF‐β–induced phosphorylation of mitogen‐activated protein kinase signalling. These results suggest that ROCK inhibitors may inhibit fibrosis by inhibiting transdifferentiation of Tenon fibroblasts into myofibroblasts and by inhibiting TGF‐β signalling after surgery through mitogen‐activated protein kinase pathway suppression. These results implicate that ROCK inhibitors may improve outcomes after filtering surgery with a potential antiscarring effect, while latanoprost and timolol may induce fibrosis.Significance of the studyScar formation is the primary cause of failure after glaucoma filtration surgery. A ROCK inhibitor, Y‐27632, has been introduced as a novel potential antiglaucoma treatment to reduce intraocular pressure. The aim of our study was to elucidate the effect of Y‐27632 on scarring formation after glaucoma filtration surgery, in direct comparison with other antiglaucoma drugs. Our findings thus suggested that Y‐27632 may inhibit fibrosis and improve outcome after glaucoma filtration surgery through inhibition of transdifferentiation of Tenon fibroblasts into myofibroblasts, and the TGF‐β and MAPK signalling after surgery, while latanoprost and timolol may induce fibrosis.

α1D-adrenoceptor involves the relaxation effect of farrerol in rat aortic vascular smooth muscle cells

The aim of this study was to investigate the relaxation effect of farrerol on rat aortic vascular smooth muscle cells (VSMCs) and its underlying mechanism. VSMCs were cultured primarily and were used to examine the relaxation effect of farrerol. Cells surface and length were measured by dynamic observation, or by rhodamine-phalloidin labeling and hematoxylin-eosin staining. Cells contractive activity were tested using collagen gel contraction assay. The [Ca2+]in was measured with molecular probe fluo-4-AM. The mRNA and protein expression of regulatory proteins for contraction were measured. In addition, rat aortic VSMCs were transfected with lentivirus-mediated α1D-adrenoceptor gene-shRNA, then the effect of farrerol were detected by the above experimental methods. The results revealed that 10 μΜ AngⅡ promoted cell contraction, increased [Ca2+]in and enhanced collagen contraction in rat aortic VSMCs. 10 μΜ AngⅡ not only increased expression of myosin light chain kinase (MLCK) and smooth muscle protein 22α (SM22α), but also increased phosphorylation level of myosin light chain (MLC) and myosin phosphatase target subunit 1 (MYPT1). The above effects induced by AngⅡ could be significantly inhibited by farrerol in a concentration dependent manner. When the cells were transfected with lentivirus mediated α1D-adrenoceptor gene-shRNA, the effects of farrerol on changes induced by AngⅡ in rat aortic VSMCs were markedly reversed. In conclusion, farrerol could produce relaxtion effect in rat aortic VSMCs precontracted by 10 μΜ AngⅡ, which was involved in downregulation expression of MLCK and SM22α, and inhibition phosphorylation level of MYPT1 and MLC via activating α1D-adrenoceptor gene.

Critical Role of mTORC2-Akt Signaling in TGF-β1-Induced Myofibroblast Differentiation of Human Pterygium Fibroblasts

Profibrotic activation is essential for pterygium development. In this study, we investigated the role of the mechanistic target of rapamycin (mTOR) in regulating TGF-β1-induced myofibroblastic responses in human pterygium fibroblasts (HPFs) and elucidated the relative contributions of mTOR signaling components. HPFs were pretreated with the mTOR inhibitors rapamycin and Torin2, and TGF-β1-induced expression of profibrotic markers, including α-smooth muscle actin (α-SMA) and fibronectin, was evaluated. RNA interference-based approaches targeting raptor and rictor, regulatory subunits of mTOR complex 1 (mTORC1) and 2 (mTORC2), respectively, were used to determine the impact of each mTOR complex on HPFs. The contractile phenotype of HPFs was assessed by a collagen gel contraction assay. The mTOR active-site inhibitor Torin2, which suppresses both mTORC1 and mTORC2 activity in HPFs, inhibited TGF-β1-induced expression of α-SMA and fibronectin. The allosteric inhibitor rapamycin only partially suppressed mTORC1 activity and exhibited a minimal effect on the induction of profibrotic markers. The induction of α-SMA and fibronectin in HPFs was abrogated by RNA interference-mediated knockdown of rictor but was only moderately affected by raptor knockdown. Akt inhibition mimicked the effect of Torin2 and rictor knockdown on myofibroblast differentiation of HPFs. mTOR inhibition potently reduced the contractile ability of HPFs in collagen gel contraction assays. This study found that mTOR signaling promoted profibrotic activation of HPFs and confirmed the importance of the mTORC2-Akt axis in TGF-β1-induced myofibroblast differentiation. Therefore, our study may open up new avenues for the development of novel therapeutic strategies involving targeting of mTOR signaling to treat pterygium.

Secretory immunoglobulin A induces human lung fibroblasts to produce inflammatory cytokines and undergo activation.

Immunoglobulin (Ig)A is the most abundant immunoglobulin in humans, and in the airway mucosa secretory IgA (sIgA) plays a pivotal role in first-line defense against invading pathogens and antigens. IgA has been reported to also have pathogenic effects, including possible worsening of the prognosis of idiopathic pulmonary fibrosis (IPF). However, the precise effects of IgA on lung fibroblasts remain unclear, and we aimed to elucidate how IgA activates human lung fibroblasts. We found that sIgA, but not monomeric IgA (mIgA), induced interleukin (IL)-6, IL-8, monocyte chemoattractant protein (MCP)-1 and granulocyte-macrophage colony-stimulating factor (GM-CSF) production by normal human lung fibroblasts (NHLFs) at both the protein and mRNA levels. sIgA also promoted proliferation of NHLFs and collagen gel contraction comparable to with transforming growth factor (TGF)-β, which is involved in fibrogenesis in IPF. Also, Western blot analysis and real-time quantitative polymerase chain reaction (PCR) revealed that sIgA enhanced production of α-smooth muscle actin (α-SMA) and collagen type I (Col I) by NHLFs. Flow cytometry showed that NHLFs bound sIgA, and among the known IgA receptors, NHLFs significantly expressed CD71 (transferrin receptor). Transfection of siRNA targeting CD71 partially but significantly suppressed cytokine production by NHLFs co-cultured with sIgA. Our findings suggest that sIgA may promote human lung inflammation and fibrosis by enhancing production of inflammatory or fibrogenic cytokines as well as extracellular matrix, inducing fibroblast differentiation into myofibroblasts and promoting human lung fibroblast proliferation. sIgA's enhancement of cytokine production may be due partially to its binding to CD71 or the secretory component.

Uncovering the dynamic precursors to motor-driven contraction of active gels.

Cells and tissues have the remarkable ability to actively generate the forces required to change their shape. This active mechanical behavior is largely mediated by the actin cytoskeleton, a crosslinked network of actin filaments that is contracted by myosin motors. Experiments and active gel theories have established that the length scale over which gel contraction occurs is governed by a balance between molecular motor activity and crosslink density. By contrast, the dynamics that govern the contractile activity of the cytoskeleton remain poorly understood. Here we investigate the microscopic dynamics of reconstituted actin-myosin networks using simultaneous real-space video microscopy and Fourier-space dynamic light scattering. Light scattering reveals different regimes of microscopic dynamics as a function of sample age. We uncover two dynamical precursors that precede macroscopic gel contraction. One is characterized by a progressive acceleration of stress-induced rearrangements, while the other consists of sudden, heterogeneous rearrangements. Intriguingly, our findings suggest a qualitative analogy between self-driven rupture and collapse of active gels and the delayed rupture of passive gels observed in earlier studies of colloidal gels under external loads.

Role of Hydrogen Sulfide in the Survival of Fibroblasts and Fibroblast-mediated Contraction of Collagen Gel

Role of Hydrogen Sulfide in the Survival of Fibroblasts and Fibroblast-mediated Contraction of Collagen Gel

Contraction of polymer gels created by the activity of molecular motors

We propose a theory based on non-equilibrium thermodynamics to describe the mechanical behavior of an active polymer gel created by the inclusion of molecular motors in its solvent. When activated, these motors attach to the chains of the polymer network and shorten them creating a global contraction of the gel, which mimics the active behavior of a cytoskeleton. The power generated by these motors is obtained by an ATP hydrolysis reaction, which transduces chemical energy into mechanical work. The latter is described by an increment of strain energy in the gel due to an increased stiffness. This effect is described with an increment of the cross-link density in the polymer network, which reduces its entropy. The theory then considers polymer network swelling and species diffusion to describe the transient passive behavior of the gel. We finally formulate the problem of uniaxial contraction of a slab of gel and compare the results with experiments, showing good agreement.

Simvastatin Modulates Extracellular Matrix Assembly by Displaying an Antifibrotic Activity in Vitro

Statins, competitive inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase, are known to possess properties beyond their cholesterol-lowering effect including anti-inflammatory, antiproliferative and anti-immunomodulatory effects. We examined the effect of simvastatin on extracellular matrix (ECM) assembly by human skin fibroblasts (HSFs) in vitro. Using collagen gel contraction (CGC) assay we showed that simvastatin inhibits contraction of type I collagen-rich gels in a dose-dependent manner. This effect of simvastatin could be overcome by co-incubating the cells with mevalonate. Actin staining revealed that inhibition of CGC by simvastatin is associated with diminished ability of the cells to form aggregates. Using whole human genome Illumina microarray we sought to search for new candidate genes whose expression is regulated by simvastatin during CGC and focused specifically on the genes related to ECM synthesis and remodeling. We found that simvastatin profoundly downregulated gene expression of 27 ECM molecules including proteoglycans decorin and versican, both of which are known to be essential constituents of proper ECM. Expression of these two molecules was further verified by Northern blot analysis. Finally, when simvastatin treated HSFs were activated with TGF-β1, the cell-mediated contraction of collagen gel was restored. Our results indicate that simvastatin markedly alters ECM assembly in vitro possessing an antifibrotic activity.