Collagen Lattice Contraction Research Articles

Roles of bovine sialoglycoproteins for anti-skin aging and accelerating skin wound healing.

Fibroblasts are the most predominant cell subpopulation in the dermal layer of human skin, they play an important role in maintaining skin architecture and function. The senescence of fibroblasts is one of major causes of skin aging and chronic wound in the elderly, which is accompanied with a reduction of α2,6-sialylation on the cell surface. In this study, we investigated the effects of the bovine sialoglycoproteins on normal human dermal fibroblasts (NHDF). The results showed that bovine sialoglycoproteins could promote the proliferation and migration of NHDF cells, and accelerate the contraction of fibroblast-populated collagen lattice (FPCL). The average doubling time of NHDF cells treated with bovine sialoglycoproteins (0.5 mg/mL) was 31.1 ± 1.0 h whereas that was 37.9 ± 2.7 h for the control (p ˂ 0.05). Moreover, the expression of basic fibroblast growth factor (FGF-2) was upregulated, while that of transforming growth factor-beta 1 (TGF-β1) and human type I collagen (COL-I) were downregulated in treated NHDF cells. Furthermore, bovine sialoglycoproteins treatment significantly enhanced the α2,6-sialylation on the cell surfaces, which was consistent with the upregulation of α2,6-sialyltransferase I (ST6GAL1) expression. These results indicated that the bovine sialoglycoproteins might be developed as a reagent against skin aging in the cosmetic industry, or as a new candidate for accelerating skin wound healing and inhibiting scar formation.

Stretching Promotes Wound Contraction Through Enhanced Expression of Endothelin Receptor B and TRPC3 in Fibroblasts.

The conditioned medium from stretched keratinocyte was added to the fibroblast populated collagen lattice. Then, we analyzed the levels of endothelin receptor in the human hypertrophic scar tissue and stretched fibroblasts. To address the function of TRPC3, we have used an overexpression system with the collagen lattice. Finally, the TRPC3 overexpressing fibroblasts were transplanted to mouse dorsal skin, and the rate of skin wound contraction was assessed. Conditioned medium from stretched keratinocytes increased the rate of contraction of fibroblast populated collagen lattice. In human hypertrophic scar and stretched fibroblasts, endothelin receptor type B was increased. Cyclic stretching of TRPC3 overexpressing fibroblasts activated NFATc4, and stretched human fibroblasts showed more activation of NFATc4 in response to ET-1. The wound treated with TRPC3 overexpressing fibroblasts showed more contraction than control wound. These findings suggest that cyclical stretching of wounds have an effect on both keratinocytes and fibroblasts, where keratinocytes secret more ET-1, and fibroblasts develop more sensitivity to ET-1 by expressing more endothelin receptors and TRPC3.

ALK5 Inhibition of Subconjunctival Scarring From Glaucoma Surgery: Effects of SB-431542 Compared to Mitomycin C in Human Tenon's Capsule Fibroblasts.

The gold standard for managing postoperative ocular fibrosis in glaucoma surgery is the chemotherapeutic mitomycin C (MMC) despite its association with significant adverse effects. This study compares in vitro the antifibrotic efficacy and cytotoxicity of the small-molecule TGFβ1 inhibitor SB-431542 (SB) to MMC. To measure collagen contraction, human Tenon's capsule fibroblasts (HTCFs) embedded in a three-dimensional collagen lattice were exposed to 0.2 mg/mL MMC or 20 µM SB followed by incubation with 2 ng/mL TGFβ1. Total protein extracted from experimentally treated HTCFs underwent immunoblotting for α-smooth muscle actin (α-SMA), matrix metallopeptidase 9 (MMP-9), and EDA splice-variant fibronectin (EDA-FN) expression. Cytotoxicity and cell metabolism were assessed using LIVE/DEAD staining, lactate dehydrogenase (LDH) assay, and methylthiazole tetrazolium (MTT) assay. Collagen lattice contraction in TGFβ1-induced HTCFs was significantly lowered by SB and MMC. Pretreatment with SB and MMC significantly lowered protein expression of α-SMA, MMP-9, and EDA-FN in HTCFs relative to TGFβ1 alone. HTCF viability in collagen lattices was significantly reduced with MMC pretreatment but not SB pretreatment. MMC-pretreated HTCFs had a significant increase in LDH release after 3 hours and a decrease in MTT activity after 20 minutes, while SB-pretreated HTCFs showed no significant changes via MTT or LDH assay during the same treatment period. SB shows comparable efficacy to MMC in reducing expression of fibrosis-promoting proteins in HTCFs and in vitro scarring activity. SB distinguishes itself from MMC by exhibiting less cytotoxicity in both two-dimensional and three-dimensional in vitro assays. This study demonstrates in vitro the potential of SB as a safer alternative ocular antifibrotic agent.

Cysteine-rich domain of type III collagen N-propeptide inhibits fibroblast activation by attenuating TGFβ signaling

TGFβ is a key regulator of the dynamic reciprocity between cells and the extracellular matrix that drives physiologic and pathologic responses in both tissue repair and tumor microenvironments. Our studies define type III Collagen (Col3) as a suppressor of scar formation and desmoplasia through its effects, in part, on myofibroblasts. TGFβ stimulates activation of myofibroblasts, and here, we demonstrate that cultured Col3-deficient fibroblasts have increased TGFβ signaling compared to wild-type fibroblasts. Moreover, kinetic binding studies show that a synthetic peptide containing a Col3 cysteine-rich (CR) domain found within its N-propeptide binds in a dose-dependent manner to TGFβ1, while a CR control peptide with mutated cysteines does not, suggesting that Col3 attenuates TGFβ signaling in part through the N-propeptide CR domain. Consistent with this hypothesis, the CR peptide attenuates TGFβ signaling in fibroblasts and 4T1 breast cancer cells and suppresses fibroblast activation and contraction, as assessed by α-smooth-muscle actin staining, cell wrinkling of deformable silicone, and stressed-fibroblast populated collagen lattice contraction assays. Finally, CR peptide treatment of orthotopically injected breast cancer cells (4T1) suppresses intratumoral fibroblast activation and inhibits primary tumor growth compared to CR control. Treatment with the CR peptide decreases both intratumoral canonical and non-canonical downstream TGFβ signaling targets, consistent with its extracellular binding to TGFβ. Taken together, our results suggest that the Col3 N-propeptide CR domain binds TGFβ1 and attenuates (but importantly does not eliminate) TGFβ signaling in fibroblasts and cancer cells. Expanding on our previous work, this study demonstrates an additional mechanism by which Col3 regulates cell behaviors in post-injury and tumor microenvironments and suggests that novel Col3-targeted strategies could effectively control biologic responses in vivo and improve anti-scarring/fibrosis and oncologic therapies.

Ellagic acid exerts anti-fibrotic effects on hypertrophic scar fibroblasts via inhibition of TGF-\u03b21/Smad2/3 pathway

Hypertrophic scar (HS) is a kind of serious pathological scar with no currently effective treatment. HS fibroblasts (HSFs) are the main effector cells for HS formation. Ellagic acid (EA) exerts regulatory effects in some diseases, but its role in HS remains unclear. This study aimed to evaluate the effect of EA on the fibrotic phenotypes of HSFs and to further investigate the downstream signaling mechanism. The cell counting kit-8 (CCK-8) assay was used to perform cytotoxicity and proliferation assays. HSFs migration was assessed using wound healing and transwell assays. HSFs contraction was measured by a collagen lattice contraction assay and detection of α-smooth muscle actin (α-SMA) expression. The levels of mRNA and protein were determined by qPCR and western blotting, respectively. The results showed that EA inhibited the proliferation, migration, and contraction of HSFs and collagen expression in HSFs in a dose-dependent manner. Furthermore, EA not only suppressed the Smad2/3 pathway but also reversed TGF-β1-induced activation of the Smad2/3 pathway and up-regulation of the fibrotic cellular phenotypes in HSFs. These findings demonstrate that EA exerts anti-fibrotic effects on HSFs by blocking the TGF-β1/Smad2/3 pathway, which indicates that EA is a potential therapeutic candidate for treatment of HS.

Synergic effect of atorvastatin and ambrisentan on sinusoidal and hemodynamic alterations in a rat model of NASH

ABSTRACTIn non-alcoholic steatohepatitis (NASH), decreased nitric oxide and increased endothelin-1 (ET-1, also known as EDN1) released by sinusoidal endothelial cells (LSEC) induce hepatic stellate cell (HSC) contraction and contribute to portal hypertension (PH). Statins improve LSEC function, and ambrisentan is a selective endothelin-receptor-A antagonist. We aimed to analyse the combined effects of atorvastatin and ambrisentan on liver histopathology and hemodynamics, together with assessing the underlying mechanism in a rat NASH model. Diet-induced NASH rats were treated with atorvastatin (10 mg/kg/day), ambrisentan (30 mg/kg/day or 2 mg/kg/day) or a combination of both for 2 weeks. Hemodynamic parameters were registered and liver histology and serum biochemical determinations analysed. Expression of proteins were studied by immunoblotting. Conditioned media experiments were performed with LSEC. HSCs were characterized by RT-PCR, and a collagen lattice contraction assay was performed. Atorvastatin and ambrisentan act synergistically in combination to completely normalize liver hemodynamics and reverse histological NASH by 75%. Atorvastatin reversed the sinusoidal contractile phenotype, thus improving endothelial function, whereas ambrisentan prevented the contractile response in HSCs by blocking ET-1 response. Additionally, ambrisentan also increased eNOS (also known as Nos3) phosphorylation levels in LSEC, via facilitating the stimulation of endothelin-receptor-B in these cells. Furthermore, the serum alanine aminotransferase of the combined treatment group decreased to normal levels, and this group exhibited a restoration of the HSC quiescent phenotype. The combination of atorvastatin and ambrisentan remarkably improves liver histology and PH in a diet-induced NASH model. By recovering LSEC function, together with inhibiting the activation and contraction of HSC, this combined treatment may be an effective treatment for NASH patients.

Dose-dependent pro- or anti-scar-preventing responses of freeze-dried amniotic membrane extract in relation to suppression of connective tissue growth factor and α-smooth muscle actin

Dose-dependent pro- or anti-scar-preventing effects of amniotic membrane extract (AME) are unclear. This study investigated the dose-dependent effects of freeze-dried AME on connective tissue growth factor (CTGF) and α-smooth muscle action (α-SMA) (key determinants of excessive scarring) expression in an attempt to develop medication for scarless wound healing. The inhibitory effect of freeze-dried AME on α-SMA was visualized through immunofluorescence confocal microscopy. The suppressive effect of 10 different concentrations of AME (0.0025–0.25 µg/mL) on CTGF and α-SMA was determined, and rates of collagen lattice contraction were measured. AME inhibited the trans-differentiation of fibroblasts to myofibroblasts, a hallmark of scar formation. AME downregulated CTGF and α-SMA in a dose-dependent manner; supplementation of 0.0175 µg/mL of AME in cell culture media downregulated CTGF (by 49%; p < 0.05) and α-SMA (by 33%; p < 0.05) relative to control values, the effect saturating with a subsequent increase in AME levels to 0.025 µg/mL. Collagen lattice contraction was maximally inhibited (75 ± 8%) upon treatment with 0.0175 µg/mL AME relative to the control (44 ± 6%). AME downregulates α-SMA and inhibits the trans-differentiation of fibroblasts to myofibroblasts in a dose-dependent manner. Treatment of cells with high AME concentrations retained high α-SMA concentration levels. A delicate balance between the wound healing properties and pro-fibrotic abilities of AME should be considered for selecting an appropriate therapeutic dose.

IL-10 alleviates lipopolysaccharide-induced skin scarring via IL-10R/STAT3 axis regulating TLR4/NF-κB pathway in dermal fibroblasts.

Hypertrophic scar (HS) is a severe fibrotic skin disease. It has always been a major problem in clinical treatment, mainly because its pathogenesis has not been well understood. The roles of bacterial contamination and prolonged wound inflammation were considered significant. IL‐10 is a potent anti‐inflammatory cytokine and plays a pivotal role in wound healing and scar formation. Here, we investigate whether IL‐10 alleviates lipopolysaccharide (LPS)‐induced inflammatory response and skin scarring and explore the possible mechanism of scar formation. Our results showed that the expression of TLR4 and pp65 was higher in HS and HS‐derived fibroblasts (HSFs) than their counterpart normal skin (NS) and NS‐derived fibroblasts (NSFs). LPS could up‐regulate the expression of TLR4, pp65, Col I, Col III and α‐SMA in NSFs, but IL‐10 could down‐regulate their expression in both HSFs and LPS‐induced NSFs. Blocking IL‐10 receptor (IL‐10R) or the phosphorylation of STAT3, their expression was up‐regulated. In addition, in vitro and in vivo models results showed that IL‐10 could alleviate LPS‐induced fibroblast‐populated collagen lattice (FPCL) contraction and scar formation. Therefore, IL‐10 alleviates LPS‐induced skin scarring via IL‐10R/STAT3 axis regulating TLR4/NF‐κB pathway in dermal fibroblasts by reducing ECM proteins deposition and the conversion of fibroblasts to myofibroblasts. Our results indicate that IL‐10 can alleviate the LPS‐induced harmful effect on wound healing, reduce scar contracture, scar formation and skin fibrosis. Therefore, the down‐regulation of inflammation may lead to a suitable scar outcome and be a better option for improving scar quality.

Lumican promotes joint fibrosis through TGF-β signaling.

Joint contracture (also known as arthrofibrosis) is a fibrotic joint disorder characterized by excessive collagen production to form fibrotic scar tissue and adhesions within joint capsules. This can severely affect day-to-day activities and quality of life because of a restricted range of motion in affected joints. The precise pathogenic mechanism underlying joint contractures is not fully understood. Lumican belongs to the class II small leucine-rich repeat proteoglycan superfamily, which makes up collagen fibrils in the extracellular matrix. Lumican is ubiquitously expressed in the skin, liver, heart, uterus and articular cartilage and has reported roles in cell migration, proliferation, angiogenesis and Toll-like receptor 4 signaling. Previous research has suggested that lumican is involved in the pathogenesis of several fibrotic diseases. Because joint contracture resembles a fibrotic disease, we aimed to investigate the role of lumican in the development of joint contracture invitro. Here, we showed that protein levels were up-regulated in the fibrotic joint capsule versus control. We observed that lumican significantly enhanced the proliferation, migration and fibroblast-myofibroblast transition of synovial fibroblasts. Moreover, lumican led to increased transcription of alpha-smooth muscle actin, matrix metallopeptidase 9, Collagen I, plasminogen activator inhibitor 1 and transforming growth factor-β invitro. Lumican treatment promoted collagen lattice contraction in a dose-dependent manner as early as 24h after treatment. Thus, our studies reveal that lumican could promote fibroblast-myofibroblast transition and joint contracture.

Minoxidil decreases collagen I deposition and tissue-like contraction in clubfoot-derived cells: a way to improve conservative treatment of relapsed clubfoot?

ABSTRACT Aim Clubfoot is a congenital deformity affecting the musculoskeletal system, resulting in contracted and stiff tissue in the medial part of the foot. Minoxidil (MXD) has an inhibitory effect on lysyl hydroxylase, which influences the quality of extracellular matrix crosslinking, and could therefore be used to reduce the stiffness and to improve the flexibility of the tissue. We assessed the in vitro antifibrotic effects of minoxidil on clubfoot-derived cells. Methods Cell viability and proliferation were quantified by xCELLigence, MTS, and LIVE/DEAD assays. The amount of collagen I deposited into the extracellular matrix was quantified using immunofluorescence with subsequent image segmentation analysis, hydroxyproline assay, and Second Harmonic Generation imaging. Extracellular matrix contraction was studied in a 3D model of cell-populated collagen gel lattices. Results MXD concentrations of 0.25, 0.5, and 0.75 mM inhibited the cell proliferation in a concentration-dependent manner without causing a cytotoxic effect. Exposure to ≥0.5 mM MXD resulted in a decrease in collagen type I accumulation after 8 and 21 days in culture. Changes in collagen fiber assembly were observed by immunofluorescence microscopy and nonlinear optical microscopy (second harmonic generation). MXD also inhibited the contraction of cell-populated collagen lattices (0.5 mM by 22%; 0.75 mM by 28%). Conclusions Minoxidil exerts an in vitro inhibitory effect on the cell proliferation, collagen accumulation, and extracellular matrix contraction processes that are associated with clubfoot fibrosis. This study provides important preliminary results demonstrating the potential relevance of MXD for adjuvant pharmacological therapy in standard treatment of relapsed clubfoot.

Zinc oxide nanoparticles induce human tenon fibroblast apoptosis through reactive oxygen species and caspase signaling pathway

Glaucoma is the leading cause of irreversible blindness in the world and trabeculectomy remains still the most commonly performed filtration surgery. Failure of trabeculectomy is due to the formation of scarring, which is associated with the increased fibroblast proliferation, activation, and collagen deposition at the site of the drainage channel with subconjunctival fibrosis. Our previous study has revealed that zinc oxide (ZnO) nanoparticles could efficiently decrease the expressions of TGF-β1 and inhibit fibroblast-mediated collagen lattice contraction. However, the mechanism underlying ZnO nanoparticle-induced fibroblast apoptosis is still unclear. In the present study, we investigated the effect of ZnO nanoparticles on the reactive oxygen species (ROS) and mitochondrial membrane potential (Δψm) in human Tenon fibroblasts (HTFs). Moreover, we also explored the influence of ZnO nanoparticles on the expression of Caspase-3, Caspase-9, apoptotic protease-activating factor-1 (Apaf-1), fibroblast-specific protein-1 (FSP-1), collagen III, and E-cadherin. The results indicated that ZnO nanoparticles markedly inhibit HTFs viability and decrease the Δψm in a concentration-dependent pattern. Exposure of HTFs to ZnO nanoparticles could also induce the elevated Caspase-3, Caspase-9, and Apaf-1 expression, decrease the levels of FSP-1, collagen III, and E-cadherin expression, leading to HTFs apoptosis. Our results suggested that elevated ROS and activated Caspase signaling play a fundamental role in ZnO nanoparticle-induced HTFs apoptosis.

IL-10 Alleviates Lipopolysaccharide-Induced Skin Scarring &lt;i&gt;via&lt;/i&gt; IL-10R/STAT3 Axis Regulating TLR4/NF-κB Pathway in Dermal Fibroblasts

Background: Hypertrophic scar (HS) is a serious fibrotic skin disease. The roles of bacterial contamination and prolonged inflammation in wound were considered to be significant. IL-10 plays a pivotal role in wound healing and scar formation. Here, we investigate whether IL-10 alleviates lipopolysaccharide (LPS)-induced inflammatory response and skin scarring, explore the possible mechanism in scar formation. Methods: RT-qPCR, Western blotting, histochemistry, immunostaining, ELISA array, electron microscope, fibroblast-populated collagen lattice (FPCL) and a rabbit ear scar model were used to investigate and validate the effect of IL-10 on LPS-stimulated scar formation. Findings: Our results showed that the expression of TLR4 and pp65 was higher in HS and HS-derived fibroblasts (HSFs) than their counterpart normal skin (NS) and NS-derived fibroblasts (NSFs). LPS could upregulate the expression of TLR4, pp65, Col I, Col III and α-SMA in NSFs, but IL-10 could downregulate their expression in both HSFs and LPS-induced NSFs. Blocking IL-10 receptor (IL-10R) or the phosphorylation of STAT3, their expression was upregulated. In addition, in vitro and in vivo models results showed that IL-10 could alleviate LPS-induced fibroblast-populated collagen lattice (FPCL) contraction and scar formation. Interpretation: Our study suggests that IL-10 may improve LPS-induced harmful to wound healing, reduce scar contracture and scar formation via IL-10R/STAT3 axis regulating TLR4/NF-κB pathway in dermal fibroblasts by reducing ECM proteins deposition and the conversion of HSFs to NSFs. Therefore, the downregulation of inflammation may be a better option for improving scar quality, and become potential therapeutic targets for scarring. Funding Statement: This research was supported by grants from the National Natural Science Foundation of China (81571914, and 81741105), and the Natural Science Foundation of Shaanxi Province (2018JQ8048). Declaration of Interests: The authors declare no conflicts of interest. Ethics Approval Statement: Written consent was obtained from all participants before surgery. All protocols used in this study were approved by the Ethics Committee of Xijing Hospital, affiliated to the Fourth Military Medical University of China. Animal experiments were approved by the Experimental Animal Committee of the Fourth Military Medical University (Xi’an, China).

Wnt4 negatively regulates the TGF-β1-induced human dermal fibroblast-to-myofibroblast transition via targeting Smad3 and ERK.

Abnormal activation of Wnt signaling has been demonstrated in the wound healing process and the pathogenesis of fibrotic disorders, with Wnt4 specifically identified as having a key role in the pathogenesis of renal, pulmonary and liver fibrosis. Wnt4 also was found to be upregulated by transforming growth factor-β1 (TGF-β1) in fetal and postnatal murine fibroblasts and bone marrow mesenchymal cells, suggesting an underlying cooperation between Wnt4 and TGF-β1 in fibrosis. However, the specific roles of Wnt4 in TGF-β1-induced skin myofibroblast transition and hypertrophic scar formation remain unclear. In the present study, we first observed reduced Wnt4 expression in hypertrophic scar tissue compared with that in normal skin tissue. Following upregulation by TGF-β1, Wnt4 inhibited the TGF-β1-induced transdifferentiation of fibroblasts into myofibroblasts. Using fibroblast-populated collagen lattice contraction assays, we showed that the increased contractility induced by TGF-β1 was significantly blocked by exogenous Wnt4 and the α-smooth muscle actin (α-SMA) expression was decreased in fibroblasts in the collagen lattices. In addition, knockdown of Wnt4 resulted in further increases in α-SMA and collagen I expressions. Further investigation showed that Wnt4 could inhibit the autocrine effect of TGF-β1 as well as block the phosphorylation of Smad3 and ERK but not of AKT or JNK. Lastly, using hypertrophic scar-derived fibroblasts, we showed that the elevated α-SMA and collagen I levels were markedly reduced after treatment with Wnt4. Taken together, our results suggest that Wnt4 negatively regulates TGF-β1-induced fibroblast activation, which may represent a novel therapeutic strategy for the treatment and prevention of hypertrophic scars.

664 Biological activities of traditional medicinal herbs on skin cells

In the field of cosmetic and dermatologic research, traditional medicinal plants gain interest, including medicinal herbs native from China. The present study aims to investigate the biological activity of Cohesium (COHE), an Ophiopogon Japonicus Root extract, in human epidermal keratinocytes and dermal fibroblasts. Primary cell cultures were established from human skin. Cell cytotoxicity was assessed by MTT assay after 2 to 5 days of culture with several dilutions of COHE (0.01-10%). Cell proliferation was measured by a cell cycle S phase analysis using Propidium Iodide staining. Wound healing was assessed by cell scratching and quantifying closure kinetics using Incucyte®. In some experiments, keratinocytes or fibroblasts were challenged by TNF-α (10 ng/mL) or TGF-β (5 ng/ml), respectively. Cytokine release was measured by ELISA assays. Synthesis of mediators and extracellular matrix by fibroblasts was determined by mRNA detection. Collagen contraction by fibroblasts was evaluated during 7 days. Regarding keratinocytes, COHE induced a slight cytotoxicity at 5%. No effect was observed on wound closure and on cell cycle S phase. The production of pro-inflammatory cytokine like IL-8 was increased with COHE in a dose dependent manner, reaching a 100% increase at 5% COHE dilution. No cytotoxicity was observed in fibroblasts whatever the COHE concentration used. Analysis of cell cycle S phase showed a significant increased proliferative state with the 5% concentration. The wound closure was not affected by COHE. The release of IL6, IL8 and VEGF cytokines as well as collagen lattice contraction was significantly increased with COHE. As suggested by its effects on fibroblast proliferation and contractile capacity and on the activation of pro-inflammatory cytokine production by both fibroblasts and keratinocytes, the Ophiopogon Japonicus Root extract might be efficient for skin repair and wound healing, in resting or activated conditions.

Zinc oxide nanoparticles ameliorate collagen lattice contraction in human tenon fibroblasts

Glaucoma is a major cause of irreversible blindness in the world and filtering surgery is commonly carried out to control intraocular pressure. Failure of filtering surgery is usually due to postoperative scarring, and fibroblast proliferation, collagen production and subconjunctival fibrosis play a prominent role in obstructing aqueous humor from the anterior chamber to the subconjunctival space. Zinc oxide (ZnO) nanoparticles have been widely applied in biomedical fields. However, the influence of ZnO nanoparticles on human tenon fibroblasts (HTFs) is still unclear. In the present study, we first explored the effects of various concentrations of ZnO nanoparticles on HTFs proliferation, reactive oxygen species (ROS) generation, cell cycle arrest, and apoptosis. Further, we determined the changes of transforming growth factor-β (TGF-β1), fibronectin (FN) extra domain A (ED-A), and procollagen I carboxyterminal propeptide (PICP) at mRNA and protein levels, explored the effect of ZnO nanoparticles on the collagen lattice contraction in HTFs. The results indicated that ZnO nanoparticles can efficiently inhibit HTFs proliferation, elevate ROS production level, and induce cell cycle arrest at G2/M phase, leading to HTFs apoptosis. ZnO nanoparticles can also decrease the expressions of TGF-β1, ED-A, and PICP at mRNA and protein levels; significantly prevent fibroblast-mediated collagen lattice contraction. Taken together, ZnO nanoparticles can efficiently ameliorate collagen lattice contraction in HTFs, and may be a promising antifibrotic agent in glaucoma filtration surgery. Our findings provide a new insight on anti-scar formation after glaucoma filtration surgery by using ZnO nanoparticles.

Telomere dysfunction promotes transdifferentiation of human fibroblasts into myofibroblasts.

Cells that had undergone telomere dysfunction‐induced senescence secrete numerous cytokines and other molecules, collectively called the senescence‐associated secretory phenotype (SASP). Although certain SASP factors have been demonstrated to promote cellular senescence in neighboring cells in a paracrine manner, the mechanisms leading to bystander senescence and the functional significance of these effects are currently unclear. Here, we demonstrate that TGF‐β1, a component of the SASP, causes telomere dysfunction in normal somatic human fibroblasts in a Smad3/NOX4/ROS‐dependent manner. Surprisingly, instead of activating cellular senescence, TGF‐β1‐induced telomere dysfunction caused fibroblasts to transdifferentiate into α‐SMA‐expressing myofibroblasts, a mesenchymal and contractile cell type that is critical for wound healing and tissue repair. Despite the presence of dysfunctional telomeres, transdifferentiated cells acquired the ability to contract collagen lattices and displayed a gene expression signature characteristic of functional myofibroblasts. Significantly, the formation of dysfunctional telomeres and downstream p53 signaling was necessary for myofibroblast transdifferentiation, as suppressing telomere dysfunction by expression of hTERT, inhibiting the signaling pathways that lead to stochastic telomere dysfunction, and suppressing p53 function prevented the generation of myofibroblasts in response to TGF‐β1 signaling. Furthermore, inducing telomere dysfunction using shRNA against TRF2 also caused cells to develop features that are characteristic of myofibroblasts, even in the absence of exogenous TGF‐β1. Overall, our data demonstrate that telomere dysfunction is not only compatible with cell functionality, but they also demonstrate that the generation of dysfunctional telomeres is an essential step for transdifferentiation of human fibroblasts into myofibroblasts.

Ascorbic acid promotes a TGFβ1-induced myofibroblast phenotype switch.

l‐Ascorbic acid (AA), generally known as vitamin C, is a crucial cofactor for a variety of enzymes, including prolyl‐3‐hydroxylase (P3H), prolyl‐4‐hydroxylase (P4H), and lysyl hydroxylase (LH)‐mediated collagen maturation. Here, we investigated whether AA has additional functions in the regulation of the myofibroblast phenotype, besides its function in collagen biosynthesis. We found that AA positively influences TGF β1‐induced expression of COL1A1,ACTA2, and COL4A1. Moreover, we demonstrated that AA promotes α SMA stress fiber formation as well as the synthesis and deposition of collagens type I and IV. Additionally, AA amplified the contractile phenotype of the myofibroblasts, as seen by increased contraction of a 3D collagen lattice. Moreover, AA increased the expression of several TGF β1‐induced genes, including DDR1 and CCN2. Finally, we demonstrated that the mechanism of AA action seems independent of Smad2/3 signaling.

An In Vitro Model for Fibroblast Photoaging Comparing Single and Repeated UVA Irradiations.

The current method for efficient evaluation of antiphotoaging compounds is an in vitro skin culture model using a single ultraviolet A (UVA) irradiation of fibroblasts. However, skin photoaging is caused by repeated exposure to UVA radiation. The objective of this study was to develop an appropriate model for in vitro skin photoaging by comparing the different effects of single (5 J cm-2 ) and repeated exposures (5 J cm-2 × 3 times) of fibroblasts to UVA irradiation. Our results demonstrated that a single and repeated exposure to UVA irradiation had different effects on fibroblasts. In the single UVA-irradiated group, collagen lattice contraction and the protein levels of type I procollagen and matrix metalloproteinase-1 (MMP-1) increased, while the levels of fibronectin and alpha-smooth muscle actin (α-SMA) were unchanged, compared to levels in the non-UVA-irradiated group (control). In contrast, repeated UVA exposure significantly induced G0/G1 cell cycle arrest, reduced collagen lattice contraction and type I procollagen and fibronectin expression, and increased MMP-1 expression. There was no difference in α-SMA expression when comparing repeatedly irradiated and non-UVA-irradiated fibroblasts. Our findings clearly indicate that repeated UVA irradiation of cells induces malfunctions found in photoaged skin and is an appropriate in vitro skin model of photoaging.

Effect of Palmitoyl-Pentapeptide (Pal-KTTKS) on Wound Contractile Process in Relation with Connective Tissue Growth Factor and α-Smooth Muscle Actin Expression.

To evaluate whether Palmitoyl-pentapeptide (Pal-KTTKS), a lipidated subfragment of type 1 pro-collagen (residues 212-216), plays a role in fibroblast contractility, the effect of Pal-KTTKS on the expression of pro-fibrotic mediators in hypertropic scarring were investigated in relation with trans-differentiation of fibroblast to myofibroblast, an icon of scar formation. α-SMA was visualized by immunofluorescence confocal microscopy with a Cy-3-conjugated monoclonal antibody. The extent of α-SMA-positive fibroblasts was determined in collagen lattices and in cell culture study. Pal-KTTKS (0-0.5µM) induced CTGF and α-SMA protein levels were determined by western blot analysis and fibroblast contractility was assessed in three-dimensional collagen lattice contraction assay. In confocal analysis, fibroblasts were observed as elongated and spindle shapes while myofibroblast observed as squamous, enlarged cells with pronounced stress fibers. Without Pal-KTTKS treatment, three quarters of the fibroblasts differentiates into the myofibroblast; α-SMA-positive stress fibers per field decreased twofold with 0.1µM Pal-KTTKS treatment (75±7.1 vs 38.6±16.1%, n=3, p<0.05). The inhibitory effect was not significant in 0.5µM Pal-KTTKS treatment. Stress fiber level and collagen contractility correlates with α-SMA expression level. In conclusion, Pal-KTTKS (0.1µM) reduces α-SMA expression and trans-differentiation of fibroblasts to myofibroblast. The degree of reduction is dose-dependent. An abundance of myofibroblast and fibrotic scarring is correlated with excessive levels of α-SMA and collagen contractility. Delicate balance between the wound healing properties and pro-fibrotic abilities of pentapeptide KTTKS should be considered for selecting therapeutic dose for scar prevention.

ILK–PI3K/AKT pathway participates in cutaneous wound contraction by regulating fibroblast migration and differentiation to myofibroblast

The interactions between fibroblasts and the extracellular matrix in wound contraction are mainly mediated via integrin signaling. Integrin-linked kinase (ILK) is a key mediator in integrin signal transduction. We investigated the role of ILK in cutaneous wound contraction. We found that ILK was involved in cutaneous wound healing in rats, and ILK and PI3K/AKT inhibitors inhibited wound contraction and re-epithelialization, consequently delaying wound healing in vivo. Further, using in vitro studies, we demonstrated that ILK and PI3K/AKT inhibitors suppressed the contraction of fibroblast-populated collagen lattices, inhibited fibroblast migration, and interrupted the effect of TGF-β1 on promoting alpha smooth muscle actin (α-SMA) expression in fibroblasts. When ILK expression was directly blocked by ILK small interfering RNA transfection, the migration and α-SMA expression of normal dermal fibroblasts were significantly suppressed as well. The data suggest that the ILK–PI3K/AKT signaling pathway mediates cutaneous wound contraction by regulating fibroblast migration and differentiation to myofibroblasts.