Collagen Gel Contractility Research Articles

Osthole mitigates the myofibroblast properties in oral submucous fibrosis by suppressing the TGF-β/smad2 signaling pathway and NCK-AS1 expression

Background/purposeNatural products are gaining increasing recognition as an alternative source for alleviating fibrosis as they can regulate various mediators or pathways against fibrosis by targeting non-coding RNAs. In the current study, we aimed to investigate the therapeutic effects of osthole in oral submucous fibrosis (OSF), a precancerous condition of the oral cavity. Materials and methodsThe cytotoxicity of osthole to normal and fibrotic buccal mucosal fibroblasts (fBMFs) derived from OSF tissues was assessed using MTT assay. Collagen gel contraction and transwell migration assays were conducted to examine the myofibroblast activities. Besides, the expression of TGF-β/Smad2 signaling as well as α-SMA and type I collagen were measured. Additionally, RNA sequencing was used to identify a potential target involved in the anti-fibrotic effect of osthole. ResultsOsthole exhibited a higher cytotoxic effect on fBMFs compared to normal BMFs and dose-dependently reduced several myofibroblast activities, including collagen gel contractility and transwell migration ability. In addition, the expression of the TGF-β/Smad2 pathway was inhibited along with a lower expression of α-SMA and type I collagen in osthole-receiving cells. Moreover, the administration of osthole downregulated the expression of NCK1-AS1 in fBMFs, which was proven to mediate the anti-fibrosis property of osthole. ConclusionOur results indicate that osthole may be a promising compound to inhibit the progression of OSF.

Down-regulation of miR-29c promotes the progression of oral submucous fibrosis through targeting tropomyosin-1

Various microRNAs (miRs) have been found to be associated with the development of the precancerous condition of the oral cavity, oral submucous fibrosis (OSF). The expression of miR-29c is dysregulated in oral cancer, but its role in OSF has not been investigated. The purpose of the study is to investigate the functional role of miR-29c and its target in OSF. The expression levels of miR-29c in OSF tissues and fibrotic buccal mucosal fibroblasts (fBMFs) were assessed using next-generation sequencing and real-time Polymerase Chain Reaction (PCR) analysis. MiR-29c mimic and inhibitors were employed to examine its functional role of myofibroblast transdifferentiation. In addition, several myofibroblast phenotypes, such as collagen gel contraction and migration were tested, and a luciferase reporter assay was conducted to confirm the relationship between miR-29c and its predicted target, tropomyosin-1 (TPM1). We observed that miR-29c expression was downregulated in fBMFs. fBMFs transfected with miR-29c mimics exhibited reduced migration ability and collagen gel contractility, whereas inhibition of miR-29c in normal BMFs induced the myofibroblast phenotypes. Results from the luciferase reporter assay showed that TPM1 was a direct target of miR-29c and the expression of TPM1 was suppressed in the fBMFs transfected with miR-29c mimics. Besides, we confirmed that the expression of miR-29c was indeed downregulated in OSF specimens. MiR-29c seems to exert an inhibitory effect on myofibroblast activation, such as collagen gel contractility and migration ability, via suppressing TPM1. These results suggested that approaches to upregulate miR-29c may be able to ameliorate the progression of OSF.

Paeonol inhibits profibrotic signaling and HOTAIR expression in fibrotic buccal mucosal fibroblasts

Betel nut chewing is the major risk factor of oral submucous fibrosis (OSF). Various studies have sought to discover alternative strategies to alleviate oral fibrogenesis. In the present study, we aimed to evaluate the anti-fibrosis effect of paeonol, a phenolic component derived from Paeonia Suffruticosa. The cytotoxicity of paeonol was tested using normal and fibrotic buccal mucosal fibroblasts (fBMFs) derived from OSF tissues. Collagen gel contraction, Transwell migration, invasion, and wound healing capacities were examined. Besides, the activation of TGF-β/Smad2 signaling and expression levels of type I collagen, α-SMA, and long non-coding RNA HOTAIR were measured as well. Paeonol exerted a higher cytotoxic effect on fBMFs compared to normal BMFs. The arecoline-induced myofibroblast activities, including collagen gel contractility, cell motility, and wound healing ability were all suppressed by paeonol treatment. In addition, the activation of the TGF-β/Smad2 pathway was inhibited along with a lower expression of α-SMA and type I collagen in paeonol-treated cells. Also, the administration of paeonol decreased the mRNA expression of HOTAIR in fBMFs. Our results indicate that paeonol may be a promising compound to attenuate the progression of oral fibrogenesis in OSF patients.

MiR-21 promotes the fibrotic properties in oral mucosa through targeting PDCD4

Background/purposeOral submucous fibrosis (OSF) has been regarded as a premalignant disorder of oral cancer, and myofibroblasts are the main cells that are responsible for pathological fibrosis. Hence, elucidation of the molecular mechanism underlying myofibroblast activation is important to treat OSF. MicroRNA-21 (miR-21) is a well-known fibrosis non-coding RNA, and its role in the development of OSF remains largely unclear. Materials and methodsLuciferase reporter assay was used to confirm the direct interaction between miR-21 and its target programmed cell death 4 (PDCD4). The expression level of PDCD4 in OSF was examined by qRT-PCR. Myofibroblast activities were assessed by collagen gel contraction and transwell migration assays. ResultsOur result validated the direct binding of miR-21 to PDCD4. We showed the expression of PDCD4 was downregulated in OSF specimens and negatively correlated with miR-21. Our results suggested that overexpression of PDCD4 in fibrotic buccal mucosal fibroblasts (fBMFs) mitigated the myofibroblast activities, including collagen gel contractility and migration capacity. Moreover, we showed miR-21 contributed to myofibroblast activation of BMFs through repression of PDCD4. ConclusionOur results suggest that the miR-21/PDCD4 axis mediates the myofibroblast activation of BMFs, and targeting this axis may exert an anti-fibrosis effect.

Honokiol inhibits arecoline-induced oral fibrogenesis through transforming growth factor-β/Smad2/3 signaling inhibition

The habit of areca nut chewing has been regarded as an etiological factor of precancerous oral submucous fibrosis (OSF). In the present study, we aimed to evaluate the anti-fibrosis effect of honokiol, a polyphenolic component derived from Magnolia officinalis. The cytotoxicity of honokiol was tested using normal and fibrotic buccal mucosal fibroblasts (fBMFs) derived from OSF tissues. Collagen gel contraction, Transwell migration, invasion, and wound healing capacities were examined. Besides, the expression of TGF-β/Smad2 signaling as well as α-SMA and type I collagen were measured as well. Honokiol exerted higher cytotoxicity of fBMFs compared to normal cells. The arecoline-induced myofibroblast activities, including collagen gel contractility, cell motility and wound healing capacities were all suppressed by honokiol treatment. In addition, the expression of the TGF-β/Smad2 pathway was downregulated along with a lower expression of α-SMA and type I collagen in honokiol-receiving cells. Our data suggest that honokiol may be a promising compound to alleviate the progression of oral fibrogenesis and prevent the transformation of OSF oral epithelium into cancer.

Targeting lncRNA H19/miR-29b/COL1A1 Axis Impedes Myofibroblast Activities of Precancerous Oral Submucous Fibrosis.

Oral submucous fibrosis (OSF) is known as a potentially malignant disorder, which may result from chemical irritation due to areca nuts (such as arecoline). Emerging evidence suggests that fibrogenesis and carcinogenesis are regulated by the interaction of long noncoding RNAs (lncRNAs) and microRNAs. Among these regulators, profibrotic lncRNA H19 has been found to be overexpressed in several fibrosis diseases. Here, we examined the expression of H19 in OSF specimens and its functional role in fibrotic buccal mucosal fibroblasts (fBMFs). Our results indicate that the aberrantly overexpressed H19 contributed to higher myofibroblast activities, such as collagen gel contractility and migration ability. We also demonstrated that H19 interacted with miR-29b, which suppressed the direct binding of miR-29b to the 3′-untranslated region of type I collagen (COL1A1). We showed that ectopic expression of miR-29b ameliorated various myofibroblast phenotypes and the expression of α-smooth muscle actin (α-SMA), COL1A1, and fibronectin (FN1) in fBMFs. In OSF tissues, we found that the expression of miR-29b was downregulated and there was a negative correlation between miR-29b and these fibrosis markers. Lastly, we demonstrate that arecoline stimulated the upregulation of H19 through the transforming growth factor (TGF)-β pathway. Altogether, this study suggests that increased TGF-β secretion following areca nut chewing may induce the upregulation of H19, which serves as a natural sponge for miR-29b and impedes its antifibrotic effects.

Amyloid Beta Peptides and Th1 Cytokines Modulate Human Brain Vascular Smooth Muscle Tonic Contractile Capacity In Vitro: Relevance to Alzheimer's Disease?

Alzheimer’s Disease (AD) is a neurodegenerative condition characterized both by the presence of tau protein neurofibrillary tangles and amyloid beta (Aβ) containing extracellular “plaques”. The cleavage of amyloid precursor protein (APP) yields several Aβ peptides. Although Aβ toxicity to neurons has been described extensively, its effects on other components of the neurovasculature such as vascular smooth muscle cells have been less well characterized. AD is now also recognized as a neurovascular disease characterized by cerebral microbleeds and disturbances in autoregulation. AD is also a neuroinflammatory condition in which several proinflammatory cytokines are elevated and may contribute to the intensification of AD severity. Cerebral autoregulation (the mechanism by which brain blood flow is maintained despite changes in perfusion pressure) is extremely tightly controlled in the brain and shows disturbances in AD. The failure of autoregulation in AD may make the brain susceptible to cerebral microbleeds through a reduced capacity to limit blood flow when pressure is increased. Conversely, reduced vasodilation during low flow might could also exacerbate tissue hypoxia. Currently, whether and how Aβ peptides and inflammatory cytokines depress brain smooth muscle cell tonic contraction is not known, but could reveal important targets in the preservation of autoregulation which is disturbed in AD. We used a collagen gel contractility assay to evaluate the influence of Aβ25-35, Aβ1-40 and Aβ1-42 peptides and inflammatory cytokines on the tonic contractility of human brain vascular smooth muscle cells (HBVSMC) as an in vitro model of cerebral autoregulation. We found that 5 and 10 μM Aβ1-42 significantly depressed HBVSM contractility, while Aβ1-40 5–20 μM had no effect on contractility. Conversely, Aβ25-35 (1–50 μM) increased contractility. Interestingly, the inflammatory cytokines TNF-α (20 ng/mL), IL-1β (20 ng/mL) and IFN-γ (1000 U/mL) also depressed HBVSM tonic contractility alone and in combination. These data suggest that both the inflammatory milieu in AD as well as the abundance of Aβ peptides may promote autoregulatory failure and increase brain susceptibility to dysregulated perfusion and microbleeds which are an important and devastating characteristic of AD.

Arecoline enhances miR-21 to promote buccal mucosal fibroblasts activation

Oral submucous fibrosis (OSF) is an irreversible fibrosis disease and a potentially malignant disorder in the oral cavity. Various studies have shown that miR-21 was implicated in the fibrogenesis and carcinogenesis, but its functional role in the development of OSF has not been investigated. The expression levels of miR-21 in arecoline-stimulated normal buccal mucosal fibroblasts (BMFs) and OSF specimens were determined by qRT-PCR. Exogenous administration of TGF-β and its inhibitor (SB431542) were utilized to examine the involvement of TGF-β signaling in miR-21 alteration. Collagen gel contraction, transwell migration, and invasion assays were used to assess the myofibroblast activities. The relationship between α-SMA and miR-21 was calculated using the Pearson correlation coefficient. MiR-21 expression was induced in BMFs by arecoline treatment in a dose-dependent manner. Our results showed that this upregulation was mediated by TGF-β signaling. Subsequently, we demonstrated that the administration of the miR-21 inhibitor suppressed the arecoline-induced myofibroblast characteristics, including a higher collagen gel contractility and cell motility, in normal BMFs. Furthermore, inhibition of miR-21 was sufficient to attenuate the myofibroblast features in fibrotic BMFs. Besides, we showed that the expression of miR-21 was aberrantly upregulated in the OSF tissues and there was a positive correlation between miR-21 and myofibroblast marker, α-SMA. MiR-21 overexpression in OSF may be due to the stimulation of areca nut, which was mediated by the TGF-β pathway. Our data suggested that the repression of miR-21 was a promising direction to palliate the development and progression of OSF.

LINC00312/YBX1 Axis Regulates Myofibroblast Activities in Oral Submucous Fibrosis.

Oral submucous fibrosis (OSF) has been recognized as a precancerous disorder in the oral cavity. Great effort has been made to inhibit the malignant progression of OSF over the past decades, but the cure of this fibrosis disease has not been discovered. In the present study, we found that a long noncoding RNA, LINC00312, was upregulated in OSF tissues, and positively associated with several fibrosis factors, such as α-SMA, type I collagen, and fibronectin. As such, we sought to investigate the role of LINC00312 in OSF progression and identify its interacting factor that mediated oral fibrogenesis. Our results showed that the inhibition of LINC00312 downregulated the myofibroblast activities, including collagen gel contractility, transwell migration, and wound healing, as well as the gene expression of myofibroblast markers. We verified that YBX1 was a downstream factor of LINC00312 and revealed that the downregulation of YBX1 repressed the gene expression of α-SMA and p-Smad2 along with the reduced myofibroblast phenotypes. Most importantly, we demonstrated that the LINC00312-induced myofibroblast activities were reverted by the knockdown of YBX1, suggesting that the LINC00312-mediated myofibroblast transdifferentiation was through YBX1. Collectively, our findings revealed that the LINC00312/ YBX1 axis may serve as a target for the development of therapies against OSF.

E3 ligase carboxyl-terminus of Hsp70-interacting protein (CHIP) suppresses fibrotic properties in oral mucosa

Oral submucous fibrosis (OSF) represents a precancerous lesion of oral mucosa that may progress into oral cancer and its major etiological factor is areca nut chewing. Carboxyl-terminus of Hsp70-interacting protein (CHIP) functions as an ubiquitin E3 ligase and is associated with fibrosis diseases. In the current study, we sought to investigate whether CHIP participated in the areca nut-mediated OSF development. The mRNA expression of CHIP in arecoline-stimulated buccal mucosal fibroblasts (BMFs) and OSF tissues was determined by qRT-PCR. Collagen gel contraction, migration and invasion assays were carried out to evaluate the myofibroblast activation. The protein expression levels of α-SMA and transglutaminase 2 (TGM2) were assessed by Western blot. The expression level of CHIP was reduced in BMFs following arecoline treatment in a dose-dependent manner, which was consistent with the observation of lower CHIP expression in OSF specimen compared to the normal counterparts. Ectopic expression of CHIP mitigated the myofibroblast activities, including elevated collagen gel contractility and cell motility. In addition, we showed that overexpression of CHIP downregulated the α-SMA and TGM-2 expression, which may lead to less fibrosis alteration. CHIP may not only function as a key regulator of protein quality control but also a critical deciding factor to oral fibrogenesis. Our findings suggested that CHIP possesses the anti-fibrotic effect, which may be mediated by TGM2 regulation. Restoration of CHIP could be a therapeutic direction to help OSF patients.

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.

Arctigenin Reduces Myofibroblast Activities in Oral Submucous Fibrosis by LINC00974 Inhibition.

Oral submucous fibrosis (OSF) is an oral precancerous condition associated with the habit of areca nut chewing and the TGF-β pathway. Currently, there is no curative treatment to completely heal OSF, and it is imperative to alleviate patients’ symptoms and prevent it from undergoing malignant transformation. Arctigenin, a lignan extracted from Arctium lappa, has been reported to have a variety of pharmacological activities, including anti-fibrosis. In the present study, we examined the effect of arctigenin on the cell proliferation of buccal mucosal fibroblasts (BMFs) and fibrotic BMFs (fBMFs), followed by assessment of myofibroblast activities. We found that arctigenin was able to abolish the arecoline-induced collagen gel contractility, migration, invasion, and wound healing capacities of BMFs and downregulate the myofibroblast characteristics of fBMFs in a dose-dependent manner. Most importantly, the production of TGF-β in fBMFs was reduced after exposure to arctigenin, along with the suppression of p-Smad2, α-smooth muscle actin, and type I collagen A1. In addition, arctigenin was shown to diminish the expression of LINC00974, which has been proven to activate TGF-β/Smad signaling for oral fibrogenesis. Taken together, we demonstrated that arctigenin may act as a suitable adjunct therapy for OSF.

MiR-200c inhibits the arecoline-associated myofibroblastic transdifferentiation in buccal mucosal fibroblasts.

MicroRNA-200c (miR-200c) recently emerged as an important regulator of tumorigenesis and cancer metastasis, however, its role in regulating oral submucous fibrosis (OSF) remains unknown. In this study, we investigated the functional role of miR-200c in myofibroblastic differentiation activity and identified its potential target. qRT-PCR was applied to assess the expression of miR-200c in OSF tissues and fibrotic buccal mucosal fibroblasts (fBMFs). Arecoline, a major areca nut alkaloid, was utilized to explore whether the expression of miR-200c would alter following stimulation. Collagen gel contraction, migration and invasion capabilities were examined in arecoline-stimulated BMFs as wells as in fBMFs. Luciferase reporter assay was conducted to show the relationship between miR-200c and ZEB1. Our results showed that the expression of miR-200c was downregulated in OSF specimen and fBMFs. Arecoline treatment dose-dependently reduced the relative expression of miR-200c in normal BMFs. Overexpression of miR-200c impeded the arecoline-induced collagen gel contraction, migration, invasion and wound healing capacities. Moreover, ectopic expression of miR-200c in fBMFs successfully reduced the increased collagen gel contractility and invasion abilities. Our results demonstrated that ZEB1 was a direct target of miR-200c, and overexpression of miR-200c inhibited the expression of ZEB1 and α-SMA. These findings suggest that downregulation of miR-200c in OSF may be involved in the pathogenesis of areca nut-associated OSF through regulation of ZEB1.

Glabridin inhibits the activation of myofibroblasts in human fibrotic buccal mucosal fibroblasts through TGF-β/smad signaling.

Oral submucous fibrosis (OSF) has been recognized as one of the oral potentially malignant disorders. Areca nut chewing is implicated in this pathological fibrosis, and it causes chronic inflammation and persistent activation of myofibroblasts. As yet, existing treatments only provide temporary symptomatic relief and there is a lack of an effective intervention to cure OSF. Therefore, development of approaches to ameliorate myofibroblast activities becomes a crucial objective to prevent the malignant progression of OSF. In this study, we examined the inhibitory effect of glabridin, an isoflavane extracted from licorice root, on the myofibroblast characteristics in human fibrotic buccal mucosal fibroblasts (fBMFs). Our results showed that myofibroblast activities, including collagen gel contractility, migration, invasion and wound healing abilities were reduced after exposure of glabridin in a dose-dependent manner. Most importantly, we demonstrated that the arecoline-induced myofiroblast activities were abolished by glabridin treatment. Additionally, the expression of the myofibroblast marker α-smooth muscle actin and other fibrogenic marker, type I collagen, in fBMFs were dose-dependently downregulated. Moreover, we showed that the production of TGF-β was suppressed by glabridin in fBMFs and the protein expression of phospho-Smad2 was decreased as well. In summary, our data suggested that glabridin repressed the myofibroblast features in fBMFs via TGF-β/Smad2 signaling pathway. Glabridin also prevented the arecoline-increased myofibroblast activities, and could serve as a natural anti-fibrosis compound for OSF.

Neutrophil elastase promotes myofibroblast differentiation in lung fibrosis.

IPF is a progressive lung disorder characterized by fibroblast proliferation and myofibroblast differentiation. Although neutrophil accumulation within IPF lungs has been negatively correlated with outcomes, the role played by neutrophils in lung fibrosis remains poorly understood. We have demonstrated previously that NE promotes lung cancer cell proliferation and hypothesized that it may have a similar effect on fibroblasts. In the current study, we show that NE(-/-) mice are protected from asbestos-induced lung fibrosis. NE(-/-) mice displayed reduced fibroblast and myofibroblast content when compared with controls. NE directly both lung fibroblast proliferation and myofibroblast differentiation in vitro, as evidenced by proliferation assays, collagen gel contractility assays, and αSMA induction. Furthermore, αSMA induction occurs in a TGF-β-independent fashion. Treatment of asbestos-recipient mice with ONO-5046, a synthetic NE antagonist, reduced hydroxyproline content. Thus, the current study points to a key role for neutrophils and NE in the progression of lung fibrosis. Lastly, the study lends rationale to use of NE-inhibitory approaches as a novel therapeutic strategy for patients with lung fibrosis.

Heparin is a promising agent for the treatment of endometriosis-associated fibrosis

To assess the use of heparin for the medical treatment of endometriosis-associated fibrosis. The effects of heparin on the endometriotic stromal cells (ECSCs)-mediated contractility were investigated. Research laboratory at a medical school. Endometriotic tissues from nine patients were used. Endometriotic stromal cells were cultured three dimensionally in the presence of heparin. The contractility of ECSCs was assessed by collagen gel contraction assay. Heparin-induced morphological changes of ECSCs were evaluated by laser scanning microscopy. The expression of contractility-related molecules in ECSCs was examined by Western blot analysis. In the presence of 10% fetal bovine serum, treated ECSCs showed significant collagen gel contractility (75.9% decrease in surface area after 48 hour vs. 0 hour controls). Endometriotic stromal cell-mediated gel contraction was significantly attenuated in the presence of heparin in a dose-dependent manner (55.7% reduction of the gel contraction at a concentration of 100 microg/mL of heparin sodium versus untreated controls after 48 hours). Heparin suppressed the ECSC attachment to collagen fibers. The expression of alpha-smooth muscle actin, Ras homology (Rho) A, Rho-associated coiled-coil-forming protein kinase (ROCK)-I, and ROCK-II was down-regulated by heparin administration. The present study suggests that heparin is a promising agent for the treatment of endometriosis-associated fibrosis. The inhibition of myofibroblastic differentiation, the attenuation of attachment to collagen fibers, and the suppression of Rho-ROCK-mediated pathway activation in ECSCs are involved in the action mechanisms of heparin.

Regulation of contractility of cultured human endometrial stromal cells by tumor necrosis factor-α

Objective The aim of this study is to evaluate the involvement of tumor necrosis factor (TNF)-α in endometrial tissue remodeling during the perimenstrual period. Study design Human endometrial stromal cells (ESCs) were isolated from eight premenopausal patients in the late secretory phase. The effects of TNF-α on the contractility of cultured ESCs were investigated by collagen gel contraction assay. The effects of TNF-α on the proliferation of ESCs were also assessed by a modified methylthiazoletetrazolium assay. Results TNF-α significantly upregulated the collagen gel contractility of ESCs in a dose-dependent manner. TNF-α did not affect ESC proliferation. Conclusion The results suggest that TNF-α may promote endometrial tissue repair by stimulating the contraction of the extracellular matrix by ESCs. By regulating ESC function during the perimenstrual period, TNF-α may be involved in the physiological tissue remodeling of the cyclic endometrium.

Collagen gel contractility is enhanced in human endometriotic stromal cells: a possible mechanism underlying the pathogenesis of endometriosis-associated fibrosis

Excessive fibrosis is frequently associated with endometriosis. To evaluate the involvement of the extracellular matrix contractility of endometriotic stromal cells (ECSCs) in the pathogenesis of endometriosis-associated fibrosis, we compared the collagen gel contractility of cultured ECSCs with that of normal endometrial stromal cells. To clarify the mechanism underlying collagen gel contraction by ECSCs, we also evaluated the effect of (+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl) cyclohexanecarboxamide dihydrochloride, monohydrate (Y-27632), a selective Rho-associated coiled-coil-forming protein kinase (ROCK) inhibitor, on the collagen gel contraction by ECSCs. ECSCs showed enhanced collagen gel contractility in comparison with NESCs. Myofibroblastic differentiation and the increased expression of fibronectin, RhoA, ROCK-I and ROCK-II proteins were observed with ECSCs using the 3D culture. Y-27632 significantly inhibited the collagen gel contractility of ECSCs without cytotoxicity. The present findings suggest that the enhanced collagen contractility in ECSCs is associated with myofibroblastic differentiation, the increased expression of fibronectin and the activation of the Rho-ROCK-mediated signalling pathway, all of which may be involved in the pathogenesis of endometriosis-associated fibrosis. These results suggest that the inhibition of the Rho-ROCK-mediated signalling pathway may provide a novel strategy for the treatment of this disease. In addition, our experimental system of ECSCs using 3D collagen gel culture would be suitable for evaluating novel treatments for endometriosis.

Regulation of Proliferation, Motility, and Contractility of Human Endometrial Stromal Cells by Platelet-Derived Growth Factor

To evaluate the involvement of platelet-derived growth factor (PDGF) isoforms (PDGF-AlphaAlpha, PDGF-AB, and PDGF-BB) on endometrial tissue remodeling during the perimenstrual period, we investigated the effects of PDGF on the proliferation, motility, invasiveness, and contractility of cultured human endometrial stromal cells (ESC) using a modified methylthiazoletetrazolium assay, a 5-bromo-2'-deoxyuridine incorporation assay, an in vitro wound repair assay, a chemotactic migration assay, a Transwell invasion assay, and a collagen gel contraction assay. All three isoforms of PDGF significantly enhanced the cell proliferation, DNA synthesis, and in vitro wound repair of ESC. Chemotactic migration assay, Transwell invasion assay, and collagen gel contraction assay demonstrated that the PDGF isoforms significantly stimulated both the motility of ESC and the collagen gel contractility of ESC. PDGF-BB showed the strongest effects on these cellular functions of ESC. The present study suggested that PDGF isoforms may promote endometrial tissue repair by enhancing the proliferation and expansion of ESC, stimulating ESC migration, and stimulating the contraction of the collagen gel matrix by ESC. By regulating ESC function during the perimenstrual period, PDGF may help to protect the endometrium from extensive fibrosis and scarring.

Correlation between ageing and collagen gel contractility of human fibroblasts.

To investigate the influence of ageing on wound healing, we cultured fibroblasts derived from human dermis in type I collagen gel, and evaluated the relationship between gel contractility and ageing. Cells were obtained from children (0-15 years old, Group A), early adulthood (16-40 years old, Group B), mid-adulthood (41-60 years old, Group C), and the elderly (61 or older, Group D). Gel contractility was determined by measuring the diameter on the second day after gel preparation. Within the tenth passage, gel contraction was the most marked in Group A, but did not differ among the other groups. Gel contraction at passages 30-40 did not differ from those within the tenth passage in Groups B, C and D, but it decreased markedly in Group A to a value similar to that in the other groups. These results show that fibroblasts in childhood are more contractile than those in adulthood and are more readily affected by passages (in vitro ageing).