Collagen Gel Contraction Assay Research Articles

Generation of an enteric smooth muscle cell line from the pig ileum.

Smooth muscle cells (SMCs) play an important role in physiology and production in farm animals such as pigs. Here, we report the generation of a pig SMC line. Our original objective was to establish an enteroendocrine cell line from the pig ileum epithelium through lentiviral transduction of the Simian Virus (SV) 40 large T antigen. However, an initial expression analysis of marker genes in nine cell clones revealed that none of them were enteroendocrine cells or absorptive enterocytes, goblet cells, or Paneth cells, some of the major cell types existing in the ileum epithelium. A more detailed characterization of one clone named PIC7 by RNA-seq showed that these cells expressed many of the known smooth muscle-specific or -enriched genes, including smooth muscle actin alpha 2, calponin 1, calponin 3, myosin heavy chain 11, myosin light chain kinase, smoothelin, tenascin C, transgelin, tropomyosin 1, and tropomyosin 2. Both quantitative PCR and RNA-seq analyses showed that the PIC7 cells had a high expression of mRNA for smooth muscle actin gamma 2, also known as enteric smooth muscle actin. A Western blot analysis confirmed the expression of SV40 T antigen in the PIC7 cells. An immunohistochemical analysis demonstrated the expression of smooth muscle actin alpha 2 filaments in the PIC7 cells. A collagen gel contraction assay showed that the PIC7 cells were capable of both spontaneous contraction and contraction in response to serotonin stimulation. We conclude that the PIC7 cells are derived from an enteric SMC from the pig ileum. These cells may be a useful model for studying the cellular and molecular physiology of pig enteric SMCs. Because pigs are similar to humans in anatomy and physiology, the PIC7 cells may be also used as a model for human intestinal SMCs.

Acetylcholine decreases formation of myofibroblasts and excessive extracellular matrix production in an in vitro human corneal fibrosis model.

Acetylcholine (ACh) has been reported to play various physiological roles, including wound healing in the cornea. Here, we study the role of ACh in the transition of corneal fibroblasts into myofibroblasts, and in consequence its role in the onset of fibrosis, in an in vitro human corneal fibrosis model. Primary human keratocytes were obtained from healthy corneas. Vitamin C (VitC) and transforming growth factor‐β1 (TGF‐β1) were used to induce fibrosis in corneal fibroblasts. qRT‐PCR and ELISA analyses showed that gene expression and production of collagen I, collagen III, collagen V, lumican, fibronectin (FN) and alpha‐smooth muscle actin (α‐SMA) were reduced by ACh in quiescent keratocytes. ACh treatment furthermore decreased gene expression and production of collagen I, collagen III, collagen V, lumican, FN and α‐SMA during the transition of corneal fibroblasts into myofibroblasts, after induction of fibrotic process. ACh inhibited corneal fibroblasts from developing contractile activity during the process of fibrosis, as assessed with collagen gel contraction assay. Moreover, the effect of ACh was dependent on activation of muscarinic ACh receptors. These results show that ACh has an anti‐fibrotic effect in an in vitro human corneal fibrosis model, as it negatively affects the transition of corneal fibroblasts into myofibroblasts. Therefore, ACh might play a role in the onset of fibrosis in the corneal stroma.

All-trans retinoic acid regulates TGF-β1-induced extracellular matrix production via p38, JNK, and NF-κB-signaling pathways in nasal polyp-derived fibroblasts.

All-trans retinoic acid (ATRA), a derivative of vitamin A, is known to have anti-fibrogenic effects and regulates cell proliferation and differentiation. Therefore, these abilities of ATRA may influence tissue remodeling in the upper airway. The aims of the present study were to investigate the effects of ATRA on the myofibroblast differentiation, extracellular matrix (ECM) production, cell migration, and collagen gel contraction and to determine the molecular mechanisms of ATRA in TGF-β1-induced nasal polyp-derived fibroblasts (NPDFs). NPDFs were isolated from nasal polyp. Cytotoxicity was evaluated by 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl-tetrazolium bromide assay. TGF-β1-induced fibroblasts were pretreated with ATRA. The expression levels of alpha-smooth muscle actin (α-SMA), collagen type 1, fibronectin, phospho-mitogen-activated protein kinase, and p-p50 (nuclear factor-kappaB [NF-κB]) were measured by Western blot analysis, real-time polymerase chain reaction, and/or immunofluorescence staining. Cell migration was analyzed with cell migration scratch assay and Transwell migration assay. Collagen contractile activity was measured using a collagen gel contraction assay. ATRA had no significant cytotoxic effect in NPDFs. Expression levels of α-SMA, collagen type 1, and fibronectin stimulated by TGF-β1 were significantly downregulated in the ATRA-pretreated fibroblasts. TGF-β1-induced cell migration and collagen gel contraction were significantly inhibited by ATRA pretreatment. ATRA also significantly inhibited phosphorylation of c-Jun N-terminal kinase (JNK), p38, and p50 in TGF-β1-induced NPDFs, but did not inhibit phosphorylation of extracellular signal-related kinase (ERK). ATRA downregulated myofibroblast differentiation, ECM production, cell migration, and collagen gel contraction via p38, JNK-dependent NF-κB-signaling pathways in TGF-β1-induced NPDFs. The findings suggest that ATRA could serve as a novel therapeutic agent to ameliorate nasal polyp development.

Abstract TP473: Investigating the Pathogenesis of Moyamoya Disease Using Patient Derived Induced Pluripotent Stem Cells

Background: Moyamoya disease (MMD) is a rare, progressive steno-occlusive cerebrovascular disorder of the internal carotid artery, leading to stroke. Affected arteries exhibit thickened intima with depleted elastic lamina and media, indicating a dysfunction of the vascular smooth muscle cells (VSMCs) and endothelial cells (ECs). However the pathogenesis of the disease is still unclear. We aim to address this gap in knowledge by using patient derived induced pluripotent stem cells (iPSCs), to generate VSMCs and ECs. Methods: Peripheral blood mononuclear cells (PBMCs) from controls and MMD patients (n=3 per group) were used for generating iPSCs. VSMC functionality was measured by collagen gel contraction assay and scratch assay. EC proliferative function was assessed by BrDU incorporation assay, and its migration capacity was evaluated by scratch assay and in vitro tube formation. VSMCs and ECs were also exposed to either hydrogen peroxide (H2O2) or normoxia/ hypoxia model (1%O 2 ) to investigate how cells respond to these insults. Hypoxia inducible factor 1α (HIF1α) activation was determined using western blot. Results: MMD VSMCs trended towards being more contractile and migrating faster than control VSMCs, in response to 10%FBS or SDF1α. On the other hand, MMD ECs migrated slower than control ECs in response to 10%FBS (p=0.0081) or VEGF (p=0.0072). MMD ECs also formed lesser tubes and exhibited fewer branch points when compared to controls. The rate of EC proliferation was similar between both groups. Cell death assays indicate that MMD VSMCs and ECs were more sensitive to the deleterious effects of H2O2 exposure when compared to control cells. Interestingly, MMD VSMCs had elevated HIF1α protein expression in normoxia, which was further increased after hypoxia. Conclusions: Our preliminary results indicate that both MMD VSMCs and ECs are dysfunctional and may be related to the elevated basal expression of HIF1α, possibly contributing to MMD pathology. We are currently investigating the interactions between VSMCs and ECs in MMD compared with controls using co-cultures. Ongoing studies also include transcriptome analysis of these differentiated cells, which will advance the understanding of the cellular and molecular mechanisms underlying MMD.

Uncoupled inflammatory, proliferative, and cytoskeletal responses in senescent human gingival fibroblasts.

Aging is characterized by a decline in tissue structure and function that may be explained by the development of cellular senescence. However, the acquisition of specific phenotypic responses in senescent gingival fibroblasts is still poorly understood. Here, we have analyzed whether proliferation of primary cultures of human gingival fibroblasts may affect different cell functions relevant to cellular senescence and tissue deterioration. Human gingival fibroblasts from five young donors were expanded until cellular senescence was achieved. Cellular senescence was evaluated by determining modifications in cell size, cell proliferation, p16 and p21 mRNA levels, H2Ax phosphorylation, cell viability, and senescence-associated beta-galactosidase staining. Inflammation was evaluated by analyzing the secretion of cytokines and nuclear translocation of NF-κB. Collagen remodeling was evaluated using a collagen gel contraction assay. Immunofluorescence and confocal microscopy were used to determine changes in the localization of the cytoskeletal proteins. Data analysis was performed to identify changes between cultures of the same donor at early and late passages using the paired sample t test or the Wilcoxon matched-pairs signed-rank test. Late passage cells showed changes compatible with cellular senescence that included increased cell size, reduced cell proliferation, staining for SA-beta gal, phosphorylated H2Ax, and increased p16 and p21 mRNA levels. Late passage cells showed a decrease in collagen contraction and reduced co-localization between the cytoskeletal proteins actin and vinculin. Importantly, late passage cells neither demonstrated changes in the secretion of inflammatory cytokines nor NF-κB activation. Our results imply that cytoskeletal changes and inhibition of cell proliferation represent early modifications in the structure and function of senescent gingival fibroblasts that are not coupled with the acquisition of an inflammatory phenotype. Further studies are needed to clarify the impact of different senescence stages during aging of the periodontium.

Endometrial stromal cell miR-29c-3p regulates uterine contraction.

Uterine peristalsis plays a vital role in fertility and female reproductive health. Although uterine peristalsis is thought to be correlated with some hormones and uterine pathologies, the physiological mechanisms underlying uterine peristalsis remain not quite clear. This study aimed to identify changes in miRNA in the endometrium of patients with abnormally high-frequency (hyper-) and low-frequency (hypo-) peristalsis to clarify whether miRNAs regulate uterine peristalsis. We used a miRNA microarray and RT-qPCR to identify changes in miRNA in endometrial tissue, a collagen gel contraction assay on co-cultured human endometrial stromal cells (ESCs) to analyze how the altered regulation of miRNAs influences uterine smooth muscle (USM) contraction, Western blots and other assays to elucidate the potential mechanisms involved. We found that among several differentially regulated miRNAs, miR-29c-3p was overexpressed in endometrial samples from patients with hypoperistalsis; oxytocin receptor (OXTR) expression was low in endometrial samples from patients with hypoperistalsis. Bioinformatic analysis and luciferase assays indicated that OXTR is a target of miR-29c-3p, which attenuates its expression. Additionally, downregulation of miR-29c-3p in ESC cultures increased the expression of aldo-keto reductase family 1, member C3 (AKR1C3) and increased the release of prostaglandin F2 alpha (PGF2α). Co-cultured ESCs overexpressing miR-29c-3p reduced USM cell contractions; the opposite tendency was found when ESCs were transfected with a miR-29c-3p inhibitor. To conclude, miR-29c-3p in endometrial cells regulates uterine contractility by attenuating the expression of OXTR and reducing PGF2α release.

Targeted Apoptosis of Myofibroblasts by Elesclomol Inhibits Hypertrophic Scar Formation

Background: Hypertrophic scar (HS) is characterized by the increased proliferation and decreased apoptosis of myofibroblasts. Myofibroblasts, the main effector cells for dermal fibrosis, develop from normal fibroblasts. Thus, the stimulation of myofibroblast apoptosis is a possible treatment for HS. We aimed to explore that whether over-activated myofibroblasts can be targeted for apoptosis by anticancer drug elesclomol. Methods: 4’,6-diamidino-2-phenylindole staining, flow cytometry, western blotting, collagen gel contraction and immunofluorescence assays were applied to demonstrate the proapoptotic effect of elesclomol in scar derived myofibroblasts and TGF-β1 induced myofibroblasts. The therapeutic potential of elesclomol was investigated by establishing rabbit ear hypertrophic scar models. Findings: Both 4′,6-diamidino-2-phenylindole staining and flow cytometry indicated that elesclomol targets myofibroblasts in vitro. Collagen gel contraction assay showed that elesclomol inhibited myofibroblast contractility. Flow cytometry and western blot analysis revealed that elesclomol resulted in excessive intracellular levels of reactive oxygen species(ROS), and caspase-3 and cytochrome c proteins. Moreover, compared with the control group, the elesclomol group had a significantly lower scar elevation index in vivo. Immunofluorescence assays for TUNEL and α-smooth muscle actin indicated that elesclomol treatment increased the number of apoptotic myofibroblasts. Interpretation: The above results indicate that elesclomol exerted a significant inhibitory effect on HS formation via targeted myofibroblast apoptosis associated with increased oxidative stress. Thus, elesclomol is a promising candidate drug for the treatment of myofibroblast-related diseases such as HS. Funding Statement: Funding for this study was provided by the Natural Science Foundation of China (81770876), Natural Science Foundation of Jiangsu Province (Grant BK20191141). Declaration of Interests: The authors declare no competing interests with relevance to this study. Ethics Approval Statement: The study protocols were approved by the ethics committee of the Affiliated Hospital of Jiangnan University. The study purpose and the procedures involved were explained to the patients, who all consented in writing to the use of their resected tissue specimens in this study. The animal experiments were approved by the Experimental Animal Committee of Jiangnan University.

Cigarette smoke extract inhibits cell migration and contraction via the reactive oxygen species/adenosine monophosphate-activated protein kinase pathway in nasal fibroblasts.

Fibroblast migration plays a significant role in wound healing after endoscopic sinonasal surgery. Cigarette smoke extract (CSE) is a potent inhibitor of fibroblast functions including cell proliferation and migration. The purpose of the study was to determine the influence of CSE on migration and collagen gel contraction in nasal fibroblasts and investigate its underlying mechanisms. Fibroblast migration was evaluated using wound healing assay and transwell migration assay. Contractile activity was assessed by collagen gel contraction assay. Reactive oxygen species (ROS) were quantified by 2',7'-dichlorofluorescein diacetate. Fibroblasts were treated with CSE and N-acetylcysteine (NAC), metformin, compound C, or transfected with small interfering RNA (siRNA) to suppress adenosine monophosphate-activated protein kinase (AMPK) expression. AMPK activation was determined by Western blot. CSE and metformin were found to significantly reduce the migration and collagen gel contraction activity of nasal fibroblasts. Conversely, pretreatment with NAC and compound C significantly enhanced the migration and collagen gel contraction activity of fibroblasts. ROS production and AMPK phosphorylation were found to be significantly induced by CSE treatment, whereas the activity was inhibited on treatment with NAC, metformin, compound C, or AMPK siRNA. Silencing of AMPK expression was found to significantly reverse the suppressive effect of CSE in nasal fibroblasts. CSE has an inhibitory effect on cell migration and collagen gel contraction activity via the ROS/AMPK signaling pathway in nasal fibroblasts.

Prostaglandin D2 stimulates phenotypic changes in vascular smooth muscle cells

Since chronic inflammation is associated with the pathogenesis of atherosclerosis, inflammatory cytokines might contribute to the phenotypic modulation of vascular smooth muscle cells (VSMCs). Tumor necrosis factor α (TNFα) facilitated the transformation of contractile VSMCs to the synthetic phenotype, as determined by the expression of marker proteins and a collagen gel contraction assay. Western blot analysis and a cyclooxygenase-2 (COX2) promoter assay revealed that TNFα stimulation resulted in the induction of COX2. The overexpression, silencing, or pharmacological inhibition of COX2 significantly affected TNFα-induced phenotypic conversion, and of the tested prostaglandins, only PGD2 significantly induced phenotypic conversion. ERK was significantly activated by PGD2 stimulation, and the pharmacological inhibition of ERK blocked the PGD2-induced phenotypic conversion of VSMCs. However, antagonists or agonists of PGD2 receptors did not affect VSMC conversion. In contrast, spontaneously dehydrated forms of PGD2, such as PGJ2, Δ12-PGJ2, and 15-d-PGJ2, strongly induced phenotypic conversion. A reporter gene assay showed that TNFα, PGD2, and 15-d-PGJ2 significantly activated the peroxisome proliferator-responsive element (PPRE) promoter. In addition, the overexpression or silencing of peroxisome proliferator-activated receptor δ (PPARδ) significantly influenced 15-d-PGJ2-induced phenotypic conversion. Finally, atherosclerotic neointima formation was significantly suppressed in mice lacking TNFα. In addition, mice fed celecoxib exhibited complete inhibition of carotid artery ligation-induced neointima formation. This study shows that PGD2 regulates the phenotypic conversion of VSMCs by generating an endogenous ligand of PPAR, and that this leads to neointima formation in occlusive arterial disease.

TGF-\u03b21-induced HSP47 regulates extracellular matrix accumulation via Smad2/3 signaling pathways in nasal fibroblasts

HSP47 is required for the production of collagen and serves an important role in tissue remodeling, a pathophysiologic mechanism of chronic rhinosinusitis (CRS). We investigated the relationship between HSP47 expression and tissue remodeling in CRS. We also determined the underlying molecular mechanisms of TGF-β1-induced HSP47 and extracellular matrix (ECM) production in nasal fibroblasts. HSP47, α-SMA, fibronectin, and collagen type I expression levels were measured using real-time PCR, western blotting, and immunofluorescence staining. Fibroblast migration was analyzed using scratch and transwell migration assays. Contractile activity was measured with a collagen gel contraction assay. HSP47 is increased in patients with CRS without nasal polyps. TGF-β1 induced HSP47 expression in nasal fibroblasts. Myofibroblast differentiation and ECM production, which are induced by TGF-β1, were inhibited by siHSP47. We also confirmed that the Smad2/3 signaling pathway is involved in TGF-β1-induced HSP47 expression in nasal fibroblasts. In a functional assay, TGF-β1-enhanced migration and contraction ability were inhibited by HSP47 knockout. Glucocorticoid reversed the stimulatory effects of TGF-β1 on HSP47 expression and ECM production in nasal fibroblasts and ex vivo organ cultures. HSP47 expression is involved in TGF-β1-induced myofibroblast differentiation and ECM production through the Smad2/3 signaling pathway, which might contribute to tissue remodeling in chronic rhinosinusitis.

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.

Direct Effects of Empagliflozin on Extracellular Matrix Remodelling in Human Cardiac Myofibroblasts: Novel Translational Clues to Explain EMPA-REG OUTCOME Results

BackgroundEmpagliflozin, an SGLT2 inhibitor, has shown remarkable reductions in cardiovascular mortality and heart failure admissions (EMPA-REG OUTCOME). However, the mechanism underlying the heart failure protective effects of empagliflozin remains largely unknown. Cardiac fibroblasts play an integral role in the progression of structural cardiac remodelling and heart failure, in part, by regulating extracellular matrix (ECM) homeostasis. The objective of this study was to determine if empagliflozin has a direct effect on human cardiac myofibroblast-mediated ECM remodelling. MethodsCardiac fibroblasts were isolated via explant culture from human atrial tissue obtained at open heart surgery. Collagen gel contraction assay was used to assess myofibroblast activity. Cell morphology and cell-mediated ECM remodelling was examined with the use of confocal microscopy. Gene expression of profibrotic markers was assessed with the use of reverse-transcription quantitative polymerase chain reaction. ResultsEmpagliflozin significantly attenuated transforming growth factor β1–induced fibroblast activation via collagen gel contraction after 72-hour exposure, with escalating concentrations (0.5 μmol/L, 1 μmol/L, and 5 μmol/L) resulting in greater attenuation. Morphologic assessment showed that myofibroblasts exposed to empagliflozin were smaller in size with shorter and fewer number of extensions, indicative of a more quiescent phenotype. Moreover, empagliflozin significantly attenuated cell-mediated ECM remodelling as measured by collagen fibre alignment index. Gene expression profiling revealed significant suppression of critical profibrotic markers by empagliflozin, including COL1A1, ACTA2, CTGF, FN1, and MMP-2. ConclusionsWe provide novel data showing a direct effect of empagliflozin on human cardiac myofibroblast phenotype and function by attenuation of myofibroblast activity and cell-mediated collagen remodelling. These data provide critical insights into the profound effects of empagliflozin as noted in the EMPA-REG OUTCOME study.

Systemic Sclerosis Serum Steers the Differentiation of Adipose-Derived Stem Cells Toward Profibrotic Myofibroblasts: Pathophysiologic Implications.

Systemic sclerosis (SSc; scleroderma) is characterized by life-threatening progressive multiorgan fibrosis orchestrated by profibrotic myofibroblasts originating from different sources. Because recent data demonstrated that the majority of myofibroblasts in a murine scleroderma model arise from adipocytic progenitors through the adipocyte-myofibroblast transition process, we sought to determine whether the SSc microenvironment may affect the differentiation potential of adipose-derived stem cells (ADSC). Normal human ADSC from three donors were treated with serum from SSc patients (n = 6), serum from healthy individuals (n = 6), or recombinant human transforming growth factor-β1 (TGFβ1) as positive control of myofibroblastic phenotype induction. ADSC were subjected to in vitro adipogenic differentiation for up to 21 days in the presence of different stimuli followed by lipid content quantification. In selected experiments, adipocytic and mesenchymal/myofibroblast marker gene and protein expression levels were assessed by Real-Time PCR, immunoblotting and immunofluorescence after administration of different stimuli for 72 and 96 h, respectively. Cell contractile phenotype was assayed by collagen gel contraction assay. Likewise stimulation with TGFβ1, SSc serum was able to significantly inhibit the adipocyte differentiation of ADSC as testified by a strong decrease in red-colored lipid droplets after 21 days of adipogenic induction. Treatment of ADSC either with SSc serum or TGFβ1 resulted in the acquisition of a myofibroblast-like phenotype characterized by a reduced expression of the adipocytic markers perilipin and adiponectin, a significant upregulation of the mesenchymal/myofibroblast markers α-SMA+ stress fibers, S100A4 and type I collagen, and an ability to effectively contract collagen gels. In SSc, the pathologic environment may favor the differentiation of ADSC into profibrotic and contractile myofibroblast-like cells. These findings strengthen the notion that the generation of myofibroblasts from ADSC may be relevant in SSc pathophysiology potentially representing a new target for the prevention/treatment of multiorgan fibrosis.

Investigating Fibroblast-Induced Collagen Gel Contraction Using a Dynamic Microscale Platform.

Mechanical forces have long been recognized as fundamental drivers in biological processes, such as embryogenesis, tissue formation and disease regulation. The collagen gel contraction (CGC) assay has served as a classic tool in the field of mechanobiology to study cell-induced contraction of extracellular matrix (ECM), which plays an important role in inflammation and wound healing. In a conventional CGC assay, cell-laden collagen is loaded into a cell culture vessel (typically a well plate) and forms a disk-shaped gel adhering to the bottom of the vessel. The decrement in diameter or surface area of the gel is used as a parameter to quantify the degree of cell contractility. In this study, we developed a microscale CGC assay with an engineered well plate insert that uses surface tension forces to load and manipulate small volumes (14 μL) of cell-laden collagen. The system is easily operated with two pipetting steps and the microscale device moves dynamically as a result of cellular forces. We used a straightforward one-dimensional measurement as the gel contraction readout. We adapted a conventional lung fibroblast CGC assay to demonstrate the functionality of the device, observing significantly more gel contraction when human lung fibroblasts were cultured in serum-containing media vs. serum-free media (p ≤ 0.05). We further cocultured eosinophils and fibroblasts in the system, two important cellular components that lead to fibrosis in asthma, and observed that soluble factors from eosinophils significantly increase fibroblast-mediated gel contraction (p ≤ 0.01). Our microscale CGC device provides a new method for studying downstream ECM effects of intercellular cross talk using 7- to 35-fold less cell-laden gel than traditional CGC assays.

Identification and Isolation of Cardiac Fibroblasts From the Adult Mouse Heart Using Two-Color Flow Cytometry.

Background: Cardiac fibroblasts represent a main stromal cell type in the healthy myocardium. Activation of cardiac fibroblasts has been implicated in the pathogenesis of many heart diseases. Profibrotic stimuli activate fibroblasts, which proliferate and differentiate into pathogenic myofibroblasts causing a fibrotic phenotype in the heart. Cardiac fibroblasts are characterized by production of type I collagen, but non-transgenic methods allowing their identification and isolation require further improvements. Herein, we present a new and simple flow cytometry-based method to identify and isolate cardiac fibroblasts from the murine heart.Methods and Results: Wild-type and reporter mice expressing enhanced green fluorescent protein (EGFP) under the murine alpha1(I) collagen promoter (Col1a1-EGFP) were used in this study. Hearts were harvested and dissociated into single cell suspensions using enzymatic digestion. Cardiac cells were stained with the erythrocyte marker Ter119, the pan-leukocyte marker CD45, the endothelial cell marker CD31 and gp38 (known also as podoplanin). Fibroblasts were defined in a two-color flow cytometry analysis as a lineage-negative (Lin: Ter119−CD45−CD31−) and gp38-positive (gp38+) population. Analysis of hearts isolated from Col1a1-EGFP reporter mice showed that cardiac Lin−gp38+ cells corresponded to type I collagen-producing cells. Lin−gp38+ cells were partially positive for the mesenchymal markers CD44, CD140a, Sca-1 and CD90.2. Sorted Lin−gp38+ cells were successfully expanded in vitro for up to four passages. Lin−gp38+ cells activated by Transforming Growth Factor Beta 1 (TGF-β1) upregulated myofibroblast-specific genes and proteins, developed stress fibers positive for alpha smooth muscle actin (αSMA) and showed increased contractility in the collagen gel contraction assay.Conclusions: Two-color flow cytometry analysis using the selected cell surface antigens allows for the identification of collagen-producing fibroblasts in unaffected mouse hearts without using specific reporter constructs. This strategy opens new perspectives to study the physiology and pathophysiology of cardiac fibroblasts in mouse models.

Inhibition of LPA1 Signaling Impedes Conversion of Human Tenon's Fibroblasts into Myofibroblasts Via Suppressing TGF-β/Smad2/3 Signaling.

Purpose: Lysophosphatidic acid (LPA) is a growth factor-like phospholipid that has been recognized as a profibrotic mediator in numerous tissues, yet, whether it plays a role in subconjunctival fibrosis remains to be investigated. Therefore, this study was designed to examine the effect of LPA1-3 signaling inhibitor, Ki16425 on the conversion of human Tenon's fibroblasts (HTFs) into myofibroblasts. Methods: Primary cultured HTFs were incubated with transforming growth factor-β1 (TGF-β1) alone or combined with Ki16425, the cell proliferation and migration were measured by Cell Counting Kit-8 and the scratch wound assay, respectively. HTFs contractility was evaluated with 3-dimensional (3D) Collagen Contraction assay. The mRNA and protein levels of α-smooth muscle actin (α-SMA), Snail and the phosphorylation levels of Smad2/3, p38MAPK, and ERK1/2 were determined by real-time quantitative polymerase chain reaction (RT-qPCR), western blot, and immunofluorescence staining. Results: Ki16425 significantly prevent the proliferation and migration of Tenon's fibroblasts (HTFs) in a dose-dependent manner. Furthermore, Ki16425 blocked HTFs myofibroblast differentiation via downregulation of mRNA and protein expression of α-SMA. 3D collagen gel contraction assay demonstrated that Ki16425 effectively inhibits myofibroblast contraction induced by TGF-β1. Mechanistically, we revealed that Ki16425 reduces Smad2/3 but not p38MAPK or ERK1/2 phosphorylation by TGF-β1. By using an LPA1-specific inhibitor, AM095, we confirmed that LPA1 signaling but not LPA2 or LPA3 is involved in TGF-β1 induced HTFs activation. Conclusions: Our results show that inhibition of LPA1 signaling presents potent antifibrotic effect in HTFs, which may serve as a promising intervention strategy for preventing subconjunctival fibrosis caused by glaucoma filtration surgery.

Effects of isocorynoxeine, from Uncaria, on lower urinary tract dysfunction caused by benign prostatic hyperplasia via antagonism of α1A-adrenoceptors

Medical therapy of lower urinary tract symptoms (LUTS) suggestive of benign prostatic hyperplasia (BPH) targets smooth muscle contraction in the prostate, for which α1A-adrenoceptor (α1A-AR) antagonists have been considered to be the primary therapeutic method. We investigated the effects and underlying mechanisms of isocorynoxeine (ICN), one of indole alkaloids from Uncaria, on the treatment of LUTS secondary to BPH via α1A-ARs in mice. The effect of ICN on prostatic contractility was studied via myographic measurements in the prostates of rabbits. The effects of ICN on bladder function, serum-hormone levels, bladder histology, and prostate histology were determined in testosterone propionate-induced prostatic hyperplasic wild-type (WT) and α1A-AR knockout (α1A-KO) mice. The cytotoxicity of ICN in cultured human prostatic stromal cells (WPMY-1) was assessed by the following: a cell-counting kit, measuring the relaxant effect on WPMY-1 by a collagen gel contraction assay, intracellular Ca2+ mobilization indicated by Fluo-4, cytoskeletal organization by phalloidin staining, and expressions of α1A-AR-mediated key messengers by western blot analyses. ICN non-competitively antagonized the contractions of prostates induced by α1A-AR agonists. ICN treatment improved bladder functions in prostatic hyperplasic WT mice, whereas it failed to ameliorate bladder functions in prostatic hyperplasic α1A-KO mice. In WPMY-1, ICN relaxed cell contractions on collagen gels, disrupted F-actin organization, inhibited α1A-AR agonist-stimulated Ca2+ mobilization, and antagonized α1A-ARs via the RhoA/ROCK2/MLC signaling pathway. Our results suggest that ICN may be a promising therapeutic drug for targeting α1A-ARs in the treatment of BPH/LUTS.

Cholangiocyte-Derived Exosomal Long Noncoding RNA H19 Promotes Hepatic Stellate Cell Activation and Cholestatic Liver Fibrosis.

Activation of hepatic stellate cells (HSCs) represents the primary driving force to promote the progression of chronic cholestatic liver diseases. We previously reported that cholangiocyte-derived exosomal long noncoding RNA-H19 (lncRNA-H19) plays a critical role in promoting cholestatic liver injury. However, it remains unclear whether cholangiocyte-derived lncRNA-H19 regulates HSC activation, which is the major focus of this study. Both bile duct ligation (BDL) and Mdr2 knockout (Mdr2-/- ) mouse models were used. Wild-type and H19maternalΔExon1/+ (H19KO) mice were subjected to BDL. Mdr2-/- H19maternalΔExon1/+ (DKO) mice were generated. Exosomes isolated from cultured mouse and human cholangiocytes or mouse serum were used for in vivo transplantation and in vitro studies. Fluorescence-labeled exosomes and flow cytometry were used to monitor exosome uptake by hepatic cells. Collagen gel contraction and bromodeoxyuridine assays were used to determine the effect of exosomal-H19 on HSC activation and proliferation. Mouse and human primary sclerosing cholangitis (PSC)/primary biliary cholangitis (PBC) liver samples were analyzed by real-time PCR, western blot analysis, histology, and immunohistochemistry. The results demonstrated that hepatic H19 level was closely correlated with the severity of liver fibrosis in both mouse models and human patients with PSC and PBC.H19 deficiency significantly protected mice from liver fibrosis in BDL and Mdr2-/- mice.Transplanted cholangiocyte-derived H19-enriched exosomes were rapidly and preferentially taken up by HSCs and HSC-derived fibroblasts, and promoted liver fibrosis in BDL-H19KO mice and DKO mice. H19-enriched exosomes enhanced transdifferentiation of cultured mouse primary HSCs and promoted proliferation and matrix formation in HSC-derived fibroblasts. Conclusion: Cholangiocyte-derived exosomal H19 plays a critical role in the progression of cholestatic liver fibrosis by promoting HSC differentiation and activation and represents a potential diagnostic biomarker and therapeutic target for cholangiopathies.

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.

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.