Monolayer Wound Healing Research Articles

Proteogenomic Identification and Analysis of KIF5B as a Prognostic Signature for Hepatocellular Carcinoma.

Metabolic disorders are significant risk factors for liver cancer, particularly Hepatocellular Carcinoma (HCC). However, the molecular genetic basis of metabolic reprogramming in the liver remains largely uncertain. This study aimed to investigate some novel prognostic biomarkers in HCC by using proteogenomic and transcriptomic analysis and explore the potential role of specific prognostic genes in HCC. Here, we have presented a proteogenomic analysis of 10 pairs of HCC. Protein co-expression and pathway analysis were performed to investigate the biological characteristics of HCC. Protein and mRNA expression profiles of multi-cohorts were integrated to detect novel prognostic protein markers of HCC. The carcinogenic roles of candidate prognostic markers were further evaluated by MTS assay, colony formation, monolayer wound healing assay, and xenograft models. A total of 2086 proteins with significantly different expressions were detected in HCC. Pathways related to oncogenic signaling and insulin-related metabolism have been found to be dysregulated and differentially regulated in HCC. We have identified the novel prognostic biomarkers, KIF5B, involved in liver metabolic reprogramming. The biomarkers were identified using multivariable COX regression analysis from two independent proteomic datasets (Fudan Cohort and our recruited cohort) and the TCGA mRNA database. Both the protein and mRNA up-regulation of KIF5B have been found to be associated with a poor clinical outcome in HCC. Insulin activated the protein expression of KIF5B in HCC. Knocking out KIF5B expression by sgRNA decreased the protein expression of FASN and SCD1 and the intracellular triglyceride concentration. Silencing KIF5B suppressed HCC cell proliferation and colony formation in vitro, as well as HCC growth in xenograft models. Our findings have suggested KIF5B protein to function as a novel prognostic biomarker in HCC. KIF5B expression has been found to activate the AKT/mTOR pathway and reprogram triglyceride metabolism, leading to HCC development. Targeting KIF5B may be an effective strategy in the clinical treatment of HCC.

Elevated expression of miR-301a and its functional roles in canine oral melanoma.

miR-301a is one of numerous dysregulated microRNAs (miRNAs) in canine oral melanoma (COM), one of which is miR-301a (upregulated). Its biological role has been described in various human cancer types, including malignant melanoma, but not in COM. Accordingly, in this study, we investigated miR-301a expression in COM in greater detail to ascertain whether it could serve as a diagnostic biomarker, elucidate its functional roles in this cancer, and predict the possible pathways by which it exerts its effects. Relative expression of miR-301a was investigated in clinical oral tissue and plasma samples and COM cell (KMeC and LMeC) lines using qRT-PCR. Knockdown of miR-301a was also validated for KMeC and LMeC cells using qRT-PCR. We performed CCK-8 assays to assess cell proliferation, monolayer wound-healing, and transwell migration assays to assess cell migration, a colony-formation assay to assess clonogenicity, a TUNEL assay and flow cytometry to assess apoptosis-related effects, and gene enrichment analyses to predict possible related pathways. miR-301a was markedly upregulated in COM oral tissue and plasma clinically, suggesting its potential as a diagnostic biomarker for COM diagnosis. In vitro assays demonstrated that miR-301 significantly inhibited apoptosis in COM cells while promoting cell migration, proliferation, and clonogenicity. We also predicted that miR-301 exerts cancer-promoting effects through the Wnt signalling pathway for COM. Our findings suggest that miR-301a is a COM oncomiR that regulates several oncogenic phenotypes with the potential to be a diagnostic biomarker.

Comparative analysis of the regenerative potential of blood derivatives on a cell model of corneal stromal injury

BACKGROUND: Agents based on blood derivatives contain substances that accelerate the healing of wound defects. Nowadays data have been accumulated that various blood derivatives have different effects on regeneration processes. Therefore, a comparative study of the effect of several types of such preparations on the corneal stromas cell culture is relevant.
 AIM: Impact assessment of 3 different blood derivatives on the processes of proliferation, migration and cell death of keratocytes in vitro.
 MATERIAL AND METHODS: This study was conducted on a primary cell culture of human corneal keratocytes. Blood samples were obtained from the cubital vein of healthy volunteers after signing informed consent to participate in the study. Monolayer wound healing test, formazan test to assess proliferation, staining for Annexin V to assess the processes of apoptosis and necrosis were perfomed. Moreover, effect evaluation of platelet-rich plasma, intact serum and processed serum were performed.
 RESULTS: It was shown that all 3 stimulants have a positive effect on the regeneration of the experimental stromal defect, but it is implemented in different ways. Processed whey showed the greatest stimulating effect on the process of cell migration. This serum reduced the level of cell death, but had almost no effect on proliferation processes. It is advisable to further study different types of stimulants to obtain processed serum. Intact serum increased the percentage of dividing cells more than others, but also proportionally increased the percentage of death in culture. Platelet-rich plasma showed the weakest effect on the migratory ability of keratocytes, but it still differed significantly from the control. This stimulator had little effect on the process of cell division, and had no effect on the overall level of cell death.
 CONCLUSION: Experimental data can be taken into account in choosing of the management in clinical practice and personalized usage of blood derivatives, specifically in prescribing some blood derivatives to accelerate regeneration, and other derivatives for reducing cell deaths processes.

Anti-inflammatory and antioxidative effects of gallic acid on experimental dry eye: in vitro and in vivo studies

BackgroundTo investigate the anti-inflammatory and antioxidative effects of gallic acid (GA) on human corneal epithelial cells (HCECs) and RAW264.7 macrophages as well as its therapeutic effects in an experimental dry eye (EDE) mouse model.MethodsA cell counting kit-8 (CCK-8) assay was used to test the cytotoxicity of GA. The effect of GA on cell migration was evaluated using a scratch wound healing assay. The anti-inflammatory and antioxidative effects of GA in vitro were tested using a hypertonic model (HCECs) and an inflammatory model (RAW264.7 cells). The in vivo biocompatibility of GA was detected by irritation tests in rabbits, whereas the preventive and therapeutic effect of GA in vivo was evaluated using a mouse model of EDE.ResultsIn the range of 0–100 μM, GA showed no cytotoxicity in RAW264.7 cells or HCECs and did not delay the HCECs monolayer wound healing within 24 h. Ocular tolerance to GA in the in vivo irritation test was good after seven days. In terms of antioxidative activity, GA significantly reduced the intracellular reactive oxygen species (ROS) in lipopolysaccharide (LPS) activated RAW264.7 macrophages and HCECs exposed to hyperosmotic stress. Furthermore, after pre-treatment with GA, the expression levels of nuclear factor E2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), and NADPH quinone oxidoreductase-1 (NQO-1) were significantly upregulated in RAW264.7 macrophages. GA also exhibits excellent anti-inflammatory properties. This is mainly demonstrated by the ability of GA to effectively downregulate the nuclear transcription factor-κB (NF-κB) pathway in LPS-activated RAW264.7 macrophages and to reduce inflammatory factors, such as nitric oxide (NO), interleukin 6 (IL-6), and tumor necrosis factor alpha (TNF-α). In vivo efficacy testing results in a mouse model of EDE showed that GA can effectively prevent and inhibit the apoptosis of corneal epithelial cells (CECs), reduce inflammatory factors in the cornea and conjunctiva as well as protect goblet cells.ConclusionIn vitro and in vivo results indicate that GA possesses potent anti-inflammatory and antioxidative properties with no apparent cytotoxicity within the range of 0–100 μM. It is a promising eye drop formulation for the effective prevention and treatment of dry eye disease (DED).

Effect of Hyaluronic Acid 35 kDa on an In Vitro Model of Preterm Small Intestinal Injury and Healing using Enteroid-derived Monolayers.

In vitro scratch wound assays are commonly used to investigate the mechanisms and characteristics of epithelial healing in a variety of tissue types. Here, we describe a protocol to generate a two-dimensional (2D) monolayer from three-dimensional (3D) non-human primate enteroids derived from intestinal crypts of the terminal ileum. These enteroid-derived monolayers were then utilized in an in vitro scratch wound assay to test the ability of hyaluronan 35 kDa (HA35), a human milk HA mimic, to promote cell migration and proliferation along the epithelial wound edge. After the monolayers were grown to confluency, they were manually scratched and treated with HA35 (50 µg/mL, 100 µg/mL, 200 µg/mL) or control (PBS). Cell migration and proliferation into the gap were imaged using a transmitted-light microscope equipped for live-cell imaging. Wound closure was quantified as percent wound healing using the Wound Healing Size Plugin in ImageJ.The scratch area and rate of cell migration and the percentage of wound closure were measured over 24 h. HA35 in vitro accelerates wound healing in small intestinal enteroid monolayers, likely through a combination of cell proliferation at the wound edge and migration to the wound area. These methods can potentially be used as a model to explore intestinal regeneration in the preterm human small intestine.

Role of β 3 ‐Adrenergic Receptors in Rat Aortic Endothelial Cell Function

Long-term success of percutaneous trans-luminal coronary angioplasty (PTCA) is limited by the frequent occurrence of restenosis, resulted from vascular smooth muscle cell (VSMC) proliferation and migration due to the procedure-induced endothelial damage. Speeding the healing of the endothelial layer has been considered as one of the major approaches to reduce the risk of restenosis. Basic fibroblast growth factor (bFGF), a major growth factor released after injury, regulates endothelial cell proliferation and migration and promotes endothelial layer healing. Our previous results revealed that β3-adrenergic receptor (β3-ARs) expression is upregulated in newly formed intima in injured blood vessels and activation of β3-ARs enhanced bFGF-induced VSMC proliferation and migration. However, the role of β3-ARs in endothelial function, specifically in injured arteries, is uncertain. The purpose of this study is to determine if activation of endothelial cell β3-ARs potentiates bFGF-induced endothelial cell proliferation and/or migration by using rat aortic endothelial cells. Cell proliferation was determined by measuring viable cell numbers using Resazurin. Cell migration will be quantified by a monolayer wound healing assay in the presence of a cell proliferation inhibitor, hydroxyurea. A dose-dependent inhibition of cell proliferation was performed to optimize the concentration of hydroxyurea in cell migration experiments. Our results revealed that pretreatment of endothelial cells with a selective β3-AR agonist, CL316,243, enhanced bFGF-induced vascular endothelial cell proliferation. We also found that 5μM of hydroxyurea completely inhibited cell proliferation induced by growth factors. In conclusion, our data suggest that activation of β3-ARs may speed the healing of endothelial layer by promoting cell proliferation and that including 5μM of hydroxyurea in cell migration experiments will allow us to measure the cell motility accurately and ensure that proliferation does not take place.

Curcuma amarissima Extract Activates Growth and Survival Signal Transduction Networks to Stimulate Proliferation of Human Keratinocyte.

Simple SummaryLike many plants in the family of Zingiberaceae, Curcuma amarissima has been traditionally used to induce healing and tissue regeneration. However, there is no scientific evidence to explain how Curcuma amarissima works to accelerate wound healing. Our data clearly proved that Curcuma amarissima extract could potentially accelerate the closure of scratch wounds of human keratinocytes by stimulating cell proliferation. The potential mechanisms underlying these effects were defined to be associated with the activated signal transduction pathways relevant to cell proliferation and survival. This strongly suggests the ability of Curcuma amarissima to enhance the process of keratinocyte reepithelization during wound healing. Our current study provides convincing evidence that supports the possibility to develop an effective wound-healing promoting agent from this plant.Many medicinal plants have been used to treat wounds. Here, we revealed the potential wound healing effects of Curcuma amarissima (CA). Our cell viability assay showed that CA extract increased the viability of HaCaT cells that were cultured in the absence of serum. This increase in cell viability was proved to be associated with the pharmacological activities of CA extract in inducing cell proliferation. To further define possible molecular mechanisms of action, we performed Western blot analysis and immunofluorescence study, and our data demonstrated that CA extract rapidly induced ERK1/2 and Akt activation. Consistently, CA extract accelerated cell migration, resulting in rapid healing of wounded human keratinocyte monolayer. Specifically, the CA-induced increase of cell monolayer wound healing was blocked by the MEK inhibitor (U0126) or the PI3K inhibitor (LY294002). Moreover, CA extract induced the expression of Mcl-1, which is an anti-apoptotic protein, supporting that CA extract enhances human keratinocyte survival. Taken together, our study provided convincing evidence that Curcuma amarissima can promote proliferation and survival of human keratinocyte through stimulating the MAPK and PI3K/Akt signaling cascades. These promising data emphasize the possibility to develop this plant as a wound healing agent for the potential application in regenerative medicine.

Angiotensin II promotes EMT of hepatocellular carcinoma cells through high mobility group protein B1 mediated by E4F1

To screen for specific transcription factors (TFs) that induce expression of the HMGB1 promoter in response to stimulation by Ang-II. A HMGB1 overexpressing vector and small interfering (si)RNA were constructed and used to transfect the three HCC cell lines used in scratched monolayer wound healing and Transwell assays. Chromatin immunoprecipitation (ChIP) assays were used to confirm the relationship between a specific TF and the HMGB1 promoter. Invasion and migration by HMGB1 overexpressing HCC cells after treatment with Ang-II were significantly increased compared to negative controls (NC); E-cadherin was down-regulated while vimentin was up-regulated. However, compared with NC, invasion and migration by HMGB1 siRNA HCC cells stimulated by Ang-II were not altered; the expression of E-cadherin and vimentin was also unaltered. Nineteen TFs were predicted by Promoter 2.0 Prediction Server and TFsitescan. Real-time qPCR was used to evaluate TF expression levels. E4F1 was the only TF abnormally elevated in all three HCC cell lines when stimulated by Ang-II. WB and ChIP assays revealed high expression of E4F1 compared to other TFs in cells stimulated by Ang-II. E4F1 is activated by Ang-II and binds to the HMGB1 promoter region to promote HMGB1 expression; it then enhances Ang-II to induce HCC cell invasion and migration, and EMT.

Organotypic & in vitro monolayer modeling of urothelial carcinoma gives different cellular responses to proteinase activated receptor (PAR) agonism/antagonism

We hypothesized that microenvironment proteases generated by urothelial carcinoma (UC) cells dictate cell invasion & metastasis by signaling via Proteinase-Activated Receptors (PARs) and evaluated this hypothesis in UC cell lines in in vitro monolayer & organotypic 3D cultures UC cell lines: RT4, SW780, T24, 5637 In vitro: PAR1/2 function was assessed via calcium signalling assays. 2 methods detected UC-secreted proteases that cleave PAR1/2: 1) N-luciferase tag fused to PAR1/2 in a non-UC cell line released upon cleavage (paracrine proteinase activity); 2) N-terminal mCherry/RFP;C-terminal eYFP-tagged PAR construct transfected into UC cells to visualize receptor autocrine cleavage (intact receptor=yellow; cleaved receptor=green). The PAR1/2 effect on UC cell migration & invasion was assessed in monolayer wound healing assays with & without PAR1/2 agonism. Organotypic Model: UC cells were cultured in a bladder-derived fibroblast-containing 3D collagen biomatrix model that maintains epithelial phenotype & supports the growth of UC cell lines. UC cells were evaluated for proliferation, apoptosis, differentiation, & invasion in this model with/without PAR activation/inhibition. UC cell lines express PAR1/2 & secrete proteases & protease inhibitors that induce &/or affect signaling. PAR1/2 activating peptides stimulated migration & invasion of UC cells in monolayer cultures with inhibition by a PAR1 antagonist. In the organotypic setting neither PAR1 or 2 agonism caused significant proliferation, apoptosis, or invasion. Uniquely in the organotypic model we observed induction of differentiation in non-invasive cells (SW780 & RT4) & increased cytokeratin CK5-6 expression switching in the cell lines induced by PAR1 agonism & antagonism. Functional PAR1/2 are expressed by UC cells. Activation of these receptors increases invasion & migration in monolayer cultures, but not in organotypic cultures. Our data illustrate significant differences between monolayer & organotypic cultures & suggest PAR1 impacts cytokeratin expression & UC cell differentiation.

Biogenesis of silver nanoparticles using leaf extracts of Asparagus racemosus and Sophora interrupta: structure characterization, antibacterial and anticancer studies

The present investigation reports simple and eco-friendly biogenesis of silver nanoparticles (AgNPs) using leaf extracts of Asparagus racemosus and Sophora interrupta. UV–Vis spectra showed absorption peaks at 413 and 420 nm respectively corresponding to A. racemosus (AR-AgNPs) and S. interrupta (SI-AgNPs). TEM, DLS, XRD and TGA studies revealed that biogenic AgNPs are monodispersed, spherical shaped, thermally stable, FCC crystals and 4–15 nm in size. Zeta potential values were found to be − 28.9 and − 18.6 mV respectively corresponding to SI-AgNPs and AR-AgNPs. SI-AgNPs exhibited strong antibacterial activities against Escherichia coli, Klebsiella pneumoniae, Bacillus subtilis and Micrococcus luteus with inhibition zones of 11.8, 10.5, 9.8 and 9.1 mm respectively. Further SI-AgNPs exhibited significant cytotoxic properties against SKOV3, DU149, PC3 and A549 cells with IC50 values of 120.87, 191.34, 235.06 and 314.27 µg/mL respectively. SI-AgNPs induce pronounced apoptotic effects on SKOV3 cells including loss of membrane integrity, chromatin condensation (pyknosis) and nuclear fragmentation (karyorrhexis). SI-AgNPs exhibited significant cell migration inhibition by reducing the wound healing of SKOV3 monolayers. Phytosynthesized AgNPs were found to be biocompatible towards normal (CHO) cells.

Sprouty2 Inhibits Migration and Invasion of Fibroblast-Like Synoviocytes in Rheumatoid Arthritis by Down-regulating ATF2 Expression and Phosphorylation.

Activating transcription factor 2(ATF2), a transcription factor belonging to the AP-1 family, plays an important role in inflammation. However, its biological functions and underlying molecular mechanisms in rheumatoid arthritis (RA) remain unclear. Western blot and immunohistochemistry were used to identify the expression of ATF2 and Sprouty2(SPRY2) in RA synovial tissues. SW982 cells were stimulated by TNF-α to establish an in vitro RA fibroblast-like synoviocyte (RA-FLS) model. Transwell and monolayer wound-healing were used to detect cell migration and invasion. RNA interference (si-ATF2) and adenovirus vector (Ad-SPRY2) methods were employed to manipulate ATF2 or SPRY2 expression in SW982 cells. The protein expression and phosphorylation levels in SW982 cells were evaluated by western blot. ATF2 expression and phosphorylation were upregulated in the RA synovial tissues. In RA-FLS model, ATF2 expression and phosphorylation were increased in a time-dependent manner. ATF2 knockdown inhibited the migration and invasion of RA-FLS model, reducing the inflammatory factors, which was consistent with the influence on cell behaviors caused by SPRY2 overexpression. Moreover, SPRY2 overexpression inhibited the TNF-α-induced phosphorylation of ERK and ATF2 in SW982 cells. The high expression and phosphorylation of ATF2 promoted migration and invasion of RA-FLSs. SPRY2 might inhibited the inflammatory responses of RA-FLSs via suppressing ERK-ATF2 pathway.

Celastrol inhibits ezrin-mediated migration of hepatocellular carcinoma cells

Progression of hepatocellular carcinoma involves multiple genetic and epigenetic alterations that promote cancer invasion and metastasis. Our recent study revealed that hyperphosphorylation of ezrin promotes intrahepatic metastasis in vivo and cell migration in vitro. Celastrol is a natural product from traditional Chinese medicine which has been used in treating liver cancer. However, the mechanism of action underlying celastrol treatment was less clear. Here we show that ROCK2 is a novel target of celastrol and inhibition of ROCK2 suppresses elicited ezrin activation and liver cancer cell migration. Using cell monolayer wound healing, we carried out a phenotype-based screen of natural products and discovered the efficacy of celastrol in inhibiting cell migration. The molecular target of celastrol was identified as ROCK2 using celastrol affinity pull-down assay. Our molecular docking analyses indicated celastrol binds to the active site of ROCK2 kinase. Mechanistically, celastrol inhibits the ROCK2-mediated phosphorylation of ezrin at Thr567 which harnesses liver cancer cell migration. Our findings suggest that targeting ROCK2-ezrin signaling is a potential therapeutic niche for celastrol-based intervention of cancer progression in hepatocellular carcinoma.

Abstract C131: The SALL2 transcription factor promotes cell migration and focal adhesion turnover via regulation of integrin expression

Abstract SALL2 is a transcription factor, member of the Spalt gene family, which has been associated with development, differentiation and cancer. Interestingly, SALL2 deficiency leads to failure of both closure of the optic fissure and neurite outgrowth, suggesting a positive role for SALL2 in cell migration. Conversely, recent studies in ovarian cancer cells suggest that SALL2 plays a negative role in cell migration. Cell migration is a multistep process that involves cellular polarization, formation of membranous protrusions, assembly and disassembly of focal adhesions and cell traction. Hence, understanding the role of SALL2 in cell migration will be relevant to comprehend its function in physiology and disease. Here, we evaluated the role of SALL2 in cell migration, by using immortalized Sall2 knockout (KO) and Sall2 wild type Mouse Embryonic Fibroblasts (MEFs), and gain/loss of function experiments. To this end, we measured the effects of SALL2 in different aspects of cell migration, including wound healing in monolayers, cell adhesion, cell detachment, focal adhesion dynamics, as well as by immunofluorescence assays. For mechanistic insights we used Western blot, real time PCR, luciferase reporter assays, and bioinformatics analysis. Data were compared in unpaired Student’s t-tests by using the GraphPad Prism software. Our data indicate that SALL2 positively regulates cell migration by promoting cell polarization, cell detachment, and increased turnover of focal adhesions. Sall2-deficiency as well as Sall2 knockdown by short hairpin RNA led to reduced cell migration and focal adhesion dynamics. Accordingly, restored SALL2 expression through a TET-ON system in the Sall2 KO MEFs recovered cell migratory capabilities. Interestingly, SALL2 accelerated focal adhesion disassembly after nocodazole- wash out treatment. In addition, in a cell spreading context and after nocodazole treatment, SALL2 augmented Focal Adhesion Kinase (FAK) autophosphorylation on Y397 and promoted the expression of integrins b1 and a6. Taken together, our data suggest that SALL2 promotes MEF cell migration by modulating focal adhesion dynamics via a mechanism involving augmented expression of integrins b1 and a6. Since deregulation of cell migration promotes tumor formation, invasion and metastasis, our findings indicate that deregulation of SALL2 may be associated with the disease. Funded by FONDECYT 1191172, FONDECYT 1180495; FONDAP 15130011 Citation Format: Elizabeth Riffo, Mario Palma, Catalina Alarcon, Ariel Castro, Vicente Torres, Roxana Pincheira. The SALL2 transcription factor promotes cell migration and focal adhesion turnover via regulation of integrin expression [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C131. doi:10.1158/1535-7163.TARG-19-C131

Small molecule FAK activator promotes human intestinal epithelial monolayer wound closure and mouse ulcer healing

GI mucosal healing requires epithelial sheet migration. The non-receptor tyrosine kinase focal adhesion kinase (FAK) stimulates epithelial motility. A virtual screen identified the small drug-like FAK mimic ZINC40099027, which activates FAK. We assessed whether ZINC40099027 promotes FAK-Tyr-397 phosphorylation and wound healing in Caco-2 monolayers and two mouse intestinal injury models. Murine small bowel ulcers were generated by topical serosal acetic acid or subcutaneous indomethacin in C57BL/6J mice. One day later, we began treatment with ZINC40099027 or DMSO, staining the mucosa for phosphorylated FAK and Ki-67 and measuring mucosal ulcer area, serum creatinine, ALT, and body weight at day 4. ZINC40099027 (10–1000 nM) dose-dependently activated FAK phosphorylation, without activating Pyk2-Tyr-402 or Src-Tyr-419. ZINC40099027 did not stimulate proliferation, and stimulated wound closure independently of proliferation. The FAK inhibitor PF-573228 prevented ZINC40099027-stimulated wound closure. In both mouse ulcer models, ZINC40099027accelerated mucosal wound healing. FAK phosphorylation was increased in jejunal epithelium at the ulcer edge, and Ki-67 staining was unchanged in jejunal mucosa. ZINC40099027 serum concentration at sacrifice resembled the effective concentration in vitro. Weight, creatinine and ALT did not differ between groups. Small molecule FAK activators can specifically promote epithelial restitution and mucosal healing and may be useful to treat gut mucosal injury.

C‑Jun/Ap‑1 is upregulated in an Ang II‑induced abdominal aortic aneurysm formation model and mediates Chop expression in mouse aortic smooth muscle cells.

Abdominal aortic aneurysm (AAA) is an asymptomatic, potentially lethal disease whose ruptures have a high mortality rate. An effective pharmacological approach to decrease expansion or prevent the rupture of AAAs in humans remains lacking. Previous studies have suggested that activator protein 1 (c-Jun/AP-1) and C/EBP homologous protein (Chop) are involved in the development of AAA. The purpose of the present study was to investigate whether c-Jun/AP-1 mediates Chop overexpression in AAA. c-Jun/AP-1 and Chop protein levels were determined in an angiotensin II (Ang II)-induced AAA model using apolipoprotein E-deficient mice. Additionally, mouse aortic smooth muscle cells (MOVAS cell line) were treated with Ang II. Apoptosis was evaluated via TUNEL assay, MOVAS cell migration ability was assessed by monolayer wound healing assay and the levels of c-Jun/AP-1 and Chop were determined by western blotting, immunofluorescence and immunocytochemical assays. Following c-Jun silencing using c-Jun-specific small interfering (si)RNA, Chop expression was evaluated. Furthermore, chromatin immunoprecipitation (ChIP) was used to investigate whether c-Jun/Ap-1 binds directly to the DNA damage-inducible transcript 3 protein (Ddit3) promoter. It was observed that c-Jun/AP-1 and Chop were synchronously overexpressed in Ang II-induced AAA and Ang II-treated cells, and that apoptosis and migration were induced by Ang II. In addition, Chop was suppressed when c-Jun was silenced by targeted siRNA. Notably, the ChIP assay demonstrated that the DNA fragments pulled down by primary antibodies against c-Jun/Ap-1 were able to be amplified by (Ddit3) promoter-specific primers. c-Jun/AP-1 may therefore mediate Chop expression in MOVAS cells via Ddit3. These results suggested that c-Jun/AP-1 may be a novel target for AAA therapy.

Inhibition of Protein Translation Initiation and Disruption of the Intestinal Epithelial Barrier in Mucosal Inflammation

Integrity of the intestinal epithelial barrier is regulated by two types of adhesive structures: intercellular junctions and focal adhesions (FA) that control epithelial cell attachment to each other and to the extracellular matrix (ECM), respectively. Disassembly of adherens junctions (AJ) and tight junctions (TJ) plays key roles in the breakdown of the intestinal epithelial barrier during mucosal inflammation. Abnormal architecture and functions of FA significantly contribute to attenuated restitution of the inflamed intestinal mucosa. Proinflammatory cytokines such as interferon (IFN)‐γ and tumor necrosis factor (TNF)‐α, are known to trigger AJ/TJ disassembly and attenuate wound healing in model intestinal epithelial cell monolayers. We hypothesized that inhibition of protein translation factors eIF4G1 and eIF4G2 could mediate cytokine‐induced disruption of the gut barrier and attenuation of epithelial restitution.Immunoblotting analysis of IFNγ and IFNγ/TNFα‐treated HT29 and T84 human colonic epithelial cells demonstrated significant downregulation of expression AJ proteins, E‐cadherin, p120 catenin, β‐catenin, and the TJ protein, occludin. By contrast quantitative RT‐PCR analysis did not reveal changes in mRNA levels of selected junctional proteins. Furthermore, expression of translation initiation factors eIF4G1 and eIF4G2 was downregulated by these proinflammatory cytokines in vitro, and eIF4G2 level was decreased in colonic mucosa of IBD patients. In order to examine functional roles of these translation initiation factors, eIF4G1 or eIF4G2 were selectively downregulated in well‐differentiated SK‐CO15 and HT29cF8 human colonic epithelial cell lines, as well as in stem‐cell like human intestinal epithelial cells (HIEC) by using either siRNA‐mediated or shRNA‐mediated knock‐down. Downregulation of either eIF4G1, or eIF4G2 increased permeability of SK‐CO15 epithelial cell monolayers and decreased expression of different adherens junction and tight junction proteins. Furthermore, depletion of these translation initiating factors inhibited collective wound healing and individual transfilter migration of both stem‐cell like and well‐differentiated intestinal epithelial cells. Interestingly, immunolabeling of newly‐translated proteins identified the epithelial leading edge as the hot‐spot site of active translation during wound healing. Finally, we observed a robust epithelial to myofibroblast transition in HIEC cells upon depletion of eIF4G1. The described results reveal a novel role for protein translation initiation in regulating intestinal epithelial barrier and epithelial restitution and suggest that this important homeostatic mechanism could be impaired during mucosal inflammation and IBD.Support or Funding InformationSupported by the Crohn's and Colitis Foundation of America grant 380973 to NGN and NIH grant DK108278 to AII.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The interaction between XBP1 and eNOS contributes to endothelial cell migration

The X-box binding protein 1 (XBP1) is a pivotal transcription factor in the endoplasmic reticulum stress response. Our previous studies have proven that XBP1 is involved in vascular endothelial growth factor (VEGF)-mediated endothelial cell (EC) proliferation and angiogenesis. In this study, we used EC monolayer wound healing, tube formation and transwell migration models to explore the role of XBP1splicing in EC migration. We found that scratching on EC monolayer triggered XBP1splicing, which was attenuated by the presence of SU5416and LY294002, suggesting that VEGF signalling pathways may be involved. Over-expression of the spliced XBP1 (XBP1s) via Ad-XBP1s gene transfer increased while knockdown of IRE1αor XBP1 by ShRNA lentivirus suppressed EC migration. Over-expression of XBP1s up-regulated the nitric oxide synthase 3 (NOS3)mRNA through the 3’UTR-mediated stabilisation and increased eNOS protein translation. Further experiments demonstrated that miR-24 participated in the XBP1s-induced eNOSup-regulation and EC migration. Further co-IP and immunofluorescence staining assays revealed that protein kinase B (Akt), eNOS andXBP1s form a complex, resulting in Akt and eNOS nucleus relocation. These results suggest that XBP1 splicing can regulate eNOS expression and cellular location, leading to EC migration and therefore contributing to wound healing and angiogenesis.

The bile acids, deoxycholic acid and ursodeoxycholic acid, regulate colonic epithelial wound healing.

The intestinal epithelium constitutes an innate barrier which, upon injury, undergoes self-repair processes known as restitution. Although bile acids are known as important regulators of epithelial function in health and disease, their effects on wound healing processes are not yet clear. Here we set out to investigate the effects of the colonic bile acids, deoxycholic acid (DCA) and ursodeoxycholic acid (UDCA), on epithelial restitution. Wound healing in T84 cell monolayers grown on transparent, permeable supports was assessed over 48 h with or without bile acids. Cell migration was measured in Boyden chambers. mRNA and protein expression were measured by RT-PCR and Western blotting. DCA (50-150 µM) significantly inhibited wound closure in cultured epithelial monolayers and attenuated cell migration in Boyden chamber assays. DCA also induced nuclear accumulation of the farnesoid X receptor (FXR), whereas an FXR agonist, GW4064 (10 µM), inhibited wound closure. Both DCA and GW4064 attenuated the expression of CFTR Cl- channels, whereas inhibition of CFTR activity with either CFTR-inh-172 (10 µM) or GlyH-101 (25 µM) also prevented wound healing. Promoter/reporter assays revealed that FXR-induced downregulation of CFTR is mediated at the transcriptional level. In contrast, UDCA (50-150 µM) enhanced wound healing in vitro and prevented the effects of DCA. Finally, DCA inhibited and UDCA promoted mucosal healing in an in vivo mouse model. In conclusion, these studies suggest bile acids are important regulators of epithelial wound healing and are therefore good targets for development of new drugs to modulate intestinal barrier function in disease treatment. NEW & NOTEWORTHY The secondary bile acid, deoxycholic acid, inhibits colonic epithelial wound healing, an effect which appears to be mediated by activation of the nuclear bile acid receptor, FXR, with subsequent downregulation of CFTR expression and activity. In contrast, ursodeoxycholic acid promotes wound healing, suggesting it may provide an alternative approach to prevent the losses of barrier function that are associated with mucosal inflammation in IBD patients.

P042 GREEN TEA EXTRACT, EGCG, INHIBITS DNMT-1, ER STRESS AND PREVENTS THE DEVELOPMENT OF INTESTINAL FIBROSIS IN MURINE CHRONIC TNBS-INDUCED COLITIS

We reported that increased endoplasmic reticulum (ER) stress signaling in subepithelial myofibroblasts (SEMF) isolated from patients with stricturing Crohn’s disease contributes to activation of TGF-β1 and fibrosis. Increased TGF-β1 results from increased interaction between GRP78 and latent TGF-beta and their translocation to the cell membrane where latent TGF-β1 is activated by αVβ3 integrin. Inhibition of ER stress by miR-199a is also lost due to DNA methyltransferase-1 (DNMT1)-induced promoter silencing of miR-199a-5p. To characterize ER stress in SEMF of mice after 8 weeks of chronic colitis, a model of fibrostenotic Crohn’s disease, and determine whether the natural DNMT-1 inhibitor, green tea extract (EGCG), inhibits ER stress and development of fibrosis. C57BL/6J mice were treated for 8 weeks with escalating doses of intrarectal TNBS. Mice were given EGCG (10mg/kg/ir) in 0.9% PBS or PBS alone twice a week for 8 weeks. Collagen content was determined by Sircol collagen assay. SEMF isolated and placed into primary culture were used to prepare RNA for qRT-PCR, cell lysates for Western blot analysis of ER stress proteins and DNMT1, or for monolayer wound healing assay. Chronic TNBS colitis in mice results in a similar ER stress response in SEMF: increased GRP78 and XBP1, and DNMT-1 as that seen in SEMF of stricturing Crohn’s disease. EGCG decreased expression of DNMT-1 by 8 fold and protein levels by 65±3% and resulted in decreased GRP78 and XBP1 protein levels indicating an inhibition of ER stress. Collagen content in EGCG treated TNBS mice was reduced to 42±2 ng/ml compared to 135±6 ng/ml in control TNBS mice. MPO assay showed no significant inflammation after 8-week of TNBS. Wound healing decreased 60±3% with siRNA GRP78 knockdown. Chronic TNBS colitis in mice is a model of ER stress in SEMF of stricturing Crohn’s disease. Inhibition of ER stress with EGCG prevents TNBS-induced intestinal fibrosis in a mouse model.

Quantitative dynamics of VE-cadherin at endothelial cell junctions at a glance: basic requirements and current concepts.

Intercellular junctions of the vascular endothelium are dynamic structures that display a high degree of plasticity, which is required to contribute to their regulation of many physiological and pathological processes including monolayer integrity, barrier function, wound healing and angiogenesis. Vascular endothelial cadherin (VE-cadherin) is connected via catenins to the actin cytoskeleton, both of which are key structures in endothelial junction regulation, and thus are the focus of much investigation. Fluorescence-based live cell imaging is the method of choice to study dynamic remodeling in living cells. Although these methods have been successfully applied to many cell types, investigations of endothelial junction dynamics were for a long time limited as they are largely resistant to transfection using many classical protocols. Application of virus-based gene transduction techniques, together with advanced microscopy, now allows both sufficient expression of fluorescence tagged junction-localized proteins in the endothelium and time-lapse recording over long periods. Using highly spatiotemporally resolved fluorescence microscopy it turned out that endothelial junctions display extensive junction heterogeneity at the subcellular level; a fact that largely limits automated quantification by available software. Recent work describes open software tools to quantitatively analyze large amounts of fluorescence-based image data in either single or confluent epithelial and endothelial cells. Based on quantitative VE-cadherin and actin dynamics novel key players, mechanisms and concepts have been suggested that control endothelial junction dynamics. Here we aim to summarize the recent developments in the field.