Monolayer Wound Research Articles

Calcitonin gene-related peptide promotes epithelial reparative and anti-colitic functions of IL-4 educated human macrophages.

Interleukin-4 activated human macrophages (M(IL4) promote epithelial wound healing and exert an anti-colitic effect in a murine model. Blood monocyte-derived M(IL4)s from healthy donors and individuals with Crohn's disease had increased mRNA expression of the calcitonin gene-related peptide (CGRP) receptor chain, RAMP1, raising the issue of neural modulation of the M(IL4)s reparative function. Thus, human (MIL4)s were treated with CGRP and the cells phagocytotic, epithelial wound repair and anti-colitic functions were assessed. Initial studies confirmed up-regulation of expression of the CGRP receptor, which was localized to the cell surface and was functional as determined by CGRP-evoked increases in cAMP. M(IL4,CGRP)s had increased mannose receptor (CD206) and FcgRIIa (CD32a) mRNA expression, a subtle, but significant increase in phagocytosis, and decreased chemokine production following exposure to E. coli. When delivered systemically (106 cells, ip.) to oxazolone-treated rag1-/- mice, M(IL4,CGRP) had an anti-colitic effect superior to M(IL4)s from the same blood donor. Conditioned medium (CM) from M(IL4,CGRP) had increased amounts of TGFb and increased wound-healing capacity compared to matched M(IL4)-CM in the human CaCo2 epithelial cell line in vitro wounding assay. Moreover, M(IL4,CGRP)s displayed increased cyclooxygenase (COX)-1 and prostaglandin D2, and CM from M(IL4,CGRP)s treated with indomethacin or SC-560 to inhibit COX1 activity failed to promote repair of wounded CaCo2 cell monolayers. These data confirm the human M(IL4)s' anti-colitic effect that was enhanced by CGRP, and may be partially dependent on macrophage COX1/PDG2 activity. Thus, input from neurone-derived molecules is a local modifier capable of boosting the anti-colitic effect of autologous M(IL4) transfer.

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.

A191 THE NEURAL SIGNAL, CALCITONIN GENE-RELATED PEPTIDE (CGRG) ENHANCES A REGULATORY PHENOTYPE IN THE HUMAN IL-4-TREATED MACROPHAGE

Abstract Background Interlukin-4 activated human macrophages (M(IL4) promote epithelial wound healing and exert an anti-colitic effect in a murine model. Blood monocyte-derived M(IL4)s from healthy donors and individuals with Crohn’s disease had increased mRNA expression of the calcitonin gene-related peptide (CGRP) receptor chain, RAMP1, raising the issue of neural modulation of the M(IL4)s reparative function. Aims To determine if CGRP-RAMP1 signalling in human IL-4 treated macrophages enhances the anti-colitic phenotype. Methods Peripheral blood mononuclear cells from healthy volunteers (HD) and individuals with Crohn’s disease were cultured on plastic (2h, 37°C) and non-adherent cells removed. The adherent cells were cultured with recombinant hM-CSF (10 ng/ml) for 7 days. The resultant macrophages (2.5×105) were differentiated with IL-4 (48h 10 ng/mL). In other cells, CGRP (10 nM) was added 24h after IL-4 and phenotype assessed by qPCR. Rag1-/- mice were treated with M(IL4)±CGRP (1x106 i.p.): 48h later, colitis was induced by oxazolone. FITC dextran was used to evaluate epithelial barrier integrity. A wound healing assay was conducted using CaCo2 epithelial cells and conditioned medium (CM,1:2 dilution) from the different treatments. Prostaglandin D2 production was evaluated by ELISA and cyclooxygenase (COX)-1 and -2 activity inhibited using SC560 and indomethacin. Results Compared to non-treated macrophages (M(0)), M(IL4)s from HD had increased RAMP1 and CLR mRNA. M(IL4)s treated with CGRP showed enhanced expression of CD206, CCL26, RAMP1 and CCL18. When delivered systemically to oxazolone-treated rag1-/- mice, M(IL4,CGRP) had an anti-colitic effect superior to M(IL4)s from the same healthy blood donor. Use of the the human colon CaCo2 cell in vitro wounding model revealed that CM from M(IL4,CGRP) had increased amounts of TGFb and increased wound-healing capacity compared to matched M(IL4)-CM. Moreover, M(IL4,CGRP)s displayed increased COX-1 and PGD2. CM from M(IL4,CGRP)s treated with indomethacin or SC-560 to inhibit COX1 activity failed to promote repair of wounded CaCo2 cell monolayers. Conclusions Neuronal input is revealed as an important local modifier capable of boosting the anti-colitic effect of autologous M(IL4) transfer to treat enteric inflammation. Funding Agencies CCCHelmsley Charity

Sustained intestinal epithelial monolayer wound closure after transient application of a FAK-activating small molecule.

M64HCl, which has drug-like properties, is a water-soluble Focal Adhesion Kinase (FAK) activator that promotes murine mucosal healing after ischemic or NSAID-induced injury. Since M64HCl has a short plasma half-life in vivo (less than two hours), it has been administered as a continuous infusion with osmotic minipumps in previous animal studies. However, the effects of more transient exposure to M64HCl on monolayer wound closure remained unclear. Herein, we compared the effects of shorter M64HCl treatment in vitro to continuous treatment for 24 hours on monolayer wound closure. We then investigated how long FAK activation and downstream ERK1/2 activation persist after two hours of M64HCl treatment in Caco-2 cells. M64HCl concentrations immediately after washing measured by mass spectrometry confirmed that M64HCl had been completely removed from the medium while intracellular concentrations had been reduced by 95%. Three-hour and four-hour M64HCl (100 nM) treatment promoted epithelial sheet migration over 24 hours similar to continuous 24-hour exposure. 100nM M64HCl did not increase cell number. Exposing cells twice with 2-hr exposures of M64HCl during a 24-hour period had a similar effect. Both FAK inhibitor PF-573228 (10 μM) and ERK kinase (MEK) inhibitor PD98059 (20 μM) reduced basal wound closure in the absence of M64HCl, and each completely prevented any stimulation of wound closure by M64HCl. Rho kinase inhibitor Y-27632 (20 μM) stimulated Caco-2 monolayer wound closure but no further increase was seen with M64HCl in the presence of Y-27632. M64HCl (100 nM) treatment for 3 hours stimulated Rho kinase activity. M64HCl decreased F-actin in Caco-2 cells. Furthermore, a two-hour treatment with M64HCl (100 nM) stimulated sustained FAK activation and ERK1/2 activation for up to 16 and hours 24 hours, respectively. These results suggest that transient M64HCl treatment promotes prolonged intestinal epithelial monolayer wound closure by stimulating sustained activation of the FAK/ERK1/2 pathway. Such molecules may be useful to promote gastrointestinal mucosal repair even with a relatively short half-life.

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.

Synthesis and characterization of rutin-loaded micelles for glioblastoma multiforme treatment: A computational and experimental study

The current experiments aimed to develop an anti-glioblastoma multiforme (GBM) formulation based on rutin-loaded nanomicelles. First, the molecular structure of water, and ethanol was constructed using Material Studio software. Then, the structure of the molecules was optimized through the Forcite module in the software. In sequence, to set the temperature of the system the MD simulations were run under the NVT ensemble (constant number of particles, volume, and temperature) with velocity scale as a thermostat and total simulation time equal to 100 ps as the initialization step. We applied the hydrophobic-hydrophobic interactions and micellization process method to encapsulate rutin into micellar nanoparticles. The computational studies showed that in the first 30 ps of the simulation the system is unstable, but after that, the system reached a stable condition. Moreover, we observed that the density of the system rapidly decreased from 0.98 g/cm3 to a stable mean value equal to 0.95 g/cm3. The characterizations using DLS and SEM imaging showed that the synthesized NPs have a size lower than 100 nm. The hemolysis evaluation showed that the Rut-micelles are hemocompatible and induced lower than 10% hemolysis in all tested groups. The highest cell lethality was observed in the group treated with the synthesized Rut-micelles with a concentration of 400 μM, which was statistically significant (p < 0.05) to the control and other test groups. The anti-metastatic effect of the Rut-micelles was confirmed via the cell monolayer wound scratch assay. These results illustrated that the synthesized rutin-loaded nanomicelles could be applied as a GBM treatment option to improve the treatment efficiency of glioblastoma multiforme.

Abstract B001: Heterogeneity of cancer network biophysical phenotypes is required for tumor muscle invasion in vivo

Abstract Bladder, colon, gastric, prostate, and uterine cancers originate from the organ’s epithelium, and each organ is surrounded by smooth muscle. In prostate cancer, organ-confined tumors, without extracapsular extension (ECE) through muscle, have a 5-year survival rate of 99% compared to 31% for metastatic disease. Previously, we modeled tumor cluster ECE and reported that a CRISPR-Cas9 integrin mutation (α6AA) blocked ECE by switching from a cell-ECM to a cell-cell biophysical phenotype. Since tumors are mosaics and migrate as heterogeneous drug-resistant populations, we tested the functional heterogeneity of the two biophysical cell phenotypes in ECE. Experimental procedures included the in vivo mouse xenograft model of ECE as a functional endpoint of muscle invasion, the MTT assay for cell survival, and using electric cell impedance sensing (ECIS) measurements to directly compare the biophysical properties of tumor cells expressing the invasion permissive α6WT integrin, cells expressing only the α6AA mutation, or a mixed population expressing both integrin types. The α6AA integrin mutation or α6KO knockout produced cells unable to invade into and through the smooth muscle of the mouse as compared to the α6WT integrin cells, as previously reported by us. The new unpublished results are that cell adhesion mediated drug resistance was detected in the α6AA population since their LD50 to Bortezomib, Gemcitabine, and Taxotere was shifted to 33.71nM, 64.66nM, and 5.77nM respectively from 16.29nM, 31.74nM, and 3.52nM in the α6WT cells as measured by MTT assay at 72 hours of incubation. In contrast, the α6AA cells were sensitized to the NAMPT inhibitor, FK866, with LD50 shifting from 27.46nM in α6WT to 7.354nM in the α6AA cells. A mixture of α6AA and α6WT cells allowed invasion into and through the muscle in the mouse of the mutant α6AA integrin cells that could not invade on their own. Examining the live cellular biophysical parameters revealed that the integrin mutation produced a 4-fold increase in cell-cell resistance (400Hz measurement) and a 12-fold recovery time delay in re-establishing a cell-cell resistance monolayer after wounding, with response complexity decreased two-fold. Cell-ECM resistance (40,000Hz measurement) in α6AA versus α6WT population was 1.5-fold decreased with a 6-fold recovery time delay in the cell-ECM resistance monolayer and response complexity decreased 2-fold. A heterogenous population containing up to a 4:1 proportion of α6AA to α6WT cells synergistically delayed cell-cell recovery time of the wounded monolayer up to 6-fold compared to the α6WT cells. Conversely, recovery of the wounded cell-ECM resistance monolayer with 1:1 or 4:1 mixture of α6AA cell only delayed recovery time by 1.5–1.7-fold. Taken together, these data suggest that a heterogenous tumor population provides functional advantages for drug-resistant tumor cells to invade and traverse the muscle barrier. Current work is ongoing to optimize the drug combinations to eliminate both phenotypes in the invasive tumor network and prevent ECE. Citation Format: Kendra D. Marr, Jaime M.C. Gard, William L. Harryman, Elijah J. Keeswood, Allan I. Paxson, Charles Wolgemuth, Lori A. Hazlehurst, Raymond B. Nagle, Anne E. Cress. Heterogeneity of cancer network biophysical phenotypes is required for tumor muscle invasion in vivo [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr B001.

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).

A novel drug-like water-soluble small molecule Focal Adhesion Kinase (FAK) activator promotes intestinal mucosal healing

Non-steroidal anti-inflammatory drugs (NSAIDs) injure the proximal and distal gut by different mechanisms. While many drugs reduce gastrointestinal injury, no drug directly stimulates mucosal wound healing. Focal adhesion kinase (FAK), a non-receptor tyrosine kinase, induces epithelial sheet migration.We synthesized and evaluated a water-soluble FAK-activating small molecule, M64HCl, with drug-like properties. Monolayer wound closure and Western blots measured migration and FAK phosphorylation in Caco-2 ​cells, in vitro kinase assays established FAK activation, and pharmacologic tests assessed drug-like properties. 30 ​mg/kg/day M64HCl was administered in two murine small intestine injury models for 4 days.M64HCl (0.1–1000 ​nM) dose-dependently increased Caco-2 FAK-Tyr 397 phosphorylation, without activating Pyk2 and accelerated Caco-2 monolayer wound closure. M64HCl dose-responsively activates the FAK kinase domain vs. the non-salt M64, increasing the Vmax of ATP-binding. Pharmacologic tests suggested M64HCl has drug-like properties and is enterally absorbed. M64HCl 25 ​mg/kg/day continuous infusion promoted healing of ischemic jejunal ulcers and indomethacin-induced small intestinal injury in C57Bl/6 mice. M64HCl-treated mice exhibited smaller ulcers 4 days after ischemic ulcer induction or indomethacin injury. Renal histology and plasma creatinine were normal. Mild hepatic inflammatory changes and ALT elevation were similar among M64HCl-treated mice and controls. M64HCl was concentrated in kidney and gastrointestinal mucosa and functional nephrectomy studies suggested predominantly urinary excretion. Little toxicity was observed in vitro or in single-dose mouse toxicity studies until >1000x higher than effective concentrations. M64HCl, a water-soluble FAK activator, promotes epithelial restitution and intestinal mucosal healing and may be useful to treat gut mucosal injury.

Spontaneous multi-scale void formation and closure in densifying epithelial and fibroblast monolayers from the sub-confluent state.

Using time-lapse phase contrast microscopy, the formation and closure of spontaneously generated voids in the densifying monolayers of isotropic epithelial cells (ECs) and elongated fibroblast cells (FCs) through proliferation from the sub-confluent state are investigated. It is found that, in both types of monolayers after forming a connected network composed of nematic patches with different orientations, numerous multi-scale voids can be spontaneously formed and gradually close with increasing time. The isotropic fluctuations of deformation and crawling of ECs and the anisotropic axial motion/alignment polarizations of FCs are the two keys leading to the following different generic dynamical behaviors. In EC monolayers, voids exhibit irregular boundary fluctuations and easier cell re-orientation of front layer cells (FLCs) surrounding void boundaries. Void closures are mainly through pinching the gap between the opposite fluctuating void boundaries, and the inward crawling of FLCs to reduce void area associated with topological rearrangement to reduce FLC number. In FC monolayers, large voids have piecewise smooth convex boundaries, and cusp-shaped concave boundaries with cells orienting toward the void at cusp tips. The extension of a thin cell bridge from the cusp tip can bisect a large void into smaller voids. For smaller FC voids dominated by convex boundaries, along which cell alignment prohibits inward crawling, the reduction of FLC number through successive outward squeezing of single FLCs by neighboring FLCs sliding along the void boundary plays an important role for topological rearrangement and void closure. Unlike those surrounding artificial wounds in dense EC monolayers, the absence of ring-like purse-strings surrounding EC and FC voids allows topological rearrangements for reducing void perimeter and void area.

Fibrin(ogen) Is Constitutively Expressed by Differentiated Intestinal Epithelial Cells and Mediates Wound Healing.

Fibrinogen is a large molecule synthesized in the liver and released in the blood. Circulating levels of fibrinogen are upregulated after bleeding or clotting events and support wound healing. In the context of an injury, thrombin activation drives conversion of fibrinogen to fibrin. Fibrin deposition contains tissue damage, stops blood loss, and prevents microbial infection. In most circumstances, fibrin needs to be removed to allow the resolution of inflammation and tissue repair, whereas failure of this may lead to the development of various disorders. However, the contribution of fibrinogen to tissue inflammation and repair is likely to be context-dependent. In this study, the concept that fibrin needs to be removed to allow tissue repair and to reduce inflammation is challenged by our observations that, in the intestine, fibrinogen is constitutively produced by a subset of intestinal epithelial cells and deposited at the basement membrane as fibrin where it serves as a substrate for wound healing under physiological conditions such as epithelial shedding at the tip of the small intestinal villus and surface epithelium of the colon as well as under pathological conditions that require rapid epithelial repair. The functional integrity of the intestine is ensured by the constant renewal of its simple epithelium. Superficial denuding of the epithelial cell layer occurs regularly and is rapidly corrected by a process called restitution that can be influenced by various soluble and insoluble factors. Epithelial cell interaction with the extracellular matrix greatly influences the healing process by acting on cell morphology, adhesion, and migration. The functional contribution of a fibrin(ogen) matrix in the intestine was studied under physiological and pathological contexts. Our results (immunofluorescence, immunoelectron microscopy, and quantitative PCR) show that fibrin(ogen) is a novel component of the basement membrane associated with the differentiated epithelial cell population in both the small intestine and colon. Fibrin(ogen) alone is a weak ligand for epithelial cells and behaves as an anti-adhesive molecule in the presence of type I collagen. Furthermore, the presence of fibrin(ogen) significantly shortens the time required to achieve closure of wounded epithelial cell monolayers and co-cultures in a PI3K-dependent manner. In human specimens with Crohn’s disease, we observed a major accumulation of fibrin(ogen) throughout the tissue and at denuded sites. In mice in which fibrin formation was inhibited with dabigatran treatment, dextran sulfate sodium administration provoked a significant increase in the disease activity index and pathological features such as mucosal ulceration and crypt abscess formation. Taken together, these results suggest that fibrin(ogen) contributes to epithelial healing under both normal and pathological conditions.

Human Amniotic Mesenchymal Stem Cells and Fibroblasts Accelerate Wound Repair of Cystic Fibrosis Epithelium.

Cystic fibrosis (CF) airways are affected by a deranged repair of the damaged epithelium resulting in altered regeneration and differentiation. Previously, we showed that human amniotic mesenchymal stem cells (hAMSCs) corrected base defects of CF airway epithelial cells via connexin (CX)43-intercellular gap junction formation. In this scenario, it is unknown whether hAMSCs, or fibroblasts sharing some common characteristics with MSCs, can operate a faster repair of a damaged airway epithelium. A tip-based scratch assay was employed to study wound repair in monolayers of CFBE14o- cells (CFBE, homozygous for the F508del mutation). hAMSCs were either co-cultured with CFBE cells before the wound or added to the wounded monolayers. NIH-3T3 fibroblasts (CX43+) were added to wounded cells. HeLa cells (CX43-) were used as controls. γ-irradiation was optimized to block CFBE cell proliferation. A specific siRNA was employed to downregulate CX43 expression in CFBE cells. CFBE cells showed a delayed repair as compared with wt-CFTR cells (16HBE41o-). hAMSCs enhanced the wound repair rate of wounded CFBE cell monolayers, especially when added post wounding. hAMSCs and NIH-3T3 fibroblasts, but not HeLa cells, increased wound closure of irradiated CFBE monolayers. CX43 downregulation accelerated CFBE wound repair rate without affecting cell proliferation. We conclude that hAMSCs and fibroblasts enhance the repair of a wounded CF airway epithelium, likely through a CX43-mediated mechanism mainly involving cell migration.

Flavored and Nicotine-Containing E-Cigarettes Induce Impaired Angiogenesis and Diabetic Wound Healing via Increased Endothelial Oxidative Stress and Reduced NO Bioavailability.

The prevalent use of electronic cigarettes (e-cigarettes) has increased exponentially in recent years, especially in youth who are attracted to flavored e-cigarettes. Indeed, e-cigarette or vaping product use-associated lung injury (EVALI) cases started to emerge in the United States in August 2019, resulting in 2807 hospitalized cases and 68 deaths as of 18 February 2020. In the present study, we investigated, for the first time, whether flavored and nicotine containing e-cigarettes induce endothelial dysfunction to result in impaired angiogenesis and wound healing particularly under diabetic condition. Nicotine containing e-cigarettes with various contents of nicotine (0, 1.2%, 2.4%), and flavored e-cigarettes of classic tobacco, mint, menthol, and vanilla or fruit from BLU (nicotine 2.4%) or JUUL (nicotine 3%), were used to treat endothelial cells in vitro and streptozotocin-induced diabetic mice in vivo. Endothelial cell superoxide production, determined by dihydroethidium (DHE) fluorescent imaging and electron spin resonance (ESR), was markedly increased by exposure to e-cigarette extract (e-CSE) in a nicotine-content dependent manner, while nitric oxide (NO) bioavailability detected by DAF-FM fluorescent imaging was substantially decreased. All of the different flavored e-cigarettes examined also showed significant effects in increasing superoxide production while diminishing NO bioavailability. Endothelial cell apoptosis evaluated by caspase 3 activity was markedly increased by exposure to e-CSE prepared from flavored and nicotine containing e-cigarettes. Endothelial monolayer wound assays revealed that nicotine-containing and flavored e-cigarettes induced impaired angiogenic wound repair of endothelial cell monolayers. Furthermore, vascular endothelial growth factor (VEGF) stimulated wound healing in diabetic mice was impaired by exposure to e-CSEs prepared from nicotine-containing and flavored e-cigarettes. Taken together, our data demonstrate for the first time that flavored and nicotine-containing e-cigarettes induce endothelial dysfunction through excessive ROS production, resulting in decreased NO bioavailability, increased endothelial cell apoptosis, and impairment in angiogenesis and wound healing, especially under diabetic condition. These responses of endothelial dysfunction likely underlie harmful effects of e-cigarettes in endothelial-rich organs, such as heart and lungs. These data also indicate that rigorous regulation on e-cigarette use should be enforced in diabetic and/or surgical patients to avoid severe consequences from impaired angiogenesis/wound healing.

Parthenolide reverses the epithelial to mesenchymal transition process in breast cancer by targeting TGFbeta1: In vitro and in silico studies

AimsBreast cancer metastasis is the leading cause of mortality among breast cancer patients. Epithelial to mesenchymal transition (EMT) is a biological process that plays a fundamental role in facilitating breast cancer metastasis. The present study assessed the efficacy of parthenolide (PTL Tanacetum parthenium) on EMT and its underlying mechanisms in both lowly metastatic, estrogen-receptor positive, MCF-7 cells and highly metastatic, triple-negative MDA-MB-231 cells. Main methodsMCF-7 and MDA-MB-231 cells were treated with PTL (2 μM and 5 μM). Cell viability was determined by MTT (3-(4,5-dimethy lthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay. Apoptosis was analyzed by the FITC (fluorescein isothiocyanate) annexin V apoptosis detection kit. The monolayer wound scratch assay was employed to evaluate cancer cell migration. Proteins were separated and identified by Western blotting. Gene expression was analyzed by quantitative real-time PCR. Key findingsPTL treatment significantly reduced cell viability and migration while inducing apoptosis in both cell lines. Also, PTL treatment reverses the EMT process by decreasing the mesenchymal marker vimentin and increasing the epithelial marker E-cadherin compared to the control treatment. Importantly, PTL downregulates TWIST1 (a transcription factor and regulator of EMT) gene expression, concomitant with the reduction of transforming growth factor beta1 (TGFβ1) protein and gene expression in both cell lines. Additionally, molecular docking studies suggest that PTL may induce anticancer properties by targeting TGFβ1 in both breast cancer cell lines. SignificanceOur findings provide insights into the therapeutic potential of PTL to mitigate EMT and breast cancer metastasis. These promising results demand in vivo studies.

Cytotoxic effects of Annona squamosa leaves against breast cancer cells via apoptotic signaling proteins

Annona squamosa L. is an important medicinal plant used in traditional medicine for the treatment of various diseases. Different parts of A. squamosa L. have various therapeutic effects; however, the anticancer activity of the leaves has not yet been identified. In vitro, MTT, nuclear staining, and LDH assays were used to evaluate cell survival and proliferation in cells exposed to the extracts. The effect of the extracts on cell migration was investigated using a monolayer wound repair assay, and the apoptotic effects were evaluated using flow cytometry. A breast cancer model was used to study the effect of the extract on the tumor size, and the expression of different proliferative and apoptotic markers was evaluated by immunohistochemical analysis. At a concentration of 100 µg/mL, A. squamosa leaf extracts exerted strong antiproliferative and cytotoxic effects against various cell lines. The extracts reduced wound closure and strongly induced apoptosis. In vivo study, rats were sacrificed 24 h after the last injection, and tumor size, as well as the expression of proliferative and apoptotic markers, were observed to be greatly affected by treatment with the extracts. Therefore, A. squamosa leaf extract may be developed as a potential novel drug to treat breast cancer in the future.

A60 THE NEURAL SIGNAL, CALCITONIN GENE-RELATED PEPTIDE (CGRG) ENHANCES A REGULATORY PHENOTYPE IN THE HUMAN IL-4 TREATED HUMAN MACROPHAGE

Abstract Background Recent studies in pre-clinical models of disease have revealed the ability of murine and human IL-4-treated macrophages (M(IL4)) to promote wound recovery and reduce the severity of colitis. An unbiased RNA-sequence analysis of human blood-derived macrophages revealed increased expression of the RAMP1 chain of the CGRP receptor in IL-4 treated cells, raising the intriguing possibility of neural control of regulatory macrophages in the context of neuroimmune interaction in colitis. Thus, we sought to address if this mRNA signal translated into increased RAMP1 protein, if/how CGPR affected M(IL4) function, and if this applied to macrophages from patients with IBD as well as those from healthy donors. Aims To determine if CRGP-RAMP1 signalling in human IL-4 treated macrophages enhances a regulatory phenotype. Methods Peripheral blood mononuclear cells from healthy volunteers and individuals with IBD were cultured on plastic (2h, 37°C) and non-adherent cells removed. The adherent cells were cultured with recombinant hM-CSF (10 ng/ml) for 7 days. The resultant macrophages (2.5×105) were differentiated with IL-4 (48h 20 ng/mL) and assessed by qPCR and immunocytochemistry. In other cells, CGRP (10 nM) was added 24h after IL-4 and (1) cAMP (2) cytokines and (3) phagocytosis of inert FITC-beads measured, and (4) the capacity of supernatant from the cells to promote healing in wounded Caco2 epithelial monolayers tested. Results Compared to non-treated macrophages (M(0)), M(IL4)s from healthy individuals had increased mRNA for both chains of the CGRP-receptor (i.e. RAMP1 and CLR), and increased surface expression of the receptor as shown by immunostaining and CGRP-evoked cAMP. The IL-4 evoked RAMP1 mRNA was only detected in macrophages from 50% of the patients with active IBD. M(IL4)s treated with CGRP showed enhanced expression of the mannose receptor (CD206, allows detection of bacteria), increase phagocytosis of inert beads/macrophages. Moreover, CGRP increases VEGF and CCL18 expression in M(IL4), and soluble mediators from these cells promoted in vitro epithelial wound repair. Conclusions Reduced expression of CGRP and its receptor has been shown in IBD. The findings herein, demonstrating how CGRP-RAMP1 signalling can reinforce and enhance a regulatory, reparatory phenotype in human macrophages reveals another aspect of IBD pathophysiology. We speculate that loss of this neuroimmune axis (i.e. CGRP/Nerve-M(IL4) interaction) has the potential to significantly impair mucosal healing in IBD. Funding Agencies CCCAlberta Innovates in Health Innovation

Oleuropein multicompartment nanovesicles enriched with collagen as a natural strategy for the treatment of skin wounds connected with oxidative stress.

Aim: Collagen-enriched transfersomes, glycerosomes andglytransfersomes were specifically tailored for skin delivery of oleuropein. Methods: Vesicles were prepared by direct sonication and their main physicochemical and technological properties were measured. Biocompatibility, protective effect and promotion of the healing of a wounded cell monolayer were tested in vitro using fibroblasts. Results: Vesicles were mainly multicompartment, small (∼108nm), slightly polydispersed (approximately0.27) and negatively charged (~-49mV). Oleuropein was incorporated in high amounts (approximately87%) and vesicles were stable during fourmonths of storage. In vitro studies confirmed the low toxicity of formulations (viability ≥95%), their effectiveness in counteracting nitric oxide generation and damages caused by free oxygen radicals, especially when collagenglytransfersomes were used (viability ~100%). These vesicles also promoted the regeneration of a wounded area by promoting the proliferation and migration of fibroblasts. Conclusion: Collagen-enriched vesicles are promising formulations capable of speeding up the healing of the wounded skin.

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.

Accessible dynamic micropatterns in monolayer cultures via modified desktop xurography

Micropatterned cell cultures provide an important tool to understand dynamic biological processes, but often require specialized equipment and expertise. Here we present subtractive bioscribing (SuBscribing), a readily accessible and inexpensive technique to generate dynamic micropatterns in biomaterial monolayers on-the-fly. We first describe our modifications to a commercially available desktop xurographer and demonstrate the utility and limits of this system in creating micropatterned cultures by mechanically scribing patterns into a brittle, non-adhesive biomaterial layer. Patterns are sufficiently small to influence cell morphology and orientation and can be extended to pattern large areas with complex reproducible shapes. We also demonstrate the use of this system as a dynamic patterning tool for cocultures. Finally, we use this technique to explore and improve upon the well-established epithelial scratch assay, and demonstrate that robotic control of the scratching tool can be used to create custom-shaped wounds in epithelial monolayers, and that the scribing direction leaves trace remnants of matrix molecules that may significantly affect conventional implementations of this common assay.