Epithelial Repair Research Articles

Abstract A154: PDLIM2 is required for regulating inflammation and cellular redox levels in mouse colorectal epithelial model

Abstract PDLIM2 is a cytoskeletal and nuclear protein present in hematopoietic and epithelial cells that regulates cell differentiation and the protein stability of transcription factors including NF-κB, STATs, and β-catenin (1). PDLIM2 expression is repressed in certain cancers, but is highly expressed in cancer cells that exhibit a metastatic phenotype (2). PDLIM2 is also highly expressed in the tumour microenvironment of breast cancer, particularly in the pro-wound healing M2 macrophage population. In line with this, bone-marrow derived macrophages from PDIM2 knockout (KO) mice have impaired differentiation towards the M2 macrophage phenotype (3). Here we investigated the effects of PDLIM2 loss in epithelial and immune cells in a colitis-associated cancer model using PDLIM2 wild type (WT) and KO mice exposed to dextran sodium sulphate (DSS) and azoxymethane (AOM). We observed that PDLIM2 KO mice exhibited a strong response to DSS alone. Colon tissue from DSS-treated mice exhibited impaired epithelium repair, sustained inflammation, and low levels of wound-healing M2 macrophages compared to WT tissues. To address the molecular mechanisms associated with PDLIM2 KO in colon epithelial cells, we suppressed or knocked out PDLIM2 in the Caco2 cell line. Caco-2 cells lacking PDLIM2 displayed dysregulated adhesion signalling, which was accompanied by elevated levels of reactive oxygen species (ROS), mitochondrial dysfunction and increased dependence on glycolysis. Overall, we conclude that PDLIM2 is required for the resolution of inflammation and to promote intestinal epithelial repair through regulating cell adhesion signalling and cellular redox balance. 1. Guo ZS, Qu Z. PDLIM2: Signaling pathways and functions in cancer suppression and host immunity. Biochim Biophys Acta Rev Cancer. 2021 Dec;1876(2):188630. doi: 10.1016/j.bbcan.2021.188630. 2. Cox OT, Edmunds SJ, Simon-Keller K, Li B, Moran B, Buckley NE, Bustamante-Garrido M, Healy N, O'Flanagan CH, Gallagher WM, Kennedy RD, Bernards R, Caldas C, Chin SF, Marx A, O'Connor R. PDLIM2 Is a Marker of Adhesion and β-Catenin Activity in Triple-Negative Breast Cancer. Cancer Res. 2019 May 15;79(10):2619-2633. doi: 10.1158/0008-5472.CAN-18-2787. 3. Cox OT, O'Sullivan N, Tresse E, Ward S, Buckley N, O'Connor R. PDLIM2 is highly expressed in Breast Cancer tumour-associated macrophages and is required for M2 macrophage polarization. Front Oncol. 2022 Nov 30;12:1028959. doi: 10.3389/fonc.2022.1028959. Citation Format: Stephanie Ward, Silvia Melgar, Orla T Cox, Raffaella Morelli, Rosemary O'Connor. PDLIM2 is required for regulating inflammation and cellular redox levels in mouse colorectal epithelial model [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A154.

Tension Remodeling Regulates Topological Transitions in Epithelial Tissues.

Cell neighbor exchanges play a critical role in regulating tissue fluidity during epithelial morphogenesis and repair. In vivo, these neighbor exchanges are often hindered by the formation of transiently stable fourfold vertices, which can develop into complex multicellular rosettes where five or more cell junctions meet. Despite their importance, the mechanical origins of multicellular rosettes have remained elusive, and current cellular models lack the ability to explain their formation and maintenance. Here we present a dynamic vertex model of epithelial tissues with strain-dependent tension remodeling and mechanical memory dissipation. We show that an increase in cell junction tension upon contraction and reduction in tension upon extension can stabilize higher-order vertices, temporarily stalling cell rearrangements. On the other hand, inducing mechanical memory dissipation via relaxation of junction strain and stress promotes the resolution of higher-order vertices, facilitating cell neighbor exchanges. We demonstrate that by tuning the rates of tension remodeling and mechanical memory dissipation, we can control topological transitions and tissue material properties, recapitulating complex cellular topologies seen in developing organisms.

Endothelial to mesenchymal transition in kidney fibrosis.

Fibrotic diseases are characterized by the uncontrolled accumulation of extracellular matrix (ECM) components leading to disruption of tissue homeostasis. Myofibroblasts as the main ECM-producing cells can originate from various differentiated cell types after injury. Particularly, the process of endothelial-to-mesenchymal transition (endMT), describing phenotypic shifts of endothelial cells to adopt a fully mesenchymal identity, may contribute to the pool of myofibroblasts in fibrosis, while leading to capillary rarefaction and exacerbation of tissue hypoxia. In renal disease, incomplete recovery from acute kidney injury (AKI) and the ensuing fibrotic reaction stand out as major contributors to chronic kidney disease (CKD) development. While the focus has largely been on impaired tubular epithelial repair as a potential fibrosis-driving mechanism, alterations in the renal microcirculation post-AKI, and in particular endMT as a maladaptive response, could hold equal significance. Dysfunctional interplays among various cell types in the kidney microenvironment can instigate endMT. Transforming growth factor beta (TGF-β) signaling, with its downstream activation of canonical/Smad-mediated and non-canonical pathways, has been identified as primary driver of this process. However, non-TGF-β-mediated pathways involving inflammatory agents and metabolic shifts in intercellular communication within the tissue microenvironment can also trigger endMT. These harmful, maladaptive cell-cell interactions and signaling pathways offer potential targets for therapeutic intervention to impede endMT and decelerate fibrogenesis such as in AKI-CKD progression. Presently, partial reduction of TGF-β signaling using anti-diabetic drugs or statins may hold therapeutic potential in renal context. Nevertheless, further investigation is warranted to validate underlying mechanisms and assess positive effects within a clinical framework.

Alveolar Type 2 Epithelial Cell Organoids: Focus on Culture Methods.

Lung diseases rank third in terms of mortality and represent a significant economic burden globally. Scientists have been conducting research to better understand respiratory diseases and find treatments for them. An ideal in vitro model must mimic the in vivo organ structure, physiology, and pathology. Organoids are self-organizing, three-dimensional (3D) structures originating from adult stem cells, embryonic lung bud progenitors, embryonic stem cells (ESCs), and induced pluripotent stem cells (iPSCs). These 3D organoid cultures may provide a platform for exploring tissue development, the regulatory mechanisms related to the repair of lung epithelia, pathophysiological and immunomodulatory responses to different respiratory conditions, and screening compounds for new drugs. To create 3D lung organoids in vitro, both co-culture and feeder-free methods have been used. However, there exists substantial heterogeneity in the organoid culture methods, including the sources of AT2 cells, media composition, and feeder cell origins. This article highlights the currently available methods for growing AT2 organoids and prospective improvements to improve the available culture techniques/conditions. Further, we discuss various applications, particularly those aimed at modeling human distal lung diseases and cell therapy.

Inactivation of Group 1B Phospholipase A2 Enhances Disease Recovery and Reduces Experimental Colitis in Mice.

Phospholipase A2 (PLA2) enzymes influence inflammatory bowel disease in both positive and negative manners depending on the type of PLA2 that is expressed. This study explored the influence of the abundantly expressed Group 1B PLA2 (PLA2G1B) on ulcerative colitis. Wild-type C57BL/6J mice and Pla2g1b-/- mice were treated with dextran sulfate sodium (DSS) for 5 days to induce epithelial injury, followed by another 5 days without DSS for recovery. The Pla2g1b-/- mice displayed significantly less body weight loss, colitis pathology, and disease activity indexes compared to the wild-type mice. The differences in colitis were not due to differences in the colonic lysophospholipid levels, but higher numbers of stem and progenitor cells were found in the intestines of Pla2g1b-/- mice compared to the wild-type mice. The DSS-treated Pla2g1b-/- mice also showed higher expressions of genes that are responsible for epithelial repair and lower expressions of proinflammatory cytokine genes in the colon, as well as reduced inflammatory cytokine levels in the plasma. In vitro experiments revealed the PLA2G1B stimulation of inflammatory cytokine expression by myeloid cells. PLA2G1B inactivation protects against DSS-induced colitis in mice by increasing the intestinal stem cell reservoir for epithelial repair and reducing myeloid cell inflammation in the diseased colon. Thus, PLA2G1B may be a target for colitis management.

An olsalazine nanoneedle-embedded inulin hydrogel reshapes intestinal homeostasis in inflammatory bowel disease

Inflammatory bowel disease (IBD) is a chronic and refractory condition characterized by disrupted epithelial barrier, dysregulated immune balance, and altered gut microbiota. Nano-enabled interventions for restoring gut homeostasis have the potential to alleviate inflammation in IBD. Herein, we developed a combination of olsalazine (Olsa)-based nanoneedles and microbiota-regulating inulin gel to reshape intestinal homeostasis and relieve inflammation. The Olsa-derived nanoneedles exhibited reactive oxygen species scavenging ability and anti-inflammatory effects in lipopolysaccharide-simulated macrophages. The composite of nanoneedles and inulin gel (Cu2(Olsa)/Gel) displayed a macroporous structure, improved bio-adhesion, and enhanced colon retention after oral administration. Mechanistically, the composite effectively downregulated pro-inflammatory cytokine levels and promoted epithelial barrier repair through anti-inflammatory and antioxidant therapies, resulting in significant alleviation of colitis in three animal models of IBD. Furthermore, analysis of gut microbiota revealed that Cu2(Olsa)/Gel treatment increased the diversity of intestinal microflora and decreased the relative abundance of pathogenic bacteria such as Proteobacteria. Overall, this study provides a self-delivering nanodrug and dietary fiber hydrogel composite for IBD therapy, offering an efficient approach to restore intestinal homeostasis.

Formononetin ameliorates airway inflammation by suppressing ESR1/NLRP3/Caspase-1 signaling in asthma

Since inhaled glucocorticoids are the first-line treatment for asthma, asthma management becomes extremely difficult when asthma does not react well to glucocorticoids. Formononetin, a bioactive isoflavone and typical phytoestrogen, has been shown to have an anti-inflammatory impact while alleviating epithelial barrier dysfunction, which plays a role in the pathogenesis of allergic illnesses like asthma. However, the biological mechanisms behind this impact are unknown. As a result, we set out to investigate the effects of formononetin on airway inflammation and epithelial barrier repair in house dust mite (HDM)-induced asthmatic mice. We further expanded on formononetin's putative mode of action in reducing airway inflammation by modifying epithelial barrier dysfunction. In the current study, researchers discovered that formononetin significantly lowered total IgE levels in serum and interleukin (IL)-4, IL-6, and IL-17A levels in bronchoalveolar lavage fluid (BALF) in HDM-challenged asthmatic mice. Experiments on cell proliferation, migration, and apoptosis were performed in vitro to determine the effect of formononetin on bronchial epithelial barrier repair. Furthermore, in lipopolysaccharide (LPS)-stimulated 16HBE cells, formononetin increased cell proliferation and migration while preventing apoptosis and lowering the Bax/Bcl-2 ratio. In vitro and in vivo, formononetin significantly inhibited toll-like receptor 4 (TLR4) and estrogen receptor (ESR1)/Nod-like receptor family pyrin domain-containing protein 3 (NLRP3)/Caspase-1 signaling. These findings show that formononetin can reduce airway inflammation in HDM-challenged asthmatic mice by promoting epithelial barrier repair and possibly by inhibiting ESR1/NLRP3/Caspase-1 signaling as the underlying mechanism; formononetin could be a promising alternative treatment for asthma.

Wound-healing plasticity enables clonal expansion of founder progenitor cells in colitis.

Chronic colonic injury and inflammation pose high risks for field cancerization, wherein injury-associated mutations promote stem cell fitness and gradual clonal expansion. However, the long-term stability of some colitis-associated mutational fields could suggest alternate origins. Here, studies of acute murine colitis reveal a punctuated mechanism of massive, neutral clonal expansion during normal wound healing. Through three-dimensional (3D) imaging, quantitative fate mapping, and single-cell transcriptomics, we show that epithelial wound repair begins with the loss of structural constraints on regeneration, forming fused labyrinthine channels containing epithelial cells reprogrammed to a non-proliferative plastic state. A small but highly proliferative set of epithelial founder progenitor cells (FPCs) subsequently emerges and undergoes extensive cell division, enabling fluid-like lineage mixing and spreading across the colonic surface. Crypt budding restores the glandular organization, imprinting the pattern of clonal expansion. The emergence and functions of FPCs within a critical window of plasticity represent regenerative targets with implications for preneoplasia.

Urban air inhalation exacerbates the oxinflammatory response after an acute lung injury, hindering alveolar epithelium repair

Urban air inhalation exacerbates the oxinflammatory response after an acute lung injury, hindering alveolar epithelium repair

24 Intestinal Function and Integrity in Health and Disease: Current Knowledge in Swine

Abstract Optimal lean tissue accretion is contingent on the ability of the pig to efficiently establish and maintain host defenses, and to digest and absorb nutrients and water. Epithelial cells in the mucosa of the gastrointestinal tract interconnect with each other by tight junctions, adherens junctions, and desmosomes, forming a complete intestinal barrier. Of the secretory cells in this epithelial barrier, mucus secreting goblet cells and hormone producing enteroendocrine cells can be found interspersed, while Paneth cells lie in the base of the crypts and secrete antimicrobial proteins/peptides into the mucus layer. In the small intestine, enterocytes lining the villi allow for efficient nutrient transport. Maintenance of the epithelial barrier and its function is dependent on intestinal stem cells residing at the crypt base. Two major signaling pathways controlling intestinal stem cell proliferation and differentiation are the β-catenin/Wnt and Notch signaling pathways. Thus, achieving and maintaining optimal nutrient absorptive capacity requires an intricate balance amongst β-catenin/Wnt and Notch signaling pathways, wherein dysregulation of either can result in inefficiency of nutrient absorption. Weaning transition, off-feed events, hypophagia, and enteric and respiratory diseases can all compromise the structural and secretory barrier of the gastrointestinal tract mucosa via changes in tight and adherens junction proteins and mucin production. As a result, these stressors can increase the incidence of gastric ulcers, villous atrophy, and colitis in pigs. Depending on the severity and duration of the mucosal damage, repair and restitution occurs due to increased stem cell proliferation, migration, and repolarization. Thus, efficiency of digestive and absorptive function depends on several factors, including the types of epithelial cells present and their functionality, the rate of cell renewal, and epithelial surface area. This paper will discuss what we know about the regulation and repair of the mucosal epithelium in growing pigs during health, stress, and disease.

CD8 T cell response and its released cytokine IFN-γ are necessary for lung alveolar epithelial repair during bacterial pneumonia.

Alveolar epithelial regeneration depends on the activity of resident quiescent progenitor cells. Alveolar epithelial type II (AT2) cells are known as the alveolar epithelial progenitor cells. They exit quiescent state, proliferate rapidly in response to injury and differentiate into alveolar epithelial type I (AT1) cells to regenerate the damaged alveolar epithelium. Although AT2 cell plasticity has been a very intense field of research, the role of CD8 T cell response and their released cytokine IFN-γ, in regulating AT2 cell plasticity and alveolar epithelial repair and regeneration after injury remains largely unknown. We used flow cytometry to quantify the amount of CD8 T cells in mouse lungs after bacterial pneumonia caused by Streptococcus pneumoniae. To determine whether CD8 T cells and their released cytokine IFN-γ are necessary for AT2 cell activity during alveolar epithelial regeneration, we performed loss of function studies using anti-CD8 or anti-IFN-γ monoclonal antibody (mAb) treatment in vivo. We assessed the effects of CD8 T cells and cytokine IFN-γ on AT2 cell differentiation capacity using the AT2- CD8 T cell co-culture system in vitro. We detected a transient wave of accumulation of CD8 T cells in mouse lungs, which coincided with the burst of AT2 cell proliferation during alveolar epithelial repair and regeneration in mice following bacterial pneumonia caused by Streptococcus pneumoniae. Depletion of CD8 T cells or neutralization of cytokine IFN-γ using anti-CD8 or anti-IFN-γ monoclonal antibody significantly reduced AT2 cell proliferation and differentiation into AT1 cells in mice after bacterial pneumonia. Furthermore, co-culture of CD8 T cells or cytokine IFN-γ with AT2 cells promoted AT2-to-AT1 cell differentiation in both murine and human systems. Conversely, blockade of IFN-γ signaling abrogated the increase in AT2-to-AT1 cell differentiation in the AT2- CD8 T cell co-culture system. Our data demonstrate that CD8 T-cell response and cytokine IFN-γ are necessary for promoting AT2 cell activity during alveolar epithelial repair and regeneration after acute lung injury caused by bacterial pneumonia.

The novel peptide DR4penA attenuates the bleomycin- and paraquat-induced pulmonary fibrosis by suppressing the TGF-β/Smad signaling pathway.

Pulmonary fibrosis (PF), which is caused by continuous alveolar epithelial cell injury and abnormal repair, is referred to as a difficult disease of the lung system by the World Health Organization due to its rapid progression, poor prognosis, and high mortality rate. However, there is still a lack of ideal therapeutic strategies. The peptide DR8 (DHNNPQIR-NH2 ), which is derived from rapeseed, exerted antifibrotic activity in the lung, liver, and kidney in our previous studies. By studying the structure-activity relationship and rational design, we introduced an unnatural hydrophobic amino acid (α-(4-pentenyl)-Ala) into DR8 and screened the novel peptide DR4penA (DHNα-(4-pentenyl)-APQIR-NH2 ), which had higher anti-PF activity, higher antioxidant activity and a longer half-life than DR8. Notably, DR4penA attenuated bleomycin- and paraquat-induced PF, and the anti-PF activity of DR4penA was equivalent to that of pirfenidone. Additionally, DR4penA suppressed the TGF-β/Smad pathway in TGF-β1-induced A549 cells and paraquat-induced rats. This study demonstrates that the novel peptide DR4penA is a potential candidate compound for PF therapy, and its antifibrotic activity in different preclinical models of PF provides a theoretical basis for further study.

IL-20 Cytokines Are Involved in the Repair of Airway Epithelial Barrier: Implication in Exposure to Cigarette Smoke and in COPD Pathology.

Dysregulated inflammation as seen in chronic obstructive pulmonary disease (COPD) is associated with impaired wound healing. IL-20 cytokines are known to be involved in wound healing processes. The purpose of this study was to use ex vivo and in vitro approaches mimicking COPD to evaluate the potential modulatory role of interleukin-20 (IL-20) on the inflammatory and healing responses to epithelial wounding. The expression of IL-20 cytokines and their receptors was investigated in lung-derived samples collected from non-COPD and COPD patients, from mice chronically exposed to cigarette smoke and from airway epithelial cells from humans and mice exposed in vitro to cigarette smoke. To investigate the role of IL-20 cytokines in wound healing, experiments were performed using a blocking anti-IL-20Rb antibody. Of interest, IL-20 cytokines and their receptors were expressed in bronchial mucosa, especially on airway epithelial cells. Their expression correlated with the disease severity. Blocking these cytokines in a COPD context improved the repair processes after a lesion induced by scratching the epithelial layer. Collectively, this study highlights the implication of IL-20 cytokines in the repair of the airway epithelium and in the pathology of COPD. IL-20 subfamily cytokines might provide therapeutic benefit for patients with COPD to improve epithelial healing.

Prednisone-hydrogen sulfide releasing hybrid shows improved therapeutic profile in asthma.

Introduction: Hydrogen sulfide (H2S) is emerging as an important potential therapeutic option for respiratory inflammatory diseases. In this study, we investigated the effectiveness of a novel corticosteroid derivative, that is chemically linked to an H2S donor, in managing asthma features. Methods: The effects of prednisone (PS), H2S donor (4-hydroxybenzamide; TBZ), and their combination (PS-TBZ) have been evaluated in vitro and in vivo. The in vitro experiments were conducted using lipopolysaccharidestimulated J774 macrophages, while the in vivo experiments utilizing an experimental asthma model. Results: In the in vitro study we found that PS-TBZ exhibited an increased effect compared to the individual parent compounds in modulating the production of inflammatory mediators. TBZ also significantly reduced bronchial contractility and enhanced bronchial relaxation. In the in vivo experiments, where we administered PS, TBZ, or PS-TBZ to ovalbumin-sensitized BALB/c mice, we confirmed that PS-TBZ had a significantly better action in controlling airway hyperreactivity as compared to TBZ or PS alone. Moreover, PS-TBZ was more effective in restoring salbutamol-induced relaxation. The immunohistochemistry analysis demonstrated a significant reduction in the production of α-SMA and procollagen III, indicating the efficacy of PS-TBZ in controlling airway remodeling. Moreover, PS-TBZ also promoted epithelial repair, recovery of the bronchial and parenchyma structure and inhibited mucin production. Discussion: In conclusion, PS-TBZ offers an important opportunity to optimize the beneficial impact of corticosteroids on asthma features.

173 Role of CFTR in airway epithelial regeneration and repair: Studies from novel human basal cell lines

173 Role of CFTR in airway epithelial regeneration and repair: Studies from novel human basal cell lines

Corneal Neurotization: Preoperative Patient Workup and Surgical Decision-making.

The use of sensory nerve transfers to the anesthetic cornea has transformed the treatment of neurotrophic keratopathy by restoring ocular surface sensation and activating dysfunctional epithelial repair mechanisms. However, despite numerous reports on surgical techniques, there is a scarcity of information on the interdisciplinary management, preoperative assessment, and surgical decision-making, which are equally critical to treatment success. This Special Topic presents a standardized, interdisciplinary preoperative workup based on our 10-year experience with corneal neurotization in 32 eyes of patients with neurotrophic keratopathy. Our assessment includes a medical history review, ophthalmic evaluation, and systematic facial sensory donor nerve mapping for light touch and pain modalities. This approach enables evidence-based patient selection, optimal surgery timing, and suitable donor nerve identification, including backup options. Based on a decade-long experience, this special topic highlights the importance of interdisciplinary collaboration and provides a practical roadmap for optimizing patient selection and surgical decision-making in patients undergoing corneal neurotization.

Rhei Radix et Rhizoma in Xuanbai-Chengqi decoction strengthens the intestinal barrier function and promotes lung barrier repair in preventing severe viral pneumonia induced by influenza A virus

Ethnopharmacological relevanceXuanbai-Chengqi decoction (XCD) is a traditional prescription for treating multiple organ injuries, which has been used to manage pneumonia caused by various pathogens. However, the effects of XCD on repairing pulmonary/intestinal barrier damage remain unclear, and there is a need to understand the compatibility mechanism of rhubarb. Aim of the studyThis work aims to investigate the protective effect and mechanism of XCD on the pulmonary/intestinal barrier guided by the theory of “gut-lung concurrent treatment”. Moreover, we elucidate the compatibility mechanism of rhubarb in XCD. Materials and methodsAn H1N1 virus-infected mouse model was adopted to investigate the reparative effects of XCD on the lung-intestinal barrier by assessing lung-intestinal permeability. Additionally, the characterization of type I alveolar epithelial cells (AT1) and type II alveolar epithelial cells (AT2) was performed to evaluate the damage to the alveolar epithelial barrier. The specific barrier-protective mechanisms of XCD were elucidated by detecting tight junction proteins and the epithelial cell repair factor IL-22. The role of rhubarb in XCD to pneumonia treatment was investigated through lung tissue transcriptome sequencing and flow cytometry. ResultsXCD significantly improved lung tissue edema, inflammation, and alveolar epithelial barrier damage by regulating IL-6, IL-10, and IL-22, which, could further improve pulmonary barrier permeability when combined with the protection of alveolar epithelial cells (AT1 and AT2) as well as inhibition of H1N1 virus replication. Simultaneously, XCD significantly reduced intestinal inflammation and barrier damage by regulating IL-6, IL-1β, and tight junction protein levels (Claudin-1 and ZO-1), improving intestinal barrier permeability. The role of rhubarb in the treatment of pneumonia is clarified for the first time. In the progression of severe pneumonia, rhubarb can significantly protect the intestinal barrier, promote the repair of AT2 cells, and inhibit the accumulation of CD11b+Ly6Gvariable aberrant neutrophils by regulating the S100A8 protein. ConclusionIn summary, our findings suggest that rhubarb in XCD plays a critical role in protecting intestinal barrier function and promoting lung barrier repair in preventing severe viral pneumonia caused by influenza A virus.

Injectable hydrogels derived from marine polysaccharides as cell carriers for large corneal epithelial defects

Injectable hydrogels have been employed for sutureless repair of corneal epithelial defects, which can perfectly fit the defect sites and minimize the associated discomfort. However, numerous hydrogels are ineffective in treating large corneal epithelial defects and still suffer from poor biocompatibility or weak applicability when used as cell carriers. Herein, hydroxypropyl chitin/carboxymethyl chitosan (HPCT/CMCS) temperature-sensitive hydrogels are fabricated, and their physicochemical properties and suitability for corneal epithelial repair are investigated. The results demonstrate that HPCT/CMCS hydrogels have excellent temperature sensitivity between 20 and 25 °C and a transparency of over 80 %. Besides, HPCT/CMCS hydrogels can promote cell proliferation and facilitate cell migration of primary rabbit corneal epithelial cells (CEpCs). A rabbit large corneal epithelial defect model (6 mm) is established, and CEpCs are transplanted into defect sites by HPCT/CMCS hydrogels. The results suggest that HPCT/CMCS/CEpCs significantly enhance the repair of large corneal epithelial defects with a healing rate of 99.6 % on day 8, while reducing inflammatory responses and scarring formation. Furthermore, HPCT/CMCS/CEpCs can contribute to the reconstruction of damaged tissues and the recovery of functional capacities. Overall, HPCT/CMCS hydrogels may be a feasible corneal cell carrier material and can provide an alternative approach to large corneal epithelial defects.

Cigarette smoke restricts the ability of mesenchymal cells to support lung epithelial organoid formation.

Adequate lung epithelial repair relies on supportive interactions within the epithelial niche, including interactions with WNT-responsive fibroblasts. In fibroblasts from patients with chronic obstructive pulmonary disease (COPD) or upon in vitro cigarette smoke exposure, Wnt/β-catenin signalling is distorted, which may affect interactions between epithelial cells and fibroblasts resulting in inadequate lung repair. We hypothesized that cigarette smoke (CS), the main risk factor for COPD, interferes with Wnt/β-catenin signalling in fibroblasts through induction of cellular stress responses, including oxidative- and endoplasmic reticulum (ER) stress, and thereby alters epithelial repair support potential. Therefore, we assessed the effect of CS-exposure and the ER stress inducer Thapsigargin (Tg) on Wnt/β-catenin signalling activation in MRC-5 fibroblasts, and on their ability to support lung epithelial organoid formation. Exposure of MRC-5 cells for 15min with 5AU/mL CS extract (CSE), and subsequent 6h incubation induced oxidative stress (HMOX1). Whereas stimulation with 100nMTg increased markers of both the integrated stress response (ISR - GADD34/PPP1R15A, CHOP) and the unfolded protein response (UPR - XBP1spl, GADD34/PPP1R15A, CHOP and HSPA5/BIP), CSE only induced GADD34/PPP1R15A expression. Strikingly, although treatment of MRC-5 cells with the Wnt activator CHIR99021 upregulated the Wnt/β-catenin target gene AXIN2, this response was diminished upon CSE or Tg pre-exposure, which was confirmed using a Wnt-reporter. Furthermore, pre-exposure of MRC-5 cells to CSE or Tg, restricted their ability to support organoid formation upon co-culture with murine pulmonary EpCam+ cells in Matrigel at day 14. This restriction was alleviated by pre-treatment with CHIR99021. We conclude that exposure of MRC-5 cells to CSE increases oxidative stress, GADD34/PPP1R15A expression and impairs their ability to support organoid formation. This inhibitory effect may be restored by activating the Wnt/β-catenin signalling pathway.

Triphenyl phosphate-induced pericardial edema in zebrafish embryos is reversible following depuration in clean water

Triphenyl phosphate (TPHP) – a widely used organophosphate-based flame retardant – blocks cardiac looping during zebrafish development in a concentration-dependent manner, a phenotype that is dependent on disruption of embryonic osmoregulation and pericardial edema formation. However, it's currently unclear whether (1) TPHP-induced effects on osmoregulation are driven by direct TPHP-induced injury to the embryonic epidermis and (2) whether TPHP-induced pericardial edema is reversible or irreversible following cessation of exposure. Therefore, the objectives of this study were to determine whether TPHP-induced pericardial edema is reversible and whether TPHP causes injury to the embryonic epidermis by quantifying the number of DAPI-positive epidermal cells and analyzing the morphology of the yolk sac epithelium using scanning electron microscopy. First, we found that exposure to 5 μM TPHP from 24–72 h post-fertilization (hpf) did not increase prolactin – a hormone that regulates ions and water levels – in embryonic zebrafish, whereas high ionic strength exposure media was associated with elevated levels of prolactin. Second, we found that exposure to 5 μM TPHP from 24–72 hpf did not decrease DAPI-positive epidermal cells within the embryonic epithelium, and that co-exposure with 2.14 μM fenretinide – a synthetic retinoid that promotes epithelial wound repair – from 24–72 hpf did not mitigate the prevalence of TPHP-induced epidermal folds within the yolk sac epithelium when embryos were exposed within high ionic strength exposure media. Finally, we found that the pericardial area and body length of embryos exposed to 5 μM TPHP from 24–72 hpf were similar to vehicle-treated embryos at 120 hpf following transfer to clean water and depuration of TPHP from 72–120 hpf. Overall, our findings suggest that (1) the ionic strength of exposure media may influence the baseline physiology of zebrafish embryos; (2) TPHP does not cause direct injury to the embryonic epidermis; and (3) TPHP-induced effects on pericardial area and body length are reversible 48 h after transferring embryos to clean water.