Cell Junction Proteins Research Articles

Altered pulmonary capillary permeability in immunosuppressed guinea pigs infected with Legionella pneumophila serogroup 1.

In immunosuppressed hosts, Legionella pneumophila (Lp) infection usually develops into severe pneumonia, which is pathologically characterized by increased vascular permeability and pulmonary edema. At present, mechanisms associated with changes in pulmonary capillary permeability (PCP) and the pathogenesis of pulmonary edema in immunosuppressed hosts with Lp infection are unclear. Therefore, in the present study an animal model of normal and immunosuppressed guinea pigs infected with Lp was established. An isolated perfused lung system was used to investigate the extent of changes in PCP. Pathological and immunofluorescence examinations were performed to explore the mechanism underlying these changes. The results indicated that PCP increased with the highest magnitude in immunosuppressed guinea pigs infected with Lp, with repeated ANOVA indicating synergism between infection and immunosuppression (P=0.0444). Hematoxylin and eosin staining and electron microscopy revealed more severe morphological damages in the lung tissues and pulmonary capillaries of the immunosuppressed animals infected with Lp compared with normal animals infected with Lp. Immunofluorescence analysis showed that immunosuppression reduced the expression of the vascular endothelial cell junction protein VE-cadherin (P=0.027). Following Lp infection, VE-cadherin expression was significantly lower in the immunosuppressed guinea pigs compared with their immunocompetent counterparts (P=0.001). These results suggest that immunosuppression combined with Lp infection induces more significant damage to pulmonary capillaries compared with Lp infection alone, resulting in a significantly increased PCP.

DPP4 inhibitor reinforces cell junction proteins in mouse model of short bowel syndrome.

Bacterial overgrowth commonly occurs and favors bacterial translocation in short bowel syndrome (SBS). Glucagon-like peptide-2 (GLP-2) is effective for treating SBS, but is rapidly inactivated by dipeptidyl peptidase IV (DPP4). DPP4 inhibitor (DPP4I) is known to be effective for treating SBS. Here, we investigated cell junction protein function following DPP4I administration in a mouse model of SBS. Mice were divided into four groups: naïve (n = 5), naïve + DPP4I (n = 6), control (n = 6), and DPP4I (n = 5). All control and DPP4I mice had 50% of their proximal small bowel resected. DPP4I or normal saline was administered orally twice daily from days 1-7 postoperatively. The functions of cell junction proteins were assessed by RT-PCR and immunohistochemistry. Body weights and blood glucose levels were recorded. E-Cadherin was significantly higher in the DPP4I group than in the control group. E-Cadherin, occludin, and claudin-4 were significantly higher in the naïve group than in the control group. Positive staining for E-cadherin and occludin varied widely between the control and DPP4I groups. Up-regulation of E-cadherin and occludin by DPP4I may be correlated with the anti-inflammatory action of DPP4I. Therefore, DPP4I may reduce bacterial translocation in SBS.

Geniposide attenuates neonatal mouse brain injury after hypoxic-ischemia involving the activation of PI3K/Akt signaling pathway

Perinatal hypoxic-ischemia (HI) is a leading cause of acute mortality and neurologic complications in newborns. Geniposide, a natural product extracted from the herb Gardenia jasminoides, has been shown to possess neuroprotective effects in neurologic deficits. This study aims to investigate whether Geniposide has therapeutic potential to HI brain injury and the underlying mechanisms. C57/bl6 mice were subjected to HI insult on postnatal day 10. Geniposide (20 mg/kg b.w.) was administered intragastrically every day after HI insult for 7 successional days. Then mice at P18 were sacrificed and brain tissues were collected for further analysis. Geniposide treatment significantly inhibited cell apoptosis, reduced serum IgG leakage into brain tissue, attenuated astrogliosis and microgliosis, prevented loss of pericytes, loss of tight junction and adherens junction proteins. The PI3K/Akt signaling pathway, which related proteins were downregulated after HI insult, was activated by Geniposide treatment. Geniposide treatment after neonatal HI insult attenuated HI-induced cell apoptosis, IgG leakage, microgliosis, astrogliosis, pericytes loss and junction protein degradation. Geniposide could protect against HI-induced brain injury, which might be through the activation of PI3K/Akt signaling pathway.

Microtubules: Evolving roles and critical cellular interactions.

Microtubules are cytoskeletal elements known as drivers of directed cell migration, vesicle and organelle trafficking, and mitosis. In this review, we discuss new research in the lens that has shed light into further roles for stable microtubules in the process of development and morphogenesis. In the lens, as well as other systems, distinct roles for characteristically dynamic microtubules and stabilized populations are coming to light. Understanding the mechanisms of microtubule stabilization and the associated microtubule post-translational modifications is an evolving field of study. Appropriate cellular homeostasis relies on not only one cytoskeletal element, but also rather an interaction between cytoskeletal proteins as well as other cellular regulators. Microtubules are key integrators with actin and intermediate filaments, as well as cell–cell junctional proteins and other cellular regulators including myosin and RhoGTPases to maintain this balance.Impact statementThe role of microtubules in cellular functioning is constantly expanding. In this review, we examine new and exciting fields of discovery for microtubule’s involvement in morphogenesis, highlight our evolving understanding of differential roles for stabilized versus dynamic subpopulations, and further understanding of microtubules as a cellular integrator.

High Mobility Group Box 1 Mediates TMAO-Induced Endothelial Dysfunction.

The intestinal microbe-derived metabolite trimethylamine N-oxide (TMAO) is implicated in the pathogenesis of cardiovascular diseases (CVDs). The molecular mechanisms of how TMAO induces atherosclerosis and CVDs’ progression are still unclear. In this regard, high-mobility group box protein 1 (HMGB1), an inflammatory mediator, has been reported to disrupt cell–cell junctions, resulting in vascular endothelial hyper permeability leading to endothelial dysfunction. The present study tested whether TMAO associated endothelial dysfunction results via HMGB1 activation. Biochemical and RT-PCR analysis showed that TMAO increased the HMGB1 expression in a dose-dependent manner in endothelial cells. However, prior treatment with glycyrrhizin, an HMGB1 binder, abolished the TMAO-induced HMGB1 production in endothelial cells. Furthermore, Western blot and immunofluorescent analysis showed significant decrease in the expression of cell–cell junction proteins ZO-2, Occludin, and VE-cadherin in TMAO treated endothelial cells compared with control cells. However, prior treatment with glycyrrhizin attenuated the TMAO-induced cell–cell junction proteins’ disruption. TMAO increased toll-like receptor 4 (TLR4) expression in endothelial cells. Inhibition of TLR4 expression by TLR4 siRNA protected the endothelial cells from TMAO associated tight junction protein disruption via HMGB1. In conclusion, our results demonstrate that HMGB1 is one of the important mediators of TMAO-induced endothelial dysfunction.

Role of lactadherin in intestinal barrier integrity in experimental neonatal necrotizing enterocolitis

Necrotizing enterocolitis (NEC) is one of the most widespread and devastating gastrointestinal diseases in neonates. Destruction of the intestinal barrier is the main underlying cause of NEC. The aim of this study was to determine the role of lactadherin in preventing NEC in a neonatal rat model and investigate the molecular mechanism of lactadherin-mediated protection of the intestinal barrier. Neonatal rats were divided into three groups: dam feeding (DF), NEC (NEC), and NEC supplemented with 10 μg/(g·day) recombinant human lactadherin (NEC+L). Intestinal permeability, tissue damage, and cell junction protein expression and localization were evaluated. We found that lactadherin reduced weight loss caused by NEC, reduced the incidence of NEC from 100% to 46.7%, and reduced the mean histological score for tissue damage to 1.40 compared with 2.53 in the NEC group. Intestinal permeability of lactadherin-treated rats was significantly reduced when compared with that of the NEC group. In addition, the expression levels of JAM-A, claudin 3, and E-calcium in the ileum of NEC group animals increased compared with those in the ileum of DF group animals, and these levels decreased in the NEC+L group. Lactadherin changed the localization of claudin 3, occludin, and E-cadherin in epithelial cells. The mechanism underlying lactadherin-mediated protection of the intestinal barrier might be restoring the correct expression levels and localization of tight junction and adherent junction proteins. These findings suggest a new candidate agent for the prevention of NEC in newborns.

Proximity biotinylation identifies a set of conformation-specific interactions between Merlin and cell junction proteins.

Neurofibromatosis type 2 is an inherited, neoplastic disease associated with schwannomas, meningiomas, and ependymomas and that is caused by inactivation of the tumor suppressor gene NF2 The NF2 gene product, Merlin, has no intrinsic catalytic activity; its tumor suppressor function is mediated through the proteins with which it interacts. We used proximity biotinylation followed by mass spectrometry and direct binding assays to identify proteins that associated with wild-type and various mutant forms of Merlin in immortalized Schwann cells. We defined a set of 52 proteins in close proximity to wild-type Merlin. Most of the Merlin-proximal proteins were components of cell junctional signaling complexes, suggesting that additional potential interaction partners may exist in adherens junctions, tight junctions, and focal adhesions. With mutant forms of Merlin that cannot bind to phosphatidylinositol 4,5-bisphosphate (PIP2) or that constitutively adopt a closed conformation, we confirmed a critical role for PIP2 binding in Merlin function and identified a large cohort of proteins that specifically interacted with Merlin in the closed conformation. Among these proteins, we identified a previously unreported Merlin-binding protein, apoptosis-stimulated p53 protein 2 (ASPP2, also called Tp53bp2), that bound to closed-conformation Merlin predominately through the FERM domain. Our results demonstrate that Merlin is a component of cell junctional mechanosensing complexes and defines a specific set of proteins through which it acts.

Ventilator-induced lung injury is alleviated by inhibiting NLRP3 inflammasome activation

BackgroundMechanical ventilation (MV) is frequently used but can aggravate or cause lung injury, known as ventilator-induced lung injury (VILI). However, the mechanisms are unclear. The NLR family pyrin domain containing 3 (NLRP3) inflammasome is a vital component of innate immunity and is closely related to VILI. MethodsMouse lung epithelial (MLE-12) cells were transfected with NLRP3 small interfering RNA (siRNA) or scramble siRNA (sc siRNA) and subjected to 20% cyclic stretch (CS). Wild-type C57BL/6 mice were injected with a liquid complex of NLRP3 siRNA/sc siRNA-Lipofectamine 2000 through the fundus venous plexus before mechanical ventilation. Western blots, immunoprecipitation, ELISAs, flow cytometry, immunofluorescence, and hematoxylin-eosin staining were used to assess the effects of the NLRP3 inflammasome on VILI and the mechanisms of those effects. ResultsCS activated the NLRP3 inflammasome by activating NIMA-related kinase 7 (NEK7). NLRP3 depletion inhibited NLRP3 inflammasome activation; alleviated the degradation of cell junction proteins, including p120-catenin (p120) and occludin; ameliorated the colocalization of p120 and E-cadherin; and mitigated the decrease in mitochondrial membrane potential caused by mechanical stretch. Furthermore, after NLRP3 depletion, VILI was attenuated by decreasing IL-1β secretion and pulmonary edema. ConclusionsInhibiting NLRP3 inflammasome activation ameliorated VILI, suggesting a potential therapeutic target for the clinical treatment of VILI.

The Caspase-3 homolog DrICE regulates endocytic trafficking during Drosophila tracheal morphogenesis

Although well known for its role in apoptosis, the executioner caspase DrICE has a non-apoptotic function that is required for elongation of the epithelial tubes of the Drosophila tracheal system. Here, we show that DrICE acts downstream of the Hippo Network to regulate endocytic trafficking of at least four cell polarity, cell junction and apical extracellular matrix proteins involved in tracheal tube size control: Crumbs, Uninflatable, Kune-Kune and Serpentine. We further show that tracheal cells are competent to undergo apoptosis, even though developmentally-regulated DrICE function rarely kills tracheal cells. Our results reveal a developmental role for caspases, a pool of DrICE that co-localizes with Clathrin, and a mechanism by which the Hippo Network controls endocytic trafficking. Given reports of in vitro regulation of endocytosis by mammalian caspases during apoptosis, we propose that caspase-mediated regulation of endocytic trafficking is an evolutionarily conserved function of caspases that can be deployed during morphogenesis.

Morphological and immunohistochemical characteristics of the equine corneal epithelium.

ObjectiveThe morphology of the corneal epithelium in two age groups of horses is described. Distribution patterns of proliferation‐, differentiation‐, stem cell‐associated markers and cell junction proteins were assessed.MethodsCorneal samples from 12 horses (six foals and six adult horses) were analyzed after H&E staining and immunohistochemistry using the following antibodies: E‐cadherin, β‐catenin, Connexin 43 (Cx43), tight junction protein 1 (TJP1), cytokeratin (CK) 14, CK 19, CK 3, CK 10, vimentin, Ki67, p63, nerve growth factor (NGF), ABCG2, and epithelial growth factor receptor. Semiquantitative analysis of crypt, limbal, peripheral, and central zone was performed. Semithin and ultrathin sections were used for ultrastructural evaluation of the epithelium.ResultsThe height of the epithelium varied between age groups and crypts were consistently present. In the peripheral and central epithelium, three types of basal cells resembling a pseudostratified epithelium were characterized. Potential stem cell markers (CK 14, p63, NGF, and ABCG2) were present in all zones with decreasing frequency toward the center. Cornea‐specific differentiation marker CK 3 was not expressed in the most basal cell layer of the limbal epithelium. E‐cadherin, β‐catenin, and Cx43 revealed a similar apico‐lateral signal pattern throughout the entire epithelium; only TJP1 was additionally seen at the basal surface.ConclusionsThis study presents a systematic semiquantitative evaluation of the equine corneal epithelium, showing the presence of crypts as potential stem cell niche with CK 14, p63, NGF, and ABCG2 as relevant markers for cells with regenerative capacity. The pseudostratified arrangement of the basal layer was a unique finding.

Cell Junction Proteins-Mimetic Artificial Nanochannel System: Basic Logic Gates Implemented by Nanofluidic Diodes.

Inspired by communication modes of cell junction proteins, an artificial bichannel nanofluidic diode system was constructed and investigated to implement basic "AND" and "OR" logic gates in different connection modes. Input conditions were set as conducting and nonconducting states of each nanofluidic diode unit. Output results were set as response current of the system at rated voltage. Besides, nanofluidic diodes with different ionic permeabilities were connected in multiple modes, and different logic operation results were obtained. This novel logic device based on nanofluidic diodes provided a new approach to establish diverse stimuli-responsive signal processing networks and has prospect to obtain nanofluidic diode intelligence chips by integrating in large scale.

Matriptase drives early-onset intestinal failure in a mouse model of congenital tufting enteropathy.

Syndromic congenital tufting enteropathy (CTE) is a life-threatening recessive human genetic disorder that is caused by mutations in SPINT2, encoding the protease inhibitor HAI-2, and is characterized by severe intestinal dysfunction. We recently reported the generation of a Spint2-deficient mouse model of CTE. Here, we show that the CTE-associated early-onset intestinal failure and lethality of Spint2-deficient mice is caused by unchecked activity of the serine protease matriptase. Macroscopic and histological defects observed in the absence of HAI-2, including villous atrophy, luminal bleeding, loss of mucin-producing goblet cells, loss of defined crypt architecture and the resulting acute inflammatory response in the large intestine, were all prevented by intestinal-specific inactivation of the St14 gene encoding matriptase. The CTE-associated loss of the cell junctional proteins EpCAM and claudin 7 was also prevented. As a result, inactivation of intestinal matriptase allowed Spint2-deficient mice to gain weight after birth and dramatically increased their lifespan. These data implicate matriptase as a causative agent in the development of CTE and may provide a new target for the treatment of CTE in individuals carrying SPINT2 mutations.This article has an associated 'The people behind the papers' interview.

Immunohistochemical expression of E–cadherin in different tissues of the teleost fish Scophthalmus maximus

E–cadherin is an evolutionary conserved protein, whose main role as the principal component of adherens junctions is supporting epithelial cell–cell adhesion. It is an essential molecule for the maintenance of the epithelial barrier function and the analysis of its immunohistochemical expression is a valuable resource in morphopathological, ontogenetic and pathogenesis studies in mammals. As well, there is an increasing understanding of the importance of E-cadherin in the physiology of the immune system and the development of the immune response. Mucosal health is a primary issue in aquaculture research; nevertheless, there is a lack of immunohistochemical studies of cell junction proteins in fish species. In this work, an immunohistochemical technique was optimized in Bouin- and formalin-fixed paraffin-embedded tissues of turbot Scophthalmus maximus, employing a commercial antibody raised against human E-cadherin. The specificity of the antibody in recognizing the molecule in this teleost species was tested by western blot and mass spectrometry-based proteomic analyses. The assays showed a good specificity and indicated that the antibody recognizes the well conserved cytoplasmic domain of the protein. Immunohistochemistry showed the localisation of E-cadherin at cell-cell contact in the epithelia of the different organs, between the hepatocytes and the pancreatic acinar cells, as well as in the reticulo-epithelial stroma of the thymus. Also, the immunoreaction was observed in the cells constituting the melano-macrophage centres in the spleen and kidney. No immunostaining was detected, as expected, only in the heart and brain. No significant difference was noticed between the two fixative used for collecting the tissues samples. This is the first description of E-cadherin immunohistochemical expression in several tissues of a teleost. The immunohistochemical technique represents a useful tool to be used in the different areas of fish health research.

MiR-218 regulated cardiomyocyte differentiation and migration in mouse embryonic stem cells by targeting PDGFRα.

MicroRNAs (miRNAs) have been identified as key players in cardiogenesis and heart pathophysiological processes. However, many miRNAs are still not recognized for their roles in cardiomyocytes differentiation. In this study, we evaluated the effects of microRNA-218 (miR-218) in cardiomyocyte differentiation of the mouse embryonic stem cells (ESCs) in vitro. The percentage of the beating embryoid bodies (EBs) in miR-218 mimic-treated cells was reduced to 32% compared with miR-218 mimic negative control (56%) on day 5 + 3. The amplitude of the intracellular Ca2+ transients in the cardiomyocytes derived from ESCs was reduced upon miR-218 overexpression, followed by the decreased calcium-related proteins and cell junction proteins expressions. Besides, miR-218 expression in ESCs was related to the directional spreading ability of EBs during differentiation. The increased expression of miR-218 could promote the migration of ESCs in vitro, while the decreased expression of miR-218 could inhibit the migration by the transwell experiment. Meanwhile, miR-218 could regulate cell migration-related proteins Cdc42 and Rac1. Platelet-derived growth factor receptor α (PDGFRα) was further confirmed to be a direct target of miR-218 both physically and functionally by dual-luciferase reporter assay. Our data further described that overexpression of PDGFRα rescued the miR-218-mediated inhibition of cardiomyocyte differentiation and restored the miR-218-mediated promotion of cell migration. In conclusion, miR-218 was demonstrated to exert an inhibitory function and promoted cell migration via targeting PDGFRα during cardiomyocyte differentiation from ESCs. The current study revealed the role of miR-218 and may provide an important hint for cardiomyocyte differentiation of ESCs and induced pluripotent stem cells.

Establishment of keratinocyte cell lines from human hair follicles

The advent of organotypic skin models advanced the understanding of complex mechanisms of keratinocyte differentiation. However, these models are limited by both availability of primary keratinocytes and donor variability. Keratinocytes derived from cultured hair follicles and interfollicular epidermis were immortalized by ectopic expression of SV40 and hTERT. The generated keratinocyte cell lines differentiated into stratified epidermis with well-defined stratum granulosum and stratum corneum in organotypic human skin models. They behaved comparable to primary keratinocytes regarding the expression of differentiation-associated proteins, cell junction components and proteins associated with cornification and formed a barrier against biotin diffusion. Mechanistically, we found that SV40 large T-antigen expression, accompanied by a strong p53 accumulation, was only detectable in the basal layer of the in vitro reconstructed epidermis. Inhibition of DNA-methylation resulted in expression of SV40 large T-antigen also in the suprabasal epidermal layers and led to incomplete differentiation of keratinocyte cell lines. Our study demonstrates the generation of keratinocyte cell lines which are able to fully differentiate in an organotypic skin model. Since hair follicles, as source for keratinocytes, can be obtained by minimally invasive procedures, our approach enables the generation of cell lines also from individuals not available for skin biopsies.

A Cell Junctional Protein Network Associated with Connexin-26.

GJB2 mutations are the leading cause of non-syndromic inherited hearing loss. GJB2 encodes connexin-26 (CX26), which is a connexin (CX) family protein expressed in cochlea, skin, liver, and brain, displaying short cytoplasmic N-termini and C-termini. We searched for CX26 C-terminus binding partners by affinity capture and identified 12 unique proteins associated with cell junctions or cytoskeleton (CGN, DAAM1, FLNB, GAPDH, HOMER2, MAP7, MAPRE2 (EB2), JUP, PTK2B, RAI14, TJP1, and VCL) by using mass spectrometry. We show that, similar to other CX family members, CX26 co-fractionates with TJP1, VCL, and EB2 (EB1 paralogue) as well as the membrane-associated protein ASS1. The adaptor protein CGN (cingulin) co-immuno-precipitates with CX26, ASS1, and TJP1. In addition, CGN co-immunoprecipitation with CX30, CX31, and CX43 indicates that CX association is independent on the CX C-terminus length or sequence. CX26, CGN, FLNB, and DAMM1 were shown to distribute to the organ of Corti and hepatocyte plasma membrane. In the mouse liver, CX26 and TJP1 co-localized at the plasma membrane. In conclusion, CX26 associates with components of other membrane junctions that integrate with the cytoskeleton.

Vitamin A Deficiency and the Lung.

Vitamin A (all-trans-retinol) is a fat-soluble micronutrient which together with its natural derivatives and synthetic analogues constitutes the group of retinoids. They are involved in a wide range of physiological processes such as embryonic development, vision, immunity and cellular differentiation and proliferation. Retinoic acid (RA) is the main active form of vitamin A and multiple genes respond to RA signalling through transcriptional and non-transcriptional mechanisms. Vitamin A deficiency (VAD) is a remarkable public health problem. An adequate vitamin A intake is required in early lung development, alveolar formation, tissue maintenance and regeneration. In fact, chronic VAD has been associated with histopathological changes in the pulmonary epithelial lining that disrupt the normal lung physiology predisposing to severe tissue dysfunction and respiratory diseases. In addition, there are important alterations of the structure and composition of extracellular matrix with thickening of the alveolar basement membrane and ectopic deposition of collagen I. In this review, we show our recent findings on the modification of cell-junction proteins in VAD lungs, summarize up-to-date information related to the effects of chronic VAD in the impairment of lung physiology and pulmonary disease which represent a major global health problem and provide an overview of possible pathways involved.

Gold Nanorod Photothermal Therapy Alters Cell Junctions and Actin Network in Inhibiting Cancer Cell Collective Migration.

Most cancer-related deaths come from metastasis. It was recently discovered that nanoparticles could inhibit cancer cell migration. Whereas most researchers focus on single-cell migration, the effect of nanoparticle treatment on collective cell migration has not been explored. Collective migration occurs commonly in many types of cancer metastasis, where a group of cancer cells move together, which requires the contractility of the cytoskeleton filaments and the connection of neighboring cells by the cell junction proteins. Here, we demonstrate that gold nanorods (AuNRs) and the introduction of near-infrared light could inhibit the cancer cell collective migration by altering the actin filaments and cell junctions with significantly triggered phosphorylation changes of essential proteins, using mass spectrometry-based phosphoproteomics. Further observation using super-resolution stochastic optical reconstruction microscopy (STORM) showed the actin cytoskeleton filament bundles were disturbed, which is difficult to differentiate under a normal fluorescence microscope. The decreased expression level of N-cadherin junctions and morphological changes of tight junction protein zonula occludens 2 were also observed. All of these results indicate possible functions of the AuNR treatments in regulating and remodeling the actin filaments and cell junction proteins, which contribute to decreasing cancer cell collective migration.

Y-27632 preconditioning enhances transplantation of human-induced pluripotent stem cell-derived cardiomyocytes in myocardial infarction mice.

The effectiveness of cell-based treatments for regenerative myocardial therapy is limited by low rates of cell engraftment. Y-27632 inhibits Rho-associated protein kinase (ROCK), which regulates the cytoskeletal changes associated with cell adhesion, and has been used to protect cultured cells during their passaging. Here, we investigated whether preconditioning of cardiomyocytes, derived from human-induced pluripotent stem cells (hiPSC-CM), with Y-27632 improves their survival and engraftment in a murine model of acute myocardial infarction (MI). After MI induction, mice were subjected to intramyocardial injections of phosphate-buffered saline, hiPSC-CM cultured under standard conditions (hiPSC-CM-RI), or Y-27632-preconditioned hiPSC-CM (hiPSC-CM+RI). The resulting engraftment rate calculated 4 weeks after implantation was significantly higher and the abundance of apoptotic transplanted cells was significantly lower in hiPSC-CM+RI recipients than in hiPSC-CM-RI animals. In cultured hiPSC-CM, Y-27632-preconditioning reversibly reduced contractile activity and the expression of troponin genes, while increasing their attachment to an underlying mouse cardiomyocyte (HL1) monolayer. Y-27632 preconditioning also increased the expression of N-cadherin and integrin ß1, the two cell junction proteins. hiPSC-CM+RI were also larger in cell area with greater cytoskeletal alignment and a more rod-like shape than hiPSC-CM-RI, both after transplantation (in vivo) and in culture. The effects of Y-27632 preconditioning on contractile activity and morphology of hiPSC-CMs in culture, as well as on their engraftment rate and apoptotic death in MI mouse grafts, could be recapitulated by hiPSC-CM treatment with the L-type calcium-channel blocker verapamil. Preconditioning with the ROCK inhibitor Y-27632 increased the engraftment of transplanted hiPSC-CM in a murine MI model, while reversibly impairing hiPSC-CM contractility and promoting adhesion.

Deficiency in class III PI3-kinase confers postnatal lethality with IBD-like features in zebrafish

The class III PI3-kinase (PIK3C3) is an enzyme responsible for the generation of phosphatidylinositol 3-phosphate (PI3P), a critical component of vesicular membrane. Here, we report that PIK3C3 deficiency in zebrafish results in intestinal injury and inflammation. In pik3c3 mutants, gut tube forms but fails to be maintained. Gene expression analysis reveals that barrier-function-related inflammatory bowel disease (IBD) susceptibility genes (e-cadherin, hnf4a, ttc7a) are suppressed, while inflammatory response genes are stimulated in the mutants. Histological analysis shows neutrophil infiltration into mutant intestinal epithelium and the clearance of gut microbiota. Yet, gut microorganisms appear dispensable as mutants cultured under germ-free condition have similar intestinal defects. Mechanistically, we show that PIK3C3 deficiency suppresses the formation of PI3P and disrupts the polarized distribution of cell-junction proteins in intestinal epithelial cells. These results not only reveal a role of PIK3C3 in gut homeostasis, but also provide a zebrafish IBD model.