Formation Of Intercellular Junctions Research Articles

Dragon's blood attenuates LPS-induced intestinal epithelial barrier dysfunction via upregulation of FAK-DOCK180-Rac1-WAVE2-Arp3 and downregulation of TLR4/NF-κB signaling pathways.

Inflammation-induced intestinal epithelial barrier (IEB) dysfunction is one of the important reasons for the occurrence and development of intestinal inflammatory-related diseases, including ulcerative colitis (UC), Crohn's disease and necrotizing enterocolitis (NEC). Dragon's blood (DB) is a traditional Chinese medicine and has been clinically used to treat UC. However, the protective mechanism of DB on intestinal inflammatory-related diseases has still not been elucidated. The present study aimed to explore the protection mechanism of DB on IEB dysfunction in rat ileum and human colorectal adenocarcinoma cells (Caco-2)/human umbilical vein endothelial cells (HUVECs) coculture system induced by lipopolysaccharide (LPS). DB could ameliorate rat ileum mucosa morphological injury, reduce the accumulation of lipid-peroxidation products and increase the expression of junction proteins. DB also alleviated LPS-induced Caco-2 cells barrier integrity destruction in Caco-2/ HUVECs coculture system, leading to increased trans-endothelial electrical resistance (TEER), reduced cell permeability, and upregulation of expressions of F-actin and junction proteins. DB contributed to the assembly of actin cytoskeleton by upregulating the FAK-DOCK180-Rac1-WAVE2-Arp3 pathway and contributed to the formation of intercellular junctions by downregulating TLR4-MyD88-NF-κB pathway, thus reversing LPS-induced IEB dysfunction. These novel findings illustrated the potential protective mechanism of DB on intestinal inflammatory-related diseases and might be useful for further clinical application of DB.

Titanium nanotopography enhances mechano-response of osteocyte three-dimensional network toward osteoblast activation

The bone turnover capability influences the acquisition and maintenance of osseointegration. The architectures of osteocyte three-dimensional (3D) networks determine the direction and activity of bone turnover through osteocyte intercellular crosstalk, which exchanges prostaglandins through gap junctions in response to mechanical loading. Titanium nanosurfaces with anisotropically patterned dense nanospikes promote the development of osteocyte lacunar-canalicular networks. We investigated the effects of titanium nanosurfaces on intercellular network development and regulatory capabilities of bone turnover in osteocytes under cyclic compressive loading. MLO-Y4 mouse osteocyte-like cell lines embedded in type I collagen 3D gels on titanium nanosurfaces promoted the formation of intercellular networks and gap junctions even under static culture conditions, in contrast to the poor intercellular connectivity in machined titanium surfaces. The osteocyte 3D network on the titanium nanosurfaces further enhanced gap junction formation after additional culturing under cyclic compressive loading simulating masticatory loading, beyond the degree observed on machined titanium surfaces. A prostaglandin synthesis inhibitor cancelled the dual effects of titanium nanosurfaces and cyclic compressive loading on the upregulation of gap junction-related genes in the osteocyte 3D culture. Supernatants from osteocyte monolayer culture on titanium nanosurfaces promoted osteocyte maturation and intercellular connections with gap junctions. With cyclic loading, titanium nanosurfaces induced expression of the regulatory factors of bone turnover in osteocyte 3D cultures, toward higher osteoblast activation than that observed on machined surfaces. Titanium nanosurfaces with anisotropically patterned dense nanospikes promoted intercellular 3D network development and regulatory function toward osteoblast activation in osteocytes activated by cyclic compressive loading, through intercellular crosstalk by prostaglandin.

Molecular and Cellular Characterization of Primary Endothelial Cells from a Familial Cavernomatosis Patient.

Cerebral cavernous malformation (CCM) or familial cavernomatosis is a rare, autosomal dominant, inherited disease characterized by the presence of vascular malformations consisting of blood vessels with an abnormal structure in the form of clusters. Based on the altered gene (CCM1/Krit1, CCM2, CCM3) and its origin (spontaneous or familial), different types of this disease can be found. In this work we have isolated and cultivated primary endothelial cells (ECs) from peripheral blood of a type 1 CCM patient. Differential functional and gene expression profiles of these cells were analyzed and compared to primary ECs from a healthy donor. The mutation of the familial index case consisted of a heterozygous point mutation in the position +1 splicing consensus between exons 15 and 16, causing failure in RNA processing and in the final protein. Furthermore, gene expression analysis by quantitative PCR revealed a decreased expression of genes involved in intercellular junction formation, angiogenesis, and vascular homeostasis. Cell biology analysis showed that CCM1 ECs were impaired in angiogenesis and cell migration. Taken together, the results obtained suggest that the alterations found in CCM1 ECs are already present in the heterozygous condition, suffering from vascular impairment and somewhat predisposed to vascular damage.

Multi-Omics Profiling of Human Endothelial Cells from the Coronary Artery and Internal Thoracic Artery Reveals Molecular but Not Functional Heterogeneity.

Major adverse cardiovascular events occurring upon coronary artery bypass graft surgery are typically accompanied by endothelial dysfunction. Total arterial revascularisation, which employs both left and right internal thoracic arteries instead of the saphenous vein to create a bypass, is associated with better mid- and long-term outcomes. We suggested that molecular profiles of human coronary artery endothelial cells (HCAECs) and human internal mammary artery endothelial cells (HITAECs) are coherent in terms of transcriptomic and proteomic signatures, which were then investigated by RNA sequencing and ultra-high performance liquid chromatography-mass spectrometry, respectively. Both HCAECs and HITAECs overexpressed molecules responsible for the synthesis of extracellular matrix (ECM) components, basement membrane assembly, cell-ECM adhesion, organisation of intercellular junctions, and secretion of extracellular vesicles. HCAECs were characterised by higher enrichment with molecular signatures of basement membrane construction, collagen biosynthesis and folding, and formation of intercellular junctions, whilst HITAECs were notable for augmented pro-inflammatory signaling, intensive synthesis of proteins and nitrogen compounds, and enhanced ribosome biogenesis. Despite HCAECs and HITAECs showing a certain degree of molecular heterogeneity, no specific markers at the protein level have been identified. Coherence of differentially expressed molecular categories in HCAECs and HITAECs suggests synergistic interactions between these ECs in a bypass surgery scenario.

The application of solution NMR spectroscopy to study dynamics of two-domain calcium-binding proteins

Protein dynamics due to flexible linkers connecting otherwise rigid domains may be critical for the functioning of a variety of biological systems, ranging from membrane transporters to calcium-signaling and the formation of intercellular junctions. Considering that NMR spectroscopy is extremely powerful to characterize dynamics at various time scales, this manuscript brings an overview of the main strategies that have been employed to characterize inter-domain dynamics in relevant biological systems. Emphasis was given to the calcium binding proteins: calmodulin, cadherin, and the Na+/Ca2+ exchanger calcium-sensor domain. The introduction of paramagnetic centers in diamagnetic proteins is seen as key to obtaining unambiguous information about inter-domain dynamics. This is because the self-alignment of one of the domains in multi-domain proteins avoids the problem of dealing with alignment tensor fluctuations in dynamic systems. The combination of residual dipolar couplings (RDCs) and pseudocontact shifts (PCSs) with computational strategies aiming to provide an ensemble description of protein dynamics is seen as the most powerful strategy to gain detailed atomistic information on inter-domain motions. It is noteworthy that the cadherin ectodomains and the Na+/Ca2+ exchanger calcium sensor respond in the same way upon calcium-binding: in the absence of calcium the two domains are flexibly linked to one another and may preferentially sample kinked inter-domain arrangements, while calcium binding stabilizes a rigid and extended inter-domain arrangement. It is thus remarkable that nature chose the same molecular mechanism to promote two very different biological functions that are triggered by calcium signaling: intercellular adhesion by the formation of cadherin dimers and the allosteric regulation of a membrane transporter in the case of the Na+/Ca2+ exchanger.

DNA methyltransferase 3A controls intestinal epithelial barrier function and regeneration in the colon

Genetic variants in the DNA methyltransferase 3 A (DNMT3A) locus have been associated with inflammatory bowel disease (IBD). DNMT3A is part of the epigenetic machinery physiologically involved in DNA methylation. We show that DNMT3A plays a critical role in maintaining intestinal homeostasis and gut barrier function. DNMT3A expression is downregulated in intestinal epithelial cells from IBD patients and upon tumor necrosis factor treatment in murine intestinal organoids. Ablation of DNMT3A in Caco-2 cells results in global DNA hypomethylation, which is linked to impaired regenerative capacity, transepithelial resistance and intercellular junction formation. Genetic deletion of Dnmt3a in intestinal epithelial cells (Dnmt3aΔIEC) in mice confirms the phenotype of an altered epithelial ultrastructure with shortened apical-junctional complexes, reduced Goblet cell numbers and increased intestinal permeability in the colon in vivo. Dnmt3aΔIEC mice suffer from increased susceptibility to experimental colitis, characterized by reduced epithelial regeneration. These data demonstrate a critical role for DNMT3A in orchestrating intestinal epithelial homeostasis and response to tissue damage and suggest an involvement of impaired epithelial DNMT3A function in the etiology of IBD.

Numerical modelling of WNT/β-catenin signal pathway in characterization of EMT of colorectal carcinoma cell lines after treatment with Pt(IV) complexes

Background and objectiveColorectal cancer (CRC) is at the top of the most common cancer types in the world, with significant mortality rates among both men and women. Deregulation of Wnt/β-catenin pathway and cell-cell junctions’ components, acquisition of invasive phenotype, epithelial-mesenchymal transition (EMT) and invasion are important for development and progression of colorectal cancer. Numerical simulation presents method for estimation of the Wnt pathway via its individual components in cells, thus providing information about EMT, migratory and invasive potential. By using this numerical model, the effectiveness of treatment in EMT suppression can be assessed. Furthermore, the model can be adapted to ``every'' cell type, application time or duration of treatment can be also modified. MethodsWe characterized colorectal cancer (CRC) cell lines (HCT-116, SW-480) from the aspect of EMT, via markers β-catenin and E-cadherin using numerical modeling. To confirm the numerical model, cells were treated with sublethal concentrations of platinum(IV) complexes and their ligands. We confirmed β-catenin regulated expression of mesenchymal markers: N-cadherin, Vimentin and MMP-9, and decreased E-cadherin expression. Treatment-induced changes were determined in the protein expression of tested markers and results showed cell-specific responses. Molecular docking was performed to investigate exact effects of treatments on E-cadherin and β-catenin in cell-cell junctions and individually in tested cells. ResultsThe application of the numerical model via β-catenin and E-cadherin (experimentally measured), is largely valid for the categorization of EMT progression in cells. This numerical modeling better characterizes cells with single cell migration, higher expression of mesenchymal markers, and advanced mesenchymal phenotype like HCT-116 cell line. The model was validated for the treatments and results show HCT-116 cells as more sensitive to applied compounds, among which ligands were more potent in reducing migration and invasiveness. Anti-migratory/invasive effects were due to increased E-cadherin, cytoplasmic β-catenin expression and suppressed mesenchymal markers. In silico methods showed higher affinity of tested chemicals towards free β-catenin, which is the key for regulation of migratory/invasive potential. ConclusionsOur study shows that, no matter individual properties of cell lines and EMT degree, de novo formation of intercellular junctions stands in the basis of anti-migratory/invasive process.

Expression, purification, and refolding of the recombinant extracellular domain β1-subunit of the dog Na+/K+-ATPase of the epithelial cells

The β1-subunit of the Na+/K+-ATPase is a cell membrane protein, beyond its classic functions, it is also a cell adhesion molecule. β1-subunits on the lateral membrane of dog kidney epithelial cells trans-interact with β1-subunits from another neighboring cells. The β-β interaction is essential for the formation and stabilization of intercellular junctions. Previous studies on site-directed mutagenesis and in silico revealed that the interaction interface involves residues 198–207 and 221–229. However, it is necessary to report the interaction interface at the structural level experimentally. Here, we describe the successful cloning, overexpression in E. coli, and purification of the extracellular domain of the β1-subunit from inclusion bodies. Experimental characterization by size exclusion chromatography and DLS indicated similar hydrodynamic properties of the protein refolded. Structural analysis by circular dichroism and Raman spectroscopy revealed the secondary structures in the folded protein of type β-sheet, α-helix, random coil, and turn. We also performed β1-β1 interaction assays with the recombinant protein, showing dimers’ formation (6xHisβ1-β1). Given our results, the recombinant extracellular domain of the β1-subunit is highly similar to the native protein, therefore the current work in our laboratory aims to characterize at the atomic level the interaction interface between EDβ1.

Rare Inherited Cholestatic Disorders and Molecular Links to Hepatocarcinogenesis

Hepatocellular carcinoma (HCC) is the most common primary liver cancer affecting adults and the second most common primary liver cancer affecting children. Recent years have seen a significant increase in our understanding of the molecular changes associated with HCC. However, HCC is a complex disease, and its molecular pathogenesis, which likely varies by aetiology, remains to be fully elucidated. Interestingly, some inherited cholestatic disorders that manifest in childhood are associated with early HCC development. This review will thus explore how three genes that are associated with liver disease in childhood (ABCB11, TJP2 and VPS33B) might play a role in the initiation and progression of HCC. Specifically, chronic bile-induced damage (caused by ABCB11 changes), disruption of intercellular junction formation (caused by TJP2 changes) and loss of normal apical–basal cell polarity (caused by VPS33B changes) will be discussed as possible mechanisms for HCC development.

Cell and Tissue Nanomechanics: From Early Development to Carcinogenesis.

Cell and tissue nanomechanics, being inspired by progress in high-resolution physical mapping, has recently burst into biomedical research, discovering not only new characteristics of normal and diseased tissues, but also unveiling previously unknown mechanisms of pathological processes. Some parallels can be drawn between early development and carcinogenesis. Early embryogenesis, up to the blastocyst stage, requires a soft microenvironment and internal mechanical signals induced by the contractility of the cortical actomyosin cytoskeleton, stimulating quick cell divisions. During further development from the blastocyst implantation to placenta formation, decidua stiffness is increased ten-fold when compared to non-pregnant endometrium. Organogenesis is mediated by mechanosignaling inspired by intercellular junction formation with the involvement of mechanotransduction from the extracellular matrix (ECM). Carcinogenesis dramatically changes the mechanical properties of cells and their microenvironment, generally reproducing the structural properties and molecular organization of embryonic tissues, but with a higher stiffness of the ECM and higher cellular softness and fluidity. These changes are associated with the complete rearrangement of the entire tissue skeleton involving the ECM, cytoskeleton, and the nuclear scaffold, all integrated with each other in a joint network. The important changes occur in the cancer stem-cell niche responsible for tumor promotion and metastatic growth. We expect that the promising concept based on the natural selection of cancer cells fixing the most invasive phenotypes and genotypes by reciprocal regulation through ECM-mediated nanomechanical feedback loop can be exploited to create new therapeutic strategies for cancer treatment.

Integrity of the Intestinal Barrier: The Involvement of Epithelial Cells and Microbiota-A Mutual Relationship.

Simple SummaryThe gastrointestinal tract is a complex organization of various types of epithelial cells forming a single layer of the mucosal barrier, the host mucosal immune system, and microorganisms termed as gut microbiota inhabiting this area. The mucosal barrier, including physical and chemical factors, spatially segregates gut microbiota and the host immune system preventing the development of immune response directed towards non-pathogenic commensals and dietary antigens. However, for the maintenance of the integrity of the mucosal surfaces, cross-talk between epithelial cells and microbiota is required. The microbiome and the intestinal epithelium developed a complex dependence necessary for sustaining intestinal homeostasis. In this review, we highlight the role of specific epithelial cell subtypes and their role in barrier arrangement, the mechanisms employed by them to control intestinal microbiota as well as the mechanisms utilized by the microbiome to regulate intestinal epithelial function. This review will provide information regarding the development of inflammatory disorders dependent on the loss of intestinal barrier function and composition of the intestinal microbiota.The gastrointestinal tract, which is constantly exposed to a multitude of stimuli, is considered responsible for maintaining the homeostasis of the host. It is inhabited by billions of microorganisms, the gut microbiota, which form a mutualistic relationship with the host. Although the microbiota is generally recognized as beneficial, at the same time, together with pathogens, they are a permanent threat to the host. Various populations of epithelial cells provide the first line of chemical and physical defense against external factors acting as the interface between luminal microorganisms and immunocompetent cells in lamina propria. In this review, we focus on some essential, innate mechanisms protecting mucosal integrity, thus responsible for maintaining intestine homeostasis. The characteristics of decisive cell populations involved in maintaining the barrier arrangement, based on mucus secretion, formation of intercellular junctions as well as production of antimicrobial peptides, responsible for shaping the gut microbiota, are presented. We emphasize the importance of cross-talk between gut microbiota and epithelial cells as a factor vital for the maintenance of the homeostasis of the GI tract. Finally, we discuss how the imbalance of these regulations leads to the compromised barrier integrity and dysbiosis considered to contribute to inflammatory disorders and metabolic diseases.

Cytomorphology of the wound healing process in the green filamentous algae, Ulothrix zonata (F. Weber & Mohr) K tzing 1833

Abstract. For the first time, we present a cytomorphological description of the self-healing (repair) process of damaged thalli sites, wound healing , in the green filamentous algae, Ulothrix zonata (F. Weber & Mohr) K tzing 1833. In the filaments of this species from Lake Baikal and the Angara (Baikal outflow), Zhilishche (Baikal inflow) and Ida (Angara inflow) rivers, light microscopy methods revealed dome-like and conical protrusions and elongations of transverse cell walls directed into adjacent dead (without protoplast) and defective (with deformed chloroplasts) cells. At the same time, there were mostly patterns where two cells formed protrusions directed into the same damaged filament site between them, i.e. towards each other. The growth of previously unconnected cells towards each other led to their convergence and adjacency. This had two important physiological consequences that ensured the restoration of the filament integrity. The first consequence was the formation of intercellular junctions. The second one was the fusion of the protoplasts and nuclei of the adjacent cells (cell fusion) with the formation of vegetative polyploid cells with increased size. During subsequent divisions of these cells, extended areas emerged with a two- to three-fold increase in the diameter of the algal filaments. It was also found that the process of wound healing promoted the development of giant hypnospores. We showed that the H-shaped septa between cells of filamentous algae were not thickenings of the outer walls but the sheaths of the dead cells preserved after this reparation process. Analysis of the wound healing patterns revealed that the Ulothrix cell nuclei did not migrate to the polarized regions of the cells but retained their central position, which testifies to their fixation in the protoplast. We observed sporadic cases of the development of lateral filaments in U. zonata were due to the self-repair of defective cells and their subsequent division during wound healing . A comparison of various cell deformations allowed us to determine the characteristic stages of the wound healing process in U. zonata, which have some similarities and differences with those in marine red filamentous algae. Our study indicates that wound healing is an evolutionarily developed and genetically programmed adaptation that may be widespread in the population of filamentous algae.

Межклеточные контакты, пролиферация и апоптоз эпителия десны у пациентов с пародонтитом после лазерной терапии

Introduction. E-cadherin participates in the formation of intercellular junctions and protects the epitheliocytes of the gums from apoptosis and regulates proliferation in them. The effect of a diode laser on intercellular contacts, proliferation, and apoptosis of gum epitheliocytes at various ages remains poorly studied. The purpose of the paper was to conduct a comparative analysis of the proliferative and apoptotic activity of human gingival epitheliocytes and their intercellular interaction in chronic inflammation, as well as in age-related diode laser therapy. Materials and methods.The study included patients with and without periodontal inflammation (30 patients in each group). The subjects were divided into 2 age groups: group I included patients aged 20–40 years, whereas group II – 41–60 years. Each age group was divided into subgroups: the control subgroup (patients without gingival inflammation); subgroup with periodontal inflammation (patients with chronic periodontitis); and subgroup after laser therapy (patients after therapy with 940-nm Prometey diode laser). We performed immunohistochemical studies using monoclonal antibodies to Ki-67, p53, E-cadherin; as well as computer morphometry and statistical data analysis. Results. Periodontitis combines a decrease in the number of both proliferating epitheliocytes and E-cadherin-positive intercellular contacts in the basal and spiny layers of the gum epithelium in both young and mature adults (p=0.00002). Exposure of a diode laser has a positive effect on the proliferative activity of epithelial cells and significantly increases the number of E-cadherin-positive intercellular contacts in the basal layer (p=0.00002), but does not affect the expression of p53 (p=0.9) in the gum epithelium in all age groups. Conclusion. Exposure of a diode laser increases the proliferation/apoptosis index in the gingival epithelium and brings the expression of E-cadherin closer to the control values. Keywords: gingival epithelium, chronic periodontitis, diode laser, Ki-67, p53, E-cadherin

Quantitative Evaluation of Cardiac Cell Interactions and Responses to Cyclic Strain.

The heart has a dynamic mechanical environment contributed by its unique cellular composition and the resultant complex tissue structure. In pathological heart tissue, both the mechanics and cell composition can change and influence each other. As a result, the interplay between the cell phenotype and mechanical stimulation needs to be considered to understand the biophysical cell interactions and organization in healthy and diseased myocardium. In this work, we hypothesized that the overall tissue organization is controlled by varying densities of cardiomyocytes and fibroblasts in the heart. In order to test this hypothesis, we utilized a combination of mechanical strain, co-cultures of different cell types, and inhibitory drugs that block intercellular junction formation. To accomplish this, an image analysis pipeline was developed to automatically measure cell type-specific organization relative to the stretch direction. The results indicated that cardiac cell type-specific densities influence the overall organization of heart tissue such that it is possible to model healthy and fibrotic heart tissue in vitro. This study provides insight into how to mimic the dynamic mechanical environment of the heart in engineered tissue as well as providing valuable information about the process of cardiac remodeling and repair in diseased hearts.

The Basolateral Polarity Module Promotes Slit Diaphragm Formation in Drosophila Nephrocytes, a Model of Vertebrate Podocytes.

Podocyte slit diaphragms (SDs) are intercellular junctions that function as size-selective filters, excluding most proteins from urine. Abnormalities in SDs cause proteinuria and nephrotic syndrome. Podocytes exhibit apicobasal polarity, which can affect fundamental aspects of cell biology, including morphology, intercellular junction formation, and asymmetric protein distribution along the plasma membrane. Apical polarity protein mutations cause nephrotic syndrome, and data suggest apical polarity proteins regulate SD formation. However, there is no evidence that basolateral polarity proteins regulate SDs. Thus, the role of apicobasal polarity in podocytes remains unclear. Genetic manipulations and transgenic reporters determined the effects of disrupting apicobasal polarity proteins in Drosophila nephrocytes, which have SDs similar to those of mammalian podocytes. Confocal and electron microscopy were used to characterize SD integrity after loss of basolateral polarity proteins, and genetic-interaction studies illuminated relationships among apicobasal polarity proteins. The study identified four novel regulators of nephrocyte SDs: Dlg, Lgl, Scrib, and Par-1. These proteins comprise the basolateral polarity module and its effector kinase. The data suggest these proteins work together, with apical polarity proteins, to regulate SDs by promoting normal endocytosis and trafficking of SD proteins. Given the recognized importance of apical polarity proteins and SD protein trafficking in podocytopathies, the findings connecting basolateral polarity proteins to these processes significantly advance our understanding of SD regulation.

Effects of Alzheimer's Disease-Related Proteins on the Chirality of Brain Endothelial Cells.

Cell chirality is an intrinsic cellular property that determines the directionality of cellular polarization along the left-right axis. We recently show that endothelial cell chirality can influence intercellular junction formation and alter trans-endothelial permeability, depending on the uniformity of the chirality of adjacent cells, which suggests a potential role for cell chirality in neurodegenerative diseases with blood-brain barrier (BBB) dysfunctions, such as Alzheimer's disease (AD). In this study, we determined the effects of AD-related proteins amyloid-β (Aβ), tau, and apolipoprotein E4 (ApoE4) on the chiral bias of the endothelial cell component in BBB. We first examined the chiral bias and effects of protein kinase C (PKC)-mediated chiral alterations of human brain microvascular endothelial cells (hBMECs) using the ring micropattern chirality assay. We then investigated the effects of Aβ, tau, and ApoE4 on hBMEC chirality using chirality assay and biased organelle positions. The hBMECs have a strong clockwise chiral bias, which can be reversed by protein kinase C (PKC) activation. Treatment with tau significantly disrupted the chiral bias of hBMECs with altered cellular polarization. In contrast, neither ApoE4 nor Aβ-42 caused significant changes in cell chirality. We conclude that tau might cause BBB dysfunction by disrupting cell polarization and chiral morphogenesis, while the effects of ApoE4 and Aβ-42 on BBB integrity might be chirality-independent. The potential involvement of chiral morphogenesis in tau-mediated BBB dysfunction in AD provides a novel perspective in vascular dysfunction in tauopathies such as AD, chronic traumatic encephalopathy, progressive supranuclear palsy, and frontotemporal dementia. The online version contains supplementary material available at 10.1007/s12195-021-00669-w.

Plectin ensures intestinal epithelial integrity and protects colon against colitis

Plectin, a highly versatile cytolinker protein, provides tissues with mechanical stability through the integration of intermediate filaments (IFs) with cell junctions. Here, we hypothesize that plectin-controlled cytoarchitecture is a critical determinant of the intestinal barrier function and homeostasis. Mice lacking plectin in an intestinal epithelial cell (IEC; PleΔIEC) spontaneously developed colitis characterized by extensive detachment of IECs from the basement membrane (BM), increased intestinal permeability, and inflammatory lesions. Moreover, plectin expression was reduced in the colons of ulcerative colitis (UC) patients and negatively correlated with the severity of colitis. Mechanistically, plectin deficiency in IECs led to aberrant keratin filament (KF) network organization and the formation of dysfunctional hemidesmosomes (HDs) and intercellular junctions. In addition, the hemidesmosomal α6β4 integrin (Itg) receptor showed attenuated association with KFs, and protein profiling revealed prominent downregulation of junctional constituents. Consistent with the effects of plectin loss in the intestinal epithelium, plectin-deficient IECs exhibited remarkably reduced mechanical stability and limited adhesion capacity in vitro. Feeding mice with a low-residue liquid diet that reduced mechanical stress and antibiotic treatment successfully mitigated epithelial damage in the PleΔIEC colon.

Abstract PO002: Microfluidic tissue culture device to mimic the bone marrow sinusoidal niche of cancer cells

Abstract The goal of this study is to develop a microfluidic tissue culture device that mimics in vitro the sinusoidal niche of cancer cells in bone marrow (BM). BM is highly vascularized by sinusoids to support the trafficking of blood, immune, and tumor cells. We envision that the microfluidic device can be developed and used as a new means of studying the trafficking of tumor cells through sinusoidal endothelium and the phenotypic development of cancer cells through interactions with stromal cells in BM. The microfluid device was designed to contain sinusoid and stroma chambers. The sinusoidal blood circulation was mimicked by culture medium flow which exerts the physiologically relevant flow-induced shear stress of 0.1 Pascals on endothelial cells (ECs). The sinusoidal endothelium was constructed by culturing ECs (EA.hy926 cell line) on a membrane (10 μm pores) placed between the sinusoid and stroma chambers. The BM stroma was mimicked by dispersing and culturing fibroblastic stromal cells (HS-5 cell line) in collagen. The device was also designed for: (1) industry-standard 96-well plate format for high-throughput culture and characterization and (2) convenient placement of cells and biomaterials to construct tissues. The device was placed inside a conventional incubator and connected to a peristaltic pump for medium circulation for dynamic culture for up to 24 days. ECs formed a confluent layer on the membrane and exhibited a cobble-stone morphology within 4 h while stroma cells could be uniformly dispersed in collagen and cultured in the stroma chamber. Upon further culture, CD31 was strongly expressed at contact regions of ECs, indicating the formation of intercellular junctions. The effects of flow-induced shear stress were evident on the morphological alignment of endothelial cells and the re-arranged cortical organization of F-actin. The effects of cancer cells on the barrier function of endothelium were examined by culturing human multiple myeloma cancer cells (MM.1S cell line) with HS-5 cells in the stroma chamber. The permeability of endothelium, which was measured using a fluorescein isothiocyanate–dextran solution (70 kDa), significantly increased from 5.8±0.2 × 10−6 to 9.3±0.1 × 10−6 cm/s with introducing MM.1S cells (p <0.0001 determined by t-test). The increased permeability was supported by the formation of larger and more gaps formed at EC junctions. Also, stromal cell-derived factor 1 was introduced as a chemoattract in the sinusoid chamber to induce the transmigration of MM.1S cells through the endothelium layer from the stroma chamber while maintaining the uniform dispersion of HS-5 cells in the stroma chamber. In conclusion, the results suggest that the device could be used to study the adverse effects of cancer cells on the barrier function of sinusoidal endothelium (e.g., less organized and more loosely connected ECs) and further mimic the trafficking of cancer cells through the BM stroma. Citation Format: Chao Sui, Jenny Zilberberg, Woo Lee. Microfluidic tissue culture device to mimic the bone marrow sinusoidal niche of cancer cells [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr PO002.

Abstract 810: Structural and Electrophysiological Maturation of Human iPSC-Derived Atrial Cardiomyocytes to Serve as a Platform to Model Atrial Fibrillation

Background: Atrial fibrillation (AF), the most globally common arrhythmia requiring treatment, is associated with significant morbidity and mortality. Although antiarrhythmic drugs (AADs) are most commonly used to treat symptomatic AF, AADs are incompletely and unpredictably effective, and fail to target underlying cellular mechanisms of AF. Previously, pluripotent stem cell derived immature atrial cardiomyocytes (iPSC-aCMs) were shown to recapitulate the structural and electrophysiologic (EP) phenotype of AF-linked mutations. However, there are gaps in structural and EP phenotype between immature and mature iPSC-aCMs to successfully evaluate cellular mechanisms. Objective: The goal is to determine optimal condition(s) to enhance maturity of iPSC-aCMs, to establish iPSC-aCMs as a novel platform to model AF in a dish, elucidate cellular mechanisms and molecular pathogenesis, and identify and assess novel, personalized mechanism based therapies. Methods: Conditions used to enhance iPSC-aCMs maturity are 1) different ECMs to culture iPSC-aCMs, 2) patterned ECM to improve alignment and formation of intercellular gap junctions, and 3) treatment with postnatal factors (T3, IGF-1 and dexamethasone; TID). Maturity was assessed with immunofluorescence and EP measurement with patch-clamping and optical voltage analysis. Results were compared to equivalent parameters in adult atrial cardiomyocytes (aCM) and untreated atrial iPSC-CMs. Results: Fibronectin and laminin were ECMs found suitable for long time culture of iPSC-aCMs. Structural maturity (α-actinin and cardiac troponin T) and EP maturity (resting membrane potential [83 ± 3%], APD 90 [67 ± 2.5%], and conduction velocity [59.6 ±3%]) with patterned ECM and TID treatment were significantly improved compared to untreated iPSC-aCMs, and closer to those of adult aCMs. Conclusion: Structural and EP maturity of iPSC-aCMs are significantly enhanced by precise microenvironmental engineering of in-vivo relevant cell-cell, cell-ECM, and cell-soluble factor interactions. Thus, our findings will provide a platform to investigate cellular mechanisms of AF.

Cellular and pathophysiological consequences of Arp2/3 complex inhibition: role of inhibitory proteins and pharmacological compounds.

The actin-related protein complex 2/3 (Arp2/3) generates branched actin networks important for many cellular processes such as motility, vesicular trafficking, cytokinesis, and intercellular junction formation and stabilization. Activation of Arp2/3 requires interaction with actin nucleation-promoting factors (NPFs). Regulation of Arp2/3 activity is achieved by endogenous inhibitory proteins through direct binding to Arp2/3 and competition with NPFs or by binding to Arp2/3-induced actin filaments and disassembly of branched actin networks. Arp2/3 inhibition has recently garnered more attention as it has been associated with attenuation of cancer progression, neurotoxic effects during drug abuse, and pathogen invasion of host cells. In this review, we summarize current knowledge on expression, inhibitory mechanisms and function of endogenous proteins able to inhibit Arp2/3 such as coronins, GMFs, PICK1, gadkin, and arpin. Moreover, we discuss cellular consequences of pharmacological Arp2/3 inhibition.