Distinct Compartmentalization of the Chemokines CXCL1 and CXCL2 and the Atypical Receptor ACKR1 Determine Discrete Stages of Neutrophil Diapedesis.

Neutrophils require directional cues to navigate through the complex structure of venular walls and into inflamed tissues. Here we applied confocal intravital microscopy to analyze neutrophil emigration in cytokine-stimulated mouse cremaster muscles. We identified differential and non-redundant roles for the chemokines CXCL1 and CXCL2, governed by their distinct cellular sources. CXCL1 was produced mainly by TNF-stimulated endothelial cells (ECs) and pericytes and supported luminal and sub-EC neutrophil crawling. Conversely, neutrophils were the main producers of CXCL2, and this chemokine was critical for correct breaching of endothelial junctions. This pro-migratory activity of CXCL2 depended on the atypical chemokine receptor 1 (ACKR1), which is enriched within endothelial junctions. Transmigrating neutrophils promoted a self-guided migration response through EC junctions, creating a junctional chemokine "depot" in the form of ACKR1-presented CXCL2 that enabled efficient unidirectional luminal-to-abluminal migration. Thus, CXCL1 and CXCL2 act in a sequential manner to guide neutrophils through venular walls as governed by their distinct cellular sources.

Leukotriene B4-Neutrophil Elastase Axis Drives Neutrophil Reverse Transendothelial Cell Migration In Vivo.

Breaching endothelial cells (ECs) is a decisive step in the migration of leukocytes from the vascular lumen to the extravascular tissue, but fundamental aspects of this response remain largely unknown. We have previously shown that neutrophils can exhibit abluminal-to-luminal migration through EC junctions within mouse cremasteric venules and that this response is elicited following reduced expression and/or functionality of the EC junctional adhesion molecule-C (JAM-C). Here we demonstrate that the lipid chemoattractant leukotriene B4 (LTB4) was efficacious at causing loss of venular JAM-C and promoting neutrophil reverse transendothelial cell migration (rTEM) in vivo. Local proteolytic cleavage of EC JAM-C by neutrophil elastase (NE) drove this cascade of events as supported by presentation of NE to JAM-C via the neutrophil adhesion molecule Mac-1. The results identify local LTB4-NE axis as a promoter of neutrophil rTEM and provide evidence that this pathway can propagate a local sterile inflammatory response to become systemic.

Inhibition of soluble epoxide hydrolase prevents diabetic retinopathy.

Diabetic retinopathy is an important cause of blindness in adults, and is characterized by progressive loss of vascular cells and slow dissolution of inter-vascular junctions, which result in vascular leakage and retinal oedema. Later stages of the disease are characterized by inflammatory cell infiltration, tissue destruction and neovascularization. Here we identify soluble epoxide hydrolase (sEH) as a key enzyme that initiates pericyte loss and breakdown of endothelial barrier function by generating the diol 19,20-dihydroxydocosapentaenoic acid, derived from docosahexaenoic acid. The expression of sEH and the accumulation of 19,20-dihydroxydocosapentaenoic acid were increased in diabetic mouse retinas and in the retinas and vitreous humour of patients with diabetes. Mechanistically, the diol targeted the cell membrane to alter the localization of cholesterol-binding proteins, and prevented the association of presenilin 1 with N-cadherin and VE-cadherin, thereby compromising pericyte-endothelial cell interactions and inter-endothelial cell junctions. Treating diabetic mice with a specific sEH inhibitor prevented the pericyte loss and vascular permeability that are characteristic of non-proliferative diabetic retinopathy. Conversely, overexpression of sEH in the retinal Müller glial cells of non-diabetic mice resulted in similar vessel abnormalities to those seen in diabetic mice with retinopathy. Thus, increased expression of sEH is a key determinant in the pathogenesis of diabetic retinopathy, and inhibition of sEH can prevent progression of the disease.

Translocation of LSR from tricellular corners causes macropinocytosis at cell-cell interface as a trigger for breaking out of contact inhibition.

Withdrawal from contact inhibition is necessary for epithelial cancer precursor cells to initiate cell growth and motility. Nevertheless, little is understood about the mechanism for the sudden initiation of cell growth under static conditions. We focused on cellular junctions as one region where breaking out of contact inhibition occurs. In well-differentiated endometrial cancer cells, Sawano, the ligand administration for tricellular tight junction protein LSR, which transiently decreased the robust junction property, caused an abrupt increase in cell motility and consequent excessive multilayered cell growth despite being under contact inhibition conditions. We observed that macropinocytosis essentially and temporarily occurred as an antecedent event for the above process at intercellular junctions without disruption of the junction apparatus but not at the apical plasma membrane. Collectively, we concluded that the formation of macropinocytosis, which is derived from tight junction-mediated signaling, was triggered for the initiation of cell growth in static precancerous epithelium.

Deoxycholic Acid Modulates Cell-Junction Gene Expression and Increases Intestinal Barrier Dysfunction.

Diet-related obesity is associated with increased intestinal hyperpermeability. High dietary fat intake causes an increase in colonic bile acids (BAs), particularly deoxycholic acid (DCA). We hypothesize that DCA modulates the gene expression of multiple cell junction pathways and increases intestinal permeability. With a human Caco-2 cell intestinal model, we used cell proliferation, PCR array, biochemical, and immunofluorescent assays to examine the impact of DCA on the integrity of the intestinal barrier and gene expression. The Caco-2 cells were grown in monolayers and challenged with DCA at physiological, sub-mM, concentrations. DCA increased transcellular and paracellular permeability (>20%). Similarly, DCA increased intracellular reactive oxidative species production (>100%) and accompanied a decrease (>40%) in extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathways. Moreover, the mRNA levels of 23 genes related to the epithelial barrier (tight junction, focal adhesion, gap junction, and adherens junction pathways) were decreased (>40%) in (0.25 mM) DCA-treated Caco-2 cells compared to untreated cells. Finally, we demonstrated that DCA decreased (>58%) the protein content of occludin present at the cellular tight junctions and the nucleus of epithelial cells. Collectively, DCA decreases the gene expression of multiple pathways related to cell junctions and increases permeability in a human intestinal barrier model.

LPAR2 receptor activation attenuates radiation-induced disruption of apical junctional complexes and mucosal barrier dysfunction in mouse colon.

The tight junction (TJ) and barrier function of colonic epithelium is highly sensitive to ionizing radiation. We evaluated the effect of lysophosphatidic acid (LPA) and its analog, Radioprotein-1, on γ-radiation-induced colonic epithelial barrier dysfunction using Caco-2 and m-ICC12 cell monolayers in vitro and mice in vivo. Mice were subjected to either total body irradiation (TBI) or partial body irradiation (PBI-BM5). Intestinal barrier function was assessed by analyzing immunofluorescence localization of TJ proteins, mucosal inulin permeability, and plasma lipopolysaccharide (LPS) levels. Oxidative stress was analyzed by measuring protein thiol oxidation and antioxidant mRNA. In Caco-2 and m-ICC12 cell monolayers, LPA attenuated radiation-induced redistribution of TJ proteins, which was blocked by a Rho-kinase inhibitor. In mice, TBI and PBI-BM5 disrupted colonic epithelial tight junction and adherens junction, increased mucosal permeability, and elevated plasma LPS; TJ disruption by TBI was more severe in Lpar2-/- mice compared to wild-type mice. RP1, administered before or after irradiation, alleviated TBI and PBI-BM5-induced TJ disruption, barrier dysfunction, and endotoxemia accompanied by protein thiol oxidation and downregulation of antioxidant gene expression, cofilin activation, and remodeling of the actin cytoskeleton. These data demonstrate that LPAR2 receptor activation prevents and mitigates γ-irradiation-induced colonic mucosal barrier dysfunction and endotoxemia.

Heterocellular Contact Can Dictate Arterial Function.

RATIONALE: Resistance arteries and conduit arteries rely on different relative contributions of endothelial-derived hyperpolarization versus nitric oxide to achieve dilatory heterocellular signaling. Anatomically, resistance arteries use myoendothelial junctions (MEJs), endothelial cell projections that make contact with smooth muscle cells. Conduit arteries have very few to no MEJs. OBJECTIVE: Determine if the presence of MEJs in conduit arteries can alter heterocellular signaling. METHODS AND RESULTS: We previously demonstrated that PAI-1 (plasminogen activator inhibitor-1) can regulate formation of MEJs. Thus, we applied pluronic gel containing PAI-1 directly to conduit arteries (carotid arteries) to determine if this could induce formation of MEJs. We found a significant increase in endothelial cell projections resembling MEJs that correlated with increased biocytin dye transfer from endothelial cells to smooth muscle cells. Next, we used pressure myography to investigate whether these structural changes were accompanied by a functional change in vasodilatory signaling. Interestingly, PAI-1-treated carotids underwent a switch from a conduit to resistance artery vasodilatory profile via diminished nitric oxide signaling and increased endothelial-derived hyperpolarization signaling in response to the endothelium-dependent agonists acetylcholine and NS309. After PAI-1 application, we also found a significant increase in carotid expression of endothelial alpha globin, a protein predominantly expressed in resistance arteries. Carotids from mice with PAI-1, but lacking alpha globin (Hba1-/-), demonstrated that l-nitro-arginine methyl ester, an inhibitor of nitric oxide signaling, was able to prevent arterial relaxation. CONCLUSIONS: The presence or absence of MEJs is an important determinant for influencing heterocellular communication in the arterial wall. In particular, alpha globin expression, induced within newly formed endothelial cell projections, may influence the balance between endothelial-derived hyperpolarization and nitric oxide-mediated vasodilation.

DRG1 Maintains Intestinal Epithelial Cell Junctions and Barrier Function by Regulating RAC1 Activity in Necrotizing Enterocolitis.

BACKGROUND: An immature intestine is a high-risk factor for necrotizing enterocolitis (NEC), which is a serious intestinal disease in newborns. The regulation of developmentally regulated GTP-binding protein 1 (DRG1) during organ development suggests a potential role of DRG1 in the maturation process of the intestine. AIM: To illustrate the function of DRG1 during the pathogenesis of NEC. METHODS: DRG1 expression in the intestine was measured using immunohistochemistry and q-PCR. Immunoprecipitation coupled with mass spectrometry was used to identify the interacting proteins of DRG1. The biological functions of the potential interactors were annotated with the Database for Annotation, Visualization and Integrated Discovery. Caco2 and FHs74Int cells with stable DRG1 silencing or overexpression were used to investigate the influence of DRG1 on cell junctions and intestinal barrier permeability and to elucidate the downstream mechanism. RESULTS: DRG1 was constitutively expressed during the intestinal maturation process but significantly decreased in the ileum in the context of NEC. Protein interaction analysis revealed that DRG1 was closely correlated with cell junctions. DRG1 deficiency destabilized the E-cadherin and occludin proteins near the cell membrane and increased the permeability of the epithelial cell monolayer, while DRG1 overexpression prevented lipopolysaccharide-induced disruption of E-cadherin and occludin expression and cell monolayer integrity. Further investigation suggested that DRG1 maintained cell junctions, especially adherens junctions, by regulating RAC1 activity, and RAC1 inhibition with NSC23766 attenuated intestinal injury and led to improved barrier integrity in experimental NEC. CONCLUSIONS: Our findings illustrate the mechanism underlying the effect of DRG1 deficiency on epithelial cell permeability regulation and provide evidence supporting the application of RAC1 inhibitors for protection against NEC.

Characterization of Estrogenic Activity and Site-Specific Accumulation of Bisphenol-A in Epididymal Fat Pad: Interfering Effects on the Endocannabinoid System and Temporal Progression of Germ Cells.

The objective of this work has been to characterize the estrogenic activity of bisphenol-A (BPA) and the adverse effects on the endocannabinoid system (ECS) in modulating germ cell progression. Male offspring exposed to BPA during the foetal-perinatal period at doses below the no-observed-adverse-effect-level were used to investigate the exposure effects in adulthood. Results showed that BPA accumulates specifically in epididymal fat rather than in abdominal fat and targets testicular expression of 3ß-hydroxysteroid dehydrogenase and cytochrome P450 aromatase, thus promoting sustained increase of estrogens and a decrease of testosterone. The exposure to BPA affects the expression levels of some ECS components, namely type-1 (CB1) and type-2 cannabinoid (CB2) receptor and monoacylglycerol-lipase (MAGL). Furthermore, it affects the temporal progression of germ cells reported to be responsive to ECS and promotes epithelial germ cell exfoliation. In particular, it increases the germ cell content (i.e., spermatogonia while reducing spermatocytes and spermatids), accelerates progression of spermatocytes and spermatids, promotes epithelial detachment of round and condensed spermatids and interferes with expression of cell-cell junction genes (i.e., zonula occcludens protein-1, vimentin and ß-catenin). Altogether, our study provides evidence that early exposure to BPA produces in adulthood sustained and site-specific BPA accumulation in epididymal fat, becoming a risk factor for the reproductive endocrine pathways associated to ECS.

Opportunities and Challenges in Tunneling Nanotubes Research: How Far from Clinical Application?

Tunneling nanotubes (TNTs) are recognized long membrane nanotubes connecting distance cells. In the last decade, growing evidence has shown that these subcellular structures mediate the specific transfer of cellular materials, pathogens, and electrical signals between cells. As intercellular bridges, they play a unique role in embryonic development, collective cell migration, injured cell recovery, cancer treatment resistance, and pathogen propagation. Although TNTs have been considered as potential drug targets for treatment, there is still a long way to go to translate the research findings into clinical practice. Herein, we emphasize the heterogeneous nature of TNTs by systemically summarizing the current knowledge on their morphology, structure, and biogenesis in different types of cells. Furthermore, we address the communication efficiency and biological outcomes of TNT-dependent transport related to diseases. Finally, we discuss the opportunities and challenges of TNTs as an exciting therapeutic approach by focusing on the development of efficient and safe drugs targeting TNTs.

Losartan protects against myocardial ischemia and reperfusion injury via vascular integrity preservation.

Vascular hyperpermeability caused by distorted endothelial cell-cell junctions is associated with the no-reflow phenomenon after opening of the occluded vessels in patients with coronary artery disease (CAD), the leading cause of death worldwide. Coronary no-reflow is observed in ∼30% of CAD patients after percutaneous coronary stenting and is associated with a worse prognosis at follow-up and a higher incidence of death. However, limited tools are available to control vascular hyperpermeability and no-reflow. Losartan, an angiotensin II (Ang II) receptor blocker acting on the Ang II type-1 receptor (AT1R) subtype, is a prescription drug for treating hypertension. Here we show that in a murine model of ischemia and reperfusion (I/R), losartan blocked vascular hyperpermeability and decreased infarct size, hemorrhages, edema, and inflammation. Mechanistically, losartan-mediated inhibition of vascular hyperpermeability is mediated by the inhibition of phosphorylation of Src and vascular endothelial cadherin (VE-cadherin), which increases VEGF receptor 2 (VEGFR2)-Src-VE-cadherin complex formation, resulting in increased cell surface VE-cadherin and inhibition of vascular hyperpermeability. On the other hand, hypoxia and reoxygenation increased the phosphorylation levels of Src and VE-cadherin and reduced the formation of the VEGFR2-Src-VE-cadherin complex, which led to reduced cell surface VE-cadherin and increased vascular hyperpermeability; all were inhibited by losartan. These data suggest that losartan may be used for blocking vascular hyperpermeability associated with I/R.-Li, Y., Yao, Y., Li, J., Chen, Q., Zhang, L., Wang, Q. K. Losartan protects against myocardial ischemia and reperfusion injury via vascular integrity preservation.

Cell membrane-related toxic responses and disruption of intercellular communication in PCB mechanisms of toxicity: A review.

An understanding of polychlorinated biphenyl (PCB) congener-specific effects on cell membrane and intercellular communication is important within the studies of PCB absorption, organ-related PCB accumulation and exertion of toxic responses. Toxic potential of PCBs is linked to various deleterious effects on human health, including neurotoxicity, immunotoxicity, reproductive toxicity and genotoxicity and, recently in 2016 International Agency for Research on Cancer (IARC) has upgraded the classification of PCBs to Group 1 "Carcinogenic to humans." Proposed mechanisms of aforementioned PCBs adverse effects at cellular membrane level are: (i) downregulation of gap junction intercellular communication and/or connexins; (ii) compromised membrane integrity; and (iii) altered tight junction barrier function. This study, based on an extensive literature survey, shows the progress in scientific research of each of these three levels with the aim of pointing out the earliest toxic events of PCBs, which can result in serious cell/tissue/organ damage.

Anisomycin, a JNK and p38 activator, suppresses cell-cell junction formation in 2D cultures of K38 mouse keratinocyte cells and reduces claudin-7 expression, with an increase of paracellular permeability in 3D cultures.

Keratinocytes in the oral mucosal epithelium, which is a non-keratinized stratified epithelium, are exposed to various stimuli from the oral cavity. JNK and p38 are stress-activated mitogen-activated protein kinases (MAPKs) that are phosphorylated by various stimuli and are involved in the assembly and disassembly of tight junctions (TJs) in keratinocytes. Therefore, we investigated the effects of stress-activated MAPKs on TJs in a mouse keratinocyte cell line during cell-cell junction formation in two-dimensional (2D) cultures or stratification to form non-keratinized epithelium in 3D cultures. In 2D cultures, calcium induced zipper-like staining for ZO-1 at 2 h and string-like staining for ZO-1 at 12 h, which indicated immature and mature cell-cell junctions, respectively. Anisomycin (AM), a JNK and p38 activator, inhibited formation of string-like staining for ZO-1, whereas inhibition of JNK, but not p38, after AM treatment restored string-like staining for ZO-1, although claudins (CLDNs) 4, 6, and 7 did not completely colocalize to ZO-1-positive sites. In 3D cultures, AM treatment for 2 weeks activated only p38, suppressed flattening of the superficial cells, removed CLDN7 from ZO-1-positive spots on the surface of 3D cultures, which represent TJs, and decreased transepithelial electrical resistance. Thus, short-term AM treatment inhibited maturation of cell-cell junctions by JNK, but not p38, activation. p38 activation by long-term AM treatment affected morphology of stratified structures and paracellular permeability, which was increased by CLDN7 removal from TJs. Various chronic stimuli that activate stress-activated MAPKs may weaken the keratinocyte barrier and be involved in TJ-related diseases.

Anthrax edema toxin disrupts distinct steps in Rab11-dependent junctional transport.

Various bacterial toxins circumvent host defenses through overproduction of cAMP. In a previous study, we showed that edema factor (EF), an adenylate cyclase from Bacillus anthracis, disrupts endocytic recycling mediated by the small GTPase Rab11. As a result, cargo proteins such as cadherins fail to reach inter-cellular junctions. In the present study, we provide further mechanistic dissection of Rab11 inhibition by EF using a combination of Drosophila and mammalian systems. EF blocks Rab11 trafficking after the GTP-loading step, preventing a constitutively active form of Rab11 from delivering cargo vesicles to the plasma membrane. Both of the primary cAMP effector pathways -PKA and Epac/Rap1- contribute to inhibition of Rab11-mediated trafficking, but act at distinct steps of the delivery process. PKA acts early, preventing Rab11 from associating with its effectors Rip11 and Sec15. In contrast, Epac functions subsequently via the small GTPase Rap1 to block fusion of recycling endosomes with the plasma membrane, and appears to be the primary effector of EF toxicity in this process. Similarly, experiments conducted in mammalian systems reveal that Epac, but not PKA, mediates the activity of EF both in cell culture and in vivo. The small GTPase Arf6, which initiates endocytic retrieval of cell adhesion components, also contributes to junctional homeostasis by counteracting Rab11-dependent delivery of cargo proteins at sites of cell-cell contact. These studies have potentially significant practical implications, since chemical inhibition of either Arf6 or Epac blocks the effect of EF in cell culture and in vivo, opening new potential therapeutic avenues for treating symptoms caused by cAMP-inducing toxins or related barrier-disrupting pathologies.

Stabilization of cell-cell junctions by active vitamin D ameliorates uraemia-induced loss of human endothelial barrier function.

Background: Uraemia induces endothelial cell (EC) injury and impaired repair capacity, for which the underlying mechanism remains unclear. Active vitamin D (VD) may promote endothelial repair, however, the mechanism that mediates the effects of VD in chronic kidney disease are poorly understood. Thus, we investigated uraemia-induced endothelial damage and the protection against such damage by active VD. Methods: We applied electric cell-substrate impedance sensing (ECIS) to study real-time responses of human ECs exposed to pooled uraemic and non-uraemic plasma with or without the addition of active VD. The effects of indoxyl sulphate and p-cresol were tested in non-uraemic plasma. Structural changes for vascular endothelial (VE)-cadherin and F-actin were assessed by immunostaining and quantified. Results: The exposure of ECs to uraemic media significantly decreased endothelial barrier function after 24 h. Cell migration after electrical wounding and recovery of the barrier after thrombin-induced loss of integrity were significantly impaired in uraemic-medium stimulated cells and cells exposed to indoxyl sulphate and p-cresol. This effect on ECIS was dependent on loss of cell-cell interaction. Mechanistically, we found that EC, exposed to uraemic media, displayed disrupted VE-cadherin interactions and F-actin reorganization. VD supplementation rescued both endothelial barrier function and cell-cell interactions in ECs exposed to uraemic media. These events were associated with an increment of VE-cadherin at intercellular junctions. Conclusions: Our data demonstrate a potentially clinically relevant mechanism for uraemia-induced endothelial damage. Furthermore, active VD rescued the uraemic medium-induced loss of cell-cell adhesion, revealing a novel role of active VD in preservation of endothelial integrity during uraemia.

Small GTPase Rap1A/B Is Required for Lymphatic Development and Adrenomedullin-Induced Stabilization of Lymphatic Endothelial Junctions.

Objective- Maintenance of lymphatic permeability is essential for normal lymphatic function during adulthood, but the precise signaling pathways that control lymphatic junctions during development are not fully elucidated. The Gs-coupled AM (adrenomedullin) signaling pathway is required for embryonic lymphangiogenesis and the maintenance of lymphatic junctions during adulthood. Thus, we sought to elucidate the downstream effectors mediating junctional stabilization in lymphatic endothelial cells. Approach and Results- We knocked-down both Rap1A and Rap1B isoforms in human neonatal dermal lymphatic cells (human lymphatic endothelial cells) and genetically deleted the mRap1 gene in lymphatic endothelial cells by producing 2 independent, conditional Rap1a/b knockout mouse lines. Rap1A/B knockdown caused disrupted junctional formation with hyperpermeability and impaired AM-induced lymphatic junctional tightening, as well as rescue of histamine-induced junctional disruption. Less than 60% of lymphatic- Rap1a/b knockout embryos survived to E13.5 exhibiting interstitial edema, blood-filled lymphatics, disrupted lymphovenous valves, and defective lymphangiogenesis. Consistently, inducible lymphatic- Rap1a/b deletion in adult animals prevented AM-rescue of histamine-induced lymphatic leakage and dilation. Conclusions- Rap1 (Ras-related protein) serves as the dominant effector downstream of AM to stabilize lymphatic junctions. Rap1 is required for maintaining lymphatic permeability and driving normal lymphatic development.

Non-Endoplasmic Reticulum-Based Calr (Calreticulin) Can Coordinate Heterocellular Calcium Signaling and Vascular Function.

OBJECTIVE: In resistance arteries, endothelial cell (EC) extensions can make contact with smooth muscle cells, forming myoendothelial junction at holes in the internal elastic lamina (HIEL). At these HIEL, calcium signaling is tightly regulated. Because Calr (calreticulin) can buffer ≈50% of endoplasmic reticulum calcium and is expressed throughout IEL holes in small arteries, the only place where myoendothelial junctions form, we investigated the effect of EC-specific Calr deletion on calcium signaling and vascular function. APPROACH AND RESULTS: We found Calr expressed in nearly every IEL hole in third-order mesenteric arteries, but not other ER markers. Because of this, we generated an EC-specific, tamoxifen inducible, Calr knockout mouse (EC Calr Δ/Δ). Using this mouse, we tested third-order mesenteric arteries for changes in calcium events at HIEL and vascular reactivity after application of CCh (carbachol) or PE (phenylephrine). We found that arteries from EC Calr Δ/Δ mice stimulated with CCh had unchanged activity of calcium signals and vasodilation; however, the same arteries were unable to increase calcium events at HIEL in response to PE. This resulted in significantly increased vasoconstriction to PE, presumably because of inhibited negative feedback. In line with these observations, the EC Calr Δ/Δ had increased blood pressure. Comparison of ER calcium in arteries and use of an ER-specific GCaMP indicator in vitro revealed no observable difference in ER calcium with Calr knockout. Using selective detergent permeabilization of the artery and inhibition of Calr translocation, we found that the observed Calr at HIEL may not be within the ER. CONCLUSIONS: Our data suggest that Calr specifically at HIEL may act in a non-ER dependent manner to regulate arteriolar heterocellular communication and blood pressure.

Functional role for stable microtubules in lens fiber cell elongation.

The process of tissue morphogenesis, especially for tissues reliant on the establishment of a specific cytoarchitecture for their functionality, depends a balanced interplay between cytoskeletal elements and their interactions with cell adhesion molecules. The microtubule cytoskeleton, which has many roles in the cell, is a determinant of directional cell migration, a process that underlies many aspects of development. We investigated the role of microtubules in development of the lens, a tissue where cell elongation underlies morphogenesis. Our studies with the microtubule depolymerizing agent nocodazole revealed an essential function for the acetylated population of stable microtubules in the elongation of lens fiber cells, which was linked to their regulation of the activation state of myosin. Suppressing myosin activation with the inhibitor blebbistatin could attenuate the loss of acetylated microtubules by nocodazole and rescue the effect of this microtubule depolymerization agent on both fiber cell elongation and lens integrity. Our results also suggest that acetylated microtubules impact lens morphogenesis through their interaction with N-cadherin junctions, with which they specifically associate in the region where lens fiber cell elongate. Disruption of the stable microtubule network increased N-cadherin junctional organization along lateral borders of differentiating lens fiber cells, which was prevented by suppression of myosin activity. These results reveal a role for the stable microtubule population in lens fiber cell elongation, acting in tandem with N-cadherin cell-cell junctions and the actomyosin network, giving insight into the cooperative role these systems play in tissue morphogenesis.

Breaking Down Barriers: How Understanding Celiac Disease Pathogenesis Informed the Development of Novel Treatments.

For decades, the pathogenesis of a variety of human diseases has been attributed to increased intestinal paracellular permeability even though scientific evidence supporting this hypothesis has been tenuous. Nevertheless, during the past decade, there have been a growing number of publications focused on human genetics, the gut microbiome, and proteomics, suggesting that loss of mucosal barrier function, particularly in the gastrointestinal tract, may substantially affect antigen trafficking, ultimately causing chronic inflammation, including autoimmunity, in genetically predisposed individuals. The gut mucosa works as a semipermeable barrier in that it permits nutrient absorption and also regulates immune surveillance while retaining potentially harmful microbes and environmental antigens within the intestinal lumen. Celiac disease (CD), a systemic, immune-mediated disorder triggered by gluten in genetically susceptible individuals, is associated with altered gut permeability. Pre-clinical and clinical studies have shown that gliadin, a prolamine component of gluten that is implicated in CD pathogenesis, is capable to disassembling intercellular junctional proteins by upregulating the zonulin pathway, which can be inhibited by the zonulin antagonist larazotide acetate. In this review, we will focus on CD as a paradigm of chronic inflammatory diseases in order to outline the contribution of gut paracellular permeability toward disease pathogenesis; moreover, we will summarize current evidence derived from available clinical trials of larazotide acetate in CD.

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.