Intercellular Openings Research Articles

Transient opening of tricellular vertices controls paracellular transport through the follicle epithelium during Drosophila oogenesis.

Paracellular permeability is regulated to allow solute transport or cell migration across epithelial or endothelial barriers. However, how cell-cell junction dynamics controls paracellular permeability is poorly understood. Here, we describe patency, a developmentally regulated process in Drosophila oogenesis, during which cell vertices in the follicular epithelium open transiently to allow paracellular transport of yolk proteins for uptake by the oocyte. We show that the sequential removal of E-cadherin, N-cadherin, NCAM/Fasciclin 2, and Sidekick from vertices precedes their basal-to-apical opening, while the subsequent assembly of tricellular occluding junctions marks the termination of patency and seals the paracellular barrier. E-cadherin-based adhesion is required to limit paracellular channel size, whereas stabilized adherens junctions, prolonged NCAM/Fasciclin 2 expression, blocked endocytosis, or increased actomyosin contractility prevent patency. Our findings reveal a key role of cell vertices as gateways controlling paracellular transport and demonstrate that dynamic regulation of adhesion and actomyosin contractility at vertices governs epithelial barrier properties.

Nanomedicines for Reactive Oxygen Species Mediated Approach: An Emerging Paradigm for Cancer Treatment.

Growth in the knowledge of cancer biology has led to the emergence and evolution of cancer nanomedicines by providing the rationale for leveraging nanotechnology to develop better treatment options. The discovery of nanometer-sized intercellular openings in the defective angiogenic tumor vasculature contributed to the development of an idea for the well-known cancer passive targeting regime, enhanced permeability and retention (EPR) effect, of the nanomedicines. Recently, reactive oxygen species (ROS) have been highlighted as one of the key players that underlie the acquisition of the various hallmarks of cancer. As ROS are associated with all stages of cancer, their applications in cancer treatment based on the following concentration-dependent implications have attracted much attention: (1) low to moderate levels of ROS as key signaling molecules, (2) elevated levels of ROS in cancer cells as one of the unique characteristics of cancer, and (3) excessive levels of ROS as cytotoxic agents. Considering ROS from a different point of view, various cancer nanomedicines have been designed to achieve spatiotemporal control of therapeutic action, the main research focus in this area. This Account includes our efforts and preclinical achievements in development of nanomedicines for a range of ROS-mediated cancer therapies. It begins with general background regarding cancer nanomedicines, the significance of ROS in cancer, and a brief overview of ROS-mediated approaches for cancer therapy. Then, this Account highlights the two key roles of ROS that define therapeutic purposes of cancer nanomedicines: (1) ROS as drug delivery enhancers and (2) ROS as cell death inducers. The former inspired us to develop nitric oxide-generating nanoparticles for improved EPR effect, endogenous ROS-responsive polymeric micelles for enhanced intracellular drug delivery, and exogenous ROS-activated micelles for subcellular localization via photochemical internalization. While refining conventional chemotherapy, recent researches also have focused on the latter, the cytotoxic ROS, to advance alternative treatment modalities such as oxidation therapy, photodynamic therapy (PDT), and sonodynamic therapy (SDT). In particular, we have been motivated to develop polymeric nanoreactors containing enzymes to produce H2O2 for oxidation therapy, photosensitizer-loaded gold-nanoclustered polymeric nanoassemblies for photothermally activated PDT overcoming the oxygen dependency of PDT, and hydrophilized TiO2 nanoparticles and Au-TiO2 nanocomposites as novel sonosensitizers for improved SDT efficiency. The integration of nanomedicine and ROS-mediated therapy has emerged as the new paradigm in the treatment of cancer, based on promising proof-of-concept demonstrations in preclinical studies. Further efforts to ensure clinical translation along with more sophisticated cancer nanomedicines to address relevant challenges are expected to be made in the coming years.

Interaction of tumor cells and lymphatic vessels in cancer progression

Metastatic spread of cancer through the lymphatic system affects hundreds of thousands of patients yearly. Growth of new lymphatic vessels, lymphangiogenesis, is activated in cancer and inflammation, but is largely inactive in normal physiology, and therefore offers therapeutic potential. Key mediators of lymphangiogenesis have been identified in developmental studies. During embryonic development, lymphatic endothelial cells derive from the blood vascular endothelium and differentiate under the guidance of lymphatic-specific regulators, such as the prospero homeobox 1 transcription factor. Vascular endothelial growth factor-C (VEGF-C) and VEGF receptor 3 signaling are essential for the further development of lymphatic vessels and therefore they provide a promising target for inhibition of tumor lymphangiogenesis. Lymphangiogenesis is important for the progression of solid tumors as shown for melanoma and breast cancer. Tumor cells may use chemokine gradients as guidance cues and enter lymphatic vessels through intercellular openings between endothelial cell junctions or, possibly, by inducing larger discontinuities in the endothelial cell layer. Tumor-draining sentinel lymph nodes show enhanced lymphangiogenesis even before cancer metastasis and they may function as a permissive 'lymphovascular niche' for the survival of metastatic cells. Although our current knowledge indicates that the development of anti-lymphangiogenic therapies may be beneficial for the treatment of cancer patients, several open questions remain with regard to the frequency, mechanisms and biological importance of lymphatic metastases.

Cellular actions of angiogenesis inhibitors on blood vessels

Cellular actions of angiogenesis inhibitors on blood vessels

Characteristics of lymphatic endothelial cells in physiological and pathological conditions.

Impairment of lymphatic structure and function, e.g., inadequate endothelial permeability and intercellular openings, abnormal lymphangiogenesis and overexpression for immunoreactive agents, will result in tumor metastasis, autoimmune response alteration and accumulation of interstitial fluid and proteins. Recently, several novel molecules have been identified that allow a more precise distinction between lymphatic and blood vascular endothelium. The differences in expression of endothelial markers on the lymphatic vessel strongly suggest the possibility that there will be important divergence in the differentiating and regenerating responses in lymphatic behavior to various pathological processes. Undoubtfully, molecular techniques would also lead to the definition of unique markers found on lymphatic endothelial cells (LECs) in lymphatic-associated diseases which are mostly involved in lymphangiogenesis. This review is mainly concentrated on the characteristics of LECs in diabetes, wound healing, lymphedema and tumor, especially in the experimental models that have offered insight into the LEC role in these diseases affecting the lymphatic system. Increased knowledge of the molecular signaling pathways driving lymphatic development and lymphangiogenesis should boost the impact of therapeutics on the diseases. Although the field about the mechanisms that control the formation and lineage-specific differentiation and function of lymphatic vessels has experienced rapid progress in the past few years, an understanding of the basis of the differences and their implications in the pathological conditions will require much more investigation.

Openings between Defective Endothelial Cells Explain Tumor Vessel Leakiness

Leakiness of blood vessels in tumors may contribute to disease progression and is key to certain forms of cancer therapy, but the structural basis of the leakiness is unclear. We sought to determine whether endothelial gaps or transcellular holes, similar to those found in leaky vessels in inflammation, could explain the leakiness of tumor vessels. Blood vessels in MCa-IV mouse mammary carcinomas, which are known to be unusually leaky (functional pore size 1.2-2 microm), were compared to vessels in three less leaky tumors and normal mammary glands. Vessels were identified by their binding of intravascularly injected fluorescent cationic liposomes and Lycopersicon esculentum lectin and by CD31 (PECAM) immunoreactivity. The luminal surface of vessels in all four tumors had a defective endothelial monolayer as revealed by scanning electron microscopy. In MCa-IV tumors, 14% of the vessel surface was lined by poorly connected, overlapping cells. The most superficial lining cells, like endothelial cells, had CD31 immunoreactivity and fenestrae with diaphragms, but they had a branched phenotype with cytoplasmic projections as long as 50 microm. Some branched cells were separated by intercellular openings (mean diameter 1.7 microm; range, 0.3-4.7 microm). Transcellular holes (mean diameter 0.6 microm) were also present but were only 8% as numerous as intercellular openings. Some CD31-positive cells protruded into the vessel lumen; others sprouted into perivascular tumor tissue. Tumors in RIP-Tag2 mice had, in addition, tumor cell-lined lakes of extravasated erythrocytes. We conclude that some tumor vessels have a defective cellular lining composed of disorganized, loosely connected, branched, overlapping or sprouting endothelial cells. Openings between these cells contribute to tumor vessel leakiness and may permit access of macromolecular therapeutic agents to tumor cells.

Mechanism of extracellular calcium regulation of intestinal epithelial tight junction permeability: role of cytoskeletal involvement.

Recent studies suggest that an abnormal increase in intestinal tight junction (TJ) permeability may be an important etiologic factor in number of diseases including Crohn's disease, NSAID-associated enteritis, and various infectious diarrheal syndromes. The intracellular processes involved in regulation of intestinal epithelial TJ permeability, however, remain poorly understood. In this study, we used cultured Caco-2 intestinal epithelial cells to examine the intracellular processes involved in extracellular Ca(++) modulation of intestinal epithelial monolayer TJ barrier. Incubation of the filter-grown Caco-2 intestinal monolayers in Ca(++)-free solution (CFS), consisting of modified Krebs-buffer solution containing 0 mM Ca(++) and 1 mM EGTA, resulted in a rapid drop in Caco-2 epithelial resistance and increase in epithelial permeability to paracellular markers mannitol and inulin, indicating an increase in TJ permeability. The increase in Caco-2 TJ permeability was rapidly reversed by the re-introduction of Ca(++) (1.8 mM) into the incubation medium. The CFS-induced increase in Caco-2 TJ permeability was associated with separation of the cytoplasmic and transmembrane TJ proteins, ZO-1 and occludin, and formation of large intercellular openings between the adjoining cells. The CFS-induced modulation of TJ barrier was associated with activation of myosin light chain kinase (MLCK) activity and centripetal retraction of peri-junctional actin and myosin filaments. The inhibition of CFS-induced activation of Caco-2 MLCK with MLCK inhibitor (ML-7) prevented the CFS-induced retraction of actin and myosin filaments and the subsequent alteration of TJ barrier function and structure. Our results suggested that the CFS-induced alteration of TJ proteins and functional increase in TJ permeability was mediated by Caco-2 MLCK activation and the resultant contraction of the peri-junctionally located actin-myosin filaments. Consistent with the role of MLCK in this process, selected inhibitors of Mg(++)-myosin ATPase and metabolic energy, but not protein synthesis inhibitors, also prevented the CFS-induced retraction of actin and myosin filaments and the subsequent increase in TJ permeability. In conclusion, our results indicate that extracellular Ca(++) is crucial for the maintenance of intestinal epithelial TJ barrier function. The removal of extracellular Ca(++) from the incubation medium causes activation of Caco-2 MLCK, which in turn leads to an increase in intestinal monolayer TJ permeability.

Modulation of cultured pulmonary microvessel and arterial endothelial cell barrier structure and function by serotonin

Pulmonary microvessel endothelial cell and pulmonary artery endothelial cell monolayers in tissue culture were treated with serotonin (5-hydroxytryptamine; 5-HT) alone or in conjunction with histamine, bradykinin, the thromboxane analog U-46619, and the actin modulating agent cytochalasin B. After agent treatment, cross-sections through endothelial cell (EC) monolayers were examined by light microscopy and the percentage and widths of intercellular openings were quantitated. To correlate structural changes in the endothelial barrier with an alteration in permeability, EC monolayers cultured on micropore filters were assayed for transit of Evan's blue albumin (EBA) following treatment with vasoactive mediators. 5-HT was found to decrease the patency of endothelial junctions by up to 94%, compared to untreated monolayers, and to prevent or reverse the appearance of interendothelial gaps induced by histamine, bradykinin, U-46619, and cytochalasin B. The 5-HT effect was dose and time dependent, with a maximal increase in junctional apposition observed at a concentration of 10 −6 M for 30 min. This response was significantly blocked by the 5-HT antagonists LSD and ketanserin. The formation or reduction of interendothelial gaps by histamine, bradykinin, and U-46619 and by 5-HT, respectively, was positively correlated to changes in monolayer permeability to EBA. These results suggest that pulmonary edema caused by inflammatory mediators in part may be a consequence of transient increases in pulmonary EC junctional gaps, and that 5-HT may contribute to the homeostatic maintenance of endothelial barrier integrity.

Endothelial cell junctional integrity modulation by serotonin: An ultrastructural analysis

We have reported previously that exogenous serotonin (5-hydroxytryptamine, 5-HT) alters cultured bovine aortic endothelial cell (BAEC) structural integrity by modulating the assembly of stress fibers. In the present study a 5-HT stimulus-coupled change in BAEC junctional integrity was quantitated by determining the width and percentage of intercellular openings in a monolayer. BAEC treated with 5-HT at concentrations of 10 −9M to 10 −3M caused a significant dose-dependent decrease in interendothelial cell junctional openings compared to controls, with the greatest reduction induced at 10 −6M (92% from control). Treatment of BAEC with histamine (10 −4M) increased the junctional openings by 82% when compared to controls. This change could be prevented by either pretreatment of the monolayers with 5-HT or by adding 5-HT in conjunction with the histamine. To assess a direct interaction of 5-HT with actin filaments, cultured BAEC monolayers were extracted, treated with 5-HT, and processed for immunocytochemical localization of 5-HT using the Avidin-Biotin method. electron microscopy revealed 5-HT antibody bound to actin filaments and dense in areas of filament intersection, which implies a role for internalized 5-HT in stimulating the assembly of an actin filament network. Collectively, these results suggest that 5-HT helps to regulate the endothelial junctional barrier by promoting actin filament formation and stability, which may in turn increase the junctional apposition between endothelial cells.

The surface activity of the phytotoxin cerato-ulmin

The glycoprotein cerato-ulmin, which shows surface activity at concentrations as low as 30 ng/mL, has been found to stabilize air bubbles, both spheroidal and of unusual morphology, in aqueous and aqueous ethanol solutions. In response to stress on a cerato-ulmin thin film, perhaps a monolayer, at an air–water interface, cylindrical and fibrilliform air bubbles are generated. They may be manipulated by application of tension, pressure, or temperature. The relationship of these observations to elm trees infected with the cerato-ulmin producing fungus Ceratocystis ulmi suggests a mechanism whereby exceedingly low concentrations of cerato-ulmin may be concentrated and delivered to intercellular openings, thereby blocking the vascular system of elm trees. The correctness of this mechanism as a major factor in any tree wilt disease remains to be tested.

Effect of Contact Lens Wear on the Conjunctival Mucous System

Biopsy specimens from the upper tarsal conjunctiva of 20 contact lens wearers with a clinically evident increase in mucus and ten non-lens wearing subjects were examined by light microscopy and scanning and transmission electron microscopy to determine the effect of contact lens wear on the mucous cell system(s). Three types of crypts associated with mucous secretion were found in all specimens: those with intracellular openings (type I, 0.1 to 0.2 μm) associated with non-goblet mucous secretory cells; those with small intercellular openings (type II, 1 to 2 μm) usually associated with goblet cells; and those with intraepithelial and intrastromal crypts with large intercellular openings (type III, 10 to 60 μm) lined with goblet and non-goblet mucous secretory cells. Contact lens wearers had increased numbers of non-goblet cells with mucous secretory vesicles lining the surface of the conjunctiva and the epithelial infoldings of type III crypts than did the normal subjects. We conclude that increased mucous secretion in contact lens wearers is associated with an increased number of cells and number of secretory vesicles involved in the non-goblet cell mucous system.