Effect of irradiation on the expression of E-cadherin and ß-catenin in early and late radiation sequelae of the urinary bladder and its modulation by NF-κB inhibitor thalidomide.

PURPOSE: In a previous study we have shown in a mouse model that administration of nuclear factor-kappa B (NF-κB) inhibitor thalidomide has promising therapeutic effects on early radiation cystitis (ERC) and late radiation sequelae (LRS) of the urinary bladder. The aim of this study was to evaluate in the same mice the effect of thalidomide on adherens junction (AJ) proteins in ERC and LRS. METHODS: Urothelial expressions of E­cadherin and ß­catenin were assessed by immunohistochemistry in formalin-fixed paraffin-embedded (FFPE) bladder specimens over 360 days post single-dose irradiation on day 0. First, the effect of irradiation on AJ expression and then effects of thalidomide on irradiation-induced AJ alterations were assessed using three different treatment times. RESULTS: Irradiation provoked a biphasic upregulation of E­cadherin and ß­catenin in the early phase. After a mild decrease of E­cadherin and a pronounced decrease of ß­catenin at the end of the early phase, both increased again in the late phase. Early administration of thalidomide (day 1-15) resulted in a steeper rise in the first days, an extended and increased expression at the end of the early phase and a higher expression of ß­catenin alone at the beginning of the late phase. CONCLUSION: Upregulation of AJ proteins is an attempt to compensate irradiation-induced impairment of urothelial barrier function. Early administration of thalidomide improves these compensatory mechanisms by inhibiting NF-κB signaling and its interfering effects.

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.

An optochemical tool for light-induced dissociation of adherens junctions to control mechanical coupling between cells.

The cadherin-catenin complex at adherens junctions (AJs) is essential for the formation of cell-cell adhesion and epithelium integrity; however, studying the dynamic regulation of AJs at high spatio-temporal resolution remains challenging. Here we present an optochemical tool which allows reconstitution of AJs by chemical dimerization of the force bearing structures and their precise light-induced dissociation. For the dimerization, we reconstitute acto-myosin connection of a tailless E-cadherin by two ways: direct recruitment of α-catenin, and linking its cytosolic tail to the transmembrane domain. Our approach enables a specific ON-OFF switch for mechanical coupling between cells that can be controlled spatially on subcellular or tissue scale via photocleavage. The combination with cell migration analysis and traction force microscopy shows a wide-range of applicability and confirms the mechanical contribution of the reconstituted AJs. Remarkably, in vivo our tool is able to control structural and functional integrity of the epidermal layer in developing Xenopus embryos.

Impairment of lymphatic endothelial barrier function by X-ray irradiation.

PURPOSE: Although the microvascular system is a significant target for radiation-induced effects, the lymphatic response to radiation has not been extensively investigated. This is one of the first investigations characterizing the lymphatic endothelial response to ionizing radiation. MATERIALS AND METHODS: Rat mesenteric lymphatic endothelial cells (RMLECs) were exposed to X-ray doses of 0, 0.5, 1, 1.5, and 2 Gy. RMLEC cellular response was assessed 24 and 72-h post-irradiation via measures of cellular morphometry and junctional adhesion markers. RMLEC functional response was characterized through permeability experiments. RESULTS: Cell morphometry showed radiation sensitivity at all doses. Notably, there was a loss of cell-to-cell adhesion with irradiated cells increasing in size and cellular roundness. This was coupled with decreased ß-catenin and VE-cadherin intensity and altered F-actin anisotropy, leading to a loss of intercellular contact. RMLEC monolayers demonstrated increased permeability at all doses 24 h post-irradiation and at 2-Gy 72 h post-irradiation. CONCLUSIONS: In summary, lymphatics show radiation sensitivity in the context of these cell culture experiments. Our results may have functional implications of lymphatics in tissue, with endothelial barrier dysfunction due to loss of cell-cell adhesion leading to leaky vessels and lymphedema. These preliminary experiments will build the framework for future investigations towards lymphatic radiation exposure response.

Persistent disruption of lateral junctional complexes and actin cytoskeleton in parotid salivary glands following radiation treatment.

Xerostomia and hyposalivation are debilitating side effects for patients treated with ionizing radiation for head and neck cancer. Despite technological advances, collateral damage to the salivary glands remains a significant problem for patients and severely diminishes their quality of life. During the wound healing process, restoration of junctional contacts is necessary to maintain polarity, structural integrity, and orientation cues for secretion. However, little is known about whether these structural molecules are impacted following radiation damage and more importantly, during tissue restoration. We evaluated changes in adherens junctions and cytoskeletal regulators in an injury model where mice were irradiated with 5 Gy and a restoration model where mice injected postradiation with insulin-like growth factor 1 (IGF1) are capable of restoring salivary function. Using coimmunoprecipitation, there is a decrease in epithelial (E)-cadherin bound to ß-catenin following damage that is restored to untreated levels with IGF1. Via its adaptor proteins, ß-catenin links the cadherins to the cytoskeleton and part of this regulation is mediated through Rho-associated coiled-coil containing kinase (ROCK) signaling. In our radiation model, filamentous (F)-actin organization is fragmented, and there is an induction of ROCK activity. However, a ROCK inhibitor, Y-27632, prevents E-cadherin/ß-catenin dissociation following radiation treatment. These findings illustrate that radiation induces a ROCK-dependent disruption of the cadherin-catenin complex and alters F-actin organization at stages of damage when hyposalivation is observed. Understanding the regulation of these components will be critical in the discovery of therapeutics that have the potential to restore function in polarized epithelium.

Magnetic forces enable controlled drug delivery by disrupting endothelial cell-cell junctions.

The vascular endothelium presents a major transport barrier to drug delivery by only allowing selective extravasation of solutes and small molecules. Therefore, enhancing drug transport across the endothelial barrier has to rely on leaky vessels arising from disease states such as pathological angiogenesis and inflammatory response. Here we show that the permeability of vascular endothelium can be increased using an external magnetic field to temporarily disrupt endothelial adherens junctions through internalized iron oxide nanoparticles, activating the paracellular transport pathway and facilitating the local extravasation of circulating substances. This approach provides a physically controlled drug delivery method harnessing the biology of endothelial adherens junction and opens a new avenue for drug delivery in a broad range of biomedical research and therapeutic applications.

Alteration of tight and adherens junctions on 50-Hz magnetic field exposure in Madin Darby canine kidney (MDCK) cells.

Adherens (AJ) and tight junctions (TJ), as integrated parts of the junctional complex, are multifunctional specialized regions of the cell membrane in epithelial cells. They are responsible for cell-to-cell interactions and also have great importance in cellular signaling processes including Wnt protein-mediated signals. As electromagnetic field (EMF) exposure is known to cause alterations in the function as well as supramolecular organization of different cell contacts, our goal was to investigate the effect of 50-Hz magnetic field (MF) exposures on the subcellular distribution of some representative structural proteins (occludin, beta-catenin, and cadherin) found in AJ and TJ. Additionally, cellular beta-catenin content was also quantified by Western blot analysis. 50-Hz MF exposures seemed to increase the staining intensity (amount) of occludin, cadherins, and beta-catenin in the junctional area of MDCK cells, while Western blot data indicated the quantity of beta-catenin was found significantly decreased at both time points after EM exposures. Our results demonstrate that MF are able to modify the distribution of TJ and AJ structural proteins, tending to stabilize these cell contacts. The quantitative changes of beta-catenin suggest a causative relationship between MF effects on the cell junctional complex and the Wnt signaling pathway.

Radiation responses in plasma membrane. Review of the present state and future trends.

Over the last several decades, the membrane system of the cell has been shown to be a fairly sensitive target for ionizing radiation. As the complex features of membrane functions and structure are revealed more and more, the interest of radiation biology grows. The present review of the biological aspects of ionizing radiation exposure suggests the importance of cell-to-cell contacts through junctions, and the signaling mechanism through receptors.