The urinary tract is exposed to a variety of possible injures that may lead to organ damage or loss, and thus, the establishment of valid in vitro urothelial models to study the mechanism of drug candidates is necessary. This study is the first to investigate the effect of chitosan on urothelia in vitro and to evaluate whether chitosan-treated urothelial models can regenerate in vitro and reestablish a functional urothelium. Biomimetic hyperplastic and normoplastic urothelial models were used to test the effect of chitosan (0.05 %) on partially and highly differentiated urothelial cells (UCs) by monitoring their molecular, ultrastructural, and physiological changes for 3 weeks. Chitosan caused an immediate and complete loss of transepithelial resistance (TER), tight junction disruption, cytopathological changes of UCs, and consequently enhanced the permeability of partially and highly differentiated urothelial models. However, 3 weeks after chitosan treatment, TER was reestablished, tight junctions resealed, permeability decreased, and progressive differentiation stages of newly exposed superficial UCs expressing uroplakins and tight junction protein claudin-8 were found. The in vitro models regenerated and reestablished urothelia with a tight barrier. The biomimetic urothelial models represent appropriate in vitro models for studying urothelial drug candidates as well as evaluating drug permeabilities and their intracellular function. Understanding the possible intracellular function of chitosan could significantly advance approaches to treating urothelial-specific diseases.
COBISS.SI-ID: 31527129
Reciprocal interactions between the epithelium and mesenchyme are essential for the establishment of proper tissue morphology during organogenesis and tissue regeneration as well as for the maintenance of cell differentiation. With this review, we highlight the importance of epithelial-mesenchymal cross-talk in healthy tissue and further discuss its significance in engineering functional tissues in vitro. We focus on urinary bladder and small intestine, organs that are often compromised by disease, and are as such in need of research that would advance effective treatment or tissue replacement. To date, the understanding of epithelial-mesenchymal reciprocal interactions has enabled the development of in vitro biomimetic tissue equivalents that have provided many possibilities in treating defective, damaged, or even cancerous tissues. Although research of the past several years has advanced the field of bladder and small intestine tissue engineering, one must be aware of its current limitations in successfully and above all safely introducing tissue-engineered constructs into clinical practice. Special attention is in particular needed when treating cancerous tissues, as initially successful tumor excision and tissue reconstruction may later on result in cancer recurrence due to oncogenic signals originating from an altered stroma. Recent rather poor outcomes in pioneering clinical trials of bladder reconstructions should serve as a reminder that recreating a functional organ to replace a dysfunctional one is an objective far more difficult to reach than initially foreseen. When considering effective tissue engineering approaches for diseased tissues in humans, it is imperative to introduce animal models with dysfunctional or, even more importantly, cancerous organs, which would greatly contribute to predicting possible complications and hence reducing risks when translating to the clinic.
COBISS.SI-ID: 32075737
The primary function of the urinary bladder is to store and periodically release urine. How the urothelium prevents permeation of water, ions, solutes, and noxious agents back into the bloodstream and underlying tissues as well as serving as a sensor and transducer of physiological and nociceptive stimuli is still not completely understood, and thus its unique functional complexity remains to be fully elucidated. This article reviews the permeation routes across urothelium as demonstrated in extensive morphological and electrophysiological studies on in vivo and in vitro urothelia. We consider the molecular and morphological structures of urothelium and how they contribute to the impermeability of the blood-urine barrier. Based on the available data, the extremely low permeability properties of urothelium can be postulated. This remarkable impermeability is necessary for the normal functioning of all mammals, but at the same time represents limitations regarding the uptake of drugs. Therefore, the current progress to overcome this most resilient barrier in our body for drug therapy purposes is also summarized in this review.
COBISS.SI-ID: 31938777
Cholesterol content can vary distinctly between normal and cancer cells, with elevated levels in cancer cells. Here, we investigated cholesterol sequestration with methyl-β-cyclodextrin (MCD), and pore-formation with the ostreolysin A/pleurotolysin B (OlyA/PlyB) protein complex that binds to cholesterol/sphingomyelin-rich membrane domains. We evaluated the effects on viability of T24 invasive and RT4 noninvasive human urothelial cancer cells and normal porcine urothelial (NPU) cells. Cholesterol content strongly correlated with cancerous transformation, as highest in the T24 high-grade invasive urothelial cancer cells, and lowest in NPU cells. MCD treatment induced prominent cell death of T24 cells, whereas OlyA/PlyB treatment resulted in greatly decreased viability of the RT4 low-grade noninvasive carcinoma cells. Biochemical and transmission electron microscopy analyses revealed that MCD and OlyA/PlyB induce necrotic cell death in these cancer cells, while viability of NPU cells was not significantly affected by either treatment. We conclude that MCD is more toxic for T24 high-grade invasive urothelial cancer cells, and OlyA/PlyB for RT4 low-grade noninvasive urothelial cancer cells, and neither is toxic for NPU cells. The cholesterol and cholesterol/sphingomyelin-rich membrane domains in urothelial cancer cells thus constitute a selective therapeutic target for elimination of urothelial cancer cells.
COBISS.SI-ID: 3572559
Cell spreading capability and cell proliferation are the major processes in wound healing of injured epithelia as well as in tumour progression. The effect of low-density lipoprotein (LDL) particles as a major extracellular source of cholesterol was evaluated in the re-epithelisation assay of in vitro induced injury. We selected two noncancer cell lines with different dependence on LDL concentrations, the kidney epithelial cells (MDCK) with higher dependence and keratinocytes (HaCaT) with lower dependence on LDL, and three cancer cell lines originating from epithelial cells: A549 (alveolar), CaCo-2 (intestinal) and RT4 (urothelial). All cells were incubated in a control medium, in an LDL-enriched medium or in an LDL-deficient medium. The LDL-enriched medium stimulated cell spreading of MDCK cells which, together with increased proliferation of these cells, resulted in an enhanced re-epithelisation of in vitro induced injury. LDL deficiency caused lower cell spreading which resulted in a decreased re-epithelisation despite the higher proliferation of MDCK cells in this medium. The re-epithelisation of keratinocytes (HaCaT) was not affected by altered LDL concentrations. In cancer cell lines A549, CaCo-2 and RT4, wide heterogeneity regarding cell proliferation and spreading capability was observed after treatment with different LDL concentrations. LDL had no influence on actin filament and tight junction distribution in any of the tested cell lines. The cholesterol content of all cell types, except for CaCo-2 cells, proved to be independent of the LDL level. Further research of the beneficial effects of LDL is needed to prove LDL as a safe enhancer of epithelial wound healing.
COBISS.SI-ID: 31875033