Mateja Erdani Kreft

Toxicological Aspects of Long‐Term Treatment of Keratinocytes with ZnO and TiO2 Nanoparticles

Sunscreens containing ZnO and TiO(2) nanoparticles (NPs) are increasingly applied to skin over long time periods to reduce the risk of skin cancer. However, long-term toxicological studies of NPs are very sparse. The in vitro toxicity of ZnO and TiO(2) NPs on keratinocytes over short- and long-term applications is reported. The effects studied are intracellular formation of radicals, alterations in cell morphology, mitochondrial activity, and cell-cycle distribution. Cellular response depends on the type of NP, concentration, and exposure time. ZnO NPs have more pronounced adverse effects on keratinocytes than TiO(2). TiO(2) has no effect on cell viability up to 100 μg mL(-1), whereas ZnO reduces viability above 15 μg mL(-1) after short-term exposure. Prolonged exposure to ZnO NPs at 10 μg mL(-1) results in decreased mitochondrial activity, loss of normal cell morphology, and disturbances in cell-cycle distribution. From this point of view TiO(2) has no harmful effect. More nanotubular intercellular structures are observed in keratinocytes exposed to either type of NP than in untreated cells. This observation may indicate cellular transformation from normal to tumor cells due to NP treatment. Transmission electron microscopy images show NPs in vesicles within the cell cytoplasm, particularly in early and late endosomes and amphisomes. Contrary to insoluble TiO(2), partially soluble ZnO stimulates generation of reactive oxygen species to swamp the cell redox defense system thus initiating the death processes, seen also in cell-cycle distribution and fluorescence imaging. Long-term exposure to NPs has adverse effects on human keratinocytes in vitro, which indicates a potential health risk.

Urothelial injuries and the early wound healing response: tight junctions and urothelial cytodifferentiation

Using primary explant cultures of mouse bladder, the early response of the urothelium after superficial and full-thickness injuries was investigated. In such an in vitro wound healing model, explant surfaces with a mostly desquamated urothelial superficial layer represented superficial wounds, and the exposed lamina propria at the cut edges of the explants represented full-thickness wounds. The urothelial cell ultrastructure, the expression and subcellular distribution of the tight junctional protein occludin, and differentiation-related proteins CK 20, uroplakins, and actin were followed. Since singular terminally differentiated superficial cells remained on the urothelium after superficial injury (i.e., original superficial cells), we sought to determine their role during the urothelial wound-healing process. Ultrastructural and immunocytochemical studies have revealed that restored tight junctions are the earliest cellular event during the urothelial superficial and full-thickness wound-healing process. Occludin-containing tight junctions are developed before the new superficial cells are terminally differentiated. New insights into the urothelium wound-healing process were provided by demonstrating that the original superficial cells contribute to the urothelium wound healing by developing tight junctions with de novo differentiated superficial cells and by stretching, thus providing a large urothelial surface with asymmetric unit membrane plaques.

Stimulation inhibits the mobility of recycling peptidergic vesicles in astrocytes

Astrocytes are increasingly viewed as playing many roles in the integration of brain function. These cells store among other gliotransmitters also neuroactive peptides in membrane bound vesicles, the trafficking and release of which, may be changed in altered conditions, therefore affecting the physiological status of neurons. In general, peptidergic membrane‐bound secretory vesicles fuse with the plasma membrane in the process of exocytosis. Some of them are retrieved from the plasma membrane to be recycled back into the cytosol. The mobility of retrieving vesicles in astrocytes was not studied yet, however, understanding the mechanisms of such trafficking would highlight the communication paths between astrocytes and neurons. We labeled vesicles with antibodies against the vesicle atrial natriuretic peptide (ANP), which is stored inside secretory vesicles. ANP‐vesicles in astrocytes have been proposed to enter Ca2+‐dependent secretion and here we show that they are associated with synaptotagmin IV (SytIV), a regulator of exocytosis in astrocytes. Moreover, the results show that recycling ANP‐vesicles are to a significant extent acidic. Their velocity (0.06 ± 0.001 μm/s) is one order of magnitude lower than the velocity of vesicles trafficking to the plasma membrane (Potokar et al. ( 2005 ) Biochem Biophys Res Commun 329:678–683; Potokar et al. ( 2007 ) Traffic 8:12–20). Interestingly, ionomycin or ATP application further attenuated ANP‐vesicle mobility to 0.02 ± 0.002 and to 0.03 ± 0.001 μm/s, respectively. In summary, the mobility of recycling peptidergic vesicles appears to be slower than the vesicle traffic to the plasma membrane and it requires an intact cytoskeleton. Physiological implications of attenuated traffic of ANP‐vesicles are considered in the discussion.

Formation and maintenance of blood–urine barrier in urothelium

Blood–urine barrier, which is formed during differentiation of superficial urothelial cells, is the tightest and most impermeable barrier in the body. In the urinary bladder, the barrier must accommodate large changes in the surface area during distensions and contractions of the organ. Tight junctions and unique apical plasma membrane of superficial urothelial cells play a critical role in the barrier maintenance. Alterations in the blood–urine barrier function accompany most of the urinary tract diseases. In this review, we discuss recent discoveries on the role of tight junctions, dynamics of Golgi apparatus and post-Golgi compartments, and intracellular membrane traffic during the biogenesis and maintenance of blood–urine barrier.

Endocytotic activity of bladder superficial urothelial cells is inversely related to their differentiation stage

The composition of the apical plasma membrane of bladder superficial urothelial cells is dramatically modified during cell differentiation, which is accompanied by the change in the dynamics of endocytosis. We studied the expression of urothelial differentiation-related proteins uroplakins and consequently the apical plasma membrane molecular composition in relation to the membrane-bound and fluid-phase endocytosis in bladder superficial urothelial cells. By using primary urothelial cultures in the environment without mechanical stimuli, we studied the constitutive endocytosis. Four new findings emerge from our study. First, in highly differentiated superficial urothelial cells with strong uroplakin expression, the endocytosis of fluid-phase endocytotic markers was 43% lower and the endocytosis of membrane-bound markers was 86% lower compared to partially differentiated cells with weak uroplakin expression. Second, superficial urothelial cells have 5-15-times lower endocytotic activity than MDCK cells. Third, in superficial urothelial cells the membrane-bound markers are delivered to lysosomes, while fluid-phase markers are seen only in early endocytotic compartments, suggesting their kiss-and-run recycling. Finally, we provide the first evidence that in highly differentiated cells the uroplakin-positive membrane regions are excluded from internalization, suggesting that uroplakins hinder endocytosis from the apical plasma membrane in superficial urothelial cells and thus maintain optimal permeability barrier function.

Apical Plasma Membrane Traffic in Superficial Cells of Bladder Urothelium

Superficial urothelial cells that line the urinary bladder accommodate cyclical changes in organ volume while maintaining a permeability barrier between urine and tissue fluids. The specific apical plasma membrane traffic is necessary for their proper function. The composition of the apical plasma membrane is dramatically modified during differentiation of bladder urothelial cells, most notably by assembly of urothelial plaques containing uroplakins. However, the assembly of uroplakins into plaques, their insertion and removal from the apical surface, and the regulation of these processes are still poorly understood. This review examines the traffic (exocytosis/endocytosis) of the apical plasma membrane during differentiation of urothelial cells and focuses on the physiological and clinical significance of the apical plasma membrane traffic in bladder superficial urothelial cells.

ANTIGENIC AND ULTRASTRUCTURAL MARKERS ASSOCIATED WITH UROTHELIAL CYTODIFFERENTIATION IN PRIMARY EXPLANT OUTGROWTHS OF MOUSE BLADDER

The purpose of this study was to establish an in vitro culture model that closely resembles whole mouse urothelial tissue. Primary explant cultures of mouse bladder were established on porous membrane supports and explant outgrowths were analysed for morphology and the presence of antigenic and ultrastructural markers associated with urothelial cytodifferentiation. When examined at the ultrastructural level, the cultured urothelium was polarized and organized as a multilayered epithelium. Differentiation was found to increase from the porous membrane towards the surface and from the explant towards the periphery of the culture. Scanning and transmission electron microscopical analysis of the most superficially‐located cells revealed four successive differentiation stages: cells with microvilli, cells with ropy microridges, cells with rounded microridges, and highly‐differentiated cells with asymmetric unit membrane (AUM) plaques forming rigid microridges and fusiform vesicles. The more highly‐differentiated cells were numerous at the periphery of the culture, but rare close to the explant. Epithelial organization was stabilized by well developed cell junctions. Immunolabeling demonstrated that superficial urothelial cells in culture: (1) develop tight junctions, E‐cadherin adherens junctions and abundant desmosomes and (2) express uroplakins and cytokeratin 20 (CK 20). Using a culture model of primary explant outgrowth we have shown that non‐differentiated mouse urothelial cells growing on a porous membrane show a high level of de novo differentiation.

Golgi apparatus fragmentation as a mechanism responsible for uniform delivery of uroplakins to the apical plasma membrane of uroepithelial cells

Background information. The GA (Golgi apparatus) has an essential role in membrane trafficking, determining the assembly and delivery of UPs (uroplakins) to the APM (apical plasma membrane) of superficial UCs (uroepithelial cells) of urinary bladder. UPs are synchronously and uniformly delivered from the GA to the APM by DFVs (discoidal‐ or fusiform‐shaped vesicles); however, the mechanism of UP delivery is not known. We have used the culture model of UCs with the capacity to undergo terminal differentiation to study the process of uniform delivery of DFVs to the APM and to elucidate the mechanisms involved.

The effect of epidermal growth factor and transforming growth factor β1 on proliferation and differentiation of urothelial cells in urinary bladder explant culture

The effects of two growth factors, EGF and TGF beta 1, on growth and differentiation of different populations of urothelial cells in explant cultures of mouse urinary bladder have been studied by electron microscopy and lectin analysis. In an explant culture 10 days after the implantation three different populations of urothelial cells can be distinguished. Original urothelial cells, which are integral part of the explant, new urothelial cells, which cover the baso-lateral sides of the explant and are organized in a multilayer epithelium, and new urothelial cells, which are not any more in direct contact with the explant and grow over the membrane in epithelium-like structure. Exogenously added EGF or TGF beta 1 did not affect either the formation or the thickness of multilayered urothelium, so cells were proliferating on the free surfaces of stroma as well as on the epithelium expanding over the membrane. In the absence of growth factors in medium, the newly formed baso-lateral multilayered epithelium bordering the stroma shows ultrastructural signs of terminal differentiation suggesting that for cell proliferation and differentiation the action of stroma is of crucial importance. On the other hand, the differentiation of the epithelium spreading over the membrane, but not its thickness, is affected by exogenously added TGF beta 1. Solely in TGF beta 1-treated cultures a differentiation similar to that in vivo takes place. The apoptosis of the urothelial cells was not increased by TGF beta 1. The lectin analysis by WGA and ConA conjugated with ferritin revealed that ConA-ferritin combines only with the surface cells which grow over the membrane in the absence of TGF beta 1.

Hyperplasia as a mechanism for rapid resealing urothelial injuries and maintaining high transepithelial resistance

When the urothelial barrier, i.e., the blood−urine barrier, is injured, rapid resealing of the injury is crucial for the normal functioning of the organism. In order to investigate the mechanisms required for rapid resealing of the barrier, we established in vitro models of hyperplastic and normoplastic urothelia. We found that hyperplastic urothelia achieve significantly higher transepithelial resistance (TER) than normoplastic urothelia. However, the expression of cell junctional (claudin-8, occludin, E-cadherin) and differentiation-related proteins (cytokeratin 20 and uroplakins) is weaker in hyperplastic urothelia. Further investigation of cell differentiation status at the ultrastructural level confirmed that superficial urothelial cells (UCs) in hyperplastic urothelial models achieve a lower differentiation stage than superficial UCs in normoplastic urothelial models. With the establishment of such in vitro models and the aid of TER measurements, flow cytometry, molecular and ultrastructural analysis, we here provide unequivocal evidence that the specific cell-cycle distribution and, consequently, the number of cell layers have a significant influence on the barrier function of urothelia. We demonstrate the importance of hyperplasia for the rapid restoration of the urothelial barrier and the maintenance of high TER until the UCs reach a highly differentiated stage and restoration of the urothelial barrier after injury is complete. The information that this approach provides is unique and we expect that further exploitation of hyperplastic and normoplastic urothelial models in future studies may advance our understanding of blood−urine barrier development and functionality.