Samo Hudoklin

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.

Recombinant Single-Chain Antibody with the Trojan Peptide Penetratin Positioned in the Linker Region Enables Cargo Transfer Across the Blood–Brain Barrier

Delivery of therapeutic proteins into tissues and across the blood–brain barrier (BBB) is limited by the size and biochemical properties of the proteins. Efficient delivery across BBB is generally restricted to small, highly lipophilic molecules. However, in the last decades, several peptides that can pass cell membranes have been identified. It has been shown that these peptides are also capable of delivering large hydrophilic cargoes into cells and are therefore a powerful biological tool for transporting drugs across cell membranes and even into the brain. We designed and prepared a single-chain antibody fragment (scFvs), specific for the pathological form of the prion protein (PrPSc), where a cell-penetrating peptide (CPP) was used as a linker between the two variable domains of the scFv. The intravenously administered recombinant scFv-CPP was successfully targeted to and delivered into mouse brain cells. Our single-chain antibody fragments are of special interest in view of possible therapeutic reagents design not only for prion diseases but also for other neurodegenerative diseases.

Urothelial Plaque Formation in Post-Golgi Compartments

Urothelial plaques are specialized membrane domains in urothelial superficial (umbrella) cells, composed of highly ordered uroplakin particles. We investigated membrane compartments involved in the formation of urothelial plaques in mouse umbrella cells. The Golgi apparatus did not contain uroplakins organized into plaques. In the post-Golgi region, three distinct membrane compartments containing uroplakins were characterized: i) Small rounded vesicles, located close to the Golgi apparatus, were labelled weakly with anti-uroplakin antibodies and they possessed no plaques; we termed them “uroplakin-positive transporting vesicles” (UPTVs). ii) Spherical-to-flattened vesicles, termed “immature fusiform vesicles” (iFVs), were uroplakin-positive in their central regions and contained small urothelial plaques. iii) Flattened “mature fusiform vesicles” (mFVs) contained large plaques, which were densely labelled with anti-uroplakin antibodies. Endoytotic marker horseradish peroxidase was not found in these post-Golgi compartments. We propose a detailed model of de novo urothelial plaque formation in post-Golgi compartments: UPTVs carrying individual 16-nm particles detach from the Golgi apparatus and subsequently fuse into iFV. Concentration of 16-nm particles into plaques and removal of uroplakin-negative membranes takes place in iFVs. With additional fusions and buddings, iFVs mature into mFVs, each carrying two urothelial plaques toward the apical surface of the umbrella cell.

Maturation of the Golgi apparatus in urothelial cells

The differentiation of urothelial cells is characterized by the synthesis of uroplakins and their assembly into the asymmetric unit membrane. The Golgi apparatus (GA) has been proposed to play a central role in asymmetric unit membrane formation. We have studied the distribution and organization of the GA in normal mouse urothelial cells and in the superficial urothelial cells that undergo differentiation following cyclophosphamide-induced regeneration, in correlation with urothelial cell differentiation. In normal urothelium, immature basal cells have a simple GA, which is small and distributed close to the nucleus. In intermediate cells, the GA starts to expand into the cytoplasm, whereas the GA of terminally differentiated umbrella cells is complex, being large and spread over the whole basal half of the cytoplasm. During early stages of regeneration after cyclophosphamide treatment, the GA of superficial cells is simple and no markers of urothelial differentiation (uroplakins or asymmetric unit membranes, discoidal or fusiform vesicles, apical surface covered with microvilli) are expressed. At a later stage, the GA expands and, in the final stage of regeneration, when cells express all markers of terminal urothelial differentiation, the GA become complex once again. Our results show that: (1) GA distribution and organization in urothelial cells is differentiation-dependent; (2) the GA matures from a simple form in partially differentiated cells to a complex form in terminally differentiated superficial cells; (3) major rearrangements of GA distribution and organization correlate with the beginning of asymmetric unit membrane production. Thus, GA maturation seems to be crucial for asymmetric unit membrane formation.

Combined cytotoxic effect of UV-irradiation and TiO2 microbeads in normal urothelial cells, low-grade and high-grade urothelial cancer cells

The differentiation of urothelial cells results in normal terminally differentiated cells or by alternative pathways in low-grade or high-grade urothelial carcinomas. Treatments with traditional surgical and chemotherapeutical approaches are still inadequate and expensive, as bladder tumours are generally highly recurrent. In such situations, alternative approaches, using irradiation of the cells and nanoparticles, are promising. The ways in which urothelial cells, at different differentiation levels, respond to UV-irradiation (photolytic treatment) or to the combination of UV-irradiation and nanoparticles (photocatalytic treatment), are unknown. Here we tested cytotoxicity of UV-irradiation on (i) normal porcine urothelial cells (NPU), (ii) human low-grade urothelial cancer cells (RT4), and (iii) human high-grade urothelial cancer cells (T24). The results have shown that 1 minute of UV-irradiation is enough to kill 90% of the cells in NPU and RT4 cultures, as determined by the live/dead viability assay. On the other hand, the majority of T24 cells survived 1 minute of UV-irradiation. Moreover, even a prolonged UV-irradiation for 30 minutes killed <50% of T24 cells. When T24 cells were pre-supplemented with mesoporous TiO2 microbeads and then UV-irradiated, the viability of these high-grade urothelial cancer cells was reduced to <10%, which points to the highly efficient cytotoxic effects of TiO2 photocatalysis. Using electron microscopy, we confirmed that the mesoporous TiO2 microbeads were internalized into T24 cells, and that the cells ultrastructure was heavily compromised after UV-irradiation. In conclusion, our results show major differences in the sensitivity to UV-irradiation among the urothelial cells with respect to cell differentiation. To achieve an increased cytotoxicity of urothelial cancer cells, the photocatalytic approach is recommended.

Electron Tomography of Fusiform Vesicles and Their Organization in Urothelial Cells

The formation of fusiform vesicles (FVs) is one of the most distinctive features in the urothelium of the urinary bladder. FVs represent compartments for intracellular transport of urothelial plaques, which modulate the surface area of the superficial urothelial (umbrella) cells during the distension-contraction cycle. We have analysed the three-dimensional (3D) structure of FVs and their organization in umbrella cells of mouse urinary bladders. Compared to chemical fixation, high pressure freezing gave a new insight into the ultrastructure of urothelial cells. Electron tomography on serial sections revealed that mature FVs had a shape of flattened discs, with a diameter of up to 1.2 µm. The lumen between the two opposing asymmetrically thickened membranes was very narrow, ranging from 5 nm to 10 nm. Freeze-fracturing and immunolabelling confirmed that FVs contain two opposing urothelial plaques connected by a hinge region that made an omega shaped curvature. In the central cytoplasm, 4–15 FVs were often organized into stacks. In the subapical cytoplasm, FVs were mainly organized as individual vesicles. Distension-contraction cycles did not affect the shape of mature FVs; however, their orientation changed from parallel in distended to perpendicular in contracted bladder with respect to the apical plasma membrane. In the intermediate cells, shorter and more dilated immature FVs were present. The salient outcome from this research is the first comprehensive, high resolution 3D view of the ultrastructure of FVs and how they are organized differently depending on their location in the cytoplasm of umbrella cells. The shape of mature FVs and their organization into tightly packed stacks makes them a perfect storage compartment, which transports large amounts of urothelial plaques while occupying a small volume of umbrella cell cytoplasm.

Gold nanoparticles as physiological markers of urine internalization into urothelial cells in vivo

Background Urothelial bladder is the reservoir of urine and the urothelium minimizes the exchange of urine constituents with this tissue. Our aim was to test 1.9 nm biocompatible gold nanoparticles as a novel marker of internalization into the urothelial cells under physiological conditions in vivo. Methods We compared normal and neoplastic mice urothelium. Neoplastic lesions were induced by 0.05% N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) in drinking water for 10 weeks. Nanoparticles, intravenously injected into normal and BBN-treated mice, were filtered through the kidneys and became constituents of the urine within 90 minutes after injection. Results Gold nanoparticles were densely accumulated in the urine, while their internalization into urothelial cells depended on the cell differentiation stage. In the terminally differentiated superficial urothelial cells of normal animals, nanoparticles were occasionally found in the endosomes, but not in the fusiform vesicles. Regions of exfoliated cells were occasionally found in the normal urothelium. Superficial urothelial cells located next to exfoliated regions contained gold nanoparticles in the endosomes and in the cytosol beneath the apical plasma membrane. The urothelium of BBN-treated animals developed fat hyperplasia with moderate dysplasia. The superficial cells of BBN-treated animals were partially differentiated as demonstrated by the lack of fusiform vesicles. These cells contained the gold nanoparticles distributed in the endosomes and throughout their cytosol. Conclusion Gold nanoparticles are a valuable marker to study urine internalization into urothelial cells in vivo. Moreover, they can be used as a sensitive marker of differentiation and functionality of urothelial cells.

Increased endocytosis of magnetic nanoparticles into cancerous urothelial cells versus normal urothelial cells

The blood–urine barrier is the tightest and most impermeable barrier in the body and as such represents a problem for intravesical drug delivery applications. Differentiation-dependent low endocytotic rate of urothelial cells has already been noted; however, the differences in endocytosis of normal and cancer urothelial cells have not been exploited yet. Here we analysed the endocytosis of rhodamine B isothiocyanate-labelled polyacrylic acid-coated cobalt ferrite nanoparticles (NPs) in biomimetic urothelial in vitro models, i.e., in highly and partially differentiated normal urothelial cells, and in cancer cells of the papillary and invasive urothelial neoplasm. We demonstrated that NPs enter papillary and invasive urothelial neoplasm cells by ruffling of the plasma membrane and engulfment of NP aggregates by macropinocytotic mechanism. Transmission electron microscopy (TEM) and spectrophotometric analyses showed that the efficacy of NPs delivery into normal urothelial cells and intercellular space is largely restricted, while it is significantly higher in cancer urothelial cells. Moreover, we showed that the quantification of fluorescent NP internalization in cells or tissues based on fluorescence detection could be misleading and overestimated without TEM analysis. Our findings contribute to the understanding of endocytosis-mediated cellular uptake of NPs in cancer urothelial cells and reveal a highly selective mechanism to distinguish cancer and normal urothelial cells.

Magnetic field contributes to the cellular uptake for effective therapy with magnetofection using plasmid DNA encoding against Mcam in B16F10 melanoma in vivo

AIM We explored the distribution and cellular uptake of intratumorally injected SPIONs-PAA-PEI-pDNA (magnetofection complexes), and antitumor effectiveness of magnetofection with plasmid DNA encoding short hairpin RNA (shRNA) against Mcam (pDNA(anti-MCAM)). MATERIALS & METHODS Analyses were made based on the histology, ultrastructure and quantitative measurements of magnetofection complexes, and quantification of the antitumor effectiveness in B16F10 melanoma in vivo. RESULTS Injected magnetofection complexes were distributed around the injection site. Exposure of tumors to external magnetic field contributed to the uptake of magnetofection complexes from extracellular matrix into melanoma cells. Three consecutive magnetofections of tumors with pDNA(anti-MCAM) resulted in significant reduction of tumor volume. CONCLUSION Magnetofection is effective for gene delivery to melanoma tumors, but requires a magnetic field for cellular uptake and antitumor effect.

Bone remodeling during orthodontic tooth movement in rats with type 2 diabetes.

INTRODUCTION Type 2 diabetes is known to affect bone metabolism. In this study, we aimed to determine the effects of type 2 diabetes on bone remodeling during orthodontic tooth movement. METHODS The 48 rats were divided into 4 groups: Wistar control group (n = 8), Goto-Kakizaki (GK) control group (n = 8), Wistar appliance group (n = 16), and GK appliance group (n = 16). The distances between the teeth were measured weekly. On day 42, maxillary alveolar bone specimens were obtained for histologic evaluation and determination of the gene expression levels of the receptor activator of nuclear factor ҡB (RANK), RANK ligand (RANKL), and osteoprotegerin (OPG). RESULTS No significant difference was observed in the levels of tooth movement between the 2 appliance groups. After orthodontic force application, the alveolar bone volume and osteoblast surface in the GK rats were diminished compared with those in the Wistar rats. The increase in the osteoclast surface relative to the control groups was 2.4-fold greater in the GK rats than in the Wistar rats. Significant upregulations of the RANK and OPG gene expression levels in the Wistar appliance group were observed. The RANKL/OPG ratio was increased in the GK appliance group compared with the Wistar appliance group. CONCLUSIONS Diminished bone formation and slightly increased bone resorption were observed during orthodontic tooth movement in the rats with type 2 diabetes.