Selective targeting of lectins and their macropinocytosis in urothelial tumours: translation from in vitro to ex vivo.

Urinary bladder cancer can be treated by intravesical application of therapeutic agents, but the specific targeting of cancer urothelial cells and the endocytotic pathways of the agents are not known. During carcinogenesis, the superficial urothelial cells exhibit changes in sugar residues on the apical plasma membranes. This can be exploited for selective targeting from the luminal side of the bladder. Here we show that the plant lectins Jacalin (from Artocarpus integrifolia), ACA (from Amaranthus caudatus) and DSA (from Datura stramonium) selectively bind to the apical plasma membrane of low- (RT4) and high-grade (T24) cancer urothelial cells in vitro and urothelial tumours ex vivo. The amount of lectin binding was significantly different between RT4 and T24 cells. Endocytosis of lectins was observed only in cancer urothelial cells and not in normal urothelial cells. Transmission electron microscopy analysis showed macropinosomes, endosome-like vesicles and multivesicular bodies filled with lectins in RT4 and T24 cells and also in cells of urothelial tumours ex vivo. Endocytosis of Jacalin and ACA in cancer cells was decreased in vitro after addition of inhibitor of macropinocytosis 5-(N-ethyl-N-isopropyl) amiloride (EIPA) and increased after stimulation of macropinocytosis with epidermal growth factor (EGF). Clathrin, caveolin and flotillin did not colocalise with lectins. These results confirm that the predominant mechanism of lectin endocytosis in cancer urothelial cells is macropinocytosis. Therefore, we propose that lectins in combination with conjugated therapeutic agents are promising tools for improved intravesical therapy by targeting cancer cells.

Attachment of Cancer Urothelial Cells to the Bladder Epithelium Occurs on Uroplakin-Negative Cells and Is Mediated by Desmosomal and Not by Classical Cadherins.

Urinary bladder cancer is often multifocal; however, the intraluminal dissemination of the urothelial cancer cells is poorly understood. The involvement of N-cadherin in the adhesion of the cancer urothelial cells to the urothelium had not previously been studied. Therefore, we herein explore the possibility of the intraluminal dissemination of the urothelial cancer cells by evaluating the role of classical cadherins in the adhesion of urothelial cancer cells to the urothelium. We used E-cadherin negative T24 cells and established a T24 Ncadlow cell line with an additionally decreased expression of N-cadherin in the plasma membrane and a decreased secretion of proform of metalloproteinase 2. The labelled T24 and T24 Ncadlow cells were seeded onto urothelial in vitro models. After 24 h in co-culture, unattached cancer cells were rinsed and urothelia with attached cancer urothelial cells were processed for fluorescence and electron microscopy. Both the T24 and T24 Ncadlow cells attached to the urothelium, yet only to the uroplakin-negative urothelial cells. The ultrastructural analysis showed that T24 and T24 Ncadlow cells adhere to poorly differentiated urothelial cells by desmosomes. To achieve this, they first disrupt tight junctions of superficial urothelial cells. This study indicates that the lack of E-cadherin expression and decreased expression of N-cadherin in the plasma membrane of T24 cells does not interfere with their adhesion to the urothelium; therefore, our results suggest that intraluminal dissemination of cancer urothelial cells along the urothelium occurs on uroplakin-negative cells and is desmosome-mediated.

Lectins as possible tools for improved urinary bladder cancer management.

Urinary bladder cancer is the ninth most common cancer in developed countries with poor prognosis and outcome for the patient due to the challenging diagnosis and limited treatment possibilities. Bladder cancer arises mainly from urothelial cells lining the lumen. Urothelial cells form a three- to five-layered urothelium, which maintains the blood-urine barrier. The carbohydrates that cover the apical surface of superficial urothelial cells, i.e. umbrella cells, are crucial for this function. The composition of the carbohydrate covering is altered during urothelial cancer transformation. These bladder cancer-associated carbohydrate changes are a promising field for diagnosis, therapy and management. Lectins, which are carbohydrate-binding proteins, can be used to detect subtle alterations in carbohydrate composition during urothelial cancer transformation. Extensive research into various lectin applications has already been conducted, but the results are often contradictory and confusing. None of these applications have reached clinical trials. We review the literature and discuss (i) current bladder cancer management, (ii) lectin-based assays for detection of various cancer subtypes, (iii) lectin-based strategies for innovative bladder cancer treatment and finally (iv) lectins in nanotheranostics for personalized bladder cancer management.

Properties of the Urothelium that Establish the Blood-Urine Barrier and Their Implications for Drug Delivery.

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.

Upregulation and axonal transport of synaptotagmin-IV in the direct-pathway medium spiny neurons in hemi-parkinsonian rats induced by dopamine D1 receptor stimulation.

Synaptotagmin-IV (Syt-IV) may function as a regulator of Ca(2+) -dependent synaptic transmission. In the hemi-parkinsonian rats with unilateral lesions of dopaminergic nigrostriatal neurons Syt-IV and substance-P (SP) mRNAs could be upregulated within the dopaminergically hypersensitive striatum of the lesioned brain hemisphere via the stimulation of striatal dopamine D1 (D1-R), but not D2 receptors. The hypersensitive D1-R-mediated transmission may be the culprit for the undesired expression of levodopa-induced dyskinesia, implying the involvement of Syt-IV and SP in the process. First, striatal cellular phenotypes expressing Syt-IV were determined. It was found to be expressed in all striatal neurons and a small population of astrocytes. Then it was examined, if the D1-R-mediated upregulation of Syt-IV mRNA may result in the upregulation of the translated protein. It was found that, after acute stimulation with a selective D1 agonist, (±)-6-chloro-7,8-dihydroxy-3-allyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrobromide (SKF-82958), Syt-IV was elevated within the SP-expressing striatal neurons of the lesioned side. This was followed by the upregulation of Syt-IV, but not of its mRNA, within the ipsilateral target nuclei of the direct-pathway medium spiny neurons, indicating axonal transport of de novo synthesized protein to their SP-positive synaptic terminals. However, despite the striatal upregulation of SP and Syt-IV following a similar time-course, their subcellular co-localization within the axonal terminals was not found. It was therefore suggested that Syt-IV may regulate the hypersensitive striatal synaptic transmission, although via a SP-independent mechanism.

The complete functional recovery of chitosan-treated biomimetic hyperplastic and normoplastic urothelial models.

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.

A novel splice-site FHOD3 founder variant is a common cause of hypertrophic cardiomyopathy in the population of the Balkans-A cohort study.

Founder variants in sarcomere protein genes account for a significant proportion of disease-causing variants in patients with hypertrophic cardiomyopathy (HCM). However, information on founder variants in non-sarcomeric protein genes, such as FHOD3, which have only recently been associated with HCM, remains scarce. In this study, we conducted a retrospective analysis of exome sequencing data of 134 probands with HCM for recurrent pathogenic variants. We discovered a novel likely pathogenic variant c.1646+2T>C in FHOD3 in heterozygous state in eight probands with HCM and confirmed its presence in seven additional relatives. Individuals with this variant had a wide range of ages at onset of the disease (4-63 years). No adverse cardiac events were observed. Haplotype analysis revealed that the individuals with this variant shared a genomic region of approximately 5 Mbp surrounding the variant, confirming the founder effect of the variant. FHOD3 c.1646+2T>C is estimated to have arisen 58 generations ago (95% CI: 45-81) in a common ancestor living on the Balkans. A founder FHOD3 c.1646+2T>C variant is the second most common genetic variant in our cohort of patients with HCM, occurring in 16% of probands with a known genetic cause of HCM, which represents a substantially higher proportion than the currently estimated 0.5-2% for causal FHOD3 variants. Our study broadens the understanding of the genetic causes of HCM and may improve the diagnosis of this condition, particularly in patients from the Balkans.

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.

Biallelic ATOH1 Gene Variant in Siblings With Pontocerebellar Hypoplasia, Developmental Delay, and Hearing Loss.

Background and Objectives: To report on the novel association of biallelic variant in atonal basic helix-loop-helix transcription factor 1 (ATOH1) gene and pontocerebellar hypoplasia (PCH), severe global developmental delay, intellectual disability, and hearing loss in a family with 2 affected siblings. Methods: A detailed clinical assessment and exome sequencing of peripheral blood sample were performed. Segregation analysis with Sanger sequencing and structural modeling of the variant was performed to support the pathogenicity of the variant. Results: A homozygous missense variant (NM_005172.1:c.481C>G) in the ATOH1 gene was identified in the proband and his affected sister. The segregation analysis subsequently confirmed its segregation with an apparently recessive PCH in this family. ATOH1 encodes for the atonal basic helix-loop-helix (bHLH) transcription factor 1, a core transcription factor in the developing cerebellum, brainstem, and dorsal spinal cord, and in the ear. The identified variant results in the p.(Arg161Gly) amino acid substitution in the evolutionarily conserved DNA-binding bHLH domain of the ATOH1 protein. Biallelic missense variants in this domain were previously reported to result in disordered cerebellar development and hearing loss in animal models. In silico homology modeling revealed that p.Arg161Gly in ATOH1 protein probably disrupts a salt bridge with DNA backbone phosphate and increases the flexibility of the bHLH helix-both of which together affect the binding capability of the bHLH domain to the DNA. Discussion: Based on the sequencing results and evidence from structural modeling of the identified variant, as well as with previous reports of ATOH1 gene disruption, we conclude that ATOH1 may represent a novel candidate gene associated with the phenotype of PCH, global developmental delay, and hearing loss in humans.

Chitosan hydrochloride has no detrimental effect on bladder urothelial cancer cells.

Bladder cancer is among the most common and aggressive human malignant carcinomas, thus targeting and removal of bladder cancer cells is still a challenge. Although it is well known that chitosan hydrochloride (CH-HCl) causes desquamation of normal urothelial cells, its effect on cancer urothelial cells has not been recognized yet. In this in vitro study, we analyzed the cytotoxicity of 0.05% CH-HCl on three urothelial models: two cancer urothelial models, i.e. invasive and papillary urothelial neoplasms, and a normal urothelial model. The cytotoxicity of CH-HCl was evaluated with viability tests, transepithelial resistance (TER) measurements, and electron microscopy. TER measurements showed that 15-minute treatment with CH-HCl caused no reduction in TER of the cancer models, whereas the TER of the normal urothelial model significantly decreased. Furthermore, after CH-HCl treatment, the viability of cancer cells was reduced by only 5%, whereas the viability of normal cells was reduced by 30%. Ultrastructural analysis revealed necrotic cell death in all cases. We have demonstrated that although CH-HCl increases the mortality of cancer urothelial cells, it increases the mortality of normal urothelial cells even more so. However, shorter 2-minute CH-HCl treatment only temporarily increases the permeability of normal urothelial model, i.e. disrupts tight junctions and reduces TER without comprising cell viability, and enables the complete recovery of the permeability barrier after 24h. Overall, our results suggest that CH-HCl cannot be used as a self-sufficient anticancer agent for urothelial bladder cancer treatment; nevertheless a possibility of its use as an enhancer of cytostatic treatment is discussed.

Uroplakin traffic through the Golgi apparatus induces its fragmentation: new insights from novel in vitro models.

Uroplakins (UPs) play an essential role in maintaining an effective urothelial permeability barrier at the level of superficial urothelial cell (UC) layer. Although the organization of UPs in the apical plasma membrane (PM) of UCs is well known, their transport in UCs is only partially understood. Here, we dissected trafficking of UPs and its differentiation-dependent impact on Golgi apparatus (GA) architecture. We demonstrated that individual subunits UPIb and UPIIIa are capable of trafficking from the endoplasmic reticulum to the GA in UCs. Moreover, UPIb, UPIIIa or UPIb/UPIIIa expressing UCs revealed fragmentation and peripheral redistribution of Golgi-units. Notably, expression of UPIb or UPIb/UPIIIa triggered similar GA fragmentation in MDCK and HeLa cells that do not express UPs endogenously. The colocalization analysis of UPIb/UPIIIa-EGFP and COPI, COPII or clathrin suggested that UPs follow constitutively the post-Golgi route to the apical PM. Depolymerisation of microtubules leads to complete blockade of the UPIb/UPIIIa-EGFP post-Golgi transport, while disassembly of actin filaments shows significantly reduced delivery of UPIb/UPIIIa-EGFP to the PM. Our findings show the significant effect of the UPs expression on the GA fragmentation, which enables secretory Golgi-outpost to be distributed as close as possible to the sites of cargo delivery at the PM.

Air-liquid and liquid-liquid interfaces influence the formation of the urothelial permeability barrier in vitro.

Optimizing culture conditions is known to be crucial for the differentiation of urothelial cell cultures and the formation of the permeability barrier. However, so far, no data exist to confirm if air-liquid (AL) and liquid-liquid (LL) interfaces are physiologically relevant during urothelial differentiation and barrier formation. To reveal the influence of interfaces on the proliferation, differentiation, and barrier formation of the urothelial cells (UCs) in vitro, we cultured UCs under four different conditions, i.e., at the AL or LL interfaces with physiological calcium concentration and without serum or without physiological calcium concentration and with serum. For each of the four models, the urothelial integrity was tested by measuring the transepithelial resistance (TER), and the differentiation stage was examined by immunolabeling of differentiation-related markers and ultrastructural analysis. We found that the UCs at a LL interface, regardless of the presence or absence of calcium or serum, form the urothelium with more cell layers and achieve a higher TER than UCs at an AL interface. However, UCs grown at an AL interface with physiological concentration of calcium in medium form only one- to two-layered urothelium of UCs, which are larger and express more differentiation-related proteins uroplakins than UCs in other models. These results demonstrate that the interface itself can play a major, although so-far neglected, role in urothelial physiology, particularly in the formation of the urothelial permeability barrier in vitro and the regulatory mechanisms related with urothelial differentiation. In the study, the culturing of UCs in three successive steps is proposed.