Causal contributors to tissue stiffness and clinical relevance in urology.

Mechanomedicine is an emerging field focused on characterizing mechanical changes in cells and tissues coupled with a specific disease. Understanding the mechanical cues that drive disease progression, and whether tissue stiffening can precede disease development, is crucial in order to define new mechanical biomarkers to improve and develop diagnostic and prognostic tools. Classically known stromal regulators, such as fibroblasts, and more recently acknowledged factors such as the microbiome and extracellular vesicles, play a crucial role in modifications to the stroma and extracellular matrix (ECM). These modifications ultimately lead to an alteration of the mechanical properties (stiffness) of the tissue, contributing to disease onset and progression. We describe here classic and emerging mediators of ECM remodeling, and discuss state-of-the-art studies characterizing mechanical fingerprints of urological diseases, showing a general trend between increased tissue stiffness and severity of disease. Finally, we point to the clinical potential of tissue stiffness as a diagnostic and prognostic factor in the urological field, as well as a possible target for new innovative drugs.

EV Separation: Release of Intact Extracellular Vesicles Immunocaptured on Magnetic Particles.

Extracellular vesicles (EVs) have attracted considerable interest due to their role in cell-cell communication, disease diagnosis, and drug delivery. Despite their potential in the medical field, there is no consensus on the best method for separating micro- and nanovesicles from cell culture supernatant and complex biological fluids. Obtaining a good recovery yield and preserving physical characteristics is critical for the diagnostic and therapeutic use of EVs. The separation of a single class of EVs, such as exosomes, is complex because blood and cell culture media contain many nanoparticles in the same size range. Methods that exploit immunoaffinity capture provide high-purity samples and overcome the issues of currently used separation methods. However, the release of captured nanovesicles usually requires harsh conditions that hinder their use in certain types of downstream analysis. A novel capture and release approach for small extracellular vesicles (sEVs) is presented based on DNA-directed immobilization of antiCD63 antibody. The flexible DNA linker increases the capture efficiency and allows for releasing EVs by exploiting the endonuclease activity of DNAse I. This separation protocol works under mild conditions, enabling the release of vesicles suitable for analysis by imaging techniques. In this study, sEVs recovered from plasma were characterized by established techniques for EV analysis, including nanoparticle tracking and transmission electron microscopy.

In vitro cultured human endometrial cells release extracellular vesicles that can be uptaken by spermatozoa.

Extracellular vesicles (EVs) derived from different parts of the male reproductive tract can be internalized by human spermatozoa affecting their maturation and regulating their functions. Here we demonstrate that EVs derived from the female tract can be uptaken by sperm and affect their competence. Primary endometrial cells release EVs with a diameter between 50 and 350 nm and bear the standard vesicle and exosome marker proteins CD63, CD9, TSG101 and ALIX. The uptake of dye-labelled endometrial cell-derived EVs by spermatozoa, quantified as fluorescence intensity, was significantly higher when EVs were derived from cells in the proliferative phase. Vital, motile fluorescent sperm could be appreciated after a 48-hour co-incubation with endometrial cells previously labelled with the Vybrant™ DiO dye. EV internalization by sperm was blocked at 4 °C and by incubation with filipin, suggesting an energy-dependent process probably attributable to the lipid-raft domain mediated-endocytosis. Sperm ability to undergo capacitation and acrosome reaction was stimulated by endometrial cell-derived EVs as manifested by the increased protein tyrosine phosphorylation and evident reactivity when stimulated with a calcium ionophore. Based on these findings, EVs exchange may be suggested as an emerging way through which female reproductive tract cells can interact with the passing spermatozoa.

Extracellular vesicles as a potential diagnostic tool in assisted reproduction.

PURPOSE OF REVIEW: Extracellular vesicles have emerged as a promising field of research for their potential to serve as biomarkers. In the pathophysiology of reproduction, they have attracted significant attention because of their diverse roles in gametogenesis and embryo-endometrial cross-talk. Advances in extracellular vesicle translational potential are herein reviewed with a particular focus in oocyte competence, semen quality diagnostics, embryo selection and detection of endometrial receptivity. RECENT FINDINGS: Specific miRNAs present in follicular fluid-derived extracellular vesicles have been associated with follicle development and oocyte maturation. Some proteins known to regulate sperm function and capacitation such as glycodelin, and CRISP1 have been found as overrepresented in semen exosomes isolated from severe asthenozoospermic compared to normozoospermic men. In vitro developed human embryos can secrete extracellular vesicles whose propitiousness for preimplantation genetic testing is being increasingly investigated. Endometrial cell-derived extracellular vesicles recovered from uterine flushings might represent a reservoir of molecular markers potentially exploited for monitoring the endometrial status. SUMMARY: Accumulated knowledge on extracellular vesicles deriving from endometrium, follicular fluid, embryos or male reproductive system may be translated to clinical practice to inform diagnostics in assisted reproduction technology (ART). Validation studies and technology developments are required to implement the profiling of extracellular vesicles as diagnostic tests in ART.

Proteomic analysis reveals the negative modulator of sperm function glycodelin as over-represented in semen exosomes isolated from asthenozoospermic patients.

STUDY QUESTION: Are there differences in the proteomic profile of exosomes isolated from seminal plasma of normozoospermic (NSP) and severe asthenozoospermic (SA) men, potentially contributing to sperm features? SUMMARY ANSWER: A relevant group of proteins known to positively regulate sperm functions were over-represented in seminal exosomes of NSP men, i.e. cysteine-rich secretory protein-1 (CRISP1), while the inhibitory protein glycodelin was enriched in exosomes of SA subjects. WHAT IS KNOWN ALREADY: Exosomes are secreted along the male reproductive tract and are thought to be involved in spermatozoa maturation and function. Ejaculated spermatozoa are still able to capture exosomes; exosomes of NSP individuals improve sperm motility and prompt capacitation, while exosomes of SA men fail to exert similar features. STUDY DESIGN, SIZE, DURATION: Semen samples from NSP and SA men, aged 18 to 55 and registered at a single IVF center, were considered for this study project. Subjects were subdivided into three groups: a discovery cohort (five NSP men and six SA patients), a validation cohort (seven NSP and seven SA men) and the 'glycodelin analysis' cohort (20 NSP and 37 SA men). Exosomes were purified from semen of every participant. PARTICIPANTS/MATERIALS, SETTING, METHODS: Exosomes were characterized by nanoparticle tracking analysis, transmission electron microscopy and western blot. Comprehensive proteomics analysis of the exosomal proteome was performed by nanoscale liquid chromatographic tandem mass spectrometry analysis. Funrich software was used to determine statistical enrichment of pathways, networks and Gene Ontology terms of the identified proteins. Validation of differentially expressed proteins was performed through ELISA and western blot analysis. MAIN RESULTS AND THE ROLE OF CHANCE: The comprehensive proteomic analysis identified a total of 2138 proteins for both groups. There were 89 proteins found to be differentially expressed in exosomes of NSP versus SA subjects, of which 37 were increased in the NSP group and 52 were increased in the SA group. One-third of the exosomes-associated proteins highly expressed in NSP samples were involved in the reproductive process; conversely, the over-expressed proteins in exosomes of SA samples were not functionally specific. Quantitative data were confirmed on seminal exosomes from different cohorts of subjects. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Transfer of the proteins from exosomes to spermatozoa has been only partially demonstrated and up-take mechanisms are still poorly defined. WIDER IMPLICATIONS OF THE FINDINGS: Seminal exosomes carry proteins that are potentially able to either favour or inhibit the reproductive process in humans. A better understanding of these phenomena might pave the way for novel intervention measures in terms of male infertility. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by the Italian Ministry of Health through an Institution Seed Grant. None of the authors has any competing interests.

Seminal plasma of men with severe asthenozoospermia contain exosomes that affect spermatozoa motility and capacitation.

OBJECTIVE: To characterize in depth and investigate the role of exosomes present in seminal plasma in affecting parameters underlying sperm activity. DESIGN: In vitro experimental study. SETTING: Research hospital. PATIENT(S): Normozoospermic, severe asthenozoospermic, and post-vasectomy azoospermic men 18-55 years of age were considered for the study. Seminal plasma was collected and processed to separate spermatozoa and exosomes. INTERVENTION(S): None. MAIN OUTCOMES MEASURE(S): Exosomes from seminal plasma were isolated and characterized by means of nanoparticle tracking analysis, transmission electron microscopy and Western blot. Exosome uptake by spermatozoa was monitored by means of immunofluorescence and flow cytometry. The effect of exosomes on spermatozoa was determined by evaluating progressive motility and capacitation, the latter assessed by means of tyrosine phosphorylation and acrosome reaction. RESULT(S): We isolated and characterized exosomes from seminal plasma of normo-, astheno-, and azoospermic patients. They display similar features in terms of shape, size, expression of canonic exosome markers and proteins involved in spermatozoa maturation, and fertilization capacity. After ejaculation, sperm cells are still receptive and are able to take up exosomes in a time- and pH-dependent manner. Exosomes derived from normozoospermic but not from asthenozoospermic individuals improve spermatozoa motility and trigger capacitation. Transfer of cysteine-rich secretory protein 1 from exosomes to spermatozoa may have a role in these phenomena. CONCLUSION(S): These findings provide evidence that: 1) sperm can still receive vesicle-derived cargo after ejaculation; 2) sperm motility and ability to undergo capacitation can benefit from exosomal transfer; and 3) semen quality is affected by male tract exosomes.

Abiraterone and Ionizing Radiation Alter the Sphingolipid Homeostasis in Prostate Cancer Cells.

Prostate cancer (PC) is one of the most common leading causes of cancer-related death in men. Currently, the main therapeutic approaches available for PC are based on the androgen deprivation and on radiotherapy. However, despite these treatments being initially effective in cancer remission, several patients undergo recurrence, developing a most aggressive and resistant PC.Emerging evidence showed that abiraterone acetate drug will reduce PC recurrence by a mechanism independent of the inhibition of Cytochrome P450 17α-hydroxylase/17,20-lyase. Here we describe the involvement in the abiraterone-mediated PC cell death of a particular class of bioactive lipids called sphingolipids (SL). Sphingolipids are components of plasma membrane (PM) that organize macromolecular complexes involved in the control of several signaling pathways including the tumor cell death induced by radiotherapy. Here, we show for the first time that both in androgen-sensitive and insensitive PC cells abiraterone and ionizing radiation induce a reorganization of the plasma membrane SL composition. This event is triggered by activation of the PM-associated glycohydrolases that induce the production of cytotoxic ceramide by the in situ hydrolyses of glycosphingolipids. Taken together our data open a new scenario on the SL involvement in the therapy of PC.

Secretome of in vitro cultured human embryos contains extracellular vesicles that are uptaken by the maternal side.

Communication between embryo and maternal endometrium occurs during a specific time frame in which implantation is possible. Here we demonstrate for the first time that conditioned media from non-manipulated human embryos cultured in vitro for 3 days or up to the blastocyst stage contain extracellular vesicles (EVs) with a diameter of 50 to 200 nm and bearing the traditional microvesicle and exosome marker proteins CD63, CD9 and ALIX. The embryonic origin of these EVs has been confirmed by the presence of stemness gene transcripts and their enrichment in the non-classical HLA-G protein. NANOG and POU5F1 transcripts were shown to be contained in vesicles deriving from embryos at different stages of development. In line with a higher detection rate of the HLA-G protein in blastocysts compared to cleavage stage embryos, a significantly higher amount of HLA-G was found in vesicles accumulated in spent media from day 3 to day 5 of development compared to those isolated from the earlier stage. Uptake of dye-labeled embryo-derived EVs by human primary endometrial epithelial and stromal cells was also demonstrated with a fluorescence intensity signal significantly higher for cells treated with vesicles derived from blastocysts. Based on these findings, EV exchange may be suggested as an emerging way of communication at the maternal-fetal interface.

Bladder cancer cell growth and motility implicate cannabinoid 2 receptor-mediated modifications of sphingolipids metabolism.

The inhibitory effects demonstrated by activation of cannabinoid receptors (CB) on cancer proliferation and migration may also play critical roles in controlling bladder cancer (BC). CB expression on human normal and BC specimens was tested by immunohistochemistry. Human BC cells RT4 and RT112 were challenged with CB agonists and assessed for proliferation, apoptosis, and motility. Cellular sphingolipids (SL) constitution and metabolism were evaluated after metabolic labelling. CB1-2 were detected in BC specimens, but only CB2 was more expressed in the tumour. Both cell lines expressed similar CB2. Exposure to CB2 agonists inhibited BC growth, down-modulated Akt, induced caspase 3-activation and modified SL metabolism. Baseline SL analysis in cell lines showed differences linked to unique migratory behaviours and cytoskeletal re-arrangements. CB2 activation changed the SL composition of more aggressive RT112 cells by reducing (p < 0.01) Gb3 ganglioside (-50 ± 3%) and sphingosine 1-phosphate (S1P, -40 ± 4%), which ended up to reduction in cell motility (-46 ± 5%) with inhibition of p-SRC. CB2-selective antagonists, gene silencing and an inhibitor of SL biosynthesis partially prevented CB2 agonist-induced effects on cell viability and motility. CB2 activation led to ceramide-mediated BC cell apoptosis independently of SL constitutive composition, which instead was modulated by CB2 agonists to reduce cell motility.

Gangliosides and cell surface ganglioside glycohydrolases in the nervous system.

Gangliosides are a large group of complex lipids found predominantly on the outer layer of the plasma membranes of cells, and they are particularly concentrated in nerve endings. Their half-life in the nervous system is short, and their membrane composition and content are strictly connected to their metabolism. Their neobiosynthesis starts in the endoplasmic reticulum and is completed in the Golgi; catabolism occurs primarily in the lysosomes. However, the final content of gangliosides in the plasma membrane is affected by other cellular processes.In this chapter structural changes in the oligosaccharide chains of gangliosides induced by the activity of glycohydrolases and in some cases by glycosyltransferases that are associated with plasma membranes are discussed. Some of the plasma membrane enzymes arise from fusion processes between intracellular fractions and the plasma membrane; however, other plasma membrane enzymes display a structure different from that of the intracellular enzymes. Several of these plasma membrane enzymes have been characterized and some of them seem to have a specific role in the nervous system.

Exploring the link between ceramide and ionizing radiation.

The aim of radiotherapy is to eradicate cancer cells with ionizing radiation; tumor cell death following irradiation can be induced by several signaling pathways, most of which are triggered as a consequence of DNA damage, the primary and major relevant cell response to radiation. Several lines of evidence demonstrated that ceramide, a crucial sensor and/or effector of different signalling pathways promoting cell cycle arrest, death and differentiation, is directly involved in the molecular mechanisms underlying cellular response to irradiation. Most of the studies strongly support a direct relationship between ceramide accumulation and radiation-induced cell death, mainly apoptosis; for this reason, defining the contribution of the multiple metabolic pathways leading to ceramide formation and the causes of its dysregulated metabolism represent the main goal in order to elucidate the ceramide-mediated signaling in radiotherapy. In this review, we summarize the current knowledge concerning the different routes leading to ceramide accumulation in radiation-induced cell response with particular regard to the role of the enzymes involved in both ceramide neogenesis and catabolism. Emphasis is placed on sphingolipid breakdown as mechanism of ceramide generation activated following cell irradiation; the functional relevance of this pathway, and the role of glycosphingolipid glycohydrolases as direct targets of ionizing radiation are also discussed. These new findings add a further attractive point of investigation to better define the complex interplay between sphingolipid metabolism and radiation therapy.

The glycosphingolipid hydrolases in the central nervous system.

Glycosphingolipids are a large group of complex lipids particularly abundant in the outer layer of the neuronal plasma membranes. Qualitative and quantitative changes in glycosphingolipids have been reported along neuronal differentiation and aging. Their half-life is short in the nervous system and their membrane composition and content are the result of a complex network of metabolic pathways involving both the de novo synthesis in the Golgi apparatus and the lysosomal catabolism. In particular, most of the enzymes of glycosphingolipid biosynthesis and catabolism have been found also at the plasma membrane level. Their action could be responsible for the fine tuning of the plasma membrane glycosphingolipid composition allowing the formation of highly specialized membrane areas, such as the synapses and the axonal growth cones. While the correlation between the changes of GSL pattern and the modulation of the expression/activity of different glycosyltransferases during the neuronal differentiation has been widely discussed, the role of the glycohydrolytic enzymes in this process is still little explored. For this reason, in the present review, we focus on the main glycolipid catabolic enzymes ß-hexosaminidases, sialidases, ß-galactosidases, and ß-glucocerebrosidases in the process of the neuronal differentiation.