Hypoxic extracellular vesicles from hiPSCs protect cardiomyocytes from oxidative damage by transferring antioxidant proteins and enhancing Akt/Erk/NRF2 signaling.

BACKGROUND: Stem cell-derived extracellular vesicles (EVs) are an emerging class of therapeutics with excellent biocompatibility, bioactivity and pro-regenerative capacity. One of the potential targets for EV-based medicines are cardiovascular diseases (CVD). In this work we used EVs derived from human induced pluripotent stem cells (hiPSCs; hiPS-EVs) cultured under different oxygen concentrations (21, 5 and 3% O2) to dissect the molecular mechanisms responsible for cardioprotection. METHODS: EVs were isolated by ultrafiltration combined with size exclusion chromatography (UF + SEC), followed by characterization by nanoparticle tracking analysis, atomic force microscopy (AFM) and Western blot methods. Liquid chromatography and tandem mass spectrometry coupled with bioinformatic analyses were used to identify differentially enriched proteins in various oxygen conditions. We directly compared the cardioprotective effects of these EVs in an oxygen-glucose deprivation/reoxygenation (OGD/R) model of cardiomyocyte (CM) injury. Using advanced molecular biology, fluorescence microscopy, atomic force spectroscopy and bioinformatics techniques, we investigated intracellular signaling pathways involved in the regulation of cell survival, apoptosis and antioxidant response. The direct effect of EVs on NRF2-regulated signaling was evaluated in CMs following NRF2 inhibition with ML385. RESULTS: We demonstrate that hiPS-EVs derived from physiological hypoxia at 5% O2 (EV-H5) exert enhanced cytoprotective function towards damaged CMs compared to EVs derived from other tested oxygen conditions (normoxia; EV-N and hypoxia 3% O2; EV-H3). This resulted from higher phosphorylation rates of Akt kinase in the recipient cells after transfer, modulation of AMPK activity and reduced apoptosis. Furthermore, we provide direct evidence for improved calcium signaling and sustained contractility in CMs treated with EV-H5 using AFM measurements. Mechanistically, our mass spectrometry and bioinformatics analyses revealed differentially enriched proteins in EV-H5 associated with the antioxidant pathway regulated by NRF2. In this regard, EV-H5 increased the nuclear translocation of NRF2 protein and enhanced its transcription in CMs upon OGD/R. In contrast, inhibition of NRF2 with ML385 abolished the protective effect of EVs on CMs. CONCLUSIONS: In this work, we demonstrate a superior cardioprotective function of EV-H5 compared to EV-N and EV-H3. Such EVs were most effective in restoring redox balance in stressed CMs, preserving their contractile function and preventing cell death. Our data support the potential use of hiPS-EVs derived from physiological hypoxia, as cell-free therapeutics with regenerative properties for the treatment of cardiac diseases.

The effect of oxidative degradation of Dopa-melanin on its basic physicochemical properties and photoreactivity.

Melanin, particularly eumelanin, is commonly viewed as an efficient antioxidant and photoprotective pigment. Nonetheless, the ability of melanin to photogenerate reactive oxygen species and sensitize the formation of cyclobutane pyrimidine dimers may contribute to melanin-dependent phototoxicity. The phototoxic potential of melanin depends on a variety of factors, including molecular composition, redox state, and degree of aggregation. Using complementary spectroscopic and analytical methods we analyzed the physicochemical properties of Dopa-melanin, a synthetic model of eumelanin, subjected to oxidative degradation induced by aerobic photolysis or exposure to 0.1 M hydrogen peroxide. Both modes of oxidative degradation were accompanied by dose-dependent bleaching of melanin and irreversible modifications of its paramagnetic, ion- and electron-exchange and antioxidant properties. Bleached melanin exhibited enhanced efficiency to photogenerate singlet oxygen in both UVA and short-wavelength visible light. Although chemical changes of melanin subunits, including a relative increase of DHICA content and disruption of melanin polymer induced by oxidative degradation were considered, these two mechanisms may not be sufficient for a satisfactory explanation of the elevated photosensitizing ability of the bleached eumelanin. This study points out possible adverse changes in the photoprotective and antioxidant properties of eumelanin that could occur in pigmented tissues after exposure to high doses of intense solar radiation.

Mesenchymal stem cell-derived extracellular vesicles exert pro-angiogenic and pro-lymphangiogenic effects in ischemic tissues by transferring various microRNAs and proteins including ITGa5 and NRP1.

Mesenchymal stem cells/stromal cells (MSCs)-derived extracellular vesicles (EVs) mediate pro-regenerative effects in damaged ischemic tissues by regulating angiogenesis. MSCs-EVs modulate functions of cells including endogenous mature cells, progenitors and stem cells, resulting in restoration of blood flow. However, the mechanisms underlying such MSC-EV activity still remain poorly understood. The present study analyzes biological effects of bone marrow (BM) MSC-EVs on endothelial cells (ECs) in ischemic tissues both in in vitro and in vivo conditions and elucidates the molecular mechanisms underlying the tissue repair. MSC-EVs were isolated from murine BM-derived MSCs and their morphological, antigenic and molecular composition regarding protein and microRNA levels were evaluated to examine their properties. Global proteomic analysis demonstrated the presence in MSC-EVs of proteins regulating pro-regenerative pathways, including integrin α5 (Itgα5) and neuropilin-1 (NRP1) involved in lymphangiogenesis. MSC-EVs were also enriched in microRNAs regulating angiogenesis, TGF-ß signaling and processes guiding cellular adhesion and interactions with extracellular matrix. The functional effects of MSC-EVs on capillary ECs in vitro included the increase of capillary-like tube formation and cytoprotection under normal and inflammatory conditions by inhibiting apoptosis. Notably, MSC-EVs enhanced also capillary-like tube formation of lymphatic ECs, which may be regulated by Itgα5 and NRP1. Moreover, in a mouse model of critical hind limb ischemia, MSC-EVs increased the recovery of blood flow in ischemic muscle tissue, which was accompanied with increased vascular density in vivo. This pro-angiogenic effect was associated with an increase in nitric oxide (NO) production via endothelial NO-synthase activation in ischemic muscles. Interestingly, MSC-EVs enhanced lymphangiogenesis, which has never been reported before. The study provides evidence on pro-angiogenic and novel pro-lymphangiogenic role of MSC-EVs on ECs in ischemic tissue mediated by their protein and miRNA molecular cargos. The results highlight Itgα5 and NRP1 carried by MSC-EVs as potential therapeutic targets to boost lymphangiogenesis.

Photoreactivity and phototoxicity of experimentally photodegraded hair melanosomes from individuals of different skin phototypes.

Even though melanin is commonly viewed as natural photoprotectant, the pigment demonstrates residual photoreactivity, which under certain conditions could contribute to UVA-dependent melanomagenesis. Skin melanin is constantly exposed to external stressors, including solar radiation, which could induce photodegradation of the pigment. Although photodegradation of melanin pigments was studied in synthetic models and RPE melanosomes, photochemical and photobiological effects of experimental photodegradation of human skin melanin of different chemical composition remain unknown. In this work, melanosomes isolated from hair of individuals of different skin phototypes (I-III, V) were exposed to high-intensity violet light and its impact on physical and chemical properties of the pigments were analyzed using electron paramagnetic resonance (EPR), spectrophotometry and dynamic light scattering (DLS). Photoreactivity of photodegraded melanins was examined by EPR oximetry, EPR spin-trapping and time-resolved singlet oxygen phosphorescence. Antioxidant potential of the pigments was measured using the EPR DPPH assay. Cellular effect of the exposure of melanosome-loaded HaCaT cells to UV-Vis light was determined by MTT assay, JC-10 assay, and iodometric assay. The data revealed that experimental photodegradation increased photoreactivity of natural melanins, while decreasing their antioxidant capacity. Photodegraded melanin was responsible for higher cell death, a decrease in mitochondrial membrane potential and elevated levels of lipid hydroperoxides.

The Potential of Congo Red Supplied Aggregates of Multitargeted Tyrosine Kinase Inhibitor (Sorafenib, BAY-43-9006) in Enhancing Therapeutic Impact on Bladder Cancer.

Bladder cancer is a common malignancy associated with high recurrence rates and potential progression to invasive forms. Sorafenib, a multi-targeted tyrosine kinase inhibitor, has shown promise in anti-cancer therapy, but its cytotoxicity to normal cells and aggregation in solution limits its clinical application. To address these challenges, we investigated the formation of supramolecular aggregates of sorafenib with Congo red (CR), a bis-azo dye known for its supramolecular interaction. We analyzed different mole ratios of CR-sorafenib aggregates and evaluated their effects on bladder cancer cells of varying levels of malignancy. In addition, we also evaluated the effect of the test compounds on normal uroepithelial cells. Our results demonstrated that sorafenib inhibits the proliferation of bladder cancer cells and induces apoptosis in a dose-dependent manner. However, high concentrations of sorafenib also showed cytotoxicity to normal uroepithelial cells. In contrast, the CR-BAY aggregates exhibited reduced cytotoxicity to normal cells while maintaining anti-cancer activity. The aggregates inhibited cancer cell migration and invasion, suggesting their potential for metastasis prevention. Dynamic light scattering and UV-VIS measurements confirmed the formation of stable co-aggregates with distinctive spectral properties. These CR-sorafenib aggregates may provide a promising approach to targeted therapy with reduced cytotoxicity and improved stability for drug delivery in bladder cancer treatment. This work shows that the drug-excipient aggregates proposed and described so far, as Congo red-sorafenib, can be a real step forward in anti-cancer therapies.

The Effect of the Topmost Layer and the Type of Bone Morphogenetic Protein-2 Immobilization on the Mesenchymal Stem Cell Response.

Recombinant human bone morphogenetic protein-2 (rhBMP-2) plays a key role in the stem cell response, not only via its influence on osteogenesis, but also on cellular adhesion, migration, and proliferation. However, when applied clinically, its supra-physiological levels cause many adverse effects. Therefore, there is a need to concomitantly retain the biological activity of BMP-2 and reduce its doses. Currently, the most promising strategies involve site-specific and site-directed immobilization of rhBMP-2. This work investigated the covalent and electrostatic binding of rhBMP-2 to ultrathin-multilayers with chondroitin sulfate (CS) or diazoresin (DR) as the topmost layer. Angle-resolved X-ray photoelectron spectroscopy was used to study the exposed chemical groups. The rhBMP-2 binding efficiency and protein state were studied with time-of-flight secondary ion mass spectrometry. Quartz crystal microbalance, atomic force microscopy, and enzyme-linked immunosorbent assay were used to analyze protein-substrate interactions. The effect of the topmost layer was tested on initial cell adhesion and short-term osteogenesis marker expression. The results show the highest expression of selected osteomarkers in cells cultured on the DR-ended layer, while the cellular flattening was rather poor compared to the CS-ended system. rhBMP-2 adhesion was observed only on negatively charged layers. Cell flattening became more prominent in the presence of the protein, even though the osteogenic gene expression decreased.

Extracellular vesicles from human iPSCs enhance reconstitution capacity of cord blood-derived hematopoietic stem and progenitor cells.

Cord blood (CB) represents a source of hematopoietic stem and progenitor cells (CB-HSPCs) for bone marrow (BM) reconstitution, but clinical CB application is limited in adult patients due to the insufficient number of CB-HSCPCs and the lack of effective ex vivo approaches to increase CB-HSPC functionality. Since human-induced pluripotent stem cells (hiPSCs) have been indicated as donor cells for bioactive extracellular vesicles (EVs) modulating properties of other cells, we are the first to employ hiPSC-derived EVs (hiPSC-EVs) to enhance the hematopoietic potential of CB-derived CD45dimLin-CD34+ cell fraction enriched in CB-HSPCs. We demonstrated that hiPSC-EVs improved functional properties of CB-HSPCs critical for their hematopoietic capacity including metabolic, hematopoietic and clonogenic potential as well as survival, chemotactic response to stromal cell-derived factor 1 and adhesion to the model components of hematopoietic niche in vitro. Moreover, hiPSC-EVs enhanced homing and engraftment of CB-HSPCs in vivo. This phenomenon might be related to activation of signaling pathways in CB-HSPCs following hiPSC-EV treatment, as shown on both gene expression and the protein kinases activity levels. In conclusion, hiPSC-EVs might be used as ex vivo modulators of CB-HSPCs capacity to enhance their functional properties and augment future practical applications of CB-derived cells in BM reconstitution.

Photoreactivity of Hair Melanin from Different Skin Phototypes-Contribution of Melanin Subunits to the Pigments Photoreactive Properties.

Photoreactivity of melanin has become a major focus of research due to the postulated involvement of the pigment in UVA-induced melanoma. However, most of the hitherto studies were carried out using synthetic melanin models. Thus, photoreactivity of natural melanins is yet to be systematically analyzed. Here, we examined the photoreactive properties of natural melanins isolated from hair samples obtained from donors of different skin phototypes (I, II, III, and V). X-band and W-band electron paramagnetic resonance (EPR) spectroscopy was used to examine the paramagnetic properties of the pigments. Alkaline hydrogen peroxide degradation and hydroiodic acid hydrolysis were used to determine the chemical composition of the melanins. EPR oximetry and spin trapping were used to examine the oxygen photoconsumption and photo-induced formation of superoxide anion, and time-resolved near infrared phosphorescence was employed to determine the singlet oxygen photogeneration by the melanins. The efficiency of superoxide and singlet oxygen photogeneration was related to the chemical composition of the studied melanins. Melanins from blond and chestnut hair (phototypes II and III) exhibited highest photoreactivity of all examined pigments. Moreover, melanins of these phototypes showed highest quantum efficiency of singlet oxygen photogeneration at 332 nm and 365 nm supporting the postulate of the pigment contribution in UVA-induced melanoma.

Melatonin exerts oncostatic capacity and decreases melanogenesis in human MNT-1 melanoma cells.

Melanogenesis is a key parameter of differentiation in melanocytes and melanoma cells; therefore, search for factors regulating this pathway are strongly desired. Herein, we investigated the effects of melatonin, a ubiquitous physiological mediator that is found throughout animals and plants. In mammals, the pineal gland secretes this indoleamine into the blood circulation to exert an extensive repertoire of biological activities. Our in vitro assessment indicates an oncostatic capacity of melatonin in time-dependent manner (24, 48, 72 hours) in highly pigmented MNT-1 melanoma cells. The similar pattern of regulation regarding cell viability was observed in amelanotic Sk-Mel-28 cells. Subsequently, MNT-1 cells were tested for the first time for evaluation of melanin/melatonin interaction. Thus primary, electron paramagnetic resonance (EPR) spectroscopy demonstrated that melatonin reduced melanin content. Artificially induced disturbances of melanogenesis by selected inhibitors (N-phenylthiourea or kojic acid) were slightly antagonized by melatonin. Additionally, analysis using transmission electron microscopy has shown that melatonin, particularly at higher dose of 10-3  mol/L, triggered the appearance of premelanosomes (stage I-II of melanosome) and MNT-1 cells synthesize de novo endogenous melatonin shown by LC-MS. In conclusion, these studies show a melanogenic-like function of melatonin suggesting it as an advantageous agent for treatment of pigmentary disorders.

Melanin presence inhibits melanoma cell spread in mice in a unique mechanical fashion.

Melanoma is a highly aggressive cancer that exhibits metastasis to various critical organs. Unlike any other cancer cells, melanoma cells can synthesize melanin in large amounts, becoming heavily pigmented. Until now the role of melanin in melanoma, particularly the effect of melanin presence on the abilities of melanoma cells to spread and metastasize remains unknown. Recently, we have shown that melanin dramatically modified elastic properties of melanoma cells and inhibited the cells invasive abilities in vitro. Here, we inoculated human melanoma cells with different melanin content into nude mice and tested the hypothesis that cell elasticity is an important property of cancer cells for their efficient spread in vivo. The obtained results clearly showed that cells containing melanin were less capable to spread in mice than cells without the pigment. Our findings indicate that the presence of melanin inhibits melanoma metastasis, emphasizing possible clinical implications of such an inhibitory effect.

Triggering NETosis via protease-activated receptor (PAR)-2 signaling as a mechanism of hijacking neutrophils function for pathogen benefits.

Neutrophil-derived networks of DNA-composed extracellular fibers covered with antimicrobial molecules, referred to as neutrophil extracellular traps (NETs), are recognized as a physiological microbicidal mechanism of innate immunity. The formation of NETs is also classified as a model of a cell death called NETosis. Despite intensive research on the NETs formation in response to pathogens, the role of specific bacteria-derived virulence factors in this process, although postulated, is still poorly understood. The aim of our study was to determine the role of gingipains, cysteine proteases responsible for the virulence of P. gingivalis, on the NETosis process induced by this major periodontopathogen. We showed that NETosis triggered by P. gingivalis is gingipain dependent since in the stark contrast to the wild-type strain (W83) the gingipain-null mutant strain only slightly induced the NETs formation. Furthermore, the direct effect of proteases on NETosis was documented using purified gingipains. Notably, the induction of NETosis was dependent on the catalytic activity of gingipains, since proteolytically inactive forms of enzymes showed reduced ability to trigger the NETs formation. Mechanistically, gingipain-induced NETosis was dependent on proteolytic activation of protease-activated receptor-2 (PAR-2). Intriguingly, both P. gingivalis and purified Arg-specific gingipains (Rgp) induced NETs that not only lacked bactericidal activity but instead stimulated the growth of bacteria species otherwise susceptible to killing in NETs. This protection was executed by proteolysis of bactericidal components of NETs. Taken together, gingipains play a dual role in NETosis: they are the potent direct inducers of NETs formation but in the same time, their activity prevents P. gingivalis entrapment and subsequent killing. This may explain a paradox that despite the massive accumulation of neutrophils and NETs formation in periodontal pockets periodontal pathogens and associated pathobionts thrive in this environment.

Increased elasticity of melanoma cells after low-LET proton beam due to actin cytoskeleton rearrangements.

Cellular response to non-lethal radiation stress include perturbations in DNA repair, angiogenesis, migration, and adhesion, among others. Low-LET proton beam radiation has been shown to induce somewhat different biological response than photon radiation. For example, we have shown that non-lethal doses of proton beam radiation inhibited migration of cells and that this effect persisted long-term. Here, we have examined cellular elasticity and actin cytoskeleton organization in BLM cutaneous melanoma and Mel270 uveal melanoma cells. Proton beam radiation increased cellular elasticity to a greater extent than X-rays and both types of radiation induced changes in actin cytoskeleton organization. Vimentin level increased in BLM cells after both types of radiation. Our data show that cell elasticity increased substantially after low-LET proton beam and persisted long after radiation. This may have significant consequences for the migratory properties of melanoma cells, as well as for the cell susceptibility to therapy.

Photobleaching of pheomelanin increases its phototoxic potential: Physicochemical studies of synthetic pheomelanin subjected to aerobic photolysis.

Although melanin is a photoprotective pigment, its elevated photochemical reactivity could lead to various phototoxic processes. Photoreactivity of synthetic pheomelanin, derived from 5-S-cysteinyldopa (5SCD-M) and its photodegradation products obtained by subjecting the melanin to aerobic irradiation with UV-visible light, was examined employing an array of advanced physicochemical methods. Extensive photolysis of 5SCD-M was accompanied by partial bleaching of the melanin, modification of its paramagnetic properties, and significant increase in the ability to photogenerate singlet oxygen. The changes correlated with a substantial decrease in the melanin content of benzothiazine (BT) units and increase of modified benzothiazole (BZ) units. Synthetically prepared BZ exhibited higher efficiency to photogenerate singlet oxygen than the synthetic BT, and the free radical form of BZ, unlike that of BT, did not show measurable spin density on nitrogen atom, which was confirmed by quantum chemical calculations. Formation of modified BZ units in the photobleached 5SCD-M is responsible for the paramagnetic and photochemical changes of the melanin and its elevated phototoxic potential. Given a relatively constant pheomelanin-eumelanin ratio, such undesirable changes could occur in individual of all skin types.

Nanomechanical Phenotype of Melanoma Cells Depends Solely on the Amount of Endogenous Pigment in the Cells.

Cancer cells have unique nanomechanical properties, i.e., they behave as if they were elastic. This property of cancer cells is believed to be one of the main reasons for their facilitated ability to spread and metastasize. Thus, the so-called nanomechanical phenotype of cancer cells is viewed as an important indicator of the cells' metastatic behavior. One of the most highly metastatic cancer cells are melanoma cells, which have a very unusual property: they can synthesize the pigment melanin in large amounts, becoming heavily pigmented. So far, the role of melanin in melanoma remains unclear, particularly the impact of the pigment on metastatic behavior of melanoma cells. Importantly, until recently the potential mechanical role of melanin in melanoma metastasis was completely ignored. In this work, we examined melanoma cells isolated from hamster tumors containing endogenous melanin pigment. Applying an array of advanced microscopy and spectroscopy techniques, we determined that melanin is the dominating factor responsible for the mechanical properties of melanoma cells. Our results indicate that the nanomechanical phenotype of melanoma cells may be a reliable marker of the cells' metastatic behavior and point to the important mechanical role of melanin in the process of metastasis of melanoma.

Induced Pluripotent Stem Cell (iPSC)-Derived Extracellular Vesicles Are Safer and More Effective for Cardiac Repair Than iPSCs.

RATIONALE: Extracellular vesicles (EVs) are tiny membrane-enclosed droplets released by cells through membrane budding or exocytosis. The myocardial reparative abilities of EVs derived from induced pluripotent stem cells (iPSCs) have not been directly compared with the source iPSCs. OBJECTIVE: To examine whether iPSC-derived EVs can influence the biological functions of cardiac cells in vitro and to compare the safety and efficacy of iPSC-derived EVs (iPSC-EVs) and iPSCs for cardiac repair in vivo. METHODS AND RESULTS: Murine iPSCs were generated, and EVs isolated from culture supernatants by sequential centrifugation. Atomic force microscopy, high-resolution flow cytometry, real-time quantitative RT-PCR, and mass spectrometry were used to characterize EV morphology and contents. iPSC-EVs were enriched in miRNAs and proteins with proangiogenic and cytoprotective properties. iPSC-EVs enhanced angiogenic, migratory, and antiapoptotic properties of murine cardiac endothelial cells in vitro. To compare the cardiac reparative capacities in vivo, vehicle, iPSCs, and iPSC-EVs were injected intramyocardially at 48 hours after a reperfused myocardial infarction in mice. Compared with vehicle-injected mice, both iPSC- and iPSC-EV-treated mice exhibited improved left ventricular function at 35 d after myocardial infarction, albeit iPSC-EVs rendered greater improvement. iPSC-EV injection also resulted in reduction in left ventricular mass and superior perfusion in the infarct zone. Both iPSCs and iPSC-EVs preserved viable myocardium in the infarct zone, whereas reduction in apoptosis was significant with iPSC-EVs. iPSC injection resulted in teratoma formation, whereas iPSC-EV injection was safe. CONCLUSIONS: iPSC-derived EVs impart cytoprotective properties to cardiac cells in vitro and induce superior cardiac repair in vivo with regard to left ventricular function, vascularization, and amelioration of apoptosis and hypertrophy. Because of their acellular nature, iPSC-EVs represent a safer alternative for potential therapeutic applications in patients with ischemic myocardial damage.

Invasive Cx43high sub-line of human prostate DU145 cells displays increased nanomechanical deformability.

Connexin(Cx)43high cells are preferentially recruited to the invasive front of prostate cancer in vitro and in vivo. To address the involvement of Cx43 in the regulation of human prostate cancer DU145 cell invasiveness, we have analysed the nanoelasticity of invasive Cx43high sub-sets of DU145 cells by atomic force microscopy (AFM). The Cx43high DU145 cells displayed considerably higher susceptibility to mechanical distortions than the wild type DU145 cells. Transient Cx43 silencing had no effect on their elastic properties. Our data confirm the relationship between the invasive potential, Cx43 expression and nanoelasticity of the DU145 cells. However, they also show that Cx43 is not directly involved in the maintenance of DU145 invasive phenotype.

A 3D model of tumour angiogenic microenvironment to monitor hypoxia effects on cell interactions and cancer stem cell selection.

Tumour microenvironment determines the fate of treatments. Reconstitution of tumour conditions is mandatory for alternative in vitro methods devoted to cancer development and the selection of therapeutic strategies. This work describes a 3D model of melanoma growth in its environment. Introducing means to mimic tumour angiogenesis, which turns on tumour progression, the model shows that melanoma tumour spheroids allow reconstitution of solid tumours with stromal cells. Angiogenesis evidenced the differential recruitment of endothelial cells (EC) from early progenitors (EEPCs) to mature ECs. Hypoxia was the key parameter that selected and stabilized melanoma cancer stem like cells (CSCs) phenotype based on aldehyde dehydrogenase expression as the best criterion. The 3D-tumour-model demonstrated the distinct reactivity of ECs toward tumour cells in terms of cellular cross-talk and humoral response. Intra-spheroid cell-to-cell membrane dye exchanges, mediated by intercellular interactions, uncovered the melanoma-to-EEPC cooperation. The resulting changes in tumour milieu were evidenced by the chemokinic composition and hypoxia-related variations in microRNA expression assessed in each cellular component of the spheroids. This method brings new tools to decipher the molecular mechanism of tumour-mediated cell recruitment and for in vitro assessment of therapeutic approaches.

The nanomechanical role of melanin granules in the retinal pigment epithelium.

Nanomechanical properties of cells and tissues, in particular their elasticity, play an important role in different physiological and pathological processes. Recently, we have demonstrated that melanin granules dramatically modify nanomechanical properties of melanoma cells making them very stiff and, as a result, less aggressive. Although the mechanical effect of melanin granules was demonstrated in pathological cells, it was never studied in the case of normal cells. In this work, we analyzed the impact of melanin granules on nanomechanical properties of primary retinal pigment epithelium tissue fragments isolated from porcine eyes. The obtained results clearly show that melanin granules are responsible for the exceptional nanomechanical properties of the tissue. Our findings suggest that melanin granules in the retinal pigment epithelium may play an important role in sustaining the stiffness of this single cell layer, which functions as a natural mechanical barrier separating the retina from the choroid.

Aerobic photoreactivity of synthetic eumelanins and pheomelanins: generation of singlet oxygen and superoxide anion.

In this work, we examined photoreactivity of synthetic eumelanins, formed by autooxidation of DOPA, or enzymatic oxidation of 5,6-dihydroxyindole-2-carboxylic acid and synthetic pheomelanins obtained by enzymatic oxidation of 5-S-cysteinyldopa or 1:1 mixture of DOPA and cysteine. Electron paramagnetic resonance oximetry and spin trapping were used to measure oxygen consumption and formation of superoxide anion induced by irradiation of melanin with blue light, and time-resolved near-infrared luminescence was employed to determine the photoformation of singlet oxygen between 300 and 600 nm. Both superoxide anion and singlet oxygen were photogenerated by the synthetic melanins albeit with different efficiency. At 450-nm, quantum yield of singlet oxygen was very low (~10-4 ) but it strongly increased in the UV region. The melanins quenched singlet oxygen efficiently, indicating that photogeneration and quenching of singlet oxygen may play an important role in aerobic photochemistry of melanin pigments and could contribute to their photodegradation and photoaging.

Roles of reactive oxygen species in UVA-induced oxidation of 5,6-dihydroxyindole-2-carboxylic acid-melanin as studied by differential spectrophotometric method.

Eumelanin photoprotects pigmented tissues from ultraviolet (UV) damage. However, UVA-induced tanning seems to result from the photooxidation of preexisting melanin and does not contribute to photoprotection. We investigated the mechanism of UVA-induced degradation of 5,6-dihydroxyindole-2-carboxylic acid (DHICA)-melanin taking advantage of its solubility in a neutral buffer and using a differential spectrophotometric method to detect subtle changes in its structure. Our methodology is suitable for examining the effects of various agents that interact with reactive oxygen species (ROS) to determine how ROS is involved in the UVA-induced oxidative modifications. The results show that UVA radiation induces the oxidation of DHICA to indole-5,6-quinone-2-carboxylic acid in eumelanin, which is then cleaved to form a photodegraded, pyrrolic moiety and finally to form free pyrrole-2,3,5-tricarboxylic acid. The possible involvement of superoxide radical and singlet oxygen in the oxidation was suggested. The generation and quenching of singlet oxygen by DHICA-melanin was confirmed by direct measurements of singlet oxygen phosphorescence.