Ochratoxin A induces hepatic and renal toxicity in mice through increased oxidative stress, mitochondrial damage, and multiple cell death mechanisms.

Ochratoxin A (OTA) is a widespread food toxin produced by Aspergillus ochraceus and other molds. In this study, we developed and established acute OTA toxicity conditions in mice, which received daily oral doses of OTA between 0.5 up to 8 mg/kg body weight up to 7 days and were subjected to histological and biochemical analysis to characterize renal and hepatic damage. Oral administration of OTA for 7 days resulted in loss of body weight in a dose-dependent manner and increased the levels of serum biomarkers of hepatic and renal damage. The kidney was more sensitive to OTA-induced damage than the liver. In addition to necrosis, OTA induced hepatic and renal apoptosis in dose- and time-dependent manners. Especially, a high dose of OTA (8 mg/kg body weight) administered for 7 days led to necroptosis in both liver and kidney tissues. OTA dose-dependently increased the oxidative stress levels, including lipid peroxidation, in the liver and kidneys. OTA disrupted mitochondrial dynamics and structure in hepatic and renal cells, leading to the dysregulation of mitochondrial homeostasis. OTA increased transferrin receptor 1 and decreased glutathione peroxidase 4 levels in a dose- and time-dependent manner. These results suggest the induction of ferroptosis. Collectively, this study highlighted the characteristics of acute OTA-induced hepatic and renal toxicity in mice in terms of oxidative stress, mitochondrial damage, and multiple cell death mechanisms, including necroptosis and ferroptosis.

Mdm1 ablation results in retinal degeneration by specific intraflagellar transport defects of photoreceptor cells.

Mouse double minute 1 (Mdm1) might be involved in the function and structure of centrioles and age-related retinal degeneration. However, the mechanism by which Mdm1 deficiency causes retinal degeneration remains unknown. We confirmed that the Mdm1 protein is localized at the connecting cilium (CC) of photoreceptor cells in the retina. The electroretinograms of 6-week-old Mdm1-/- mice revealed decreased vision, which was eventually lost, and outer segment (OS) photoreceptor degeneration was evident on postnatal day 7, with complete loss of the outer nuclear layer (ONL) observed at 35 weeks. Mdm1-/- mouse retinas showed mislocalization of opsins in the photoreceptor cells, indicating particular intraflagellar transport (IFT) defects, and entrapment of the nuclei in the ONL by microvilli of retinal pigment epithelial cells, leading to apoptosis in the ONL. These results suggest that Mdm1 ablation causes specific IFT defects, which prevents the OS from continuously replenishing new discs, resulting in retinal degeneration.

Engineering of hybrid spheroids of mesenchymal stem cells and drug depots for immunomodulating effect in islet xenotransplantation.

Immunomodulation is an essential consideration for cell replacement procedures. Unfortunately, lifelong exposure to nonspecific systemic immunosuppression results in immunodeficiency and has toxic effects on nonimmune cells. Here, we engineered hybrid spheroids of mesenchymal stem cells (MSCs) with rapamycin-releasing poly(lactic-co-glycolic acid) microparticles (RAP-MPs) to prevent immune rejection of islet xenografts in diabetic C57BL/6 mice. Hybrid spheroids were rapidly formed by incubating cell-particle mixture in methylcellulose solution while maintaining high cell viability. RAP-MPs were uniformly distributed in hybrid spheroids and sustainably released RAP for ~3 weeks. Locoregional transplantation of hybrid spheroids containing low doses of RAP-MPs (200- to 4000-ng RAP per recipient) significantly prolonged islet survival times and promoted the generation of regional regulatory T cells. Enhanced programmed death-ligand 1 expression by MSCs was found to be responsible for the immunomodulatory performance of hybrid spheroids. Our results suggest that these hybrid spheroids offer a promising platform for the efficient use of MSCs in the transplantation field.

CD9 induces cellular senescence and aggravates atherosclerotic plaque formation.

CD9, a 24 kDa tetraspanin membrane protein, is known to regulate cell adhesion and migration, cancer progression and metastasis, immune and allergic responses, and viral infection. CD9 is upregulated in senescent endothelial cells, neointima hyperplasia, and atherosclerotic plaques. However, its role in cellular senescence and atherosclerosis remains undefined. We investigated the potential mechanism for CD9-mediated cellular senescence and its role in atherosclerotic plaque formation. CD9 knockdown in senescent human umbilical vein endothelial cells significantly rescued senescence phenotypes, while CD9 upregulation in young cells accelerated senescence. CD9 regulated cellular senescence through a phosphatidylinositide 3 kinase-AKT-mTOR-p53 signal pathway. CD9 expression increased in arterial tissues from humans and rats with age, and in atherosclerotic plaques in humans and mice. Anti-mouse CD9 antibody noticeably prevented the formation of atherosclerotic lesions in ApoE-/- mice and Ldlr-/- mice. Furthermore, CD9 ablation in ApoE-/- mice decreased atherosclerotic lesions in aorta and aortic sinus. These results suggest that CD9 plays critical roles in endothelial cell senescence and consequently the pathogenesis of atherosclerosis, implying that CD9 is a novel target for prevention and treatment of vascular aging and atherosclerosis.

Fully Automated, Label-Free Isolation of Extracellular Vesicles from Whole Blood for Cancer Diagnosis and Monitoring.

Extracellular vesicles (EVs) that circulate in body fluids possess significant potential for disease diagnosis. Their use in clinical settings, however, has been limited owing to lack of simple and robust isolation methods. To rectify this problem, a centrifugal device for automatic, fast, and efficient isolation of EVs from whole-blood, called Exodisc-B is presented in this paper. Methods: The device comprises a built-in chamber to facilitate plasma separation and two nanoporous filters-one for removing larger particles and the other for enriching EVs. The performance of the device in comparison to ultracentrifugation (UC) was evaluated by analyzing the yield, purity, protein and RNA content of the isolated EVs. Additionally, the EV protein marker expressions were measured by ELISA and statistically analyzed to differentiate prostate cancer patients from healthy donors. Results: Compared with the UC technique, the proposed device is capable of isolating at least an order of magnitude higher number of EVs with about 30-fold higher mRNA count within 40 min. Sandwich ELISA of EV-specific membrane proteins-CD9-CD81-confirmed that Exodisc-B can isolate EVs from a volume of whole blood as low as 30 µL with a capture efficiency exceeding 75%. The device also facilitates temporal monitoring of tumor progression within live mouse xenograft models over a period of 13 weeks while using minimal volumes of weekly collected blood samples. Further, in ELISA analyses of multiple cancer-related proteins, such as prostate-specific antigen (PSA), prostate-specific membrane antigen (PSMA), epithelial cell adhesion molecule (EpCAM), epidermal growth factor receptor 1 (EGFR1), and heat shock protein 90 (HSP90), extracted from EVs isolated from human plasma, 43 patients were differentiated from 30 healthy donors. Conclusion: The results demonstrated the ability of Exodisc-B to provide a rapid, sensitive, and point-of-care-type method for extracting intact EVs from small volumes of clinical blood samples for disease diagnosis and monitoring.

Embryonic Stem Cell-Derived mmu-miR-291a-3p Inhibits Cellular Senescence in Human Dermal Fibroblasts Through the TGF-ß Receptor 2 Pathway.

Senescent cells accumulate in various tissues over time and contribute to tissue dysfunction and aging-associated phenotypes. Accumulating evidence suggests that cellular senescence can be inhibited through pharmacological intervention, as well as through treatment with soluble factors derived from embryonic stem cells (ESCs). In an attempt to investigate the anti-senescence factors secreted by ESCs, we analyzed mouse ESC-derived extracellular microRNAs in conditioned medium via microRNA array analysis. We selected mmu-miR-291a-3p as a putative anti-senescence factor via bioinformatics analysis. We validated its inhibitory effects on replicative, Adriamycin-induced, and ionizing radiation-induced senescence in human dermal fibroblasts. Treatment of senescent cells with mmu-miR-291a-3p decreased senescence-associated ß-galactosidase activity, enhanced proliferative potential, and reduced mRNA and protein expression of TGF-ß receptor 2, p53, and p21. mmu-miR-291a-3p in conditioned medium was enclosed in ESC-derived exosomes and exosomes purified from ESC conditioned medium inhibited cellular senescence. The inhibitory effects of mmu-miR-291a-3p were mediated through the TGF-ß receptor 2 signaling pathway. Hsa-miR-371a-3p and hsa-miR-520e, the human homologs of mmu-miR-291a-3p, showed similar anti-senescence activity. Furthermore, mmu-miR-291a-3p accelerated the excisional skin wound healing process in aged mice. Our results indicate that the ESC-derived mmu-miR-291a-3p is a novel candidate agent that can be utilized for cell-free therapeutic intervention against aging and aging-related diseases.

Polymeric microsphere-facilitated site-specific delivery of quercetin prevents senescence of pancreatic islets in vivo and improves transplantation outcomes in mouse model of diabetes.

Attenuation of senescence progression may be attractive way to preserve the functionality of pancreatic islets (PI) after transplantation. In this study, we developed a model for in vitro induction of premature senescence in rat PI and showed the effectiveness of quercetin (QU) to prevent the senescence. To provide targeted-delivery of QU to the PI after transplantation, we prepared the hybrid clusters (HC) of islet single cells (ISC) and QU-loaded polymeric microspheres (QU; ∼7.55 ng HC-1). Long-term culture of the HC revealed reduced levels of reactive oxygen species and decreased expression of senescence-associated beta galactosidase, Rb, p53, p16, and p21 compared to that of the control islets. Transplantation of HC into subcutaneous space of the immune-deficient mice produced better glycemic control compared to the control islets or the ICC-transplanted mice. SA-ß-Gal staining of the in vivo transplanted HC sample showed lower intensity compared to that of the control islets or the islet cell clusters. Thus, in situ delivery of therapeutic agent may be a promising approach to improve therapeutic outcomes in cell therapy. STATEMENT OF SIGNIFICANCE: In this study, we aimed to improve outcomes in islet transplantation using in situ delivery of quercetin to pancreatic islets, using polymeric microspheres. We prepared prolonged release-type microspheres and constructed hybrid clusters of pancreatic islets and the microspheres using hanging drop method. The presence of quercetin in the cellular microenvironment attenuated the progression of senescence in the pancreatic islets in a long-term in vitro culture. Moreover, transplantation of the hybrid clusters in the diabetic mice produced better glycemic control compared to that of the control islets. In addition, quercetin delayed the progression of senescence in the pancreatic islets after in vivo transplantation. Thus, local delivery of antioxidants like quercetin may be an attractive way to improve outcomes in cell therapy.

Development of Bioactive PEGylated Nanostructured Platforms for Sequential Delivery of Doxorubicin and Imatinib to Overcome Drug Resistance in Metastatic Tumors.

Metastasis of cancers accounts for almost all cancer-related deaths. In this study, we report a PEGylated nanostructured platform for coadministration of doxorubicin (DOX) and imatinib (IMT) intended to effectively inhibit metastatic tumors. The DOX and IMT coloaded nanostructured system (DOX/IMT-N) is characterized by an excellent encapsulation potential for both drugs and shows sequential and sustained drug release in vitro. DOX/IMT-N significantly inhibited the in vitro proliferation of MDA-MB-231 and SK-MEL-28 cells. The inhibitory effect on in vitro proliferation of the cells was significantly greater than the effect of free DOX, DOX/IMT cocktail, or the nanostructured system housing DOX only (DOX-N). DOX/IMT-N remarkably enhanced cellular drug uptake, resulting in enhanced apoptosis, caused by significant increases in the expression levels of apoptotic marker proteins. Intravenous administration of DOX/IMT-N to MBA-MB-231 xenograft tumor-bearing mice resulted in significantly improved inhibition of tumor progression compared to that with DOX, DOX/IMT, or DOX-N. Therefore, the nanostructured DOX/IMT-N system could potentially aid in overcoming drug resistance in metastatic tumors and improve the effectiveness of metastatic tumor therapeutics.

Prognostic significance of CD9 expression differs between tumour cells and stromal immune cells, and depends on the molecular subtype of the invasive breast carcinoma.

AIMS: CD9, a tetraspanin transmembrane protein, modulates cell motility, migration, and proliferation. The aim of this study was to investigate the prognostic significance of CD9 expression in patients with invasive breast carcinoma (IBC). METHODS AND RESULTS: CD9 expression was evaluated in tissue microarrays of 1349 IBC samples via immunohistochemistry. CD9 expression in tumour cells (T-CD9 expression) and CD9 expression in stromal immune cells (S-CD9 expression) were analysed separately. T-CD9 expression was observed in 732 (54.3%) cases, and was associated with lymph node metastasis, histological type, lymphovascular invasion, high histological grade, HER2 positivity, a high Ki67 labelling index, and distant metastasis. S-CD9 expression was observed in 833 (61.7%) cases, and was associated with large tumour size, histological type, high histological grade, negative hormone receptors, HER2 positivity, a high Ki67 labelling index, and tumour-infiltrating lymphocytes. Patients with T-CD9 expression had shorter disease-free survival (DFS) than those without T-CD9 expression in the univariate and multivariate analyses. However, S-CD9 expression correlated significantly with a favourable DFS in the univariate and multivariate analyses. In the subgroup analysis, T-CD9 expression and S-CD9 expression were independent markers for DFS in luminal A and luminal B (HER2-negative) subgroups, respectively. CONCLUSIONS: T-CD9 expression could be a biomarker for poor prognosis in luminal A IBC, whereas S-CD9 expression could be a marker of good prognosis in luminal B (HER2-negative) IBC. Therefore, tumour compartment-specific analyses considering molecular subtypes are necessary to study the prognostic significance of CD9 expression in IBC.