Preclinical study of a new matrix to help the ocular surface in dry eye disease.

Dry eye disease (DED), a multifactorial disease of the tears and ocular system, causes loss of tear film homeostasis with damage to the ocular surface. This study aimed to assess whether a peculiar matrix based on sodium hyaluronate (HA), xanthan gum (XNT), glycine (GLY) and betaine (BET) as osmoprotectants, could be involved in biological responses. Wound healing assay on human corneal epithelial (HCE) cells in monolayer showed a synergistic effect of the combination of HA + XNT (**p ≤ 0.01) together with an efficient extracellular matrix remodeling of the formulation in SkinEthic™ HCE 3D-model sought by integrin beta-1 (ITGß1) expression and morphological analysis by hematoxylin and eosin (H&E), compared to a reference marketed product. The synergistic effect of HA + XNT + GLY + BET showed an antioxidant effect on HCE cells (***p ≤ 0.001). Real-time PCR analysis showed that the combination of GLY + BET seemed to ameliorate the effect exhibited by the single osmoprotectants in reducing tumor necrosis factor-alpha (TNFα, #p ≤ 0.05), interleukin-1 beta (IL1ß, ####p ≤ 0.0001) and cyclooxygenases-2 (COX2, ####p ≤ 0.0001) genes in SIRC cells under hyperosmotic stress. Furthermore, pretreatment with XNT, alone and in combination (##p ≤ 0.01), reduced COX2 expression in human non-small cell lung cancer cells (A549). Finally, the formulation was well-tolerated following q.i.d. ocular administration in rabbits during a 28-day study. Due to the synergistic effect of its components, the matrix proved able to repair the ocular surface restoring cell homeostasis and to protect the ocular surface from pro-inflammatory pathways activation and oxidative damage, thus behaving as a reactive oxygen species (ROS) scavenger.

Assessment of a New Nanostructured Microemulsion System for Ocular Delivery of Sorafenib to Posterior Segment of the Eye.

Eye drop formulations allowing topical treatment of retinal pathologies have long been sought as alternatives to intravitreal administration. This study aimed to assess whether a novel nanostructured microemulsions system (NaMESys) could be usefully employed to deliver sorafenib to the retina following topical instillation. NaMESys carrying 0.3% sorafenib (NaMESys-SOR) proved to be cytocompatible in vitro on rabbit corneal cells, and well-tolerated following b.i.d. ocular administration to rabbits during a 3-month study. In rats subject to retinal ischemia-reperfusion, NaMESys-SOR significantly inhibited retinal expression of tumor necrosis factor-alpha (TNFα, 20.7%) and inducible nitric oxide synthase (iNos, 87.3%) mRNAs in comparison to controls. Similarly, in streptozotocin-induced diabetic rats, NaMESys-SOR inhibited retinal expression of nuclear factor kappa B (NFκB), TNFα, insulin like growth factor 1 (IGF1), IGF1 receptor (IGF1R), vascular endothelial growth factor receptor 1 (VEGFR1) and 2 (VEGFR2) mRNAs by three-fold on average compared to controls. Furthermore, a reduction in TNFα, VEGFR1 and VEGFR2 protein expression was observed by western blot. Moreover, in mice subject to laser-induced choroidal neovascularization, NaMESys-SOR significantly inhibited neovascular lesions by 54%. In conclusion, NaMESys-SOR was shown to be a well-tolerated ophthalmic formulation able to deliver effective amounts of sorafenib to the retina, reducing proinflammatory and pro-angiogenic mediators in reliable models of proliferative retinopathies. These findings warrant further investigations on the full therapeutic potential of NaMESys-SOR eye drops, aiming to address unmet needs in the pharmacotherapy of retinal neovascular diseases.

Molecular characterization of exosomes and their microRNA cargo in human follicular fluid: bioinformatic analysis reveals that exosomal microRNAs control pathways involved in follicular maturation.

OBJECTIVE: To characterize well-represented microRNAs in human follicular fluid (FF) and to ascertain whether they are cargo of FF exosomes and whether they are involved in the regulation of follicle maturation. DESIGN: FF exosomes were characterized by nanosight, flow cytometry, and exosome-specific surface markers. Expression microRNA profiles from total and exosomal FF were compared with those from plasma of the same women. SETTING: University laboratory and an IVF center. PATIENT(S): Fifteen healthy women who had undergone intracytoplasmic sperm injection. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): TaqMan low-density array to investigate the expression profile of 384 microRNAs; DataAssist and geNorm for endogenous control identification; significance analysis of microarrays to identify differentially expressed microRNAs; nanosight, flow-cytometry, and bioanalyzer for exosome characterization; bioinformatic tools for microRNAs target prediction, gene ontology, and pathway analysis. RESULT(S): We identified 37 microRNAs upregulated in FF as compared with plasma from the same women. Thirty-two were carried by microvesicles that showed the well-characterized exosomal markers CD63 and CD81. These FF microRNAs are involved in critically important pathways for follicle growth and oocyte maturation. Specifically, nine of them target and negatively regulate mRNAs expressed in the follicular microenvironment encoding inhibitors of follicle maturation and meiosis resumption. CONCLUSION(S): This study identified a series of exosomal microRNAs that are highly represented in human FF and are involved in follicular maturation. They could represent noninvasive biomarkers of oocyte quality in assisted reproductive technology.

The apoptotic transcriptome of the human MII oocyte: characterization and age-related changes.

Fully competent oocytes represent the final outcome of a highly selective process. The decline of oocyte competence with ageing, coupled to quantitative decrease of ovarian follicles has been well established; on the contrary, its molecular bases are still poorly understood. Through quantitative high throughput PCR, we investigated the role of apoptotic machinery (AM) in this process. To this aim, we determined AM transcriptome in mature MII oocyte pools from women aged more than 38 years (cohort A), and compared to women aged up to 35 years (cohort B). Subsequently, 10 representative AM genes were selected and analyzed in 33 single oocytes (15 from cohort A and 18 from cohort B). These investigations led us to identify: (1) the significant upregulation of proapoptotic genes such us CD40, TNFRSF10A, TNFRSF21 and the downregulation of antiapoptotic genes such as BCL2 and CFLAR in cohort A respect to cohort B; (2) AM transcripts that have not previously been reported in human oocytes (BAG3, CD40, CFLAR, TNFRSF21, TRAF2, TRAF3). Our results demonstrated that during maturation the oocytes from older women selectively accumulate mRNAs that are able to trigger the extrinsic apoptotic pathway. These data contribute to clarify the molecular mechanisms of AM involvement in the natural selection strategy of removing low quality oocytes and preventing unfit or poorly fit embryos.

Specific alterations of the microRNA transcriptome and global network structure in colorectal cancer after treatment with MAPK/ERK inhibitors.

The mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway has a master control role in various cancer-related biological processes as cell growth, proliferation, differentiation, migration, and apoptosis. It also regulates many transcription factors that control microRNAs (miRNAs) and their biosynthetic machinery. To investigate on the still poorly characterised global involvement of miRNAs within the pathway, we profiled the expression of 745 miRNAs in three colorectal cancer (CRC) cell lines after blocking the pathway with three different inhibitors. This allowed the identification of two classes of post-treatment differentially expressed (DE) miRNAs: (1) common DE miRNAs in all CRC lines after treatment with a specific inhibitor (class A); (2) DE miRNAs in a single CRC line after treatment with all three inhibitors (class B). By determining the molecular targets, biological roles, network position of chosen miRNAs from class A (miR-372, miR-663b, miR-1226*) and class B (miR-92a-1*, miR-135b*, miR-720), we experimentally demonstrated that they are involved in cell proliferation, migration, apoptosis, and globally affect the regulation circuits centred on MAPK/ERK signaling. Interestingly, the levels of miR-92a-1*, miR-135b*, miR-372, miR-720 are significantly higher in biopsies from CRC patients than in normal controls; they also are significantly higher in CRC patients with mutated KRAS than in those with wild-type genotypes (Wilcoxon test, p < 0.05): the latter could be a downstream effect of ERK pathway overactivation, triggered by KRAS mutations. Finally, our functional data strongly suggest the following miRNA/target pairs: miR-92a-1*/PTEN-SOCS5; miR-135b*/LATS2; miR-372/TXNIP; miR-663b/CCND2. Altogether, these results contribute to deepen current knowledge on still uncharacterized features of MAPK/ERK pathway, pinpointing new oncomiRs in CRC and allowing their translation into clinical practice and CRC therapy.

TAp73 is downregulated in oocytes from women of advanced reproductive age.

Studies on oocyte transcriptome are important to understand the biological pathways involved in oogenesis, totipotence and early embryonic development. Moreover, genes regulating physiological pathways in gametes could represent potential candidates for reproductive disorders. In addition to oocyte specific transcription factors, also the members of the p53 family could be etiologically involved due to their biological functions. In fact, their role in the control of cell cycle, apoptosis, and germ-line genome stability is well known. Female reproductive aging is one of the causes of fertility reduction and it is often associated with egg aneuploidy increase. In order to verify the potential involvement of p73 in reproductive aging, we determined its expression in single mature MII oocytes from two groups of women, younger than 35 or older than 38 years, respectively. We found that TAp73 isoforms are down regulated in oocytes from women older than 38 years. We confirmed these data in pools of mouse oocytes. TAp73 down regulation in oocytes from women of advanced reproductive age could explain both the reduction of fertility and the increase of newborns with chromosomal abnormalities.

Molecular profiling of human oocytes after vitrification strongly suggests that they are biologically comparable with freshly isolated gametes.

To assess the effects of vitrification on the biomolecular profile of oocytes, we analyzed through real-time reverse transcriptase-polymerase chain reaction eight genes encoding critically important proteins for embryo development and compared this partial transcriptome with that of freshly collected gametes isolated from the same women. The comparison of the molecular profiles demonstrated that our vitrification protocol does not alter the biomolecular quality of oocytes: in fact, between the two groups we found the absence of statistically significant variations. Accordingly, this cryopreservation technique might be helpful in preserving women's fertility.