This would, in turn, activate the signaling cascade within the target cell. blot analysis indicated that this nanovesicles were positive for classic exosome markers, including Tsg101 and Alix. Isolated nanoparticles were Vericiguat found in sucrose density fractions common of exosomes (1.118C1.188 g/mL sucrose). Using confocal microscopy, we exhibited time-dependent specific accumulation of the NPCE-derived exosomes in NTM cells. Other cell lines investigated hardly revealed any exosome uptake. We further showed that exosomes induced changes in Wnt signaling protein expression in the TM cells. Western blot analysis further revealed decreased phosphorylation of GKS3 and reduced -catenin levels. Finally, we found that treatment of NTM Vericiguat cells with exosomes resulted in a greater than 2-fold decrease in the level of -catenin in the cytosolic portion. In contrast, no amazing difference in the amount of -catenin in the nuclear portion was noted, relative to the control. Conclusions The data suggest that NPCE cells release exosome-like vesicles and that these nanoparticles impact canonical Wnt signaling in TM cells. These findings may have therapeutic relevance since canonical Wnt pathway is usually involved in intra-ocular pressure regulation. Further understanding of NPCE-derived exosome-responsive signaling pathways may reveal new targets for pharmacological intervention within the drainage system as a target for glaucoma therapy. Introduction Glaucoma and the ocular drainage system The privilege of age-related diseases is predicted to increase in the coming year and glaucoma patient number worldwide is usually expected reach 80 million by 2020, despite current improvements in therapy[1]. Glaucoma is usually characterized by the ongoing deterioration of the retinal ganglion layer and worsening of visual field defects, accompanied changes in the optic nerve head. High intraocular pressure (IOP) has long been Rabbit Polyclonal to p38 MAPK (phospho-Thr179+Tyr181) considered the most important risk factor for the onset and progression of glaucoma, and therefore pharmacological and surgical treatments have focused on lowering the IOP. However, even with treatment to lower IOP and even in normal tension glaucoma [2], optic nerve damage may progress. The ocular drainage system comprises a number of unique tissues providing specialized functions. In glaucoma diseases, the delicate balance between the tissue generating the aqueous humor (AH), i.e., the ciliary epithelium, and the AH-draining tissues, i.e., the trabecular meshwork (TM) and Schlem’s canal, is essential for maintenance of the intra-ocular pressure (IOP). Communication between these tissues was first suggested by Coca-Prados and colleagues [3]. Still, the details of the hypothesized communication remain to be defined. Active proteins [4] have been detected in the AH, supporting the idea of tissue communication within the drainage system. Among AH proteins, some were uniquely recognized in the AH, while many others, such as cytokines [5], kinases [6], phosphatases [7], growth factors [8], are general participants of cellular communication. Further support for communication between the ocular drainage systems came from cell culture experiments where co-cultured non-pigmented ciliary epithelium (NPCE) and TM cells induced significant increases in the activity of some phosphatases and MMPs (matrix metalloproteinases), MMP-2 and MMP-9,) in TM cells [9]. Still, it remains mechanistically unclear how active molecule proteins Vericiguat and enzymes known to be located in the intracellular moiety, could function in the AH which is found in the extracellular milieu. The involvement of Extracellular vesicles (EV) offers one possible answer. EV and exosomes Exosomes are 30C140 nm-diameter membrane-bound extracellular vesicles that are shed by various types of cells under both physiological and pathological conditions [10]. Exosomes are a part of a larger group of vesicles known as EV. Biological extraction methods of exosomes cannot exclude the presence of some larger vesicles and the exosomes size.