Nitric Oxide and miRNAs - Modulators of glaucoma relevant genes in the outflow tissues

Glaucoma is one of the leading causes of blindness in the western world (Quigley 1996; Resnikoff et al. 2004). Its pathogenesis is characterized by a chronic degenerative neuropathy leading to the loss of axons of the retinal ganglion cells (Allingham et al. 2009; Kwon et al. 2009) and Primary Open Angle Glaucoma (POAG) is its most prevalent form. Patients suffer from visual field loss, ending in total blindness if not appropriately treated. Intraocular pressure (IOP) has been found to be a main risk factor for POAG (Gordon et al. 2002; Higginbotham et al. 2004). During the pathogenesis of POAG, the outflow of aqueous humor (AH) is reduced due to an increased outflow resistance in the JCT of the TM (Grant 1963; Johnson 2006). Even if molecular mechanisms causing this increase are still unclear, changes in amount and quality of extracellular matrix (ECM) are likely to be involved (Lütjen-Drecoll et al. 1986; Rohen et al. 1993). Connective Tissue Growth Factor (CTGF) has been found to be elevated in the AH (Browne et al. 2011; Fahmy et al. 2008) as well as in SC cells of POAG patients (Overby et al. 2014), and potent to induce ECM expression in TM cells (Junglas et al. 2009). A CTGF overexpressing mouse model, developing elevated IOP and axon loss over time, underlined the role of CTGF in the pathogenesis of POAG (Junglas et al. 2012). Next to changes in the composition of the ECM in the JCT, endothelial cells of Schlemm’s Canal (SC) inner wall seem to have the potential to modulate outflow resistance (Stamer et al. 2015). A negative correlation of pore forming ability and SC cell stiffness was found (Overby et al. 2014). If that pore forming ability becomes disrupted in POAG, it would lead to impaired AH outflow and IOP elevation. When IOP increases, it results in increased fluid shear stress for the affected cells of the outflow pathway (Ethier et al. 2004; Stamer et al. 2011). Under healthy conditions, increasing IOP leads to increasing NO signaling and - by its cell relaxing effect - to an increased outflow facility (Buys et al. 2014). Under glaucomatous conditions, SC cells stiffen (Overby et al. 2014; Vahabikashi et al. 2019) and a dysregulation of NO release is suggested to result in ocular hypertension (Emam et al. 2014). Reducing cell stiffness trough cell relaxation is therefore an optional aim to treat POAG. Therefore, DETA-NO was chosen as exogenous NO donor in this study to investigate its effect on outflow cells in vitro. Micro ribonucleic acids (miRNAs) have emerged as key regulators of gene expression by post-transcriptionally binding target messenger RNA (mRNA) according to their nucleotide sequences (Wienholds and Plasterk 2005; Krol et al. 2010; Huntzinger and Izaurralde 2011). Alterations in miRNAs have been implicated in many different diseases (Esteller 2011; Gutschner and Diederichs 2012) and it just started to investigate its role in the pathogenesis of POAG. In the present study, the premise was that aberrant biomechanical behavior of outflow cells might be due to altered expression of miRNAs that are associated to the NO regulatory system. The present study investigated a glaucoma mouse model overexpressing CTGF. It was found that macroglia activation and IOP increase are specific for the transgenic βB1-CTGF mice, independent on their genetic background. A potential antagonist of the CTGF effects was investigated by treating SC cells with exogenous NO. There, DETA-NO treatment not only reduced the expression of CTGF and ECM components but also caused a significant reduction of cell stiffness. Furthermore, in vitro flow conditions induced NO production. By a miRNA-microarray, 39 miRNAs associated to the NO signaling pathway were found to be differentially expressed in healthy and glaucomatous SC cell strains. By correlation analysis, four of them were found to have a positive correlation of miRNA abundance and cell stiffness. For miR-222-3p and miR-455-3p, the increase was confirmed and miRNA alteration by transfection experiments led to significant impact on CTGF expression. To further investigate a potential delivery system for miRNAs towards outflow tissue, the distribution of gold-core nanoparticles (AuNPs) within the anterior chamber was assessed after ex vivo perfusion of porcine eyes and gold content of ciliary body, cornea, iris, lens, and the TM was analyzed. ICP-MS analysis exhibited that independent on AuNP size and surface, most gold was targeted to the TM. Therefore, NPs can be used as delivery system to specifically target therapeutic agents to outflow tissue. By revealing the effect of alterations in NO signaling and miRNA abundance on glaucoma-associated factors, the molecular mechanisms underlying glaucoma could be better understood