ALK5 inhibitor acts on trabecular meshwork cell and reduces intraocular pressure.

Intraocular pressure (IOP) is the most important risk factor for the onset and progression of glaucoma. IOP reduction has been proven effective in the treatment of glaucoma. IOP is controlled by the production and outflow of the aqueous humor (AH), and the trabecular meshwork (TM) is the main pathway for AH drainage from the eye. However, there are few conventional IOP-lowering treatments that target TM, and there is a need for such treatments. In this study, we screened for the expression level of fibronectin as an indicator and identified an activin receptor-like kinase (ALK) 5 inhibitor. Western blot analysis showed that SB431542, an ALK 5 inhibitor, reduced fibronectin and α-SMA expression. Moreover, a single dose of the ALK5 inhibitor SB431542 reduced IOP in mice, and the IOP-lowering effect of the ALK5 inhibitor was greater than that of a Rho-associated coiled-coil-containing protein kinase inhibitor (Y-27632). Repeated dosing with ALK5 inhibitor eye drops (once daily) enhanced the murine IOP-lowering effect. Furthermore, ALK5 inhibition decreased the expression of extracellular matrix (ECM) mRNA and suppressed ECM production. These findings suggest that ALK5 inhibitors may contribute to the development of new treatments for glaucoma that target the TM.

Timolol maleate, a ß blocker eye drop, improved edema in a retinal vein occlusion model.

Purpose: To investigate the therapeutic effects of eye drops, namely, timolol maleate, a ß-adrenergic receptor antagonist, and latanoprost, a prostaglandin F2α analog, on retinal edema in a murine retinal vein occlusion (RVO) model. Methods: An RVO model was established using laser-induced RVO in mice, which were administered timolol maleate and latanoprost eye drops several times after venous occlusion. Subsequently, the thickness of the inner nuclear layer (INL) and the expression levels of such genes as Vegf and Atf4, which are stress markers of the endoplasmic reticulum, were examined. Primary human cultured retinal microvascular endothelial cells (HRMECs) were treated with timolol under hypoxic conditions, after which the gene expression pattern was investigated. Importantly, an integrated stress response inhibitor (ISRIB) was used in the RVO model, he known ISRIB, which suppresses the expression of ATF4 in retinal edema. Results: Increased INL thickness was suppressed by timolol eye drops, as were the expressions of Vegf and Atf4, in the RVO model. However, latanoprost eye drops did not induce any change in INL thickness. In HRMECs, hypoxic stress and serum deprivation increased the Vegf and Atf4 expressions; in response, treatment with timolol suppressed the Vegf expression. Furthermore, the ISRIB decreased the Vegf expression pattern and edema formation, which are associated with RVO. Conclusions: These results indicate that timolol eye drops may be a potential option for RVO treatment.

The pathological association between the anterior eye segment and the retina in a murine model of neovascular glaucoma.

Neovascular glaucoma (NVG) is caused by the formation of new blood vessels in the angle, iris, and cornea in retinal ischemic disease, such as proliferative diabetic retinopathy (PDR) and retinal vein occlusion (RVO), which can reduce the visual acuity. However, the pathophysiological symptoms of NVG are still not well understood because there is no model for the formation of NVG in the angle, iris, and cornea. The aim of this study was to investigate the involvement of NVG during ischemic disease, in a murine model of retinal ischemia. We evaluated the changes of the intraocular pressure (IOP) and pathological symptoms in the anterior eye segment and retina in this model, and the changes in the RNA or protein expression of vascular endothelial growth factor (VEGF) and fibrosis-related factors were analyzed in the retina and cornea by quantitative real-time polymerase chain reaction or western blot, respectively. Furthermore, we examined the changes in IOP after intravitreal injection of an anti-VEGF antibody. First, NVG formed in the retinal ischemic murine model, and the IOP was elevated in mice with NVG formation. Interestingly, VEGF expression was decreased in the retina but increased in the cornea in the murine model of NVG. On the other hand, fibrosis-related factors were increased in the retina and also significantly increased in the cornea in NVG. Moreover, the administration of anti-VEGF antibody immediately after vessel occlusion suppressed the increase in IOP, but administration at 7 days after vessel occlusion accelerated the increase in IOP. These findings suggest that the formation of NVG may be correlated with the pathological symptoms of retinal ischemic disease, via changes in VEGF and fibrosis-related factor expression.

Piezo1 activation induces fibronectin reduction and PGF2α secretion via arachidonic acid cascade.

Glaucoma is a neurodegenerative disease that leads to blindness, and lowering intraocular pressure (IOP) is very important in glaucoma treatment. The trabecular meshwork is responsible for aqueous humor outflow, and the accumulation of fibronectin in trabecular meshwork is known to cause ocular hypertension. We have already shown that Piezo1 activation has an IOP lowering effect in mice and suppresses fibronectin expression level in human trabecular meshwork cells (HTMC). In this study, we report the mechanism of the reduction of fibronectin caused by Piezo1 activation. Activation of Piezo1 in HTMC showed increased expression of matrix metalloproteinase-2 (MMP-2) and cyclooxygenase (COX)-2, and decreased fibronectin expression. In addition, Piezo1 activation enhanced phosphorylation of cytosolic phospholipase A2 (cPLA2), and inhibitors targeting cPLA2 and COX-2 suppressed Yoda 1, a Piezo1 agonist, induced fibronectin reduction. These results indicate that the arachidonic acid cascade underlies this reaction, and, in support of this hypothesis, activation of Piezo1 promoted secretion of prostaglandin F2α (PGF2α) in HTMC. These results indicate that the activation of Piezo1 in HTMC promotes the degrading of fibronectin by promoting the arachidonic acid cascade and increasing the expression of PGF2α and MMP-2.

Involvement of endoplasmic reticulum stress in rotenone-induced leber hereditary optic neuropathy model and the discovery of new therapeutic agents.

Leber hereditary optic neuropathy (LHON) is caused by mitochondrial DNA mutations and is the most common inherited mitochondrial disease. It is responsible for central vision loss in young adulthood. However, the precise mechanisms of onset are unknown. This study aimed to elucidate the mechanisms underlying LHON pathology and to discover new therapeutic agents. First, we assessed whether rotenone, a mitochondrial complex Ⅰ inhibitor, induced retinal degeneration such as that in LHON in a mouse model. Rotenone decreased the thickness of the inner retina and increased the expression levels of 8-hydroxy-2'-deoxyguanosine (8-OHdG) and immunoglobulin heavy-chain binding protein (BiP). Second, we assessed whether rotenone reproduces LHON pathologies on RGC-5, a neural progenitor cell derived from the retina. Rotenone increased the cell death rate, ROS production and the expression levels of ER stress markers. During chemical compounds screening, we used anti-oxidative compounds, ER stress inhibitors and anti-inflammatory compounds in a rotenone-induced in vitro model. We found that SUN N8075, an ER stress inhibitor, reduced mitochondrial ROS production and improved the mitochondrial membrane potential. Consequently, the ER stress response is strongly related to the pathologies of LHON, and ER stress inhibitors may have a protective effect against LHON.

Piezo 1 is involved in intraocular pressure regulation.

Trabecular meshwork (TM) regulates the intraocular pressure (IOP) through the control of aqueous humor outflow. Previous reports show that TM cells express 11 types of mechanosensitive molecules, including Piezo 1, which sense mechanical stimuli. However, the role of Piezo 1 on TM remains unclear. Thus, in this study, we focused on the Piezo 1 and examined its role in TM cells. Immunostaining showed that Piezo 1 was expressed in mouse TM and human TM cells. Moreover, the eye drops containing Piezo 1 agonist Yoda 1 reduced the IOP in mice, and also reduced fibronectin expression level around the TM. In addition, Piezo 1 activation suppressed human TM cells migration/proliferation, and decreased fibronectin expression level. On the other hand, Piezo 1 activation increased matrix metalloproteinase (MMP)-2 expression responsible for fibronectin degradation. These findings could contribute to the development of new treatments for glaucoma.