Role Of Mechanosensitive Channels Piezo In Intraocular Pressure And Aqueous Humor Circulation

Glaucoma is a type of ophthalmic disease characterized by optic nerve atrophy and visual field defect.An elevated intraocular pressure(IOP)is the defined glaucoma risk factor and controlling IOP is the mainstay of glaucoma therapy.Stable IOP primarily depends on the balance of equal rate between the production and outflow of aqueous humor(AH).The epithelium of ciliary body,consisting of the internal pigmented epithelium(PE)and the external non-pigmented epithelium(NPE)layers,is responsible for AH production.The trabecular meshwork(TM)-Schlemm's canal pathway and uveoscleral pathway are responsible for AH outflow.Currently,there is no effective treatment to lower the elevated IOP because the complexity of regulating aqueous humor production and outflow.Previous investigations suggest that TREK-1 and TRPV4,as an important type of mechanical responder,are expressed in the TM and NPE cells and palyed an important role in regulating the production and oufflow of aqueous humor.These results suggest that mechanicanosensitive ion channels play an important physiological and pathological role in regulating IOP.As a novel class of mechanosensitive channel,Piezo channels exhibit crucial roles in transducing mechanical force under physiological situation,and they have been reported to function in different organs.However,it is still elusive about the expression pattern of Piezo channels in the eye and their function in maintaining IOP and aqueous humor dynamics.Objective: The goal of this study was to investigate the precise expression pattern of the mechanically sensitive ion channels,Piezo1 and Piezo2 in AH circulation pathways and also was to examine the any effect of Piezo channels modulations on IOP homeostasis and AH circulation.Methods: Immunofluorescence and Western blot were used to detect the expression of Piezo channels on the AH production and outflow pathways.The function of Piezo channels was inhibited through intracameral injection of GsMTx4,a mechanosensitive ion channel blocker or shRNAs.The effects of the Piezo channels on IOP and AH circulation were evaluated by using Tonometer and the Syring-Pump system respectively.Myocilin transgenic mouse glaucoma model was used to evaluate the therapeutic effects of Piezo1 on glaucoma.Results: Immunofluorescence and Western blot results showed that Piezo1 and Piezo2 were highly expressed in human and mouse tissues relevant to aqueous humor production and outflow pathway,such as ciliary body,TM,Schlemm's canal and ciliary muscle.We also found a precise expression pattern for Piezo2 in human TM,with a high expression in the uveal meshwork,and a moderate expression in the corneoscleral meshwork and JCT.Inhibition of Piezo1 by intracameral injection of 20 ?M GsMTx4,a short peptide toxin of 34 amino acids,significantly reduced the IOP in normal and high IOP mice,likely through increasing the AH outflow facility.Intracameral injection of Ad5 with shRNA-mPiezo1 to knockdown Piezo1 specificly can also reduce IOP and increase AH outflow facility.These results indicated that Piezo1 played an important role in regulating IOP and AH circulation.However,no significant differences in IOP and AH outflow facility were observed after down-regulating the expression of Piezo2 through using the Cre-loxP system.Based on the modified Goldmann equation,down-regulation of Piezo2 expression leads to a significantly decrease of the flow rate through the uveoscleral outflow pathway.Conclusions:1.Piezo1 and Piezo2 channels are robustly expressed in human and mouse aqueous humor production and outflow pathway tissues.Piezo1 has a uniform expression in human trabecular meshwork,Piezo2 expression is robust in the uveal meshwork,and moderate in the corneoscleral meshwork and JCT.2.Inhibition of Piezo1 significantly reduces the IOP of normal and high IOP mice,likely through increasing the aqueous humor outflow facility.3.Although down-regulation of Piezo2 expression exhibits a mild effect on IOP and the facility of aqueous humor,it can significantly reduce the uveaoscleral outflow rate of aqueous humor.4.Piezo channels play an important fuction in IOP regulation and aqueous humor circulation.Piezo channels may be a potential target for the therapy of glaucoma treatment.