| BackgroundsProgressive loss of optic nerve axons and retinal ganglion cells(RGCs)result in characteristic optic nerve atrophy and visual field defects in glaucoma patients.The prevalence of this disease was 1.5% ～3.6%in adults aged 40 years and older living in China.Primary open-angle glaucoma(POAG)accounts for 0.7%to 2.6%,and 10% was bilateral blindness [1].It seriously affects the quality of life and increases the burdens of family and sociality.IOP is usually regarded as a key risk factor [2],which is regulated through the formation and drainage of the aqueous humor.It is essential to keep homeostasis and shape of the eyes.There are two different pathways through which aqueous humor leaves the anterior chamber.The major outlet for approximately 80% of aqueous humor in humans is the conventional outflow pathway,consisting of the trabecular meshwork and Schlemm’s canal.The other route is through the uveoscleral pathway,which is referred to as the unconventional pathway.Aqueous humor exits the anterior chamber in this pathway by diffusing through the intercellular spaces among ciliary muscle fibers [3].Most of the resistance to aqueous humor outflow is thought to be due to the extracellular matrix(ECM)within the deepest portion of the trabecular meshwork and the basement lamina of Schlemm’s canal inner wall endothelium.These regions are a complex network composed of collagen,proteoglycans,fibronectin,and other matricellular proteins and many others [4-6].During normal tissue homeostasis,the ECM is remodeled and trabecular cell behavior is modified,permitting increased aqueous fluid outflow to maintain IOP within a relatively narrow physiological pressure.Dysfunction in the normal homeostatic process leads to increased outflow resistance and elevated IOP.Therefore,restoring ECM homeostasis is a rational approach to prevent disease progression.The renin-angiotensin system(RAS)plays a crucial role in the regulation of blood pressure,as well as fluid and electrolyte balance.The local RAS also has been studied to have ECM remodeling in many organs,such as the heart [7],liver [8],and eyes [9].Accumulating evidence strongly implicates components of RAS existing in ocular tissues including renin,angiotensin-converting enzyme(ACE),and angiotensin Ⅱ(Ang Ⅱ)[10-12].Ang Ⅱ,the major bioactive peptide of the RAS,has widely known various properties,including ECM remodeling,vasoconstriction,fibrosis,inflammation,increased generation of reactive oxygen species(ROS),and ion channel dysfunction.Increased amounts of Ang Ⅱ may be involved in IOP dysregulation and pathology of POAG [13].However,the association between Ang Ⅱ and IOP in POAG is unknown and further studies are investigated.PURPOSEThe level of Ang Ⅱ was detected in the aqueous humor of POAG patients and cataract patients.The association between elevated Ang Ⅱ and IOP dysregulation was unclear.Our study sought to dissect the underlying mechanisms linking Ang Ⅱ to POAG and provide supporting evidence and actionable opportunities for the treatment of glaucoma.METHODS1.(1)Aqueous humor was collected from cataract patients,and the protein content of AGT was detected by western blotting;TM,sclera,and corneal cells were isolated and extracted from one patient’s donated eyeball.The protein expression of AGT was measured by western blotting.(2)Aqueous humor was obtained from patients with cataracts and POAG,that was analyzed the correlation between IOP and the concentration of Ang II;2.The isolated and transformed HTMCs were identified by immunofluorescence staining for myocilin(MYOC)expression after cultured HTMCs with or without DEX at the concentration of 400 n M.3.Primary and transformed HTMCs were used to experiment as follows:3.1 To investigate the effect of Ang Ⅱ on ECM fibrosis.Method: HTMCs were randomly divided into the blank group and the Ang Ⅱ at the concentration of 10μM.Col1,FN and a-SMA expression were investigated by western blot,q PCR and immunofluorescence after treatment with 48 h.3.2 Effect of Ang Ⅱ on the ROS level and NADPH oxidase activity in HTMCs.Methods:(1)Primary and transformed HTMCs were treated with or without 10μM Ang Ⅱ respectively.After 48 h,the DCFH-DA fluorescent probe was used to detect the fluorescent intensity of ROS.All five known m RNA isoforms of NOX oxidase were quantified by q PCR,and the protein level of NOX4 was detected by Western blot.3.3 To further investigate whether NOX4 mediates the enhancing effect of Ang Ⅱ on ECM fibrosis.Methods: HTMCs transfected with si RNA targeting NOX4 followed exposure to Ang Ⅱ was applied to perform Western blotting and immunofluorescence to explore the expression of the related protein.DCFHDA fluorescent probe was used to detect the ROS level.3.4 To assess the effect of GLX351322 and NAC on the Ang-induced ROS and the indexes of ECM fibrosis.Methods: HTMCs were distributed into different groups,including the control,Ang Ⅱ,AngⅡ++GLX351322(an inhibitor of NADPH oxidase 4,10μM)group and Ang Ⅱ+ NAC(ROS inhibitor,40μM)groups.After 48 h,DCFH-DA fluorescent probe was used to detect the ROS level.And the protein level of Col1,FN and a-SMA in HTMCs were detected by Western blot and immunofluorescence.3.5 To study the influence of Ang Ⅱ on the protein expression of Smad3 and p-Smad3.Methods:(1)HTMCs incubated with or without 10μM Ang Ⅱ,the protein expression of Smad3 and p-Smad3 were measured by Western blot.(2)HTMCs were divided into the control group,Ang Ⅱ group,and Ang Ⅱ+GLX351322 group.Western blot was used to detect the protein expression of Smad3,p-Smad3,and indexes of ECM fibrosis.(3)HTMCs were divided into 3 groups: the control group,Ang Ⅱ,and AngⅡ+SIS3 group.SIS3 was a Smad3 inhibitor.The protein level of Smad3,p-Smad3,Col1,FN and aSMA were measured by Western blot.4.Animal experiments:4.1 To investigate whether the inhibitor of NOX4 attenuates trabecular meshwork fibrosis induced by Ang Ⅱ in mice.Methods: Established cultured human eye anterior segment model.Mice were randomly divided into the control group,the Ang Ⅱ group,and the Ang Ⅱ+ GLX351322 group,and the control group was injected with normal saline.The IOP was recorded before injection every week.The expression of Col1,FN in HTMCs was detected by immunohistochemical staining.4.2 To evaluate the effect of Smad3 inhibitors on IOP and trabecular meshwork fibrosis induced by Ang Ⅱ.Methods: Established cultured human eye anterior segment model.Mice were randomly divided into the control group,the Ang Ⅱ group,and the AngⅡ+SIS3 group.And the normal saline was injected into the fornix conjunctival as the control.The IOP was recorded before injection every week.During the4-week experimental period,the expression of Col1,FN in HTMCs was detected by immunohistochemical staining.Results1.(1)The AGT protein,a component of RAS in aqueous humor could be detected in patients with cataracts.AGT can be expressed in the HTMCs,sclera and corneal cells,and the content of AGT is highest in the HTMCs among them.(2)The concentration of Ang Ⅱ in the aqueous humor of POAG patients was significantly higher than in cataract patients;And elevated Ang Ⅱ was positively correlated with IOP in POAG(R=0.422,P<0.05).2.After treatment with DEX,MYOC was positive for immunocytochemistry stains in HTMCs(P<0.05).HTMCs were cultured successfully in vitro.3.Compared with the control group,m RNA and protein levels of Col1,FN,and a-SMA in primary and transformed HTMCs were elevated significantly after the Ang Ⅱ treatment(P<0.05).4.Compared with the control group,ROS level was increased after treatment with Ang Ⅱ at the concentration of 10umol/L(P<0.05).Primary and transformed HTMCs cultured in the presence of Ang Ⅱexhibited a marked selective increase in NOX4 m RNA and Protein expression(P<0.05);5.HTMCs reverse transformed with si RNA against NOX4 prevented Ang Ⅱ mediated increases in ROS expression(P<0.05).Western blotting and immunofluorescence analysis were used to confirm the repressive effect of NOX4 in the ECM fibrosis induced by Ang Ⅱ(P<0.05).6.Incubating transformed HTMCs with GLX351322 or NAC attenuated Ang Ⅱ induced increased ROS,Col1,FN and a-SMA protein expression(P<0.05).7.(1)After treatment of transformed HTMCs with Ang Ⅱ for 48 h,we observed that the p-Smad3 protein level was significantly increased(P<0.05),and the total Smad3 was the same as before(P>0.05).(2)Compared with control cells,the upregulation of Col1,FN,a-SMA,and the ratio of p-Smad3/ Smad3 induced by Ang Ⅱ were significantly attenuated in the knockdown of the NOX4 group(P<0.05).(3)The protein levels of p-Smad3,Col1,FN and a-SMA were significantly increased in cells by treatment with Ang Ⅱ alone compared with the control,and decreased by co-treatment with Ang Ⅱ and SIS3(P<0.05).8.The IOP fluctuated in a range of 10-15 mm Hg in mice before injection.In the fornix conjunctival with Ang Ⅱ injection for 4 weeks,the IOP was elevated significantly than the mixture group of Ang Ⅱ and GLX351322(P < 0.05).Representative immunohistochemical images also showed that GLX351322 inhibited the expression of Col1 and FN induced by Ang Ⅱ(P<0.05).9.The basal IOP ranges from 10-15 mm Hg.After 4 weeks,SIS3 inhibited the elevated IOP and expression of Col1 and FN in trabecular meshwork induced by Ang Ⅱ(P<0.05).No significant difference existed between the control group and co-treatment with Ang Ⅱ and SIS3(P>0.05).CONCLUSIONS1.The concentration of Ang Ⅱ in the aqueous humor of POAG patients was higher than the control and was positively correlated with IOP.2.Ang Ⅱ promotes fibrosis of ECM in the trabecular meshwork.3.Ang Ⅱ activates the Smad3 through upregulated NOX4,which promotes fibrosis of ECM in trabecular meshwork. |
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