October 2017
Volume 58, Issue 12
Open Access
Glaucoma  |   September 2017
Genetic Deletion of the NOS3 Gene in CAV1−/− Mice Restores Aqueous Humor Outflow Function
Author Affiliations & Notes
  • Maomao Song
    Department of Ophthalmology & Visual Science, Eye & ENT Hospital, Shanghai Medical College, Fudan University, Shanghai, China
    Key Laboratory of Myopia, National Health and Family Planning Commission, and Shanghai Key Laboratory of Visual Impairment and Restoration, Fudan University, Shanghai, China
  • Jihong Wu
    Department of Ophthalmology & Visual Science, Eye & ENT Hospital, Shanghai Medical College, Fudan University, Shanghai, China
    Key Laboratory of Myopia, National Health and Family Planning Commission, and Shanghai Key Laboratory of Visual Impairment and Restoration, Fudan University, Shanghai, China
  • Yuan Lei
    Department of Ophthalmology & Visual Science, Eye & ENT Hospital, Shanghai Medical College, Fudan University, Shanghai, China
    Key Laboratory of Myopia, National Health and Family Planning Commission, and Shanghai Key Laboratory of Visual Impairment and Restoration, Fudan University, Shanghai, China
  • Xinghuai Sun
    Department of Ophthalmology & Visual Science, Eye & ENT Hospital, Shanghai Medical College, Fudan University, Shanghai, China
    Key Laboratory of Myopia, National Health and Family Planning Commission, and Shanghai Key Laboratory of Visual Impairment and Restoration, Fudan University, Shanghai, China
    State Key Laboratory of Medical Neurobiology, Institutes of Brain Science and Collaborative Innovation Center for Brain Science, Fudan University, Shanghai, China
  • Correspondence: Xinghuai Sun, Department of Ophthalmology and Visual Sciences, Eye and ENT Hospital of Fudan University, Shanghai 200031, China; xhsun@shmu.edu.cn
  • Yuan Lei, Department of Ophthalmology and Visual Sciences, Eye and ENT Hospital of Fudan University, Shanghai 200031, China; lilian0167@hotmail.com
Investigative Ophthalmology & Visual Science September 2017, Vol.58, 4976-4987. doi:10.1167/iovs.16-21072
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      Maomao Song, Jihong Wu, Yuan Lei, Xinghuai Sun; Genetic Deletion of the NOS3 Gene in CAV1−/− Mice Restores Aqueous Humor Outflow Function. Invest. Ophthalmol. Vis. Sci. 2017;58(12):4976-4987. doi: 10.1167/iovs.16-21072.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: The purpose of this study was to investigate the impact of genetic deletion of NOS3 in CAV1−/− mice on aqueous humor outflow function using a mouse genetic double knockout model (DKO, NOS3−/− CAV1−/−).

Methods: IOP was measured in DKO, NOS3 KO, CAV1 KO, and wild-type (WT) mice by rebound tonometry. Outflow facility was measured by perfusing enucleated mouse eyes at multiple pressure steps. Sodium nitroprusside (SNP) and L-NG-nitroarginine methyl ester (L-NAME) was administered topically, whereas the contralateral eyes served as vehicle controls. IOP was measured in both eyes before drug treatment and 1 hour after the last drug treatment. Mock aqueous humor ± the nitric oxide (NO) donor SNP or NOS inhibitor L-NAME was perfused into enucleated eyes.

Results: IOP was 11 ± 0.23 mm Hg in DKO mice, which was similar to WT mice and significantly lower than CAV1 KO mice (n = 18, P > 0.05). NOS3 deletion in CAV1−/− mice resulted in a 1.9-fold increase in conventional outflow facility (Ccon) compared with CAV1 KO mice (n = 7, P < 0.05). Topical application of NO donor SNP did not significantly change IOP (n = 18, P > 0.05) or Ccon in DKO mice (SNP, n = 20; vehicle, n = 11, P > 0.05). Topical application of L-NAME significantly increased IOP in WT, DKO, and CAV1 mice by reducing Ccon. Nitrotyrosine and PKG levels of DKO mice were similar to, whereas sGC was lower than, WT mice (P < 0.05).

Conclusions: Genetic deletion of NOS3 in CAV1-deficient mice restored IOP and conventional aqueous humor drainage to WT level. NOS3 and CAV1 interaction is important to IOP regulation.

Glaucoma is a complex and genetically heterogeneous disease.1 Elevated IOP and aging are the most important risk factors for glaucoma. NO is an important regulator of IOP.2,3 The NO-cGMP signaling pathway maintains the IOP homeostasis by regulating aqueous humor dynamics.47 In line with this observation, the risk of developing primary open angle glaucoma (POAG) was shown to be associated with NOS3 gene polymorphisms.810 The common variations in endothelial NOS (eNOS) are also involved in the pathogenesis of primary angle closure glaucoma.11 Further, genome-wide association studies identified a single nucleotide polymorphism, rs4236601, located between caveolin 1 (CAV1) and caveolin 2 (CAV2) on chromosome 7q31,12 which was significantly associated with elevated IOP and POAG.1316 
eNOS and CAV1 are important molecules in regulating IOP.2,1722 eNOS is an enzyme that converts L-arginine to L-citrulline and produces nitric oxide (NO). NO is a critical endogenous signaling mediator initially studied in the cardiovascular system where it played a key role in regulating smooth muscle relaxation and vasodilation. Later it is evident that the NO pathway is also important for multiple functions in the eye.2326 Recently, several studies supported the important role of NO in the control of IOP.2,22,27,28 Our previous work showed that eNOS overexpression resulted in lower IOP and increased conventional outflow,2 and eNOS knockout (KO) elevated IOP possibly by reducing conventional outflow. 
Preclinical data showed that NO-donating drugs had superior IOP lowering effect than the equal molar concentration conventional compounds (without NO releasing).4,7,29 Excitingly, latanoprostene bunod (VESNEO; Nicox, France), a topically administered NO donating prostaglandin F2-α analogue, is currently under review by the US Food and Drug Administration (FDA) as an IOP-lowering single-agent eye drop.3036 
CAV1 is a scaffolding protein that orchestrates many signaling molecules, including eNOS, tyrosine kinase receptor, G protein–coupled receptor, GTPase, and components of the mitogen-activated protein kinases, and regulates their function.3739 The binding of eNOS to CAV1 leads to its inactivation. When the cells are subjected to stress or other stimulation, intracellular calcium level increases, which leads to disruption of the CAV1–eNOS interaction.37,38,40 CAV1 is expressed in trabecular meshwork (TM) cells41 and Schlemm's canal (SC) endothelial cells.20,21 It plays an important role in regulating aqueous outflow resistance.20,21,42 In a CAV1 KO mouse model, it was shown that CAV1 deficiency resulted in IOP elevation and aqueous humor outflow reduction (Elliott MH, et al. IOVS 2014;55:ARVO E-Abstract 2888). We further showed that, despite increased IOP, eNOS activity was elevated in CAV1 KO mice.18 However, the role of CAV1 together with eNOS in regulating IOP is not completely understood. 
In view of the important role of CAV1 and NOS3 in regulating aqueous humor outflow,2,1722 this study will investigate the impact of NOS3 deletion in CAV1−/− mice using a mouse genetic double KO (DKO). NOS3 KO mice and CAV1 KO mice both have elevated IOP,18,22 even though CAV1 KO mice also have increased eNOS activity. The increased eNOS activation in CAV1 KO mice was associated with excessive protein nitration, which might have damaged the outflow tissue including TM and SC endothelial cells.18 The aim of this study is to investigate the impact of genetic deletion of NOS3 in CAV1−/− mice on aqueous humor outflow function; we hypothesize that increased IOP in CAV1 mice is due to eNOS activation, and the deletion of NOS3 in mice will restore aqueous humor outflow and normalize IOP. 
Methods
Animals
All experiments were in compliance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. All the mice were professionally bred by Model Animal Research Center (Nanjing University, Nanjing, China). DKO mice were obtained by crossing the NOS3−/− mice (B6.129P2-Nos3<tm1Unc>/J, stock no. 002684; Jackson Laboratory, MN, USA) and CAV1−/− mice (B6.Cg-Cav1<tm1Mls>/J, stock no. 007083; Jackson Laboratory). Heterozygous CAV1+/−eNOS+/− mice were first produced, and then the mice were crossed again to select for CAV1 and eNOS DKO (CAV1−/−eNOS−/−, wild-type [WT] littermates from the cross were used as controls). Both strains are C57BL/6J in genetic background. For B6.129P2-Nos3<tm1Unc>/J (https://www.jax.org/strain/002684), a targeting vector containing neomycin resistance and herpes simplex virus thymidine kinase genes was used to replace 129 bp of exon 12, which disrupted the calmodulin binding domain. The construct was electroporated into 129P2/OlaHsd-derived E14TG2a embryonic stem (ES) cells. Correctly targeted ES cells were injected into C57BL/6J blastocysts, and the resulting chimeric males were crossed to C57BL/6 female mice. Heterozygotes were intercrossed to generate homozygotes. The mice were subsequently backcrossed onto the C57BL/6J background for 12 generations. For B6.Cg-Cav1<tm1Mls>/J (https://www.jax.org/strain/007083), a targeting vector containing a neomycin resistance gene was used to disrupt 2.2 kb of sequence-containing exons 1 and 2. The construct was electroporated into WW6 ES cells (75% 129/Sv, 20% C57BL/6J, 5% SJL). Correctly targeted ES cells were injected into C57BL/6 blastocysts. The resulting chimeric animals were crossed to C57BL/6J mice for approximately five generations. All genotypes (including WT mice) were generated from the same cross. WT C57BL/6J littermates were used as WT controls. Mice were bred and housed in clear cages covered loosely with air filters and containing white pine shavings for bedding. For all four stains of animals, mice aged 8 to 10 weeks were used in this study. 
Mouse Genotyping
Genotyping was performed on ear tissue samples obtained at weaning. Ear tissue was lysed with 0.3 mg/mL proteinase K (Sigma-Aldrich, St. Louis, MO, USA). Normalized to weight, precleared ear lysate solution was added directly to the PCR. A hot-start mix (KAPA2G Robust HotStartReadyMix; KapaBiosystems, Shanghai, China) was used in PCRs, run for 35 cycles at annealing temperatures of 65°C for primers directed against the sense and antisense strands according to Jackson Laboratory genotyping protocols for the respective genes. The following primers were used: CAV1 WT, CTA GTG AGA CGT GCT ACT TCC (sense), CTT GAG TTC TGT TAG CCC AG (antisense); CAV1 mutant, CTT GAG TTC TGT TAG CCC AG (sense), GTG TAT GAC GCG CAC ACC AAG (antisense); eNOS WT, AGG GGA ACA AGC CCA GTA GT (sense), CTT GTC CCC TAG GCA CCT CT (antisense); eNOS mutant, CTT GTC CCC TAG GCA CCT CT (sense), AAT TCG CCA ATG ACA AGA CG (antisense). PCR products were resolved by gel electrophoresis (2% agarose) in the presence of DNA gel stain (SYBR Safe; Invitrogen, Shanghai, China). 
Western Blot
Outflow tissue containing iris, TM, SC, and possible some iris root was dissected following an established method.22 The outflow tissue was prepared using RIPA solution, 50 μg protein was loaded into each lane of gel, and proteins were separated by SDS-PAGE (10% or 12.5% acrylamide). The resolved proteins were transferred by electrophoresis to nitrocellulose membranes. The membranes were blocked with 5% nonfat dry milk in Tris-buffered saline with 0.05% Tween-20 for 2 hours. Membranes were then probed with antibodies that specifically recognize eNOS (1:1000; Abcam, Shanghai, China), CAV1 (1:1000; Abcam), protein kinase G-1 (PKG-1, 1:1000; Abcam), soluble guanylyl cyclase (sGC, 1:1000; Abcam), and nitrotyrosine (1:1000; Abcam), followed by incubation with peroxidase-linked secondary antibodies. GAPDH was used as a loading control. Signals in the linear range of the X-ray film were captured digitally, and densitometry performed using Kodak Molecular Imaging Software (Kodak, Shinkawa, Japan). 
IOP Measurements in Mice
We measured IOP in both mouse eyes using rebound tonometry in live animals (TonoLab; ICare, Espoo, Finland). IOP was measured without anesthesia at the same time of day (10 to 11 AM). The pressures of both eyes were measured three times, and the average was counted as a single measurement. 
Topical Drug Application
Mouse eyes were treated with sodium nitroprusside (SNP; 4 × 2-μL drops; total dose, 160 μg) and L-NG-nitroarginine methyl ester (L-NAME; 4 × 2-μL drops; total dose, 0.431 μg) by topical application at 0, 0.5, 1, and 1.5 hours. At each time point, two 1-μL drops were given 1 minute apart. For all eye drop administrations, the drug was administered to the central cornea. The contralateral eyes were treated with drug vehicle diluted in PBS. IOP was measured in both eyes before drug treatment and 1 hour after the last drug treatment. 
Mouse Eye Perfusion
Outflow facility was measured by perfusing enucleated mouse eyes according to an established method.43 After IOP measurements, mice were killed by cervical dislocation, and the eyes were enucleated for perfusion. The experimental setup was developed by our laboratory and is described in detail elsewhere.44 The eyes were cannulated and the eye pressure was stabilized using a perfusion reservoir containing PBS for 30 minutes, and the total infusion volume was recorded after a 10-minute interval. At each pressure, the measurement was repeated twice. Throughout the experiment, the eyes were moisturized with drops of saline placed on top of the cornea. Then the eyes were perfused at four different levels of IOP (5, 12, 19, and 27 mm Hg). We assume that at equilibrium, the total inflow rate equals the total outflow rate. Conventional out flow facility (Ccon) was calculated according Goldman's equation:  
\(\def\upalpha{\unicode[Times]{x3B1}}\)\(\def\upbeta{\unicode[Times]{x3B2}}\)\(\def\upgamma{\unicode[Times]{x3B3}}\)\(\def\updelta{\unicode[Times]{x3B4}}\)\(\def\upvarepsilon{\unicode[Times]{x3B5}}\)\(\def\upzeta{\unicode[Times]{x3B6}}\)\(\def\upeta{\unicode[Times]{x3B7}}\)\(\def\uptheta{\unicode[Times]{x3B8}}\)\(\def\upiota{\unicode[Times]{x3B9}}\)\(\def\upkappa{\unicode[Times]{x3BA}}\)\(\def\uplambda{\unicode[Times]{x3BB}}\)\(\def\upmu{\unicode[Times]{x3BC}}\)\(\def\upnu{\unicode[Times]{x3BD}}\)\(\def\upxi{\unicode[Times]{x3BE}}\)\(\def\upomicron{\unicode[Times]{x3BF}}\)\(\def\uppi{\unicode[Times]{x3C0}}\)\(\def\uprho{\unicode[Times]{x3C1}}\)\(\def\upsigma{\unicode[Times]{x3C3}}\)\(\def\uptau{\unicode[Times]{x3C4}}\)\(\def\upupsilon{\unicode[Times]{x3C5}}\)\(\def\upphi{\unicode[Times]{x3C6}}\)\(\def\upchi{\unicode[Times]{x3C7}}\)\(\def\uppsy{\unicode[Times]{x3C8}}\)\(\def\upomega{\unicode[Times]{x3C9}}\)\(\def\bialpha{\boldsymbol{\alpha}}\)\(\def\bibeta{\boldsymbol{\beta}}\)\(\def\bigamma{\boldsymbol{\gamma}}\)\(\def\bidelta{\boldsymbol{\delta}}\)\(\def\bivarepsilon{\boldsymbol{\varepsilon}}\)\(\def\bizeta{\boldsymbol{\zeta}}\)\(\def\bieta{\boldsymbol{\eta}}\)\(\def\bitheta{\boldsymbol{\theta}}\)\(\def\biiota{\boldsymbol{\iota}}\)\(\def\bikappa{\boldsymbol{\kappa}}\)\(\def\bilambda{\boldsymbol{\lambda}}\)\(\def\bimu{\boldsymbol{\mu}}\)\(\def\binu{\boldsymbol{\nu}}\)\(\def\bixi{\boldsymbol{\xi}}\)\(\def\biomicron{\boldsymbol{\micron}}\)\(\def\bipi{\boldsymbol{\pi}}\)\(\def\birho{\boldsymbol{\rho}}\)\(\def\bisigma{\boldsymbol{\sigma}}\)\(\def\bitau{\boldsymbol{\tau}}\)\(\def\biupsilon{\boldsymbol{\upsilon}}\)\(\def\biphi{\boldsymbol{\phi}}\)\(\def\bichi{\boldsymbol{\chi}}\)\(\def\bipsy{\boldsymbol{\psy}}\)\(\def\biomega{\boldsymbol{\omega}}\)\(\def\bupalpha{\bf{\alpha}}\)\(\def\bupbeta{\bf{\beta}}\)\(\def\bupgamma{\bf{\gamma}}\)\(\def\bupdelta{\bf{\delta}}\)\(\def\bupvarepsilon{\bf{\varepsilon}}\)\(\def\bupzeta{\bf{\zeta}}\)\(\def\bupeta{\bf{\eta}}\)\(\def\buptheta{\bf{\theta}}\)\(\def\bupiota{\bf{\iota}}\)\(\def\bupkappa{\bf{\kappa}}\)\(\def\buplambda{\bf{\lambda}}\)\(\def\bupmu{\bf{\mu}}\)\(\def\bupnu{\bf{\nu}}\)\(\def\bupxi{\bf{\xi}}\)\(\def\bupomicron{\bf{\micron}}\)\(\def\buppi{\bf{\pi}}\)\(\def\buprho{\bf{\rho}}\)\(\def\bupsigma{\bf{\sigma}}\)\(\def\buptau{\bf{\tau}}\)\(\def\bupupsilon{\bf{\upsilon}}\)\(\def\bupphi{\bf{\phi}}\)\(\def\bupchi{\bf{\chi}}\)\(\def\buppsy{\bf{\psy}}\)\(\def\bupomega{\bf{\omega}}\)\(\def\bGamma{\bf{\Gamma}}\)\(\def\bDelta{\bf{\Delta}}\)\(\def\bTheta{\bf{\Theta}}\)\(\def\bLambda{\bf{\Lambda}}\)\(\def\bXi{\bf{\Xi}}\)\(\def\bPi{\bf{\Pi}}\)\(\def\bSigma{\bf{\Sigma}}\)\(\def\bPhi{\bf{\Phi}}\)\(\def\bPsi{\bf{\Psi}}\)\(\def\bOmega{\bf{\Omega}}\)\begin{equation}F = \left( {IOP - EVP} \right){C_{con}} + {F_u}\end{equation}
where Fu is the pressure-independent (unconventional) outflow rate, EVP is episcleral venous pressure, and Ccon is the conventional (pressure dependent) outflow facility. As the mouse eyes were enucleated at the time of the experiment, EVP equals zero. A regression was fit in a flow rate–pressure response graph, and the slope of the regression line was Ccon.  
To test the effect of the NO donor SNP (10−3 M) and NOS inhibitor L-NAME (100 μM), the mouse eyes were perfused with these drug solutions for 60 minutes before the measurements were taken to exchange the aqueous humor and ensure that the drug concentration was uniform throughout the experiment. Then the eyes were perfused at four different levels of IOP (5, 12, 19, and 27 mm Hg). The contralateral control eyes were treated in the same way but perfused with drug vehicle. The perfusion with SNP was performed in the dark as it was known to degrade under light exposure. The drug concentrations used were similar to that in previous studies using monkeys and mice.2,22,28 
Statistics
IOP data were analyzed by a nonparametric test for related samples. Conventional outflow data were analyzed by a nonparametric test for independent samples (SPSS 16 for Windows; IBM-SPSS, Chicago, IL, USA). In all cases, differences were considered significant at P < 0.05. 
Results
The goal of this project was to determine the impact of NOS3 KO on CAV1-deficient mice in their IOP and conventional outflow function. To accomplish our goal, we used a gene DKO mouse model where both NOS3 and CAV1 genes were deleted by interbreeding the two strains of single gene KO mice. Genotyping confirmed the absence of NOS3 and CAV1 in this model (Fig. 1A). This result was confirmed by Western blot (Fig. 1B). 
Figure 1
 
Genotyping and protein expression of a typical litter of pups in NOS3 and CAV1 DKO mice by PCR analyzed using electrophoresis of a stained agarose gel (A) and Western blot analysis (B). (A) In this case, three animals were found to have the deletion of the WT CAV1 gene (315, 317, and 320; size, 690 bp) and the expression of mutant gene (size, 410 bp). The other four littermates express CAV1 WT gene (314, 316, 318, and 319). NOS3 WT bands of 337 bp were absent in all the animals (314 to 320). Mutant positive control, WT negative controls and WT control for the PCR are indicated by P, N, and B6 above the three lanes. Mut, mutant. (B) Western blot confirmed the absence of eNOS and CAV1. y-axis is the band density normalized to loading control as a ratio.
Figure 1
 
Genotyping and protein expression of a typical litter of pups in NOS3 and CAV1 DKO mice by PCR analyzed using electrophoresis of a stained agarose gel (A) and Western blot analysis (B). (A) In this case, three animals were found to have the deletion of the WT CAV1 gene (315, 317, and 320; size, 690 bp) and the expression of mutant gene (size, 410 bp). The other four littermates express CAV1 WT gene (314, 316, 318, and 319). NOS3 WT bands of 337 bp were absent in all the animals (314 to 320). Mutant positive control, WT negative controls and WT control for the PCR are indicated by P, N, and B6 above the three lanes. Mut, mutant. (B) Western blot confirmed the absence of eNOS and CAV1. y-axis is the band density normalized to loading control as a ratio.
Iridocorneal Morphology
To determine whether DKO affected the anatomic structure of the iridocorneal angle tissues responsible for IOP generation, mouse eyes were sectioned and H&E stained. Similar to control mice, DKO mice displayed an open iridocorneal angle plus normal gross morphology of the trabecular meshwork and SC inner wall endothelium (Fig. 2). 
Figure 2
 
Hematoxylin and eosin–stained sections of mouse eye anterior chamber (AD) and outflow tissue (EH).
Figure 2
 
Hematoxylin and eosin–stained sections of mouse eye anterior chamber (AD) and outflow tissue (EH).
Genetic Deletion of NOS3 in CAV1−/− Mice Prevents Ocular Hypertension
IOP was 11 ± 0.23 mm Hg (mean ± SEM, n = 18 eyes) in DKO mice, which was similar to WT mice of 10 ± 0.28 mm Hg (n = 20 eyes, P > 0.05; Fig. 3). Genetic deletion of NOS3 in CAV1−/− mice restored the IOP to WT mice level. IOP was 15 ± 0.51 mm Hg (n = 16 eyes) and 15 ± 0.29 mm Hg (n = 16 eyes) in CAV1 KO and NOS3 KO mice, respectively. IOP in CAV1 KO and NOS3 KO mice was significantly higher than DKO mice and WT mice (P < 0.05). 
Figure 3
 
Intraocular pressure (IOP) of wildtype (WT), CAV1 and NOS3 double knockout (DKO), NOS3 knockout (NOS3 KO), CAV1 knockout (CAV1 KO) mice. (A) Individual IOP readings in the four strains of mice. (B) Mean IOP in the four strains of mice (error bars denote SEM). DKO, n = 18; WT, n = 20; CAV1, n = 16; NOS3 KO, n = 16. *P < 0.05.
Figure 3
 
Intraocular pressure (IOP) of wildtype (WT), CAV1 and NOS3 double knockout (DKO), NOS3 knockout (NOS3 KO), CAV1 knockout (CAV1 KO) mice. (A) Individual IOP readings in the four strains of mice. (B) Mean IOP in the four strains of mice (error bars denote SEM). DKO, n = 18; WT, n = 20; CAV1, n = 16; NOS3 KO, n = 16. *P < 0.05.
Restoration of Aqueous Humor Outflow Function in DKO Mice
We then examined if/how genetic deletion of NOS3 in CAV1−/− mice impacted aqueous humor outflow. The flow rate data were plotted as a function of IOP and compared with the data in DKO, WT, NOS3 KO, and CAV1 KO mice (Fig. 4). In DKO mice, the outflow rate at 5, 12, 15, 19, and 27 mm Hg was 0.09 ± 0.01, 0.20 ± 0.01, 0.27 ± 0.04, 0.33 ± 0.03, and 0.44 ± 0.03 μL/min, respectively. In comparison, in CAV1 KO mice, the outflow rate was 0.05 ± 0.01, 0.13 ± 0.01, 0.16 ± 0.01, 0.19 ± 0.01, and 0.25 ± 0.01 μL/min, respectively. The conventional outflow facility (Ccon), which is the slope of the flow rate versus pressure graph, was 0.0161 ± 0.0022 mm Hg (n = 19), 0.0125 ± 0.0014 mm Hg (n = 7), 0.0088 ± 0.0010 mm Hg (n = 7), and 0.0084 ± 0.0008 μL/min/mm Hg (n = 7) for the DKO, WT, NOS3 KO, and CAV1 KO mice, respectively. Ccon of DKO mice was similar to WT controls (P > 0.05). NOS3 deletion in CAV1−/− mice resulted in a 1.9-fold increase in Ccon in DKO mice compared with CAV1 KO mice. 
Figure 4
 
Relation between the mean flow rate and IOP in all perfused DKO (n = 19), WT (n = 7), NOS3 KO (n = 7), and CAV1 KO (n = 7) mouse eyes. Mouse eyes were perfused at sequential pressures of 5, 12, 15, 19, and 27 mm Hg. (A) Outflow rate and pressure relationship of the four strains of mice. The slope of the regression line (solid line) is the conventional outflow facility (Ccon). (B) Comparison of Ccon of the four strains of mice that Ccon of DKO mice is similar to WT mice, but significantly lower than NOS3 KO and CAV1 KO mouse eyes. In the box plots, the bottom and top of the box are the first and third quartiles, and the band inside the box is the second quartile (the median). The ends of the whiskers are the minimum and maximum values. The circle represents a data outlier, and the number next to the outlier is the observation number. An outlier is defined as a score that is between 1.5 and 3 box lengths away from the upper or lower edge of the box. *P < 0.05, error bars denote SEM.
Figure 4
 
Relation between the mean flow rate and IOP in all perfused DKO (n = 19), WT (n = 7), NOS3 KO (n = 7), and CAV1 KO (n = 7) mouse eyes. Mouse eyes were perfused at sequential pressures of 5, 12, 15, 19, and 27 mm Hg. (A) Outflow rate and pressure relationship of the four strains of mice. The slope of the regression line (solid line) is the conventional outflow facility (Ccon). (B) Comparison of Ccon of the four strains of mice that Ccon of DKO mice is similar to WT mice, but significantly lower than NOS3 KO and CAV1 KO mouse eyes. In the box plots, the bottom and top of the box are the first and third quartiles, and the band inside the box is the second quartile (the median). The ends of the whiskers are the minimum and maximum values. The circle represents a data outlier, and the number next to the outlier is the observation number. An outlier is defined as a score that is between 1.5 and 3 box lengths away from the upper or lower edge of the box. *P < 0.05, error bars denote SEM.
Response to NO Donor
To assess whether NOS3 and CAV1 double-deficient mice were able to respond to the NO donor, we gave SNP to DKO mice both topically (Fig. 5) and by anterior chamber perfusion (Fig. 6). Topical application of SNP did not significantly lower IOP compared with contralateral vehicle-treated eyes (9.8 ± 0.2 vs. 8.7 ± 0.2 mm Hg, n = 15, P > 0.05; Fig. 5A). However, SNP significantly reduced IOP in NOS3 KO, CAV1 KO, and WT mice, and the magnitude of reduction was 16%, 12%, and 18%, respectively (P < 0.05; Figs. 5B–5D). 
Figure 5
 
IOP of four strains of mice treated with topical application of NO donor SNP. SNP did not significantly affect IOP in DKO mice (A). SNP significantly reduced IOP in WT mice (B), NOS3 KO mice (C), and CAV1 KO mice (D). Error bars denote SEM. DKO, n = 15; WT, n = 6; CAV1 KO, n = 6; NOS3 KO, n = 8. *P < 0.05.
Figure 5
 
IOP of four strains of mice treated with topical application of NO donor SNP. SNP did not significantly affect IOP in DKO mice (A). SNP significantly reduced IOP in WT mice (B), NOS3 KO mice (C), and CAV1 KO mice (D). Error bars denote SEM. DKO, n = 15; WT, n = 6; CAV1 KO, n = 6; NOS3 KO, n = 8. *P < 0.05.
Figure 6
 
Mouse eyes perfused with NO donor SNP. (A) Comparison of outflow data in SNP-treated eyes and control eyes perfused with the drug vehicle (PBS) in DKO (A, B), WT (C, D), NOS3 KO (E, F), and CAV1 KO (G, H) mice. Conventional outflow (Ccon) facility was significantly higher in SNP treated WT, NOS3 KO, and CAV1 KO mice compared with vehicle controls (*P < 0.05). Error bars denote SEM. In the box plots, the bottom and top of the box are the first and third quartiles, and the band inside the box is the second quartile (the median). The ends of the whiskers are the minimum and maximum values. The circle represents a data outlier, and the number next to the outlier is the observation number. DKO: SNP, n = 20; vehicle, n = 11. WT: SNP, n = 6, vehicle, n = 6; CAV1 KO: SNP, n = 7, vehicle, n = 8; NOS3 KO: SNP, n = 6, vehicle, n = 6.
Figure 6
 
Mouse eyes perfused with NO donor SNP. (A) Comparison of outflow data in SNP-treated eyes and control eyes perfused with the drug vehicle (PBS) in DKO (A, B), WT (C, D), NOS3 KO (E, F), and CAV1 KO (G, H) mice. Conventional outflow (Ccon) facility was significantly higher in SNP treated WT, NOS3 KO, and CAV1 KO mice compared with vehicle controls (*P < 0.05). Error bars denote SEM. In the box plots, the bottom and top of the box are the first and third quartiles, and the band inside the box is the second quartile (the median). The ends of the whiskers are the minimum and maximum values. The circle represents a data outlier, and the number next to the outlier is the observation number. DKO: SNP, n = 20; vehicle, n = 11. WT: SNP, n = 6, vehicle, n = 6; CAV1 KO: SNP, n = 7, vehicle, n = 8; NOS3 KO: SNP, n = 6, vehicle, n = 6.
Following IOP measurements, we further investigated the effects of NO donors on outflow rate and facility (Fig. 6). In DKO mice, the outflow rates of SNP treated eyes at 5, 12, 19, and 27 mm Hg were 0.14 ± 0.01, 0.28 ± 0.03, 0.44 ± 0.04, and 0.64 ± 0.05 μL/min, respectively, compared with 0.08 ± 0.01, 0.20 ± 0.01, 0.29 ± 0.03, and 0.39 ± 0.03 μL/min in the vehicle-treated eyes (Fig. 6A). SNP-treated eyes showed a linear relationship in the flow-pressure graphs. Ccon in SNP-treated and vehicle-treated eyes was 0.0207 ± 0.0018 mm Hg (n = 20) and 0.0151 ± 0.0011 μL/min/mm Hg (n = 11), respectively, which was not significantly different (Fig. 6A; P > 0.05). In WT, NOS3 KO, and CAV1 KO mice, SNP significantly increased Ccon from 0.0096 ± 0.0010 to 0.0204 ± 0.0030 mm Hg, from 0.0128 ± 0.0005 to 0.0185 ± 0.0018 mm Hg, and from 0.0096 ± 0.0016 to 0.0171 ± 0.0014 μL/min/mm Hg, respectively (P < 0.05; Figs. 6B–6D). 
Response to NOS Inhibitor
In DKO mice, the NOS inhibitor L-NAME significantly increased IOP from 10.6 ± 0.4 mm Hg to 12.4 ± 0.4 mm Hg by 1.2-fold (mean ± SEM, n = 6, P < 0.05; Fig. 7A). L-NAME significantly increased IOP by 1.2-fold in CAV1 KO mice (n = 6, P < 0.05; Fig. 7D). The magnitude of IOP increase was similar to WT mice, which was also 1.2-fold (n = 6, P < 0.05; Fig. 7B). However, NOS3 KO mice were unresponsive to L-NAME (n = 6, P > 0.05; Fig. 7C). 
Figure 7
 
IOP of four strains of mice treated with topical application of NOS inhibitor L-NAME. L-NAME significantly increased IOP in DKO (A), WT mice (B), and CAV1 KO mice (D). Error bars denote SEM. DKO, n = 6; WT, n = 6, NOS3 KO, n = 6; CAV1 KO, n = 6. *P < 0.05.
Figure 7
 
IOP of four strains of mice treated with topical application of NOS inhibitor L-NAME. L-NAME significantly increased IOP in DKO (A), WT mice (B), and CAV1 KO mice (D). Error bars denote SEM. DKO, n = 6; WT, n = 6, NOS3 KO, n = 6; CAV1 KO, n = 6. *P < 0.05.
We confirmed the effect of L-NAME in regulating conventional outflow (Fig. 8). L-NAME reduced Ccon by 47%, 34%, and 48% in DKO (n = 6; Fig. 8A), CAV1 KO (n = 6; Fig. 8D), and WT (n = 6; Fig. 8A) mice, respectively. However, in NOS3 KO mice, Ccon was similar in L-NAME–treated and drug vehicle-treated eyes (n = 6, P > 0.05; Fig. 8C). 
Figure 8
 
Mouse eyes perfused with NOS inhibitor L-NAME. Flow rate and conventional outflow facility (Ccon) in L-NAME–treated eyes and drug vehicle-treated eyes in DKO (A), WT (B), NOS3 KO (C), and CAV1 KO mice (D). It significantly reduced Ccon in DKO, WT, and CAV1 KO mice (n = 6, P < 0.05). However, in NOS3 KO mice, Ccon was similar in L-NAME– and drug vehicle-treated eyes (n = 6, P > 0.05). Error bars denote SEM. Box plot showed that the conventional outflow was significantly higher in drug-treated eyes compared with paired controls in all strains of mice. In the box plots, the bottom and top of the box is the first and third quartiles, and the band inside the box is the second quartile (the median). The ends of the whiskers are the minimum and maximum values. DKO, n = 6; WT, n = 6, NOS3 KO, n = 6; CAV1 KO, n = 6. *P < 0.05.
Figure 8
 
Mouse eyes perfused with NOS inhibitor L-NAME. Flow rate and conventional outflow facility (Ccon) in L-NAME–treated eyes and drug vehicle-treated eyes in DKO (A), WT (B), NOS3 KO (C), and CAV1 KO mice (D). It significantly reduced Ccon in DKO, WT, and CAV1 KO mice (n = 6, P < 0.05). However, in NOS3 KO mice, Ccon was similar in L-NAME– and drug vehicle-treated eyes (n = 6, P > 0.05). Error bars denote SEM. Box plot showed that the conventional outflow was significantly higher in drug-treated eyes compared with paired controls in all strains of mice. In the box plots, the bottom and top of the box is the first and third quartiles, and the band inside the box is the second quartile (the median). The ends of the whiskers are the minimum and maximum values. DKO, n = 6; WT, n = 6, NOS3 KO, n = 6; CAV1 KO, n = 6. *P < 0.05.
Nitrotyrosine Expression
Western blot further revealed different nitrotyrosine expression level in WT, DKO, CAV1, and eNOS mice (Fig. 9). Nitrotyrosine expression in DKO mice (n = 6 eyes) was similar to WT mice (n = 6 eyes; P > 0.05) but was significantly lower than CAV1 mice (n = 6 eyes; P < 0.05). The data show that NOS3 deletion in CAV1-deficient mice restored protein nitration to the WT level. There is very little nitrotyrosine in eNOS KO mice. 
Figure 9
 
Tyrosine nitration of proteins in WT, NOS3, and CAV1 DKO mice, and eNOS KO mice. Representative blots (A) and densitometry analyses (B) of the mouse outflow tissue in WT, NOS3, and CAV1 DKO mice are shown. DKO mice express similar level of nitrotyrosine to WT mice, but lower level nitrotyrosine than CAV1 KO mice. eNOS KO mice express a low level of nitrotyrosine. Error bar denotes SEM. n = 6 eyes. *P < 0.05.
Figure 9
 
Tyrosine nitration of proteins in WT, NOS3, and CAV1 DKO mice, and eNOS KO mice. Representative blots (A) and densitometry analyses (B) of the mouse outflow tissue in WT, NOS3, and CAV1 DKO mice are shown. DKO mice express similar level of nitrotyrosine to WT mice, but lower level nitrotyrosine than CAV1 KO mice. eNOS KO mice express a low level of nitrotyrosine. Error bar denotes SEM. n = 6 eyes. *P < 0.05.
Signaling Molecule Expression
sGC level of DKO mice was significantly reduced by 87% compared with WT mice (P < 0.05, n = 6 eyes; Fig. 10A). PKG level in DKO mice was not significantly different from WT mice (P > 0.05, n = 6 eyes; Fig. 10B). 
Figure 10
 
sGC and PKG expression in WT, NOS3, and CAV1 DKO mice. Representative blots and densitometry analyses of the mouse outflow tissue in WT, NOS3, and CAV1 DKO mice are shown. DKO mice express lower level of sGC than WT mice and similar level of PKG to WT mice. Error bar denotes SEM. n = 4. *P < 0.05.
Figure 10
 
sGC and PKG expression in WT, NOS3, and CAV1 DKO mice. Representative blots and densitometry analyses of the mouse outflow tissue in WT, NOS3, and CAV1 DKO mice are shown. DKO mice express lower level of sGC than WT mice and similar level of PKG to WT mice. Error bar denotes SEM. n = 4. *P < 0.05.
Discussion
Our goal was to investigate the effect of genetic deletion of NOS3 in CAV1−/− mice on aqueous humor outflow function. We established a double gene KO mouse model by interbreeding NOS3 KO and CAV1 KO mice. PCR and Western blot analysis confirmed the absence of both genes and proteins (Fig. 1). CAV1 KO mice have increased IOP, and we showed that genetic deletion of NOS3 in CAV1−/− mice restored the IOP and conventional outflow function to WT level. DKO mice were unresponsive to NO donor SNP, but the NOS inhibitor L-NAME increased its IOP by decreasing conventional outflow facility. NO signaling molecules sGC appeared compromised in DKO mice. 
It is interesting that genetic deletion of NOS3 in CAV1 KO mice restored IOP and conventional outflow facility to WT level and prevented ocular hypertension seen in CAV1 KO and NOS3 KO mice (Figs. 3, 4). Normalization of IOP in DKO mice was consistent with previous finding in the cardiovascular system that, although CAV1 KO mice had significantly increased right ventricular systolic pressure (RVSP) compared with WT mice, RVSP in DKO mice was the same as in WT.45 A possible explanation for IOP and outflow facility normalization was that, in CAV1 KO mice, CAV1 deficiency led to eNOS activation, and then the uncoupled eNOS could be rapidly converted into a source of damaging superoxide radicals such as peroxynitrite,4648 which may impair the activity of NO downstream signaling molecule PKG and lead to pressure elevation. However, this could not happen in DKO mice, as eNOS was knocked out and its activity depressed. Western blot showed that nitrotyrosine expression in the outflow tissue of DKO mice was similar to WT mice but was significantly lower than CAV1 KO mice. This observation was consistent with results from microvascular endothelial cells.48 For cells depleted of CAV1, it produced markedly greater amount of peroxynitrite than WT controls. However, deletion of NOS3 in CAV1-deficient mice attenuates the detrimental effects of CAV1 deficiency.48 In the lung, there was increased PKG nitration in the lung tissue of CAV1 KO mice; however, in the aqueous humor outflow tissue, PKG nitration in CAV1 KO mice was similar to WT mice. 
IOP data correlated well with conventional outflow facility data in that where a significant difference of IOP was observed, there was also a significant difference in the conventional outflow facility (aqueous humor outflow through the TM/SC pathway). However, from these data, we could not conclude whether the IOP changes are mediated by the TM/SC or the ciliary body, and unconventional outflow facility was not measured. 
eNOS is an important regulator of IOP and blood pressure. The deletion of eNOS resulted in increased IOP and systolic blood pressure22,49 but does not result in pulmonary hypertension.45 NOS3 deletion corrected nitrative stress induced by NOS3 hyperactivity in CAV1 KO mice, but the loss of NOS3 does not induce ocular hypertension. This suggests that the mechanism by which NOS3 deletion elevates IOP (presumably reduced NO) is not acting in the CAV1 KO background. In CAV1 KO mice, increased eNOS activity led to excessive protein nitration, which may be one of the reasons of elevated IOP.3 In comparison, although eNOS was knocked out in DKO mice, the expression of nitrotyrosine and IOP was at a similar level to WT mice (Fig. 9). In eNOS KO mice, although eNOS was absent, iNOS and nNOS are still present, and NO downstream signaling was intact.4 
NO donor SNP did not significantly change IOP or conventional outflow facility in DKO mice. However, SNP significantly reduced IOP in CAV1 KO, NOS3 KO, and WT mice (Figs. 5, 6). SNP did not lower IOP in DKO mice. Similarly, NO donor S-nitroso-N-acetylpenicillamine had no effect on outflow facility in NOS3-overexpressed mice.50 NO released by SNP may stimulate sGC and increase the intracellular levels of cGMP, which is an important pathway for IOP regulation.5,6 The vasodilation action of NO is mediated through stimulation of sGC, which appeared compromised in DKO mice (Fig. 10A). 
The NOS inhibitor L-NAME increased IOP in DKO, CAV1 KO, and WT mice, which was consistent with L-NAME reduced conventional outflow facility (Figs. 7, 8). A recent study confirmed that L-NAME reduced outflow facility in CAV1 KO mice, Elliot et al. further showed that CAV1 KO mice were more sensitive to NOS inhibition by L-NAME.51 However, NOS3 KO mice were unresponsive to this drug, which may be due to the absence of NOS3 in this strain of mouse. Although the DKO mice did not express the NOS3 gene, it still responded to L-NAME. This might be due to inhibition of other isoform of NOS by L-NAME, a nonselective inhibitor of NOS, and showed a biphasic effect on outflow facility.50 NOS is an enzyme that converts l-arginine to l-citruline and produces NO. In vertebrates, three isoforms of NOS have been identified: endothelial NOS (eNOS, also referred as NOS3), neuronal NOS (nNOS, also referred as NOS1), and inducible NOS (iNOS, also referred as NOS2). The activity of eNOS and nNOS is controlled by the calcium level via calmodulin–calcium interaction, but iNOS has calmodulin bound permanently so its function depends solely on the expression level. NOS1, NOS2, and NOS3 were expressed in the outflow tissue including the uveal vascular endothelium, the TM, the SC, and the ciliary body.5254 In eNOS KO mice, iNOS was expressed at a low level.22 However, in DKO mice, iNOS was markedly upregulated (unpublished data by our laboratory). L-NAME could lower IOP by inhibiting iNOS in DKO mice. 
Nitrotyrosine is a surrogate measure of peroxynitrite. Increased tyrosine nitration indicated the formation of peroxynitrite. NO can relax TM and SC endothelial cells and facilitate aqueous humor conventional outflow. On the other hand, excessive NO can react with superoxide to form the damaging reactive nitrogen species peroxynitrite (ONOO), which modifies proteins and may interfere with their function through tyrosine nitration.55 Formation of nitrotyrosine in proteins will cause protein malfunction.56 In our previous study, we showed that CAV1 KO mice had increased eNOS activity, peroxynitrite formation, and IOP elevation compared with WT mice.18 This study further showed that NOS3 deletion in CAV1 KO mice reduced protein nitration (Fig. 9), which could contribute to the restoration of aqueous humor outflow function. The implication that increased nitrative stress in a model with eNOS hyperactivity (CAV1 KO) might suggest that the use of NO donors as chronic therapeutics could be problematic. PKG nitration was higher in the lung tissue of CAV1 KO mice45; however, in the outflow tissue, PKG nitration level was similar to WT mice and DKO mice (Supplementary Materials). 
In conclusion, the current study shows that CAV1 and NOS3 interaction is important to IOP regulation. CAV1 gene knockout–related abnormalities in IOP and aqueous humor conventional outflow function was restored in CAV1 and NOS3 DKO mice. 
Acknowledgments
The authors thank Xing Chao for excellent assistant with the animal work. They also thank W. D. Stamer, PhD, for insightful comments on the manuscript. 
Supported by grants from the State Key Program of National Natural Science Foundation of China (81430007), the National Major Scientific Equipment program (2012YQ12008003), the Ministry of Science and Technology, China, and the Young Scientist Program of EENT hospital of Fudan University (PP00124). 
Disclosure: M. Song, None; J. Wu, None; Y. Lei, None; X. Sun, None 
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Figure 1
 
Genotyping and protein expression of a typical litter of pups in NOS3 and CAV1 DKO mice by PCR analyzed using electrophoresis of a stained agarose gel (A) and Western blot analysis (B). (A) In this case, three animals were found to have the deletion of the WT CAV1 gene (315, 317, and 320; size, 690 bp) and the expression of mutant gene (size, 410 bp). The other four littermates express CAV1 WT gene (314, 316, 318, and 319). NOS3 WT bands of 337 bp were absent in all the animals (314 to 320). Mutant positive control, WT negative controls and WT control for the PCR are indicated by P, N, and B6 above the three lanes. Mut, mutant. (B) Western blot confirmed the absence of eNOS and CAV1. y-axis is the band density normalized to loading control as a ratio.
Figure 1
 
Genotyping and protein expression of a typical litter of pups in NOS3 and CAV1 DKO mice by PCR analyzed using electrophoresis of a stained agarose gel (A) and Western blot analysis (B). (A) In this case, three animals were found to have the deletion of the WT CAV1 gene (315, 317, and 320; size, 690 bp) and the expression of mutant gene (size, 410 bp). The other four littermates express CAV1 WT gene (314, 316, 318, and 319). NOS3 WT bands of 337 bp were absent in all the animals (314 to 320). Mutant positive control, WT negative controls and WT control for the PCR are indicated by P, N, and B6 above the three lanes. Mut, mutant. (B) Western blot confirmed the absence of eNOS and CAV1. y-axis is the band density normalized to loading control as a ratio.
Figure 2
 
Hematoxylin and eosin–stained sections of mouse eye anterior chamber (AD) and outflow tissue (EH).
Figure 2
 
Hematoxylin and eosin–stained sections of mouse eye anterior chamber (AD) and outflow tissue (EH).
Figure 3
 
Intraocular pressure (IOP) of wildtype (WT), CAV1 and NOS3 double knockout (DKO), NOS3 knockout (NOS3 KO), CAV1 knockout (CAV1 KO) mice. (A) Individual IOP readings in the four strains of mice. (B) Mean IOP in the four strains of mice (error bars denote SEM). DKO, n = 18; WT, n = 20; CAV1, n = 16; NOS3 KO, n = 16. *P < 0.05.
Figure 3
 
Intraocular pressure (IOP) of wildtype (WT), CAV1 and NOS3 double knockout (DKO), NOS3 knockout (NOS3 KO), CAV1 knockout (CAV1 KO) mice. (A) Individual IOP readings in the four strains of mice. (B) Mean IOP in the four strains of mice (error bars denote SEM). DKO, n = 18; WT, n = 20; CAV1, n = 16; NOS3 KO, n = 16. *P < 0.05.
Figure 4
 
Relation between the mean flow rate and IOP in all perfused DKO (n = 19), WT (n = 7), NOS3 KO (n = 7), and CAV1 KO (n = 7) mouse eyes. Mouse eyes were perfused at sequential pressures of 5, 12, 15, 19, and 27 mm Hg. (A) Outflow rate and pressure relationship of the four strains of mice. The slope of the regression line (solid line) is the conventional outflow facility (Ccon). (B) Comparison of Ccon of the four strains of mice that Ccon of DKO mice is similar to WT mice, but significantly lower than NOS3 KO and CAV1 KO mouse eyes. In the box plots, the bottom and top of the box are the first and third quartiles, and the band inside the box is the second quartile (the median). The ends of the whiskers are the minimum and maximum values. The circle represents a data outlier, and the number next to the outlier is the observation number. An outlier is defined as a score that is between 1.5 and 3 box lengths away from the upper or lower edge of the box. *P < 0.05, error bars denote SEM.
Figure 4
 
Relation between the mean flow rate and IOP in all perfused DKO (n = 19), WT (n = 7), NOS3 KO (n = 7), and CAV1 KO (n = 7) mouse eyes. Mouse eyes were perfused at sequential pressures of 5, 12, 15, 19, and 27 mm Hg. (A) Outflow rate and pressure relationship of the four strains of mice. The slope of the regression line (solid line) is the conventional outflow facility (Ccon). (B) Comparison of Ccon of the four strains of mice that Ccon of DKO mice is similar to WT mice, but significantly lower than NOS3 KO and CAV1 KO mouse eyes. In the box plots, the bottom and top of the box are the first and third quartiles, and the band inside the box is the second quartile (the median). The ends of the whiskers are the minimum and maximum values. The circle represents a data outlier, and the number next to the outlier is the observation number. An outlier is defined as a score that is between 1.5 and 3 box lengths away from the upper or lower edge of the box. *P < 0.05, error bars denote SEM.
Figure 5
 
IOP of four strains of mice treated with topical application of NO donor SNP. SNP did not significantly affect IOP in DKO mice (A). SNP significantly reduced IOP in WT mice (B), NOS3 KO mice (C), and CAV1 KO mice (D). Error bars denote SEM. DKO, n = 15; WT, n = 6; CAV1 KO, n = 6; NOS3 KO, n = 8. *P < 0.05.
Figure 5
 
IOP of four strains of mice treated with topical application of NO donor SNP. SNP did not significantly affect IOP in DKO mice (A). SNP significantly reduced IOP in WT mice (B), NOS3 KO mice (C), and CAV1 KO mice (D). Error bars denote SEM. DKO, n = 15; WT, n = 6; CAV1 KO, n = 6; NOS3 KO, n = 8. *P < 0.05.
Figure 6
 
Mouse eyes perfused with NO donor SNP. (A) Comparison of outflow data in SNP-treated eyes and control eyes perfused with the drug vehicle (PBS) in DKO (A, B), WT (C, D), NOS3 KO (E, F), and CAV1 KO (G, H) mice. Conventional outflow (Ccon) facility was significantly higher in SNP treated WT, NOS3 KO, and CAV1 KO mice compared with vehicle controls (*P < 0.05). Error bars denote SEM. In the box plots, the bottom and top of the box are the first and third quartiles, and the band inside the box is the second quartile (the median). The ends of the whiskers are the minimum and maximum values. The circle represents a data outlier, and the number next to the outlier is the observation number. DKO: SNP, n = 20; vehicle, n = 11. WT: SNP, n = 6, vehicle, n = 6; CAV1 KO: SNP, n = 7, vehicle, n = 8; NOS3 KO: SNP, n = 6, vehicle, n = 6.
Figure 6
 
Mouse eyes perfused with NO donor SNP. (A) Comparison of outflow data in SNP-treated eyes and control eyes perfused with the drug vehicle (PBS) in DKO (A, B), WT (C, D), NOS3 KO (E, F), and CAV1 KO (G, H) mice. Conventional outflow (Ccon) facility was significantly higher in SNP treated WT, NOS3 KO, and CAV1 KO mice compared with vehicle controls (*P < 0.05). Error bars denote SEM. In the box plots, the bottom and top of the box are the first and third quartiles, and the band inside the box is the second quartile (the median). The ends of the whiskers are the minimum and maximum values. The circle represents a data outlier, and the number next to the outlier is the observation number. DKO: SNP, n = 20; vehicle, n = 11. WT: SNP, n = 6, vehicle, n = 6; CAV1 KO: SNP, n = 7, vehicle, n = 8; NOS3 KO: SNP, n = 6, vehicle, n = 6.
Figure 7
 
IOP of four strains of mice treated with topical application of NOS inhibitor L-NAME. L-NAME significantly increased IOP in DKO (A), WT mice (B), and CAV1 KO mice (D). Error bars denote SEM. DKO, n = 6; WT, n = 6, NOS3 KO, n = 6; CAV1 KO, n = 6. *P < 0.05.
Figure 7
 
IOP of four strains of mice treated with topical application of NOS inhibitor L-NAME. L-NAME significantly increased IOP in DKO (A), WT mice (B), and CAV1 KO mice (D). Error bars denote SEM. DKO, n = 6; WT, n = 6, NOS3 KO, n = 6; CAV1 KO, n = 6. *P < 0.05.
Figure 8
 
Mouse eyes perfused with NOS inhibitor L-NAME. Flow rate and conventional outflow facility (Ccon) in L-NAME–treated eyes and drug vehicle-treated eyes in DKO (A), WT (B), NOS3 KO (C), and CAV1 KO mice (D). It significantly reduced Ccon in DKO, WT, and CAV1 KO mice (n = 6, P < 0.05). However, in NOS3 KO mice, Ccon was similar in L-NAME– and drug vehicle-treated eyes (n = 6, P > 0.05). Error bars denote SEM. Box plot showed that the conventional outflow was significantly higher in drug-treated eyes compared with paired controls in all strains of mice. In the box plots, the bottom and top of the box is the first and third quartiles, and the band inside the box is the second quartile (the median). The ends of the whiskers are the minimum and maximum values. DKO, n = 6; WT, n = 6, NOS3 KO, n = 6; CAV1 KO, n = 6. *P < 0.05.
Figure 8
 
Mouse eyes perfused with NOS inhibitor L-NAME. Flow rate and conventional outflow facility (Ccon) in L-NAME–treated eyes and drug vehicle-treated eyes in DKO (A), WT (B), NOS3 KO (C), and CAV1 KO mice (D). It significantly reduced Ccon in DKO, WT, and CAV1 KO mice (n = 6, P < 0.05). However, in NOS3 KO mice, Ccon was similar in L-NAME– and drug vehicle-treated eyes (n = 6, P > 0.05). Error bars denote SEM. Box plot showed that the conventional outflow was significantly higher in drug-treated eyes compared with paired controls in all strains of mice. In the box plots, the bottom and top of the box is the first and third quartiles, and the band inside the box is the second quartile (the median). The ends of the whiskers are the minimum and maximum values. DKO, n = 6; WT, n = 6, NOS3 KO, n = 6; CAV1 KO, n = 6. *P < 0.05.
Figure 9
 
Tyrosine nitration of proteins in WT, NOS3, and CAV1 DKO mice, and eNOS KO mice. Representative blots (A) and densitometry analyses (B) of the mouse outflow tissue in WT, NOS3, and CAV1 DKO mice are shown. DKO mice express similar level of nitrotyrosine to WT mice, but lower level nitrotyrosine than CAV1 KO mice. eNOS KO mice express a low level of nitrotyrosine. Error bar denotes SEM. n = 6 eyes. *P < 0.05.
Figure 9
 
Tyrosine nitration of proteins in WT, NOS3, and CAV1 DKO mice, and eNOS KO mice. Representative blots (A) and densitometry analyses (B) of the mouse outflow tissue in WT, NOS3, and CAV1 DKO mice are shown. DKO mice express similar level of nitrotyrosine to WT mice, but lower level nitrotyrosine than CAV1 KO mice. eNOS KO mice express a low level of nitrotyrosine. Error bar denotes SEM. n = 6 eyes. *P < 0.05.
Figure 10
 
sGC and PKG expression in WT, NOS3, and CAV1 DKO mice. Representative blots and densitometry analyses of the mouse outflow tissue in WT, NOS3, and CAV1 DKO mice are shown. DKO mice express lower level of sGC than WT mice and similar level of PKG to WT mice. Error bar denotes SEM. n = 4. *P < 0.05.
Figure 10
 
sGC and PKG expression in WT, NOS3, and CAV1 DKO mice. Representative blots and densitometry analyses of the mouse outflow tissue in WT, NOS3, and CAV1 DKO mice are shown. DKO mice express lower level of sGC than WT mice and similar level of PKG to WT mice. Error bar denotes SEM. n = 4. *P < 0.05.
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