VIP Regulates Morphology and F-Actin Distribution of Schlemm's Canal in a Chronic Intraocular Pressure Hypertension Model via the VPAC2 Receptor

Abstract

Purpose: To investigate the roles of vasoactive intestinal peptides (VIPs) in regulating the morphology and F-actin distribution of Schlemm's canal (SC) of rat eyes.

Methods: Chronic intraocular pressure (IOP) hypertension models with episcleral venous cauterization (EVC) were treated with topical VIP or PG99-465 (vasoactive intestinal peptide receptors 2 [VPAC2] antagonist). IOPs were measured with Tono-Pen, and the SC parameters, including the cross-section area, circumference, and length, were statistically evaluated by hematoxylin-eosin and CD31 immunohistochemical staining. Immunofluorescence was performed to detect the distribution of F-actin in the SC. Moreover, the distribution of filamentous actin (F-actin) and globular actin (G-actin) in human umbilical vein endothelial cells (HUVECs) was studied under a pressure system by immunofluorescence and Western blotting.

Results: Increased expressions of VIP and VPAC2 receptors, as well as a disordered distribution of F-actin were found in SC endothelial cells (SCEs) in the EVC model. Moreover, topical VIP maintained the normal distribution of F-actin in SCEs, expanded the collapsed SC, and induced a significant decrease in IOP in the EVC model. In in vitro HUVECs, the F-actin/G-actin ratio increased significantly under stress stimulation for 30 minutes. A total of 50 μM VIP helped maintain the normal F-actin/G-actin ratio of HUVECs against stress stimulation.

Conclusions: VIP regulates the distribution of F-actin in SCEs via the VPAC2 receptor in order to induce a decrease in IOP. VIP may represent a new target for antiglaucoma drugs.

Primary open angle glaucoma (POAG) is a chronic, progressive ocular disease associated with optic nerve degeneration and retinal ganglion cell death, leading to visual field loss and eventual blindness.¹ POAG patients suffer from elevated intraocular pressure (IOP) and abnormal IOP fluctuations, with an open anterior chamber angle according to gonioscopy.² It is widely recognized that the obstruction of the aqueous humor outflow pathway results in increased aqueous outflow resistance, which is the key factor in the pathogenesis of elevated IOP.³ The main resistance of the conventional route is located between the juxtacanalicular tissue and Schlemm's canal (SC).^3,4 Remarkably, there is currently no drug treatment in clinical use that directly targets these two sites, mainly because the precise mechanisms of increased flow resistance remain elusive. 

SC plays a critical role in the increase in outflow resistance. The dimensions of the SC lumen are smaller in glaucomatous eyes, and these changes are positively correlated with outflow resistance and IOP.^5,6 The volume of SC is also smaller in POAG patients, as determined by 3D microcomputed tomography under low and high perfusion pressure.⁷ In addition, there is a lower pore density in the SC inner wall compared with that in nonglaucomatous eyes.⁸ Thus, pathologic changes in the SC may be involved in the pathogenesis of POAG. 

It has been shown that the stiffness of SC endothelial cells (SCEs) is determined by the cell cytoskeleton and accounts for the outflow resistance.⁹ Filamentous actin (F-actin) is one of the three major components of the cytoskeleton and participates in many important cellular processes, including cell shape, cell motility, cell adhesion, and endocytosis.^10,11 Glaucomatous tissue shows a more “disordered” F-actin architecture overall. In glaucomatous eyes, peripheral F-actin bands are less common in the inner walls of SCEs; instead, F-actin is more centrally located within the cell and appears “tangled”.¹² Based on these results, a promising antiglaucoma therapy may involve targeting SCE stiffness, which could directly modify outflow resistance. 

Our previous studies have found smaller SC diameters in POAG patients than in normal subjects by using 80-MHz ultrasound biomicroscopy, and the expansion or collapse of SC may be under automatic regulation.^13,14 Our further unpublished investigations have shown that vasoactive intestinal peptide (VIP)-PGP9.5 double-labeled nerve fibers are observed circumferentially surrounding the SC wall in cadaver eyes. Remarkably, there is also positive expression of VIP focally in SCEs. VIP is a neuropeptide of 28 amino acid residues that belongs to a glucagon/secretin superfamily, and it exerts its actions through three G-protein-coupled receptors (PAC1, VPAC1, and VPAC2).¹⁵ VIP is widely distributed in the central nervous system and peripheral nervous system.¹⁶ In the peripheral nervous system, VIP is released by parasympathetic postganglionic fibers that colocalize with acetylcholine.¹⁷ Postganglionic neurons within the pterygopalatine ganglion are cholinergic, but many also express VIP.¹⁸ It is well known that VIP acts as a potent systemic vasodilator and lowers arterial blood pressure.¹⁹ Hence, we wondered whether similar dilation also affects the local SC, and, if so, how VIP educes this effect by binding with its receptors and whether VIP can directly regulate the morphology and distribution of F-actin. 

In order to answer these questions, we first compared SC measurements and the intensity of F-actin, VIP, and its receptors (VPAC1 and VPAC2) in the SCEs of control eyes and those under a model of chronic IOP hypertension. Then, we observed the conformation of F-actin and globular actin (G-actin), with VIP under stress stimulation in vitro in human umbilical vein endothelial cells (HUVECs). Finally, we examined the effect of VIP and a specific VPAC2 antagonist, PG 99-465, on IOP in a chronic IOP hypertension rat model. 

Materials and Methods

Animals

Male Sprague-Dawley rats from Tongji Medical College weighing 220 to 280 g were used in this study. All animals were raised in a 12 hour light/dark cycle environment, with free access to food and water. Animals were cared for and handled according to the ARVO Statement for the Use of Animals in Vision and Ophthalmic Research and the Use Committee of Huazhong University of Science and Technology. Animals were randomly divided into 12 groups (each consisted of eight eyes; Fig. 1), according to the various posttreatments. At day 28 postcauterization, the rats were killed, both eyes were fixed with 10% formalin as whole globes at 4°C for 2 days, and each eye was subsequently divided into two sections. All divided specimens were embedded in paraffin to enable SC to be cut perpendicularly to its longitudinal axis. 

Figure 1

 

Subgrouping of the study.

Rat Model of Chronic IOP Hypertension

High IOP was induced by cauterizing three episcleral vessels of right eyes, essentially according to a method developed by Shareef,²⁰ and the left eyes were used as controls. In brief, rats were anaesthetized by intraperitoneal injection of 10% chloral hydrate (4 mL/kg). Following a 1.5- to 2-mm-long incision through the conjunctiva and Tenon's capsule, three episcleral veins near the superior and temporal rectus muscles were cauterized by ophthalmic cautery under an operation microscope (66 Vision Tech YZ20T9; Suzhou, China). The ophthalmic cautery was applied to a point (each lasted about 1 second) on the trunk and branch of the veins. The right eyes were flushed with gatifloxacin eyedrops and treated with antibiotic ointment at the end of the operation. The IOP of cauterized right eyes was higher than noncauterized left eyes for 1 month (Fig. 2A). 

Figure 2

 

Histologic measurements analyses of SC in a chronic IOP hypertension model and control rat eyes. (A) Mean IOP values ± standard deviation (n = 8). Cauterized eyes exhibit sustained elevation of IOP up to 28 days by using two-side paired t-test. (B) Schematic representation of SC measurements. Cross-section area and circumference were drawn freehand based on the outline of SC. SC length was measured from anterior to posterior (a to b) distance. (C) Representative light microscopy images of HE staining (n = 8), and CD31 immunohistochemical staining (n = 8) showed the location of the SC and TM at the corneoscleral junction of the iridocorneal angle. (D–F) Comparisons of different SC measurements in chronic IOP hypertension and control groups (n = 8). Eyes with EVC had a smaller SC area, circumference, and length than contralateral normal eyes by using the two-tailed paired t-test. Scale bars: 50 μM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Peptides and Drugs Administration

VIP (HSDAVFTDNYTRLRKQMAVKKYLNSILN) and PG 99-465 (Myr-HSDAVFTDNYTKLRKQMAVKKLNSIKKGGT) with purities of 98% were synthesized by Bioyeargene Biosciences (Wuhan, China). VIP with N-terminal fluorescein isothiocyanate (FITC)-Acp was synthesized by Sangon Biotech (Shanghai, China). For topical administration, one 40-μL drop VIP (10 or 100 μM) in 0.2% Azone (Yick Vic Chemicals and Pharmaceuticals [HK] Ltd., Shanghai, China) or PG 99-465 (10 or 100 μM) in 0.2% Azone or 0.2% Azone was administered to the central cornea of both eyes in all groups, with blinking prevented at intervals of 30 seconds. The prepared drugs were stored in a dark and dry place at room temperature (Fig. 3A). Both eyes received drug interventions through eye dropping three times a day (9:00 AM/3:00 PM/9:00 PM) during day 14 to 27 postcauterization (Fig. 3B). The peptides-azone could penetrate through the cornea and reach into the anterior chamber, iris, trabecular meshwork (TM), and SC (Fig. 3C), traced by the fluorescence of VIP-FITC (10 μM) (Fig. 3D). For cell experiments, VIP (1, 10, or 50 μM) or vehicle double distilled water (ddH₂O) was added in Dulbecco's modified Eagle's medium under different treatments. 

Figure 3

 

Experimental protocol of VIP or PG 99-465 preparation and administration. (A) Schematic processes of VIP or PG 99-465 preparations. The five drugs were stored in a cool, dark place and then extracted with injectors as needed. (B) Protocol of VIP or PG 99-465 administration. The right eyes of rats received EVC treatment (red arrow). The treatments 0.2% Azone, VIP (10 μM/100 μM)-0.2% Azone, or PG 99-465 (10 μM/100 μM)-0.2% Azone were given by eye dropping for both eyes during day 14 to 27 postcauterization (blue line, ter in die, 9:00 AM/3:00 PM/9:00 PM). The rats were killed at day 28 for histology and immunostaining (black arrow). IOP was measured at day 0, 1, 7, 10, 14, 21, and 28 postcauterization (hollow arrow). (C) Schematic diagram of VIP or PG 99-465 distributions via the cornea. (D) Representative fluorescence distributions of VIP-FITC in corneal epithelium, corneal endothelium, iris, TM, and SC after eye dropping for three days. Green: VIP-FITC. White scale bar: 50 μM.

IOP Measurements

IOP was measured in both eyes by using a TONO-PEN XL Tonometer (Reichert, NY, USA) in awake rats. Eyes were topically anesthetized with 20% lidocaine hydrochloride before IOP measurements. The accuracy of the Tonopen results, even under anesthesia, has been established.²¹ Measurement of IOP was always performed between 10 and 11 AM at day 0, 1, 7, 10, 14, 21, and 28 postcauterization (Fig. 3B). Seven measurements were taken from each eye and averaged. All repeated measurements reached the level with a coefficient of variation less than 5%. 

SC Measurements

Sections of hydrated and deparaffinized tissues were stained with hematoxylin-eosin and CD31. The SC parameters included cross-section area, circumference, and SC length (Fig. 2B). These parameters were measured by two analyzers by using the ImageJ software (http://imagej.nih.gov/ij/; provided in the public domain by the National Institutes of Health, Bethesda, MD, USA) and who were masked to the sample information. Optimum image light and contrast were subjectively defined in order to maximize the visualization of the SC. The cross-section area and circumference were drawn freehand based on the outline of SC. SC length was measured from both ends of the SC. Each measurement was repeated two times and averaged. 

Cell Culture of HUVECs

HUVECs were given as a gift from the Department of Obstetrics and Gynecology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology. HUVECs were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, USA), penicillin (100 U/mL)/streptomycin (100 μg/mL), and 2 mM L-glutamine at 37°C in a 5% CO₂. Confluent cell layers were split two times per week. For different experiment designs, the cells on coverslips were incubated with VIP under pressure stimulations and received recovery experiments. In all cases, the changes of F-actin and G-actin distribution were examined by immunofluorescence. 

Pressure System

HUVECs were placed within the pressure chamber and the incubation gas mix was pressurized. The cells were exposed to the elevated ambient hydrostatic pressure (30 and 40 mm Hg) and maintained under conditions for 30, 60, or 120 minutes. Following this, the pressure was restored to the normal atmospheric pressure and cells were incubated for the additional 1, 2, or 4 hours. 

Immunofluorescence and Immunohistochemistry

For immunofluorescence, SC sections (5 μM) were gently washed three times with phosphate-buffered saline (PBS) and then treated with 5% donkey serum albumin for 1 hour to block nonspecific binding. The F-actin in SC sections and HUVECs on coverslips were labeled with phalloidin (1:1000; ab112127; Abcam, Cambridge, UK) overnight at 4°C. The G-actin in the HUVECs was stained by Deoxyribonuclease I (300 nM; Alexa Fluor 488 conjugate; Invitrogen, Carlsbad, CA, USA). The sections and coverlips were examined with a laser-scanning confocal microscope (Zeiss LSM 710; Zeiss, Oberkochen, Germany) under excitation wave lengths of 405 nm for 4′,6-diamidino-2-phenylindole (DAPI), 488 nm for FITC, and 594 nm for phalloidin. For immunohistochemical staining, the primary antibodies included CD31 (1:100; BA2966; Boster Biological Technology, Co., Ltd., Wuhan, China), VIP (1:500; ab78536; Abcam, Cambridge, MA, USA), VPAC1 (1:100; MAB5468; Millipore, Temecula, CA, USA), and VPAC2 (1:100; AB2266; Millipore). The SC sections were incubated with biotinylated secondary antibody. As a peroxidase substrate, 3′,3′-diaminobenzidine (DakoCytomation, Carpinteria, CA, USA) was used for developing a brown color, and hematoxylin (Merck Ltd., Taipei, Taiwan, Republic of China) was used as a counter stain. The immunohistochemical sections were observed by light microscopy. The anatomic details of SC were photographed using the 40× objective lenses. For the purpose of publication, images have resized and subjected to digital contract enhancement. 

F-actin to G-actin Ratio and Western Blotting

In Figure 4, the F-actin to G-actin ratio was quantified by immunofluorescence by using ImageJ software (n = 100 cells per group). In Supplementary Figure S1, the F-actin to G-actin ratio was determined by Western blotting. Radio immunoprecipitation assay lysis buffer (P0013K; Beyotime, Beijing, China) was used to lyse HUVEC cells from control and VIP-treated (1, 10, and 50 mmol/L) groups on ice for 10 minutes and then centrifuged at 15,000g for 30 minutes. Soluble actin (G-actin) was collected in the supernatant. The insoluble F-actin in the pellet was resuspended in lysis buffer plus an equal volume of buffer 2 (1.5 mM guanidine hydrochloride, 1 mM sodium acetate, 1 mM CaCl₂, 1 mM adenosine triphosphate, and 20 mM Tris-HCl; pH 7.5) and incubated on ice for 1 hour to convert F-actin into soluble G-actin, with gentle mixing every 15 minutes The samples were centrifuged at 15,000g for 30 minutes, and F-actin was collected in the supernatant. Samples from the supernatant (G-actin) and pellet (F-actin) fractions were proportionally loaded and analyzed by Western blotting using a specific actin antibody (1:5000; ab179467; Abcam, Cambridge, UK). 

Figure 4

 

Immunofluorescence staining of F-actin and G-actin in HUVECs with pressure or VIP stimulations. F-actin/G-actin ratio quantified by immunofluorescence using the ImageJ program (n = 100 cells per group). (A) Representative staining of F-actin and G-actin in HUVECs under 30-mm Hg compressive stress stimulations for 0, 30, 60, and 120 minutes. The F-actin/G-actin ratio began to increase at 30 minutes, and then significantly decreased with prolonged stress stimulation. (B) Representative staining of F-actin and G-actin in HUVECs under 0-, 1-, 10-, or 50-μM VIP treatments for 30 minutes without compressive stress stimulation. The F-actin/G-actin ratio decreased in the 50 μM VIP group. (C) Representative staining of F-actin and G-actin in HUVECs under 0-, 10-, or 50-μM VIP treatments with 30-mm Hg compressive stress stimulation for 30 minutes at the same time. The increased F-actin/G-actin ratio gradually decreased in the VIP group. Control, without any intervention. All groups compared with the control. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Scale bars: 50 μM.

The expression of the VPAC2 receptor was also determined by Western blotting. Radio immunoprecipitation assay lysis buffer (P0013K; Beyotime, Beijing, China) was used to lyse HUVEC cells from control and pressure-treated (30 mm Hg for 30, 60, and 120 minutes, respectively) groups on ice for 10 minutes and then centrifuged at 12,000g for 10 minutes. The supernatant was collected and the VPAC2 receptor was analyzed by Western blotting using a specific actin antibody (1:2000; AB2266; Millipore). 

Data Analysis

Statistic analyses were performed using two-tailed pair/unpaired t tests and ordinary 1-way ANOVA with Dunnett's multiple comparisons test by GraphPad Prism version 6.0 (GraphPad, San Diego, CA, USA). All data for SC measurements was reported with mean ± standard deviation. A level of 5% was considered significant. 

Results

Elevated IOP and Reduced SC Parameters in a Chronic IOP Hypertension Model

As shown in Figure 2A, a significant rise in IOP was noted 10 days after episcleral venous cauterization (EVC) compared with that in contralateral control eyes (16.4 ± 1.9 mm Hg vs. 12.4 ± 1.7 mm Hg; P = 0.0005). The evaluated IOP state was continuously sustained until 28 days postoperation. To confirm the SC localization and measurements in rat eyes, serial sections were stained with HE and CD31 and revealed the cross-sectional structure of SC at the corneoscleral junction of the iridocorneal angle (Fig. 2C). Both with hematoxylin-eosin and CD31 immunohistologic staining, we found reduced SC parameters after EVC treatment (Fig. 2D–F). The SC measurements of contralateral control eyes and those under chronic IOP hypertension according to HE staining were the following: (1) cross-section area, 1121.09 ± 138.54 μM² vs. 879.18 ± 192.14 μM²; P = 0.0352, (2) circumference, 344.47 ± 27.91 μM vs. 273.37 ± 31.20 μM; P = 0.0026, and (3) SC length, 157.05 ± 13.40 μM vs. 123.99 ± 16.56 μM; P = 0.0043. Similar results were found with CD31 immunohistologic staining: (1) cross-section area, 940.29 ± 163.67 μM² vs. 620.24 ± 95.45 μM²; P = 0.0027, (2) circumference, 290.19 ± 40.18 μM vs. 220.49 ± 56.38 μM; P = 0.0069, and (3) SC length, 129.54 ± 13.62 μM vs. 100.76 ± 26.55 μM; P = 0.0124. These results demonstrated the IOP-elevating effects and morphologic changes in the SC in rat eyes after EVC treatment. 

Disordered F-actin Distribution in SC in a Chronic IOP Hypertension Model

To compare the in situ F-actin distribution of the SC in EVC eyes and normal eyes, we observed the morphology of F-actin under oil microscopy (100×) by immunostaining (Fig. 5A). In normal eyes, F-actin was typically observed in a band that ran around the periphery of the SCEs and TM cells. In EVC eyes, the F-actin distribution became disordered, with few peripheral actin bands compared to normal eyes. Focal concentrations of F-actin were sometimes present, giving the actin labeling a punctate appearance. These actin “dots” or “clumps” were located in the SC and TM with a random distribution. Besides, the intensity of F-actin significantly decreased in EVC eyes (Fig. 5E; 11.84% ± 0.57% vs. 15.61% ± 0.60%; P < 0.0001). The disordered F-actin distribution in SCEs is likely to be associated with increasing outflow resistance, leading to high IOP in rats after EVC treatment. 

Figure 5

 

Immunostaining of F-actin, VIP, VPAC1, and VPAC2 in SC of a chronic IOP hypertension model and control rat eyes. (A) Representative immunofluorescence distribution of F-actin in SC (n = 6). Red: F-actin; Blue: DAPI for nuclear staining. White scale bar: 20 μM. (B–D) Representative immunohistochemical staining of VIP, VPAC1 and VPAC2 in SCEs (n = 6). VIP was focally detected in SC and SCEs only expressed VPAC2, but not VPAC1. Black scale bars: 50 μM. (E) Quantification of F-actin positive area by immunofluorescence by using ImageJ software (n = 5). (F, G) Scatter diagram depicted the quantifications of positive staining SCEs of VIP and VPAC2 on the basis of immunohistochemical results (n = 6). The comparisons were analyses using the two-tailed pair t-test. ***P < 0.001, ****P < 0.0001.

Increased Expression of VIP and the VPAC2 Receptor in SC in a Chronic IOP Hypertension Model

To confirm the local detection of VIP in SCEs, we first explored the expression of VIP, VPAC1, and VPAC2 in SCEs and then quantified the positive-labeled SCEs in each group. As shown in Figure 5B, VIP was focally detected in SCEs and along the luminal surface, with higher percentages of VIP-positive SCEs in EVC eyes than in control eyes (Fig. 5F; 46.71% ± 4.99% vs. 27.17% ± 1.50%; P = 0.0004). Remarkably, SCEs only expressed VPAC2 receptors (Fig. 5D) and not VPAC1 receptors (Fig. 5C). Consistent with the expression pattern of VIP, a higher percentage of VPAC2-positive SCEs was found in eyes treated with EVC (Fig. 5G; 42.06 ± 5.59% vs. 18.99 ± 5.03%; P = 0.0003). In consideration of these expression changes, subsequent experiments were designed to focus on the relationship between VIP and F-actin, both in vitro and in vivo. 

Distribution of F-actin and G-actin in HUVECs Under Compressive Stress or VIP Stimulation

Based on the disordered F-actin distribution in SCEs in the animal experiments with continuously high IOP, we further investigated whether similar changes would be observed in the expression and conformation of F-actin in HUVECs under 30-mm Hg pressure or VIP stimulation. We found that the distribution of F-actin was dramatically altered in a time-dependent manner (Fig. 4A). In control cells, F-actin was clearly expressed at the outer membrane and cytoplasm of each HUVEC, whereas G-actin was mainly located at the perinuclear space. Some long actin filaments were also distributed throughout the whole cell. Under 30-mm Hg compression for 30 minutes, the linear F-actin became abundant and typically observed in a band that ran around the periphery of the cell. The linear F-actin network was then obscure with increased punctate G-actin after 60 minutes. Furthermore, after stimulation for 120 minutes, the cells lost most of their filament beams. The F-actin network was absent with focal concentrated G-actin. Correspondingly, the F-actin/G-actin ratio began to increase and then significantly decrease with prolonged stress stimulation. However, the expressions of the VPAC2 receptor remained unchanged under the pressure stimulation (Supplementary Fig. S1B). 

To examine whether the F-actin structure was also affected by VIP, HUVECs were treated with 1, 10, or 50 μM VIP under 30-mm Hg pressure for 30 minutes. The appearance of the F-actin network remained virtually unchanged under different concentrations of VIP without pressure stimulation (Fig. 4B), although there was obvious punctate G-actin localized in the cytoplasm, especially in the perinuclear compartment, with a high concentration of VIP. Correspondingly, the F-actin/G-actin ratio significantly decreased in the 50-μM VIP group. The Western blotting analysis showed similar results (Supplementary Fig. S1A). 

When giving pressure and VIP at the same time for 30 minutes, there was obvious punctate G-actin localized in HUVECs, especially in the 50-μM VIP group (Fig. 4C). Correspondingly, the increased F-actin/G-actin ratio gradually decreased in the VIP group. All of these results indicated the depolymerizing effect of VIP on F-actin. 

Distribution of F-actin and G-actin in HUVECs With Recovery Regimens

To determine whether VIP could reverse the stiffening effect of pressure on HUVECs, recovery experiments were then carried out. We found that perinuclear G-actin became obvious during the recovery. The F-actin/G-actin ratio in the 2-hour group was still greater than the control, but was able to return back within 4 hours of recovery (Fig. 6A) after 30-mm Hg stress stimulation for 30 minutes. In contrast, when 50 μM VIP was administrated during the recovery, the F-actin/G-actin ratio was able to return back within 2 hours of recovery (Fig. 6B). This further corroborated the depolymerizing effect of VIP on HUVECs under pressure stimulation. 

Figure 6

 

Immunofluorescence staining of F-actin and G-actin in HUVECs with recovery regimens. (A) Representative staining of F-actin and G-actin in HUVECs under 30-mm Hg compressive stress stimulations for 30 minutes and recovery for 0, 2, and 4 hours. The F-actin/G-actin ratio in 2-hour group was still more than the control, but was able to return back within 4 hour of recovery. (B) Representative staining of F-actin and G-actin in HUVECs under 30-mm Hg compressive stress stimulations for 30 minutes and then recovery for 2 hours with 0-, 10-, or 50-μM VIP treatments. The F-actin/G-actin ratio was able to return back in the 50-μM VIP group. Control, without any intervention. All groups compared with the control. *P < 0.05, **P < 0.01, ****P < 0.0001. Scale bars: 50 μM.

Increased Cross-Sectional Area of SC With VIP Administration and Decreased Cross-Sectional Area of SC With PG 99-465 Administration After EVC Treatment

Because increased expressions of VIP and the VPAC2 receptor were observed in SCEs with high IOP, we next sought to determine the effects of VIP on SC measurements via treatment with synthesized peptides that activated (VIP) or inhibited (PG 99-465) the VPAC2 receptor (Fig. 7). Although normal eyes received drug treatment, no significant differences were observed in SC measurements using either staining method (Fig. 7A–J). Eyes treated with EVC of different groups all exhibited smaller SC measurements than contralateral normal eyes. Overall, the changes in the SC parameters in eyes under a model of chronic IOP hypertension were remarkably dose dependent. As shown in Figure 7K, the cross-section area of the SC in eyes treated with VIP (100 μM) and Azone-EVC was significantly larger than that in eyes treated with Azone-EVC alone (HE staining, 1075.84 ± 140.73 μM² vs. 894.97 ± 107.25 μM²; P = 0.0118 and CD31 staining, 836.54 ± 140.15 μM² vs. 657.43 ± 90.00 μM²; P = 0.0088). As shown in Figure 7L, the cross-section area of the SC in eyes treated with PG 99-465 (100 μM) and Azone-EVC was significantly smaller than that in eyes treated with Azone-EVC alone (HE staining, 705.34 ± 164.28 μM² vs. 894.97 ± 107.25 μM²; P = 0.0161 and CD31 staining, 522.52 ± 139.55 μM² vs. 657.43 ± 90.00 μM²; P = 0.0375). All data confirmed that VIP could expand the collapsed SC in eyes under a model of chronic IOP hypertension. 

Figure 7

 

Histologic measurement analyses of SC in a chronic IOP hypertension model and control rat eyes after VIP or PG 99-465 was administered through eye drops. (A–J) Representative light microscopy images of HE staining (n = 8) and CD31 immunohistochemical staining (n = 8) showed the SC morphologies of different models. (K) Comparisons of different SC measurements in VIP groups (n = 8). Normal eyes with drug treatment showed no difference to each other in SC measurements (all P > 0.05, ordinary 1-way ANOVA, data not shown). Eyes treated with EVC of different groups all exhibited smaller SC measurements than contralateral normal eyes (all P < 0.05, two-tailed paired t-test; data not shown). The cross-section area of SC in VIP (100 μM)-Azone-EVC eyes was larger than that in Azone-EVC eyes with two methods. (L) Comparisons of different SC measurements in PG 99-465 groups (n = 8). Normal eyes with drug treatment showed no difference to each other in SC measurements (all P > 0.05, ordinary 1-way ANOVA, data not shown). Eyes treated with EVC of different groups all exhibited smaller SC measurements than contralateral normal eyes (all P < 0.001, two-tailed paired t-test; data not shown). The cross-section area of SC in PG 99-465 (100 μM)-Azone-EVC eyes was smaller than that in Azone-EVC eyes with two methods. Scale bars: 50 μM. #Multiple groups comparison using ordinary 1-way ANOVA; *Two groups comparison using two-tailed unpaired t-test. #P < 0.05, *P < 0.05, **P < 0.01. ns, no statistical significance.

Distribution of F-actin in the SC After Treatment With VIP, PG 99-465, and EVC

Having demonstrated that VIP could regulate the intensity of F-actin in vitro in cultured HUVECs, further investigations into the effects of VIP on F-actin in vivo were carried out in SCEs under oil microscopy (100×) by immunostaining (Fig. 8A–E). In the VIP (10/100 μM)-Azone-EVC groups (Fig. 8B and C), linear and continuous F-actin was labeled peripherally in SC and TM cells, which was similar to that observed in the normal control group. In the PG 99-465 (10/100 μM)-Azone-EVC groups (Fig. 8D, 8E), the expression of linear F-actin was reduced and the rest of the punctate F-actin accumulated in a dense cluster in the outflow tissue, with an obviously collapsed SC lumen. Correspondingly, compared with the Azone-EVC group, the intensity of F-actin significantly increased in the VIP (100 μM)-Azone-EVC group (Fig. 8K; 15.89% ± 1.17% vs. 11.44% ± 1.15%; P < 0.01), but decreased in the PG 99-465 (100 μM)-Azone-EVC group (Fig. 8L; 8.05% ± 1.71% vs. 11.44% ± 1.15%; P < 0.05). Treatment with VIP and PG 99-465 led to different appearances of F-actin in SCEs under EVC treatment. 

Figure 8

 

Immunostaining of F-actin and VPAC2 in SC of a chronic IOP hypertension model and control rat eyes after VIP or PG 99-465 was administered through eye drops. (A–E) Representative immunofluorescence distribution of F-actin in SC (n = 5). Red: F-actin; Blue: DAPI for nuclear staining. White scale bar: 20 μM. (F–J) Representative immunohistochemical staining of VPAC2 in SCEs with different treatments (n = 5). Black scale bars: 50 μM. (K, L) Quantification of F-actin positive area by immunofluorescence using ImageJ software (n = 4). (M, N) Scatter diagram depicted the quantifications of positive staining SCEs of VPAC2 on the basis of immunohistochemical results (n = 5). #Multiple groups comparison using ordinary 1-way ANOVA; *Two groups comparison using two-tailed unpaired t-test. ##P < 0.01, ####P < 0.0001, *P < 0.05, **P < 0.01, ****P < 0.0001.

Increased Expression of the VPAC2 Receptor in SCEs After VIP Administration and Decreased Expression of the VPAC2 Receptor in SCEs After PG 99-465 Administration Following EVC Treatment

To further demonstrate that VIP regulates SC morphology and the F-actin distribution via the VPAC2 receptor, we compared the expression of the VPAC2 receptor in all groups by immunohistologic staining (Fig. 8F–J). In accordance with drug concentrations, the percentages of VPAC2-positive SCEs in the VIP (10 or 100 μM)-Azone-EVC groups were higher than that in the Azone-EVC group (Fig. 8M; 60.75% ± 7.29% [EVC + 100 μM VIP] vs. 54.99% ± 10.01% [EVC + 10 μM VIP] vs. 42.64% ± 5.28% [EVC]; P = 0.0093, according to 1-way ANOVA). Conversely, the percentages of VPAC2-positive SCEs in the PG 99-465 (10 or 100 μM)-Azone-EVC groups were significantly lower than that in the Azone-EVC group (Fig. 8N; 9.65% ± 4.28% [EVC + 100 μM PG99-465] vs. 11.77% ± 5.40% [EVC + 10 μM PG99-465] vs. 42.64% ± 5.28% [EVC]; P < 0.0001, according to 1-way ANOVA). These results also showed that the daily topical treatment used in our experiments was a valid method for drug administration into the anterior ocular segment. 

Reduced IOP After VIP Administration and Increased IOP After PG 99-465 Administration After EVC Treatment

Because a significant rise in IOP was noted 10 days after cauterization, daily drug application was started at day 14 postcauterization of both eyes. Therefore, there were no significant differences in cauterized eye IOP before 14 days. Prominent changes in IOP were first observed at day 21 postcauterization (Fig. 9). Reduced IOPs were observed in those receiving VIP at day 21 and 28 postoperation. A larger reduction was found in the VIP (100 μM)-Azone-EVC group compared to that in the Azone-EVC group (day 21, 14.3 ± 0.9 mm Hg vs. 16.2 ± 0.5 mm Hg; P < 0.0001 and day 28, 13.9 ± 0.6 mm Hg vs. 16.1 ± 0.7 mm Hg; P < 0.0001). In contrast, higher IOPs were observed in the PG 99-465 treatment groups at day 21 and 28 postoperation. A greater increase was found in the PG 99-465 (100 μM)-Azone-EVC group than in the Azone-EVC group (day 21, 20.7 ± 1.1 mm Hg vs. 16.2 ± 0.5 mm Hg; P < 0.0001 and day 28, 21.9 ± 1.4 mm Hg vs. 16.1 ± 0.7 mm Hg; P < 0.0001).The final pharmacologic effect of VIP was that of reducing IOP, making it a potential antiglaucoma drug. 

Figure 9

 

IOP of a chronic IOP hypertension model and control rat eyes after VIP or PG 99-465 was administered through eye drops. The average IOP of normal eyes with drug treatment fluctuated from 12 to 13 mm Hg. At day 14 postcauterization, the IOP of EVC eyes in different groups rose to approximately 16 to 17 mm Hg (higher than the contralateral normal eyes, all P < 0.0001, two-tailed paired t-test; data not shown). The drugs were given from day 14 to 27 postcauterization. (A) A reduction in IOP of VIP-Azone-EVC groups was noted at days 21 and 28. (B) Conversely, a significant rise in IOP of PG 99-465-Azone-EVC groups was noted at days 21 and 28. All groups n = 8. *All drug groups compared with the Azone-EVC group (purple solid square) using the two-tailed unpaired t-test. *P < 0.05, ***P < 0.001, ****P < 0.0001.

Discussion

In this study, we aimed to investigate the roles of VIP expressed in the SC in a model of chronic IOP hypertension. We demonstrated that EVC caused increased expression of VIP and VPAC2 receptor, with a disordered distribution and decreased intensity of F-actin in SCEs. Furthermore, the linear F-actin became abundant and the F-actin/G-actin ratio significantly increased in vitro with pressure stimulation for 30 minutes. VIP induced F-actin depolymerization and kept the normal stiffness of HUVECs against stress stimulation. Additionally, topical administration of VIP resulted in maintenance of the normal distribution and intensity of F-actin in SCEs, expanded the collapsed SC, and induced a significant reduction in IOP in EVC model animals. 

Many researchers have found abnormal SC morphometry in vivo in glaucomatous eyes, including reduced SC area, length, and longitudinal minimum width.¹⁴ Other studies of histologic sections have resulted in similar conclusions, and SCs in POAG patients with a family history of POAG may easily collapse at middle age.²² In our experiments, reductions in SC measurements were also shown in a chronic IOP hypertension model. CD31 is a standard marker for blood vessel endothelial cells, and it is also detected in SCEs.²³ CD31 immunohistochemical staining therefore provides a clear profile of the SC. In the chronic IOP hypertension model, we found that both ends of CD31-labeled SCEs lacked a lumen structure, suggesting that this part of the SC was occluded. Therefore, the cross-section area, circumference, and length of the SC were significantly reduced in EVC eyes. Besides, the increased IOP itself may cause the SC to collapse. SC measurements, especially the cross-section area, may reflect the outflow facility and indirectly reflect the aqueous outflow resistance.^24,25 

The outflow resistance is closely related to the stiffness of SCEs, which is determined by the conformation and arrangement of F-actin in the cell. Immunofluorescence staining of F-actin in our study was in accordance with previous studies.¹² Punctate and clump-like F-actin was focally detected in the SC and TM in a disordered distribution, presumably making cells less able to deform in response to a pressure gradient. The disordered F-actin potentially implied an increase in outflow resistance in the high IOP models. We then observed the distribution and expression of F-actin and G-actin in vitro in HUVECs with pressure stimulation. The spatial architecture of the cytoskeletal network plays a central role in transmitting compressive and tensile stresses and in sensing the mechanical microenvironment.²⁶ The initial stiffened state of cells with an increased F-actin/G-actin ratio encountered the inward force of compression, which always resulted in the formation of stress fibers.²⁷ However, after a long-term treatment, the F-actin network depolymerized, and the decrease of actin stress fibers was correlated with morphologic changes in cell shape.^27,28 These phenomena have been explained by several studies. When stresses are applied, the F-actin network stiffens and prevents additional deformation as a result of filament entanglement and the entropic elasticity of individual filaments.²⁹ Additionally, the branched F-actin network shows nonlinear stress stiffening, followed by stress softening at high compressive forces.³⁰ Elevated hydrostatic pressure blocks cytokinesis, stops active translocation of organelles and cytoplasmic streaming, disrupts intercellular adhesions, and, finally, results in cell death.²⁷ Significant changes in the F-actin/G-actin ratio have been observed under elevated hydrostatic pressure, but whether they do so in situ remains to be determined. Because IOP is derived from the aqueous humor flow, an ideal model should be designed to mimic the hydrodynamic pressure. 

In addition to a disordered F-actin architecture, increased expression of VIP was first shown. We further found that SCEs expressed only the VPAC2 receptor and not the VPAC1 receptor. In the central nervous system, the VPAC1 receptor is widely distributed in the cerebral cortex and hippocampus, whereas the VPAC2 receptor is highly expressed in the thalamus and suprachiasmatic nucleus and seldom in the hippocampus, brainstem, spinal cord, or dorsal root ganglia.^31,32 In peripheral tissues, both receptors are found in the gastrointestinal, cardiovascular, and respiratory systems, whereas VPAC2 is predominantly expressed in blood vessels.^31,33,34 Previous studies have found VIP-positive neurons in the pterygopalatine ganglion, with the postganglionic fibers targeting the ciliary epithelium and TM.^18,35 Our findings expand the distribution of VIP and VPAC2 in the eye. Based on the above results, we inferred that increased VIP may regulate the intensity of F-actin via VPAC2. In order to confirm this hypothesis, interventions were carried out using in vitro and in vivo experiments. 

In vitro experiments, high concentrations of VIP induced F-actin depolymerization and maintained the normal stiffness of HUVECs under pressure stimulation. Despite the significant effect of VIP on HUVECs, this study did not involve investigation of the cellular pathway and molecular mechanisms of actin rearrangement. It has been confirmed that the cAMP/protein kinase A (PKA) pathway mediates the endothelial cell cytoskeleton organization and barrier regulation.³⁶ Moreover, the regulation of actin polymerization is orchestrated by Rho/Rac/Cdc42 proteins.³⁷ VIP binds to the VPAC2 receptor and predominantly leads to the stimulation of adenylate cyclase/PKA.³⁸ PKA induces phosphorylation of RhoA at Ser-188 and inhibits RhoA/ROCK signaling, which minimizes the formation of cellular stress fibers.^39,40 A recent comprehensive review has summarized the biomechanical properties of SCEs and states that dibutyryl-cAMP and Rho kinase inhibitors decrease outflow resistance.⁴¹ Thus, we inferred that VIP may also participate in the regulation of outflow resistance. 

Following the acylation of the amino-terminus of the extended VIP (1–26) KKGGT peptide with myristic acid, the resulting peptide, named PG 99-465, is reported to be the first selective VPAC2 receptor antagonist, suppressing the activation of adenylate cyclase.⁴² We used synthetic VIP as an agonist and PG 99-465 as an antagonist of the VPAC2 receptor to explore the effects of VIP on the SC. In combination with IOP monitoring and SC measurements, we demonstrated that VIP could increase the SC area of EVC eyes and expand the collapsed SC. The pharmacologic action of highly concentrated VIP was stronger, with a greater reduction in IOP. In normal eyes, VIP had little influence on the IOP, with IOPs fluctuating between 12 and 14 mm Hg. In contrast, PG 99-465 blocked the effect of VIP and led to the further shrinkage of the SC, and the IOPs of EVC eyes were considerably elevated. The expression of VPAC2 and F-actin in SCEs of various treatment groups were also in accordance with the drug treatments. The in vivo results showed that a specificn concentration of VIP bound with the VPAC2 receptor and maintained the normal distribution of F-actin in SCEs to help resist the damage induced by a high IOP. A similar conclusion was drawn in the in vitro experiments above. 

In EVC models, the disordered distribution of F-actin and collapsed SC implied increased outflow resistance. It was interesting that the expressions of VIP and the VPAC2 receptor were also increased in the cauterized eyes at the same time. When we increased the local concentration by topical VIP administration in EVC eyes, the distribution of F-actin recovered to normal and the collapsed SC expanded, which suggested the decline of increased resistance and thus lowered the elevated IOP. However, the relaxation effect of VIP on episcleral vessels could not be ignored, which may also account for the decrease of outflow resistance. In addition, previous studies have proven that VIP increases vascular endothelial growth factor expression to enhance the angiogenesis in prostate or breast cancer cells and in rats with focal cerebral ischemia.^43–47 Accordingly, in our present models, VIP could presumably increase angiogenesis at the cauterized area to restore circulation and, hence, lower IOP. All of these effects might explain the mechanism of IOP decline. The effect of VIP on the cauterized episcleral vessels should be further confirmed and could be an interesting future research area. 

Inhibiting aqueous inflow or increasing unconventional outflow results in a consequent deprivation of aqueous humor through the conventional outflow pathway, which can cause further damage to the abnormal TM and SC.^48,49 Therefore, the identification of drugs that lower IOP by decreasing outflow resistance is a promising strategy for the treatment of glaucoma. 

In summary, we found that VIP regulates the distribution of F-actin in SCEs via the VPAC2 receptor in order to resist damage induced by IOP hypertension. Furthermore, VIP can also expand the collapsed SC and lower IOP. These results suggest that the VIP-VPAC2 pathway may be developed into a novel medical treatment for glaucoma. 

Acknowledgments

The authors thank Xin Wang (Department of Obstetrics and Gynecology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology) for providing HUVECs. 

Disclosure: L. Chen, None; M. Li, None; Z. Luo, None; X. Yan, None; K. Yao, None; Y. Zhao, None; H. Zhang, None