In diabetic retinopathy, vision loss is usually secondary to macular edema, which is thought to depend on the functional integrity of the blood-retina barrier. The levels of advanced glycation end products in the vitreous correlate with the progression of diabetic retinopathy. Natriuretic peptides (NP) are expressed in the eye and their receptors are present in the retinal pigment epithelium (RPE). Here, we investigated the effect of glycated-albumin (Glyc-alb), an advanced glycation end product model, on RPE-barrier function and the ability of NP to suppress this response. Transepithelial electrical resistance (TEER) measurements were used to assess the barrier function of ARPE-19 and human fetal RPE (hfRPE) monolayers. The monolayers were treated with 0.1–100 μg/ml Glyc-alb in the absence or presence of 1 pM to 100 nM apical atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), or C-type natriuretic peptide (CNP). Glyc-alb induced a significant reduction in TEER within 2 hours. This response was concentration-dependent (EC50= 2.3 μg/ml) with a maximal reduction of 40 ± 2% for ARPE-19 and 27 ± 7% for hfRPE at 100 μg/ml 6 hours post-treatment. One hour pretreatment with ANP, BNP, or CNP blocked the reduction in TEER induced by Glyc-alb (100 μg/ml). The suppression of the Glyc-alb response by NP was dependent on the generation of cyclic guanosine monophosphate and exhibited a rank order of agonist potency consistent with the activation of natriuretic-peptide-receptor-2 (NPR2) subtype (CNP >> BNP ≥ ANP). Our data demonstrate that Glyc-alb is effective in reducing RPE-barrier function, and this response is suppressed by NP. Moreover, these studies support the idea that NPR2 agonists can be potential candidates for treating retinal edema in diabetic patients.
Advanced glycation end products (AGE) are formed by nonenzymatic glycation reactions between reducing sugars and the free amino groups on proteins, lipids, and DNA (Li et al., 1996). Although the accumulation of AGE is a consequence of aging, the rate of AGE formation is accelerated in diabetic patients. Diabetic retinopathy is a major complication of diabetes mellitus and a leading cause of visual impairment and blindness in the United States (Aiello, 2003). The progression of diabetic retinopathy is associated with the accumulation of AGE in the retina and vitreous (Yokoi et al., 2005; Yamagishi et al., 2006; Kakehashi et al., 2008).
The responses to AGE are mediated by pattern-recognition receptors (Yamada et al., 2006). Previous studies have shown that the retina expresses several of these receptors, including the AGE receptor (RAGE) (Yamada et al., 2006), the AGE receptor complex (AGE R1–R3) (Wautier and Guillausseau, 2001), class A scavenger receptor, class B scavenger receptors, and class D scavenger receptors (Horiuchi et al., 2003; Tamura et al., 2003). However, simply blocking the RAGE receptor was not effective in reducing the complications associated with diabetic retinopathy (Peyroux and Sternberg, 2006).
In diabetic retinopathy, visual loss is often secondary to disruption of the blood-retina barrier and the development of macular edema (Aiello, 2003). The retinal pigment epithelium (RPE) and blood vessels of the inner retina form the outer and inner blood-retina barrier, respectively. These barriers control the movement of fluid and solutes into the extracellular spaces of the neural retina (Maepea, 1992). Although RPE function is essential to maintaining a dehydrated neural retinal environment, the regulation of the RPE barrier in macular edema has received relatively limited attention.
Natriuretic peptides [atrial (ANP), brain (BNP), and C-type (CNP)] primarily control diuresis, natriuresis, and vasodilatory functions of the cardiovascular system (Potter et al., 2006). ANP and BNP activate natriuretic peptide receptor 1 (NPR1), while CNP activates natriuretic peptide receptor 2 (NPR2) (Potter et al., 2006). NPR1 and NPR2 are transmembrane guanylyl cyclase receptors and catalyze the synthesis of cyclic guanosine monophosphate (cGMP), which in turn activates protein kinase G (PKG) and subsequent target genes (Levin et al., 1998). A third natriuretic peptide receptor, NPR3, clears natriuretic peptides through receptor-mediated internalization and degradation (Potter et al., 2006).
Natriuretic peptides and their receptors are expressed in the neural retina and the RPE (Rollin et al., 2004). Although the roles of these peptides and their receptors in the retina are not clear, induction of diabetes in rats causes a down-regulation of ANP and NPR3 expression in the retina (Rollin et al., 2005). Moreover, it has been shown recently that ANP can suppress endothelial (Xing et al., 2011) and RPE (Lara-Castillo et al., 2009) leakage. Together these indicate that the natriuretic peptide system in the retina may influence diabetic retinopathy and blood-retina barrier dysfunction. In the research described here, using the ARPE-19 cell line to perform initial experiments and confirming the results in the more RPE-like primary hfRPE-cell model (Ablonczy et al., 2011), we provide new evidence that the administration of glycated-albumin (Glyc-alb), a RAGE agonist, decreased RPE transepithelial resistance and that this response was prevented by pretreatment with natriuretic peptides through a cGMP-dependent pathway.
Materials and Methods
ARPE-19 cells were obtained from the American Type Culture Collection (Manassas, VA) and hfRPE cells were isolated from human fetal eyes acquired from Advanced Bioscience Resources (Alameda, CA). Confluent monolayers of both cell types were established and maintained on permeable membrane inserts (Costar Clear Transwell, 0.4 μm pore, 24 mm; Thermo Fisher Scientific, Waltham, MA), as described previously (Ablonczy et al., 2011). Transepithelial electrical resistance (TEER), which is inversely proportional to the paracellular permeability of cultured RPE cells and is a reliable assay for the assessment of RPE barrier function (Dunn et al., 1996; Ablonczy and Crosson, 2007; Ablonczy et al., 2009; Ablonczy et al., 2011), was measured by means of a volt-ohm meter equipped with an STX2 electrode or 24-mm EndOhm chamber (World Precision Instruments, Sarasota, FL). Resistance values for each condition were determined from a minimum of four individual cultures and corrected for the inherent Transwell resistance within 3 minutes after removing the plates from the incubator. All values represent the mean ± S.E. Data were analyzed using the Student t test, and were considered statistically significant at P < 0.05. Concentration curves were analyzed using Prism 4.02 software (GraphPad Software, San Diego, CA). Only confluent monolayer cultures with stable TEER values were used in these experiments (40–50 Ω-cm2 for ARPE-19 cells and >800 Ω-cm2 for hfRPE cells).
Cells were treated with various concentrations of 0.1–100 μg/ml human albumin or human Glyc-alb (Sigma-Aldrich, St. Louis, MO), the latter of which is a widely used RAGE agonist (Fritz, 2011), apically and basolaterally. Change in TEER was then followed for 6 hours postadministration. ANP, BNP, and CNP were obtained from Sigma-Aldrich. Natriuretic peptides were administered apically or basolaterally 1 hour prior to the administration of 100 μg/ml Glyc-alb at concentrations that ranged from 1 pM to 100 nM. In peptide studies, TEER was measured prior to NP treatment, 1 hour after treatment, and then followed for 6 hours post-Glyc-alb administration. Selected cultures were pretreated with isatin (100 μmol/l; Sigma-Aldrich) at a concentration high enough to antagonize all three NP receptors (Telegdy et al., 2000; Potter et al., 2004), or KT5823 (5 μmol/l; Cayman Chemical, Ann Arbor, MI) 1 hour before the addition of NP and Glyc-alb. 8-Bromoguanosine 3′,5′-cyclic monophosphate (8-Br-cGMP; Sigma-Aldrich), a cell-permeable cGMP analog, was administered in a similar manner to natriuretic peptides.
cGMP Enzyme-Linked Immunoassay.
Twenty-four-hour–starved hfRPE monolayers from two different donors were treated with 500 μM 3-isobutyl-1-methylxanthine (IBMX; Sigma-Aldrich), 15 minutes prior to the addition of ANP or CNP (100 nM). Fifteen minutes later, cGMP was extracted and quantified using a cGMP ELISA kit (Cayman Chemical) per provider instructions. cGMP concentrations were then normalized to the cellular protein concentration as determined by protein assay (Bio-Rad, Hercules, CA). Selected cultures were pretreated with isatin (100 μmol/l) 15 minutes before the administration of IBMX.
Monolayers of hfRPE cells were stained as described previously (Ablonczy et al., 2011). The primary antibodies were mouse anti-ZO1 (diluted 1:100; Chemicon, Temecula, CA), rabbit anti-NPR2 (1:50; Sigma-Aldrich). The secondary antibodies were Alexa 594-conjugated goat anti-mouse (diluted 1:100; Invitrogen, Grand Island, NY), and Alexa 488-conjugated goat anti-rabbit (1:100; Invitrogen). Draq5 (Cell Signaling Technology, Danvers, MA) was used as a nuclear stain. The resulting samples were analyzed in a Leica TCS RM confocal microscope (Leica Microsystems, Wetzlar, Germany) at 488- and 594-nm excitation using Leica Confocal Software. Stacks of 200 confocal images were collected at successive focal planes (0.11 μm apart) throughout the entire cell monolayer (22 μm).
Monolayers of ARPE-19 and hfRPE cultures were washed with ice-cold phosphate-buffered saline and lysed [100 μl; pH 7.5; 2.42 g/l Tris Base, 1 mM EGTA, 2.5 mM EDTA, 5 mM dithiothreitol, 0.3 M sucrose, 1 mM sodium orthovanadate (Na3VO4), and 20 mM NaF (all from Sigma); one complete mini-protease-8-inhibitor tablet (Roche Applied Science, Indianapolis, IN)], scraped, and collected in a centrifuge tube. The samples were sonicated twice for 10 seconds each, centrifuged for 5 minutes at 10,000g, and the supernatant collected and centrifuged at 50,000g for 90 minutes. Equal quantities of the samples (determined by protein assay; Bio-Rad) were separated on 4–12% Bis-Tris Gel, transferred to a blotting membrane, blocked with 5% nonfat dry milk, and incubated with anti-NPR2 (Sigma-Aldrich) overnight at 4°C. After washing, the membranes were incubated with horseradish peroxidase–conjugated secondary antibody for 1 hour, and the lanes were visualized with a VersaDoc 5000 imager (Bio-Rad) after treatment with chemiluminescent reagent (Thermo Fisher Scientific).
Glycated-Albumin Decreases TEER in Both ARPE-19 and hfRPE Cells.
To investigate if AGE products alter RPE-barrier function, the RAGE agonist, Glyc-alb, was used. The mean basal TEER for confluent ARPE-19 monolayers was 40 ± 4 Ω-cm2. Apical administration of Glyc-alb (100 μg/ml) caused a rapid decline in TEER with a maximal response of 40 ± 2% drop after 6 hours (Fig. 1A). This decline in TEER was concentration-dependent with an EC50 of 2.3 μg/ml (Fig. 1B). Basolateral administration of human Glyc-alb (100 μg/ml) did not significantly alter TEER (Fig. 1B). The administration of 100 μg/ml albumin to the apical or basolateral solution did not significantly alter the TEER compared with untreated controls (Fig. 1A).
In primary cultures of hfRPE the mean basal TEER for confluent monolayers was 916 ± 40 Ω-cm2. Apical administration of 100 μg/ml human Glyc-alb produced a 27 ± 7% drop in TEER 6 hours after treatment. Again, basolateral administration of Glyc-alb or the administration of albumin to apical or basolateral surfaces did not significantly alter TEER (Fig. 2).
Natriuretic Peptides Suppress the AGE-Induced Decrease in TEER.
In ARPE-19 monolayers, the administration of individual natriuretic peptides alone did not significantly alter the TEER (Fig. 3A). However, 1 hour apical pretreatment with 1 nM ANP, BNP, or CNP inhibited the reduction in TEER caused by apical Glyc-alb by 52, 60, and 100%, respectively (Fig. 3A). For each NP, the inhibitory response was concentration-dependent. The IC50 for ANP, BNP, and CNP responses were 2.5 nM, 0.9 nM, and 9.5 pM, respectively (Fig. 3B).
In hfRPE cells, apical pretreatment with CNP (100 nM) completely blocked the decrease in TEER induced by Glyc-alb. Pretreatment with ANP or BNP apically at concentrations of 100 nM produced a partial inhibition of 52% (11 ± 6% reduction in TEER) and 33% (21 ± 2% reduction in TEER), respectively (Fig. 4).
To confirm this evidence that the suppressive action of natriuretic peptides was mediated by NP receptors, we evaluated the effect of isatin, a nonselective natriuretic peptide receptor antagonist (Telegdy et al., 2000; Potter et al., 2004), on CNP-induced changes in TEER in ARPE-19 cells. In the presence of isatin (100 μM), pretreatment with CNP (10 nM) did not significantly alter the reduction in TEER induced by Glyc-alb. The administration of isatin alone did not significantly change the TEER in ARPE-19 monolayers (Fig. 5A). To determine if the response to natriuretic peptides was polarized, we compared apical and basolateral pretreatments of 1 nM CNP. Basolateral administration of 1 nM CNP did not significantly alter the reduction in TEER induced by Glyc-alb. However, apical pretreatment with 1 nM CNP completely blocked the response to Glyc-alb in ARPE-19 monolayers (Fig. 5B).
Natriuretic Peptide Effect Is Mediated by cGMP.
The involvement of cGMP in NP-induced suppression of Glyc-alb was investigated by treating ARPE-19 monolayers with 100 μM 8-Br-cGMP. Pretreatment with 8-Br-cGMP suppressed the Glyc-alb–induced reduction in TEER (Fig. 6A). Additional studies demonstrated that pretreatment with KT5823 (5 μM), a PKG inhibitor, also reversed the protective effect of CNP on the Glyc-alb–induced barrier breakdown.
Treating hfRPE monolayers with 100 nM CNP for 15 minutes showed a fourfold increase in cGMP levels compared with the statistically nonsignificant increase when treated with ANP. The production of cGMP increased depending on the concentration of CNP (1–100 nM), resulting in an EC50 of 8.69 nM (data not shown). This effect of CNP on cGMP production was reversed by pretreatment with isatin (100 μM) (Fig. 6B).
Immunoanalysis Shows the Apical Localization of NPR2.
The rank order of potency for the suppression of AGE-induced reduction in TEER by natriuretic peptides and the synthesis of cGMP provides evidence that this response is mediated by the NPR2 subtype. To confirm the presence of these receptors in ARPE-19 and hfRPE, Western blot and immunofluorescence were conducted. Fig. 7, A–H shows the immunofluorescence staining for NPR2 in hfRPE cells (green) with the tight junction marker ZO1 (red) indicating cellular membranes and Draq5 (blue) labeling the nuclei. Confocal analysis demonstrated that NPR2 is mainly localized on the apical surface of the cells above ZO1, which delineates the apical and basolateral sides of the cell, and above the basolateral cell nuclei (Fig. 7I). Western blot analysis for the membranous and the cytosolic fractions against the NPR2 showed a single band at ∼130 kDa only in the membranous fraction, and not in the cytosolic fraction (Fig. 7J).
Advanced glycation end products have been associated with numerous complications of diabetes (Ahmed, 2005). The levels of AGEs in the blood and vitreous humor of diabetic patients have been correlated with the clinical progression of diabetic retinopathy (Yokoi et al., 2005). Although the RPE expresses several pattern-recognizing receptors activated by AGEs, a direct causal relationship between AGEs and RPE dysfunction has not been addressed before. Using human glycated-albumin, we determined the effect of AGEs on the barrier function of the RPE. The RPE constitutes the outer blood-retina barrier and is responsible for fluid transport from the neural retina to the choroid. This transport counters the Starling forces across the RPE that drive fluid toward the retina (Maepea, 1992). As a result, increasing RPE permeability can contribute to the development of macular edema, a key component of diabetic retinopathy.
Our experiments demonstrated that the administration of human Glyc-alb reduced TEER in both ARPE-19 and hfRPE monolayers only when administered to the apical surface, and the response was concentration-dependent (EC50 of 2.3 μg/ml and log EC50 = –5.63 ± 0.4). These data support the idea that this effect is receptor-mediated, and that these receptors for AGE products are localized on the apical side of the RPE monolayers. The EC50 for glycated-albumin is consistent with the results for a dose-dependent increase in permeability (log EC50 = –5.88 ± 0.3) seen in retinal microvasculature (Warboys et al., 2009), and is in the same range of Glyc-alb increase seen in the vitreous of streptozotocin-induced diabetic rats (1.92 μg/ml) (Cohen et al., 2008).
While our current studies were not designed to characterize the pattern-recognition receptor(s) involved in the response, the polarized nature of the response would argue that the receptor for AGE, RAGE, was on the apical side of the cells and is affected by any AGE increase in the vitreous (Yokoi et al., 2005; Yamagishi et al., 2006; Kakehashi et al., 2008). Increase in intravitreal AGEs (which can originate from leakage of blood through the inner retina vessels, or can be generated in the retina in situ) would then in turn disrupt RPE-barrier function, causing further increase in AGE accumulation in the ocular environment. AGE can also accumulate at the basolateral surface of the RPE most likely diffusing from the choroid (Yamada et al., 2006). Moreover in vivo RAGE was found to colocalize with AGE at the basal deposits; however, its physiologic implications have not been shown. Our polarized acute AGE response may indicate just a difference between short- and long-term AGE exposure or that the expression of RAGE might be under the control of its agonist.
Natriuretic peptides are important regulators of cardiovascular function influencing fluid balance, vasodilatation, and vascular permeability (Potter et al., 2006). In the eye, studies have shown that NP receptors are present in the mammalian neural retina (Rollin et al., 2004) and RPE (Fujiseki et al., 1999), and the activation of these receptors can influence vascular endothelial growth factor–induced permeability changes in the RPE (Ablonczy and Crosson, 2007; Lara-Castillo et al., 2009). However, the receptor subtype responsible for this response has not been investigated. In the current study we investigated if natriuretic peptides can suppress the permeability changes induced by AGEs. As shown in Figs. 3 and 4, pretreating RPE monolayers with ANP, BNP, or CNP suppressed the decrease in TEER induced by Glyc-alb in a concentration-dependent fashion. While all three NP peptides (100 nM) were able to reverse the effect of Glyc-alb on the TEER in ARPE-19 cells, only 100 nM CNP was able to do so in hfRPE cultures, indicating that the two cell lines respond a little differently to NPs. Although the responses in hfRPE cells are expected to better represent properties in vivo, both models showed the same rank order of potency with CNP >> BNP ≥ ANP. In addition, the nonselective NPR antagonist, isatin, blocked this response. Together these data provide pharmacological evidence that response is primarily mediated through the NPR2 subtype.
We used isatin primarily to show that the action of NPs was a receptor-mediated process. Although several studies have used isatin as a selective NPR1 antagonist (Katoli et al., 2010), it has also been shown that at high enough concentration it can antagonize all three NP receptors (Telegdy et al., 2000; Potter et al., 2004; Katoli et al., 2010). Isatin is an indole molecule; nevertheless, it has little effect on the many neurotransmitter and hormonal receptors in the rat hippocampus, acting primarily as an inhibitor of atrial natriuretic peptide binding (Telegdy et al., 2000). Although changes in serotonin and melatonin levels or the activation of their receptors have not been investigated, isatin by itself had no effect on RPE survival or barrier function (Fig. 5), implying that any effect, other than blocking NP receptors, was not relevant.
As only apical treatment with natriuretic peptides suppressed the AGE-induced reduction in RPE resistance, we conclude that the receptors (NPR2) are localized on the apical surface of the RPE. Consistent with this conclusion and results from other laboratories (Wistow et al., 2002), Western blot analysis confirmed that NPR2 receptors are expressed in RPE monolayers. Confocal analysis of immune-localization studies confirmed the apical orientation (Fig. 7I).
The primary second messenger linked to the NPR2 receptor is cGMP (Levin et al., 1998; Potter et al., 2006). Our data provided evidence that natriuretic peptides are effective in stimulating cGMP synthesis in the RPE and that the cell-permeable cGMP analog can suppress AGE-induced RPE barrier dysfunction. Consistent with the data that NPR2 receptor subtype is the primary receptor involved in the NP effect, 100 nM CNP for 15 minutes showed a fourfold increase in cGMP levels compared with the statistically nonsignificant increase when treated with 100 nM ANP. Although ANP at 100 nM showed half protection (52%) against the Glyc-alb–induced decrease in TEER, the reason why we do not see a significant increase in cGMP might be as simple as a timing difference between the two experiments. cGMP levels were evaluated 15 minutes after adding 100 nM ANP, while we evaluated the pharmacological effect on TEER after 2 hours. Moreover, cGMP might be compartmentalized, resulting in an increase in cGMP with ANP treatment that is too low to be detected by our assay.
Previous studies have shown that ANP and cGMP alone can stimulate the pumping activity of the RPE and increase the rate of reabsorption of subretinal fluid (Marmor and Negi, 1986; Baetz et al., 2012). These data provide evidence that the activation of the cGMP-PKG pathway is important for maintaining the functional integrity of the RPE and removal of fluid from the subretinal environment.
Preclinical studies targeting agents that suppress AGE formation or RAGE-signaling showed encouraging results; however, subsequent clinical trials were disappointing (Peyroux and Sternberg, 2006). In the current study, we demonstrated that administration of RAGE agonist, Glyc-alb, produced significant reductions in RPE monolayer resistance and that pretreatment with natriuretic peptides suppressed this response. Pharmacological and structural studies provided evidence that the response to natriuretic peptides was mediated by NPR2 receptors in the apical membranes of the RPE and is dependent on cGMP. Thus, NPR2 agonists or agents that increase cGMP may provide alternative treatment options for diabetic macular edema.
The authors thank Dr. Luanna Bartholomew for critical review (Medical University of South Carolina, Charleston, South Carolina).
Participated in research design: Dahrouj, Crosson, Ablonczy.
Conducted experiments: Dahrouj, Liu.
Performed data analysis: Dahrouj, Crosson, Ablonczy.
Wrote or contributed to the writing of the manuscript: Dahrouj, Alsarraf, Crosson, Ablonczy.
This work was supported by in part by the National Institutes of Health National Eye Institute [Grants EY009741 (to C.E.C.) and EY019065 (to Z.A.)], the Ola B. Williams Foundation, and an unrestricted grant to Medical University of South Carolina, Storm Eye Institute, from Research to Prevent Blindness, New York, New York.
Portions of this work were presented as an abstract at the 2011 Annual Meeting of the Association for Research in Vision and Ophthalmology, Fort Lauderdale, Florida, as Dahrouj M, Ablonczy Z, and Crosson CE. Natriuretic peptides protect the RPE from AGE-induced barrier breakdown. Invest Ophthalmol Vis Sci 2011; 52:E-Abstract PN 5659.
- advanced glycation end product
- atrial natriuretic peptide
- brain natriuretic peptide
- 8-bromoguanosine 3′,5′-cyclic monophosphate
- C-type natriuretic peptide
- human fetal retinal pigment epithelium
- natriuretic peptide
- NPR2, NPR3, natriuretic peptide receptors 1, 2, and 3
- protein kinase G
- AGE receptor
- retinal pigment epithelium
- transepithelial electrical resistance
- Received August 14, 2012.
- Accepted October 18, 2012.
- Copyright © 2013 by The American Society for Pharmacology and Experimental Therapeutics