Molecular Vision 2022; 28:500-506
Received 04 August 2022 | Accepted 29 December 2022 | Published 31 December 2022
Li Liu, Youde Jiang, Jena J. Steinle
Department of Ophthalmology, Visual and Anatomical Sciences, Wayne State University School of Medicine, Detroit, MI
Correspondence to: Jena J. Steinle, Department of Ophthalmology, Visual and Anatomical Sciences, 9314 Scott Hall, Detroit,
MI 48202; Phone: (313) 577-9731; FAX: (313) 577-3125 email:
Purpose: Reactive oxygen species (ROS) activate inflammatory pathways in several organs, including the retina. More recent work has shown that ROS activate the NOD-like receptor protein 3 (NLRP3) inflammasome pathway proteins. We recently showed that the exchange protein activated by cAMP 1 (Epac1) and protein kinase A (PKA) regulates NLRP3 proteins in the retina. Our goal was to determine whether Epac1 and PKA reduce ROS and NLRP3 inflammasome proteins.
Methods: We used human primary retinal endothelial cells (RECs) grown in normal glucose (5 mM) and stimulated in normal glucose with hydrogen peroxide (H2O2) to induce ROS and measured NLRP3 pathway proteins. In some groups, we treated cells with an Epac1 or a PKA agonist in addition to H2O2 treatment to determine whether Epac1 and PKA reduced ROS and induced NLRP3 pathway proteins.
Results: The data showed that 500 µM H2O2 was the optimal dose to increase ROS in RECs. In RECs treated with H2O2, NLRP3 pathway proteins were increased, which were significantly reduced by cotreatment with PKA or Epac1 agonists. H2O2 significantly increased NIMA-related kinase 7 (Nek7) and purinergic 2X7 receptor 7 (P2X7) levels, which were blocked by Epac1 and PKA agonists.
Conclusions: Taken together, these data suggest that Epac1 and PKA reduce retinal inflammation through the reduced ROS-induced activation of NLRP3 pathway proteins.
Increased levels of reactive oxygen species (ROS) are a key factor in retinal damage due to diabetes . Studies on the role of ROS remain ongoing, as they provide a target for therapeutic development . Using both cell cultures and diabetic animal models, a large number of studies have focused on reducing ROS in retinal cells [3,4].
One potential way that ROS can cause retinal damage is through the activation of the NOD-like receptor protein 3 (NLRP3) inflammasome. Several studies have focused on the mechanisms by which ROS induce NLRP3 actions. Using a rat diabetes model and arising retinal pigment epithelia (ARPE)-19 cells, studies have shown that both ghrelin and proanthocyanidins significantly reduce ROS, leading to the inhibition of NLRP3 proteins and apoptosis [5,6]. Similarly, studies on retinal endothelial cells (RECs) and diabetic rats showed that vitamin D3 was protective to the retina through reduced ROS and NLRP3 pathways . Most studies have agreed that ROS is one mechanism leading to the activation of the NLRP3 inflammasome in diabetic retinas and retinal cells grown in high glucose.
We reported that the exchange protein activated by cAMP 1 (Epac1) can regulate the NLRP3 inflammasome in primary human RECs . More recently, we showed that both protein kinase A (PKA) and Epac1 agonists can regulate NLRP3 proteins in the retina and in REC grown in high glucose . The role of ROS in this regulation by Epac1 and PKA agonists was unclear. Epac1 can reduce ROS in the tubular epithelium in models of ischemia/reperfusion . Work in vascular injury models also demonstrated that Epac1 is key to neointima formation through reduced ROS levels . Studies using glucagon-like peptide 1-receptor agonists showed that their protective effects on cardiomyoblasts occurred through PKA- and Epac1-mediated reduction in ROS actions . Based on studies demonstrating that PKA and Epac1 can reduce ROS in other cell types, we hypothesized that PKA and Epac1 agonists would reduce ROS levels in REC, leading to decreased NLRP3 pathway proteins.
Primary human RECs were purchased from Cell Systems Corp. (Kirkland, WA). The cells were grown in basal glucose medium (5 mM glucose) for all cell culture studies. Cell culture was performed as previously described [13,14]. The cells were maintained in the appropriate medium for a minimum of 3 days.
Some of the cells were treated with a protein kinase A agonist (forskolin, 20 μm for 2 h, Tocris, UK) , or an Epac1 agonist (10 μM for 2 h, Tocris) . Some were also treated with H2O2 (Sigma) for a dose–response curve. Once the optimal dose was determined, the cells were treated with 500 µM H2O2 alone or in combination with the Epac1 agonist or forskolin. H2O2 was administered for 2 h before stimulation with the Epac1 or PKA agonists, with cells in treatment for a total of 4 h. Six replicates for cell culture work were done for cell treatments, except for the dose response, where n = 9 was used.
ROS levels were measured using a fluorescent probe, 2,7-dichloroflurescein diacetate (DCF-DA; Invitrogen, Waltham, MA). In brief, cell lysates with 1 μg/μl proteinase inhibitor diluted in PBS were collected, and protein concentrations were calculated. Protein samples (10 μg) were loaded in triplicate into a black 96-well plate. A proteinase inhibitor (100 μl) diluted in PBS and containing 5 μM fresh DCF-DA was added to the plate and incubated in 37 °C for 1 h. Fluorescence intensity was read on a plate reader at excitation and emission wavelengths of 485 and 530 nm, respectively.
Western blotting was performed as previously described . The primary antibodies used were Epac1 (Ab124162, 1:1000), PKA (Ab75991, 1:500), NLRP3 (Ab263899, 1:500), ASC1 (Ab70627, 1:600), Nek7 (Ab133514, 1:500), P2X7 receptor (Ab109054, 1:500; Abcam, Cambridge, MA), cleaved caspase 1 (Asp297, ThermoFisher PA5–77886, 1:200), and beta-actin (Santa Cruz). The primary antibodies were incubated overnight. Secondary antibodies were conjugated to horseradish peroxide (HRP; Promega, Madison, WI). Bands were visualized using an Azure C500 machine (Azure, Dublin, CA). IL-1β ELISA. IL-1β ELISA (R&D Systems, Menomomie, WI) was performed according to the manufacturer’s instructions, with the exception that the ELISA was run overnight at 4 °C.
Data are presented as mean ± SEM. Statistics were measured using Prism 8.0 (GraphPad, San Diego, CA). One-way ANOVA with Tukey’s post-hoc test was used. p < 0.05 was considered statistically significant. In the case of Western blotting, one representative blot is shown. The molecular weight is shown by the representative blot.
To dissect potential mechanisms of NLRP3 regulation in REC, we used REC in normal glucose and treated them with varying doses of H2O2 to determine the optimal dose to increase ROS levels. Figure 1 shows a dose–response curve for the levels of ROS after H2O2 treatment in RECs grown in normal glucose. All subsequent experiments were performed with 500 µM H2O2.
We recently reported that Epac1 and PKA agonists reduce NLRP3 signaling proteins [8,9,17]. We wanted to ascertain whether this occurred through a reduction in ROS. Figure 2 demonstrates that H2O2 significantly increased ROS. Both the Epac1 (A) agonist and the PKA (B) agonist were able to significantly reduce the H2O2-induced increase in ROS.
The Epac1 agonist could reduce ROS (Figure 2) and NLRP3 proteins , the goal was to determine whether Epac1 could overcome H2O2 to reduce NLRP3 proteins. Figure 3A shows that Epac1 levels were reduced in REC treated with H2O2 and that the Epac1 agonist was able to increase Epac1 levels. Figure 3B–E shows that RECs grown in normal glucose and treated with H2O2 have increased NLRP3 pathway protein levels. These levels are reduced when the cells are treated with the Epac1 agonist and H2O2, demonstrating that Epac1 can overcome the ROS produced by H2O2 to reduce NLRP3 signaling proteins.
We have shown that, similar to Epac1, PKA agonists can reduce ROS. In these experiments, we demonstrated that forskolin, a PKA agonist, can decrease H2O2-induced activation of the NLRP3 pathway. Figure 4A is a control to show that H2O2 decreases PKA levels, and forskolin significantly increases PKA levels. Figure 4B–E shows that H2O2 increased NLRP3 (B), ASC1 (C), and cleaved caspase 1 (D) levels and the activation of IL-1β (E). Forskolin treatment combined with H2O2 significantly reduced the levels of all proteins compared to H2O2 alone.
We recently showed that Nek7 and P2X7R caused the activation of the NLRP3 inflammasome [9,17]. To further these findings, we explored whether the Epac1 agonist and forskolin could reduce H2O2-mediated increases in NLRP3 proteins. Figure 5A,B show that H2O2 increased both Nek7 (A) and P2X7 (B) levels. These increases were inhibited by the Epac1 agonist. Similarly, PKA significantly reduced H2O2-mediated increases in Nek7 (Figure 5C) and P2X7R (Figure 5D) when RECs were treated with forskolin combined with H2O2.
The role of ROS in the retina has been studied for decades [18,19]. Whereas it is clear that ROS play a role, more recent work has focused on the potential mechanisms by which ROS induce retinal damage in response to high glucose levels. One of these mechanisms is likely the activation of the NLRP3 inflammasome. An abundance of literature supports the idea that ROS activates the NLRP3 inflammasome to induce damage to retinal cells [20-22]. Our data support these findings, showing that H2O2, a known ROS activator, caused significant increases in NLRP3 pathway proteins in RECs. We showed that a reduction in ROS led to decreased levels of NLRP3 inflammasome proteins in REC, similar to what has been shown in retinal pigment epithelium (RPE) cells . Thus, our findings on RECs agree with the literature.
The novel aspects of our studies were the experiments showing that Epac1 and PKA reduce ROS to block NLRP3 pathway proteins. In addition, we expanded our recent work to show that Nek7 and P2X7 receptors are regulated by ROS in RECs. Studies of other targets have suggested that Epac1 and PKA could reduce ROS in other targets [11,12]. We were the first to demonstrate a role for Epac1 and PKA in these actions in REC. In addition, we recently reported that Epac1 and PKA reduced Nek7 and P2X7 to block NLRP3 actions in RECs [9,17]; however, we did not explore the role of ROS in these actions. These studies expanded our work to show that both Epac1 and PKA can reduce ROS levels in RECs, which was correlated with a significant reduction in NLRP3 pathway proteins.
In conclusion, we showed that ROS led to increased levels of NLRP3 pathway proteins, which corroborates the existing literature on retinal cells. We added to the existing knowledge with our data showing that Epac1 and PKA reduce H2O2-induced ROS levels. This reduction was associated with a significant decrease in NLRP3 pathway proteins. Future work will expand these studies into mouse work.
These studies were funded by R01EY0028442 (JJS), R01EY030284 (JJS), and P30EY04068 (LDH) and a unrestricted grant from Research to Prevent Blindness.