In the present study, we investigated the pharmacological actions of both fast- and slow-releasing H2S compounds, as well as polysulfides, on acute inflammation induced by the bacterial lipopolysaccharide (LPS) in an ex vivo porcine model of anterior uveitis. Isolated porcine iris-ciliary bodies (ICB) were maintained in oxygenated Krebs-Henseleit solution and then incubated in separate wells of RPMI 1640 supplemented media with an antibiotic. ICBs were exposed to sodium hydrosulfide (NaHS), GYY4137, S-allyl-cysteine (SAC), and polysulfides, diallyl disulfide (DADS), and diallyl tetrasulfide (DATTS) four hours before the end of incubation. We observed that LPS (5-200 ng/ml) produced increases in the release of pro-inflammatory mediators (TNF-α, IL-6, and PGE2) and a corresponding decrease in the anti-inflammatory marker, IL-10, in the ICB explants. Both fast- (NaHS, 0.001-100 μM) and slow-releasing H2S compounds (GYY4137, 0.001-10 μM) and the organosulfur H2S-releasing compounds, DADS (0.001-300 μM), SAC (0.1-1000 nM), and DATTS (0.001 pM - 1 μM), caused a concentration-dependent attenuation of LPS-induced increases in the levels of TNF-α, IL-6, and PGE2. DATTS (0.001 pM - 10 nM) reversed the LPS (25 ng/ml)-induced decrease in IL-10 production in the explants. We conclude that both fast- and slow-releasing H2S compounds, as well as polysulfides, were effective in reducing LPS-induced acute inflammation in porcine anterior uveal explants, ex vivo. Inhibition of endogenous H2S biosynthesis exacerbates LPS-induced production of an inflammatory mediator, a response that was reversed by an H2S-releasing compound.

Background: Low retinal blood flow and/or vasospasm represent major risk factors for the development of glaucomatous optic neuropathy (GON), a potentially blinding eye disease. Bradykinin (BK), a nonapeptide, is endogenously produced and released, which can cause smooth muscles to contract and relax in different tissues depending on the physiological/pathological situation and the presence or absence of vascular tone. Several reports have shown the presence of BK receptor mRNAs, and in some cases, B1- and B2-receptor proteins, in ocular tissues, including the retina. However, the function of these receptors remains to be determined, especially in retinal blood vessels. Methods: We pharmacologically characterized the ability of BK and any related peptide agonists to promote the contraction of isolated bovine posterior ciliary arteries (PCAs) in an organ bath setup using a cumulative compound addition and tension development recording process. Receptor-selective kinin agonists and subtype-selective BK receptor antagonists were utilized to define the possible heterogeneity in the functional BK receptors for PCAs. Results: All agonist kinin peptides concentration-dependently contracted the PCA rings bi-phasically over a 5-log unit range (0.1 nM–10 μM). The relative potencies (EC50 values; n = 4–5) regarding the high-affinity receptor site were: Lys–BK = 0.9 ± 0.4 nM; Des–Arg9–BK = 0.9 ± 0.4 nM; RMP-7 = 1.1 ± 0.6 nM; Met–Lys–BK = 1.3 ± 0.5 nM; Hyp3–BK = 2.7 ± 0.5 nM; BK = 3.0 ± 0.7 nM. The low-affinity receptor site activated by these peptides mostly exhibited EC50 values ranging from 0.3 μM to 3 μM. The concentration–response curves to Des–Arg9–BK (B1-selective agonist) were shifted to the left in the presence of increasing concentrations of a B1-receptor antagonist (R715: 1–10 μM; n = 3). Similarly, WIN-64338 (a B2-receptor antagonist: 1–10 μM; n = 3) moved the BK concentration–response curves to the left. Conclusions: The pharmacological characteristics of BK and analog-induced contractions, and their inhibition by receptor-selective antagonists, indicated the presence of both B1- and B2-receptors, and perhaps another subtype, which mediate the PCA contractions. These results have potential implications for the involvement of heterogeneous kinin receptors, narrowing PCA diameters in vivo, restricting blood flow to the retina, causing GON, and subsequent visual impairment that can eventually cause blindness.

PURPOSE:To study the ability of five kinin peptides to relax phenylephrine-contracted bovine posterior ciliary artery (PCA; major retinal blood supplier) and carbachol-contracted ciliary muscle (CM) (involved in accommodation and aqueous humor drainage) in vitro. METHODS:Isolated bovine CM strips and PCA rings were mounted in small organ baths and perfused with oxygenated Krebs' solution containing 3 µM flurbiprofen. The tissues were then contracted with 10 µM carbachol (for CM) and 10 µM phenylephrine (for PCA), and the relaxant effects of kinins (0.3 nM to 10 µM) were determined. RESULTS:All tested kinin peptides concentration-dependently relaxed precontracted CM and PCA in a biphasic manner. The concentrations of the peptides (BK [bradykinin], Hyp3-BK, Lys-BK, Met-Lys-BK, and Des-Arg9-BK) yielding half-maximal relaxation (EC50) of PCA via the high potency receptor site ranged between 0.1 nM and 9.0 nM (n = 5-6). Precontracted PCA relaxation by the same kinins via the low potency receptor site occurred with EC50 values of 39.5-399 nM (n = 5-6). In contrast, EC50 values for these peptides relaxing the precontracted CM strips were 0.25-30 nM (high potency receptor site) and 100-3,000 nM (low potency receptor site) (n = 3-6). Precontracted PCA rings were significantly (generally P < 0.005-0.05) more sensitive to the relaxant activity of the kinin peptides than precontracted CM strips. CONCLUSIONS:The tissue relaxant agonist profile of the kinins in both precontracted CM strips and PCA rings, as judged by the 2-site-fit data, indicated the involvement of both B1- and B2-receptor subtypes.

Both hydrogen sulfide and endocannabinoids can protect the neural retina from toxic insults under in vitro and in vivo conditions. Purpose: The aim of the present study was two-fold: (a) to examine the neuroprotective action of cannabinoids [methanandamide and 2-arachidonyl glycerol (2-AG)] against hydrogen peroxide (H2O2)-induced oxidative damage in the isolated bovine retina and (b) to evaluate the role of endogenously biosynthesized hydrogen sulfide (H2S) in the inhibitory actions of cannabinoids on the oxidative stress in the bovine retina. Methods: Isolated neural retinas from cows were exposed to oxidative damage using H2O2 (100 µM) for 10 min. When used, tissues were pretreated with methanandamide (1 nM–100 nM) and 2-AG (1–10 µM) for 30 min before a 10 min treatment with H2O2 (100 µM). In some experiments, retinas were pretreated with inhibitors of the biosynthesis of H2S [cystathionine β-synthase/cystathionine γ-lyase (CBS/CSE), aminooxyacetic acid, AOAA 30 µM, or 3-mercaptopyruvate sulfurtransferase (3MST), α-keto-butyric acid, KBA 1 mM] and the CB1-receptor antagonist, AM251 (100 nM) for 30 min before treatment with methanandamide (1 nM–100 µM). Enzyme immunoassay measurement of 8-epi PGF2α (8-isoprostane) levels was performed to assess lipid peroxidation in retinal tissues. Results: In the presence of H2O2 (100 µM), methanandamide (1 nM–100 µM) and 2-AG (1–10 µM) significantly (p < 0.001) blocked the H2O2-induced elevation in 8-isoprostane levels in the isolated bovine retina. In the presence of the CB1 antagonist AM251 (100 nM), the effect of methanandamide (1 nM) on the H2O2-induced 8-isoprostane production was significantly (p < 0.001) attenuated. While AOAA (30 µM) had no significant (p > 0.05) effect on the inhibition of H2O2-induced oxidative stress elicited by methanandamide, KBA (1 mM) reversed the neuroprotective action of methanandamide. Conclusions: The cannabinoids, methanandamide and 2-AG can prevent H2O2-induced oxidative stress in the isolated bovine retina. The neuroprotective actions of cannabinoids are partially dependent upon the activation of the CB1 receptors and endogenous production of H2S via the 3-MST/CAT pathway.

Background: We have evidence that hydrogen sulfide (H2S)-releasing compounds can reduce intraocular pressure in normotensive and glaucomatous rabbits by increasing the aqueous humor (AH) outflow through the trabecular meshwork. Since H2S has been reported to possess neuroprotective actions, the prevention of retinal ganglion cell loss is an important strategy in the pharmacotherapy of glaucoma. Consequently, the present study aimed to investigate the neuroprotective actions of H2S-releasing compounds against hydrogen peroxide (H2O2)-induced oxidative stress in an isolated bovine retina. Materials and Methods: The isolated neural retinae were pretreated with a substrate for H2S biosynthesis called L-cysteine, with the fast H2S-releasing compound sodium hydrosulfide, and with a mitochondrial-targeting H2S-releasing compound, AP123, for thirty minutes before a 30-min oxidative insult with H2O2 (100 µM). Lipid peroxidation was assessed via an enzyme immunoassay by measuring the stable oxidative stress marker, 8-epi PGF2α (8-isoprostane), levels in the retinal tissues. To determine the role of endogenous H2S, studies were performed using the following biosynthesis enzyme inhibitors: aminooxyacetic acid (AOAA, 30 µM); a cystathione-β-synthase/cystathionine-γ-lyase (CBS/CSE) inhibitor, α–ketobutyric acid (KBA, 1 mM); and a 3-mercaptopyruvate-s-sulfurtransferase (3-MST) inhibitor, in the absence and presence of H2S-releasing compounds. Results: Exposure of the isolated retinas to H2O2 produced a time-dependent (10–40 min) and concentration-dependent (30–300 µM) increase in the 8-isoprostane levels when compared to the untreated tissues. L-cysteine (10 nM–1 µM) and NaHS (30 –100 µM) significantly (p < 0.001; n = 12) prevented H2O2-induced oxidative damage in a concentration-dependent manner. Furthermore, AP123 (100 nM–1 µM) attenuated oxidative H2O2 damage resulted in an approximated 60% reduction in 8-isoprostane levels compared to the tissues treated with H2O2 alone. While AOAA (30 µM) and KBA (1 mM) did not affect the L-cysteine evoked attenuation of H2O2-induced oxidative stress, KBA reversed the antioxidant responses caused by AP123. Conclusions: In conclusion, various forms of H2S-releasing compounds and the substrate, L-cysteine, can prevent H2O2-induced lipid peroxidation in an isolated bovine retina.

In the original publication [...].

Abstract ID 100665Poster Board 530PurposeThere is evidence that the slow-releasing hydrogen sulfide compound, GYY 4137 (GYY) can lower intraocular pressure (IOP) in normotensive rabbits (Salvi A et al. Ocul Pharmacol Ther. 2016) and protect retinal ganglion cells (RGC) from glaucomatous loss in rats (Liu H et al., IOVS, 2017) in vivo. In the present study, we investigated the pharmacological actions of GYY on IOP and retinal degeneration in carbomer-induced ocular hypertensive (OHT), male New Zealand albino rabbits, in vivo.MethodsOHT was induced by bilateral intracameral injection of carbomer (100 μL; 0.3%) into rabbit eyes. Acute studies: GYY (2%; 50 μL) was topically instilled into one eye while the contralateral eye received normal saline (control). IOP was assessed hourly using a pneumotonometer (Model 30 classic) until baseline IOP was attained. Chronic studies: 15 animals were divided into 3 groups (n = 5 each) and treated twice daily as follows: Group I: normotensive, saline-treated; Group II: OHT-saline treated; Group III: OHT-GYY-treated. IOP was measured daily for up to 25 days post-OHT induction. Animals were then humanely euthanized and retina isolated for morphological (H&E) and cell death (TUNEL) assays.ResultsIntracameral injection of carbomer into rabbit eyes induced OHT that was sustained for up to 25 days (maximum elevation of 123.62 ± 19.25% (p > 0.001) 9 days post-OHT induction). Acute: GYY (2%) reduced IOP up to 9 h, with a maximum reduction of 29.27 ± 5.23 % (p > 0.001) observed after 5 h. Chronic: GYY reduced IOP in the treated eye, producing a significant (p<0.05) reduction of 31.85 ± 1.47% 9 days post-OHT induction. Moreover, OHT-induced loss in RGC (18.51%; p<0.05) and Photoreceptor:Outer Nuclear Layer ratio (37.66%; p<0.001) was reduced by GYY.ConclusionGYY reduced IOP and protected the retina from OHT-induced damage in rabbit eyes, in vivo.1R15EY032274-01

Background: Bradykinin is an endogenously produced nonapeptide with many physiological and pathological functions that are mediated by two pharmacologically defined receptor subtypes, B1- and B2-receptors. Current studies sought to characterize the functional bradykinin (BK) receptors present in freshly isolated bovine ciliary muscle (BCM) using an organ-bath tissue contraction system. Methods: Cumulative longitudinal isometric tension responses of BCM strips (4–5 mm) were recorded before and after the addition of test compounds to BCM strips hooked up to an isometric strain gauge transducer system. Results: BK and its analogs (7–11 concentrations) contracted BCM in a biphasic concentration-dependent manner. The first high affinity/potency phase accounted for 40–60% of the maximal contraction by each of BK (potency, EC50 = 0.9 ± 0.3 nM), Lys-BK (EC50 = 0.7 ± 0.1 nM), Met-Lys-BK (EC50 = 1 ± 0.1 nM), Hyp3-BK (EC50 = 1 ± 0.2 nM), RMP-7 (EC50 = 3.5 ± 0.5 nM), and Des-Arg9-BK (EC50 = 10 ± 0.4nM) (mean ± SEM, n = 3–8). The second lower activity phase of contraction potency values for these peptides ranged between 100 nM and 3 µM. In the presence of a selective B1-receptor antagonist (R715; 0.1–10 µM), the concentration–response curves to Des-Arg9-BK (B1-receptor agonist) were still observed, indicating activation of B2-receptors by this kinin. Likewise, when B2-receptors were completely blocked by using a B2-selective antagonist (WIN-64338; 1–10 µM), BK still induced BCM contraction, now by stimulating B1-receptors. Conclusions: This agonist/antagonist profile of BCM receptors indicated the presence of both B1- and B2-receptor subtypes, both being responsible for contracting this smooth muscle. The BCM kinin receptors may be involved in changing the shape of the ocular lens to influence accommodation, and since the ciliary muscle is attached to the trabecular meshwork through which aqueous humor drains, endogenously released kinins may regulate intraocular pressure.

Background: Hydrogen sulfide (H2S)-releasing compounds can reduce intraocular pressure in normotensive rabbits by increasing aqueous humor (AH) outflow through the trabecular meshwork. In the present study, we investigated the contribution of endogenous H2S and the role of intramurally generated prostaglandins in the observed increase in AH outflow facility in an ex vivo porcine ocular anterior segment model. Material and Methods: Porcine ocular anterior segment explants were perfused with Dulbecco’s Modified Eagle’s Medium maintained at 37 °C and gassed with 5% CO2 and 95% air under an elevated pressure of 15 mmHg for four hours. Perfusates from the anterior segment explants were collected and immediately assayed for their H2S and prostaglandin E2 content. Results: Elevating perfusion pressure from 7.35 to 15 mm Hg significantly (p < 0.001) increased H2S concentration in the perfusate from 0.4 ± 0.1 to 67.6 ± 3.6 nM/µg protein. In the presence of an inhibitor of cystathionine ß-synthase/cystathionine γ-lyase, aminooxyacetic acid (AOAA, 30 µM), or an inhibitor of 3-mercaptopyruvate sulfurtransferase, α-ketobutyric acid (KBA, 1 mM), the effects of elevated pressure on H2S levels in the perfusate was significant (p < 0.001). Furthermore, flurbiprofen (30 µM) and indomethacin (10 µM) attenuated the elevated pressure-induced increase in H2S levels in the perfusate. Interestingly, elevating perfusion pressure had no significant effect on PGE2 concentrations in the perfusate. While the inhibition of H2S biosynthesis by AOAA or KBA did not affect PGE2 levels in perfusate, flurbiprofen (30 µM) caused a significant (p < 0.05) decrease in the concentration of PGE2 under conditions of elevated perfusion pressure. Conclusions: We conclude that the elevated perfusion pressure-induced increase in H2S concentrations depends upon the endogenous biosynthesis of H2S and intramurally produced prostaglandins in the porcine anterior segment explants. While the concentration of PGE2 in the perfusate under elevated perfusion pressure was unaffected by pretreatment with inhibitors of H2S biosynthesis, it was reduced in the presence of an inhibitor of cyclooxygenase.

Ocular health has emerged as one of the major issues of global health concern with a decline in quality of life in an aging population, in particular and rise in the number of associated morbidities and mortalities. One of the chief reasons for vision impairment is oxidative damage inflicted to photoreceptors in rods and cone cells by blue light as well as UV radiation. The scenario has been aggravated by unprecedented rise in screen-time during the COVID and post-COVID era. Lutein and Zeaxanthin are oxygenated carotenoids with proven roles in augmentation of ocular health largely by virtue of their antioxidant properties and protective effects against photobleaching of retinal pigments, age-linked macular degeneration, cataract, and retinitis pigmentosa. These molecules are characterized by their characteristic yellow-orange colored pigmentation and are found in significant amounts in vegetables such as corn, spinach, broccoli, carrots as well as fish and eggs. Unique structural signatures including tetraterpenoid skeleton with extensive conjugation and the presence of hydroxyl groups at the end rings have made these molecules evolutionarily adapted to localize in the membrane of the photoreceptor cells and prevent their free radical induced peroxidation. Apart from the benefits imparted to ocular health, lutein and zeaxanthin are also known to improve cognitive function, cardiovascular physiology, and arrest the development of malignancy. Although abundant in many natural sources, bioavailability of these compounds is low owing to their long aliphatic backbones. Under the circumstances, there has been a concerted effort to develop vegetable oil-based carriers such as lipid nano-emulsions for therapeutic administration of carotenoids. This review presents a comprehensive update of the therapeutic potential of the carotenoids along with the challenges in achieving an optimized delivery tool for maximizing their effectiveness inside the body.

There are limited studies that report the physiological levels of H2S in the eye. The currently available UV/Vis methods lack the required sensitivity and precision. Hence, the purpose of this study was to develop and validate a sensitive and robust pre-column derivatization LC-MS/MS method to measure changes in H2S levels in tissues from isolated porcine eyes. H2S was derivatized and an LC-MS/MS method was developed to monitor the derivatized product, Sulfide-dibimane (Sdb) using a reverse phase Waters Acquity BEH C18 column (1.7 mu m, 2.1 x 100 mm). H2S quantification was performed using multiple-ion reaction monitoring (MRM) in positive mode, with the transitions of m/z 415.0 -* m/z 223.0 for Sdb and m/z 353.0 -* m/z 285.0 for internal standard (griseofulvin). This method provided a suitable way to quantify H2S and was then successfully adapted to measure H2S levels in isolated porcine iris-ciliary body tissues previously treated in the presence or absence of varying concentrations of lipopolysaccharide (LPS, 5-100 ng/ml), a pro-inflammatory agent. Isolated iris-ciliary bodies (ICB) from porcine eyes were cut into quadrants of approximately 50 mg and homogenized using a 1:3 volume of homogenizing buffer. H2S in the supernatant was then derivatized with monobromobimane and quantified.

Background: Hydrogen sulfide (H2S) is an endogenous transmitter with the potential to regulate aqueous humor dynamics and protect retinal neurons from degeneration. The aim of the present study was two-fold: (a) to evaluate the release of H2S from two polysulfides, diallyl disulfide (DADS), and diallyl trisulfide (DATS); and (b) to investigate their ocular hypotensive actions in normotensive male and female rabbits in the presence and absence of GSH. Materials and Methods: H2S was quantified hourly for up to 6 h using a H2S-Biosensor (World Precision Instruments, Sarasota, Fl). Intraocular pressure (IOP) was assessed in normotensive New Zealand Albino rabbits using a pneumotonometer (model 30 classic; Reichert Ophthalmic Instruments, Depew, NY, USA). Results: In the presence of GSH, there was an increase in the in vitro release of H2S produced by DADS and DATS. Both DADS and DATS also caused a dose-dependent reduction in IOP in male and female rabbits, in both treated and untreated eyes. For instance, in male animals, the presence of GSH (3% and 5%) significantly (p < 0.05, n = 5) enhanced the ocular hypotensive action of DADS (2%) and DATS (2%) from 14.02 ± 2.89% to 18.67 ± 5.6% and from 16.22 ± 3.48 to 23.62 ± 5.79%, respectively. Conclusions: GSH enhanced both H2S release and ocular hypotensive action of the polysulfides in a manner that was dependent on the number of sulfur atoms present in each polysulfide. Furthermore, female animals were less sensitive to the IOP-lowering action of the polysulfides, when compared to their male counterparts.

Lutein, zeaxanthin, and mesozeaxanthin, collectively termed as macular pigments, are key carotenoids integral to optimized central vision of the eye. Therefore, nutraceuticals and functional foods have been developed commercially using carotenoid rich flowers, such as marigold and calendula or single celled photosynthetic algae, such as the Dunaliella. Industrial formulation of such products enriched in macular pigments have often suffered from serious bottlenecks in stability, delivery, and bioavailability. The two chief factors largely responsible for decreasing the shelf-life have been solubility and oxidation of these pigments owing to their strong lipophilic nature and presence of conjugated double bonds. In this regard, oil-based formulations have often been found to be more suitable than powder-based formulations in terms of shelf life and targeted delivery. In some cases, addition of phenolic acids in the formulations have also augmented the product value by enhancing micellization. In this regard, a novel proprietary formulation of these pigments has been developed in our laboratory utilizing marigold extracts in a colloidal solution of extra virgin olive oil and canola oil fortified with antioxidants like thyme oil, tocopherol, and ascorbyl palmitate. This review article presents an updated insight into the stability and bioavailability of industrially manufactured macular carotenoids together with their safety and solubility issues.