Inflammation In Endotoxin-induced Uveitis Research Articles

Serum amyloid A aggravates endotoxin-induced ocular inflammation through the regulation of retinal microglial activation.

Serum amyloid A (SAA) are major acute-phase response proteins which actively participate in many inflammatory diseases. This study was designed to explore the function of SAA in acute ocular inflammation and the underlying mechanism. We found that SAA3 was upregulated in endotoxin-induced uveitis (EIU) mouse model, and it was primarily expressed in microglia. Recombinant SAA protein augmented intraocular inflammation in EIU, while the inhibition of Saa3 by siRNA effectively alleviated the inflammatory responses and rescued the retina from EIU-induced structural and functional damage. Further study showed that the recombinant SAA protein activated microglia, causing characteristic morphological changes and driving them further to pro-inflammatory status. The downregulation of Saa3 halted the amoeboid change of microglia, reduced the secretion of pro-inflammatory factors, and increased the expression of tissue-reparative genes. SAA3 also regulated the autophagic activity of microglial cells. Finally, we showed that the above effect of SAA on microglial cells was at least partially mediated through the expression and signaling of Toll-like receptor 4 (TLR4). Collectively, our study suggested that microglial cell-expressed SAA could be a potential target in treating acute ocular inflammation.

LY294002 attenuates inflammatory response in endotoxin-induced uveitis by downregulating JAK3 and inactivating the PI3K/Akt signaling

Context Uveitis is a prevalent inflammatory eye disease that damages the vision of patients and even leads to blindness. LY294002, an inhibitor of PI3K, was reported to suppress the inflammation and alleviate the progression of many diseases. However, the function of LY294002 in uveitis is unclear. Objective This study aimed to explore the function of LY294002 in endotoxin-induced uveitis (EIU). Materials and methods EIU rat models were established via a single intravitreal injection of LPS. At 24 h after LPS injection, the rats received LY294002 treatment for 14 days. The histopathology was observed by H&E staining. The concentration of proinflammatory cytokines in aqueous humor was tested by ELISA. The expression of proinflammatory cytokines in the iris ciliary body (ICB) and retina of EIU rats were detected by RT-qPCR. JAK3, PI3K, and Akt expression were assessed by RT-qPCR and western blotting. Translocation of Akt in rat retinal Müller cells (rMC-1) was evaluated by immunofluorescence staining. Results LY294002 alleviated ocular inflammation and decreased inflammatory cell infiltration in the anterior chamber, iris, ciliary body, vitreous cavity, and retina of EIU rats. LY294002 decreased the concentration of proinflammatory cytokines INF-γ, IL-17, IL-6, TNF-α, and IL-1β in aqueous humor and their expression in the ICB and retina of EIU rats. LY294002 downregulated JAK3 expression in EIU rats. LY294002 inhibited p-PI3K and p-Akt expression in EIU rats and restrained Akt translocation from cytoplasm to cell membrane in LPS-treated rMC-1 cells. Conclusion LY294002 ameliorates inflammation in EIU by downregulating JAK3 and inactivating the PI3K/Akt signaling.

Amelioration of Endotoxin-Induced Inflammatory Toxic Response by a Metal Chelator in Rat Eyes.

PurposeMetal ions play a key role in exacerbating toxicity associated with oxidative stress and inflammation. This study examines the effects of a formulation containing the metal chelator ethylenediaminetetraacetic acid (EDTA) and permeability enhancer methyl sulfonyl methane (MSM) on the early course of inflammation in endotoxin-induced uveitis (EIU). The proprietary MSM/EDTA formulation of Livionex, Inc., which was used for this study, is covered by several patents and pending patent applications.MethodsEIU was induced by using subcutaneous injection of lipopolysaccharide (LPS) into the thighs of Lewis rats. Treatment consisted of topical application to the eyes of either PBS or eye drops designated as ME that contain EDTA and MSM. Clinical signs of uveitis were monitored at 6 and 24 hours postinjection. Oxidative and inflammatory markers were evaluated by ELISA or immunohistochemistry.ResultsRats treated with ME showed fewer clinical signs of uveitis including reduced miosis, fibrinous exudates, and dilated blood vessels. The aqueous humor of treated rats contained fewer leukocytes, lower protein levels, and less PGE2. Formation of protein adducts with the lipid peroxidation end-product, 4-hydroxynonenal, expression of NF-κB, TNF-α, and MMP-9 were all reduced in rats treated with ME.ConclusionsOur results indicate that ME eye drops downregulate the ocular inflammatory response in LPS treated rats, suggesting that induction of EIU involves metal ions and chelation therapy with ME is a potential treatment for uveitis.

Green tea extract treatment alleviates ocular inflammation in a rat model of endotoxin-induced uveitis.

Green tea extract (GTE) ingested by rats exerted anti-oxidative activities in various ocular tissues as shown in our previous studies. The present work investigated anti-inflammatory effects of GTE on endotoxin-induced uveitis (EIU). EIU was generated in adult rats by a footpad injection of 1 mg/kg lipopolysaccharide (LPS). Oral administration of GTE (550 mg/kg) was given one, two or four times after LPS injection. Twenty-four hours later, LPS produced severe hyperemia and edema in the iris. Immunocytochemical examinations showed an accumulation of infiltrating cells in the aqueous humor that were immunopositive for cluster of differentiation 43 (CD43) and CD68, markers for leucocytes and macrophages, respectively. Analyses of the aqueous humor showed an increase in pro-inflammatory mediators including tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1). GTE treatments improved the clinical manifestations and reduced infiltrating cells and protein exudation in the aqueous humor, which were not observed under half dose of GTE (275 mg/kg). The number of CD68 positive macrophages residing in the iris and ciliary was also reduced. GTE suppressed production of TNF-α, IL-6 and MCP-1 in the aqueous humor, which was associated with a down-regulation of LPS receptor complex subunits, Toll-like receptor 4 (TLR-4) and CD14, and suppression of nuclear factor-kappa Bp65 (NF-κBp65) in the iris and ciliary body. Our findings show that GTE is a potent anti-inflammatory agent against the inflammation of EIU, and suggest a potential use in treatment of acute uveitis.

Effects of Instillation of Eyedrops Containing Disulfiram and Hydroxypropyl-β-Cyclodextrin Inclusion Complex on Endotoxin-Induced Uveitis in Rats

Objective: To investigate the anti-inflammatory effects of the instillation of disulfirum (DSF) eyedrops that enhance solubility using 2-hydroxypropyl-β-cyclodextrin (HPβCD) and hydroxypropylmethylcellulose (HPMC) on endotoxin-induced uveitis (EIU) in rats and mechanisms related to ocular inflammation.Methods: EIU was induced in male Lewis rats by subcutaneous injection of 200 μg lipopolysaccharide (LPS). DSF (0.125%, 0.25% and 0.5%) or commercially available 0.05% dexamethasone (Dexa) was topically applied to both eyes of rats 1 hour before, immediately after, and 1 and 2 hours after injection of LPS. The aqueous humor (AqH) was collected 24 hours after LPS injection, and the number of infiltrating cells, protein concentration, and levels of tumor necrosis factor-α (TNF-α), nitric oxide (NO) and prostaglandin E2 (PGE2) were determined. Immunohistochemical analysis of the iris ciliary body (ICB) cells was performed to determine the expression of activated nuclear factor κB (NF-κB), inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2).Results: The topical administration with DSF suppressed, in a dose-dependent manner, the number of inflammatory cells, the protein concentration, and the levels of NO, TNF-α and PGE2 in the AqH and improved the histologic status of the ocular tissue. The anti-inflammatory potency of 0.5% DSF treatment was as strong as that of 0.05% Dexa. Topical DSF treatment also suppressed the activated NF-κB 3 hours after LPS injection, and iNOS and COX-2 expression in the ICB 24 hours after LPS injection.Conclusions: The present results demonstrate that the topical instillation of DSF eyedrops suppresses the inflammation in EIU, suggesting a possible novel approach for the treatment of ocular inflammation.

Calcium channel blocker nilvadipine, but not diltiazem, inhibits ocular inflammation in endotoxin-induced uveitis

Calcium channel blockers (CCBs), widely used for hypertensive patients, have recently been shown to inhibit atherosclerosis by their antioxidative action. The aim of the present study was to examine whether the CCBs nilvadipine and diltiazem reduce ocular inflammation in endotoxin-induced uveitis (EIU). EIU was induced in male C57/B6 mice with a single intraperitoneal injection of lipopolysaccharide (LPS). The animals received intraperitoneal injections of either nilvadipine, diltiazem, or vehicle for 5 days before the LPS application. Twenty-four hours after EIU induction, adherent leukocytes to the retinal vasculature were counted with a concanavalin A lectin perfusion-labeling technique. The protein concentration in the aqueous humor was measured to assess blood-ocular barrier breakdown. Retinal levels of intercellular adhesion molecule (ICAM)-1 and monocyte chemotactic protein (MCP)-1 were analyzed by enzyme-linked immunosorbent assay. LPS-stimulated generation of superoxide in murine microvascular endothelial cells was examined with a nitroblue tetrazolium assay. Compared to vehicle treatment, application of nilvadipine, but not diltiazem, led to significant suppression of EIU-associated retinal leukocyte adhesion, together with anterior-chamber protein leakage, retinal expression of ICAM-1 and MCP-1, and LPS-induced superoxide generation in vitro. The CCB nilvadipine exercises an inhibitory effect on the pathogenesis of ocular inflammation through the suppression of inflammation-related molecules.

Inhibitory Effects of Oral Disulfiram on Endotoxin-Induced Uveitis in Rats

Purpose: Disulfiram (DSF) exhibits a wide variety of biological activities, including an anti-inflammatory action, on which we focused our attention. The aim of the present study was to investigate the effect of oral DSF on endotoxin-induced uveitis (EIU) in rats.Methods: We investigated its effect upon cellular infiltration and protein leakage, as well as on the concentration of tumor necrosis factor-α (TNF-α), nitric oxide (NO), and prostaglandin E2 (PGE2) in the anterior chamber. Some eyes were enucleated for histologic examination and immunohistochemical analysis. EIU was induced in male Lewis rats by a footpad injection of lipopolysaccharide (LPS). One hour before the LPS injection, either 250, 500, or 750 mg/kg DSF was administered orally. Twenty-four hours later, the aqueous humor was collected from both eyes, and the number of infiltrating cells and protein concentration in the aqueous humor were determined. Levels of TNF-α, NO, and PGE2 were determined by enzyme-linked immunosorbent assay. Immunohistochemical analysis in the iris ciliary body (ICB) cells was perfomed to determine the expression of activated nuclear factor kappa B (NF-κB), inducible-nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2).Results: The oral administration with DSF suppressed, in a dose-dependent manner, the number of inflammatory cells, the protein concentration, and the levels of TNF-α, NO, and PGE2 in the aqueous humor and improved the histiologic status of the ocular tissue. The expression of activated NF-κB-positive cells in the ICB was significantly inhibited by oral administrated with DSF 3 hr after the LPS injection. The LPS-induced increased expressions of iNOS and COX-2 proteins in the ICB were also inhibited by oral DSF 24 hr after LPS injection.Conclusions: The present results indicate that oral DSF suppresses the inflammation in EIU by inhibiting the NF-κB-dependent pathway and the subsequent production of pro-inflammatory mediators.

Prevention of Endotoxin-Induced Uveitis in Rats by Plant Sterol Guggulsterone

To investigate the anti-inflammatory effects of guggulsterone, an antioxidant and antitumor agent, in endotoxin-induced uveitis (EIU) in rats and to elucidate the underlying molecular mechanism or mechanisms related to ocular inflammation. EIU was induced by subcutaneous injection of lipopolysaccharide (LPS; 150 μg) into Lewis rats treated with guggulsterone (30 mg/kg body weight, intraperitoneally) or its carrier. After 24 hours the rats were killed, eyes were enucleated, and aqueous humor (AqH) was collected. Numbers of infiltrating cells and levels of matrix metalloproteinase-2 (MMP-2), nitric oxide (NO), and prostaglandin E(2) (PGE(2)) were determined in AqH by specific ELISAs. An antibody array was used to measure the expression of various inflammatory cytokines in AqH. The expression of MMP-2, iNOS, Cox-2, phospho-IκB, and phospho-NF-κB was determined immunohistochemically. Human primary nonpigment ciliary epithelial cells (HNPECs) were used to determine the in vitro efficacy of guggulsterone on the LPS-induced inflammatory response. Compared with control, the EIU rat eye AqH had a significantly higher number of infiltrating cells, total protein, and inflammatory markers, such as MMP-2, NO, and PGE(2), and the treatment of guggulsterone prevented EIU-induced increases. Guggulsterone also prevented the expression of MMP-2, iNOS, and Cox-2 proteins and of IκB and NF-κB in various eye tissues. Moreover, in cultured HNPECs, guggulsterone inhibited LPS-induced expression of inflammatory proteins. These results for the first time demonstrate that the plant sterol guggulsterone suppresses ocular inflammation in EIU, suggesting that the supplementation of guggulsterone could be a novel approach for the treatment of ocular inflammation.

A Novel Therapeutic Target in Inflammatory Uveitis: Transglutaminase 2 Inhibitor

PurposeOur goal was to investigate the effects of inhibition of transglutaminase 2 (TGase 2) on endotoxin-induced uveitis (EIU)MethodsEIU was induced in female Lewis rats by single footpad injections of 200 µg of lipopolysaccharide (LPS). TGase 2 inhibitors were administered intraperitoneally 30 minutes before and at the time of LPS administration. Rats were sacrificed 24 hours after injection, and the effects of the TGase 2 inhibitors were evaluated by the number of intraocular inflammatory cells present on histologic sections and by measuring the TGase 2 activity and TGase products in the aqueous humor (AqH). TGase 2 substrates were also assayed in AqH from uveitis patients.ResultsClinical indications of EIU, the number of cells present on histologic sections, and TGase 2 activity in AqH increased in a time-dependent manner, peaking 24 hours after LPS injection. Inflammation in EIU was significantly reversed by treatment with TGase inhibitors. A 23-kDa cross-linked TGase substrate was identified in the AqH from EIU rats and uveitis patients. MALDI-TOF analysis showed that this substrate in uveitis patients was human Ig kappa chain C region.ConclusionsTGase 2 activity and its catalytic product were increased in the AqH of EIU rats. TGase 2 inhibition attenuated the degree of inflammation in EIU. Safe and stable TGase inhibitors may have great potential for the treatment of inflammatory uveitis.

Arginase Activity Mediates Retinal Inflammation in Endotoxin-Induced Uveitis

Arginase has been reported to reduce nitric oxide bioavailability in cardiovascular disease. However, its specific role in retinopathy has not been studied. In this study, we assessed the role of arginase in a mouse model of endotoxin-induced uveitis induced by lipopolysaccharide (LPS) treatment. Measurement of arginase expression and activity in the retina revealed a significant increase in arginase activity that was associated with increases in both mRNA and protein levels of arginase (Arg)1 but not Arg2. Immunofluorescence and flow cytometry confirmed this increase in Arg1, which was localized to glia and microglia. Arg1 expression and activity were also increased in cultured Muller cells and microglia treated with LPS. To test whether arginase has a role in the development of retinal inflammation, experiments were performed in mice deficient in one copy of the Arg1 gene and both copies of the Arg2 gene or in mice treated with a selective arginase inhibitor. These studies showed that LPS-induced increases in inflammatory protein production, leukostasis, retinal damage, signs of anterior uveitis, and uncoupling of nitric oxide synthase were blocked by either knockdown or inhibition of arginase. Furthermore, the LPS-induced increase in Arg1 expression was abrogated by blocking NADPH oxidase. In conclusion, these studies suggest that LPS-induced retinal inflammation in endotoxin-induced uveitis is mediated by NADPH oxidase-dependent increases in arginase activity.

Prevention of Ocular Inflammation in Endotoxin-Induced Uveitis with Resveratrol by Inhibiting Oxidative Damage and Nuclear Factor–κB Activation

Resveratrol is known as one of the antioxidant polyphenols contained in red wine and grape skin. The purpose of the present study was to investigate the role of resveratrol in ocular inflammation in endotoxin-induced uveitis (EIU). EIU was induced in male C57/B6 mice at the age of 6 weeks by a single intraperitoneal injection of lipopolysaccharide (LPS). Animals had received oral supplementation of resveratrol at the doses of 5, 50, 100, or 200 mg/kg for 5 days until LPS injection. Twenty-four hours after LPS administration, leukocyte adhesion to the retinal vasculature was examined with a concanavalin A lectin perfusion-labeling technique. Retinal and retinal pigment epithelium (RPE)-choroidal levels of intercellular adhesion molecule (ICAM)-1, monocyte chemotactic protein (MCP)-1, and 8-hydroxy-2'-deoxyguanosine (8-OHdG) and nuclear translocation of nuclear factor (NF)-kappaB p65 were evaluated by enzyme-linked immunosorbent assay. Retinal and RPE-choroidal activities of silent information regulator two ortholog (SIRT) 1 were measured by deacetylase fluorometric assay. Resveratrol pretreatment led to significant and dose-dependent suppression of leukocyte adhesion to retinal vessels of EIU mice compared with vehicle application. Protein levels of MCP-1 and ICAM-1 in the retina and the RPE-choroid of EIU animals were significantly reduced by resveratrol administration. Importantly, resveratrol-treated animals showed significant decline of retinal 8-OHdG generation and nuclear NF-kappaB P65 translocation, both of which were upregulated after EIU induction. RPE-choroidal SIRT1 activity, reduced in EIU animals, was significantly augmented by treatment with resveratrol. Resveratrol prevented EIU-associated cellular and molecular inflammatory responses by inhibiting oxidative damage and redox-sensitive NF-kappaB activation.

Prevention of Endotoxin-Induced Uveitis in Rats by Benfotiamine, a Lipophilic Analogue of Vitamin B1

To study the amelioration of ocular inflammation in endotoxin-induced uveitis (EIU) in rats by benfotiamine, a lipid-soluble analogue of thiamine. EIU in Lewis rats was induced by subcutaneous injection of lipopolysaccharide (LPS) followed by treatment with benfotiamine. The rats were killed 3 or 24 hours after LPS injection, eyes were enucleated, aqueous humor (AqH) was collected, and the number of infiltrating cells, protein concentration, and inflammatory marker levels were determined. Immunohistochemical analysis of eye sections was performed to determine the expression of inducible-nitric oxide synthase (iNOS), cyclooxygenase (Cox)-2, protein kinase C (PKC), and transcription factor NF-kappaB. Infiltrating leukocytes, protein concentrations, and inflammatory cytokines and chemokines were significantly elevated in the AqH of EIU rats compared with control rats, and benfotiamine treatment suppressed these increases. Similarly increased expression of inflammatory markers iNOS and Cox-2 in ciliary body and retinal wall was also significantly inhibited by benfotiamine. The increased phosphorylation of PKC and the activation of NF-kappaB in the ciliary body and in the retinal wall of EIU rat eyes were suppressed by benfotiamine. These results suggest that benfotiamine suppresses oxidative stress-induced NF-kappaB-dependent inflammatory signaling leading to uveitis. Therefore, benfotiamine could be used as a novel therapeutic agent for the treatment of ocular inflammation, especially uveitis.

Anti-inflammatory effect of angiotensin type 1 receptor antagonist on endotoxin-induced uveitis in rats

Angiotensin II type 1 (AT1) receptor-antagonists are widely used for treatment of hypertension. Recent studies have demonstrated a protective effect of renin angiotensin system (RAS) antagonism against immune-mediated inflammatory diseases such as myocarditis, chronic allograft rejection, antiglomerular basement membrane nephritis, colitis, and arthritis. However, only a few reports have demonstrated the effect of RAS in ocular inflammatory conditions. The purpose of this study was to investigate the anti-inflammatory effect of a selective AT1 receptor antagonist, losartan, on endotoxin-induced uveitis (EIU) and compare the effect on experimental autoimmune uveoretinitis (EAU). To induce EIU, 7-week-old Lewis rats were injected subcutaneously with 200 microg lipopolysaccharide (LPS). Losartan was administered intravenously at the same time. The aqueous humor was collected from eyes 24 h after LPS injection. The number of infiltrating cells, protein concentration, and levels of tumor necrosis factor (TNF)-alpha and monocyte chemoattractant protein-1 (MCP-1) in the aqueous humor were determined. The collected eyes were immunohistochemically stained with monoclonal antibody for activated nuclear factor (NF)-kappaB. To induce EAU, C57BL/6 mice (6-8 weeks old) were immunized with human interphotoreceptor retinoid binding protein (hIRBP)-derived peptide emulsified in complete Freund's adjuvant (CFA) and concomitantly injected with purified Bordetella pertussis toxin (PTX). Clinical severity of EAU and T cell proliferative response were analyzed. Losartan significantly suppressed the development of EIU. Numbers of aqueous cells of control EIU rats, those from EIU rats treated with 1 or 10 mg/kg of losartan were 75.3+/-45.6 x 10(5), 27.9+/-8.1 x 10(5), or 41.3+/-30.9 x 10(5) cells/ml respectively (p<0.01 vs control). Aqueous protein, TNF-alpha, and MCP-1 levels were also significantly decreased in a manner dependent on the amount of losartan administered (p<0.01). Treatment of EIU rats with losartan suppressed activation of NF-kappaB at the iris ciliary body. Thus, the suppressive effect of losartan on ocular inflammation in EIU appeared to result from down-regulation of NF-kappaB activation and reduction of inflammatory cytokine production. On the other hand, in the EAU model, neither the clinical score nor the antigen-specific T cell proliferative response was significantly influenced by the treatment with losartan. The present findings indicate that RAS may be involved in the acute inflammation of the eye, but not in T cell-dependent ocular autoimmunity. Antagonism of the RAS may be a potential prophylactic strategy for treatment of the human acute ocular inflammation.

Aldose Reductase Inhibition Prevents Endotoxin-Induced Uveitis in Rats

The purpose of the present study was to elucidate the role of the polyol pathway enzyme aldose reductase (AR) in the mediation of ocular inflammation in a rat model of endotoxin-induced uveitis (EIU). EIU was induced by a subcutaneous injection of 200 microg lipopolysaccharide (LPS) in male Lewis rats treated with the AR inhibitor, zopolrestat (25 mg/kg body weight, intraperitoneally) or its carrier. The rats were killed 24 hours after LPS injection, the eyes were enucleated immediately, and aqueous humor (AqH) was collected. The number of infiltrating cells, protein concentration, and levels of nitric oxide (NO), tumor necrosis factor (TNF)-alpha, and prostaglandin E(2) (PGE(2)) in the AqH were determined. Immunohistochemical analysis was performed in paraformaldehyde-fixed eye sections by staining with antibodies against iNOS, COX-2, TNF-alpha, NF-kappaB, and AR. The levels of reactive oxygen species (ROS) in rat eye sections were determined by dihydroethidium (hydroethidine) fluorescence staining. In the EIU rat eye AqH, both the number of infiltrating cells and protein concentrations of the inflammatory markers, TNF-alpha, NO, and PGE(2) were significantly higher than in the control rats, and inhibition of AR by zopolrestat suppressed the LPS-induced increases. The LPS-induced increased expression of AR, TNF-alpha, iNOS, and COX-2 proteins in the ciliary body, corneal epithelium, and retinal wall was also significantly inhibited by zopolrestat. Furthermore, AR inhibition prevented the LPS-induced increased levels of ROS and activation of NF-kappaB in the ciliary body, corneal epithelium, and retinal wall of the rat eye. AR inhibition also prevented the LPS-induced activation of NF-kappaB and expression of COX-2 and iNOS in the human monocyte cell line U-937. The results indicate that AR inhibition suppresses the inflammation in EIU by blocking the expression and release of inflammatory markers in ocular tissues, along with the attenuation of NF-kappaB activation. This finding suggests that AR inhibition could be a novel therapeutic target for the treatment of uveitis and associated ocular inflammation.

Role of Nonproteolytically Activated Prorenin in Pathologic, but Not Physiologic, Retinal Neovascularization

Recently, it was revealed that the inhibition of nonproteolytic activation of prorenin led to significant suppression of ocular inflammation in endotoxin-induced uveitis. The purpose of the present study was to investigate whether nonproteolytically activated prorenin plays a role in ischemia-induced retinal neovascularization. C57BL/6 neonatal mice were reared in an 80% concentration of oxygen from postnatal (P) day 7 to P12, followed by room-air breathing to P17 to induce ischemia-initiated retinal neovascularization. Tissue localization of activated prorenin and prorenin receptor was examined by immunohistochemistry. Animals received intraperitoneal injections of handle-region peptide (HRP), a decoy peptide corresponding to the handle region of prorenin, which inhibits prorenin receptor-mediated upregulation of the renin-angiotensin system (RAS). A concanavalin A lectin perfusion-labeling technique was used to evaluate the areas of physiologic and pathologic retinal new vessels and the number of leukocytes adhering to the vasculature. Retinal mRNA expression and protein levels of intercellular adhesion molecule (ICAM)-1, vascular endothelial growth factor (VEGF), VEGF receptor (VEGFR)-1, and VEGFR-2 were examined by RT-PCR and ELISA. Retinal vessels in ischemic retinopathy eyes were positive for activated prorenin and prorenin receptor. Pathologic, but not physiologic, retinal neovascularization was significantly attenuated in HRP-treated mice compared with vehicle- or control peptide-treated animals. The number of adherent leukocytes was also significantly reduced. Retinal mRNA expression and protein levels of ICAM-1, VEGF, VEGFR-1, and VEGFR-2 in ischemic retinopathy were also significantly suppressed by the application of HRP. The present findings suggest that nonproteolytic activation of prorenin selectively promotes pathologic, but not physiologic, retinal neovascularization through the inflammatory processes related to pathologic neovascularization.

Suppression of Ocular Inflammation in Endotoxin-Induced Uveitis by Inhibiting Nonproteolytic Activation of Prorenin

A recent study revealed that angiotensin receptor signaling mediates ocular inflammation and neovascularization. It was also found that prorenin undergoes nonproteolytic activation leading to upregulation of the renin-angiotensin system (RAS) when prorenin receptor interacts specifically with the handle region of prorenin. The purpose of the present study was to elucidate the role of the receptor-dependent nonproteolytic activation of prorenin in ocular inflammation in endotoxin-induced uveitis (EIU). EIU was induced in Long-Evans rats by a single intraperitoneal injection of 100 microg lipopolysaccharide (LPS). Tissue localization of total prorenin, prorenin receptor, and activated prorenin in the EIU retina was examined by immunohistochemistry. To inhibit the prorenin receptor-mediated upregulation of the RAS, a decoy handle-region peptide (HRP) was intraperitoneally administered 24 hours before and immediately after the injection of LPS. Twenty-four hours after LPS injection, leukocyte adhesion to the retinal vasculature was evaluated with a concanavalin A lectin perfusion-labeling technique. In addition, leukocyte infiltration into the vitreous cavity and protein concentration in the anterior chamber were also measured. Retinal mRNA and protein levels of intercellular adhesion molecule (ICAM)-1, interleukin (IL)-6, and C-C chemokine ligand (CCL) 2/monocyte chemotactic protein (MCP)-1 were examined by RT-PCR and ELISA. Retinal vessels in rats with EIU were strongly positive for total prorenin, prorenin receptor, and activated prorenin. Systemic treatment with HRP resulted in dose- and time-dependent inhibition of the leukocyte adhesion and infiltration and the protein leakage, all of which were increased by the induction of EIU. Retinal mRNA expression and protein levels of ICAM-1, CCL2/MCP-1 and IL-6, induced in rats with EIU, were also significantly suppressed with application of HRP. These findings demonstrate for the first time that nonproteolytically activated prorenin plays a significant role in the development of ocular inflammation in the EIU model. The present study suggests the potential use of HRP, a decoy peptide binding to the prorenin receptor, as a therapeutic agent to reduce ocular inflammation.

Captopril suppresses inflammation in endotoxin-induced uveitis in rats

Captopril is an inhibitor of angiotensin-converting enzyme (ACE) that is largely used in the treatment of cardiovascular diseases. Several previous studies have demonstrated that captopril exhibits a wide variety of biological activities, including an anti-inflammatory action, on which we focused our attention. The aim of the present study was to investigate the efficacy of captopril on endotoxin induced uveitis (EIU) in rats. We investigated its effect upon cellular infiltration and protein leakage, as well as on the concentration of tumor necrosis factor-α (TNF-α), nitric oxide (NO), prostaglandin E2 (PGE2), monocyte chemoattractant protein-1 (MCP-1) in the anterior chamber. In addition, we checked its effect on activation of nuclear factor kappa B (NF-κB) in iris and ciliary body (ICB) cells in vivo. EIU was induced in male Lewis rats by a footpad injection of lipopolysaccharide (LPS). One hour after the LPS inoculation, either 1 mg/kg, 10 mg/kg or 100 mg/kg captopril were injected intravenously. 24 h later, the aqueous humor was collected from both eyes, and the number of infiltrating cells and protein concentration in the aqueous humor were determined. Levels of TNF-α, PGE2, NO and MCP-1 were determined by enzyme-linked immunosorbent assay. On some eyes, after enucleation, immunohistochemical staining with a monoclonal antibody against activated NF-κB was performed. Captopril treatment significantly decreased the inflammatory cells infiltration, the level of protein, concentrations of TNF-α, PGE2, NO and MCP-1 in the aqueous humor. The number of activated NF-κB-positive cells was lower in ICB of the rats treated with captopril 3 h after the LPS injection. The present results indicate that captopril suppresses the inflammation in EIU by inhibiting the NF-κB-dependent pathway and the subsequent production of pro-inflammatory mediators.

Angiotensin II and Reactive Oxygen Species

Antioxidants & Redox SignalingVol. 7, No. 9-10 Forum EditorialAngiotensin II and Reactive Oxygen SpeciesMarta Ruiz-Ortega and Alberto OrtizMarta Ruiz-OrtegaSearch for more papers by this author and Alberto OrtizSearch for more papers by this authorPublished Online:22 Aug 2005https://doi.org/10.1089/ars.2005.7.1258AboutSectionsPDF/EPUB ToolsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail FiguresReferencesRelatedDetailsCited ByThe C-Terminal Module IV of Connective Tissue Growth Factor, Through EGFR/Nox1 Signaling, Activates the NF-κB Pathway and Proinflammatory Factors in Vascular Smooth Muscle Cells Raúl R. Rodrigues-Diez, Ana Belen Garcia-Redondo, Macarena Orejudo, Raquel Rodrigues-Diez, Ana Maria Briones, Enrique Bosch-Panadero, Gyorgy Kery, Janos Pato, Alberto Ortiz, Mercedes Salaices, Jesus Egido, and Marta Ruiz-Ortega17 December 2014 | Antioxidants & Redox Signaling, Vol. 22, No. 1Integrin-linked kinase plays a key role in the regulation of angiotensin II-induced renal inflammation10 March 2014 | Clinical Science, Vol. 127, No. 1Angiotensin II downregulates ACE2-mediated enhancement of MMP-2 activity in human cardiofibroblastsBiochemistry and Cell Biology, Vol. 91, No. 6The Protective Effects of Cobra Venom from Naja naja atra on Acute and Chronic NephropathyEvidence-Based Complementary and Alternative Medicine, Vol. 2013Antioxidants in Kidney Diseases: The Impact of Bardoxolone MethylInternational Journal of Nephrology, Vol. 2012Lisinopril attenuates renal oxidative injury in l-NAME-induced hypertensive rats11 April 2011 | Molecular and Cellular Biochemistry, Vol. 352, No. 1-2Intracardiac renin–angiotensin system and myocardial repair/remodeling following infarctionJournal of Molecular and Cellular Cardiology, Vol. 48, No. 3HMG-CoA Reductase Inhibitors Decrease Angiotensin II–Induced Vascular FibrosisHypertension, Vol. 50, No. 2Captopril suppresses inflammation in endotoxin-induced uveitis in ratsExperimental Eye Research, Vol. 83, No. 3 Volume 7Issue 9-10Sep 2005 InformationCopyright 2005, Mary Ann Liebert, Inc.To cite this article:Marta Ruiz-Ortega and Alberto Ortiz.Angiotensin II and Reactive Oxygen Species.Antioxidants & Redox Signaling.Sep 2005.1258-1260.http://doi.org/10.1089/ars.2005.7.1258Published in Volume: 7 Issue 9-10: August 22, 2005PDF download

Suppression of Ocular Inflammation in Endotoxin-Induced Uveitis by Blocking the Angiotensin II Type 1 Receptor

To examine whether the angiotensin II type 1 receptor (AT1-R) signaling plays a role in ocular inflammation in endotoxin-induced uveitis (EIU). EIU was induced in C57BL/6 mice by a single intraperitoneal injection of 150 mug lipopolysaccharide (LPS). Tissue localization, mRNA expression, and protein levels of AT1-R in murine retinas were examined by immunohistochemistry, RT-PCR, and Western blot analyses, respectively. Telmisartan, an AT1-R antagonist widely used as an antihypertensive agent, was administered intraperitoneally at a dose of 10 mg/kg daily for 5 days until the injection of LPS. Twenty-four hours after administration, leukocyte adhesion to the retinal vasculature was evaluated with a concanavalin A lectin perfusion-labeling technique. Retinal mRNA and protein levels of intercellular adhesion molecule (ICAM)-1 were examined by RT-PCR and ELISA, respectively. Protein concentration and inflammatory cells in the aqueous humor were also measured. Retinal vessels were positive for AT1-R. In mice with EIU, retinal AT1-R mRNA and protein levels were significantly increased when compared to the normal control. EIU animals also showed significant increases in the number of inflammatory cells infiltrating the anterior chamber and adhering to the retinal vessels and in retinal ICAM-1 levels. Administration of telmisartan to EIU mice resulted in significant suppression of retinal ICAM-1 expression and leukocyte adhesion and infiltration compared with vehicle treatment. Protein concentration in the aqueous humor of telmisartan-treated EIU mice tended to be lower than that of vehicle-treated EIU mice, but the difference was not statistically significant. AT1-R signaling blockade inhibited retinal ICAM-1 upregulation and leukocyte adhesion and infiltration in the EIU model. These results suggest the potential use of an AT1-R antagonist as a therapeutic agent to reduce ocular inflammation.

Immunopharmacological analysis of endotoxin-induced uveitis in the rat

Footpad injection of endotoxin causes exclusive ocular inflammation in the rat. In order to clarify its physiopathologic mechanism, we studied the effect of different treatments on endotoxin-induced uveitis (EIU). Salmonella endotoxin was injected into the footpads of Lewis rats. 18 hr later. inflammation was assessed by evaluating proteins and cells in the anterior chamber; arachidonic acid (AA) metabolites, prostaglandin E 2 and leukotriene B 4, as well as substance P were measured by radioimmunoassay, and Ia-(MHC class II)-antigen expression in ciliary body was assessed by immunohistochemistry. The effect of inhibitors of phospholipase A 2 (EPC), of lipoxygenase (azelastine) and of cyclo-oxygenase (diclofenac), as well as dexamethasone, cyclosporine (CsA) and anti-Ia antibody, were evaluated on these parameters. Phospholipase A2 inhibitor EPC and dexamethasone were most effective on inflammation: they also reduced AA metabolites very effectively and prevented Ia-expression. Lipoxygenase and cyclo-oxygenase inhibitors were partially effective on inflammation and on AA metabolites but failed to prevent Ia-expression. Immunosuppressive treatments (CsA and anti-Ia-antibody) also reduced inflammation. Our findings suggest that inflammation mediators initiate inflammation in EIU, Ia-Ag-expression is secondarily produced by mediators leading to additional inflammation due to immune mediated mechanisms.