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AAV8-Mediated Angiotensin-Converting Enzyme 2 Gene Delivery Prevents Experimental Autoimmune Uveitis by Regulating MAPK, NF-?B and STAT3 Pathways.

ABSTRACT: Renin angiotensin system (RAS) is a key hormonal system which regulates the cardiovascular function and is implicated in several autoimmune diseases. With the discovery of the angiotensin-converting enzyme 2 (ACE2), a protective axis of RAS namely ACE2/Ang-(1-7)/Mas that counteracts the deleterious ACE/AngII/AT1R axis has been established. This axis is emerging as a novel target to attenuate ocular inflammation. However, the underlying molecular mechanisms remain unclear. We investigated the hypothesis that enhancing the activity of the protective axis of RAS by subretinal delivery of an AAV8 (Y733F)-ACE2 vector would protect against the ocular inflammation in experimental autoimmune uveitis (EAU) mice through regulating the local immune responses. Our studies demonstrated that increased ACE2 expression exerts protective effects on inflammation in EAU mouse by modulating ocular immune responses, including the differentiation of Th1/Th17 cells and the polarization of M1/M2 macrophages; whereas the systemic immune responses appeared not affected. These effects were mediated by activating the Ang-(1-7)/Mas and inhibiting the MAPK, NF-?B and STAT3 signaling pathways. This proof-of-concept study suggests that activation of ocular ACE2/Ang-(1-7)/Mas axis with AAV gene transfer modulates local immune responses and may be a promising, long-lasting therapeutic strategy for refractory and recurrent uveitis, as well as other inflammatory eye diseases.

SUBMITTER: Qiu Y

PROVIDER: S-EPMC4997264 | biostudies-literature | 2016 Aug

REPOSITORIES: biostudies-literature

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AAV8-Mediated Angiotensin-Converting Enzyme 2 Gene Delivery Prevents Experimental Autoimmune Uveitis by Regulating MAPK, NF-κB and STAT3 Pathways.

Qiu Yiguo Y Tao Lifei L Zheng Shijie S Lin Ru R Fu Xinyu X Chen Zihe Z Lei Chunyan C Wang Jiaming J Li Hongwei H Li Qiuhong Q Lei Bo B

Scientific reports 20160825

Renin angiotensin system (RAS) is a key hormonal system which regulates the cardiovascular function and is implicated in several autoimmune diseases. With the discovery of the angiotensin-converting enzyme 2 (ACE2), a protective axis of RAS namely ACE2/Ang-(1-7)/Mas that counteracts the deleterious ACE/AngII/AT1R axis has been established. This axis is emerging as a novel target to attenuate ocular inflammation. However, the underlying molecular mechanisms remain unclear. We investigated the hyp ...[more]

PMID: 27558087

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Project description:BackgroundRetinal inflammation is a devastating pathological process in ocular diseases. Functional impairment of retinal pigment epithelium (RPE) is associated with inflammatory retinal diseases. Enhancing the protective axis namely ACE2/Ang-(1-7)/Mas by activation of ACE2 presents anti-inflammatory properties. We investigated whether diminazene aceturate (DIZE), an angiotensin-converting enzyme 2 (ACE2) activator, prevented lipopolysaccharide (LPS)-induced inflammatory response by activating the protective axis and whether the effect was mediated by inhibiting the mitogen-activated protein kinase (MAPK) and the nuclear factor-κB (NF-κB) pathways.MethodsCell counting kit-8 (CCK-8) assay and real-time PCR were used to determine the optimum concentration and incubation time of DIZE. ARPE-19 cells and primary cultured human retinal pigment epithelia (hRPE) were incubated with or without 10 μg/mL DIZE for 6 h before stimulated with 5 μg/mL LPS for 24 h. The mRNA expression of inflammatory cytokines, AT1R, and AT2R was analyzed. The protein level of inflammatory cytokines, Ang II, and Ang-(1-7) was detected. Phosphorylation of p38 MAPK, extracellular signal-regulated kinase (ERK)1/2, c-Jun N-terminal kinase (JNK) and phosphorylated transcription inhibition factor-κB-α (p-IκB-α) were measured. Inhibitors of MAPKs and NF-κB were added to verify the involvement of these pathways. A small interfering RNA (siRNA) targeted to ACE2 and a selective Ang-(1-7) antagonist A779 was used to confirm the role of ACE2 and the involvement of ACE2/Ang-(1-7)/Mas axis.ResultsDIZE remarkably increased the expression of ACE2 and inhibited the expression of IL-6, IL-8, and MCP-1 at both mRNA and protein levels in both RPE cell lines stimulated with LPS. Inhibitors of p38, ERK1/2, JNK, and NF-κB significantly decreased LPS-induced overproduction of IL-6, IL-8, and MCP-1. DIZE reduced the expression of Ang II and AT1R, whereas increased Ang-(1-7). Furthermore, DIZE downregulated the phosphorylation of p38MAPK, ERK1/2, JNK, and the activation of NF-κB upon stimulation with LPS. Downregulating ACE2 and pre-treatment with A779 abrogated the effects of DIZE on production of cytokines, the expression of Ang II, Ang-(1-7), AT1R, phosphorylation of MAPKs and activation of NF-κB.ConclusionsDIZE inhibits LPS-induced inflammatory response by activating ACE2/Ang-(1-7)/Mas axis in human RPE cells. The protective effect is mediated by inhibiting the p38MAPK, ERK1/2, JNK, and NF-κB pathways.

Project description:PurposeUveitis is a common cause of vision loss. The renin angiotensin system (RAS), which plays a vital role in cardiovascular system, is a potent mediator of inflammation and has been implicated in the pathogenesis of uveitis. A newly identified axis of RAS, ACE2/Ang-(1-7)/Mas, has emerged as a novel target because it counteracts the deleterious effect of angiotensin II. The purpose of this study was to investigate the effect of endogenous ACE2 activation in preventing endotoxin-induced uveitis (EIU) in mice.MethodsACE2 activator diminazene aceturate (DIZE) was administered both systemically and locally. For systemic administration, female BALB/c mice received intraperitoneal injection of DIZE (60 mg/kg body weight [BW]) for 2 days prior to lipopolysaccharide (LPS) intravitreal injection (125 ng) to induce uveitis. For local study, DIZE was given at 0.5, 0.1, and 0 mg/mL as eyedrops six times per day for 2 days before LPS injection. The anterior segment of the mice was examined at 12, 24, 48, and 72 hours after LPS injection, and clinical scores were determined at the same time. Morphology and infiltrating inflammatory cells were evaluated after 24 hours. The mRNA levels of inflammatory cytokines were analyzed by real-time RT-PCR. ACE2 activity was determined using a self-quenching fluorescent substrate.ResultsAt 24 hours, the clinical score of mice treated with DIZE systemically was significantly lower (mean, ∼1.75) than the saline vehicle group (mean, ∼4) (P < 0.001). Histological examination showed 63.4% reduction of infiltrating inflammatory cells in the anterior segment and 57.4% reduction in the posterior segment of DIZE-treated eyes. The number of CD45(+) inflammatory cells in the vitreous of the DIZE-treated group was decreased (43.3%) compared to the vehicle group (P < 0.01). The mRNA levels of inflammatory cytokines were significantly reduced in the DIZE-treated group (P < 0.01, P < 0.001). The number of infiltrating inflammatory cells was also significantly reduced in eyes that received topical administration of DIZE: 73.8% reduction in the 0.5 mg/mL group and 51.7% reduction in the 0.1mg/mL group compared to the control group. DIZE treatment resulted in significantly increased ACE2 activity in the retina (P < 0.001).ConclusionsEndogenous ACE2 activation by DIZE has a preventive effect on LPS-induced ocular inflammation in the EIU mouse model. These results support the notions that RAS plays a role in modulating ocular immune response and that enhancing ACE2 provides a novel therapeutic strategy for uveitis.

Dataset Information

AAV8-Mediated Angiotensin-Converting Enzyme 2 Gene Delivery Prevents Experimental Autoimmune Uveitis by Regulating MAPK, NF-?B and STAT3 Pathways.

Publications

AAV8-Mediated Angiotensin-Converting Enzyme 2 Gene Delivery Prevents Experimental Autoimmune Uveitis by Regulating MAPK, NF-κB and STAT3 Pathways.

Similar Datasets

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