Angiotensin-converting enzyme 2 activator diminazene aceturate prevents lipopolysaccharide-induced inflammation by inhibiting MAPK and NF-κB pathways in human retinal pigment epithelium

Abstract

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.

The Angiotensin-(1-7)/Mas Axis Counteracts Angiotensin II-Dependent and -Independent Pro-inflammatory Signaling in Human Vascular Smooth Muscle Cells

“…In the present study, the stimulation of Ang-(1-7)/Mas axis revealed itself as an alternate strategy to minimize the impact of IL-1β on human VSMC pro-inflammatory activation. This is in line with other reports showing that in the context of neuroinflammation the pharmacological activation of the ACE2/Ang-(1-7) can reduce the inflammatory response of human retinal pigment epitelial cells to lipopolysaccharide (Tao et al, 2016). Furthermore, Ang-(1-7) may prevent the inflammation induced in astrocytes by physical stressors, such as radiation (Moore et al, 2013).…”

Role of Tissue Renin-angiotensin System and the Chymase/angiotensin-( 1-12) Axis in the Pathogenesis of Diabetic Retinopathy

“…Recent study shows that diabetic microvascular complications can be prevented and reversed by overexpressing the ACE2 level in diabetic rodent retina [72]. Another study reported that the inflammatory response induced by lipopolysaccharide in retinal pigment epithelium (RPE) cells were decreased with diminazene aceturate (DIZE, an ACE2 activator) treatment [73]. They also found that DIZE reduced the expression of Ang II and AT1R, whereas it increased the Ang-(1-7) level in RPE cells.…”

Overexpression of Angiotensin-Converting Enzyme 2 Ameliorates Amyloid β-Induced Inflammatory Response in Human Primary Retinal Pigment Epithelium

“…67 In line with our previous studies, activation of the ACE2/Ang-(1-7)/Mas axis downregulated phosphorylation of MAPKs and NF-jB signaling. 33,36 These data indicated that ACE2 may ameliorate the inflammatory response by regulating MAPKs and NF-jB pathways.…”

“…[27][28][29][30] Most components of RAS, including ACE2/Ang-(1-7)/Mas axis, have been confirmed in various tissues and organs, including the eye. 21,31,32 Our previous study showed that activation of ACE2 alleviated the lipopolysaccharide (LPS)-induced inflammatory response in both human RPE cells 33 and in endotoxin-induced uveitis (EIU) mouse/rat models. 34,35 Moreover, the most recent study from our laboratory demonstrated that adeno-associated virus (AAV)-mediated ACE2 gene delivery alleviated the ocular inflammation in experimental autoimmune uveitis (EAU) mouse model by activating the local ACE2/Ang-(1-7)/Mas axis.…”

“…The concentration of A779 was chosen as previously described. 33 Both hRPE and ARPE-19 cells, whether normal or transfected, were pretreated (or not) with 10 lM Ang-(1-7) antagonist A779 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 2 hours and then stimulated with Ab1-42 (1 lM per well) for 48 hours.…”