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Soluble RAGE attenuates AngII-induced endothelial hyperpermeability by disrupting HMGB1-mediated crosstalk between AT1R and RAGE.

ABSTRACT: Increased endothelial permeability, one of the earliest signs of endothelial dysfunction, is associated with the development of cardiovascular diseases such as hypertension and atherosclerosis. Recent studies suggest that the receptor for advanced glycation end products (RAGE) regulates endothelial permeability in inflammation. In the present study, we investigated the regulatory mechanism of RAGE in endothelial hyperpermeability induced by angiotensin II (Ang II), a well-known inflammatory mediator, and the potential therapeutic effect of soluble RAGE (sRAGE), a decoy receptor for RAGE ligands. For in vitro studies, Ang II-treated human umbilical vein endothelial cells (HUVECs) were treated with siRNA specific to either RAGE or sRAGE to disrupt RAGE-mediated signaling. Endothelial permeability was estimated using FITC-labeled dextran 40 and a resistance meter. To evaluate intercellular junction disruption, VE-cadherin expression was examined by western blotting and immunocytochemistry. Ang II increased the expression of the Ang II type 1 receptor (AT1R) and RAGE, and this increase was inhibited by sRAGE. sRAGE prevented Ang II-induced VE-cadherin disruption in HUVECs. For in vivo studies, Ang II-infused, atherosclerosis-prone apolipoprotein E knockout mice were utilized. Endothelial permeability was assessed by Evans blue staining of the aorta. Ang II increased endothelial barrier permeability, and this effect was significantly attenuated by sRAGE. Our data demonstrate that blockade of RAGE signaling using sRAGE attenuates Ang II-induced endothelial barrier permeability in vitro and in vivo and indicate the therapeutic potential of sRAGE in controlling vascular permeability under pathological conditions.

SUBMITTER: Jeong J

PROVIDER: S-EPMC6802637 | biostudies-literature | 2019 Sep

REPOSITORIES: biostudies-literature

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Soluble RAGE attenuates AngII-induced endothelial hyperpermeability by disrupting HMGB1-mediated crosstalk between AT1R and RAGE.

Jeong Jisu J Lee Jiye J Lim Juyeon J Cho Soyoung S An Soyoung S Lee Myungeun M Yoon Nara N Seo Miran M Lim Soyeon S Park Sungha S

Experimental & molecular medicine 20190927 9

Increased endothelial permeability, one of the earliest signs of endothelial dysfunction, is associated with the development of cardiovascular diseases such as hypertension and atherosclerosis. Recent studies suggest that the receptor for advanced glycation end products (RAGE) regulates endothelial permeability in inflammation. In the present study, we investigated the regulatory mechanism of RAGE in endothelial hyperpermeability induced by angiotensin II (Ang II), a well-known inflammatory medi ...[more]

PMID: 31562296

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Project description:BackgroundThe blood-spinal cord barrier (BSCB) is composed of a monolayer of endothelium linked with tight junctions and extracellular matrix (ECM)-rich basement membranes and is surrounded by astrocyte foot processes. Endothelial permeability is regulated by interaction between endothelial cells and ECM proteins. Fibronectin (FN) is a principal ECM component of microvessels. Excessive FN deposition disrupts cell-cell adhesion in fibroblasts through β1 integrin ligation. To determine whether excessive FN deposition contributes to the disruption of endothelial integrity, we used an in vitro model of the endothelial monolayer to investigate whether the FN inhibitor pUR4 prevents FN deposition into the subendothelial matrix and attenuates endothelial leakage.MethodsTo correlate the effects of excessive FN accumulation in microvessels on BSCB disruption, spinal nerve ligation-which induces BSCB leakage-was applied, and FN expression in the spinal cord was evaluated through immunohistochemistry and immunoblotting. To elucidate the effects by which pUR4 modulates endothelial permeability, brain-derived endothelial (bEND.3) cells treated with tumor necrosis factor (TNF)-α were used to mimic a leaky BSCB. A bEND.3 monolayer was preincubated with pUR4 before TNF-α treatment. The transendothelial electrical resistance (TEER) measurement and transendothelial permeability assay were applied to assess the endothelial integrity of the bEND.3 monolayer. Immunofluorescence analysis and immunoblotting were performed to evaluate the inhibitory effects of pUR4 on TNF-α-induced FN deposition. To determine the mechanisms underlying pUR4-mediated endothelial permeability, cell morphology, stress fiber formation, myosin light chain (MLC) phosphorylation, and β1 integrin-mediated signaling were evaluated through immunofluorescence analysis and immunoblotting.ResultsExcessive FN was accumulated in the microvessels of the spinal cord after spinal nerve ligation; moreover, pUR4 inhibited TNF-α-induced FN deposition in the bEND.3 monolayer and maintained intact TEER and endothelial permeability. Furthermore, pUR4 reduced cell morphology alteration, actin stress fiber formation, and MLC phosphorylation, thereby attenuating paracellular gap formation. Moreover, pUR4 reduced β1 integrin activation and downstream signaling.ConclusionspUR4 reduces TNF-α-induced β1 integrin activation by depleting ECM FN, leading to a decrease in endothelial hyperpermeability and maintenance of monolayer integrity. These findings suggest therapeutic benefits of pUR4 in pathological vascular leakage treatment.

Project description:Antiphospholipid antibody syndrome (APS) is a systemic autoimmune disease characterized by arterial and/or venous thrombosis, pregnancy morbidity in the presence of circulating "anti-phospholipid antibodies" (aPL). One of the main target antigens of aPL is β2-glycoprotein I (β2-GPI). APS may occur as a primary syndrome or associated with Systemic Lupus Erythematosus (SLE). High Mobility Group Box 1 (HMGB1) is a nuclear non-histone protein which is secreted from different type of cells during activation and/or cell death and may act as a proinflammatory mediator through ligation to its receptors, including RAGE. There is accumulating evidence that HMGB1 contributes to the pathogenesis of inflammatory and autoimmune diseases, especially SLE. In a previous study we demonstrated increased serum levels of HMGB1 in both primary and secondary APS patients. In this work we analyzed: (i) in vitro whether anti-β2-GPI antibodies from APS patients may induce both a HMGB1 cellular relocation by activation of its putative receptor RAGE in platelets and monocytes and, (ii) ex vivo, serum levels of HMGB1/soluble RAGE (sRAGE) in APS patients and their possible correlation with clinical manifestations. Platelets and monocytes from healthy donors were incubated with affinity purified anti-β2-GPI antibodies. HMGB1 and RAGE expression were analyzed by Western Blot. Sera from 60 consecutive APS patients (primary or secondary), diagnosed according to the Sydney Classification Criteria, were enrolled. As a control, 30 matched healthy subjects were studied. Serum levels of HMGB1 and sRAGE were analyzed by Western Blot. In vitro results showed that anti-β2-GPI antibodies were able to induce RAGE activation and HMGB1 cellular relocation in both monocytes and platelets. HMGB1 and sRAGE serum levels were significantly increased in APS patients in comparison with healthy subjects (p<0.0001). Interestingly, APS patients with spontaneous recurrent abortion showed significantly higher levels of sRAGE; moreover, in APS patients a direct correlation between serum levels of HMGB1 and disease duration was detected. Our observations suggest that anti-β2-GPI antibodies may trigger RAGE activation and HMGB1 cellular relocation during APS. Monitoring these molecules serum levels may represent an useful tool to evaluate the pathogenesis and risk stratification of clinical manifestations in APS.

Dataset Information

Soluble RAGE attenuates AngII-induced endothelial hyperpermeability by disrupting HMGB1-mediated crosstalk between AT1R and RAGE.

Publications

Soluble RAGE attenuates AngII-induced endothelial hyperpermeability by disrupting HMGB1-mediated crosstalk between AT1R and RAGE.

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