Blockade of NOX2 and STIM1 signaling limits lipopolysaccharide-induced vascular inflammation
Rajesh Kumar Gandhirajan, … , Donald L. Gill, Muniswamy Madesh
Rajesh Kumar Gandhirajan, … , Donald L. Gill, Muniswamy Madesh
Published January 25, 2013
Citation Information: J Clin Invest. 2013;123(2):887-902. https://doi.org/10.1172/JCI65647.
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Research Article

Blockade of NOX2 and STIM1 signaling limits lipopolysaccharide-induced vascular inflammation

Abstract

During sepsis, acute lung injury (ALI) results from activation of innate immune cells and endothelial cells by endotoxins, leading to systemic inflammation through proinflammatory cytokine overproduction, oxidative stress, and intracellular Ca2+ overload. Despite considerable investigation, the underlying molecular mechanism(s) leading to LPS-induced ALI remain elusive. To determine whether stromal interaction molecule 1–dependent (STIM1-dependent) signaling drives endothelial dysfunction in response to LPS, we investigated oxidative and STIM1 signaling of EC-specific Stim1-knockout mice. Here we report that LPS-mediated Ca2+ oscillations are ablated in ECs deficient in Nox2, Stim1, and type II inositol triphosphate receptor (Itpr2). LPS-induced nuclear factor of activated T cells (NFAT) nuclear accumulation was abrogated by either antioxidant supplementation or Ca2+ chelation. Moreover, ECs lacking either Nox2 or Stim1 failed to trigger store-operated Ca2+ entry (SOCe) and NFAT nuclear accumulation. LPS-induced vascular permeability changes were reduced in EC-specific Stim1–/– mice, despite elevation of systemic cytokine levels. Additionally, inhibition of STIM1 signaling prevented receptor-interacting protein 3–dependent (RIP3-dependent) EC death. Remarkably, BTP2, a small-molecule calcium release–activated calcium (CRAC) channel blocker administered after insult, halted LPS-induced vascular leakage and pulmonary edema. These results indicate that ROS-driven Ca2+ signaling promotes vascular barrier dysfunction and that the SOCe machinery may provide crucial therapeutic targets to limit sepsis-induced ALI.

Authors

Rajesh Kumar Gandhirajan, Shu Meng, Harish C. Chandramoorthy, Karthik Mallilankaraman, Salvatore Mancarella, Hui Gao, Roshanak Razmpour, Xiao-Feng Yang, Steven R. Houser, Ju Chen, Walter J. Koch, Hong Wang, Jonathan Soboloff, Donald L. Gill, Muniswamy Madesh

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Figure 9

BTP2 attenuates LPS-induced lung inflammation and vascular endothelial integrity loss.

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BTP2 attenuates LPS-induced lung inflammation and vascular endothelial i...
C57BL/6 mice were challenged with LPS (1 mg/kg; i.p.), and BTP2 (1 mg/kg; i.p.) was delivered 2 hours after challenge. Saline was used as a vehicle. Samples were collected after 24 hours. (A) IL-1α, IL-1β, IL-2, IL-6, TNF-α, and G-CSF were measured in sera from mice. (B) Representative photomicrographs of H&E-stained lung sections. Original magnification, ×400. (C) Quantification of alveolar leukocytes infiltration. (D) Representative Western blot of ICAM-1 induction in ECs treated with LPS and/or BTP2. (E) Representative immunohistochemistry images of ICAM-1 using PE in lung sections from treated mice. Scale bar: 20 μm. (F) Quantification of ICAM-1 fluorescence intensity. (G and H) BAL protein content (G) and lung weight (H) changes are a functional measure of EC activation associated with increased vascular permeability. (I) A 0.1- to 0.15-ml bolus of FITC-dextran (70 kDa, 5%w/v) was injected into the animals via facial vein. Anesthetized animals were placed under an intravital 2-photon imaging system, and images were acquired. Vascular permeability was assessed based on fluorescence intensity in the extravascular space around 4–5 regions per mouse. Scale bar: 100 μm. (J) Quantification of extravascular FITC-dextran fluorescence. Cumulative data are the mean ± SEM of triplicates and are representative of 3 independent experiments with 3–6 per group as indicated. *P < 0.05, **P < 0.01, ***P < 0.001.