Endothelial C3a receptor mediates vascular inflammation and blood-brain barrier permeability during aging
Nicholas E. Propson, Ethan R. Roy, Alexandra Litvinchuk, Jörg Köhl, Hui Zheng
Nicholas E. Propson, Ethan R. Roy, Alexandra Litvinchuk, Jörg Köhl, Hui Zheng
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Research Article Aging

Endothelial C3a receptor mediates vascular inflammation and blood-brain barrier permeability during aging

Abstract

Dysfunction of immune and vascular systems has been implicated in aging and Alzheimer disease; however, their interrelatedness remains poorly understood. The complement pathway is a well-established regulator of innate immunity in the brain. Here, we report robust age-dependent increases in vascular inflammation, peripheral lymphocyte infiltration, and blood-brain barrier (BBB) permeability. These phenotypes were subdued by global inactivation and by endothelial cell–specific ablation of C3ar1. Using an in vitro model of the BBB, we identified intracellular Ca2+ as a downstream effector of C3a/C3aR signaling and a functional mediator of vascular endothelial cadherin junction and barrier integrity. Endothelial C3ar1 inactivation also dampened microglia reactivity and improved hippocampal and cortical volumes in the aging brain, demonstrating a crosstalk between brain vasculature dysfunction and immune cell activation and neurodegeneration. Further, prominent C3aR-dependent vascular inflammation was also observed in a tau-transgenic mouse model. Our studies suggest that heightened C3a/C3aR signaling through endothelial cells promotes vascular inflammation and BBB dysfunction and contributes to overall neuroinflammation in aging and neurodegenerative disease.

Authors

Nicholas E. Propson, Ethan R. Roy, Alexandra Litvinchuk, Jörg Köhl, Hui Zheng

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Figure 5

C3a-mediated barrier disruption is dependent on Ca2+ mobilization and alters VE-cadherin through cytoskeletal activation.

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C3a-mediated barrier disruption is dependent on Ca2+ mobilization and al...
(A) Schematic of TEER analysis using a coculture of astrocytes and endothelial cells. (B) TEER values in cocultures treated with vehicle, C3a, C3a with C3aRA, or IL-1β for 0, 1, 4, 12, and 24 hours. (C) Quantification of percentage reduction of TEER at 24 hours from treatments recorded in B. (D) TEER values in cocultures treated with vehicle, C3a, or C3a with C3aRA or BAPTA-AM over 24 hours. (E) Quantification of the percentage of reduction in TEER at 24 hours from the treatments in D. All TEER experiments were performed 2 times with duplicates and normalized to time-point control wells of cell-free membranes. (F) Representative immunofluorescence images of human brain microvascular endothelial cells (HBMECs) treated with vehicle, C3a, or a combination of C3a plus C3aRA (C3a/C3aRA), BAPTA-AM (C3a/BAPTA), or W7 (C3a/W7) for 2 hours and stained with anti-pMLC and anti–VE-cadherin antibodies. (G) Quantification of pMLC or VE-cadherin MFI showed an increase in pMLC but not VE-cadherin (n = 7 areas from 3 replicates of 250–300 cells/condition). (H) Representative immunofluorescence images of HBMECs treated as stated in F using anti-pMLC and anti–VE-cadherin antibodies 24 hours after treatment. (K) Quantification of pMLC or VE-cadherin MFI showed normalized pMLC levels but decreased VE-cadherin (n = 7 areas from 3 replicates of 250–300 cells/condition). All data represent the mean ± SEM. Analysis was performed on average percentage decrease in TEER using 1-way ANOVA with Tukey’s post hoc test (*P < 0.05, **P < 0.01, ***P < 0.001). Scale bar: 10 μm.