TIMP2 ameliorates blood-brain barrier disruption in traumatic brain injury by inhibiting Src-dependent VE-cadherin internalization
Jingshu Tang, … , Lei Wu, Ying Peng
Jingshu Tang, … , Lei Wu, Ying Peng
Published November 28, 2023
Citation Information: J Clin Invest. 2024;134(3):e164199. https://doi.org/10.1172/JCI164199.
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Research Article Neuroscience Vascular biology

TIMP2 ameliorates blood-brain barrier disruption in traumatic brain injury by inhibiting Src-dependent VE-cadherin internalization

Abstract

Blood-brain barrier (BBB) disruption is a serious pathological consequence of traumatic brain injury (TBI), for which there are limited therapeutic strategies. Tissue inhibitor of metalloproteinase-2 (TIMP2), a molecule with dual functions of inhibiting MMP activity and displaying cytokine-like activity through receptor binding, has been reported to inhibit VEGF-induced vascular hyperpermeability. Here, we investigate the ability of TIMP2 to ameliorate BBB disruption in TBI and the underlying molecular mechanisms. Both TIMP2 and AlaTIMP2, a TIMP2 mutant without MMP-inhibiting activity, attenuated neurological deficits and BBB leakage in TBI mice; they also inhibited junctional protein degradation and translocation to reduce paracellular permeability in human brain microvascular endothelial cells (ECs) exposed to hypoxic plus inflammatory insult. Mechanistic studies revealed that TIMP2 interacted with α3β1 integrin on ECs, inhibiting Src activation–dependent VE-cadherin phosphorylation, VE-cadherin/catenin complex destabilization, and subsequent VE-cadherin internalization. Notably, localization of VE-cadherin on the membrane was critical for TIMP2-mediated EC barrier integrity. Furthermore, TIMP2-mediated increased membrane localization of VE-cadherin enhanced the level of active Rac1, thereby inhibiting stress fiber formation. All together, our studies have identified an MMP-independent mechanism by which TIMP2 regulates EC barrier integrity after TBI. TIMP2 may be a therapeutic agent for TBI and other neurological disorders involving BBB breakdown.

Authors

Jingshu Tang, Yuying Kang, Yujun Zhou, Nianying Shang, Xinnan Li, Hongyue Wang, Jiaqi Lan, Shuai Wang, Lei Wu, Ying Peng

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Figure 5

TIMP2 interacts with α3β1 integrin to alleviate TBI-induced BBB disruption.

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TIMP2 interacts with α3β1 integrin to alleviate TBI-induced BBB disrupti...
(A) Experimental scheme for AAV-BR1 targeting cerebrovascular knockdown of α3 integrin, establishment of TBI, and analysis of BBB integrity in mice. (B–H) Mice were intravenously injected with AAV-BR1-FLAG-shITGA3 or AAV-BR1-FLAG-shNC. Three weeks after AAV injection, experimental TBI was established in mice, which were treated with 100 μg/kg rmTIMP2, 100 μg/kg rmAlaTIMP2, or PBS, and neurological function was assessed at 72 hours after TBI. (B) FLAG-shITGA3–transduced cells were analyzed by immunofluorescence staining using FLAG (green) and the endothelial marker CD31 (red) in brain cortex 21 days after AAV injection. Scale bars: 75 μm. (C) Microvasculature isolated from brain 21 days after AAV injection and subjected to Western blot analysis for the indicated proteins. Neurological function, including fall latency of accelerated rotarod (D), beam balance (E), and mNSS (F), was assessed at 72 hours after TBI (n = 9–11 per group). (G and H) Representative images (G) and quantification (H) of Evans blue dye leakage into the ipsilateral cerebral hemisphere at 72 hours after TBI (n = 9–10 per group). ##P < 0.01 vs. sham group, **P < 0.01 vs. model group, $P < 0.05, $$P < 0.01, by 1-way ANOVA.