Upregulation of C Terminus of Hsc70-Interacting Protein Attenuates Apoptosis and Procoagulant Activity and Facilitates Brain Repair After Traumatic Brain Injury.

Hao Chen,Yan Guo,Dianxu Yang,Zhiming Xu,Hengli Tian,Shiming Ju,Lixia Xue,Yao Jing

doi:10.3389/fnins.2020.00925

Abstract

Traumatic brain injury (TBI) could highly induce coagulopathy through breaking the dynamic balance between coagulation and fibrinolysis systems, which may be a major contributor to the progressive secondary injury cascade that occurs after TBI. Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) inhibition is reported to exert neuroprotection in TBI, making it a potential regulatory target involved in TBI-induced coagulation disorder. PTEN level is controlled in a major way by E3 ligase-mediated degradation through the ubiquitin–proteasome system. The C terminus of Hsc70-interacting protein (CHIP) has been shown to regulate proteasomal degradation and ubiquitination level of PTEN. In the present study, CHIP was overexpressed and knocked down in mouse brain microvascular endothelial cells (bEnd.3) and tissues during the early phase of TBI. In vitro cell proliferation, cell apoptosis, migration capacity, and invasion capacity were determined. The changes of procoagulant and apoptosis molecules after TBI were also detected as well as the micrangium density and blood–brain barrier permeability after in vivo TBI. In vitro results demonstrated that CHIP overexpression facilitated bEnd.3 cell proliferation, migration, and invasion and downregulated cell apoptosis and the expressions of procoagulant molecules through promoting PTEN ubiquitination in a simulated TBI model with stretch-induced injury treatment. In vivo experiments also demonstrated that CHIP overexpression suppressed post-TBI apoptosis and procoagulant protein expressions, as well as increased microvessel density, reduced hemorrhagic injury, and blood–brain barrier permeability. These findings suggested that the upregulation of CHIP may attenuate apoptosis and procoagulant activity, facilitate brain repair, and thus exerts neuroprotective effects in TBI.

Highlights

Traumatic brain injury (TBI) is an important public health problem worldwide
To generate stable expression and interference constructions, polymerase chain reaction (PCR)-amplified mouse C terminus of Hsc70-interacting protein (CHIP) and short hairpin RNA1, shRNA2, shRNA3, and shRNA4 were cloned into lentivirus expression vectors, which contained green fluorescent protein to indicate transfection efficiency, detailed information for shRNA is provided in Table 1. bEnd.3 cells were seeded into a 12-well plate at 5 × 104 cells and infected with lentivirus at a multiplicity of infection of 100
The results showed that CHIP overexpression (CHIP-OE) slightly attenuated cell apoptosis of bEnd.3 cells after stretchinduced injury treatment; the apoptosis rates of normal control (NC)-OE cells and CHIP-OE were 1.991 and 0.072%, respectively (p < 0.01), whereas the cell apoptosis of injured bEnd.3 cells was induced after CHIP interference (Figures 2B–D)

Summary

Introduction

Traumatic brain injury (TBI) is an important public health problem worldwide. It is a major cause of morbidity and mortality, which is greatly contributed by the concomitant of coagulation disorders to brain injury (Zhang et al, 2012). In our previous clinical study, the mentioned coagulopathy parameters (elevated D-dimer, abnormal PT, and thrombocytopenia), or disseminated intravascular coagulation scores, which reflect thrombus formation, were each independently associated with an increased risk of PTCI, further indicating that TIC was associated with post-traumatic cerebral ischemia (Chen et al, 2013)

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Results

Conclusion