Inhibition of BRD4 decreases fibrous scarring after ischemic stroke in rats by inhibiting the phosphorylation of Smad2/3

Aims: Fibrous scarring may play a much more important role in preventing secondary expansion of tissue damage and hindering repair and regeneration than glial scarring after central nervous system (CNS) injury. However, relatively little is known about how fibrous scars form and how fibrous scar formation is regulated after CNS injury. Bromodomain-containing protein 4 (BRD4) is involved in fibrosis in many tissues, and transforming growth factor-β1 (TGF-β1)/SMAD2/3 signaling is one of the critical pathways of fibrosis. However, it is unclear whether and how BRD4 affects fibrous scar formation after ischemicbraininjury. In the present study, whether BRD4 can regulate the formation of fibrous scars after ischemic stroke via TGF-β1/SMAD2/3 signaling was assessed.

Materials and methods: Primary meningeal fibroblasts isolated from neonatal SD rats were treated with TGF-β1, SB431542 (a TGF-β1 receptor inhibitor) and JQ1 (a small-molecule BET inhibitor that can also inhibit BRD4). BRD4 was knocked down in adult Sprague-Dawley (SD) rats by using adenovirus before middle cerebral artery occlusion/reperfusion (MCAO/R) injury. The proliferation and migration of meningeal fibroblasts in vitro were evaluated with the Cell Counting Kit-8 (CCK-8) assay and scratch test, respectively. Neurological function was assessed with Longa scores, modified Bederson Scores and modified neurological severity scores (mNSSs). The infarct volume was assessed with TTC staining. The protein expression of synaptophysin (SY), BRD4, SMAD2/3, p-Smad2/3, α-smooth muscle actin (α-SMA), collagen-1 (COL1) and fibronectin (FN) in vivo and in vitro was examined with immunocytochemistry, immunofluorescence, and Western blotting.

Key findings: BRD4 expression was upregulated in a TGF-β1-induced meningeal fibroblast fibrosis model and was downregulated by the TGF-β1 receptor inhibitor SB431542 in vitro. JQ1, a small-molecule BET inhibitor, inhibited BRD4 and decreased TGF-β1-induced meningeal fibroblast proliferation, migration and activation. Furthermore, MCAO/R injury induced fibrosis and upregulated BRD4 expression in the cerebral infarct center. BRD4 knockdown by adenovirus inhibited fibrous scarring, promoted synaptogenesis, decreased the infarct volume, and improved neurological function after MCAO/R injury. Moreover, inhibition of BRD4, either by JQ1 in vitro or adenovirus in vivo, decreased the phosphorylation of SMAD2/3.

Conclusions: This study is the first to indicate that inhibition of BRD4 delays fibrous scarring after ischemic stroke through mechanisms involving the phosphorylation of SMAD2/3.

BRD4; MCAO/R injury; fibroblast; fibrotic scar.