Neovascularization and vascular remodeling are functionally important for brain repair after stroke. We show that neutrophils accumulate in the peri-infarct cortex during all stages of ischemic stroke. Neutrophils producing intravascular and intraparenchymal neutrophil extracellular traps (NETs) peak at 3–5 days. Neutrophil depletion reduces blood-brain barrier (BBB) breakdown and enhances neovascularization at 14 days. Peptidylarginine deiminase 4 (PAD4), an enzyme essential for NET formation, is upregulated in peri-ischemic brains. Overexpression of PAD4 induces an increase in NET formation that is accompanied by reduced neovascularization and increased BBB damage. Disruption of NETs by DNase 1 and inhibition of NET formation by genetic ablation or pharmacologic inhibition of PAD increases neovascularization and vascular repair and improves functional recovery. Furthermore, PAD inhibition reduces stroke-induced STING-mediated production of IFN-β, and STING knockdown and IFN receptor-neutralizing antibody treatment reduces BBB breakdown and increases vascular plasticity. Collectively, our results indicate that NET release impairs vascular remodeling during stroke recovery.

Intracerebral hemorrhage associated with thrombolytic therapy with tissue plasminogen activator (tPA) in acute ischemic stroke continues to present a major clinical problem. Here, we report that infusion of tPA resulted in a significant increase in markers of neutrophil extracellular traps (NETs) in the ischemic cortex and plasma of mice subjected to photothrombotic middle cerebral artery occlusion. Peptidylarginine deiminase 4 (PAD4), a critical enzyme for NET formation, is also significantly upregulated in the ischemic brains of tPA-treated mice. Blood–brain barrier (BBB) disruption after ischemic challenge in an in vitro model of BBB was exacerbated after exposure to NETs. Importantly, disruption of NETs by DNase I or inhibition of NET production by PAD4 deficiency restored tPA-induced loss of BBB integrity and consequently decreased tPA-associated brain hemorrhage after ischemic stroke. Furthermore, either DNase I or PAD4 deficiency reversed tPA-mediated upregulation of the DNA sensor cyclic GMP-AMP (cGAMP) synthase (cGAS). Administration of cGAMP after stroke abolished DNase I–mediated downregulation of the STING pathway and type 1 interferon production and blocked the antihemorrhagic effect of DNase I in tPA-treated mice. We also show that tPA-associated brain hemorrhage after ischemic stroke was significantly reduced in cGas−/− mice. Collectively, these findings demonstrate that NETs significantly contribute to tPA-induced BBB breakdown in the ischemic brain and suggest that targeting NETs or cGAS may ameliorate thrombolytic therapy for ischemic stroke by reducing tPA-associated hemorrhage.

Stroke is a leading cause of long‐lasting disability in humans. However, currently there are still no effective therapies available for promoting stroke recovery. Recent studies have shown that the adult brain has the capacity to regenerate neurons after stroke. Although this neurogenic response may be functionally important for brain repair after injury, the mechanisms underlying stroke‐induced neurogenesis are not known. Caspase‐3 is a major executioner and has been identified as a key mediator of neuronal death in the acute stage of stroke. Recently, however, accumulating data indicate that caspase‐3 also participates in various biological processes that do not cause cell death. Here, we show that cleaved caspase‐3 was increased in newborn neuronal precursor cells (NPCs) in the subventricular zone (SVZ) and the dentate gyrus during the period of stroke recovery, with no evidence of apoptosis. We observed that cleaved caspase‐3 was expressed by NPCs and limited its self‐renewal without triggering apoptosis in cultured NPCs from the SVZ of ischemic mice. Moreover, we revealed that caspase‐3 negatively regulated the proliferation of NPCs through reducing the phosphorylation of Akt. Importantly, we demonstrated that peptide inhibition of caspase‐3 activity significantly promoted the proliferation and migration of SVZ NPCs and resulted in a significant increase in subsequent neuronal regeneration and functional recovery after stroke. Together, our data identify a previously unknown caspase‐3‐dependent mechanism that constrains stroke‐induced endogenous neurogenesis and should revitalize interest in targeting caspase‐3 for treatment of stroke. Stem Cells 2014;32:473–486

Blood–brain barrier (BBB) breakdown and inflammation occurring at the BBB have a key, mainly a deleterious role in the pathophysiology of ischemic stroke. Neddylation is a ubiquitylation-like pathway that is critical in various cellular functions by conjugating neuronal precursor cell-expressed developmentally down-regulated protein 8 (NEDD8) to target proteins. However, the roles of neddylation pathway in ischemic stroke remain elusive. Here, we report that NEDD8 conjugation increased during acute phase after ischemic stroke and was present in intravascular and intraparenchymal neutrophils. Inhibition of neddylation by MLN4924, also known as pevonedistat, inactivated cullin-RING E3 ligase (CRL), and reduced brain infarction and improved functional outcomes. MLN4924 treatment induced the accumulation of the CRL substrate neurofibromatosis 1 (NF1). By using virus-mediated NF1 silencing, we show that NF1 knockdown abolished MLN4924-dependent inhibition of neutrophil trafficking. These effects were mediated through activation of endothelial P-selectin and intercellular adhesion molecule-1 (ICAM-1), and blocking antibodies against P-selectin or anti–ICAM-1 antibodies reversed NF1 silencing-induced increase in neutrophil infiltration in MLN4924-treated mice. Furthermore, we found that NF1 silencing blocked MLN4924-afforded BBB protection and neuroprotection through activation of protein kinase C δ (PKCδ), myristoylated alanine-rich C-kinase substrate (MARCKS), and myosin light chain (MLC) in cerebral microvessels after ischemic stroke, and treatment of mice with the PKCδ inhibitor rottlerin reduced this increased BBB permeability. Our study demonstrated that increased neddylation promoted neutrophil trafficking and thus exacerbated injury of the BBB and stroke outcomes. We suggest that the neddylation inhibition may be beneficial in ischemic stroke.

Background: Growth differentiation factor 11 (GDF11), a member of transforming growth factor-β (TGF-β) superfamily, was shown to rejuvenate cardiac and skeletal muscle function and to improve cerebral vasculature and neurogenesis in old mice. However, recent experimental data reported that raising GDF11 levels inhibited skeletal muscle regeneration and had no effect on cardiac hypertrophy. Our aim was to investigate the effects of GDF11 on brain repair during the recovery phase after stroke. Methods: Mice were subjected to distal middle cerebral artery occlusion, and recombinant GDF11 (rGDF11) was injected intraperitoneally once a day during days 7–13 after stroke. Neuronal precursor cells (NPCs) proliferation and angiogenesis were assayed at 14 days. Neuronal regeneration was assayed at 42 days. The beam-walking test and CatWalk were used to evaluate behavioral functions. Downstream pathways of GDF11 were also investigated. Results: GDF11 was upregulated in the ipsilateral peri-infarct cortex and subventricular zone (SVZ) at 14 days after stroke. Treatment with rGDF11 enhanced the number of newborn NPCs and endothelial cells, microvascular length and area, and brain capillary perfusion. Western blots showed that rGDF11 upregulated brain-derived neurotrophic factor (BDNF) and increased the levels of proangiogenic factor angiopoietin-2 (Ang-2) and phosphorylation of vascular endothelial growth factor receptor-2 (VEGFR-2). We also found that rGDF11 upregulated the transcription factors Smad2 and Smad3 phosphorylation, but these activations were blocked by a TGF-β receptor inhibitor SB431542. Moreover, rGDF11-induced angiogenic remodeling and NPCs proliferation were reversed by injection of SB431542, suggesting that GDF11 may exert its effect via the TGF-β/Smad2/3 signaling pathway. Finally, treating mice with rGDF11 resulted in a significant increase in neuronal regeneration and functional recovery. Conclusion: GDF11 promoted neurogenesis and angiogenesis and contributed to functional recovery after stroke in mice.