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.

Key Points• ADAMTS13 controls key steps of vascular remodeling during stroke recovery.• Recombinant ADAMTS13 enhances ischemic neovascularization and vascular repair.Angiogenic response is essential for ischemic brain repair. The von Willebrand factor (VWF)-cleaving protease disintegrin and metalloprotease with thrombospondin type I motif, member 13 (ADAMTS13) is required for endothelial tube formation in vitro, but there is currently no in vivo evidence supporting a function of ADAMTS13 in angiogenesis. Here we show that mice deficient in ADAMTS13 exhibited reduced neovascularization, brain capillary perfusion, pericyte and smooth muscle cell coverage on microvessels, expression of the tight junction and basement membrane proteins, and accelerated blood-brain barrier (BBB) breakdown and extravascular deposits of serum proteins in the peri-infarct cortex at 14 days after stroke. Deficiency of VWF or anti-VWF antibody treatment significantly increased microvessels, perfused capillary length, and reversed pericyte loss and BBB changes in Adamts13 2/2 mice. Furthermore, we observed that ADAMTS13 deficiency decreased angiopoietin-2 and galectin-3 levels in the isolated brain microvessels, whereas VWF deficiency had the opposite effect. Correlating with this, overexpression of angiopoietin-2 by adenoviruses treatment or administration of recombinant galectin-3 normalized microvascular reductions, pericyte loss, and BBB breakdown in Adamts13 2/2 mice. The vascular changes induced by angiopoietin-2 overexpression and recombinant galectin-3 treatment in Adamts13 2/2 mice were abolished by the vascular endothelial growth factor receptor-2 antagonist SU1498. Importantly, treating wild-type mice with recombinant ADAMTS13 at 7 days after stroke markedly increased neovascularization and vascular repair and improved functional recovery at 14 days. Our results suggest that ADAMTS13 controls key steps of ischemic vascular remodeling and that recombinant ADAMTS13 is a putative therapeutic avenue for promoting stroke recovery. (Blood. 2017;130(1):11-22)

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 in tPA-treated mice. Blood-brain barrier (BBB) disruption following ischemic challenge in an in vitro model of BBB was exacerbated after exposure to NETs. Importantly, disruption of NETs by DNase 1 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 1 or PAD4 deficiency reversed tPA-mediated upregulation of the DNA sensor cyclic GMP-AMP (cGAMP) synthase (cGAS). Administration of cGAMP after stroke abolished DNase 1-mediated downregulation of the STING pathway and type I interferon (IFN) production, and blocked the antihemorrhagic effect of DNase 1 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 ischemic brain, and suggest that targeting NETs or cGAS may ameliorate thrombolytic therapy for ischemic stroke by reducing tPA-associated hemorrhage.

Glycosylphosphatidylinositol (GPI) is a glycolipid membrane anchor found on surface proteins in all eukaryotes. It is synthesized in the ER membrane. Each GPI anchor requires three molecules of ethanolamine phosphate (P-Etn), which are derived from phosphatidylethanolamine (PE). We found that efficient GPI anchor synthesis in Saccharomyces cerevisiae requires Csf1; cells lacking Csf1 accumulate GPI precursors lacking P-Etn. Structure predictions suggest Csf1 is a tube-forming lipid transport protein like Vps13. Csf1 is found at contact sites between the ER and other organelles. It interacts with the ER protein Mcd4, an enzyme that adds P-Etn to nascent GPI anchors, suggesting Csf1 channels PE to Mcd4 in the ER at contact sites to support GPI anchor biosynthesis. CSF1 has orthologues in Caenorhabditis elegans (lpd-3) and humans (KIAA1109/TWEEK); mutations in KIAA1109 cause the autosomal recessive neurodevelopmental disorder Alkuraya-Kučinskas syndrome. Knockout of lpd-3 and knockdown of KIAA1109 reduced GPI-anchored proteins on the surface of cells, suggesting Csf1 orthologues in human cells support GPI anchor biosynthesis.