DNA characterization reveals potential operon-unit packaging of extracellular vesicle cargo from a gut bacterial symbiont.

Extracellular vesicles (EVs) are lipid bilayer-bound entities secreted by cells across all domains of life, known to contain a range of components, including protein complexes, RNA, and DNA. Recent studies on microbial extracellular vesicles indicate that these virus-sized nanoparticles, 40-90nm in diameter, readily cross the epithelial barrier and reach systemic circulation, can be detected in tissues throughout the body in mice and that 1mL of plasma from healthy humans contains up to one million bacterial EVs. They have been recently recognized for their biologically functional roles, including modulation of bacterial physiology and host-microbe interactions, hence their gain in the microbiome research community's attention. However, the exact understanding of their functionality is still a subject of active research and debate. Here, we employ long-read DNA sequencing on purified extracellular vesicles from a common mammalian gut symbiont, Parabacteroides goldsteinii, to characterize the genomic component within EV cargos. Our findings challenge the notion of DNA packaging into EVs as a stochastic event. Instead, our data demonstrate that the DNA packaging is non-random. Here, we suggest a novel hypothesis of selective EV-DNA packaging, potentially arranged in operon units, hence providing new insights into our understanding of its genetic makeup and its potential role, underlining the importance of our findings in microbial community dynamics.

Plasma Kallikrein Contributes to Intracerebral Hemorrhage and Hypertension in Stroke-Prone Spontaneously Hypertensive Rats.

Plasma kallikrein (PKa) has been implicated in contributing to hemorrhage following thrombolytic therapy; however, its role in spontaneous intracerebral hemorrhage is currently not available. This report investigates the role of PKa on hemorrhage and hypertension in stroke-prone spontaneously hypertensive rats (SHRSP). SHRSP were fed with a high salt-containing stroke-prone diet to increase blood pressure and induce intracerebral hemorrhage. The roles of PKa on blood pressure, hemorrhage, and survival in SHRSP were examined in rats receiving a PKa inhibitor or plasma prekallikrein antisense oligonucleotide (PK ASO) compared with rats receiving control ASO. Effects on PKa on the proteolytic cleavage of atrial natriuretic peptide (ANP) were analyzed by tandem mass spectrometry. We show that SHRSP on high-salt diet displayed increased levels of PKa activity compared with control rats. Cleaved kininogen was increased in plasma during stroke compared to SHRSP without stroke. Systemic administration of a PKa inhibitor or PK ASO to SHRSP reduced hemorrhage and blood pressure, and improved neurological function and survival compared with SHRSP receiving control ASO. Since PKa inhibition was associated with reduced blood pressure in hypertensive rats, we investigated the effects of PKa on the cleavage of ANP. Incubation of PKa with ANP resulted in the generation fragment ANP5-28, which displayed reduced effects on blood pressure lowering compared with full length ANP. PKa contributes to increased blood pressure in SHRSP, which is associated with hemorrhage and reduced survival. PKa-mediated cleavage of ANP reduces its blood pressure lowering effects and thereby may contribute to hypertension-induced intracerebral hemorrhage.

E. coli Nissle 1917 modulates host glucose metabolism without directly acting on glucose.

Managing postprandial glycemic response, or the increase in blood sugar following a meal, is a crucial component to maintaining healthy blood sugar in patients with diabetes. To test whether oral probiotics can impact postprandial glycemic response, E. coli Nissle 1917 (EcN) was evaluated in an oral glucose tolerance test. Oral gavage of EcN concurrent with a glucose bolus reduced the post-gavage glycemic response in mice. However, there was no difference in glycemic response when comparing EcN to a mutant deficient in glucose metabolism. This suggests that while EcN can alter glycemic response to a glucose bolus, this effect is not mediated by direct uptake of glucose. Of the possible indirect effects EcN could have, gastric emptying rate was highlighted as a likely cause, but EcN had no effect on gastric emptying rate in mice. This leaves many more possible indirect explanations for the interaction between EcN and host glucose metabolism to be explored in future work.

Early molecular oxidative stress biomarkers of ischemic penumbra in acute stroke.

OBJECTIVES: To assess whether plasma biomarkers of oxidative stress predict diffusion-perfusion mismatch in patients with acute ischemic stroke (AIS). METHODS: We measured plasma levels of oxidative stress biomarkers such as F2-isoprostanes (F2-isoPs), total and perchloric acid Oxygen Radical Absorbance Capacity (ORACTOT and ORACPCA), urinary levels of 8-oxo-7,8-dihydro-2'-deoxyguoanosine, and inflammatory and tissue-damage biomarkers (high-sensitivity C-reactive protein, matrix metalloproteinase-2 and -9) in a prospective study of patients with AIS presenting within 9 hours of symptom onset. Diffusion-weighted (DWI) and perfusion-weighted (PWI) MRI sequences were analyzed with a semiautomated volumetric method. Mismatch was defined as baseline mean transit time volume minus DWI volume. A percent mismatch cutoff of >20% was considered clinically significant. A stricter definition of mismatch was also used. Mismatch salvage was the region free of overlap by final infarction. RESULTS: Mismatch >20% was present in 153 of 216 (70.8%) patients (mean [±SD] age 69.2 ± 14.3 years, 41.2% women). Patients with mismatch >20% were more likely to have higher baseline plasma levels of ORACPCA (p = 0.020) and F2-isoPs (p = 0.145). Multivariate binary logistic regression demonstrated that lnF2-isoP (odds ratio [OR] 2.44, 95% confidence interval [CI] 1.19-4.98, p = 0.014) and lnORACPCA (OR 4.18, 95% CI 1.41-12.41, p = 0.010) were independent predictors of >20% PWI-DWI mismatch and the stricter mismatch definition, respectively. lnORACTOT significantly predicted mismatch salvage volume (>20% mismatch p = 0.010, stricter mismatch definition p = 0.003). CONCLUSIONS: Elevated hyperacute plasma levels of F2-isoP and ORAC are associated with radiographic evidence of mismatch and mismatch salvage in patients with AIS. If validated, these findings may add to our understanding of the role of oxidative stress in cerebral tissue fate during acute ischemia.

Meta-omics analysis of elite athletes identifies a performance-enhancing microbe that functions via lactate metabolism.

The human gut microbiome is linked to many states of human health and disease1. The metabolic repertoire of the gut microbiome is vast, but the health implications of these bacterial pathways are poorly understood. In this study, we identify a link between members of the genus Veillonella and exercise performance. We observed an increase in Veillonella relative abundance in marathon runners postmarathon and isolated a strain of Veillonella atypica from stool samples. Inoculation of this strain into mice significantly increased exhaustive treadmill run time. Veillonella utilize lactate as their sole carbon source, which prompted us to perform a shotgun metagenomic analysis in a cohort of elite athletes, finding that every gene in a major pathway metabolizing lactate to propionate is at higher relative abundance postexercise. Using 13C3-labeled lactate in mice, we demonstrate that serum lactate crosses the epithelial barrier into the lumen of the gut. We also show that intrarectal instillation of propionate is sufficient to reproduce the increased treadmill run time performance observed with V. atypica gavage. Taken together, these studies reveal that V. atypica improves run time via its metabolic conversion of exercise-induced lactate into propionate, thereby identifying a natural, microbiome-encoded enzymatic process that enhances athletic performance.

Protective effects of a radical scavenger edaravone on oligodendrocyte precursor cells against oxidative stress.

Oligodendrocyte precursor cells (OPCs) play critical roles in maintaining the number of oligodendrocytes in white matter. Previously, we have shown that oxidative stress dampens oligodendrocyte regeneration after white matter damage, while a clinically proven radical scavenger, edaravone, supports oligodendrocyte repopulation. However, it is not known how edaravone exerts this beneficial effect against oxidative stress. Using in vivo and in vitro experiments, we have examined whether edaravone exhibits direct OPC-protective effects. For in vivo experiments, prolonged cerebral hypoperfusion was induced by bilateral common carotid artery stenosis in mice. OPC damage was observed on day 14 after the onset of cerebral hypoperfusion, and edaravone was demonstrated to decrease OPC death in cerebral white matter. In vitro experiments also confirmed that edaravone reduced oxidative-stress-induced OPC death. Because white matter damage is a major hallmark of many neurological diseases, and OPCs are instrumental in white matter repair after injury, our current study supports the idea that radical scavengers may provide a potential therapeutic approach for white matter related diseases.

Oxidative Stress Biomarkers of Brain Damage: Hyperacute Plasma F2-Isoprostane Predicts Infarct Growth in Stroke.

BACKGROUND AND PURPOSE: Oxidative stress is an early response to cerebral ischemia and is likely to play an important role in the pathogenesis of cerebral ischemic injury. We sought to evaluate whether hyperacute plasma concentrations of biomarkers of oxidative stress, inflammation, and tissue damage predict infarct growth (IG). METHODS: We prospectively measured plasma F2-isoprostane (F2-isoP), urinary 8-oxo-7,8-dihydro-2'-deoxyguoanosine, plasma oxygen radical absorbance capacity assay, high sensitivity C reactive protein, and matrix metalloproteinase 2 and 9 in consecutive patients with acute ischemic stroke presenting within 9 hours of symptom onset. Patients with baseline diffusion-weighted magnetic resonance imaging and follow-up diffusion-weighted imaging or computed tomographic scan were included to evaluate the final infarct volume. Baseline diffusion-weighted imaging volume and final infarct volume were analyzed using semiautomated volumetric method. IG volume was defined as the difference between final infarct volume and baseline diffusion-weighted imaging volume. RESULTS: A total of 220 acute ischemic stroke subjects were included in the final analysis. One hundred seventy of these had IG. Baseline F2-isoP significantly correlated with IG volume (Spearman ρ=0.20; P=0.005) and final infarct volume (Spearman ρ=0.19; P=0.009). In a multivariate binary logistic regression model, baseline F2-isoP emerged as an independent predictor of the occurrence of IG (odds ratio, 2.57; 95% confidence interval, 1.37-4.83; P=0.007). In a multivariate linear regression model, baseline F2-isoP was independently associated with IG volume (B, 0.38; 95% confidence interval, 0.04-0.72; P=0.03). CONCLUSIONS: Elevated hyperacute plasma F2-isoP concentrations independently predict the occurrence of IG and IG volume in patients with acute ischemic stroke. If validated in future studies, measuring plasma F2-isoP might be helpful in the acute setting to stratify patients with acute ischemic stroke for relative severity of ischemic injury and expected progression.

A free radical scavenger edaravone suppresses systemic inflammatory responses in a rat transient focal ischemia model.

A free radical scavenger edaravone is clinically used in Japan for acute stroke, and several basic researches have carefully examined the mechanisms of edaravone's protective effects. However, its actions on pro-inflammatory responses under stroke are still understudied. In this study, we subjected adult male Sprague-Dawley rats to 90-min middle cerebral artery (MCA) occlusion followed by reperfusion. Edaravone was treated twice via tail vein; after MCA occlusion and after reperfusion. As expected, edaravone-treated group showed less infarct volume and edema formation compared with control group at 24-h after an ischemic onset. Furthermore, edaravone reduced the levels of plasma interleukin (IL)-1ß and matrix metalloproteinase-9 at 3-h after ischemic onset. Several molecules besides IL-1ß and MMP-9 are involved in inflammatory responses under stroke conditions. Therefore, we also examined whether edaravone treatment could decrease a wide range of pro-inflammatory cytokines/chemokines by testing rat plasma samples with a rat cytokine array. MCAO rats showed elevations in plasma levels of CINC-1, Fractalkine, IL-1α, IL-1ra, IL-6, IL-10, IP-10, MIG, MIP-1α, and MIP-3α, and all these increases were reduced by edaravone treatment. These data suggest that free radical scavengers may reduce systemic inflammatory responses under acute stroke conditions, and therefore, oxidative stress can be still a viable target for acute stroke therapy.

CD200 restrains macrophage attack on oligodendrocyte precursors via toll-like receptor 4 downregulation.

There are numerous barriers to white matter repair after central nervous system injury and the underlying mechanisms remain to be fully understood. In this study, we propose the hypothesis that inflammatory macrophages in damaged white matter attack oligodendrocyte precursor cells via toll-like receptor 4 signaling thus interfering with this endogenous progenitor recovery mechanism. Primary cell culture experiments demonstrate that peritoneal macrophages can attack and digest oligodendrocyte precursor cells via toll-like receptor 4 signaling, and this phagocytosis of oligodendrocyte precursor cells can be inhibited by using CD200-Fc to downregulate toll-like receptor 4. In an in vivo model of white matter ischemia induced by endothelin-1, treatment with CD200-Fc suppressed toll-like receptor 4 expression in peripherally circulating macrophages, thus restraining macrophage phagocytosis of oligodendrocyte precursor cells and leading to improved myelination. Taken together, these findings suggest that deleterious macrophage effects may occur after white matter ischemia, whereby macrophages attack oligodendrocyte precursor cells and interfere with endogenous recovery responses. Targeting this pathway with CD200 may offer a novel therapeutic approach to amplify endogenous oligodendrocyte precursor cell-mediated repair of white matter damage in mammalian brain.

Oligodendrocyte precursor cells support blood-brain barrier integrity via TGF-ß signaling.

Trophic coupling between cerebral endothelium and their neighboring cells is required for the development and maintenance of blood-brain barrier (BBB) function. Here we report that oligodendrocyte precursor cells (OPCs) secrete soluble factor TGF-ß1 to support BBB integrity. Firstly, we prepared conditioned media from OPC cultures and added them to cerebral endothelial cultures. Our pharmacological experiments showed that OPC-conditioned media increased expressions of tight-junction proteins and decreased in vitro BBB permeability by activating TGB-ß-receptor-MEK/ERK signaling pathway. Secondly, our immuno-electron microscopic observation revealed that in neonatal mouse brains, OPCs attach to cerebral endothelial cells via basal lamina. And finally, we developed a novel transgenic mouse line that TGF-ß1 is knocked down specifically in OPCs. Neonates of these OPC-specific TGF-ß1 deficient mice (OPC-specific TGF-ß1 partial KO mice: PdgfraCre/Tgfb1flox/wt mice or OPC-specific TGF-ß1 total KO mice: PdgfraCre/Tgfb1flox/flox mice) exhibited cerebral hemorrhage and loss of BBB function. Taken together, our current study demonstrates that OPCs increase BBB tightness by upregulating tight junction proteins via TGF-ß signaling. Although astrocytes and pericytes are well-known regulators of BBB maturation and maintenance, these findings indicate that OPCs also play a pivotal role in promoting BBB integrity.

A radical scavenger edaravone inhibits matrix metalloproteinase-9 upregulation and blood-brain barrier breakdown in a mouse model of prolonged cerebral hypoperfusion.

Matrix metalloproteinase-9 (MMP-9) plays key roles in the brain pathophysiology, especially in blood-brain barrier (BBB) breakdown. Therefore, inhibiting MMP-9 activity may be a promising therapy for protecting brains in cerebrovascular diseases. Here we show that in a mouse prolonged cerebral hypoperfusion model, a clinically proven radical scavenger edaravone suppressed MMP-9 and reduced BBB damage in cerebral white matter. Prolonged cerebral hypoperfusion was induced by bilateral common carotid artery stenosis in male adult C57BL/6J mice (10 weeks old). After 7 days of cerebral hypoperfusion, white matter region (e.g. corpus callosum) exhibited significant BBB leakage, assessed by IgG staining. Correspondingly, immunostaining and western blotting showed that MMP-9 was upregulated in the white matter. Edaravone treatment (3mg/kg, i.p. at days 0 and 3) inhibited both BBB leakage and MMP-9 increase. Under the early phase of cerebral hypoperfusion conditions, oligodendrocyte precursor cells (OPCs) mainly contribute to the MMP-9 increase, but our immunostaining data showed that very little OPCs expressed MMP-9 in the edaravone-treated animals at day 7. Therefore, in vitro studies with primary rat OPCs were conducted to examine whether edaravone would directly suppressed MMP-9 expressions in OPCs. OPC cultures were exposed to sub-lethal CoCl2 for 7 days to induce prolonged chemical hypoxic stress. Prolonged chemical hypoxic stress increased MMP-9 expression in OPCs, and radical scavenging with edaravone (10µM for 7 days) ameliorated the increase. Taken together, our proof-of-concept study demonstrates that radical scavengers may provide a potential therapeutic approach for white matter injury by suppressing BBB damage.

Reactive astrocytes promote adhesive interactions between brain endothelium and endothelial progenitor cells via HMGB1 and beta-2 integrin signaling.

Endothelial progenitor cells (EPCs) may contribute to neurovascular repair after stroke and neurodegeneration. A key step in this process should involve adhesive interactions between EPCs and the targeted cerebral endothelium. Here, we tested the hypothesis that reactive astrocytes may play a critical role in enhancing adhesive interactions and transmigration of EPCs across cerebral endothelial cells. Transiently seeding EPCs onto a monolayer of RBE.4 rat brain endothelial cells resulted in a time-dependent adherence between the two cell types. Blocking ß2 integrins on EPCs or blocking the receptor for advanced glycation endproducts (RAGE) on endothelial cells significantly decreased EPC-endothelial adherence. Next, we tested whether reactive astrocytes can enhance this process by growing EPCs, brain endothelial cells and astrocytes together in a transwell co-culture system. The presence of reactive astrocytes in the lower chamber significantly promoted adherence between EPCs and endothelial cells in the upper chamber. This process involved the release of soluble HMGB1 from reactive astrocytes that then upregulated endothelial expression of RAGE via Egr1 signaling. Directly adding HMGB1 to the transwell system also promoted EPC-endothelial adhesion and accelerated EPC transmigration into the lower chamber. These initial findings provide proof-of-concept that reactive astrocytes promote crosstalk between cerebral endothelium and EPCs. Further investigation of this phenomenon may lead to a better understanding of cell-cell interactions required for neurovascular recovery after stroke.

Crosstalk between cerebral endothelium and oligodendrocyte.

It is now relatively well accepted that the cerebrovascular system does not merely provide inert pipes for blood delivery to the brain. Cerebral endothelial cells may compose an embedded bunker of trophic factors that contribute to brain homeostasis and function. Recent findings suggest that soluble factors from cerebral endothelial cells nourish neighboring cells, such as neurons and astrocytes. Although data are strongest in supporting mechanisms of endothelial-neuron and/or endothelial-astrocyte trophic coupling, it is likely that similar interactions also exist between cerebral endothelial cells and oligodendrocyte lineage cells. In this mini-review, we summarize current advances in the field of endothelial-oligodendrocyte trophic coupling. These endothelial-oligodendrocyte interactions may comprise the oligovascular niche to maintain their cellular functions and sustain ongoing angiogenesis/oligodendrogenesis. Importantly, it should be noted that the cell-cell interactions are not static-the trophic coupling is disturbed under acute phase after brain injury, but would be recovered in the chronic phase to promote brain remodeling and repair. Oligodendrocyte lineage cells play critical roles in white matter function, and under pathological conditions, oligodendrocyte dysfunction lead to white matter damage. Therefore, a deeper understanding of the mechanisms of endothelial-oligodendrocyte trophic coupling may lead to new therapeutic approaches for white matter-related diseases, such as stroke or vascular dementia.

Oxidative stress interferes with white matter renewal after prolonged cerebral hypoperfusion in mice.

BACKGROUND AND PURPOSE: White matter injury caused by cerebral hypoperfusion may contribute to the pathophysiology of vascular dementia and stroke, but the underlying mechanisms remain to be fully defined. Here, we test the hypothesis that oxidative stress interferes with endogenous white matter repair by disrupting renewal processes mediated by oligodendrocyte precursor cells (OPCs). METHODS: In vitro, primary rat OPCs were exposed to sublethal CoCl2 for 7 days to induce prolonged chemical hypoxic stress. Then, OPC proliferation/differentiation was assessed. In vivo, prolonged cerebral hypoperfusion was induced by bilateral common carotid artery stenosis in mice. Then, reactive oxygen species production, myelin density, oligodendrocyte versus OPC counts, and cognitive function were evaluated. To block oxidative stress, OPCs and mice were treated with the radical scavenger edaravone. RESULTS: Prolonged chemical hypoxic stress suppressed OPC differentiation in vitro. Radical scavenging with edaravone ameliorated these effects. After 28 days of cerebral hypoperfusion in vivo, reactive oxygen species levels were increased in damaged white matter, along with the suppression of OPC-to-oligodendrocyte differentiation and loss of myelin staining. Concomitantly, mice showed functional deficits in working memory. Radical scavenging with edaravone rescued OPC differentiation, ameliorated myelin loss, and restored working memory function. CONCLUSIONS: Our proof-of-concept study demonstrates that after prolonged cerebral hypoperfusion, oxidative stress interferes with white matter repair by disrupting OPC renewal mechanisms. Radical scavengers may provide a potential therapeutic approach for white matter injury in vascular dementia and stroke.

Age-related decline in oligodendrogenesis retards white matter repair in mice.

BACKGROUND AND PURPOSE: Aging is one of the major risk factors for white matter injury in cerebrovascular disease. However, the effects of age on the mechanisms of injury/repair in white matter remain to be fully elucidated. Here, we ask whether, compared with young brains, white matter regions in older brains may be more vulnerable in part because of decreased rates of compensatory oligodendrogenesis after injury. METHODS: A mouse model of prolonged cerebral hypoperfusion was prepared by bilateral common carotid artery stenosis in 2-month and 8-month-old mice. Matching in vitro studies were performed by subjecting oligodendrocyte precursor cells to sublethal 7-day CoCl2 treatment to induce chemical hypoxic stress. RESULTS: Baseline myelin density in the corpus callosum was similar in 2-month and 8-month-old mice. But after induction of prolonged cerebral hypoperfusion, older mice showed more severe white matter injury together with worse deficits in working memory. The numbers of newborn oligodendrocytes and their precursors were increased by cerebral hypoperfusion in young mice, whereas these endogenous responses were significantly dampened in older mice. Defects in cyclic AMP response element-binding protein signaling may be involved because activating cyclic AMP response element-binding protein with the type-III phosphodiesterase inhibitor cilostazol in older mice restored the differentiation of oligodendrocyte precursor cells, alleviated myelin loss, and improved cognitive dysfunction during cerebral hypoperfusion. Cell culture systems confirmed that cilostazol promoted the differentiation of oligodendrocyte precursor cells. CONCLUSIONS: An age-related decline in cyclic AMP response element-binding protein-mediated oligodendrogenesis may compromise endogenous white matter repair mechanisms, and therefore, drugs that activate cyclic AMP response element-binding protein signaling provide a potential therapeutic approach for treating white matter injury in aging brains.

High-mobility group box 1: an amplifier of stem and progenitor cell activity after stroke.

Stroke induces a highly complex web of pathophysiology that usually leads to serious long-term -disability. Molecules from the damage-associated molecular pattern (DAMP) family immediately increase after stroke. DAMPs are known to cause massive inflammation and brain damage. Thus, they may be targets for neuroprotection. However, emerging data now suggest that DAMPs may not always be detrimental. The high-mobility group box1 (HMGB1) protein is discussed as an example of this idea. During the acute phase after stroke, HMGB1 amplifies neuroinflammation. But during the brain remodeling phase of stroke recovery, HMGB1 can mediate beneficial plasticity and enhance stem and progenitor cell recruitment, proliferation, and differentiation within damaged brain. These emerging findings support the hypothesis that HMGB1 might be an important molecule for regulating stem and progenitor cell therapies in stroke patients.

Biphasic mechanisms of neurovascular unit injury and protection in CNS diseases.

In the past decade, evidence has emerged that there is a variety of bidirectional cell-cell and/or cell-extracellular matrix interactions within the neurovascular unit (NVU), which is composed of neuronal, glial, and vascular cells along with extracellular matrix. Many central nervous system diseases, which lead to NVU dysfunction, have common features such as glial activation/transformation and vascular/blood-brain-barrier alteration. These phenomena show dual opposite roles, harmful at acute phase and beneficial at chronic phase. This diverse heterogeneity may induce biphasic clinical courses, i.e. degenerative and regenerative processes in the context of dynamically coordinated cellcell/ cell-matrix interactions in the NVU. A deeper understanding of the seemingly contradictory actions in cellular levels is essential for NVU protection or regeneration to suppress the deleterious inflammatory reactions and promote adaptive remodeling after central nervous system injury. This mini-review will present an overview of recent progress in the biphasic roles of the NVU and discuss the clinical relevance of NVU responses associated with central nervous system diseases, such as stroke and other chronic neurodegenerative diseases.

Oligodendrocyte precursors induce early blood-brain barrier opening after white matter injury.

Oligodendrocyte precursor cells (OPCs) are thought to maintain homeostasis and contribute to long-term repair in adult white matter; however, their roles in the acute phase after brain injury remain unclear. Mice that were subjected to prolonged cerebral hypoperfusion stress developed white matter demyelination over time. Prior to demyelination, we detected increased MMP9 expression, blood-brain barrier (BBB) leakage, and neutrophil infiltration in damaged white matter. Notably, at this early stage, OPCs made up the majority of MMP9-expressing cells. The standard MMP inhibitor GM6001 reduced the early BBB leakage and neutrophil infiltration, indicating that OPC-derived MMP9 induced early BBB disruption after white matter injury. Cell-culture experiments confirmed that OPCs secreted MMP9 under pathological conditions, and conditioned medium prepared from the stressed OPCs weakened endothelial barrier tightness in vitro. Our study reveals that OPCs can rapidly respond to white matter injury and produce MMP9 that disrupts the BBB, indicating that OPCs may mediate injury in white matter under disease conditions.

High-mobility group box 1 from reactive astrocytes enhances the accumulation of endothelial progenitor cells in damaged white matter.

High-mobility group box 1 (HMGB1) was initially described as a damage-associated-molecular-pattern (DAMP) mediator that worsens acute brain injury after stroke. But, recent findings suggest that HMGB1 can play a surprisingly beneficial role during stroke recovery by promoting endothelial progenitor cell (EPC) function and vascular remodeling in cortical gray matter. Here, we ask whether HMGB1 may also influence EPC responses in white matter injury. The standard lysophosphatidylcholine (LPC) injection model was used to induce focal demyelination in the corpus callosum of mice. Immunostaining showed that within the focal white matter lesions, HMGB1 was up-regulated in GFAP-positive reactive astrocytes, along with the accumulation of Flk1/CD34-double-positive EPCs that expressed pro-recovery mediators such as brain-derived neurotrophic factor and basic fibroblast growth factor. Astrocyte-EPC signaling required the HMGB1 receptor RAGE as treatment with RAGE-neutralizing antibody significantly decreased EPC accumulation. Moreover, suppression of HMGB1 with siRNA in vivo significantly decreased EPC numbers in damaged white matter as well as proliferated endothelial cell numbers. Finally, in vitro cell culture systems confirmed that HMGB1 directly affected EPC function such as migration and tube formation. Taken together, our findings suggest that HMGB1 from reactive astrocytes may attract EPCs to promote recovery after white matter injury.

Astrocytic high-mobility group box 1 promotes endothelial progenitor cell-mediated neurovascular remodeling during stroke recovery.

Crosstalk between the brain and systemic responses in blood is increasingly suspected of playing critical roles in stroke. However, how this communication takes place remains to be fully understood. Here, we show that reactive astrocytes can release a damage-associated molecular-pattern molecule called high-mobility-group-box-1 (HMGB1) that promotes endothelial progenitor cell (EPC)-mediated neurovascular remodeling during stroke recovery. Conditioned media from reactive astrocytes increase EPC proliferation in vitro. siRNA suppression of HMGB1 in astrocytes or blockade of the HMGB1 receptor for advanced glycation endproducts in EPCs prevents this effect. In a mouse model of focal cerebral ischemia, reactive astrocytes in the peri-infarct cortex up-regulate HMGB1 at 14 d poststroke, along with an accumulation of endogenous EPCs. In vivo siRNA suppression of HMGB1 blocks this EPC response, reduces peri-infact angiogenesis, and worsens neurological deficits. Taken together, these molecular and in vivo findings support a previously undescribed mechanism of crosstalk between reactive astrocytes and EPCs wherein HMGB1 promotes neurovascular remodeling and functional recovery after stroke and brain injury.