Endothelial peroxiredoxin-4 is indispensable for blood-brain barrier integrity and long-term functional recovery after ischemic stroke.

The endothelial lining of cerebral microvessels is damaged relatively early after cerebral ischemia/reperfusion (I/R) injury and mediates blood-brain barrier (BBB) disruption, neurovascular injury, and long-term neurological deficits. I/R induces BBB leakage within 1 h due to subtle structural alterations in endothelial cells (ECs), including reorganization of the actin cytoskeleton and subcellular redistribution of junctional proteins. Herein, we show that the protein peroxiredoxin-4 (Prx4) is an endogenous protectant against endothelial dysfunction and BBB damage in a murine I/R model. We observed a transient upregulation of Prx4 in brain ECs 6 h after I/R in wild-type (WT) mice, whereas tamoxifen-induced, selective knockout of Prx4 from endothelial cells (eKO) mice dramatically raised vulnerability to I/R. Specifically, eKO mice displayed more BBB damage than WT mice within 1 to 24 h after I/R and worse long-term neurological deficits and focal brain atrophy by 35 d. Conversely, endothelium-targeted transgenic (eTG) mice overexpressing Prx4 were resistant to I/R-induced early BBB damage and had better long-term functional outcomes. As demonstrated in cultures of human brain endothelial cells and in animal models of I/R, Prx4 suppresses actin polymerization and stress fiber formation in brain ECs, at least in part by inhibiting phosphorylation/activation of myosin light chain. The latter cascade prevents redistribution of junctional proteins and BBB leakage under conditions of Prx4 repletion. Prx4 also tempers microvascular inflammation and infiltration of destructive neutrophils and proinflammatory macrophages into the brain parenchyma after I/R. Thus, the evidence supports an indispensable role for endothelial Prx4 in safeguarding the BBB and promoting functional recovery after I/R brain injury.

Novel insight into the etiology of ischemic stroke gained by integrative multiome-wide association study.

Stroke, characterized by sudden neurological deficits, is the second leading cause of death worldwide. Although genome-wide association studies (GWAS) have successfully identified many genomic regions associated with ischemic stroke (IS), the genes underlying risk and their regulatory mechanisms remain elusive. Here, we integrate a large-scale GWAS (N = 1 296 908) for IS together with molecular QTLs data, including mRNA, splicing, enhancer RNA (eRNA), and protein expression data from up to 50 tissues (total N = 11 588). We identify 136 genes/eRNA/proteins associated with IS risk across 60 independent genomic regions and find IS risk is most enriched for eQTLs in arterial and brain-related tissues. Focusing on IS-relevant tissues, we prioritize 9 genes/proteins using probabilistic fine-mapping TWAS analyses. In addition, we discover that blood cell traits, particularly reticulocyte cells, have shared genetic contributions with IS using TWAS-based pheWAS and genetic correlation analysis. Lastly, we integrate our findings with a large-scale pharmacological database and identify a secondary bile acid, deoxycholic acid, as a potential therapeutic component. Our work highlights IS risk genes/splicing-sites/enhancer activity/proteins with their phenotypic consequences using relevant tissues as well as identify potential therapeutic candidates for IS.

Mitofusin-2 Regulates Platelet Mitochondria and Function.

BACKGROUND: Single-nucleotide polymorphisms linked with the rs1474868 T allele (MFN2 [mitofusin-2] T/T) in the human mitochondrial fusion protein MFN2 gene are associated with reduced platelet MFN2 RNA expression and platelet counts. This study investigates the impact of MFN2 on megakaryocyte and platelet biology. METHODS: Mice with megakaryocyte/platelet deletion of Mfn2 (Mfn2-/- [Mfn2 conditional knockout]) were generated using Pf4-Cre crossed with floxed Mfn2 mice. Human megakaryocytes were generated from cord blood and platelets isolated from healthy subjects genotyped for rs1474868. Ex vivo approaches assessed mitochondrial morphology, function, and platelet activation responses. In vivo measurements included endogenous/transfused platelet life span, tail bleed time, transient middle cerebral artery occlusion, and pulmonary vascular permeability/hemorrhage following lipopolysaccharide-induced acute lung injury. RESULTS: Mitochondria was more fragmented in megakaryocytes derived from Mfn2-/- mice and from human cord blood with MFN2 T/T genotype compared with control megakaryocytes. Human resting platelets of MFN2 T/T genotype had reduced MFN2 protein, diminished mitochondrial membrane potential, and an increased rate of phosphatidylserine exposure during ex vivo culture. Platelet counts and platelet life span were reduced in Mfn2-/- mice accompanied by an increased rate of phosphatidylserine exposure in resting platelets, especially aged platelets, during ex vivo culture. Mfn2-/- also decreased platelet mitochondrial membrane potential (basal) and activated mitochondrial oxygen consumption rate, reactive oxygen species generation, calcium flux, platelet-neutrophil aggregate formation, and phosphatidylserine exposure following dual agonist activation. Ultimately, Mfn2-/- mice showed prolonged tail bleed times, decreased ischemic stroke infarct size after cerebral ischemia-reperfusion, and exacerbated pulmonary inflammatory hemorrhage following lipopolysaccharide-induced acute lung injury. Analysis of MFN2 SNPs in the iSPAAR study (Identification of SNPs Predisposing to Altered ALI Risk) identified a significant association between MFN2 and 28-day mortality in patients with acute respiratory distress syndrome. CONCLUSIONS: Mfn2 preserves mitochondrial phenotypes in megakaryocytes and platelets and influences platelet life span, function, and outcomes of stroke and lung injury.

LOX-1 and MMP-9 Inhibition Attenuates the Detrimental Effects of Delayed rt-PA Therapy and Improves Outcomes After Acute Ischemic Stroke.

BACKGROUND: Acute ischemic stroke triggers endothelial activation that disrupts vascular integrity and increases hemorrhagic transformation leading to worsened stroke outcomes. rt-PA (recombinant tissue-type plasminogen activator) is an effective treatment; however, its use is limited due to a restricted time window and hemorrhagic transformation risk, which in part may involve activation of MMPs (matrix metalloproteinases) mediated through LOX-1 (lectin-like oxLDL [oxidized low-density lipoprotein] receptor 1). This study's overall aim was to evaluate the therapeutic potential of novel MMP-9 (matrix metalloproteinase 9) ± LOX-1 inhibitors in combination with rt-PA to improve stroke outcomes. METHODS: A rat thromboembolic stroke model was utilized to investigate the impact of rt-PA delivered 4 hours poststroke onset as well as selective MMP-9 (JNJ0966) ±LOX-1 (BI-0115) inhibitors given before rt-PA administration. Infarct size, perfusion, and hemorrhagic transformation were evaluated by 9.4-T magnetic resonance imaging, vascular and parenchymal MMP-9 activity via zymography, and neurological function was assessed using sensorimotor function testing. Human brain microvascular endothelial cells were exposed to hypoxia plus glucose deprivation/reperfusion (hypoxia plus glucose deprivation 3 hours/R 24 hours) and treated with ±tPA and ±MMP-9 ±LOX-1 inhibitors. Barrier function was assessed via transendothelial electrical resistance, MMP-9 activity was determined with zymography, and LOX-1 and barrier gene expression/levels were measured using qRT-PCR (quantitative reverse transcription PCR) and Western blot. RESULTS: Stroke and subsequent rt-PA treatment increased edema, hemorrhage, MMP-9 activity, LOX-1 expression, and worsened neurological outcomes. LOX-1 inhibition improved neurological function, reduced edema, and improved endothelial barrier integrity. Elevated MMP-9 activity correlated with increased edema, infarct volume, and decreased neurological function. MMP-9 inhibition reduced MMP-9 activity and LOX-1 expression. In human brain microvascular endothelial cells, LOX-1/MMP-9 inhibition differentially attenuated MMP-9 levels, inflammation, and activation following hypoxia plus glucose deprivation/R. CONCLUSIONS: Our findings indicate that LOX-1 inhibition and ± MMP-9 inhibition attenuate negative aspects of ischemic stroke with rt-PA therapy, thus resulting in improved neurological function. While no synergistic effect was observed with simultaneous LOX-1 and MMP-9 inhibition, a distinct interaction is evident.

Leveraging single-cell RNA sequencing to unravel the impact of aging on stroke recovery mechanisms in mice.

Aging compromises the repair and regrowth of brain vasculature and white matter during stroke recovery, but the underlying mechanisms remain elusive. To understand how aging jeopardizes brain tissue repair after stroke, we performed single-cell transcriptomic profiling of young adult and aged mouse brains at acute (3 d) and chronic (14 d) stages after ischemic injury, focusing a priori on the expression of angiogenesis- and oligodendrogenesis-related genes. We identified unique subsets of endothelial cells (ECs) and oligodendrocyte (OL) progenitors in proangiogenesis and pro-oligodendrogenesis phenotypic states 3 d after stroke in young mice. However, this early prorepair transcriptomic reprogramming was negligible in aged stroke mice, consistent with the impairment of angiogenesis and oligodendrogenesis observed during the chronic injury stages after ischemia. In the stroke brain, microglia and macrophages (MG/MΦ) may drive angiogenesis and oligodendrogenesis through a paracrine mechanism. However, this reparative cell-cell cross talk between MG/MΦ and ECs or OLs is impeded in aged brains. In support of these findings, permanent depletion of MG/MΦ via antagonism of the colony-stimulating factor 1 receptor resulted in remarkably poor neurological recovery and loss of poststroke angiogenesis and oligodendrogenesis. Finally, transplantation of MG/MΦ from young, but not aged, mouse brains into the cerebral cortices of aged stroke mice partially restored angiogenesis and oligodendrogenesis and rejuvenated sensorimotor function and spatial learning and memory. Together, these data reveal fundamental mechanisms underlying the age-related decay in brain repair and highlight MG/MΦ as effective targets for promoting stroke recovery.

Early Versus Late Initiation of Direct Oral Anticoagulants After Ischemic Stroke in People With Atrial Fibrillation and Hemorrhagic Transformation: Prespecified Subanalysis of the Randomized Controlled ELAN Trial.

BACKGROUND: Whether hemorrhagic transformation (HT) modifies the treatment effect of early compared with late initiation of direct oral anticoagulation in people with ischemic stroke and atrial fibrillation is unknown. METHODS: This is a post hoc analysis of the ELAN trial (Early Versus Late Initiation of Direct Oral Anticoagulants in Post-Ischaemic Stroke Patients With Atrial Fibrillation). The primary outcome was a composite of recurrent ischemic stroke, symptomatic intracranial hemorrhage, major extracranial bleeding, systemic embolism, or vascular death within 30 days. Secondary outcomes were the individual components, 30- and 90-day functional outcome. We estimated outcomes based on HT, subclassified as hemorrhagic infarction (HI) or parenchymal hemorrhage (PH) on prerandomization imaging (core laboratory rating) using adjusted risk differences between treatment arms. RESULTS: Overall, 247 of 1970 participants (12.5%) had HT (114 HI 1, 77 HI 2, 34 PH 1, 22 PH 2). For the primary outcome, the estimated adjusted risk difference (early versus late) was -2.2% (95% CI, -7.8% to 3.5%) in people with HT (HI: -4.7% [95% CI, -10.8% to 1.4%]; PH: 6.1% [95% CI, -8.5% to 20.6%]) and -0.9% (95% CI, -2.6% to 0.8%) in people without HT. Numbers of symptomatic intracranial hemorrhage were identical in people with and without HT. With early treatment, the estimated adjusted risk difference for poor 90-day functional outcome (modified Rankin Scale score, 3-6) was 11.5% (95% CI, -0.8% to 23.8%) in participants with HT (HI: 7.4% [95% CI, -6.4% to 21.2%]; PH: 25.1% [95% CI, 0.2% to 50.0%]) and -2.6% (95% CI, -7.1% to 1.8%) in people without HT. CONCLUSIONS: We found no evidence of major treatment effect heterogeneity or safety concerns with early compared with late direct oral anticoagulation initiation in people with and without HT. However, early direct oral anticoagulation initiation may worsen functional outcomes in people with PH. REGISTRATION: URL: http://www.clinicaltrials.gov; Unique identifier: NCT03148457.

Arterial Thromboembolism in Patients With Atrial Fibrillation and CHA2DS2-VASc Score 1: A Nationwide Study.

BACKGROUND: Oral anticoagulation is suggested in patients with atrial fibrillation and a CHA2DS2-VASc score ≥1 (congestive heart failure, hypertension, age ≥75 years, diabetes, stroke, vascular disease, age 65-74 years, and sex score). To assess granular differences within CHA2DS2-VASc 1, the incidence of arterial thromboembolism according to CHA2DS2-VASc 1 subgroups was examined. METHODS: The Danish National Patient Registry and the Danish Prescription Registry were linked on a nationwide level to identify patients with atrial fibrillation from 2000 to 2021 without oral anticoagulation and categorized according to CHA2DS2-VASc score: CHA2DS2-VASc 0 (male and female subjects); CHA2DS2-VASc 1 (hypertension, heart failure, diabetes, vascular disease, and age 65-74 years); or CHA2DS2-VASc 2 (age ≥75 years without other risk factors). Female sex was not considered a risk factor in any risk group. The outcome was arterial thromboembolism (ischemic stroke, embolism of extremity, or transient cerebral ischemia). Study groups were compared using Cox regression analysis. RESULTS: We included 26 701 patients with a CHA2DS2-VASc 0 score; 22 915 with CHA2DS2-VASc 1 (1483 patients with heart failure, 9066 with hypertension, 843 with diabetes, 770 with vascular disease, and 10 753 who were 65 to 74 years of age); and 14 525 patients with CHA2DS2-VASc 2 (≥75 years of age without other risk factors). With a median of 1 year of observation time, the cumulative incidence of arterial thromboembolism was 0.6% (n=154 [95% CI, 0.6%-0.8%]), 1.4% (n=16 [95% CI, 0.8%-2.2%]), 1.9% (n=141 [95% CI, 1.6%-2.2%]), 1.7% (n=12 [95% CI, 0.9%-2.9%]), 2.0% (n=13 [95% CI, 1.1%-3.4%]), 2.3% (n=187 [95% CI, 2.0%-2.7%]), and 4.4% (n=533 [95% CI, 4.1%-4.8%]) for CHA2DS2-VASc 0, heart failure, hypertension, diabetes, vascular disease, age 65 to 74 years (CHA2DS2-VASc 1), and age ≥75 years (CHA2DS2-VASc 2), respectively. No statistically significant difference was identified among subgroups of CHA2DS2-VASc 1 (P=0.15 for difference). CONCLUSIONS: For patients with atrial fibrillation, all subgroups of CHA2DS2-VASc 1 were associated with lower incidence of arterial thromboembolism compared with age ≥75 years without other risk factors (ie, CHA2DS2-VASc 2) and a higher incidence compared with CHA2DS2-VASc 0. No statistically significant difference was identified between the subgroups of CHA2DS2-VASc 1. These findings support current recommendations that patients within this intermediate risk group could be identified with a similar risk of arterial thromboembolism.

Comparison of Incidence and Prognostic Impact of Ischemic, Major Bleeding and Heart Failure Events in Patients With Chronic Coronary Syndrome: Insights From the CORONOR Registry.

BACKGROUND: Evaluation of the residual risk in patient with chronic coronary syndrome is challenging in daily practice. Several types of events (myocardial infarction, ischemic stroke, bleeding, and heart failure [HF]) may occur, and their impact on subsequent mortality is unclear in the era of modern evidence-based pharmacotherapy. METHODS: CORONOR (Suivi d'une cohorte de patients Coronariens stables en région Nord-pas-de-Calais) is a prospective multicenter cohort that enrolled 4184 consecutive unselected outpatients with chronic coronary syndrome. We analyzed the incidence, correlates, and impact of ischemic events (a composite of myocardial infarction and ischemic stroke), major bleeding (Bleeding Academic Research Consortium 3 or higher), and hospitalization for HF on subsequent patient mortality. RESULTS: During follow-up (median, 4.9 years), 677 patients (16.5%) died. The 5-year cumulative incidences (death as competing event) of ischemic events, major bleeding, and HF hospitalization were 6.3% (5.6%-7.1%), 3.1% (2.5%-3.6%), and 8.1% (7.3%-9%), respectively. Ischemic events, major bleeding, and HF hospitalization were each associated with all-cause mortality. Major bleeding and hospitalization for HF were associated with the highest mortality rates in the postevent period (42.4%/y and 34.7%/y, respectively) compared with incident ischemic events (13.1%/y). The age- and sex-adjusted hazard ratios for all-cause mortality were 3.57 (95% CI, 2.77-4.61), 9.88 (95% CI, 7.55-12.93), and 8.60 (95% CI, 7.15-10.35) for ischemic events, major bleeding, and hospitalization for HF, respectively (all P<0.001). CONCLUSIONS: Hospitalization for HF has become both the most frequent and one of the most ominous events among patients with chronic coronary syndrome. Although less frequent, major bleeding is strongly associated with worse patient survival. Secondary prevention should not be limited to preventing ischemic events. Minimizing bleeding and preventing HF may be at least as important.

A cross-species approach using an in vivo evaluation platform in mice demonstrates that sequence variation in human RABEP2 modulates ischemic stroke outcomes.

Ischemic stroke, caused by vessel blockage, results in cerebral infarction, the death of brain tissue. Previously, quantitative trait locus (QTL) mapping of cerebral infarct volume and collateral vessel number identified a single, strong genetic locus regulating both phenotypes. Additional studies identified RAB GTPase-binding effector protein 2 (Rabep2) as the casual gene. However, there is yet no evidence that variation in the human ortholog of this gene plays any role in ischemic stroke outcomes. We established an in vivo evaluation platform in mice by using adeno-associated virus (AAV) gene replacement and verified that both mouse and human RABEP2 rescue the mouse Rabep2 knockout ischemic stroke volume and collateral vessel phenotypes. Importantly, this cross-species complementation enabled us to experimentally investigate the functional effects of coding sequence variation in human RABEP2. We chose four coding variants from the human population that are predicted by multiple in silico algorithms to be damaging to RABEP2 function. In vitro and in vivo analyses verify that all four led to decreased collateral vessel connections and increased infarct volume. Thus, there are naturally occurring loss-of-function alleles. This cross-species approach will expand the number of targets for therapeutics development for ischemic stroke.

HuR deficiency abrogated the enhanced NLRP3 signaling in experimental ischemic stroke.

Human antigen R (HuR) is a universally expressed RNA-binding protein that plays an essential role in governing the fate of mRNA transcripts. Accumulating evidence indicated that HuR is involved in the development and functions of several cell types. However, its role in cerebral ischemia/reperfusion injury (CIRI) remains unclear. In this study, we found that HuR was significantly upregulated after CIRI. Moreover, we found that silencing HuR could inhibit the inflammatory response of microglia and reduce the damage to neurons caused by oxygen-glucose deprivation/reperfusion treatment. In vivo, we found that microglial HuR deficiency significantly ameliorated CIRI and reduced NLRP3-mediated inflammasome activation. Mechanistically, we found that HuR could regulate NLRP3 mRNA stability by binding to the AU-rich element (ARE) region within the 3' untranslated region (UTR) of NLRP3 mRNA. In addition, we found that the upregulation of HuR was dependent on the upregulation of NADPH oxidase-mediated ROS accumulation. Collectively, our studies revealed that HuR could regulate NLRP3 expression and that HuR deficiency abrogated the enhanced NLRP3 signaling in experimental ischemic stroke. Targeting HuR may be a novel therapeutic strategy for cerebral ischemic stroke treatment.

Poly(ADP-ribose) mediates bioenergetic defects and redox imbalance in neurons following oxygen and glucose deprivation.

PARP-1 over-activation results in cell death via excessive PAR generation in different cell types, including neurons following brain ischemia. Glycolysis, mitochondrial function, and redox balance are key cellular processes altered in brain ischemia. Studies show that PAR generated after PARP-1 over-activation can bind hexokinase-1 (HK-1) and result in glycolytic defects and subsequent mitochondrial dysfunction. HK-1 is the neuronal hexokinase and catalyzes the first reaction of glycolysis, converting glucose to glucose-6-phosphate (G6P), a common substrate for glycolysis, and the pentose phosphate pathway (PPP). PPP is critical in maintaining NADPH and GSH levels via G6P dehydrogenase activity. Therefore, defects in HK-1 will not only decrease cellular bioenergetics but will also cause redox imbalance due to the depletion of GSH. In brain ischemia, whether PAR-mediated inhibition of HK-1 results in bioenergetics defects and redox imbalance is not known. We used oxygen-glucose deprivation (OGD) in mouse cortical neurons to mimic brain ischemia in neuronal cultures and observed that PARP-1 activation via PAR formation alters glycolysis, mitochondrial function, and redox homeostasis in neurons. We used pharmacological inhibition of PARP-1 and adenoviral-mediated overexpression of wild-type HK-1 (wtHK-1) and PAR-binding mutant HK-1 (pbmHK-1). Our data show that PAR inhibition or overexpression of HK-1 significantly improves glycolysis, mitochondrial function, redox homeostasis, and cell survival in mouse cortical neurons exposed to OGD. These results suggest that PAR binding and inhibition of HK-1 during OGD drive bioenergetic defects in neurons due to inhibition of glycolysis and impairment of mitochondrial function.

A Critical Role for ERO1α in Arterial Thrombosis and Ischemic Stroke.

BACKGROUND: Platelet adhesion and aggregation play a crucial role in arterial thrombosis and ischemic stroke. Here, we identify platelet ERO1α (endoplasmic reticulum oxidoreductase 1α) as a novel regulator of Ca2+ signaling and a potential pharmacological target for treating thrombotic diseases. METHODS: Intravital microscopy, animal disease models, and a wide range of cell biological studies were utilized to demonstrate the pathophysiological role of ERO1α in arteriolar and arterial thrombosis and to prove the importance of platelet ERO1α in platelet activation and aggregation. Mass spectrometry, electron microscopy, and biochemical studies were used to investigate the molecular mechanism. We used novel blocking antibodies and small-molecule inhibitors to study whether ERO1α can be targeted to attenuate thrombotic conditions. RESULTS: Megakaryocyte-specific or global deletion of Ero1α in mice similarly reduced platelet thrombus formation in arteriolar and arterial thrombosis without affecting tail bleeding times and blood loss following vascular injury. We observed that platelet ERO1α localized exclusively in the dense tubular system and promoted Ca2+ mobilization, platelet activation, and aggregation. Platelet ERO1α directly interacted with STIM1 (stromal interaction molecule 1) and SERCA2 (sarco/endoplasmic reticulum Ca2+-ATPase 2) and regulated their functions. Such interactions were impaired in mutant STIM1-Cys49/56Ser and mutant SERCA2-Cys875/887Ser. We found that ERO1α modified an allosteric Cys49-Cys56 disulfide bond in STIM1 and a Cys875-Cys887 disulfide bond in SERCA2, contributing to Ca2+ store content and increasing cytosolic Ca2+ levels during platelet activation. Inhibition of Ero1α with small-molecule inhibitors but not blocking antibodies attenuated arteriolar and arterial thrombosis and reduced infarct volume following focal brain ischemia in mice. CONCLUSIONS: Our results suggest that ERO1α acts as a thiol oxidase for Ca2+ signaling molecules, STIM1 and SERCA2, and enhances cytosolic Ca2+ levels, promoting platelet activation and aggregation. Our study provides evidence that ERO1α may be a potential target to reduce thrombotic events.

Microvascular Network Remodeling in the Ischemic Mouse Brain Defined by Light Sheet Microscopy.

BACKGROUND: Until now, the analysis of microvascular networks in the reperfused ischemic brain has been limited due to tissue transparency challenges. METHODS: Using light sheet microscopy, we assessed microvascular network remodeling in the striatum from 3 hours to 56 days post-ischemia in 2 mouse models of transient middle cerebral artery occlusion lasting 20 or 40 minutes, resulting in mild ischemic brain injury or brain infarction, respectively. We also examined the effect of a clinically applicable S1P (sphingosine-1-phosphate) analog, FTY720 (fingolimod), on microvascular network remodeling. RESULTS: Over 56 days, we observed progressive microvascular degeneration in the reperfused striatum, that is, the lesion core, which was followed by robust angiogenesis after mild ischemic injury induced by 20-minute middle cerebral artery occlusion. However, more severe ischemic injury elicited by 40-minute middle cerebral artery occlusion resulted in incomplete microvascular remodeling. In both cases, microvascular networks did not return to their preischemic state but displayed a chronically altered pattern characterized by higher branching point density, shorter branches, higher unconnected branch density, and lower tortuosity, indicating enhanced network connectivity. FTY720 effectively increased microvascular length density, branching point density, and volume density in both models, indicating an angiogenic effect of this drug. CONCLUSIONS: Utilizing light sheet microscopy together with automated image analysis, we characterized microvascular remodeling in the ischemic lesion core in unprecedented detail. This technology will significantly advance our understanding of microvascular restorative processes and pave the way for novel treatment developments in the stroke field.

CEBPD aggravates apoptosis and oxidative stress of neuron after ischemic stroke by Nrf2/HO-1 pathway.

CCAAT enhancer binding protein delta (CEBPD) is a transcription factor and plays an important role in apoptosis and oxidative stress, which are the main pathogenesis of ischemic stroke. However, whether CEBPD regulates ischemic stroke through targeting apoptosis and oxidative stress is unclear. Therefore, to answer this question, rat middle cerebral artery occlusion (MCAO) reperfusion model and oxygen-glucose deprivation/reoxygenation (OGD/R) primary cortical neuron were established to mimic ischemic reperfusion injury. We found that CEBPD was upregulated and accompanied with increased neurological deficit scores and infarct size, and decreased neuron in MCAO rats. The siRNA targeted CEBPD inhibited CEBPD expression in rats, and meanwhile lentivirus system was used to blocked CEBPD expression in primary neuron. CEBPD degeneration decreased neurological deficit scores, infarct size and brain water content of MCAO rats. Knockdown of CEBPD enhanced cell viability and reduced apoptosis as well as oxidative stress in vivo and in vitro. CEBPD silencing promoted the translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) to the nucleus and the expression of heme oxygenase 1 (HO-1). Newly, CEBPD facilitated the transcription of cullin 3 (CUL3), which intensified ischemic stroke through Nrf2/HO-1 pathway that was proposed by our team in the past. In conclusion, targeting CEBPD-CUL3-Nrf2/HO-1 axis may be contributed to cerebral ischemia therapy.

White matter fiber integrity and structural brain network topology: implications for balance function in postischemic stroke patients.

Previous studies have suggested that ischemic stroke can result in white matter fiber injury and modifications in the structural brain network. However, the relationship with balance function scores remains insufficiently explored. Therefore, this study aims to explore the alterations in the microstructural properties of brain white matter and the topological characteristics of the structural brain network in postischemic stroke patients and their potential correlations with balance function. We enrolled 21 postischemic stroke patients and 21 age, sex, and education-matched healthy controls (HC). All participants underwent balance function assessment and brain diffusion tensor imaging. Tract-based spatial statistics (TBSS) were used to compare the fractional anisotropy, mean diffusivity, axial diffusivity, and radial diffusivity of white matter fibers between the two groups. The white matter structural brain network was constructed based on the automated anatomical labeling atlas, and we conducted a graph theory-based analysis of its topological properties, including global network properties and local node properties. Additionally, the correlation between the significant structural differences and balance function score was analyzed. The TBSS results showed that in comparison to the HC, postischemic stroke patients exhibited extensive damage to their whole-brain white matter fiber tracts (P < 0.05). Graph theory analysis showed that in comparison to the HC, postischemic stroke patients exhibited statistically significant reductions in the values of global efficiency, local efficiency, and clustering coefficient, as well as an increase in characteristic path length (P < 0.05). In addition, the degree centrality and nodal efficiency of some nodes in postischemic stroke patients were significantly reduced (P < 0.05). The white matter fibers of the entire brain in postischemic stroke patients are extensively damaged, and the topological properties of the structural brain network are altered, which are closely related to balance function. This study is helpful in further understanding the neural mechanism of balance function after ischemic stroke from the white matter fiber and structural brain network topological properties.

Targeting lipid nanoparticles to the blood-brain barrier to ameliorate acute ischemic stroke.

Effective delivery of mRNA or small molecule drugs to the brain is a significant challenge in developing treatment for acute ischemic stroke (AIS). To address the problem, we have developed targeted nanomedicine to increase drug concentrations in endothelial cells of the blood-brain barrier (BBB) of the injured brain. Inflammation during ischemic stroke causes continuous neuronal death and an increase in the infarct volume. To enable targeted delivery to the inflamed BBB, we conjugated lipid nanocarriers (NCs) with antibodies that bind cell adhesion molecules expressed at the BBB. In the transient middle cerebral artery occlusion mouse model, NCs targeted to vascular cellular adhesion molecule-1 (VCAM) achieved the highest level of brain delivery, nearly two orders of magnitude higher than untargeted ones. VCAM-targeted lipid nanoparticles with luciferase-encoding mRNA and Cre-recombinase showed selective expression in the ischemic brain. Anti-inflammatory drugs administered intravenously after ischemic stroke reduced cerebral infarct volume by 62% (interleukin-10 mRNA) or 35% (dexamethasone) only when they were encapsulated in VCAM-targeted NCs. Thus, VCAM-targeted lipid NCs represent a new platform for strongly concentrating drugs within the compromised BBB of penumbra, thereby ameliorating AIS.

Targeting SRSF3 restores immune mRNA translation in microglia/macrophages following cerebral ischemia.

We recently described a novel ribosome-based regulatory mechanism/checkpoint that controls innate immune gene translation and microglial activation in non-sterile inflammation orchestrated by RNA binding protein SRSF3. Here we describe a role of SRSF3 in the regulation of microglia/macrophage activation phenotypes after experimental stroke. Using a model-system for analysis of the dynamic translational state of microglial ribosomes we show that 24 h after stroke highly upregulated immune mRNAs are not translated resulting in a marked dissociation of mRNA and protein networks in activated microglia/macrophages. Next, microglial activation after stroke was characterized by a robust increase in pSRSF3/SRSF3 expression levels. Targeted knockdown of SRSF3 using intranasal delivery of siRNA 24 h after stroke caused a marked knockdown of endogenous protein. Further analyses revealed that treatment with SRSF3-siRNA alleviated translational arrest of selected genes and induced a transient but significant increase in innate immune signaling and IBA1+ immunoreactivity peaking 5 days after initial injury. Importantly, delayed SRSF3-mediated increase in immune signaling markedly reduced the size of ischemic lesion measured 7 days after stroke. Together, our findings suggest that targeting SRSF3 and immune mRNA translation may open new avenues for molecular/therapeutic reprogramming of innate immune response after ischemic injury.

Endothelial PDGF-D contributes to neurovascular protection after ischemic stroke by rescuing pericyte functions.

Ischemic stroke induces neovascularization of the injured tissue as an attempt to promote structural repair and neurological recovery. Angiogenesis is regulated by pericytes that potently react to ischemic stroke stressors, ranging from death to dysfunction. Platelet-derived growth factor (PDGF) receptor (PDGFR)ß controls pericyte survival, migration, and interaction with brain endothelial cells. PDGF-D a specific ligand of PDGFRß is expressed in the brain, yet its regulation and role in ischemic stroke pathobiology remains unexplored. Using experimental ischemic stroke mouse model, we found that PDGF-D is transiently induced in brain endothelial cells at the injury site in the subacute phase. To investigate the biological significance of PDGF-D post-ischemic stroke regulation, its subacute expression was either downregulated using siRNA or upregulated using an active recombinant form. Attenuation of PDGF-D subacute induction exacerbates neuronal loss, impairs microvascular density, alters vascular permeability, and increases microvascular stalling. Increasing PDGF-D subacute bioavailability rescues neuronal survival and improves neurological recovery. PDGF-D subacute enhanced bioavailability promotes stable neovascularization of the injured tissue and improves brain perfusion. Notably, PDGF-D enhanced bioavailability improves pericyte association with brain endothelial cells. Cell-based assays using human brain pericyte and brain endothelial cells exposed to ischemia-like conditions were applied to investigate the underlying mechanisms. PDGF-D stimulation attenuates pericyte loss and fibrotic transition, while increasing the secretion of pro-angiogenic and vascular protective factors. Moreover, PDGF-D stimulates pericyte migration required for optimal endothelial coverage and promotes angiogenesis. Our study unravels new insights into PDGF-D contribution to neurovascular protection after ischemic stroke by rescuing the functions of pericytes.

The NEDD8-activating enzyme inhibitor MLN4924 reduces ischemic brain injury in mice.

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.

Sulfated Polysaccharide-Based Nanocarrier Drives Microenvironment-Mediated Cerebral Neurovascular Remodeling for Ischemic Stroke Treatment.

Stroke is a leading cause of global mortality and severe disability. However, current strategies used for treating ischemic stroke lack specific targeting capabilities, exhibit poor immune escape ability, and have limited drug release control. Herein, we developed an ROS-responsive nanocarrier for targeted delivery of the neuroprotective agent rapamycin (RAPA) to mitigate ischemic brain damage. The nanocarrier consisted of a sulfated chitosan (SCS) polymer core modified with a ROS-responsive boronic ester enveloped by a red blood cell membrane shell incorporating a stroke homing peptide. When encountering high levels of intracellular ROS in ischemic brain tissues, the release of SCS combined with RAPA from nanoparticle disintegration facilitates effective microglia polarization and, in turn, maintains blood-brain barrier integrity, reduces cerebral infarction, and promotes cerebral neurovascular remodeling in a mouse stroke model involving transient middle cerebral artery occlusion (tMCAO). This work offers a promising strategy to treat ischemic stroke therapy.