Molecular Pathogenesis of Ischemic and Hemorrhagic Strokes: Background and Therapeutic Approaches.

Stroke represents one of the neurological diseases most responsible for death and permanent disability in the world. Different factors, such as thrombus, emboli and atherosclerosis, take part in the intricate pathophysiology of stroke. Comprehending the molecular processes involved in this mechanism is crucial to developing new, specific and efficient treatments. Some common mechanisms are excitotoxicity and calcium overload, oxidative stress and neuroinflammation. Furthermore, non-coding RNAs (ncRNAs) are critical in pathophysiology and recovery after cerebral ischemia. ncRNAs, particularly microRNAs, and long non-coding RNAs (lncRNAs) are essential for angiogenesis and neuroprotection, and they have been suggested to be therapeutic, diagnostic and prognostic tools in cerebrovascular diseases, including stroke. This review summarizes the intricate molecular mechanisms underlying ischemic and hemorrhagic stroke and delves into the function of miRNAs in the development of brain damage. Furthermore, we will analyze new perspectives on treatment based on molecular mechanisms in addition to traditional stroke therapies.

Hemorrhagic stroke-induced subtype of inflammatory reactive astrocytes disrupts blood-brain barrier.

Astrocytes undergo disease-specific transcriptomic changes upon brain injury. However, phenotypic changes of astrocytes and their functions remain unclear after hemorrhagic stroke. Here we reported hemorrhagic stroke induced a group of inflammatory reactive astrocytes with high expression of Gfap and Vimentin, as well as inflammation-related genes lipocalin-2 (Lcn2), Complement component 3 (C3), and Serpina3n. In addition, we demonstrated that depletion of microglia but not macrophages inhibited the expression of inflammation-related genes in inflammatory reactive astrocytes. RNA sequencing showed that blood-brain barrier (BBB) disruption-related gene matrix metalloproteinase-3 (MMP3) was highly upregulated in inflammatory reactive astrocytes. Pharmacological inhibition of MMP3 in astrocytes or specific deletion of astrocytic MMP3 reduced BBB disruption and improved neurological outcomes of hemorrhagic stroke mice. Our study demonstrated that hemorrhagic stroke induced a group of inflammatory reactive astrocytes that were actively involved in disrupting BBB through MMP3, highlighting a specific group of inflammatory reactive astrocytes as a critical driver for BBB disruption in neurological diseases.

Iron/ROS/Itga3 mediated accelerated depletion of hippocampal neural stem cell pool contributes to cognitive impairment after hemorrhagic stroke.

Hemorrhagic stroke, specifically intracerebral hemorrhage (ICH), has been implicated in the development of persistent cognitive impairment, significantly compromising the quality of life for affected individuals. Nevertheless, the precise underlying mechanism remains elusive. Here, we report for the first time that the accumulation of iron within the hippocampus, distal to the site of ICH in the striatum, is causally linked to the observed cognitive impairment with both clinical patient data and animal model. Both susceptibility-weighted imaging (SWI) and quantitative susceptibility mapping (QSM) demonstrated significant iron accumulation in the hippocampus of ICH patients, which is far from the actual hematoma. Logistical regression analysis and multiple linear regression analysis identified iron level as an independent risk factor with a negative correlation with post-ICH cognitive impairment. Using a mouse model of ICH, we demonstrated that iron accumulation triggers an excessive activation of neural stem cells (NSCs). This overactivation subsequently leads to the depletion of the NSC pool, diminished neurogenesis, and the onset of progressive cognitive dysfunction. Mechanistically, iron accumulation elevated the levels of reactive oxygen species (ROS), which downregulated the expression of Itga3. Notably, pharmacological chelation of iron accumulation or scavenger of aberrant ROS levels, as well as conditionally overexpressed Itga3 in NSCs, remarkably attenuated the exhaustion of NSC pool, abnormal neurogenesis and cognitive decline in the mouse model of ICH. Together, these results provide molecular insights into ICH-induced cognitive impairment, shedding light on the value of maintaining NSC pool in preventing cognitive dysfunction in patients with hemorrhagic stroke or related conditions.

Targeted macrophage phagocytosis by Irg1/itaconate axis improves the prognosis of intracerebral hemorrhagic stroke and peritonitis.

BACKGROUND: Macrophages are innate immune cells whose phagocytosis function is critical to the prognosis of stroke and peritonitis. cis-aconitic decarboxylase immune-responsive gene 1 (Irg1) and its metabolic product itaconate inhibit bacterial infection, intracellular viral replication, and inflammation in macrophages. Here we explore whether itaconate regulates phagocytosis. METHODS: Phagocytosis of macrophages was investigated by time-lapse video recording, flow cytometry, and immunofluorescence staining in macrophage/microglia cultures isolated from mouse tissue. Unbiased RNA-sequencing and ChIP-sequencing assays were used to explore the underlying mechanisms. The effects of Irg1/itaconate axis on the prognosis of intracerebral hemorrhagic stroke (ICH) and peritonitis was observed in transgenic (Irg1flox/flox; Cx3cr1creERT/+, cKO) mice or control mice in vivo. FINDINGS: In a mouse model of ICH, depletion of Irg1 in macrophage/microglia decreased its phagocytosis of erythrocytes, thereby exacerbating outcomes (n = 10 animals/group, p < 0.05). Administration of sodium itaconate/4-octyl itaconate (4-OI) promoted macrophage phagocytosis (n = 7 animals/group, p < 0.05). In addition, in a mouse model of peritonitis, Irg1 deficiency in macrophages also inhibited phagocytosis of Staphylococcus aureus (n = 5 animals/group, p < 0.05) and aggravated outcomes (n = 9 animals/group, p < 0.05). Mechanistically, 4-OI alkylated cysteine 155 on the Kelch-like ECH-associated protein 1 (Keap1), consequent in nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) and transcriptional activation of Cd36 gene. Blocking the function of CD36 completely abolished the phagocytosis-promoting effects of Irg1/itaconate axis in vitro and in vivo. INTERPRETATION: Our findings provide a potential therapeutic target for phagocytosis-deficiency disorders, supporting further development towards clinical application for the benefit of stroke and peritonitis patients. FUNDING: The National Natural Science Foundation of China (32070735, 82371321 to Q. Li, 82271240 to F. Yang) and the Beijing Natural Science Foundation Program and Scientific Research Key Program of Beijing Municipal Commission of Education (KZ202010025033 to Q. Li).

Ischemic and hemorrhagic stroke lesion environments differentially alter the glia repair potential of neural progenitor cell and immature astrocyte grafts.

Using cell grafting to direct glia-based repair mechanisms in adult CNS injuries represents a potential therapeutic strategy for supporting functional neural parenchymal repair. However, glia repair directed by neural progenitor cell (NPC) grafts is dramatically altered by increasing lesion size, severity, and mode of injury. To address this, we studied the interplay between astrocyte differentiation and cell proliferation of NPC in vitro to generate proliferating immature astrocytes (ImA) using hysteretic conditioning. ImA maintain proliferation rates at comparable levels to NPC but showed robust immature astrocyte marker expression including Gfap and Vimentin. ImA demonstrated enhanced resistance to myofibroblast-like phenotypic transformations upon exposure to serum enriched environments in vitro compared to NPC and were more effective at scratch wound closure in vitro compared to quiescent astrocytes. Glia repair directed by ImA at acute ischemic striatal stroke lesions was equivalent to NPC but better than quiescent astrocyte grafts. While ischemic injury environments supported enhanced survival of grafts compared to healthy striatum, hemorrhagic lesions were hostile towards both NPC and ImA grafts leading to poor survival and ineffective modulation of natural wound repair processes. Our findings demonstrate that lesion environments, rather than transcriptional pre-graft states, determine the survival, cell-fate, and glia repair competency of cell grafts applied to acute CNS injuries.

CCL5 mediated astrocyte-T cell interaction disrupts blood-brain barrier in mice after hemorrhagic stroke.

The crosstalk between reactive astrocytes and infiltrated immune cells plays a critical role in maintaining blood-brain barrier (BBB) integrity. However, how astrocytes interact with immune cells and the effect of their interaction on BBB integrity after hemorrhagic stroke are still unclear. By performing RNA sequencing in astrocytes that were activated by interleukin-1α (IL-1α), tumor necrosis factor α (TNFα), and complement component 1q (C1q) treatment, we found CCL5 was among the top upregulated genes. Immunostaining and western blot results demonstrated that CCL5 was increased in mice brain after hemorrhagic stroke. Flow cytometry showed that knockout of astrocytic CCL5 reduced the infiltration of CD8+ but not CD4+ T and myeloid cells into the brain (p < 0.05). In addition, knockout CCL5 in astrocytes increased tight junction-related proteins ZO-1 and Occludin expression; reduced Evans blue leakage, perforin and granzyme B expression; improved neurobehavioral outcomes in hemorrhagic stroke mice (p < 0.05), while transplantation of CD8+ T cells reversed these protective effects. Moreover, co-culture of CD8+ T cells with bEnd.3 cells induced the apoptosis of bEnd.3 cells, which was rescued by inhibiting perforin. In conclusion, our study suggests that CCL5 mediated crosstalk between astrocytes and CD8+ T cells represents an important therapeutic target for protecting BBB in stroke.

An MMP-9 exclusive neutralizing antibody attenuates blood-brain barrier breakdown in mice with stroke and reduces stroke patient-derived MMP-9 activity.

Rapid upregulation of matrix metalloproteinase 9 (MMP-9) leads to blood-brain barrier (BBB) breakdown following stroke, but no MMP-9 inhibitors have been approved in clinic largely due to their low specificities and side effects. Here, we explored the therapeutic potential of a human IgG monoclonal antibody (mAb), L13, which was recently developed with exclusive neutralizing specificity to MMP-9, nanomolar potency, and biological function, using mouse stroke models and stroke patient samples. We found that L13 treatment at the onset of reperfusion following cerebral ischemia or after intracranial hemorrhage (ICH) significantly reduced brain tissue injury and improved the neurological outcomes of mice. Compared to control IgG, L13 substantially attenuated BBB breakdown in both types of stroke model by inhibiting MMP-9 activity-mediated degradations of basement membrane and endothelial tight junction proteins. Importantly, these BBB-protective and neuroprotective effects of L13 in wild-type mice were comparable to Mmp9 genetic deletion and fully abolished in Mmp9 knockout mice, highlighting the in vivo target specificity of L13. Meanwhile, ex vivo co-incubation with L13 significantly neutralized the enzymatic activities of human MMP-9 in the sera of ischemic and hemorrhagic stroke patients, or in the peri-hematoma brain tissues from hemorrhagic stroke patients. Overall, we demonstrated that MMP-9 exclusive neutralizing mAbs constitute a potential feasible therapeutic approach for both ischemic and hemorrhagic stroke.

Endogenous zinc protoporphyrin formation critically contributes to hemorrhagic stroke-induced brain damage.

Hemorrhagic stroke is a leading cause of death. The causes of intracerebral hemorrhage (ICH)-induced brain damage are thought to include lysis of red blood cells, hemin release and iron overload. These mechanisms, however, have not proven very amenable to therapeutic intervention, and so other mechanistic targets are being sought. Here we report that accumulation of endogenously formed zinc protoporphyrin (ZnPP) also critically contributes to ICH-induced brain damage. ICH caused a significant accumulation of ZnPP in brain tissue surrounding hematoma, as evidenced by fluorescence microscopy of ZnPP, and further confirmed by fluorescence spectroscopy and supercritical fluid chromatography-mass spectrometry. ZnPP formation was dependent upon both ICH-induced hypoxia and an increase in free zinc accumulation. Notably, inhibiting ferrochelatase, which catalyzes insertion of zinc into protoporphyrin, greatly decreased ICH-induced endogenous ZnPP generation. Moreover, a significant decrease in brain damage was observed upon ferrochelatase inhibition, suggesting that endogenous ZnPP contributes to the damage in ICH. Our findings reveal a novel mechanism of ICH-induced brain damage through ferrochelatase-mediated formation of ZnPP in ICH tissue. Since ferrochelatase can be readily inhibited by small molecules, such as protein kinase inhibitors, this may provide a promising new and druggable target for ICH therapy.

Effect of a Coordinated Community and Chronic Care Model Team Intervention vs Usual Care on Systolic Blood Pressure in Patients With Stroke or Transient Ischemic Attack: The SUCCEED Randomized Clinical Trial.

Importance: Few stroke survivors meet recommended cardiovascular goals, particularly among racial/ethnic minority populations, such as Black or Hispanic individuals, or socioeconomically disadvantaged populations. Objective: To determine if a chronic care model-based, community health worker (CHW), advanced practice clinician (APC; including nurse practitioners or physician assistants), and physician team intervention improves risk factor control after stroke in a safety-net setting (ie, health care setting where all individuals receive care, regardless of health insurance status or ability to pay). Design, Setting, and Participants: This randomized clinical trial included participants recruited from 5 hospitals serving low-income populations in Los Angeles County, California, as part of the Secondary Stroke Prevention by Uniting Community and Chronic Care Model Teams Early to End Disparities (SUCCEED) clinical trial. Inclusion criteria were age 40 years or older; experience of ischemic or hemorrhagic stroke or transient ischemic attack (TIA) no more than 90 days prior; systolic blood pressure (BP) of 130 mm Hg or greater or 120 to 130 mm Hg with history of hypertension or using hypertensive medications; and English or Spanish language proficiency. The exclusion criterion was inability to consent. Among 887 individuals screened for eligibility, 542 individuals were eligible, and 487 individuals were enrolled and randomized, stratified by stroke type (ischemic or TIA vs hemorrhagic), language (English vs Spanish), and site to usual care vs intervention in a 1:1 fashion. The study was conducted from February 2014 to September 2018, and data were analyzed from October 2018 to November 2020. Interventions: Participants randomized to intervention were offered a multimodal coordinated care intervention, including hypothesized core components (ie, ≥3 APC clinic visits, ≥3 CHW home visits, and Chronic Disease Self-Management Program workshops), and additional telephone visits, protocol-driven risk factor management, culturally and linguistically tailored education materials, and self-management tools. Participants randomized to the control group received usual care, which varied by site but frequently included a free BP monitor, self-management tools, and linguistically tailored information materials. Main Outcomes and Measures: The primary outcome was change in systolic BP at 12 months. Secondary outcomes were non-high density lipoprotein cholesterol, hemoglobin A1c, and C-reactive protein (CRP) levels, body mass index, antithrombotic adherence, physical activity level, diet, and smoking status at 12 months. Potential mediators assessed included access to care, health and stroke literacy, self-efficacy, perceptions of care, and BP monitor use. Results: Among 487 participants included, the mean (SD) age was 57.1 (8.9) years; 317 (65.1%) were men, and 347 participants (71.3%) were Hispanic, 87 participants (18.3%) were Black, and 30 participants (6.3%) were Asian. A total of 246 participants were randomized to usual care, and 241 participants were randomized to the intervention. Mean (SD) systolic BP improved from 143 (17) mm Hg at baseline to 133 (20) mm Hg at 12 months in the intervention group and from 146 (19) mm Hg at baseline to 137 (22) mm Hg at 12 months in the usual care group, with no significant differences in the change between groups. Compared with the control group, participants in the intervention group had greater improvements in self-reported salt intake (difference, 15.4 [95% CI, 4.4 to 26.0]; P = .004) and serum CRP level (difference in log CRP, -0.4 [95% CI, -0.7 to -0.1] mg/dL; P = .003); there were no differences in other secondary outcomes. Although 216 participants (89.6%) in the intervention group received some of the 3 core components, only 35 participants (14.5%) received the intended full dose. Conclusions and Relevance: This randomized clinical trial of a complex multilevel, multimodal intervention did not find vascular risk factor improvements beyond that of usual care; however, further studies may consider testing the SUCCEED intervention with modifications to enhance implementation and participant engagement. Trial Registration: ClinicalTrials.gov Identifier: NCT01763203.

IL-4/STAT6 signaling facilitates innate hematoma resolution and neurological recovery after hemorrhagic stroke in mice.

Intracerebral hemorrhage (ICH) is a devastating form of stroke affecting millions of people worldwide. Parenchymal hematoma triggers a series of reactions leading to primary and secondary brain injuries and permanent neurological deficits. Microglia and macrophages carry out hematoma clearance, thereby facilitating functional recovery after ICH. Here, we elucidate a pivotal role for the interleukin (IL)-4)/signal transducer and activator of transcription 6 (STAT6) axis in promoting long-term recovery in both blood- and collagenase-injection mouse models of ICH, through modulation of microglia/macrophage functions. In both ICH models, STAT6 was activated in microglia/macrophages (i.e., enhanced expression of phospho-STAT6 in Iba1+ cells). Intranasal delivery of IL-4 nanoparticles after ICH hastened STAT6 activation and facilitated hematoma resolution. IL-4 treatment improved long-term functional recovery in young and aged male and young female mice. In contrast, STAT6 knockout (KO) mice exhibited worse outcomes than WT mice in both ICH models and were less responsive to IL-4 treatment. The construction of bone marrow chimera mice demonstrated that STAT6 KO in either the CNS or periphery exacerbated ICH outcomes. STAT6 KO impaired the capacity of phagocytes to engulf red blood cells in the ICH brain and in primary cultures. Transcriptional analyses identified lower level of IL-1 receptor-like 1 (ST2) expression in microglia/macrophages of STAT6 KO mice after ICH. ST2 KO diminished the beneficial effects of IL-4 after ICH. Collectively, these data confirm the importance of IL-4/STAT6/ST2 signaling in hematoma resolution and functional recovery after ICH. Intranasal IL-4 treatment warrants further investigation as a clinically feasible therapy for ICH.

Iron Measured in Nasal Exudate Samples as a New and Useful Biomarker in the Differential Diagnosis of Patients with Acute Stroke.

INTRODUCTION: Differential diagnosis between ischemic and hemorrhagic strokes in the acute stage is one of the major challenges of neurovascular research. Several biomarkers have been studied, but attempts to date have focused on determining their blood levels. Recently, cerebral lymphatic drainage toward the nostrils has been discovered, giving us the chance to study nasal exudate looking for biomarkers of neural damage. We sought to confirm whether iron levels in nasal exudate could identify the hemorrhagic nature of acute stroke. METHODS: We studied iron nasal exudate levels in 32 ischemic and 43 hemorrhagic stroke patients. All patients underwent neurological examination assessed by the National Institutes of Health Stroke Scale (NIHSS), brain computed tomography to the differential diagnosis of stroke subtype, laboratory tests, and measurement of iron levels in nasal exudate. RESULTS: The iron levels in nasal exudate were higher in hemorrhagic stroke patients. The area under the receiver operating characteristic curve for ischemic/hemorrhagic stroke discrimination was 0.896 (95% confidence interval 0.823-0.970) and cutoff point of 0.078 nmol/mg (sensitivity 93%, specificity 73%). CONCLUSIONS: Our findings suggest that iron levels in nasal exudate may be useful in the acute stage for the differential diagnosis between ischemic and hemorrhagic damage in acute stroke patients. They also open a potential field to study other biomarkers in nasal exudate in several neurological disorders. Clinical studies must be performed to confirm our results.

Neurovascular manifestations in connective tissue diseases: The case of Marfan Syndrome.

Patients with connective tissue diseases (CTDs) are suspected to be at higher risk for cerebrovascular involvement, such as intracranial aneurysms, dissections and strokes, than the general population. Particularly, Marfan Syndrome (MFS) has been reported as associated with an increased risk of cerebrovascular alterations. Literature data report different prevalence of intracranial aneurysms in MFS, ranging from 4 % to 29 %, suggesting a role of genetic cause that involves the regulation of the TGF-ß signaling. Ischemic and hemorrhagic strokes have been also reported in MFS, but with an estimated prevalence from 3 % to 4 %. However, the aetiology of both events appears to be reliable more to a cardiac source than to the primary connective tissue defect. Finally, the available literature suggests that MFS patients have a higher prevalence of arterial tortuosity of neck and head vessels and these findings may be related to an enhanced chance of dissection. Overall, despite of the lack of studies, we could affirm that it may exists an increased prevalence of some neurovascular findings in MFS patients. Nevertheless, further studies are required to determine the true prevalence of these features and investigate specific gene mutations involved in MFS.

Systematic Understanding of Mechanism of Yi-Qi-Huo-Xue Decoction Against Intracerebral Hemorrhagic Stroke Using a Network Pharmacology Approach.

BACKGROUND Intracerebral hemorrhage (ICH), a fatal type of stroke, profoundly affects public health. Yi-Qi-Huo-Xue decoction (YQHXD), a traditional Chinese medicine (TCM) prescription, is verified to be an efficient method to treat ICH stroke among the Chinese population. Nevertheless, the pharmacological mechanisms of YQHXD have been unclear. MATERIAL AND METHODS We used a strategy based on network pharmacology to explore the possible multi-component, multi-target, and multi-pathway pattern of YQHXD in treating ICH. First, candidate targets for YQHXD were identified using the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP). Then, these candidate YQHXD targets were used in combination with the known targets for the treatment of ICH stroke to construct the core network (cPPI) using data on protein-protein interaction (PPI). We calculated 5 topological parameters for identification of the main hubs. Pathway enrichment and GO biological process enrichment analyses were performed after the incorporation of the main hubs into ClueGO. RESULTS In total, 55 candidate YQHXD targets for ICH were recognized to be the major hubs in accordance with their topological importance. As suggested by enrichment analysis, the YQHXD targets for ICH were roughly classified into several biological processes (related to redox equilibrium, cell-cell communication, adhesion and collagen biosynthesis, cytokine generation, lymphocyte differentiation and activation, neurocyte apoptosis and development, neuroendocrine system, and vascular development) and related pathways (VEGF, mTOR, NF-kappaB, RAS/MAPK, JAK/STAT and cytokine-cytokine receptors interaction), indicating those mechanisms underlying the therapeutic effect of YQHXD. CONCLUSIONS The present results may serve as a pharmacological framework for TCM studies in the future, helping to promote the use of YQHXD in clinical treatment of ICH.

VEGF signalling enhances lesion burden in KRIT1 deficient mice.

The exact molecular mechanisms underlying CCM pathogenesis remain a complicated and controversial topic. Our previous work illustrated an important VEGF signalling loop in KRIT1 depleted endothelial cells. As VEGF is a major mediator of many vascular pathologies, we asked whether the increased VEGF signalling downstream of KRIT1 depletion was involved in CCM formation. Using an inducible KRIT1 endothelial-specific knockout mouse that models CCM, we show that VEGFR2 activation plays a role in CCM pathogenesis in mice. Inhibition of VEGFR2 using a specific inhibitor, SU5416, significantly decreased the number of lesions formed and slightly lowered the average lesion size. Notably, VEGFR2 inhibition also decreased the appearance of lesion haemorrhage as denoted by the presence of free iron in adjacent tissues. The presence of free iron correlated with increased microvessel permeability in both skeletal muscle and brain, which was completely reversed by SU5416 treatment. Finally, we show that VEGFR2 activation is a common downstream consequence of KRIT1, CCM2 and CCM3 loss of function, though the mechanism by which VEGFR2 activation occurs likely varies. Thus, our study clearly shows that VEGFR2 activation downstream of KRIT1 depletion enhances the severity of CCM formation in mice, and suggests that targeting VEGF signalling may be a potential future therapy for CCM.