HAND2-AS1 Promotes Ferroptosis to Reverse Lenvatinib Resistance in Hepatocellular Carcinoma by TLR4/NOX2/DUOX2 Axis.

INTRODUCTION: Lenvatinib resistance causes less than 40% of the objective response rate. Therefore, it is urgent to explore new therapeutic targets to reverse the lenvatinib resistance for HCC. HAND2-AS1 is a critical tumor suppressor gene in various cancers. METHODS: Here, we investigated the role of HAND2-AS1 in the molecular mechanism of lenvatinib resistance in HCC. It was found that HAND2-AS1 was lowly expressed in the HepG2 lenvatinib resistance (HepG2-LR) cells and HCC tissues and associated with progression-free intervals via TCGA. Overexpression of HAND2-AS1 (OE-HAND2-AS1) decreased the IC50 of lenvatinib in HepG2-LR cells to reverse lenvatinib resistance. Moreover, OE-HAND2-AS1 induced intracellular concentrations of malondialdehyde (MDA) and lipid ROS and decreased the ratio of glutathione to glutathione disulfide (GSH/GSSG) to promote ferroptosis. RESULTS: A xenograft model in which nude mice were injected with OE-HAND2-AS1 HepG2-LR cells confirmed that OE-HAND2-AS1 could reverse lenvatinib resistance and decrease tumor formation in vivo. HAND2-AS1 promoted the expression of ferroptosis-related genes (TLR4, NOX2, and DUOX2) and promoted ferroptosis to reverse lenvatinib resistance by increasing TLR4/ NOX2/DUOX2 via competing endogenous miR-219a-1-3p in HCC cells. Besides, patients with a low HAND2-AS1 level had early recurrence after resection. CONCLUSION: These findings suggested that HAND2-AS1 may be a potential therapeutic target and an indicator of early recurrence for HCC.

Serum Exosome-Derived microRNA-193a-5p and miR-381-3p Regulate Adenosine 5'-Monophosphate-Activated Protein Kinase/Transforming Growth Factor Beta/Smad2/3 Signaling Pathway and Promote Fibrogenesis.

INTRODUCTION: Liver fibrosis results from chronic liver injury and inflammation, often leading to cirrhosis, liver failure, portal hypertension, and hepatocellular carcinoma. Progress has been made in understanding the molecular mechanisms underlying hepatic fibrosis; however, translating this knowledge into effective therapies for disease regression remains a challenge, with considerably few interventions having entered clinical validation. The roles of exosomes during fibrogenesis and their potential as a therapeutic approach for reversing fibrosis have gained significant interest. This study aimed to investigate the association between microRNAs (miRNAs) derived from serum exosomes and liver fibrosis and to evaluate the effect of serum exosomes on fibrogenesis and fibrosis reversal, while identifying the underlying mechanism. METHODS: Using serum samples collected from healthy adults and paired histologic patients with advanced fibrosis or cirrhosis, we extracted human serum exosomes by ultrahigh-speed centrifugation. Transcriptomic analysis was conducted to identify dysregulated exosome-derived miRNAs. Liver fibrosis-related molecules were determined by qRT-PCR, Western blot, Masson staining, and immunohistochemical staining. In addition, we analyzed the importance of serum exosome-derived miRNA expression levels in 42 patients with advanced fibrosis or cirrhosis. RESULTS: Exosome-derived miR-193a-5p and miR-381-3p were associated with fibrogenesis, as determined by transcriptomic screening. Compared with healthy control group, the high expression of serum exosome-derived miR-193a-5p and miR-381-3 in chronic hepatitis B (n = 42) was closely associated with advanced liver fibrosis and cirrhosis. In vitro , exosome-derived miRNA-193a-5p and miR-381-3p upregulated the expression of α-smooth muscle actin, collagen 1a1, and tissue inhibitors of metalloproteinase 1 in the human hepatic stellate cell line at both mRNA and protein levels. DISCUSSION: Serum exosome-derived miR-193a-5p and miR-381-3p regulated the adenosine 5'-monophosphate-activated protein kinase/transforming growth factor beta/Smad2/3 signaling pathway and promoted fibrogenesis.

Association of white matter hyperintensities with long-term EGFR-TKI treatment and prediction of progression risk.

PURPOSE: The purpose of this study was to test the hypothesis that brain white matter hyperintensities (WMH) are more common in patients receiving epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) and identify clinical risk factors associated with WMH. EXPERIMENTAL DESIGN: This multiple-center, prospective cohort study was conducted from March 2017 to July 2020. Two groups of patients with non-small cell lung cancer (NSCLC) who received or did not receive EGFR-TKI were included and followed up for more than 24 months. The progression of WMH was defined as an increase of ≥1 point on the Fazekas visual rating scale between the baseline and at the 2-year follow-up. A modified Poisson regression model was performed to evaluate risk factors on increased WMH load. RESULTS: Among 286 patients with NSCLC, 194 (68%) patients with NSCLC who received EGFR-TKI and 92 (32%) patients with NSCLC without EGFR-TKI treatment were analyzed. Modified Poisson regression analysis showed that EGFR-TKI treatment was independently associated with the WMH progression (EGFR-TKI: aRR 2.72, 95% confidence interval [CI] 1.46-5.06, p = .002). Interleukin (IL)-2, IL-4, and IL-10 were associated with increased WMH in the adjusted model (IL-2: aRR 1.55 [95% CI 1.06-2.25], p = .023; IL-4: aRR 1.66 [95% CI 1.13-2.43], p = .010; IL-10: aRR 1.48 [95% CI 1.06-2.06], p = .020). CONCLUSION: Patients with NSCLC who received EGFR-TKI may be at higher risk of developing WMH or worsening of WMH burden. The impact of increased WMH lesions in these patients is to be further assessed. IL-2, IL-4, and IL-10 may be used as potential biomarkers to monitor the risk of increased WMH burden.

Ferroptosis and Iron Metabolism after Intracerebral Hemorrhage.

The method of iron-dependent cell death known as ferroptosis is distinct from apoptosis. The suppression of ferroptosis after intracerebral hemorrhage (ICH) will effectively treat ICH and improve prognosis. This paper primarily summarizes the mechanism of ferroptosis after ICH, with an emphasis on lipid peroxidation, the antioxidant system, iron metabolism, and other pathways. In addition, regulatory targets and drug molecules were described. Although there has been some progress in the field of study, there are still numerous gaps. The mechanism by which non-heme iron enters neurons through the blood-brain barrier (BBB), the mitochondrial role in ferroptosis, and the specific mechanism by which lipid peroxidation induces ferroptosis remain unclear and require further study. In addition, the inhibitory effect of many drugs on ferroptosis after ICH has only been demonstrated in basic experiments and must be translated into clinical trials. In summary, research on ferroptosis following ICH will play an important role in the treatment of ICH.

The P2RY12 receptor promotes VSMC-derived foam cell formation by inhibiting autophagy in advanced atherosclerosis.

Vascular smooth muscle cells (VSMCs) are an important source of foam cells in atherosclerosis. The mechanism for VSMC-derived foam cell formation is, however, poorly understood. Here, we demonstrate that the P2RY12/P2Y12 receptor is important in regulating macroautophagy/autophagy and VSMC-derived foam cell formation in advanced atherosclerosis. Inhibition of the P2RY12 receptor ameliorated lipid accumulation and VSMC-derived foam cell formation in high-fat diet-fed apoe-/- mice (atherosclerosis model) independent of LDL-c levels. Activation of the P2RY12 receptor blocked cholesterol efflux via PI3K-AKT, while genetic knockdown or pharmacological inhibition of the P2RY12 receptor inhibited this effect in VSMCs. Phosphoproteomic analysis showed that the P2RY12 receptor regulated the autophagy pathway in VSMCs. Additionally, activation of the P2RY12 receptor inhibited MAP1LC3/LC3 maturation, SQSTM1 degradation, and autophagosome formation in VSMCs. Genetic knockdown of the essential autophagy gene Atg5 significantly attenuated P2RY12 receptor inhibitor-induced cholesterol efflux in VSMCs. Furthermore, activation of the P2RY12 receptor led to the activation of MTOR through PI3K-AKT in VSMCs, whereas blocking MTOR activity (rapamycin) or reducing MTOR expression reversed the inhibition of cholesterol efflux mediated by the P2RY12 receptor in VSMCs. In vivo, inhibition of the P2RY12 receptor promoted autophagy of VSMCs through PI3K-AKT-MTOR in advanced atherosclerosis in apoe-/- mice, which could be impeded by an autophagy inhibitor (chloroquine). Therefore, we conclude that activation of the P2RY12 receptor decreases cholesterol efflux and promotes VSMC-derived foam cell formation by blocking autophagy in advanced atherosclerosis. Our study thus suggests that the P2RY12 receptor is a therapeutic target for treating atherosclerosis.Abbreviations: 2-MeSAMP: 2-methylthioadenosine 5'-monophosphate; 8-CPT-cAMP: 8-(4-chlorophenylthio)-adenosine-3',5'-cyclic-monophosphate; ABCA1: ATP binding cassette subfamily A member 1; ABCG1: ATP binding cassette subfamily G member 1; ACTB: actin beta; ADPßs: adenosine 5'-(alpha, beta-methylene) diphosphate; ALs: autolysosomes; AMPK: AMP-activated protein kinase; APOA1: apolipoprotein A1; APs: autophagosomes; ATG5: autophagy related 5; ATV: atorvastatin; AVs: autophagic vacuoles; CD: chow diet; CDL: clopidogrel; CQ: chloroquine; DAPI: 4',6-diamidino-2-phenylindole; dbcAMP: dibutyryl-cAMP; DIL-oxLDL: dioctadecyl-3,3,3,3-tetramethylin docarbocyanine-oxLDL; EIF4EBP1/4E-BP1: eukaryotic translation initiation factor 4E binding protein 1; EVG: elastic van gieson; HE: hematoxylin-eosin; HDL: high-density lipoprotein; HFD: high-fat diet; KEGG: Kyoto Encyclopedia of Genes and Genomes; LDL-c: low-density lipoprotein cholesterol; LDs: lipid droplets; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; Masson: masson trichrome; MCPT: maximal carotid plaque thickness; MK2206: MK-2206 2HCL; NBD-cholesterol: 22-(N-[7-nitrobenz-2-oxa-1,3-diazol-4-yl] amino)-23,24-bisnor-5-cholen-3ß-ol; OLR1/LOX-1: oxidized low density lipoprotein receptor 1; ORO: oil Red O; ox-LDL: oxidized low-density lipoprotein; SQSTM1/p62: sequestosome 1; TEM: transmission electron microscopy; TIC: ticagrelor; ULK1: unc-51 like autophagy activating kinase 1; VSMCs: vascular smooth muscle cells.

Neurologic Manifestations of Hospitalized Patients With Coronavirus Disease 2019 in Wuhan, China.

Importance: The outbreak of coronavirus disease 2019 (COVID-19) in Wuhan, China, is serious and has the potential to become an epidemic worldwide. Several studies have described typical clinical manifestations including fever, cough, diarrhea, and fatigue. However, to our knowledge, it has not been reported that patients with COVID-19 had any neurologic manifestations. Objective: To study the neurologic manifestations of patients with COVID-19. Design, Setting, and Participants: This is a retrospective, observational case series. Data were collected from January 16, 2020, to February 19, 2020, at 3 designated special care centers for COVID-19 (Main District, West Branch, and Tumor Center) of the Union Hospital of Huazhong University of Science and Technology in Wuhan, China. The study included 214 consecutive hospitalized patients with laboratory-confirmed diagnosis of severe acute respiratory syndrome coronavirus 2 infection. Main Outcomes and Measures: Clinical data were extracted from electronic medical records, and data of all neurologic symptoms were checked by 2 trained neurologists. Neurologic manifestations fell into 3 categories: central nervous system manifestations (dizziness, headache, impaired consciousness, acute cerebrovascular disease, ataxia, and seizure), peripheral nervous system manifestations (taste impairment, smell impairment, vision impairment, and nerve pain), and skeletal muscular injury manifestations. Results: Of 214 patients (mean [SD] age, 52.7 [15.5] years; 87 men [40.7%]) with COVID-19, 126 patients (58.9%) had nonsevere infection and 88 patients (41.1%) had severe infection according to their respiratory status. Overall, 78 patients (36.4%) had neurologic manifestations. Compared with patients with nonsevere infection, patients with severe infection were older, had more underlying disorders, especially hypertension, and showed fewer typical symptoms of COVID-19, such as fever and cough. Patients with more severe infection had neurologic manifestations, such as acute cerebrovascular diseases (5 [5.7%] vs 1 [0.8%]), impaired consciousness (13 [14.8%] vs 3 [2.4%]), and skeletal muscle injury (17 [19.3%] vs 6 [4.8%]). Conclusions and Relevance: Patients with COVID-19 commonly have neurologic manifestations. During the epidemic period of COVID-19, when seeing patients with neurologic manifestations, clinicians should suspect severe acute respiratory syndrome coronavirus 2 infection as a differential diagnosis to avoid delayed diagnosis or misdiagnosis and lose the chance to treat and prevent further transmission.

Microglia-derived TNF-α mediates endothelial necroptosis aggravating blood brain-barrier disruption after ischemic stroke.

Endothelium (EC) is a key component of blood-brain barrier (BBB), and has an important position in the neurovascular unit. Its dysfunction and death after cerebral ischemic/reperfusion (I/R) injury not only promote evolution of neuroinflammation and brain edema, but also increase the risk of intracerebral hemorrhage of thrombolytic therapies. However, the mechanism and specific interventions of EC death after I/R injury are poorly understood. Here we showed that necroptosis was a mechanism underlying EC death, which promoted BBB breakdown after I/R injury. Treatment of rats with receptor interacting protein kinase 1 (RIPK1)-inhibitor, necrostatin-1 reduced endothelial necroptosis and BBB leakage. We furthermore showed that perivascular M1-like microglia-induced endothelial necroptosis leading to BBB disruption requires tumor necrosis factor-α (TNF-α) secreted by M1 type microglia and its receptor, TNF receptor 1 (TNFR1), on endothelium as the primary mediators of these effects. More importantly, anti-TNFα (infliximab, a potent clinically used drug) treatment significantly ameliorate endothelial necroptosis, BBB destruction and improve stroke outcomes. Our data identify a previously unexplored role for endothelial necroptosis in BBB disruption and suggest infliximab might serve as a potential drug for stroke therapy.

Complement C3a receptor antagonist attenuates tau hyperphosphorylation via glycogen synthase kinase 3ß signaling pathways.

Neurofibrillary tangles aggregated from hyperphosphorylated tau protein are the main pathological feature of Alzheimer's disease (AD). Complement C3 (or C3a) is the core component of the complement system and is associated with AD pathological processes. However, it remains unclear whether C3a or the C3a receptor has any effect on tau phosphorylation. In this study, we found that exposure of SH-SY5Y cells to okadaic acid (OA) decreased cell viabilities and induced tau hyperphosphorylation. These effects were alleviated by C3a receptor antagonist SB290157 and were further validated by C3a receptor siRNA in OA-treated SH-SY5Y cells. In addition, our results demonstrated that SB290157 markedly inhibited the activities of glycogen synthase kinase 3ß (GSK3ß), but had no effect on protein phosphatase 2A C subunit (PP2Ac) and cyclin-dependent kinases 5 (CDK5). Our findings here indicate the unique role of the C3a receptor in regulating tau phosphorylation via GSK3ß signaling pathways and suggest that the C3a receptor may be a viable target for treating AD.

Sema3E/PlexinD1 signaling inhibits postischemic angiogenesis by regulating endothelial DLL4 and filopodia formation in a rat model of ischemic stroke.

Angiogenesis is a crucial defense response to hypoxia that regulates the process of raising the promise of long-term neurologic recovery during the management of stroke. A high expression of antiangiogenic factors leads to the loss of neovascularization capacity in pathologic conditions. We have previously documented an impairment of the cerebral vessel perfusion and neovascularization in the cortex neighboring the stroke-induced lesion, which was accompanied by an activation of semaphorin 3E (Sema3E)/PlexinD1 after ischemic stroke. In this study, we employed micro-optical sectioning tomography to fully investigate the details of the vascular pattern, including the capillaries. We found that after transient middle cerebral artery occlusion, inhibiting PlexinD1 signaling led to an organized recovery of the vascular network in the ischemic area. We then further explored the possible mechanisms. In vivo, Sema3E substantially decreased dynamic delta-like 4 (DLL4) expression. In cultured brain microvascular endothelial cells, Sema3E down-regulated DLL4 expression via inhibiting Ras-related C3 botulinum toxin substrate 1-induced JNK phosphorylation. At the microcosmic level, Sema3E/PlexinD1 signaling promoted F-actin disassembly and focal adhesion reduction by activating the small guanosine triphosphatase Ras homolog family member J by releasing RhoGEF Tuba from direct binding to PlexinD1, thus mediating endothelial cell motility and filopodia retraction. Our study reveals that Sema3E/PlexinD1 signaling, which suppressed endothelial DLL4 expression, cell motility, and filopodia formation, is expected to be a novel druggable target for angiogenesis during poststroke progression.-Zhou, Y.-F., Chen, A.-Q., Wu, J.-H., Mao, L., Xia, Y.-P., Jin, H.-J., He, Q.-W., Miao, Q. R., Yue, Z.-Y., Liu, X.-L., Huang, M., Li, Y.-N., Hu, B. Sema3E/PlexinD1 signaling inhibits postischemic angiogenesis by regulating endothelial DLL4 and filopodia formation in a rat model of ischemic stroke.

Acute stroke patients' knowledge of stroke at discharge in China: a cross-sectional study.

OBJECTIVES: A good mastery of stroke-related knowledge can be of great benefit in developing healthy behaviours. This study surveyed the knowledge about stroke and influencing factors among patients with acute ischaemic stroke (AIS) at discharge in a Chinese province. METHODS: A cross-section study was conducted from November 1, 2014 to January 31, 2015. A total of 1531 AIS patients in Hubei Province completed a questionnaire at discharge. Multivariate linear regression was used to identify the influencing factors of their knowledge of stroke. RESULTS: About 31.2% of the respondents did not know that stroke is caused by blockage or rupture of cerebral blood vessels and 20.3% did not realise they need immediate medical attention after onset. Approximately 50% did not know that sudden blurred vision, dizziness, headache and unconsciousness are the warning signs of stroke. Over 40% were not aware of the risk factors of the condition, such as hypertension, hyperlipidaemia, diabetes mellitus, smoking and obesity. Over 20% had no idea that they need long-term medication and strict control of blood pressure, blood lipids and blood sugar. Their knowledge levels were correlated with regions of residence (P < 0.0001), socioeconomic status (P < 0.05), physical condition (P < 0.01), previous stroke (P < 0.0001) and family members and friends having had a stroke (P < 0.01). CONCLUSIONS: Most AIS patients in Hubei Province, China, had little knowledge of stroke at discharge. Further efforts should be devoted to strengthening the in-hospital education of stroke patients, especially those with a low income and those from rural areas.

MicroRNA-149-5p regulates blood-brain barrier permeability after transient middle cerebral artery occlusion in rats by targeting S1PR2 of pericytes.

Blood-brain barrier (BBB) disruption caused by reperfusion injury after ischemic stroke is an intractable event conducive to further injury. Brain pericytes play a vital role in maintaining BBB integrity by interacting with other components of the BBB. In this study, we found that sphingosine-1-phosphate receptor (S1PR)2 expressed in pericytes was significantly up-regulated after ischemia in vivo and in vitro. By using a S1PR2 antagonist (JTE-013), we showed that S1PR2 plays a critical role in the induction of BBB permeability of transient middle cerebral artery occlusion (tMCAO) rats and the in vitro BBB model. Furthermore, we discovered that S1PR2 may decrease N-cadherin expression and increase pericyte migration via NF-κB p65 signal and found that S1PR2 could be regulated by miR-149-5p negatively, which was decreased in the ischemic boundary zone and cultured pericytes after ischemia. Overexpression of miR-149-5p in cultured pericytes substantially increased N-cadherin expression and decreased pericyte migration, which decreased BBB leakage in the in vitro model. Up-regulating miR-149-5p by intracerebroventricular injection of agomir-149-5p attenuated BBB permeability and improved the outcomes of tMCAO rats significantly. Thus, our data suggest that miR-149-5p may serve as a potential target for treatment of BBB disruption after ischemic stroke.-Wan, Y., Jin, H.-J., Zhu, Y.-Y., Fang, Z., Mao, L., He, Q., Xia, Y.-P., Li, M., Li, Y., Chen, X., Hu, B. MicroRNA-149-5p regulates blood-brain barrier permeability after transient middle cerebral artery occlusion in rats by targeting S1PR2 of pericytes.

Administration of sonic hedgehog protein induces angiogenesis and has therapeutic effects after stroke in rats.

The Sonic hedgehog (Shh) signaling pathway is recapitulated in response to ischemic injury. Here, we investigated the clinical implications of Shh protein in the ischemic stroke and explored the underlying mechanism. Intracerebroventricular injection of Shh, Cyclopamine, or anti-vascular endothelial growth factor (VEGF) was performed immediately after permanent middle cerebral artery occlusion (pMCAO) surgery and lasted for 7days (d). Phosphate-buffered saline (PBS) was used as control. Neurological deficits and infarct volume were examined 7d after pMCAO. Microvascular density with fluorescein-iso-thiocyanate (FITC) assay and double staining with CD31 and Ki-67 was measured at 7d. To observe in vitro angiogenesis, rat brain microvascular endothelial cells (RBMECs) were incubated under oxygen glucose deprivation (OGD) for 6h (h) and treated with Shh/anti-VEGF. We found that (1) Shh improved neurological scores and reduced infarct volume, which was blocked by Cyclopamine, (2) Shh improved the microvascular density and promoted angiogenesis and neuron survival in the ischemic boundary zone, (3) Shh enhanced VEGF expression and VEGF antibody could reverse angiogenic and protective effect of Shh in vivo and in vitro. These data demonstrate that the administration of Shh protein could protect brain from ischemic injury, in part by promoting angiogenic repair.

P2Y12 Promotes Migration of Vascular Smooth Muscle Cells Through Cofilin Dephosphorylation During Atherogenesis.

OBJECTIVE: P2Y12 is a well-recognized receptor expressed on platelets and the target of thienopyridine-type antiplatelet drugs. However, recent evidence suggests that P2Y12 expressed in vessel wall plays a role in atherogenesis, but the mechanisms remain elusive. In this study, we examined the molecular mechanisms of how vessel wall P2Y12 mediates vascular smooth muscle cells (VSMCs) migration and promotes the progression of atherosclerosis. APPROACH AND RESULTS: Using a high-fat diet-fed apolipoprotein E-deficient mice model, we found that the expression of P2Y12 in VSMCs increased in a time-dependent manner and had a linear relationship with the plaque area. Moreover, administration of P2Y12 receptor antagonist for 12 weeks caused significant reduction in atheroma and decreased the abundance of VSMCs in plaque. In cultured VSMCs, we found that activation of P2Y12 receptor inhibited cAMP/protein kinase A signaling pathway, which induced cofilin dephosphorylation and filamentous actin disassembly, thereby enhancing VSMCs motility and migration. In addition, the number of P2Y12-positive VSMCs was decreased in the carotid artery plaque from patients receiving clopidogrel. CONCLUSIONS: Vessel wall P2Y12 receptor, which promotes VSMCs migration through cofilin dephosphorylation, plays a critical role in the development of atherosclerotic lesion and may be used as a therapeutic target for atherosclerosis.

MiR-181b Antagonizes Atherosclerotic Plaque Vulnerability Through Modulating Macrophage Polarization by Directly Targeting Notch1.

Atherosclerotic plaque vulnerability is the major cause for acute stroke and could be regulated by macrophage polarization. MicroRNA-181b (miR-181b) was involved in macrophage differential. Here, we explore whether miR-181b could regulate atherosclerotic plaque vulnerability by modulating macrophage polarization and the underline mechanisms. In acute stroke patients with atherosclerotic plaque, we found that the serum level of miR-181b was decreased. Eight-week apolipoprotein E knockout (ApoE-/-) mice were randomly divided into three groups (N = 10): mice fed with normal saline (Ctrl), mice fed with high-fat diet, and tail vein injection with miRNA agomir negative control (AG-NC)/miR-181b agomir (181b-AG, a synthetic miR-181b agonist). We found that the serum level of miR-181b in AG-NC group was lower than that in Ctrl group. Moreover, 181b-AG could upregulate miR-181b expression, reduce artery burden and attenuate atherosclerotic plaque vulnerability by modulating macrophage polarization. In RAW264.7 cells treated with oxidized low-density lipoprotein (ox-LDL), we found miR-181b could reverse the function of ox-LDL on M1/M2 markers at both mRNA and protein levels. Furthermore, by employing luciferase reporter assay, we found that Notch1 was a direct target of miR-181b and could be regulated by miR-181b in vivo and in vitro. Finally, inhibition of Notch1 could abolish the function of downregulating miR-181b on increasing M2 phenotype macrophages. Our study demonstrates that administration of miR-181b could reduce atherosclerotic plaque vulnerability partially through modulating macrophage phenotype by directly targeting Notch1.

MicroRNA-493 regulates angiogenesis in a rat model of ischemic stroke by targeting MIF.

MicroRNA-493 (miR-493) is known to suppress tumour metastasis and angiogenesis and its expression is decreased in stroke patients. In the present study, we investigated a role for miR-493 in regulating post-stroke angiogenesis. We found decreased expression of miR-493 in the ischemic boundary zone (IBZ) of rats subjected to middle cerebral artery occlusion (MCAO), and in rat brain microvascular endothelial cells (RBMECs) exposed to oxygen glucose deprivation. Down-regulating miR-493 with a lateral ventricular injection of antagomir-493, a synthetic miR-493 inhibitor, increased capillary density in the IBZ, decreased focal infarct volume and ameliorated neurologic deficits in rats subjected to MCAO. Intriguingly, MCAO also increased the expression of macrophage migration inhibitory factor (MIF) in the IBZ of rats; MIF expression was also increased in RBMECs exposed to oxygen glucose deprivation. We found that miR-493 directly targeted MIF, and that the protective effect of miR-493 inhibition in angiogenesis was attenuated by knocking down MIF. This effect could then be rescued by administration of recombinant MIF. Our findings highlight the importance of miR-493 in regulating angiogenesis after MCAO, and indicate that miR-493 is a potential therapeutic target in the treatment of stroke.

MiR-150 Regulates Poststroke Cerebral Angiogenesis via Vascular Endothelial Growth Factor in Rats.

AIMS: Angiogenesis is a harmonized target for poststroke recovery. Therefore, exploring the mechanisms involved in angiogenesis after stroke is vitally significant. In this study, we are reporting a miR-150-based mechanism underlying cerebral poststroke angiogenesis. METHODS: Rat models of middle cerebral artery occlusion (MCAO) and cell models of oxygen-glucose deprivation were conducted. Capillary density, tube formation, cell proliferation, and cell migration were measured by FITC-dextran assay, matrigel assay, Ki-67 staining, and wound healing assay, respectively. The expression of miR-150 and vascular endothelial growth factor (VEGF) was, respectively, measured by RT-PCR and Western blotting. Dual-luciferase assay was conducted to confirm the binding sites between miR-150 and VEGF. RESULTS: We found that miR-150 expression in the brain and serum of rats subjected to cerebral ischemia, and in oxygen-glucose-deprived brain microvascular endothelial cells (BMVECs) and astrocytes. Upregulation of miR-150 expression could decrease vascular density of infarct border zone in rat after MCAO and decrease tube formation, proliferation, and migration of BMVECs. We also found that miR-150 could negatively regulate the expression of VEGF, and VEGF was confirmed to be a direct target of miR-150. Moreover, VEGF mediated the function of miR-150 on tube formation, proliferation, and migration of BMVECs. CONCLUSIONS: Our data suggested that miR-150 could regulate cerebral poststroke angiogenesis in rats through VEGF.

Endogenous endothelial progenitor cells participate in neovascularization via CXCR4/SDF-1 axis and improve outcome after stroke.

AIM: To study whether endogenous endothelial progenitor cells (EPCs) are involved in neovascularization after stroke. MATERIALS AND METHODS: Animal stroke models were established by subjecting male SD rats to permanent middle cerebral artery occlusion (pMCAO). Vessels in ischemic boundary zone (IBZ) were stained with antibody against laminin at 1 to 21 days after pMCAO. EPCs and newly formed vessels were identified by staining with special markers. After inhibiting recruitment of EPCs with AMD3100, a CXCR4 antagonist, endogenous EPCs, capillary density, cerebral blood flow (CBF) in IBZ, and neurobehavioral functions were assessed by staining, FITC-dextran, laser-Doppler perfusion monitor, and neurologic severity score. RESULTS: After pMCAO, vessels were found in IBZ at day 3, reaching a peak at day 14. The change in number of laminin-positive cells showed a similar pattern with that of vessels. Apart from few endothelial cells, most of laminin-positive cells were endogenous EPCs. After treatment with AMD3100, the number of endogenous EPCs, capillary density, and CBF in IBZ were significantly reduced, and neurobehavioral functions were worse as compared with the normal saline group. CONCLUSIONS: Our findings suggested that endogenous EPCs participated in the neovascularization via CXCR4/SDF-1 axis after pMCAO and mobilizing endogenous EPCs could be a treatment alternative for stroke.

Sonic hedgehog (Shh) regulates the expression of angiogenic growth factors in oxygen-glucose-deprived astrocytes by mediating the nuclear receptor NR2F2.

Sonic hedgehog (Shh) has been found to regulate the angiogenic growth factor such as VEGF, Ang-1, and Ang-2 during ischemic insults, but the underlying mechanism is not fully understood. In this study, we employed oxygen-glucose deprivation (OGD) in astrocytes to mimic the ischemia in vitro. We found that OGD could induce the expressions of VEGF, Ang-1, and Ang-2, with the expression of Shh signaling components increased. Moreover, inhibiting the Shh signaling pathway with 5EI, a specific antibody, could decrease the expressions of VEGF, Ang-1, and Ang-2. Furthermore, the administration of exogenous Shh could induce the expressions of VEGF, Ang-1, and Ang-2 in astrocytes. The results of silencing Gli-1, or NR2F2, exhibited that exogenous Shh could regulate the expressions of VEGF, Ang-1, and Ang-2 in astrocytes by activating the NR2F2, but not the Gli-1. These results suggested that Shh could regulate the angiogenic growth factor after ischemic insults in astrocytes, and the regulation was partially mediated by the NR2F2.