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1.  MicroRNAs in Cerebral Ischemia 
Stroke Research and Treatment  2013;2013:276540.
The risk of ischemic stroke increases substantially with age, making it the third leading cause of death and the leading cause of long-term disability in the world. Numerous studies demonstrated that genes, RNAs, and proteins are involved in the occurrence and development of stroke. Current studies found that microRNAs (miRNAs or miRs) are also closely related to the pathological process of stroke. miRNAs are a group of short, noncoding RNA molecules playing important role in posttranscriptional regulation of gene expression and they have emerged as regulators of ischemic preconditioning and ischemic postconditioning. Here we give an overview of the expression and function of miRNAs in the brain, miRNAs as biomarkers during cerebral ischemia, and clinical applications and limitations of miRNAs. Future prospects of miRNAs are also discussed.
doi:10.1155/2013/276540
PMCID: PMC3606790  PMID: 23533957
2.  Reorganization of Brain Networks in Aging and Age-related Diseases 
Aging and Disease  2011;3(2):181-193.
Aging is associated with reorganization of brain in both structure and function. In recent years, graph theoretical analysis of brain organization has drawn increasing attention, and reorganization of brain in aging has been investigated in terms of connectivity and networks in topology such as modular organization, global and local efficiency, and small-worldness. Beyond studying on abnormity in local brain regions, connectivity quantifies alternations of correlation between two regions that may be spatially far separated, and graph theoretical analysis of brain network examines the complex interactions among multiple regions. This article reviewed complex brain networks of human in normal aging or with age-related diseases such as stroke and Alzheimer’s disease after a technical introduction of brain networks and graph theoretical analysis. We further discussed the relationship between the functional and the structural brain networks of subjects in aging or with age-related diseases. Finally, we proposed several interesting topics for future research in this field.
PMCID: PMC3377830  PMID: 22724079
Aging; Brain network; Connectivity; Functional; Neuroimaging; Small-world; Structural
3.  DEVELOPMENT OF CEREBRAL MICROVASCULAR DYSPLASIA MODEL IN RODENTS 
SUMMARY
Normal vasculature development of the central nervous system is extremely important because patients with vascular malformations are at life-threatening risk of intracranial hemorrhage or cerebral ischemia. The etiology and pathogenesis of abnormal vasculature development in the central nervous system are unknown, and progress is hampered by the lack of animal models of the human cerebrovascular diseases. Here, we reported our current study on cerebral microvascular dysplasia (CMVD) development. Using vascular endothelial growth factor hyper-stimulation, we demonstrated that aberrant microvessels could be developed in the rodent brain in certain conditions (such as genetic deficient background, local cytokine and chemokine release, or exogenous vessel dilating stimulation) that may speed up focal angiogenesis and lead to cerebral vascular dysplasia.
PMCID: PMC2677755  PMID: 19066107
cerebral; dysplasia; microvascular; mice; VEGF
4.  Comparison of doxycycline and minocycline in the inhibition of VEGF-induced smooth muscle cell migration 
Neurochemistry international  2006;50(3):524-530.
Smooth muscle migration plays an important role during angiogenesis and vascular remodeling. In this study, we examined the effects of doxycycline and minocycline on vascular endothelial growth factor (VEGF)-induced human aortic smooth muscle cell (HASMCs) migration, and explored the mechanisms in which doxycycline or minocycline inhibit HASMC migration. We demonstrated that both doxycycline and minocycline attain consistent anti-angiogenic effects in the inhibition of HASMC migration via a different signal pathway (p<0.05). This effect is through attenuating VEGF-induced matrix metalloproteinase-9 (MMP-9) activity (p<0.05). Doxycycline could increase tissue inhibitors of metalloproteinases-1 (TIMP-1) expression while minocycline down-regulated PI3K/Akt phosphorylation in HASMC. Our study suggests that doxycycline has a stronger ability to inhibit MMP secretion in HASMC by up-regulating endogenous MMPs inhibitor TIMP-1, while minocycline implements anti-angiogenic effect through inhibiting HASMC migration by down-regulating PI3K/Akt pathway.
doi:10.1016/j.neuint.2006.10.008
PMCID: PMC1876824  PMID: 17145119
Doxycycline; Matrix metalloproteinase; Minocycline; Migration; Smooth muscle cell; Vascular endothelial growth factor
5.  Correction: Genetic Associations of Angiotensin-Converting Enzyme with Primary Intracerebral Hemorrhage: A Meta-analysis 
PLoS ONE  2014;9(1):10.1371/annotation/7f2d85e0-c41a-40cf-80e1-6ce4b0470eff.
doi:10.1371/annotation/7f2d85e0-c41a-40cf-80e1-6ce4b0470eff
PMCID: PMC3883730
6.  microRNAs: innovative targets for cerebral ischemia and stroke 
Current drug targets  2013;14(1):90-101.
Stroke is one of the leading causes of death and disability worldwide. Because stroke is a multifactorial disease with a short therapeutic window many clinical stroke trials have failed and the only currently approved therapy is thrombolysis. MicroRNAs (miRNA) are endogenously expressed noncoding short single-stranded RNAs that play a role in the regulation of gene expression at the post-transcriptional level, via degradation or translational inhibition of their target mRNAs. The study of miRNAs is rapidly growing and recent studies have revealed a significant role of miRNAs in ischemic disease. miRNAs are especially important candidates for stroke therapeutics because of their ability to simultaneously regulate many target genes and since to date targeting single genes for therapeutic intervention has not yet succeeded in the clinic. Although there are already quite a few review articles about miRNA in ischemic heart disease, much less is currently known about miRNAs in cerebral ischemia. This review summarizes current knowledge about miRNAs and cerebral ischemia, focusing on the role of miRNAs in ischemia, both changes in expression and identification of potential targets, as well as the potential of miRNAs as biomarkers and therapeutic targets in cerebral ischemia.
PMCID: PMC3673881  PMID: 23170800
BCL2 family; cerebral ischemia; heat shock protein; microRNA; mitochondria
7.  In-Line Phase Contrast Imaging of Hepatic Portal Vein Embolization with Radiolucent Embolic Agents in Mice: A Preliminary Study 
PLoS ONE  2013;8(12):e80919.
It is crucial to understand the distribution of embolic agents inside target liver during and after the hepatic portal vein embolization (PVE) procedure. For a long time, the problem has not been well solved due to the radiolucency of embolic agents and the resolution limitation of conventional radiography. In this study, we first reported use of fluorescent carboxyl microspheres (FCM) as radiolucent embolic agents for embolizing hepatic portal veins. The fluorescent characteristic of FCM could help to determine their approximate location easily. Additionally, the microspheres were found to be fairly good embolizing agents for PVE. After the livers were excised and fixed, they were imaged by in-line phase contrast imaging (PCI), which greatly improved the detection of the radiolucent embolic agents as compared to absorption contrast imaging (ACI). The preliminary study has for the first time shown that PCI has great potential in the pre-clinical investigation of PVE with radiolucent embolic agents.
doi:10.1371/journal.pone.0080919
PMCID: PMC3851775  PMID: 24324646
8.  Surgery-Related Thrombosis Critically Affects the Brain Infarct Volume in Mice Following Transient Middle Cerebral Artery Occlusion 
PLoS ONE  2013;8(9):e75561.
Transient middle cerebral artery occlusion (tMCAO) model is widely used to mimic human focal ischemic stroke in order to study ischemia/reperfusion brain injury in rodents. In tMCAO model, intraluminal suture technique is widely used to achieve ischemia and reperfusion. However, variation of infarct volume in this model often requires large sample size, which hinders the progress of preclinical research. Our previous study demonstrated that infarct volume was related to the success of reperfusion although the reason remained unclear. The aim of present study is to explore the relationship between focal thrombus formation and model reproducibility with respect to infarct volume. We hypothesize that suture-induced thrombosis causes infarct volume variability due to insufficient reperfusion after suture withdrawal. Seventy-two adult male CD-1 mice underwent 90 minutes of tMCAO with or without intraperitoneal administration of heparin. Dynamic synchrotron radiation microangiography (SRA) and laser speckle contrast imaging (LSCI) were performed before and after tMCAO to observe the cerebral vascular morphology and to measure the cerebral blood flow in vivo. Infarct volume and neurological score were examined to evaluate severity of ischemic brain injury. We found that the rate of successful reperfusion was much higher in heparin-treated mice compared to that in heparin-free mice according to the result of SRA and LSCI at 1 and 3 hours after suture withdrawal (p<0.05). Pathological features and SRA revealed that thrombus formed in the internal carotid artery, middle cerebral artery or anterior cerebral artery, which blocked reperfusion following tMCAO. LSCI showed that cortical collateral circulation could be disturbed by thrombi. Our results demonstrated that suture-induced thrombosis was a critical element, which affects the success of reperfusion. Appropriate heparin management provides a useful approach for improving reproducibility of reperfusion model in mice.
doi:10.1371/journal.pone.0075561
PMCID: PMC3782513  PMID: 24086572
9.  CO2-based in-line phase contrast imaging of small intestine in mice 
Scientific Reports  2013;3:2313.
The objective of this study was to explore the potential of CO2 single contrast in-line phase contrast imaging (PCI) for pre-clinical small intestine investigation. The absorption and phase contrast images of CO2 gas production were attained and compared. A further increase in image contrast was observed in PCI. Compared with CO2-based absorption contrast imaging (ACI), CO2-based PCI significantly enhanced the detection of mucosal microstructures, such as pits and folds. The CO2-based PCI could provide sufficient image contrast for clearly showing the intestinal mucosa in living mice without using barium. We concluded that CO2-based PCI might be a novel and promising imaging method for future studies of gastrointestinal disorders.
doi:10.1038/srep02313
PMCID: PMC3727055  PMID: 23896957
10.  Genetic Associations of Angiotensin-Converting Enzyme with Primary Intracerebral Hemorrhage: A Meta-analysis 
PLoS ONE  2013;8(6):e67402.
Background
A number of studies have reported an association of angiotensin-converting enzyme (ACE) gene polymorphism with primary intracerebral hemorrhage (PICH), however the reports have demonstrated inconclusive results. To clarify this conflict, we updated the previously performed meta-analysis by Peck et al., which revealed negative results, by investigating the ACE polymorphism and its correlation to PICH.
Methods
PubMed and Embase databases (through Dec 2012) were searched for English articles on the relationship of the I/D polymorphism in ACE with PICH in humans. Summary odds ratios (ORs) were estimated and potential sources of heterogeneity and bias were explored.
Results
A total of 805 PICH cases and 1641 control cases obtained from 8 case-control studies were included. The results suggest that in dominant genetic models, the ACE I/D polymorphic variant was associated with a 58% increase in susceptibility risk of PICH (OR = 1.58; 95% CI = 1.07–2.35 for DD vs. DI+II). However, in the subgroup analysis based on race, a significant increased risk was found in Asian DD homozygote carriers (OR = 1.76 and 95% CI = 1.16–2.66 for DD vs. DI+II), but not in Caucasian DD homozygote carriers (OR = 1.18, 95% CI = 0.36–3.88, P = 0.784 for DD vs. DI+II). The heterogeneity between studies was remarkable, and its major sources of heterogeneity were due to the year in which the study was published. No potential publication bias was observed in dominant genetic models.
Conclusions
These data demonstrated evidence of a positive association between ACE I/D polymorphism with PICH, and suggested that the ACE gene is a PICH susceptible gene in Asian populations.
doi:10.1371/journal.pone.0067402
PMCID: PMC3694901  PMID: 23826288
11.  Differences of Circulating Inflammatory Markers between Large- and Small Vessel Disease in Patients with Acute Ischemic Stroke 
Background: The difference of inflammatory response between the pathogenesis of cerebral large- and small vessel disease after stroke remains unclear. In present study, we aim to determine the association of circulating inflammatory markers with different stroke subtype.
Methods: 99 patients with non-cardioembolic stroke were divided into large artery atherosclerosis (LAA) and small-artery occlusion (SAO) according to TOAST classification. A panel of plasma inflammatory markers including leukocyte, lymphocyte, CRP, fibrinogen, D-dimer, CD40L, IFN-γ, IL-1α, IL-1β, IL-6, IL-8, IL-17 and TNF-α were measured within 72 hours following cerebral ischemia. The relation of their levels in plasma with stroke subtype was further studied. All statistical data analysis was performed by SPSS 17.0 software.
Results: We found that only CRP were closely associated with stroke subtype (p<0.05). Compared to SAO subgroup, the plasma levels of CRP was higher in LAA subgroup (p<0.05). The predictive efficiency of CRP more than 3.2 for LAA was 85.7% sensitivity. The influencing factor of CRP includes IL-6, lymphocyte, fibrinogen and D-dimer.
Conclusion: LAA had a stronger activation of inflammation than SAO in the pathogenesis, which was associated with the changes of CRP.
doi:10.7150/ijms.6652
PMCID: PMC3753418  PMID: 23983602
CRP; Cytokine; Inflammation; Stroke subtype
12.  Neuroprotection and Sensorimotor Functional Improvement by Curcumin after Intracerebral Hemorrhage in Mice 
Journal of Neurotrauma  2011;28(12):2513-2521.
Abstract
Previous studies revealed that curcumin is neuroprotective in diseases of the central nervous system such as cerebral ischemia and traumatic brain injury. However, the effect of curcumin on intracerebral hemorrhage remains unclear. We, therefore, investigated the pre-clinical effect of curcumin treatment on neurological outcomes following intracerebral hemorrhage, using a mouse model. Intracerebral hemorrhage was induced by autologous blood injection into the right basal ganglia. Curcumin (150 mg/kg) was administered 15 min after intracerebral hemorrhage. Grid walk and neurological scores were evaluated at 1, 3, 7, and 14 days post-injury. Mice were killed at 24 h or 28 days following injury, for histological examination. Evans Blue and water content in the ipsilateral and contralateral hemispheres were measured to evaluate the extent of blood–brain barrier disruption and brain edema. Zonula occludens-1 was detected by immunostaining. In situ zymography was used to measure the localization and focal enzymatic activity of matrix metalloproteinase. Our results demonstrated that curcumin reduced brain edema, measured by alleviated water content and Evans Blue leakage at 24 h (p<0.05). Lateral ventricle measurements indicated that curcumin reduced brain tissue loss in the ipsilateral hemisphere (p<0.05). The same dose of curcumin also significantly attenuated neurological deficits at 1 and 3 days of intracerebral hemorrhage (p<0.05). Immunostaining showed that tight junction continuity around the hematoma was better sustained in curcumin-treated mice than in vehicle-treated mice. At 24 h, the number of matrix metalloproteinase-positive cells was significantly reduced by curcumin (p<0.05). Our study suggests that curcumin ameliorates intracerebral hemorrhage damage by preventing matrix metalloproteinase-mediated blood–brain barrier damage and brain edema, which might provide therapeutic potential for intracerebral hemorrhage.
doi:10.1089/neu.2011.1958
PMCID: PMC3235343  PMID: 21770745
13.  X-ray Phase Contrast Imaging of Cell Isolation with Super-Paramagnetic Microbeads 
PLoS ONE  2012;7(9):e45597.
Super-paramagnetic microbeads are widely used for cell isolation. Evaluation of the binding affinity of microbeads to cells using optical microscopy has been limited by its small scope. Here, magnetic property of microbeads was first investigated by using synchrotron radiation (SR) in-line x-ray phase contrast imaging (PCI). The cell line mouse LLC (Lewis lung carcinoma) was selected for cell adhesion studies. Targeted microbeads were prepared by attaching anti-VEGFR2 (vascular endothelial growth factor receptor-2) antibody to the shell of the microbeads. The bound microbeads were found to better adhere to LLC cells than unbound ones. PCI dynamically and clearly showed the magnetization and demagnetization of microbeads in PE-50 tube. The cells incubated with different types of microbeads were imaged by PCI, which provided clear and real-time visualization of the cell isolation. Therefore, PCI might be considered as a novel and efficient tool for further cell isolation studies.
doi:10.1371/journal.pone.0045597
PMCID: PMC3454406  PMID: 23029126
14.  A Novel Intravital Method to Evaluate Cerebral Vasospasm in Rat Models of Subarachnoid Hemorrhage: A Study with Synchrotron Radiation Angiography 
PLoS ONE  2012;7(3):e33366.
Precise in vivo evaluation of cerebral vasospasm caused by subarachnoid hemorrhage has remained a critical but unsolved issue in experimental small animal models. In this study, we used synchrotron radiation angiography to study the vasospasm of anterior circulation arteries in two subarachnoid hemorrhage models in rats. Synchrotron radiation angiography, laser Doppler flowmetry-cerebral blood flow measurement, [125I]N-isopropyl-p-iodoamphetamine cerebral blood flow measurement and terminal examinations were applied to evaluate the changes of anterior circulation arteries in two subarachnoid hemorrhage models made by blood injection into cisterna magna and prechiasmatic cistern. Using synchrotron radiation angiography technique, we detected cerebral vasospasm in subarachnoid hemorrhage rats compared to the controls (p<0.05). We also identified two interesting findings: 1) both middle cerebral artery and anterior cerebral artery shrunk the most at day 3 after subarachnoid hemorrhage; 2) the diameter of anterior cerebral artery in the prechiasmatic cistern injection group was smaller than that in the cisterna magna injection group (p<0.05), but not for middle cerebral artery. We concluded that synchrotron radiation angiography provided a novel technique, which could directly evaluate cerebral vasospasm in small animal experimental subarachnoid hemorrhage models. The courses of vasospasm in these two injection models are similar; however, the model produced by prechiasmatic cistern injection is more suitable for study of anterior circulation vasospasm.
doi:10.1371/journal.pone.0033366
PMCID: PMC3299776  PMID: 22428033
15.  Bone Marrow-Derived Cells Contribute to VEGF-Induced Angiogenesis in the Adult Mouse Brain by Supplying MMP-9 
Background and Purpose
Our previous studies have shown that bone marrow-derived cells (BMDCs) home to the brain angiogenic focus. Angiogenic response to vascular endothelial growth factor (VEGF) stimulation is reduced in matrix metalloproteinase-9 (MMP-9) knockout mice. We hypothesized that BMDCs contribute to VEGF-induced angiogenesis by supplying MMP-9.
Methods
Bone marrow (BM) transplantation was conducted using MMP-9 knockout (MMP-9 KO) or wild-type (WT) mice as donor and recipient. Adeno-associated viral vectors expressing VEGF or LacZ was injected into the striatum 4 weeks after BM transplantation. Circulating white blood cells (WBCs), microvessel density, number of BMDCs, and MMP-9 activity around the injection site were analyzed.
Results
Circulating WBCs increased in WT mice but not in MMP-9 KO mice 2 weeks after vector injection. Overexpressing VEGF increased microvessel density by 38% in WT mice 4 weeks after vector injection. Transplantation of MMP-9 KO BM to WT mice reduced angiogenic response by 80% of that in WT mice. The microvessel density only increased 18% after VEGF stimulation with MMP-9 activity reduced to 35% of the level of WT mice. There was no significant angiogenic response in the brain of MMP-9 KO. Transplantation of WT BM to MMP-9 KO mice restored their brain angiogenic response to 92% of the WT level, indicated by a 30% increase of microvessel density in the VEGF-treated group with MMP-9 activity close to the level of WT mice.
Conclusion
BM-derived MMP-9 plays an important role in BM cell mobilization and focal angiogenesis in the brain in response to VEGF stimulation.
doi:10.1161/STROKEAHA.110.596452
PMCID: PMC3026909  PMID: 21164138
angiogenesis; bone marrow-derived cells; brain angiogenesis; metalloproteinase-9; vascular endothelial growth factor
16.  Surgical treatment and pathologic analysis of posterior lentiglobus with cataract 
doi:10.3980/j.issn.2222-3959.2011.05.21
PMCID: PMC3340720  PMID: 22553723
17.  Evidence of endothelial progenitor cells in the human brain and spinal cord arteriovenous malformations 
Neurosurgery  2010;67(4):1029-1035.
Objective
Brain and spinal cord arteriovenous malformations (AVMs) are characterized by aberrant angiogenesis and vascular remodeling. Endothelial progenitor cells (EPCs) can be recruited by stromal cell-derived factor-1 (SDF-1), and participate in vascular remodeling in both physiological and pathological settings. We hypothesized that there was increased EPC levels in the brain and spinal cord AVM nidus.
Methods
Microsurgical specimens without endovascular embolization and radiosurgery from the brain (n=12) and spinal cord (n=5) AVMs were examined. Hemangioblastoma, meningioma, cerebral cortex obtained from epilepsy surgery, and the basilar artery (BA) from the autopsy were chosen for control comparisons. EPCs were identified as cells that were double-positive for the stem cell marker CD133 and the endothelial cell marker VEGFR-2 (vascular endothelial growth factor receptor-2 or KDR). In addition, SDF-1 was characterized by immunohistochemistry.
Results
Both brain and spinal AVM tissues displayed more CD133, SDF-1, and CD68-positive signals than epilepsy and basilar artery control tissues. The level of EPCs was increased in the brain and spinal cord AVM nidus, mainly at the edge of the vessel wall. The expression of SDF-1 was co-localized with CD31-positive and α-smooth muscle cells, and was predominantly found within the vessel wall.
Conclusion
Our data demonstrate that EPCs are present in the nidus of the brain and spinal cord AVMs, which may mediate pathological vascular remodeling and impact the clinical course of AVMs.
doi:10.1227/NEU.0b013e3181ecc49e
PMCID: PMC2949301  PMID: 20881566
Angiogenesis; Endothelial progenitor cell; Stromal cell-derived factor-1; Vascular malformation
18.  VEGF Induces More Severe Cerebrovascular Dysplasia in Endoglin+/- than in Alk1+/- Mice 
Translational stroke research  2010;1(3):197-201.
Brain arteriovenous malformations (BAVMs) are an important cause of intracranial hemorrhage (ICH) in young adults. A small percent of BAVMs is due to hereditary hemorrhagic telangiectasia 1 and 2 (HHT1 and 2), which are caused by mutations in two genes involved in TGF-β signaling: endoglin (ENG) and activin-like kinase 1 (ALK1). The BAVM phenotype is an incomplete penetrant in HHT patients, and the mechanism is unknown. We tested the hypothesis that a “response-to-injury” triggers abnormal vascular (dysplasia) development, using Eng and Alk1 haploinsufficient mice. Adeno-associated virus (AAV) expressing vascular endothelial growth factor (VEGF) was used to mimic the injury conditions. VEGF overexpression caused a similar degree of angiogenesis in the brain of all groups, except that the cortex of Alk1+/- mice had a 33% higher capillary density than other groups. There were different levels of cerebrovascular dysplasia in haploinsufficient mice (Eng+/>Alk1+/-), which simulates the relative penetrance of BAVM in HHT patients (HHT1>HHT2). Few dysplastic capillaries were observed in AAV-LacZ-injected mice. Our data indicate that both angiogenic stimulation and genetic alteration are necessary for the development of dysplasia, suggesting that anti-angiogenic therapies might be adapted to slow the progression of the disease and decrease the risk of spontaneous ICH.
doi:10.1007/s12975-010-0020-x
PMCID: PMC2902730  PMID: 20640035
intracranial hemorrhage; hereditary hemorrhagic telangiectasia 1 and 2; brain AVM; angiogenic stimulation
19.  MicroRNA-9 Coordinates Proliferation and Migration of Human Embryonic Stem Cell–Derived Neural Progenitors 
Cell stem cell  2010;6(4):323-335.
Summary
Human pluripotent stem cells offer promise for use in cell-based therapies for brain injury and diseases. However, their cellular behavior is poorly understood. Here we show that the expression of the brain-specific microRNA-9 (miR-9) is turned on in human neural progenitor cells (hNPCs) derived from human embryonic stem cells. Loss of miR-9 suppressed proliferation but promoted migration of hNPCs cultured in vitro. hNPCs without miR-9 activity also showed enhanced migration when transplanted into mouse embryonic brains or adult brains of a mouse model of stroke. These effects were not due to precocious differentiation of hNPCs. One of the key targets directly regulated by miR-9 encodes stathmin, which increases microtubule instability and whose expression in hNPCs correlates inversely with that of miR-9. Partial inhibition of stathmin activity suppressed the effects of miR-9 loss on proliferation and migration of human or embryonic rat neural progenitors. These results identify miR-9 as a novel regulator that coordinates the proliferation and migration of hNPCs.
doi:10.1016/j.stem.2010.02.015
PMCID: PMC2851637  PMID: 20362537
Human Embryonic Stem Cells; Neural Progenitors; Migration; Proliferation; MicroRNA-9; Stathmin
20.  Endothelial progenitor cell transplantation improves long-term outcome in mice 
Annals of neurology  2010;67(4):488-497.
Endothelial progenitor cells (EPCs) have been implicated in playing an important role in vascular repair and revascularization in ischemic organs including brain tissue. However, the cause of EPC migration and the function of EPC playing following post-ischemia are unclear. Here, we reported EPC therapy in a mouse model of transient middle cerebral artery occlusion (tMCAO) to explore the roles of EPC following ischemic brain injury.
Human EPCs were cultured, characterized, and confirmed with flow cytometry. Ex vivo expanded EPCs (1×106) were injected via jugular vein after 1 hour of tMCAO. Histological and behavioral analyses were performed from day 1 to 28 days after tMCAO.
EPCs were detected in ischemic brain region 24 hours after MCAO. EPC transplantation significantly reduced ischemic infarct volume at 3 days following MCAO compared to the control (p<0.05). CXCR4 was expressed on majority of EPCs and SDF-1-induced EPC migration was blocked by AMD3100 in vitro. SDF-1 was up-regulated in ischemic brain and AMD3100 could reduce EPCs migration to the ischemic region in vivo, suggesting that SDF-1/CXCR4 was involved in EPC-mediated neuroprotection. Compared to the control, EPC therapy reduced mouse cortex atrophy 4 weeks after tMCAO, which was accompanied by improved neurobehavioral outcomes (p<0.05). In addition, EPC injection potently increased angiogenesis in the peri-infarction area (p<0.05).
We conclude that systemic delivery of EPC protect against cerebral ischemic injury, promote neurovascular repair, and improve long-term neurobehavioral outcomes. Our data suggests that SDF-1/CXCR4 plays a critical role in EPC-mediated neuroprotection.
doi:10.1002/ana.21919
PMCID: PMC3026588  PMID: 20437584
angiogenesis; EPCs; ischemia; mice; neuroprotection
21.  Notch-1 signalling is activated in brain arteriovenous malformations in humans 
Brain  2009;132(12):3231-3241.
A role for the Notch signalling pathway in the formation of arteriovenous malformations during development has been suggested. However, whether Notch signalling is involved in brain arteriovenous malformations in humans remains unclear. Here, we performed immunohistochemistry on surgically resected brain arteriovenous malformations and found that, compared with control brain vascular tissue, Notch-1 signalling was activated in smooth muscle and endothelial cells of the lesional tissue. Western blotting showed an activated form of Notch-1 in brain arteriovenous malformations, irrespective of clinical presentation and with or without preoperative embolization, but not in normal cerebral vessels from controls. In addition, the Notch-1 ligands Jagged-1 and Delta-like-4 and the downstream Notch-1 target Hes-1 were increased in abundance and activated in human brain arteriovenous malformations. Finally, increased angiogenesis was found in adult rats treated with a Notch-1 activator. Our findings suggest that activation of Notch-1 signalling is a phenotypic feature of brain arteriovenous malformations, and that activation of Notch-1 in normal vasculature induces a pro-angiogenic state, which may contribute to the development of vascular malformations.
doi:10.1093/brain/awp246
PMCID: PMC2792368  PMID: 19812212
Notch-1; AVM; human; brain; signalling; angiogenesis
22.  Attenuation of Brain Response to VEGF-mediated Angiogenesis and Neurogenesis in Aged Mice 
Background and Purpose
Alterations of neuro-angiogenic response play important roles in the development of aging-related neurodisorders, and affect gene-based therapies. We tested brain response to vascular endothelial growth factor (VEGF) in aged mice.
Methods
AAV-VEGF, an adeno-associated viral vector expressing VEGF, was injected into the brain of 3-, 12- and, 24-month old mice. AAV-LacZ-injected mice were used as control (n=6). Before euthanization at 6 weeks after vector injection, the mice were intraperitoneally injected with BrdU for 3 consecutive days. The vascular density and the number of neuroprogenitors were analyzed.
Results
Injection of AAV-VEGF increased the vascular density in the brain of 3-, 12-and 24-month old mice by 22±7% (AAV-VEGF: 320±15 per 10X field vs. AAV-LacZ: 263±8, p<0.05), 20%±8 (AAV-VEGF: 300±9 vs. AAV-LacZ: 250±11, p<0.05), and 7±16% (AAV-VEGF: 257±27 vs. AAV-LacZ: 236±13, p=0.283), respectively. There were more VEGF receptor positive neuroprogenitors in the subventricular zone of AAV-VEGF injected 3- (22±2) and 12-month old mice (21±5) than that of 24-month old mice (7±1). More BrdU positive endothelial cells and neuroprogenitors were detected around the injection site and SVZ of 3- (13±4) and 12-month old mice (14±5) than that of 24-month old mice (1±1). VEGF receptor 2 was upregulated in AAV-VEGF-injected brains of 3- and 12-month old mice, but not in 24-month old mice.
Conclusion
The angiogenic and neurogenic response to VEGF stimulation is attenuated in the aged mouse brain, which may be due to reduced VEGF receptor activity.
doi:10.1161/STROKEAHA.109.561050
PMCID: PMC2783321  PMID: 19745179
aging; brain; angiogenesis; neurogenesis; VEGF
23.  Postischemic IGF-1 gene transfer promotes neurovascular regeneration after experimental stroke 
Promoting neural regeneration after cerebral infarction has emerged as a potential approach for the treatment of stroke. Insulin-like growth factor 1 (IGF-1) possesses both neurotrophic and angiogenic properties. The aim of this study is to determine whether postischemic gene transfer of IGF-1 enhances neurovascular regeneration in a mouse model of permanent focal cerebral ischemia. Long-term cerebral IGF-1 overexpression was achieved with adeno-associated viral vector (AAV) via stereotaxic injection at 24 h after stroke. AAV-GFP or saline was injected as control. The success of postischemic gene transduction was confirmed by a strong green fluorescent protein signal and by increased IGF-1 protein expression in the peri-infarct region. Postischemic gene transfer of IGF-1 significantly enhanced vascular density at 8 weeks post stroke in the peri-infarct and injection needle tract area compared to AAV-GFP or saline treatment, as shown by immunohistochemical staining with vascular marker lectin. Furthermore, increased vascular density was associated with improved local vascular perfusion. Immunohistochemical staining with neuronal progenitor marker DCX and cell proliferation marker BrdU indicated that AAV-IGF-1 treatment potently increased neurogenesis compared to AAV-GFP injection. These data demonstrate that postischemic treatment of IGF-1 effectively promoted neural and vascular regeneration in the chronic stage of cerebral infarction.
doi:10.1038/jcbfm.2009.75
PMCID: PMC2763573  PMID: 19513085
angiogenesis; IGF-1; neurovascular; regeneration; stroke
24.  Nitric Oxide in VEGF-induced Focal Angiogenesis and Matrix Metalloproteinase-9 Activity in the Mouse Brain 
Background
Vascular endothelial growth factor (VEGF) can induce matrix metalloproteinase (MMP)-9 activities and focal angiogenesis. We hypothesized that VEGF activation of cerebral MMP-9 would require nitric oxide (NO) participation.
Methods
We compared the in vivo effects of: (1) NG-monomethyl-L-arginine (L-NMMA), a non-specific NO synthase (NOS) inhibitor; (2) L-N6-(1-iminoethyl)lysine (L-NIL), an inducible NOS (iNOS) selective inhibitor; and (3) doxycycline, a known non-specific inhibitor of MMP in the mouse brain, using in situ zymography and endothelial marker CD31. 3-nitrotyrosine (3-NT) was used as a surrogate for NO activity. Inflammatory cell markers CD68 and MPO were used to confirm leukocyte infiltration.
Results
VEGF-stimulated MMP-9 activity expressed primarily around cerebral microvessels. L-NMMA suppressed cerebral angiogenesis (p<0.05), especially those microvessels associated with MMP-9 activation (p<0.02) induced by VEGF, comparable to the effect of doxycycline. L-NIL showed similar inhibitory effects. 3-NT confirmed NO levels in the brain. Compared to the lacZ control, VEGF increased inflammatory cell infiltration, especially macrophages, in the induced brain angiogenic focuses.
Conclusions
Inhibition of NO production decreased MMP-9 activity and focal angiogenesis in the VEGF-stimulated brain. Both specific and non-specific inhibition of NOS resulted in similar reductions, suggesting that VEGF-stimulated cerebral MMP activity and angiogenesis are predominantly mediated through iNOS, a specific NOS isoform mediating inflammatory responses.
doi:10.1161/STROKEAHA.109.552059
PMCID: PMC2717176  PMID: 19498186
nitric oxide; vascular endothelial growth factor; matrix metalloproteinase
25.  Nonischemic cerebral venous hypertension promotes a pro-angiogenic stage via HIF-1 downstream genes and leukocyte-derived MMP-9 
Cerebral venous hypertension (VH) and angiogenesis are implicated in the pathogenesis of brain arteriovenous malformation and dural arteriovenous fistulae. We studied the association of VH and angiogenesis using a mouse brain VH model. Sixty mice underwent external jugular vein and common carotid artery (CCA) anastomosis (VH model), CCA ligation, or sham dissection (n=20). Hypoxia-inducible factor-1α (HIF-1α), vascular endothelial growth factor (VEGF) and stromal cell-derived factor-1α (SDF-1α) expression, and matrix metalloproteinase (MMP) activity were analyzed. We found VH animals had higher (p<0.05) SSP pressure (8±1 mmHg) than control groups (1±1 mmHg). Surface cerebral blood flow and mean arterial pressure did not change. HIF-1, VEGF and SDF-1α expression increased (p<0.05). Neutrophils and MMP-9 activity increased 10-fold 1 day after surgery, gradually decreased afterwards, and returned to baseline 2 weeks after surgery. Macrophages began to increase 3 days after surgery (p<0.05), which coincided with the changes in SDF-1α expression. Capillary density in the parasagittal cortex increased 17% compared to the controls. Our findings suggest that mild nonischemic VH results in a pro-angiogenic stage in the brain by upregulating HIF-1 and its downstream targets, VEGF and SDF-1α, increasing leukocyte-infiltration and MMP-9 activity.
doi:10.1038/jcbfm.2009.67
PMCID: PMC2745831  PMID: 19471278
Mouse brain venous hypertension; HIF-1; MMP-9; angiogenesis; inflammatory cells

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