Zheng Gang Zhang

VEGF enhances angiogenesis and promotes blood-brain barrier leakage in the ischemic brain

VEGF is a secreted mitogen associated with angiogenesis and is also a potent vascular permeability factor. The biological role of VEGF in the ischemic brain remains unknown. This study was undertaken to investigate whether VEGF enhances cerebral microvascular perfusion and increases blood-brain barrier (BBB) leakage in the ischemic brain. Using magnetic resonance imaging (MRI), three-dimensional laser-scanning confocal microscope, and functional neurological tests, we measured the effects of administrating recombinant human VEGF(165) (rhVEGF(165)) on angiogenesis, functional neurological outcome, and BBB leakage in a rat model of focal cerebral embolic ischemia. Late (48 hours) administration of rhVEGF(165) to the ischemic rats enhanced angiogenesis in the ischemic penumbra and significantly improved neurological recovery. However, early postischemic (1 hour) administration of rhVEGF(165) to ischemic rats significantly increased BBB leakage, hemorrhagic transformation, and ischemic lesions. Administration of rhVEGF(165) to ischemic rats did not change BBB leakage and cerebral plasma perfusion in the contralateral hemisphere. Our results indicate that VEGF can markedly enhance angiogenesis in the ischemic brain and reduce neurological deficits during stroke recovery and that inhibition of VEGF at the acute stage of stroke may reduce the BBB permeability and the risk of hemorrhagic transformation after focal cerebral ischemia.

Intravenous Administration of Human Bone Marrow Stromal Cells Induces Angiogenesis in the Ischemic Boundary Zone After Stroke in Rats

Abstract— We tested the hypothesis that intravenous infusion of human bone marrow stromal cells (hMSCs) promotes vascular endothelial growth factor (VEGF) secretion, VEGF receptor 2 (VEGFR2) expression and angiogenesis in the ischemic boundary zone (IBZ) after stroke. hMSCs (1×106) were intravenously injected into rats 24 hours after middle cerebral artery occlusion (MCAo). Laser scanning confocal microscopy (LSCM), immunohistochemistry and ELISA were performed to assay angiogenesis and levels of human and rat VEGF in the host brain, respectively. In addition, capillary-like tube formation was measured using mouse brain-derived endothelial cells (MBDECs). Morphological and three dimensional image analyses revealed significant (P <0.05) increases in numbers of enlarged and thin walled blood vessels and numbers of newly formed capillaries at the boundary of the ischemic lesion in rats (n=12) treated with hMSCs compared with numbers in rats (n=12) treated with PBS. ELISA measurements showed that treatment with hMSCs significantly (P <0.05) raised endogenous rat VEGF levels in the IBZ from 10.5±1.7 ng/mL in the control group to 17.5±1.6 ng/mL in the hMSC-treated group. In addition, treatment with hMSCs increased endogenous VEGFR2 immunoreactivity. In vitro, when MBDECs were incubated with the supernatant obtained from cultured hMSCs, capillary-like tube formation was significantly (P <0.01) induced. However, hMSC-induced capillary-like tube formation was significantly (P <0.01) inhibited when the endothelial cells were incubated with the supernatant from hMSCs in the presence of a neutralizing anti-VEGFR2. These data suggest that treatment of stroke with hMSCs enhances angiogenesis in the host brain and hMSC-enhanced angiogenesis is mediated by increases in levels of endogenous rat VEGF and VEGFR2.

Statins induce angiogenesis, neurogenesis, and synaptogenesis after stroke

We demonstrate that the 3‐hydroxy‐3‐methyl‐glutaryl‐coenzyme A (HMG‐CoA) reductase inhibitors atorvastatin and simvastatin enhance functional outcome and induce brain plasticity when administered after stroke to rats. With atorvastatin treatment initiated 1 day after stroke, animals exhibited significant increases in vascular endothelial growth factor, cyclic guanosine monophosphate, angiogenesis, endogenous cell proliferation and neurogenesis, and an increase in the synaptic protein, synaptophysin. Atorvastatin‐induced angiogenesis in a tube formation assay was reduced by an antibody against the vascular endothelial growth factor receptor 2 (FIK‐1) and by the nitric oxide synthase inhibitor, N‐mono‐methyl‐L‐arginine (L‐NAME). Atorvastatin also induced phosphorylation of Akt and Erk in cultured primary cortical neurons. These data indicate that atorvastatin induced brain plasticity and has neurorestorative activity after experimental stroke. Ann Neurol 2003

Neurorestorative therapies for stroke: underlying mechanisms and translation to the clinic

Restorative cell-based and pharmacological therapies for experimental stroke substantially improve functional outcome. These therapies target several types of parenchymal cells (including neural stem cells, cerebral endothelial cells, astrocytes, oligodendrocytes, and neurons), leading to enhancement of endogenous neurogenesis, angiogenesis, axonal sprouting, and synaptogenesis in the ischaemic brain. Interaction between these restorative events probably underpins the improvement in functional outcome. This Review provides examples of cell-based and pharmacological restorative treatments for stroke that stimulate brain plasticity and functional recovery. The molecular pathways activated by these therapies, which induce remodelling of the injured brain via angiogenesis, neurogenesis, and axonal and dendritic plasticity, are discussed. The ease of treating intact brain tissue to stimulate functional benefit in restorative therapy compared with treating injured brain tissue in neuroprotective therapy might more readily help with translation of restorative therapy from the laboratory to the clinic.

Exosome-mediated transfer of miR-133b from multipotent mesenchymal stromal cells to neural cells contributes to neurite outgrowth.

Multipotent mesenchymal stromal cells (MSCs) have potential therapeutic benefit for the treatment of neurological diseases and injury. MSCs interact with and alter brain parenchymal cells by direct cell‐cell communication and/or by indirect secretion of factors and thereby promote functional recovery. In this study, we found that MSC treatment of rats subjected to middle cerebral artery occlusion (MCAo) significantly increased microRNA 133b (miR‐133b) level in the ipsilateral hemisphere. In vitro, miR‐133b levels in MSCs and in their exosomes increased after MSCs were exposed to ipsilateral ischemic tissue extracts from rats subjected to MCAo. miR‐133b levels were also increased in primary cultured neurons and astrocytes treated with the exosome‐enriched fractions released from these MSCs. Knockdown of miR‐133b in MSCs confirmed that the increased miR‐133b level in astrocytes is attributed to their transfer from MSCs. Further verification of this exosome‐mediated intercellular communication was performed using a cel‐miR‐67 luciferase reporter system and an MSC‐astrocyte coculture model. Cel‐miR‐67 in MSCs was transferred to astrocytes via exosomes between 50 and 100 nm in diameter. Our data suggest that the cel‐miR‐67 released from MSCs was primarily contained in exosomes. A gap junction intercellular communication inhibitor arrested the exosomal microRNA communication by inhibiting exosome release. Cultured neurons treated with exosome‐enriched fractions from MSCs exposed to 72 hours post‐MCAo brain extracts significantly increased the neurite branch number and total neurite length. This study provides the first demonstration that MSCs communicate with brain parenchymal cells and may regulate neurite outgrowth by transfer of miR‐133b to neural cells via exosomes. STEM CELLS2012;30:1556–1564

Correlation of VEGF and Angiopoietin Expression with Disruption of Blood–Brain Barrier and Angiogenesis after Focal Cerebral Ischemia:

In an effort to elucidate the molecular mechanisms underlying cerebral vascular alteration after stroke, the authors measured the spatial and temporal profiles of blood–brain barrier (BBB) leakage, angiogenesis, vascular endothelial growth factor (VEGF), associated receptors, and angiopoietins and receptors after embolic stroke in the rat. Two to four hours after onset of ischemia, VEGF mRNA increased, whereas angiopoietin 1 (Ang 1) mRNA decreased. Three-dimensional immunofluorescent analysis revealed spatial coincidence between increases of VEGF immunoreactivity and BBB leakage in the ischemic core. Two to 28 days after the onset of stroke, increased expression of VEGF/VEGF receptors and Ang/Tie2 was detected at the boundary of the ischemic lesion. Concurrently, enlarged and thin-walled vessels were detected at the boundary of the ischemic lesion, and these vessels developed into smaller vessels via sprouting and intussusception. Three-dimensional quantitative analysis of cerebral vessels at the boundary zone 14 days after ischemia revealed a significant (P < 0.05) increase in numbers of vessels (n = 365) compared with numbers (n = 66) in the homologous tissue of the contralateral hemisphere. Furthermore, capillaries in the penumbra had a significantly smaller diameter (4.8 ± 2.0 μm) than capillaries (5.4 ± 1.5 μm) in the homologous regions of the contralateral hemisphere. Together, these data suggest that acute alteration of VEGF and Ang 1 in the ischemic core may mediate BBB leakage, whereas upregulation of VEGF/VEGF receptors and Ang/Tie2 at the boundary zone may regulate neovascularization in ischemic brain.

A test for detecting long-term sensorimotor dysfunction in the mouse after focal cerebral ischemia

The mouse is an excellent model for investigations of stroke and neural injury. However, there is a paucity of long term functional outcome measurements for the mouse. We, therefore, developed a sensorimotor functional test (corner test) and applied this test to a model of focal cerebral ischemia in the mouse. Male C57/6J mice (n=20) were subjected to embolic middle cerebral artery (MCA) occlusion. Reduction of cerebral blood flow (CBF) was measured by perfusion weighted MRI at 1 h after ischemia. The corner test, which is sensitive to chronic sensorimotor and postural symmetries, a general neurological test battery, and a foot fault test were performed between 2 and 90 days after ischemia. Infarct volume was measured at 90 days after ischemia. Multivariable analysis revealed that the corner test was highly predictive for infarct volume measured at 90 days after stroke, with R(2) values ranging from 0.73 to 0.93. The foot-fault test and neurological score did not detect chronic behavioral impairments. A significant (P<0.001) correlation between the infarct volume and the corner test was detected at 90 days after mild focal cerebral ischemia, whereas, there was no correlation between the infarct volume and neurological score or foot-fault. The data demonstrate that the corner test is a sensitive and objective test, which can be applied to evaluate long term functional outcome after stroke in the mouse.

Systemic Administration of Exosomes Released from Mesenchymal Stromal Cells Promote Functional Recovery and Neurovascular Plasticity After Stroke in Rats

Here, for the first time, we test a novel hypothesis that systemic treatment of stroke with exosomes derived from multipotent mesenchymal stromal cells (MSCs) promote neurovascular remodeling and functional recovery after stroke in rats. Adult male Wistar rats were subjected to 2 hours of middle cerebral artery occlusion (MCAo) followed by tail vein injection of 100 μg protein from MSC exosome precipitates or an equal volume of vehicle phosphate-buffered saline (PBS) (n = 6/group) 24 hours later. Animals were killed at 28 days after stroke and histopathology and immunohistochemistry were employed to identify neurite remodeling, neurogenesis, and angiogenesis. Systemic administration of MSC-generated exosomes significantly improved functional recovery in stroke rats compared with PBS-treated controls. Axonal density and synaptophysin-positive areas were significantly increased along the ischemic boundary zone of the cortex and striatum in MCAo rats treated with exosomes compared with PBS control. Exosome treatment significantly increased the number of newly formed doublecortin (a marker of neuroblasts) and von Willebrand factor (a marker of endothelial cells) cells. Our results suggest that intravenous administration of cell-free MSC-generated exosomes post stroke improves functional recovery and enhances neurite remodeling, neurogenesis, and angiogenesis and represents a novel treatment for stroke.

Induction of DNA Fragmentation After 10 to 120 Minutes of Focal Cerebral Ischemia in Rats

BACKGROUND AND PURPOSE The induction of neuronal necrosis has been studied after various durations of transient middle cerebral artery (MCA) occlusion in the rat. The objective of the present study was to measure the numbers and anatomic distribution of cells exhibiting apoptotic bodies as an indication of DNA fragmentation and apoptotic cell death as a function of duration of transient MCA occlusion in the rat. METHODS The MCA of male Wistar rats (n = 24) was occluded for 10, 20, 30, 60, 90, and 120 minutes (n = 4 per group) with the use of an intraluminal monofilament, and reperfusion was instituted for 48 hours. DNA fragmentation was measured in paraffin sections with the use of a terminal deoxynucleotidyl-transferase (TdT)-mediated dUTP-biotin nick end-labeling (TUNEL) method. Adjacent sections were stained with hematoxylin and eosin for analysis of ischemic cell damage, and immunohistochemical double staining methods were used for cell identification. Sham-operated rats (n = 4) and normal rats not subjected to any surgical procedure (n = 4) were used as controls for apoptosis detection. RESULTS Within 5-microns-thick coronal sections, DNA fragmentation was present in 0 to 3 apoptotic cells in each hemisphere of normal, sham-operated rats as well as in the contralateral hemisphere of ischemic rats. After 10 to 20 minutes of MCA occlusion, apoptotic cells exhibiting DNA fragmentation (10 to 20) increased in the regions of selective neuronal necrosis in the preoptic area and in the striatum. After 30 to 60 minutes of ischemia, scattered apoptotic cells (30 to 60) exhibited DNA fragmentation and expanded into areas of selective neuronal necrosis in the cortex. After 90 to 120 minutes of occlusion, groups of apoptotic cells (70 to 200, > 95% neurons) were primarily localized to the inner boundary zone of the infarct. CONCLUSIONS A range of mild to severe ischemia-reperfusion stimuli induce internucleosomal DNA cleavage. The presence and anatomic location of apoptotic cells exhibiting DNA fragmentation after transient cerebral occlusion indicate that apoptosis accompanies neuronal necrosis.

Matrix Metalloproteinase 2 (MMP2) and MMP9 Secreted by Erythropoietin-Activated Endothelial Cells Promote Neural Progenitor Cell Migration

We investigated the hypothesis that endothelial cells activated by erythropoietin (EPO) promote the migration of neuroblasts. This hypothesis is based on observations in vivo that treatment of focal cerebral ischemia with EPO enhances the migration of neuroblasts to the ischemic boundary, a site containing activated endothelial cells and angiogenic microvasculature. To model the microenvironment within the ischemic boundary zone, we used a coculture system of mouse brain endothelial cells (MBECs) and neural progenitor cells derived from the subventricular zone of the adult mouse. Treatment of MBECs with recombinant human EPO (rhEPO) significantly increased secretion of matrix metalloproteinase 2 (MMP2) and MMP9. rhEPO-treated MBEC supernatant as conditioned medium significantly increased the migration of neural progenitor cells. Application of an MMP inhibitor abolished the supernatant-enhanced migration. Incubation of neurospheres alone with rhEPO failed to increase progenitor cell migration. rhEPO activated phosphatidylinositol 3-kinase/Akt (PI3K/Akt) and extracellular signal-regulated kinase (ERK1/2) in MBECs. Selective inhibition of the PI3K/Akt and ERK1/2 pathways significantly attenuated the rhEPO-induced MMP2 and MMP9, which suppressed neural progenitor cell migration promoted by the rhEPO-activated MBECs. Collectively, our data show that rhEPO-activated endothelial cells enhance neural progenitor cell migration by secreting MMP2 and MMP9 via the PI3K/Akt and ERK1/2 signaling pathways. These data demonstrate that activated endothelial cells can promote neural progenitor cell migration, and provide insight into the molecular mechanisms underlying the attraction of newly generated neurons to injured areas in brain.