Yongfeng Fan

Endothelial progenitor cell transplantation improves long‐term stroke outcome in mice

Endothelial progenitor cells (EPCs) play an important role in tissue repairing and regeneration in ischemic organs, including the brain. However, the cause of EPC migration and the function of EPCs after ischemia are unclear. In this study, we demonstrated the effects of EPCs on ischemic brain injury in a mouse model of transient middle cerebral artery occlusion (tMCAO).

Interleukin-6 stimulates circulating blood-derived endothelial progenitor cell angiogenesis in vitro

Circulating blood endothelial progenitor cells (EPCs) contribute to postnatal vasculogenesis, providing a novel therapeutic target for vascular diseases. However, the molecular mechanism of EPC-induced vasculogenesis is unknown. Interleukin-6 plays multiple functions in angiogenesis and vascular remodeling. Our previous study demonstrated that the polymorphism (174G > C) in IL-6 gene promoter was associated with brain vascular disease. In this study, we investigated if IL-6 receptor is expressed in human EPCs derived from circulating mononuclear cells, and if interleukin-6 (IL-6) stimulates EPC angiogenesis in vitro. First, we isolated and cultured mononuclear cells from adult human circulating blood. We obtained EPC clones that were further cultured and expended for the angiogenesis study. We found that the EPCs possessed human mature endothelial cell phenotypes; however, they proliferated much faster than mature endothelial cells (P <0.05). We then found that IL-6 receptor (gp-80) was expressed in the EPCs, and that administration of IL-6 could activate receptor gp80/gp130 signaling pathways including downstream extracellular signal-regulated kinase 1/2 and STAT3 phosphorylation in EPCs. Furthermore, IL-6 stimulated EPC proliferation, migration, and matrigel tube formation in a dose-dependent manner (P <0.05); anti-IL-6 antibodies or IL-6 receptor could abolish these effects (P <0.05). These results suggest that IL-6 plays a crucial role in the biologic behavior of blood-derived EPCs, which may help clarify the mechanism of IL-6 inflammatory-related diseases.

Insulin Growth Factor-1 Gene Transfer Enhances Neurovascular Remodeling and Improves Long-Term Stroke Outcome in Mice

Background and Purpose— Insulin-like growth factor I (IGF-1) is a pleiotropic growth factor that has been demonstrated to protect against acute ischemic brain injury. Whether IGF-1 improves long-term functional outcome after ischemic stroke is not known. The aim of this study is to examine whether IGF-1 overexpression through adeno-associated virus (AAV) -mediated gene transfer enhances neurovascular remodeling and improves functional outcome in a mouse model of focal cerebral ischemia. Methods— Long-term cerebral IGF-1 overexpression was achieved with the AAV transduction system through stereotaxic injection. Control mice were injected with AAV–green fluorescent protein or saline. Three weeks after gene transfer, the mice underwent permanent distal middle cerebral artery occlusion. Histological and behavioral analyses were performed at day 21 after middle cerebral artery occlusion. Results— IGF-1 gene transfer compared with control treatment significantly improved motor performance assessed by sensorimotor tests. The functional recovery was accompanied by reduced volume of cerebral infarction. Immunohistochemical analysis with endothelial cell marker CD31 revealed that IGF-1 gene transfer potently increased neovessel formation in the periinfarct and injection needle tract area compared with AAV–green fluorescent protein transduction. Increased vascular density was associated with increased local vascular perfusion. Additionally, AAV-IGF-1 treatment enhanced neurogenesis in the subventricular zone compared with AAV–green fluorescent protein treatment. Conclusions— These data demonstrate that IGF-1 overexpression promoted long-lasting functional recovery after cerebral infarction. The improved functional performance was paralleled by enhanced neovascularization and neurogenesis.

Adeno-associated viral-vector-mediated hypoxia-inducible vascular endothelial growth factor gene expression attenuates ischemic brain injury after focal cerebral ischemia in mice.

Background and Purpose— Exogenous delivery of vascular endothelial growth factor gene (VEGF) may provide a useful approach to the treatment of brain ischemia. We investigated the use of a hypoxia-responsive element to control VEGF expression given for neuroprotection. Methods— Three groups (n=36) of mice received AAVH9-VEGF, AAVH9-lacZ, or saline injection. Five days after gene transfer, the mice underwent 45 minutes of transient middle cerebral artery occlusion (tMCAO) followed by 1 to 7 days of reperfusion. Infarct volume was determined using cresyl violet staining; neuronal injury was examined using TUNEL, cleaved caspase-3, and fluoro-Jade B staining. Results— Hypoxia-inducible factor-1 (HIF-1) was overexpressed after tMCAO in the ischemic hemisphere in the brain. Expression of lacZ, mediated by AAV-lacZ, was seen before and after tMCAO; however, AAVH9-lacZ-mediated lacZ expression was detected only after tMCAO. Infarct volume was smaller in the AAVH9-VEGF-transduced group compared with AAVH9-lacZ and saline groups (55% reduction, P<0.05) with reduced TUNEL (29±5% and 30±7% versus 12±3%, P<0.05), cleaved caspase-3 (20±3% and 21±5% versus 13±4%, P<0.05) and fluoro-Jade B (23±3% and 24±5% versus 12±5%, P<0.05) -positive neurons, respectively. Conclusion— Exogenous expression of VEGF through AAVH9-VEGF gene transfer 5 days before the onset of ischemia provides neuroprotection. Hypoxia-responsive element is a viable strategy of restricting VEGF expression to areas of ischemia to minimize adverse effects of therapy on adjacent normal parenchyma.

A Single Domain Llama Antibody Potently Inhibits the Enzymatic Activity of Botulinum Neurotoxin by Binding to the Non-Catalytic Alpha-Exosite Binding Region

Ingestion or inhalation of botulinum neurotoxin (BoNT) results in botulism, a severe and frequently fatal disease. Current treatments rely on antitoxins, which, while effective, cannot reverse symptoms once BoNT has entered the neuron. For treatments that can reverse intoxication, interest has focused on developing inhibitors of the enzymatic BoNT light chain (BoNT Lc). Such inhibitors typically mimic substrate and bind in or around the substrate cleavage pocket. To explore the full range of binding sites for serotype A light chain (BoNT/A Lc) inhibitors, we created a library of non-immune llama single-domain VHH (camelid heavy-chain variable region derived from heavy-chain-only antibody) antibodies displayed on the surface of the yeast Saccharomyces cerevisiae. Library selection on BoNT/A Lc yielded 15 yeast-displayed VHH with equilibrium dissociation constants (K(d)) from 230 to 0.03 nM measured by flow cytometry. Eight of 15 VHH inhibited the cleavage of substrate SNAP25 (synaptosome-associated protein of 25,000 Da) by BoNT/A Lc. The most potent VHH (Aa1) had a solution K(d) for BoNT/A Lc of 1.47 x 10(-)(10) M and an IC(50) (50% inhibitory concentration) of 4.7 x 10(-)(10) M and was resistant to heat denaturation and reducing conditions. To understand the mechanism by which Aa1 inhibited catalysis, we solved the X-ray crystal structure of the BoNT/A Lc-Aa1 VHH complex at 2.6 A resolution. The structure reveals that the Aa1 VHH binds in the alpha-exosite of the BoNT/A Lc, far from the active site for catalysis. The study validates the utility of non-immune llama VHH libraries as a source of enzyme inhibitors and identifies the BoNT/A Lc alpha-exosite as a target for inhibitor development.

Therapeutic angiogenesis for brain ischemia: a brief review.

In the normal mature brain, blood vessel formation is tightly downregulated. However, pathologic processes such as ischemia can induce cerebral vascular regeneration. Angiogenesis is one of the major styles of new vessel formation. In this article, we summarize the major angiogenic factors in the brain, discuss the significant changes of angiogenic factors and endothelial progenitor cells (EPCs) in response to brain ischemia, and finally, review the therapeutic potential of angiogenic factors and EPCs in experimental cerebral ischemia based on the concept of neurovascular unit.

Neutrophil depletion decreases VEGF-induced focal angiogenesis in the mature mouse brain.

To explore the role of neutrophil-derived matrix metalloproteinases (MMPs) during angiogenesis in the brain, we hypothesized that transient neutrophil depletion attenuates the angiogenic response to focal hyperstimulation with vascular endothelial growth factor (VEGF). Brain focal angiogenesis was achieved using an adeno-associated virus delivered VEGF (AAV-VEGF) gene transfer in the mature mouse. Four groups of mice underwent AAV vector injection in the brain parenchyma: (1) AAV-LacZ; (2) AAV-VEGF; (3) AAV-VEGF plus anti-polymorphonuclear (PMN) antibody; and (4) AAV-VEGF plus serum. Animals in groups 3 and 4 underwent 4 days of PMN antibody or serum treatment before transfection; treatment was sustained for an additional 14 days. Anti-PMN treatment decreased circulating neutrophils to 9% of baseline (P < 0.001). Microvessels in the AAV-VEGF-group increased 25% compared with the AAV-lacZ-transduced group (256 ± 15 versus 208 ± 16; P < 0.05). Anti-PMN treatment attenuated the increase to 10% compared with control serum treatment (234 ± 16 versus 255 ± 22; P < 0.05). Similarly, compared with control serum treatment, anti-PMN treatment also reduced MMP-9 by 50% (2 ± 0.9 versus 4 ± 1.4; P < 0.05) and MPO expression by 25% (2 ± 0.8 versus 3 ± 0.9; P < 0.05); MMP-9 activity correlated with MPO expression (R2 = 0.8, P < 0.05). Our study demonstrated that transient depletion of neutrophils suppressed VEGF-induced angiogenesis, indicating that circulating neutrophils contribute to VEGF-induced focal angiogenesis. In addition, brain MMP-9 activity was attenuated after neutrophil depletion, suggesting that neutrophil is an important source of MMP-9.

Overexpression of Netrin-1 Induces Neovascularization in the Adult Mouse Brain:

Netrin-1 is a critical molecule for axonal pathfinding during embryo development, and because of its structural homology to the endothelial mitogens, it may share its effects on vascular network formation. Using an adeno-associated viral netrin-1 vector (AAV-NT-1) gene transfer, we demonstrated that netrin-1 was able to stimulate the proliferation and migration of human cerebral endothelial cells (HCECs) and human aortic smooth muscle cells (HASMCs) compared with the control (P < 0.05), and could also promote HCEC tube formation on matrigel (P < 0.05) in vitro. Moreover, netrin-1 hyperstimulation could promote focal neovascularization (P < 0.05) in the adult brain in vivo. Unlike VEGF-induced microvessel increase, netrin-1-induced newly formed vessels showed an artery-like phenotype, with an intact endothelial cell monolayer surrounded by multiple cell layers, including smooth muscle cells and an astrocyte-connected outer layer. Our findings suggest that netrin-1 plays an important role in promoting blood vessel formation in the adult rodent central nervous system, and could have broad implication in cerebrovascular development and remodeling.

MMP-9 expression is associated with leukocytic but not endothelial markers in brain arteriovenous malformations.

Brain arteriovenous malformations (BAVM) have high matrix metalloproteinase-9 (MMP-9) expression, the source of which is unclear. We hypothesized MMP-9 production might be due to inflammation in BAVM. Compared to control brain tissues (n = 5), BAVM tissue (n = 139) had a higher expression (by ELISA) of myeloperoxidase (MPO) (193 +/- 189 vs. 6 +/- 3, ng/mg, P < .001), MMP-9 (28 +/- 32 vs. 0.7 +/- 0.6, ng/mg, P < .001), and IL-6 (102 +/- 218 vs. 0.1 +/- 0.1, pg/mg, P < .001), but not eNOS (114 +/- 87 vs. 65 +/- 9, pg/mg, P = .09). MMP-9 expression in BAVM highly correlated with myeloperoxidase (R2 = .76, P < .001), as well as with IL-6 (R2 = .32, P < .001). In contrast, MMP-9 in BAVM poorly correlated with the endothelial marker, eNOS (R2 = .03, P = .05), and CD31 (R2 = .004, P = .57). Compared to non-embolized patients (n = 46), patients with pre-operative embolization (n = 93) had higher levels of myeloperoxidase (236 +/- 205 vs. 106 +/- 108, ng/mg, P < .001) and MMP-9 (33 +/- 35 vs. 16 +/- 20, ng/mg, P < .001), however the correlation between MMP-9 and myeloperoxidase was equally strong for both groups (R2 = .69, n = 93, P < .001, for both). MMP-9 expression correlated with the lipocalin-MMP-9 complex, suggesting neutrophils as the MMP-9 source. MPO co-localized with majority of MMP-9 signal by immunohistochemistry. Our data suggest that inflammation is a prominent feature of BAVM lesional phenotype, and neutrophils appear to be a major source of MMP-9 in these lesions.

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 was 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) by stereotaxic injection at 24 h after a stroke. Adeno-associated viral vector-green fluorescent protein (GFP) or saline was injected as a control. The success of postischemic gene transduction was confirmed by a strong GFP 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 after a stroke in the peri-infarct and injection needle tract area compared with AAV-GFP or saline treatment, as shown by immunohistochemical staining with the vascular marker lectin. Furthermore, increased vascular density was associated with improved local vascular perfusion. Immunohistochemical staining with the neuronal progenitor marker, DCX (doublecortin), and the cell proliferation marker, BrdU (5-bromo-2-deoxyuridine-5-monophosphate), indicated that AAV-IGF-1 treatment potently increased neurogenesis compared with AAV-GFP injection. These data show that postischemic treatment of IGF-1 effectively promoted neural and vascular regeneration in the chronic stage of cerebral infarction.