Vascular endothelial growth factor enhances migration of astroglial cells in subventricular zone neurosphere cultures.

Vascular endothelial growth factor (VEGF) is an endothelial and neuronal survival factor and a mitogen for endothelial cells and astrocytes in both explant and in vivo injury models. In the CNS, interplay between the vasculature and neural stem progenitor (NSP) cells is required for the maintenance of angiogenic/neurogenic coordination in the germinal niche in the subventricular zone (SVZ) of the lateral ventricle. Using an in vitro SVZ neurosphere (NS) model, this study aimed to understand the direct effects of VEGF and its receptor signaling on neonatal NSP cell growth and migration. Our data indicate that VEGF administration, compared with untreated or brain-derived neurotrophic factor-treated NS, significantly increased growth and migratory capacity of glial fibrillary acidic protein (GFAP)(+) and nestin(+) NSP cells and in secondary cultures induced a stellate astrocyte morphology. Blockade of both VEGF, which is normally expressed in some NS cells, and its flt-1 receptor signaling by neutralizing antibodies caused morphological changes specifically in GFAP(+) cells and disrupted sphere formation and outward migration. These cells did not appear as conventional polygonal astrocytes; their process growth was severely restricted, and overall migration was reduced by up to 76% of control cultures. Blockade of VEGF's flk-1 receptor reduced VEGF expression and caused a lesser, though significant, decrease (29%) in NSP (GFAP(+)) cell migration. The results show that both VEGF and, in particular, flt-1 receptor signaling are critical to the proper configuration of the NS and its subsequent development. VEGF is also an important growth and migratory factor particularly for GFAP(+) cells developing in SVZ-derived NS in culture.

Neurotrophic effects of vascular endothelial growth factor on organotypic cortical explants and primary cortical neurons.

Vascular endothelial growth factor (VEGF) is well known to play an important regulatory role in vascular growth and development. Because gene knock-outs of VEGF and its receptors flk-1 and flt-1 result in early embryonic lethality, determining roles for VEGF in CNS development has been particularly difficult. Recent studies have shown that VEGF is upregulated after various injuries to the adult brain and that the cytokine affords protection to cultured neurons affected by oxidative or excitotoxic stress. The present study demonstrates, for the first time, that VEGF is directly neurotrophic to CNS neurons in culture. We applied VEGF to normoxic fetal organotypic cortical explants as a model of CNS neuropil, in addition to primary cortical neurons, to assess direct growth effects absent vascular or astroglial activity. We found that VEGF provided a significant dose-responsive increase in the neuronal microtubule markers TUJ1 and MAP-2, as well as mRNA for MAP-2 and flk-1. Antisense oligodeoxynucleotides to flk-1, but not flt-1, inhibited neuritic outgrowth, whereas inhibitors of the signaling pathways MEK1 and P13-AKT both abrogated VEGF-induced growth. VEGF applied to primary cortical neurons produced significant increases in neuronal cell body diameter and the number of emerging neurites mediated by flk-1. Possibly, VEGF achieves its effects by acting on the neuronal microtubular content, which is involved with growth, stability and maturation. Several studies have now shown that VEGF is neurotrophic and neuroprotective independent of a vascular component; we suggest that VEGF plays seminal pleiotrophic roles in CNS development and repair.

Activation of receptor-mediated angiogenesis and signaling pathways after VEGF administration in fetal rat CNS explants.

SUMMARY: The angiogenic role of vascular endothelial growth factor (VEGF) receptors, flk-1 and flt-1, and their downstream signaling pathways, MAPK/ERK and PI-3 kinase, were examined in a fetal rat cortical explant model after exposure to exogenous VEGF. Treatment with VEGF resulted in substantial neovascularization characterized by increased vascular flk-1 receptor expression, whereas flt-1 receptor protein expression was absent. The specific role of flk-1 receptors in the angiogenic process was confirmed by the addition of antisense oligonucleotides (AS-ODNs) to flk-1, which blocked angiogenesis, whereas AS-ODNs to flt-1 had no effect. These results were further supported by the finding that specific chemical inhibition of flk-1 receptors caused disruption of the angiogenic response, whereas inhibition of the flt-1 receptors had no effect. Application of either MAPK/ERK or PI-3 kinase pathway inhibitors disrupted VEGF-induced angiogenesis, thereby indicating that both signaling pathways mediate this process. Thus VEGF binding to the endothelial flk-1 receptor activates the MAPK/ERK and PI-3 kinase pathways, resulting in neoangiogenic events. Of interest is the fact that although VEGF is regarded as a vascular permeability factor, its application to nascent cortical tissue caused an increase in a key physiologic protein of the blood-brain barrier function, glucose transporter-1, suggesting that the cytokine may have a role in blood-brain barrier development.

Vascular endothelial growth factor promotes neurite maturation in primary CNS neuronal cultures.

Recent studies have demonstrated that vascular endothelial growth factor (VEGF) and its receptor VEGFR2 (flk-1) are expressed by neurons during development and following hypoxic-ischemic events. Moreover, fetal CNS tissue explants exposed to exogenous VEGF exhibit increased neuronal Map-2 expression, suggesting that VEGF could have an effect on neuronal maturation. To determine whether this effect is of a direct nature, we examined the expression of Map-2 in the presence of VEGF in primary CNS neuronal cultures. After 3 days in culture, a statistically significant dose-dependent increase in the length of Map-2(+) processes was observed, with the peak occurring at 10 ng/ml of VEGF. Immunohistochemical analysis of the cultures demonstrated the presence of VEGFR2 after VEGF treatment, as well as the expression of the VEGF receptor VEGFR1 (flt-1). Treatment of the cultures with antisense oligonucleotides against VEGFR2, but not against VEGFR1, abolished the effect of VEGF on the length of Map-2(+) processes. RT-PCR analyses of Map-2 and VEGFR1 indicated that mRNAs of these two genes are upregulated in the presence of VEGF. The addition of wortmannin, an inhibitor of PI3K/Akt signal-transduction pathway, to the media did not affect the VEGF-dependent increase in Map-2(+) length. In contrast PD98059, which inhibits the MAPK pathway, partially abolished this effect of VEGF. These experiments suggest that VEGF has a direct effect on neuronal growth and maturation under normoxic conditions during CNS development, which is mediated by the VEGFR2 receptor via the MAPK pathway.

VEGF in the nervous system.

Vascular endothelial growth factor (VEGF, VEGFA) is critical for blood vessel growth in the developing and adult nervous system of vertebrates. Several recent studies demonstrate that VEGF also promotes neurogenesis, neuronal patterning, neuroprotection and glial growth. For example, VEGF treatment of cultured neurons enhances survival and neurite growth independently of blood vessels. Moreover, evidence is emerging that VEGF guides neuronal migration in the embryonic brain and supports axonal and arterial co-patterning in the developing skin. Even though further work is needed to understand the various roles of VEGF in the nervous system and to distinguish direct neuronal effects from indirect, vessel-mediated effects, VEGF can be considered a promising tool to promote neuronal health and nerve repair.

Roles of the endogenous VEGF receptors flt-1 and flk-1 in astroglial and vascular remodeling after brain injury.

Following trauma to the brain significant changes occur in both the astroglial and vascular components of the neuropil. Angiogenesis is required to re-establish metabolic support and astrocyte activation encompasses several functions including scar formation and the production of growth factors. VEGF has seminal involvement in the process of brain repair and is upregulated during many pathological events. VEGF signaling is regulated mainly through its two primary receptors: flk-1 (KDR/VEGF-R2) is expressed on vascular endothelium and some neurons and flt-1 (VEGF-R1) in the CNS, is expressed predominantly by activated astrocytes. Using an injury model of chronic minipump infusion of neutralizing antibodies (NA) to block VEGF receptor signaling, this study takes advantage of these differences in VEGF receptor distribution in order to understand the role the cytokine plays after brain injury. Infusion of NA to flk-1 caused a significant decrease in vascular proliferation and increased endothelial cell degeneration compared to control IgG infusions but had no effect on astrogliosis. By contrast infusion of NA to flt-1 significantly decreased astroglial mitogenicity and scar formation and caused some increase in endothelial degeneration. Neutralization of the flt-1 receptor function, but not flk-1, caused significant reduction in the astroglial expression of the growth factors, CNTF and FGF by 7days. These data suggest that after CNS injury, endogenous VEGF upregulation (by astrocytes) induces angiogenesis and, by autocrine signaling, increases both astrocyte proliferation and facilitates expression of growth factors. It is likely that VEGF plays an important role in aspects of astroglial scar formation.

Astrocyte growth effects of vascular endothelial growth factor (VEGF) application to perinatal neocortical explants: receptor mediation and signal transduction pathways.

The non-angiogenic role of vascular endothelial growth factor (VEGF), and its receptors flt-1 and flk-1, together with downstream signaling pathways were examined in fetal and postnatal rat cerebral cortical organotypic explants. VEGF application in both paradigms caused a significant increase in astroglial proliferation and a dose-dependent increase in GFAP and nestin immunoreactivity. The VEGF receptor flt-1 was observed on most, though not all astrocytes, while flk-1 receptor immunoexpression was absent. Treatment with antisense oligonucleotides (AS-ODNs) to flt-1 resulted in a dramatic decrease in GFAP and nestin immunoreactivity, which further confirmed the role of flt-1 in mediating VEGF's gliotrophic effects, while AS-ODNs to flk-1 had no effect. VEGF-induced gliotrophic effects were found to be mediated by the MAPK/ERK and PI-3 kinase signaling pathways, since the both the MEK1 inhibitor, PD98059 and the PI-3 kinase inhibitor, Wortmannin abolished VEGF-induced astrocytic GFAP(+) expression. Although high dose VEGF application resulted in strong upregulation of both GFAP and nestin immunoreactivity in astrocytes, overlap of the two proteins was not observed in all cells, suggesting that some of the nestin(+) cells might be neural progenitors. Exposure to VEGF resulted in upregulation of both VEGF and bFGF mRNA at the one-day time point, and bFGF protein by 3 days; VEGF activated astrocytes expressed bFGF to a much greater degree than those in untreated explants. The increased expression of bFGF induced by VEGF, may serve in the proliferation of multipotential neural stem/progenitor cells in vitro. VEGF, an established angiogenic factor, appears to play a significant role in the growth and differentiation of astrocytes in the CNS.

New roles for VEGF in nervous tissue--beyond blood vessels.

Vascular endothelial growth factor (VEGF) is a secreted dimeric polypeptide that until recently has been believed to be a specific mitogen for endothelial cells subserving angiogenesis and permeability in development and after injury. Recent studies have depicted the localization of VEGF and its receptors on neurons and astrocytes and it has been shown to induce neuritic growth and to provide neuroprotection particularly after ischemia or spinal cord injuries. VEGF also shares common receptor signaling with the guidance molecule SEMA3A and thus could have an additional role linking the coordinated patterning of developing vascular and nervous tissue. It is now apparent that VEGF's role in nervous tissue is pleiotropic in nature, and further elucidation of its mechanisms of action may serve as a key substrate in understanding aspects of neural repair and development.

Inhibition of endogenous VEGF impedes revascularization and astroglial proliferation: roles for VEGF in brain repair.

Vascular endothelial growth factor (VEGF) is upregulated following injury to the CNS. Our previous work has shown that exogenous application of VEGF promotes angiogenesis, blood-brain barrier permeability, and astroglial mitogenicity in the traumatized brain. To develop a model that could link endogenously secreted VEGF to brain tissue repair, a specific neutralizing antibody to VEGF was infused by osmotic minipump directly into the neocortex and striatum for up to 1 week. Tissues adjacent to the infusion/wound site were analyzed for specific vascular and astroglial protein markers and proliferation, necrosis/apoptosis (via TUNEL staining), VEGF, the VEGF receptors flt-1 and flk-1, and bFGF expression using immunohistochemistry and semi-quantitative RT-PCR. Neutralization of native VEGF caused significant decreases in angiogenic activity, astroglial proliferation, and nestin immunoexpression, while vascular and astroglial degeneration was substantially increased, resulting in much larger wound cavities when compared to controls. The hindrance of brain tissue repair occurred despite an increase in bFGF expression at the wound sites. VEGF appears to be an integral factor in CNS wound healing that is essential for vascular endothelial proliferation and survival and may also be necessary for astroglial proliferation and maintenance during the repair of brain injury.

Changes in astroglial GLT-1 expression after neural transplantation or stab wounds.

Uncontrolled release of glutamate from damaged brain initiates events that result in excitotoxic neuronal death. Glutamate uptake by specialized astroglial transporters is essential for control of extracellular glutamate levels. Many studies have demonstrated a reduction in astrocytic GLT-1 expression after different forms of injury. Because extensive neuronal death does not occur after direct cortical stab wounds and viable developing neurons populate fetal CNS grafts, we hypothesized that reactive astroglia associated with these procedures might maintain or up-regulate GLT-1. We examined the temporal and spatial distribution of GLT-1, GFAP and nestin proteins by confocal double-label immunohistochemistry combined with a new methodology in which precise brain areas are microdissected and analyzed for protein content by immunoaffinity chromatography. In stab wounds, GLT-1 protein content did not change compared to normal cortex, as determined by direct protein measurements; GLT-1 colocalized with nestin- and GFAP(+) astroglia adjacent to the lesion. In contrast, host reactive astroglia adjacent to grafts significantly upregulated GLT-1 by 3 days postoperative. The GFAP protein analysis suggests that increased GLT-1 is not the result of greater numbers of activated astroglia around grafts, but that developing graft tissue influences adjacent host astroglia to upregulate GLT-1. GLT-1 protein within grafts was rapidly accelerated to mature levels by just three days, and was expressed by the nestin(+) cell population. These data, which demonstrate immunoexpression of GLT-1 protein combined with a new method for protein measurement in situ indicate that, in contrast to other injury models, astroglial GLT-1 is upregulated or maintained following invasive CNS procedures. (c)2002 Elsevier Science (USA).