Research reportVascular endothelial growth factor promotes neurite maturation in primary CNS neuronal cultures
Introduction
Vascular endothelial growth factor (VEGF) plays a major regulatory role in developmental and pathological angiogenesis [24]. It is a 46-kDa heparin-binding homodimeric glycoprotein, which exerts its biological actions through binding to the receptor tyrosine kinases VEGFR1 (Flt-1) and VEGFR2 (Flk-1) [47]. Until recently, VEGF was presumed to be a highly selective endothelial cell mitogen that promotes angiogenesis and increases vascular permeability and survival [10], [14]. In the past several years, increased evidence of the pleiotropic nature of VEGF has emerged, particularly with regard to nervous tissue. The earliest in situ hybridization studies of adult brains and embryonic neural tubes (E7.5–E12.5) were unable to detect either VEGFR1 mRNA [33] or VEGFR2 mRNA in non-endothelial cells [36]. In contrast, a number of later studies demonstrated expression of VEGF receptor protein kinases in non-endothelial cells of the peripheral and central nervous systems. VEGF-mediated stimulation of axonal outgrowth was observed in adult superior cervical ganglia and dorsal root ganglia in vitro via VEGFR2 [43]. Expression of both VEGFR1 and VEGFR2 mRNAs has been reported in Muller cells and neural progenitors of the developing retina [38], [50]. Reactive astrocytes express VEGFR1 following VEGF infusions [39], neural grafting or injury of the mature rat brain [21], [22]. In CNS neurons, VEGF exerts VEGFR2-mediated neuroprotective effects against excitotoxic damage [27], [45] and injury caused by hypoxia and glucose deprivation in vitro [18], [19], [45].
Previous studies have demonstrated that exposure of fetal neocortical or mesencephalic explants to VEGF results in increased perikaryal and neuritic immunoexpression of microtubule-associated protein-2 (Map-2) in neurons [26], [42]. There has been only one other report showing that VEGF stimulates growth in CNS neurons. VEGF treatment resulted in increased neurite outgrowth from ganglion cells within retinal explants, apparently via the VEGFR2 receptor [3]. However, explant systems are not conducive to determining unequivocally if VEGF's effects are indirect, i.e. the result of interactions with other cell types within the explants, or if neurons respond directly via their expression of specific VEGF receptors. To determine if VEGF's stimulatory effect on Map-2 expression is directly mediated by neuronal VEGF receptors, we have further examined the effects of exogenous VEGF on primary neuronal cultures derived from P0 rat neocortex. Map-2 is expressed in cell bodies and in processes that are committed to a dendritic fate and reflects the level of dendritic maturation [15], [28]. Primary neuronal cell cultures were isolated from P0 rat pups, incubated for 3 days in serum-free Neurobasal medium with or without VEGF (1–100 ng/ml), and the length of Map-2(+) neurites was measured. To determine if the MAPK and PI3-kinase pathways play a role in neuronal maturation effects of VEGF, their respective inhibitors PD98059 and wortmannin were used. The involvement of VEGF receptors in the outgrowth of Map-2(+) processes was analyzed using antisense oligonucleotides against VEGFR1 and VEGFR2 genes. In addition, neuronal VEGF receptor expression was analyzed by immunohistochemistry and RT-PCR.
The present study demonstrates that VEGF directly affects Map-2 expression by cultured neurons and this effect is mediated by VEGFR2 and the MAPK pathway. In addition, VEGF regulates the expression of VEGFR1 by neurons through the PI3-K pathway. These data provide evidence of a possible developmental role of VEGF during neuronal maturation and that VEGF's actions are not restricted to the endothelial cell lineage in the CNS.
Section snippets
Cell culture
Primary serum-free neuronal cultures were obtained from dissociated cerebral cortices. Newborn (P0) Wistar rats were rendered unconscious after inhalation of Halothane vapors and were decapitated, in accordance with the George Washington University IACUC and the NIH Guide for the Care and Use of Laboratory Animals. Brains were removed under sterile conditions and placed in Neurobasal medium (Gibco). The cerebral cortices were dissected out, cut into 1 mm cubes, briefly trypsinized and
VEGF administration stimulates neurite outgrowth and Map-2 expression
After 3 days of incubation, β-tubulin (III) and Map-2 expression in cultured cerebral cortical neurons were examined by immunohistochemistry (Fig. 1). TuJ1 antibody is raised against neuron specific Class III β-tubulin and indiscriminately labels the neuronal cytoplasm and all processes. Cultures treated with 25 ng/ml of VEGF had neuronal diameters that averaged 40% larger than those of control cultures; control neurons also had fewer processes than those exposed to VEGF (Fig. 1A,B).
In contrast
Discussion
The major finding of this study is that VEGF exposure directly enhances MAP-2 expression and neurite maturation in neocortical neurons in vitro. Stage 4 of neuronal development in culture is characterized by dendritic fate determination [8], the hallmark of which is the increase in the expression of Map-2 mRNA and protein. In the presence of VEGF, the degree of Map-2 spatial extension and the levels of Map-2 mRNA are higher compared to the untreated controls under normoxic, serum-free culture
Acknowledgements
We thank Newton More for his technical expertise in immunofluorescence methods and Gauri Tadvalkar for the help with the RT-PCR technique. This work was supported by National Institutes of Health Grants NS 38128 and 39282 (JMK and JMR).
References (50)
- et al.
Migration of human monocytes in response to vascular endothelial growth factor (VEGF) is mediated via the VEGF receptor flt-1
Blood
(1996) - et al.
Vascular endothelial growth factor effect on endothelial cell proliferation, migration, and platelet-activating factor synthesis is Flk-1-dependent
J. Biol. Chem.
(1999) - et al.
Numbers, time and neocortical neuronogenesis: a general developmental and evolutionary model
Trends Neurosci.
(1995) - et al.
The vascular endothelial growth factor receptor Flt-1 mediates biological activities. Implications for a functional role of placenta growth factor in monocyte activation and chemotaxis
J. Biol. Chem.
(1996) - et al.
The interaction of neuropilin-1 with vascular endothelial growth factor and its receptor flt-1
J. Biol. Chem.
(2000) - et al.
Vascular endothelial growth factor regulates endothelial cell survival through the phosphatidylinositol 3′-kinase/Akt signal transduction pathway. Requirement for Flk-1/KDR activation
J. Biol. Chem.
(1998) - et al.
Caspase-3 and the regulation of hypoxic neuronal death by vascular endothelial growth factor
Neuroscience
(2001) - et al.
VEGF mRNA and its receptor flt-1 are expressed in reactive astrocytes following neural grafting and tumor cell implantation in the adult CNS
Exp. Neurol.
(1998) - et al.
Transient coexpression of nestin, GFAP, and vascular endothelial growth factor in mature reactive astroglia following neural grafting or brain wounds
Exp. Neurol.
(1999) - et al.
Angiogenic and astroglial responses to vascular endothelial growth factor administration in adult rat brain
Neuroscience
(2002)
Coordinated interaction of neurogenesis and angiogenesis in the adult songbird brain
Neuron
Neuronal VEGF expression correlates with angiogenesis in postnatal developing rat brain
Dev. Brain Res.
Paracrine and autocrine functions of neuronal vascular endothelial growth factor (VEGF) in the central nervous system
J. Biol. Chem.
Angiogenesis in developing rat brain: an in vivo and in vitro study
Brain Res.
Vascular, glial and neuronal effects of vascular endothelial growth factor in mesencephalic explant cultures
Neuroscience
The roles of microtubule-associated proteins in brain morphogenesis: a review
Brains Res. Rev.
Different signal transduction properties of KDR and Flt1, two receptors for vascular endothelial growth factor
J. Biol. Chem.
Quantitative analysis of mRNA expression of neuron-specific growth-associated genes in rat primary neurons by competitive RT-PCR
Brain Res. Brain Res. Protoc.
Erythropoietin and VEGF promote neural outgrowth from retinal explants in postnatal rats
Invest. Ophthalmol. Vis. Sci.
Expression of vascular endothelial growth factor during embryonic angiogenesis and endothelial cell differentiation
Development
Abnormal blood vessel development and lethality in embryos lacking a single VEGF allele
Nature
The establishment of polarity by hippocampal neurons in culture
J. Neurosci.
Astrocytes are the primary source of tissue factor in the murine central nervous system. A role for astrocytes in cerebral hemostasis
J. Clin. Invest.
The biology of vascular endothelial growth factor
Endocr. Rev.
Role of the Flt-1 receptor tyrosine kinase in regulating the assembly of vascular endothelium
Nature
Cited by (110)
Restoration of early deficiency of axonal guidance signaling by guanxinning injection as a novel therapeutic option for acute ischemic stroke
2021, Pharmacological ResearchCitation Excerpt :It is well known that vascular endothelial growth factor A (VEGF-A) is critical in blood vessel growth [78,79]. However, in vitro and in vivo experiments have shown that VEGF-A has a direct impact on neurogenesis, neuronal migration, neuronal survival and axon guidance [80–82]. It plays an increasingly prominent role in promoting nerve repair and recovery of nerve function [80,83].
Vascular endothelial growth factor and neurodevelopment
2021, Factors Affecting Neurodevelopment: Genetics, Neurology, Behavior, and DietThe rat hippocampal gliovascular system following one week vortioxetine and fluoxetine
2021, European NeuropsychopharmacologyNeurocognitive Function in Adult Cancer Patients
2018, Neurologic Clinics