Janette M. Krum

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 VEGFs 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.

Angiogenic and astroglial responses to vascular endothelial growth factor administration in adult rat brain.

The effects of exogenous vascular endothelial growth factor (VEGF) on angiogenesis, blood-brain barrier permeability and astroglial proliferation in the adult rat CNS in situ were investigated. Recombinant human VEGF(165) (25 or 50 ng/ml) was delivered for up to 1 week using either intracerebral osmotic minipumps or less traumatic subdural gelatin sponge placement. By 3 days, VEGF delivery caused significantly increased cerebral angiogenesis (25 ng/ml was most effective) in both experimental models when compared to saline controls; VEGF infusion resulted in a 100% increase in an index of vascular proliferation, and gelatin sponge delivery produced a 65% increase. The blood-brain barrier hallmark endothelial glucose transporter-1 was not present in nascent vascular sprouts. Infusion of VEGF produced extensive protein leakage that persisted after saline-induced permeability was mostly resolved, while gelatin sponge administration caused milder barrier dysfunction. Administration of the angiogenic factor had unexpected proliferative effects on astroglia in both models, resulting in an 80-85% increase in mitotically active astroglia when compared to controls. Immunohistochemical results and semi-quantitative reverse transcriptase-polymerase chain reaction indicated that the VEGF receptors flk-1 and flt-1 were up-regulated in response to the infusion trauma; flt-1 was localized to reactive astroglia, while flk-1 was expressed in vascular endothelium but predominantly in neuronal somata and processes adjacent to the delivery site. mRNA for the VEGF(121), VEGF(165) and VEGF(188) isoforms was also increased after delivery of the recombinant protein. These data show that VEGF application has substantial proliferative effects on CNS endothelium and astroglia and causes up-regulation of its own message. Flt-1 and flk-1 receptor mRNAs and proteins are up-regulated in both vascular and non-vascular cell types following infusion trauma. From these results we suggest that administered VEGF has heretofore unanticipated pleiotrophic effects in the adult CNS.

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. Note: Previously published in VEGF in Development, edited by Christiana Ruhrberg. Landes Bioscience and Springer Science+Business Media 2008; pp. 91-103.

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.

VEGF mRNA and its receptor flt-1 are expressed in reactive astrocytes following neural grafting and tumor cell implantation in the adult CNS.

Significant angiogenesis occurs only after injury in the adult mammalian brain; capillaries proliferate and astrocytes are activated by presently unresolved cellular mechanisms. Because of the intimate relationship between astrocytes and brain capillaries we examined the expression of the specific endothelial mitogen vascular endothelial growth factor (VEGF) in reactive astrocytes following CNS trauma models: neural grafting, stab wounds, and glioma implantation. In situ hybridization was combined with GFAP immunohistochemistry to delineate VEGF mRNA expression in reactive astrocytes. In addition, VEGF and its receptor flt-1 protein expression were detected immunohistochemically. In all three models we found unexpectedly that only reactive astrocytes, not endothelium, expressed the VEGF receptor flt-1, VEGF mRNA, and VEGF protein in a spatiotemporal manner, suggesting that activated astroglia may have a direct role in the induction of angiogenesis or permeability in mature brain. In addition, secreted VEGF may play a part in astroglial signalling by the induction of its own receptor in reactive astroglia following injury. These findings may have significant implications with regard to growth and reparative mechanisms of the adult cerebrovasculature.

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 VEGFs 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.

Immunocytochemical expression of the endothelial barrier antigen (EBA) during brain angiogenesis.

The antibody to the endothelial barrier antigen (anti-EBA) is localized to the luminal plasma membrane of endothelia that have a blood-brain barrier (BBB) but not to other vessels, for instance those in the circumventricular organs, which lack barrier function. We have examined EBA expression in the rat in certain tissues and in brain microvessels in models of brain angiogenesis such as development, wound healing and neural transplantation. All brain microvessels including pial ones stained for anti-EBA whereas those of the dura, median eminence and choroid plexus did not. Vessels of the iris which are characterized by tight junctions and barrier function expressed EBA strongly. Embryonic day 18 brain did not stain at all for anti-EBA although vessels were readily localized with anti-laminin. Following stab wounds to mature brain, directly injured and adjacent microvessels lacked EBA expression for a period of approximately 2 weeks which is a similar time frame of BBB breakdown. Following this period, EBA expression gradually returned to a normal pattern by 3-4 weeks. Likewise, in intraparenchymal transplants of fetal neocortex EBA expression was not observed for 2 weeks and while at later times transplant vessels expressed EBA whereas some interface vessels associated with inflammatory cells did not. Permeable choroid plexus vessels vascularizing intraventricular transplants did not stain for anti-EBA at any time period and neither did vessels in adrenal medulla transplants. The present study shows that while EBA expression is a postnatal event unlike the development of a barrier to serum protein, its expression may be lost or delayed in injured vessels or ones associated with inflammatory cells or reactive astrocytes.

Brain angiogenesis: Variations in vascular basement membrane glycoprotein immunoreactivity

Changes in the distribution and quantity of laminin and fibronectin within the basement membranes of developing or regenerating CNS blood vessels were investigated using two immunocytochemical techniques. Three models of angiogenesis were studied: normal pre- and postnatal development, wound healing, and vascularization of fetal neocortical transplants placed in the adult rat brain. Although all brain vessels were stained in enzymatically pretreated immunoreacted paraffin sections, those associated with wound and transplant sites were the most intensely reactive with both antisera during the first postoperative week. When 40-microns vibratome sections of normal adult brains were immunoprocessed, only the meninges and vessels of the circumventricular organs were stained. The remainder of the brain vasculature was immunoreactive only if sections were enzymatically treated prior to immunoprocessing. In contrast, the nascent vasculature in developing brain and the regenerating vessels at wound and transplant sites were reactive to both antisera without enzymatic pretreatment of the sections. This immunoreactivity decreased by 11 days postnatal in normal animals and 4 weeks postoperative in experimental animals, coinciding with the period of astrocytic contact and complete vascular basement membrane formation in both cases. The variations in staining pattern and intensity may be reflections of differences in the quantity of laminin and fibronectin within the basement membranes of proliferating and/or non-blood-brain barrier vasculature. However, the results of the different experimental protocols suggest that immature vascular basement membranes may have a molecular configuration that does not require an enzymatic unmasking step to react with the antisera. Alternatively, the looseness of the surrounding neuropil inherent in developing and injured CNS could allow the antisera greater access to basement membrane antigens.

Expression of Blood–Brain Barrier Characteristics Following Neuronal Loss and Astroglial Damage after Administration of Anti-Thy-1 Immunotoxin

In most regions of the CNS, vascular endothelial cells play an important role in maintaining the composition of the neuronal microenvironment by virtue of their blood-brain barrier (BBB) characteristics. The maintenance of the endothelial BBB phenotype in vitro has been attributed primarily to astrocytes but little attention has been paid the potential role of neurons. In this study we have attempted to injure or destroy neurons and fibers of passage in a circumscribed area while leaving vascular and glial elements intact in order to determine if neurons are involved in BBB maintenance in situ. The immunotoxin OX7-SAP, a conjugate of the Thy-1 antibody OX7 and the ribosome-inactivating protein saporin, was injected into the adult rat striatum to effect neuronal death at the injection site. Although neurons and fibers of passage were destroyed within the lesion, glial cells unexpectedly were also severely injured as determined by immunohistochemical expression of several neuronal and astroglial marker proteins and ultrastructural analysis. The microvasculature remained intact, allowing a qualitative immunohistochemical analysis of several BBB markers at time points ranging from 3 to 28 days postinjection. Despite the loss of both neurons and astroglia within the lesions, the microvasculature continued to express the brain-type endothelial glucose transporter GLUT-1 at all time points examined. In contrast, the barrier to endogenous protein (rat serum albumin) and the expression of endothelial barrier antigen (EBA) decreased initially but recovered even in areas that contained minimal numbers of astroglia and neuronal elements. We conclude that intact neuronal or glial cells do not appear to be necessary for the maintenance in situ of the BBB properties examined herein.