The striking structural and anatomical parallels between the vasculature and the nervous system are reflected by the fact that these two organ systems appear to use related mechanisms during their development.¹ Thus, it is not surprising that an increasing number of vascular biologists and researchers in the neuroscience field are fascinated by molecules that play an important role in both systems.

Vascular endothelial growth factor (VEGF) is a major regulator of new blood vessel growth and an important inducer of vascular permeability. However, during the last few years, it has become apparent that VEGF has additional non-vascular functions. In particular the identification of the neuropilins, receptors for the semaphorin family of proteins that mediate neuronal axon pathfinding, as coreceptors for various members of the VEGF family, as well as the detection of VEGF receptors on neurons and astrocytes, suggests that VEGF can act as a neurotrophic and neuroprotective factor in the central as well as in the peripheral nervous system. Thus, VEGF is a pleiotrophic factor with many important functions in the brain. This Chapter summarizes the current knowledge on VEGF expression, regulation and function in the adult nervous system.

The Members of the VEGF Family

VEGF (also designated as VEGF-A) is the founding member of a family of homodimeric glycoproteins that are structurally related to the platelet-derived growth factors (PDGF); this family also includes placenta growth factor (PlGF), VEGF-B, VEGF-C, VEGF-D and VEGF-E. This VEGF family of proteins binds selectively with different affinities to at least five distinct receptors (Fig. 1). Three of them belong to the superfamily of receptor tyrosine kinases and are termed VEGF receptor-1 (VEGFR-1), also called Flt-1 (fms-like tyrosine kinase 1), VEGFR-2, also called KDR (kinase insert-domain containing receptor) in humans, and Flk-1 (fetal liver kinase 1) in rodents, respectively, as well as VEGFR-3, also called Flt-4. The fourth and fifth receptors are neuropilin-1 and neuropilin-2.² Most of the research on the VEGF family so far, especially with respect to the expression pattern and function in the nervous system, has focused on VEGF-A.

Figure 1

The receptors for VEGF. Neuropilin receptors have five extracellular domains, two each with homologies to complement components C1r and C1s (circles) and coagulation factors V and VIII (rectangles), as well as a MAM (meprin, A5, receptor tyrosine phosphatase (more...)

The secreted PlGF shows a strong structural homology to VEGF⁶ and exists as three isoforms, PlGF-1, PlGF-2 and PlGF-3, which result from alternative splicing from a single gene locus.⁵ PlGF is predominantly expressed in placenta and binds exclusively to VEGFR-1.⁷ Although no PlGF expression has been detected in human cortical brain tissue, expression was demonstrated in various tumors of the CNS.^8,9 PlGF induces both proliferation and chemotaxis of endothelial cells in vitro and is angiogenic in vivo.^6,10,11 These results support the proposal that PlGF is involved in tumor angiogenesis and that it is not required for vascular development in the brain. A recent report further demonstrates that embryonic angiogenesis in mice is not affected by PlGF deficiency, but that PlGF contributes to angiogenesis under pathological conditions.¹²

VEGF-B binds to both VEGFR-1 and neuropilin-1. VEGF-B is widely expressed, most prominently in heart and skeletal muscle, but also in mouse and human brain.^13,14 The highest levels of VEGF-B in the brain are detected in neuronal-like cells of the hippocampus and the cerebral cortex.¹⁵ VEGF-B is implicated in angiogenesis by its role in the regulation of extracellular matrix degradation, cell adhesion and migration of endothelial cells.¹⁴

VEGF-C is a ligand for both VEGFR-2 and VEGFR-3.¹⁶ VEGF-C is synthesized as a prepropeptide and subsequently undergoes proteolytic maturation.¹⁷ VEGF-C mRNA is found in several tissues including heart, placenta, ovary, and small intestine,¹⁸ although it is undetectable in the normal brain.¹⁵ As VEGFR-3 is the main receptor for VEGF-C and is predominantly expressed on lymphatic endothelium, VEGF-C was therefore considered to be the prototypic lymphangiogenic factor.¹⁹ However, VEGF-C has also been shown to act on vascular endothelial cells both in vitro and in vivo.^20–22 Indeed, mice which lacked a functional vegfr-3 gene showed defective blood vessel development in early stage mouse embryos.²³ Thus, the VEGF-C/VEGFR-3 system has an essential role not only for lymphatic vessel formation but also for angiogenesis.

VEGF-D is structurally very similar to VEGF-C and it also binds to VEGFR-2 and VEGFR-3.²⁴ VEGF-D is mitogenic for endothelial cells and thus may play a role in endothelial cell regulation. The expression of VEGF-D is prominent in heart and skeletal muscle, as well as in mesenchymal cells of the lung and skin, but shows only very weak expression in the brain.^24,25

VEGF-E is the collective term for a group of proteins with homology to VEGF-A that are encoded by certain strains of the orf parapoxvirus, which affects goats, sheep and occasionally humans.⁵ It possesses about 25% amino acid identity to mammalian VEGF.²⁶ VEGF-E binds with high affinity to VEGFR-2 and neuropilin-1, but neither to VEGFR-1 nor to VEGFR-3, inducing vascular permeability and potent angiogenic activity both in vitro and in vivo.^27-29

Finally, VEGF-A is ubiquitously expressed at low levels within the CNS by astrocytes and neurons.³⁰ VEGF-A exists as several isoforms, derived from a single gene by alternative splicing (Fig. 2). The smallest form, VEGF₁₂₁, is 121 amino acids long and does not bind to heparin, while the four larger forms, VEGF₁₄₅, VEGF₁₆₅, VEGF₁₈₉ and VEGF₂₀₆, all bind heparin with increasing affinity. VEGF₁₈₉ and VEGF₂₀₆ remain cell-attached while the smaller isoforms are secreted.^5,10,31 VEGF was first isolated in 1983 as a factor leading to increased vascular permeability in tumors and was thus called vascular permeability factor (VPF).³² In 1989, VPF was independently isolated and cloned as an endothelial cell-specific mitogen.³³ VEGF is expressed in virtually all cells in the body, however; expression in endothelial cells has mainly been found in vitro in cell culture, while it is undetectable in vivo.³⁴ These findings indicate that VEGF acts as a paracrine factor on neighboring endothelial cells carrying the VEGF receptors rather than in an autocrine fashion. VEGF is such a potent regulator of vascular development that its dosage must be tightly regulated. Disruption of even a single allele of the VEGF gene in mice results in embryonic lethality due to severe vascular defects.^35,36 VEGF-A binds to receptors, VEGFR-1 and VEGFR-2. Recently, the neuropilins have been identified as coreceptors for specific VEGF isoforms. While neuropilin-1 is a receptor only for the VEGF₁₆₅ isoform,³⁷ neuropilin-2 binds both VEGF₁₆₅ and VEGF₁₄₅.³⁸ Targeted inactivation of VEGFR-1 and VEGFR-2 as well as neuropilin-1 in mice resulted in defects of blood vessel formation and embryonic lethality, demonstrating further the importance of VEGF-A for appropriate vascular development.^39–41

Figure 2

The human isoforms of VEGF-A. At least five different isoforms are generated by alternative splicing from a single gene. Exon I, encoding for a signal peptide, exons IIV and exon VIII are present in all isoforms. Exons III and IV are required for binding (more...)

In summary, VEGF-A and VEGF-B appear to be the only VEGF members expressed at significant levels in the CNS, whereas PlGF may be activated during pathological situations. While VEGF-A fulfills an important role in vascular development of the brain,⁴² it remains to be established whether VEGF-B has any physiological function in the CNS. I will, therefore, for the remaining part of this Chapter focus on the regulation and function of VEGF-A in the nervous system.

Regulation of VEGF and VEGF Receptor Expression

A variety of physiological and pathological processes are associated with upregulation of components of the VEGF/VEGFR-system, including embryogenesis, the female reproductive cycle, pregnancy, wound healing, tumor growth, diabetic retinopathy and ischemic diseases (for a review see refs. ^3,43). In the search for mechanisms and factors capable of influencing VEGF expression during these processes, many cytokines and growth factors have been shown to modulate VEGF gene expression.⁵ For example, TNFa and bFGF are able to induce VEGF gene expression in vitro;^44,45 also glucose deficiency has been shown to increase VEGF expression.⁴⁶ The most important and intensively studied inducer of VEGF gene expression, however, is hypoxia, which was demonstrated for the first time in the perinecrotic areas of glioblastomas.^47,48 Under hypoxic conditions VEGF expression is mediated through the activation of specific hypoxia-inducible transcription factors, HIF-1 and HIF-2.^49,50 In addition, VEGF upregulation during hypoxia is also achieved by an increase in the stability of its mRNA⁵¹ and by the efficient hypoxic translation of the VEGF mRNA which is mediated by an internal ribosomal entry site.⁵²

HIF-1 is an ubiquitously expressed master regulator of oxygen homeostasis. It is a heterodimeric transcription factor that is composed of HIF-1α and HIF-1β (also known as the arylhydrocarbon receptor nuclear translocator ARNT) protein subunits. Whereas HIF-1β/ARNT is constitutively expressed, HIF-1α expression is induced in hypoxic cells. HIF-1 binds to the promoter/enhancer elements of hitherto more than 20 known hypoxia-inducible genes and stimulates their transcription. These genes play essential roles in the physiologic adaptation to hypoxia, including glycolysis, erythropoiesis, angiogenesis and vascular remodeling.⁵³ Furthermore, it became evident that HIF-1 plays a central role in many human diseases, including myocardial ischemia, stroke and tumor growth.⁵⁴ Recently, a close homologue of HIF-1α was identified and termed HIF-2α.^50,55–57 In addition, it has been shown that ARNT2, a conserved ARNT homologue that is highly expressed in neurons, forms functional HIF complexes in vivo, leading to the hypothesis that HIF-1α/ARNT2 heterodimers may specifically mediate transcriptional responses in the nervous system.⁵⁸

HIF-1α mRNA is constitutively expressed in virtually all organs and cells⁵⁹ implicating a regulation of HIF-1 activity at a posttranscriptional level.⁶⁰ HIF-1α protein levels are downregulated in normoxic cells by ubiquitination and rapid proteasomal degradation, a process that is mediated by the von Hippel-Lindau tumor suppressor protein (pVHL).^53,61 The binding of pVHL to the α subunit requires a specific, oxygen-dependent, prolyl hydroxylation within the degradation domain of HIF-1α (Fig. 3).^62,63 The involved prolyl-hydroxylase might therefore act as an oxygen sensor. As a result HIF-1α and also HIF-2α proteins are undetectable in normoxic tissues.⁶⁴ However, expression of HIF-1α increases dramatically in hypoxic tissues, including the brain (e.g., during chronic hypoxia or ischemia).^65–67

Figure 3

Model of oxygen sensing, signaling and gene regulation. Under hypoxic conditions, HIF-1α (α) is stable and can translocate to the nucleus where it heterodimerizes with HIF-1β/ARNT (β) and after binding to hypoxia-response (more...)

As a consequence of HIF-1 activation, VEGF transcription is increased during hypoxia.⁴⁹ VEGF expression is upregulated mainly in astrocytes and neurons during hypoxic and ischemic events,^34,67,68 but also in injured brain or spinal cord tissue.^69–71 In addition, gene expression of both VEGFR-1 and VEGFR-2 is activated in hypoxic tissues.^68,72,73 Whereas VEGFR-1 is a target gene for HIF-1 and is therefore directly upregulated by hypoxia,^34,74 VEGFR-2 is not induced after short term hypoxic exposure³⁴ but rather after prolonged periods of hypoxia, such as those occurring during ischemic events.^68,75 VEGFR-2 has no HIF-1 consensus site in its promoter region⁷⁴ but may be activated by HIF-2.⁷⁶ Increased VEGF levels may activate VEGFR-2 expression through a positive feedback loop indicating a further regulatory pathway.^77,78 The third receptor for VEGF, neuropilin-1, is upregulated in endothelial cells of cerebral blood vessels from hypoxic tissue after focal cerebral ischemia.⁷⁹ For the other members of the VEGF family, as well as for VEGFR-3, upregulation by hypoxia has not been demonstrated conclusively, and it is so far unknown whether hypoxia-response elements allowing transcriptional regulation by either HIF-1 or HIF-2 are present in these genes.^80,81

Pleiotropic Action of VEGF in the CNS

VEGF has long been considered a selective endothelial cell mitogen that promotes vasculogenesis and angiogenesis. In addition, it initiates endothelial procoagulant activity and induces vascular permeability. These effects are compatible with the expression of VEGF receptors specifically on vascular endothelial cells. However, other cell types, including hematopoietic progenitor cells, monocytes and trophoblasts also express functional VEGF receptors (for a review see ref. ²). More recently, additional cellular targets of VEGF were discovered in the CNS. Astrocytes and neurons have been reported to express either or both VEGFR-1 and VEGFR-2 during cerebral ischemia.⁸³ VEGFR-2 expression has also been described in neural progenitor cells of the retina,⁸⁴ and expression of VEGFR-1 was found on reactive astrocytes after VEGF stimulation,⁸⁵ and in other models involving CNS trauma.⁸⁶ These findings led to the hypothesis that VEGF might act as a direct neurotrophic or even neuroprotective factor. This concept is supported by the finding that other pro-angiogenic growth factors such as bFGF and PDGF have been shown to be neurotrophic as well.⁸⁷ Furthermore, the identification of the neuropilins as coreceptors for different isoforms of VEGF^37,38 implicates VEGF in the processes of neuronal growth and repair. Thus, VEGF may affect neuronal survival in a variety of pathological conditions indirectly by inducing angiogenesis and vascular permeability but also by a direct action on neurons and astrocytes.

Direct Neurotrophic/Neuroprotective Actions of VEGF

The first evidence for a neurotrophic function for VEGF came from a series of experiments performed in the laboratory of Jeffrey Rosenstein, using murine brain explant cultures in vitro. The study was initially designed to investigate angiogenic reactions in the CNS after VEGF administration. This group found an increase in enlarged, dilated and branching vessels after VEGF stimulation in rat cortical slice explant cultures but also expression of VEGFR-1 on reactive astrocytes.⁸⁵ Subsequently, in mesencephalic explant cultures they detected that VEGF administration led to the proliferation of astrocytes and also prevented dopaminergic neurons from cell death.⁸⁸ The significance of VEGF-mediated astrocyte proliferation is unknown. Recent studies support the assertion that astrocytes are actively involved in the generation and maintenance of neuronal synapses.⁸⁹ VEGF may sustain these effects on neurons by providing the necessary glial bed and thus lead to an indirect stabilization of neuronal function. This idea is supported by the finding that expression of both VEGFR-1 and VEGFR-2 in Müller cells of the developing retina is required for normal retinal development, from which it is surmised that the development of blood vessels and neural tissue takes place in a coordinated fashion, guided by the expression of VEGF receptors in glial cells.⁹⁰ In addition, direct effects of VEGF on neuronal cell lines and primary neurons were also reported, including the role of VEGF in promoting the development and survival of rat photoreceptor cells.⁹¹ Also, addition of VEGF to the immortalized hippocampal neuronal cell line HN33 resulted in reduced cell death that was associated with an in vitro model of cerebral ischemia by doubling the number of surviving cells after 24 hours of combined oxygen and glucose deprivation.⁹² Furthermore, VEGF protects rat primary embryonic hippocampal neurons against glutamate-induced neurotoxicity.⁹³ Six hours of pretreatment with VEGF before exposure to glutamate resulted in increased cell survival, inhibited upregulation of caspase-3-like activity and also attenuated DNA laddering. These results imply that VEGF is able to block neuronal apoptosis in specific cell lineages. Further evidence in support of this hypothesis comes from a recent in vivo study, where topical application of VEGF to the surface of an ischemic brain led to a significant reduction of infarct volume and neuronal damage some 24 hours after onset of experimentally induced stroke.⁹⁴ The positive effect of VEGF administration after 24 hours is supportive of a direct protective effect of VEGF on the neurons itself. An indirect neuroprotective effect mediated by inducing angiogenesis (see below) would need more time, as new vessel growth during cerebral ischemia is initiated only 24 to 48 hours after occlusion of the artery.⁶⁸ Alternatively, VEGF may improve survival of hypoxic endothelial cells in the penumbra surrounding the infarcted area, thus preserving a residual blood flow which then contributes to better neuronal survival.

In the peripheral nervous system, VEGF has also been implicated in neurotrophic and neuroprotective effects. VEGF is expressed by neurons of the superior cervical ganglia (SCG) and dorsal root ganglia (DRG), and VEGFR-2 is present on neurons within SCG and DRG, and also on Schwann cells.⁸⁷ In these different cell types, VEGF and VEGFR-2 expression demonstrates temporal changes during early postnatal life, from which it may be concluded that there is developmental regulation of VEGF activity in peripheral ganglia.⁹⁵ Addition of VEGF to explanted ganglia promoted cell survival, axonal outgrowth, and proliferation of Schwann cells.⁸⁷ Topical application of VEGF to injured peripheral nerves, therefore, could have a beneficial effect on nerve regeneration by promoting the invasion of Schwann cells and new vessel growth.^96,97 In addition, it has been shown that VEGF gene transfer in a rabbit ischemic hindlimb model, as well as in diabetic rats, had favorable effects on peripheral nerve function.^98,99 The role of VEGF, and especially its inducibility by hypoxia, on neuronal survival was very recently underlined by a fascinating study in the laboratory of Peter Carmeliet. Transgenic mice with a deletion of the hypoxia-response element in the promoter of the VEGF gene demonstrated a reduction of baseline levels of VEGF specifically in neural tissue, and hypoxia-induced VEGF expression in the spinal cord and the brain was blunted. At the age of five months, the transgenic animals developed a progressive motor neuron degeneration, which was reminiscent of human amyotrophic lateral sclerosis.¹⁰⁰

In the search for mechanisms that mediate the neuronal functions of VEGF, the interaction between VEGF and the neuropilins deserves attention. Neuropilins were initially identified according to their ability to bind secreted semaphorins of subclass 3.¹ However, this semaphorin-neuropilin complex is unable to transmit a signal to the cell because neuropilins lack a signaling-competent cytoplasmic domain. Therefore, the binding of an additional class of receptors, the plexins, is required to form a fully functional semaphorin receptor complex. Alternatively, neuropilins can use VEGFR-2 as a coreceptor by interacting with specific isoforms of VEGF. The presence of neuropilin-1 on endothelial cells enhances the affinity of VEGF₁₆₅ for its signaling receptor VEGFR-2, leading to increased endothelial cell chemotaxis and mitogenesis.³⁷ Furthermore, neuropilin-1 may also use the tyrosine kinase activity of VEGFR-1 for signaling after semaphorin binding.¹⁰¹ In nervous tissue, secreted semaphorins control cell migration by forming tissue gradients that are sensed by receptor complexes consisting of plexins and neuropilins.⁴ Semaphorins act as repelling cues for the guidance of axons in developing nervous tissue. In the adult organism, semaphorins seem to inhibit axon growth after injury and maintain established neural pathways. Secreted semaphorins and VEGF compete with each other for binding to their natural receptors neuropilin-1 and VEGFR-2, respectively. To further complicate the situation, it has been demonstrated that neuropilin-1 binds with high affinity to VEGFR-1, thereby inhibiting the binding of neuropilin-1 to VEGF₁₆₅.¹⁰² VEGFR-1 lacks a significant signaling activity and has therefore been considered to negatively regulate the VEGF-dependent activity by functioning as a sink receptor.² Based on these results, a number of scenarios emerge, as to how VEGF action on neuronal cells could be mediated (Fig. 4). 1) VEGF can bind to VEGFR-2 present on neurons and signal via the kinase domain of this receptor. An additional complex formation with neuropilin-1 could modulate or enhance this signal. 2) As VEGFR-1 can act as a sink receptor, preventing VEGF binding to the signaling VEGFR-2, the binding of neuropilin-1 to VEGFR-1 could block this sink function and thereby increase the availability of VEGF for VEGFR-2 leading to an increased activity via this receptor. 3) The neurotrophic action of VEGF could be an indirect one. As semaphorins inhibit axonal growth, VEGF could compete with semaphorins for neuropilin binding, thereby blocking the repellent function of semaphorins on neurons. This would result in an increased axonal outgrowth and better survival of neurons as previously demonstrated.^87,97,101 4) Regulated expression of VEGFR-1 could modulate the level of available neuropilin-1 and thus influence the affinity of VEGFR-2 for VEGF as well as signaling via the semaphorin-plexin complexes. The balance between VEGF and semaphorin availability at their common receptors VEGFR-1, VEGFR-2 and neuropilin-1 may therefore modulate the migration, apoptosis, survival and proliferation of neural cells.¹⁰¹ The situation may be even more complex, as it is currently unknown whether neuropilin-1 and VEGFR-2 are able to directly interact. Furthermore, neuropilin-2 and the plexins may interfere with the other receptors as well. On the other hand, it is evident that semaphorins by competing with VEGF for binding to neuropilin-1 could influence the angiogenesis reaction mediated by VEGF.³⁷

Figure 4

Hypothetical model of VEGF-mediated neuroprotection. Various VEGF receptors are expressed on glial, neuronal and endothelial cells. 1) VEGF signals through binding to VEGFR-2 on neurons. Additional complex formation with neuropilin-1 enhances this signal. (more...)

Intracellular Signaling Events

An important question in the field of neuroprotective aspects of VEGF is: through which intracellular signaling cascades are the various VEGF effects mediated? With regard to the possible therapeutic use of VEGF in the CNS, it would be helpful to know whether these several different VEGF-mediated effects use different signaling pathways which can be specifically activated (e.g., by low molecular compounds interfering with one pathway but not affecting the other). For example, it has been shown that distinct members of the Src kinase family (Src, Yes, Fyn) are required for different VEGF-mediated processes.¹¹⁷ Mice lacking Src or Yes, but not Fyn, showed no vascular permeability in response to VEGF administration, whereas induction of angiogenesis and endothelial cell survival required Src family kinase activity in general. In accordance with these results, blockage of Src, but not Fyn activity in mice provided cerebral protection following stroke, probably via a reduction in brain edema formation.¹¹⁸

In endothelial cells, VEGFR-2 is considered to be the major signaling receptor, while VEGFR-1 acts as a sink to trap an excess of VEGF. Endothelial proliferation is mediated via the Ras-Raf-MAP (mitogen-activated protein)-kinase pathway, while protein kinase C (PKC) activation is involved in endothelial migration and vascular permeability (for a review see ref. ²). The role of VEGF as a survival factor for endothelial cells is mediated by the phosphoinositol 3 (PI₃) kinase-AKT signaling pathway.¹¹⁹ This situation may be similar in neuronal cells. A recent study, using antisense oligonucleotides for VEGFR-1 or VEGFR-2, supported the hypothesis that VEGFR-2, but not VEGFR-1, mediate neuroprotective effects. However, involvement of neuropilins was not investigated in this study. Neuroprotection was associated with the activation of PI₃ kinase-AKT pathway as well as MEK/ERK signaling pathway.⁹³ In the setting of cerebral ischemia in vivo and in various in vitro models of neuronal death, the neuroprotective function of VEGF is associated with an activation of VEGFR-2 expression and increased levels of phosphorylated AKT, implicating the PI₃ kinase-AKT pathway.^73,92,120 Figure 5 summarizes the major signal transduction pathways that are induced by activated VEGFR-2 in endothelial cells and neurons.

Figure 5

Major signal transduction pathways induced by VEGF. In endothelial cells and neurons, VEGF signaling through activated VEGFR-2 involves the MAP-kinase pathway (MEK-ERK) as well as the PI₃ kinase-AKT pathway. In endothelial cells, activation of the MAP-kinase (more...)

Conclusion

The VEGF/VEGFR receptor system is activated in a variety of pathological conditions in the CNS, most prominent during cerebral ischemia. But also in brain injury and neurodegenerative diseases, such as Alzheimer's disease, involvement of VEGF is discussed. VEGF expression is markedly enhanced in Alzheimer's disease implicating compensatory mechanisms to counter insufficient vascularity or reduced perfusion apparent in this condition.¹²¹ VEGF might therefore be an important factor in the pathophysiology of diseases that are associated with tissue hypoxia and may has also an important role for their treatment. VEGF can exert its neuroprotective function by at least two mechanisms. VEGF acts directly on neurons expressing various VEGF receptor types, thereby mediating its neuroprotective effect. Alternatively, VEGF induces angiogenesis, thereby allowing increased oxygen and nutrient transportation to ischemic or hypoxic tissues, which ultimately leads to protection of stressed neurons and possibly other cell types in the area. VEGF or VEGF-like agents may therefore be useful for the treatment of neurodegenerative as well as ischemic disorders of the CNS. A major risk of this form of therapy is that VEGF may also damage neurons by its effect of increasing permeability leading to brain edema formation. This precludes VEGF therapy as an efficient treatment for ischemic conditions such as stroke because serious adverse side effects may occur. As indicated above, a potential stroke therapy could therefore consist of early VEGF inhibition followed by a period of active VEGF treatment. Alternatively, one might search for additional factors capable of inducing angiogenesis and increasing neuronal survival, but devoid of the harmful effects associated with edema formation. The hematopoietic growth factor erythropoietin might be such a factor. Erythropoietin is expressed in the CNS, is induced by hypoxia like VEGF and has a very similar pattern of expression and functional role during cerebral ischemia. Indeed, erythropoietin has been shown to be neuroprotective as well as angiogenic in the brain,¹¹² but does not seem to induce vascular permeability. Thus, therapeutic application of erythropoietin, which has been widely used as human therapeutic in the clinic, might be a more promising therapeutic approach. An alternative to the treatment of ischemic brain injury by application of VEGF alone, might be the combination of different angiogenic and neuroprotective growth factors. The combined application of VEGF with angiopoietins has been shown to protect vessels from VEGF-induced vascular leakage.¹²² However, the use of VEGF to treat patients with diseases of the CNS may lead to the induced growth of dormant tumors or facilitate atherosclerotic plaque progression.¹²³ Thus, much more research is needed to establish the role of VEGF as neuroprotective agent, and whether the concept of therapeutic angiogenesis using VEGF is applicable as therapy for the treatment of neurodegenerative or ischemic diseases such as Alzheimer's disease or stroke. Furthermore, the potential damage to nervous tissue which may be caused by blocking an essential survival signal for neuronal cells mediated through the VEGF pathway should be avoided under any circumstances. The rapidly increasing number of reports demonstrating neurotrophic or neuroprotective functions of VEGF therefore warrants a careful re-evaluation of all antiangiogenic strategies (e.g., for treatment of solid tumor growth).

Acknowledgments

I gratefully acknowledge Sabine Raab and Marcel Groot for helpful discussion and Christopher Mitchell and Friedemann Kiefer for critically reading the manuscript. I was supported by a fellowship from the Max Planck Society.

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