In the past decade, there has been a surge of interest in vascular growth factors. During development, these factors have potent effects on endothelial cells, and are thought to regulate proliferation, migration, endothelial tube formation, vascular differentiation, permeability, and regression (for reviews see 29
). Although much still remains to be understood regarding the effects of these factors on adult vasculature, current data suggest that they play similar roles in the changes that occur in both normal and pathological states (for review see 30
). Cerebral ischemia is one such pathological state in which vascular changes are striking. During and after cerebral ischemia, alterations in the cerebral vasculature include blood-brain barrier breakdown, endothelial cell apoptosis, upregulation of adhesion molecules, and angiogenesis (the development of new blood vessels from existing blood vessels).1,2,3,4,5,6,7,31,32,33,34,35,36,37,38,39
Vascular abnormalities, such as blood-brain barrier breakdown, have been observed after seizures also. Because these vascular alterations might contribute to the brain pathology observed after ischemia or seizures, it is important to understand how changes in the levels of various vascular growth factors might contribute to these pathologies.
There are a large number of protein factors that act on vasculature including, but not limited to, fibroblast growth factor, platelet-derived growth factor, transforming growth factor, hepatocyte growth factor (scatter factor), vascular endothelial growth factor (VEGF), and the angiopoietins. Proteins in the VEGF family of factors have potent vascular effects, and will be the topic of this chapter. These factors modulate the structure and function of vasculature in both developing and adult organisms.
VEGF was originally described as vascular permeability factor (VPF) because of its potent permeabilizing effects on endothelium.40
Since the discovery of VEGF, four additional VEGF-like family members have been described. These additional VEGF-like proteins are placental growth factor (PlGF), VEGFB, VEGFC, and VEGFD (the original VEGF has been termed “VEGFA”) (for review see 41
; refer to ). The receptors currently described for the VEGF family are VEGFR1 (Flt-1), VEGFR2 (Flk-1 or KDR), VEGFR3 (Flt-4), and the neuropilins (see ).41,42,43,44
The primary receptors for VEGFA, VEGFR1 and VEGFR2, are localized predominantly to the vascular endothelium, including cerebral endothelium. However, several recent papers have reported neuronal localization of VEGFR2 in cultured hippocampal or dorsal root ganglion cells.45,46
In addition, neurons located in peri-infarct regions after focal cerebral ischemia or in VEGF-treated brain express VEGFR2.47,48
It is possible that VEGFR2, while not normally detectable in resting neurons, is upregulated during neuronal perturbation. One could argue that cultured neurons are in some way “perturbed,” having been removed from their normal neural microenvironment. VEGFR2 has also been described on glial cells, particularly after cerebral ischemia.49
VEGFR1 has been localized almost exclusively to vascular endothelium, but has been described on circulating inflammatory cells and VEGF-treated astroglia.48,50
In addition to VEGFR1 and VEGFR2, members of the neuropilin receptor family bind to VEGFA. Although neuropilin can be found on vascular endothelium, it is most densely expressed in nonendothelial cells, especially in the nervous system.51,52
Schematic illustrating the VEGF family of proteins and their receptors.
There are 5 known isoforms of VEGFA in humans which are termed VEGF 121, 145, 165, 189 and 206, corresponding to the number of amino acids found in each isoform (each isoform has 1 amino acid fewer in rodents).53
While all isoforms of VEGFA bind with high affinity to both VEGFR1 and VEGFR2, binding of VEGFA to the neuropilins is isoform-specific (e.g., the 165 amino acid isoform of VEGFA binds to neuropilin-1, while the 121 amino acid isoform does not).44
In addition, the various VEGF family members have different receptor specificities (e.g., VEGFA binds to VEGFR1 and 2, but PlGF only binds to VEGFR1; (); for review see 30
). There is still much to be learned about the roles of the diverse members of the VEGF family of proteins, as well as of the various VEGFA isoforms. Because more is currently known about VEGFA than its protein relatives, the remainder of this chapter will be devoted to discussions of VEGFA (hereafter referred to simply as “VEGF”).
In recent years, most of the in vivo research on VEGF has focused on its role as a potent angiogenic factor, responsible for the development of new vascular sprouts (for reviews see 54
). Gene deletion studies have shown that VEGF is critical for the development of new blood vessels during development. Mutant mice lacking even a single VEGF allele die during embryonic development with a striking lack of secondary vasculature (i.e., deficient angiogenesis).56,57
Both VEGFR1and VEGFR2 null mutants are early embryonic lethal and show a lack of vasculature (i.e., deficient vasculogenesis).58,59
In adult animals, application of recombinant VEGF protein induces the formation of new blood vessels from preexisting blood vessels (angiogenesis) in a variety of tissues, including brain.48,60,61,62,63,64,65
However, the blood vessels formed by application of VEGF to adult tissues are grossly abnormal, characterized by profound permeability and a disorganized, dilated, tortuous morphology.48,63,64,65,66,67
Because of the leaky nature of the new blood vessels formed by exogenous administration of VEGF, increasing attention has been given to VEGF's originally-described function as a vascular permeabilizing agent. Application of VEGF to adult tissues or cells results in edema and vascular leak. VEGF results in vascular leak in every tissue to which it has been applied including, but not limited to, brain, lung, testis, bladder, skin, duodenum, mesentery, and intestine.65,66,68,69,70,71
VEGF's effects on vascular leak in the brain occur rapidly, within 30 minutes of exposure to VEGF.68
In ischemic brain, the timing of VEGF mRNA and protein upregulation corresponds closely to the peak of vasogenic edema.35,47,49,72,73,74,75,76,77,78,79
VEGF expression is increased in both glia and neurons of the ischemic brain, as determined by both immunostaining and in situ hybridization.47,49,75,76
Presumably, the VEGF is secreted by the neurons and glia, and binds to VEGF receptors on local vasculature to mediate the increases in vascular permeability. Upregulation of VEGF mRNA occurs both in vivo and in vitro under hypoxic conditions.36,80,81,82,83
Hypoxia-induced upregulation of VEGF mRNA is associated with increases in the transcription factor hypoxia inducible factor (HIF)1-α, which is also upregulated after cerebral ischemia.36,80,81,82,84
Interestingly, upregulation of VEGF mRNA after cerebral ischemia sometimes occurs in cells not directly affected by the ischemic insult, such as the cingulate cortex and hippocampus, suggesting that triggers other than hypoxia could lead to VEGF upregulation.47
Possible secondary mechanisms of VEGF upregulation could include damage to neuronal afferents or efferents, pressure effects of edema, or ischemia-related physiological phenomena such as cortical spreading depression (for review see 85
). Cortical spreading depression has been shown to induce increases in other cytokines.86
Because some phenomena observed during cerebral ischemia can also be observed in association with seizures, we hypothesized that VEGF might increase in neural cells after seizures. Specifically, post-ischemic cortical spreading depression follows increases in synchronous neuronal firing such as those observed during seizures. Additionally, prolonged seizures can lead to hypoxic states due to breathing compromise during tonus, and hypoxia is the most consistently confirmed trigger of VEGF upregulation.