Vascular endothelial growth factors (VEGFs) and stroke

Abstract

Vascular endothelial growth factors (VEGFs) have been shown to participate in atherosclerosis, arteriogenesis, cerebral edema, neuroprotection, neurogenesis, angiogenesis, postischemic brain and vessel repair, and the effects of transplanted stem cells in experimental stroke. Most of these actions involve VEGF-A and the VEGFR-2 receptor, but VEGF-B, placental growth factor, and VEGFR-1 have been implicated in some cases as well. VEGF signaling pathways represent important potential targets for the acute and chronic treatment of stroke.

Introduction

Vascular endothelial growth factors (VEGFs, including VEGF-A, VEGF-B, and placental growth factor, or PlGF) have important roles in the development and function of the circulatory and nervous systems, so it should not be surprising to find them involved in stroke, which occurs at the interface of these systems. VEGFs have been implicated in all phases of vascular, including neurovascular, development: vasculogenesis, or the de novo production of blood vessels from mesenchymal precursor cells [1]; angiogenesis, or the hypoxia-driven sprouting of new capillaries from existing vessels [2]; and arteriogenesis, or the enlargement of anastomotic arteriolar channels in response to blood-pressure gradients [3]. In addition, VEGFs exert direct trophic and protective effects on neurons [4], so that both their vascular and neuronal actions are relevant to stroke.

This review will consider the induction of VEGFs by cerebral ischemia and their role in atherosclerosis, collateral cerebral circulation, cerebral edema, neuroprotection, neurogenesis, cerebral angiogenesis, postischemic brain repair, postischemic vascular repair, and stroke therapeutics.

Atherosclerosis

Stroke results from focal cerebral ischemia or, less commonly, hemorrhage. The causes of focal cerebral ischemia include thrombosis of large or small blood vessels, usually arteries, and artery-to-artery or cardiogenic embolus. Among these processes, VEGF-A has been implicated most clearly in arterial thrombosis due to atherosclerosis (Fig. 1).

Fig. 1.

VEGF-A and atherosclerosis. Hypoxia and inflammation in the atherosclerotic plaque trigger VEGF-A expression in vascular smooth muscle cells (VSMC) and macrophages. VEGF-A, in turn, acts on vasa vasorum to promote angiogenesis, which may be associated with hemorrhage, and promotes migration of VSMC from the tunica media of the vessel wall into the plaque

Atherosclerosis is a complex inflammatory and degenerative disorder that affects primarily large and medium-sized arteries, especially at branch points. Atherosclerotic plaques cause clinical ischemic syndromes such a stroke when they rupture, releasing thrombogenic and embolic material, or when they directly occlude an artery. Plaques are subject to at least two processes associated with enhanced expression of VEGF-A—hypoxia and inflammation [5]. These increase levels of hypoxia-inducible factor-1 and other transcription factors associated with VEGF-A expression in plaque macrophages and vascular smooth muscle cells. VEGF-A acts on vasa vasorum (“vessels of the vessels”) of atherosclerotic arteries to promote angiogenesis, which in some cases leads to intraplaque hemorrhage and plaque rupture, although whether hemorrhage causes rupture is unresolved. However, increased VEGF-A expression, angiogenesis, and intraplaque hemorrhage have been observed in carotid endarterectomy specimens from symptomatic compared to asymptomatic patients [6].

VEGF-A may also contribute to atherogenesis by stimulating the migration of vascular smooth muscle cells, which has been attributed to its activation of phosphatidylinositol 3-kinase (PI3 K) and extracellular signal-related kinase (ERK) 1/2 [7]. VEGF-B has been implicated in the regulation of fatty acid uptake into endothelial cells [8], which could also influence atherogenesis.

The role of VEGFs in atherosclerosis has been tested in a variety of animal models. In low-density lipoprotein receptor-knockout mice fed an atherogenic diet, who develop hyperlipidemia and atherosclerosis, vaccination against VEGF receptor 2 (VEGFR-2/Flk-1) reduced the size and microvessel density of aortic atherosclerotic lesions [9]. In another study, administration of a plasmid vector coding for the decoy receptor, soluble VEGF receptor 1 (VEGFR-1/Flt-1), to rabbits given a high-lipid diet and subjected to balloon-catheter injury of the iliac artery decreased plaque size and neovascularity [10].

Collateral circulation

Collateral vessels constitute the first line of defense against tissue ischemia, by providing alternative pathways for arterial blood flow. Flow through preexisting collaterals is activated by blood pressure gradients between patent and occluded arteries, and is established almost instantaneously upon occlusion. Sources of collateral blood supply in the cerebral circulation include both extracranial and intracranial vessels, and the adequacy of collateral circulation helps to determine the severity of stroke and response to treatment [11].

The channels through which blood flow is redirected in response to focal hypoperfusion develop through a process termed arteriogenesis, in which arterioles are remodeled and enlarged to accommodate increased flow [3]. In rats, this occurs over days to weeks following middle cerebral artery occlusion leading to infarction [12, 13]. The molecular mechanisms underlying cerebral arteriogenesis are poorly understood, but in a study in rats, granulocyte-macrophage colony-stimulating factor potentiated enlargement of the ipsilateral posterior cerebral artery following occlusion of the middle cerebral artery (MCA) and both vertebral arteries [14].

A study of myocardial ischemia in rats showed that administration of an anti-VEGF neutralizing antibody, which had no acute effect on coronary blood flow, inhibited the growth of collateral coronary vessels [15]. This observation was extended to the cerebral circulation in a study involving VEGF-A hypermorphic, hypomorphic, and wild-type mice. Counting of pial collaterals showed a correlation with VEGF-A expression, and VEGF-A hypermorphs had smaller cerebral infarcts after MCA occlusion [16]. It seems likely, therefore, that VEGF-A helps to mediate the developmental and postischemic growth of collateral vessels in the brain, but not the acute increase in collateral blood flow triggered by cerebral ischemia.

Ischemic induction

Several studies have documented effects of cerebral ischemia on the expression of VEGFs and VEGF receptors, primarily in the ischemic border zone, or penumbra. This region, which surrounds the infarct core, remains salvageable pending reperfusion, and clinical outcome from stroke is strongly influenced by the fate of the penumbra.

VEGF-A protein expression increased in neurons, astrocytes and macrophages, and VEGFR-1 protein expression increased in endothelial cells, over days to weeks following MCA occlusion in rats [17]. VEGF-A mRNA and protein also increased within hours of MCA occlusion in rats, with subsequent rapid decline in neurons and more sustained expression in pial cells [18]. A comparison of transient and permanent MCA occlusion in rats showed elevations of VEGF-A (in neurons and endothelial cells), VEGFR-1 (in neurons, endothelial cells, and astrocytes), and VEGFR-2 (in endothelial cells and astrocytes), which were detectable at 1–3 days, and were generally more prominent in the ipsilateral hemisphere and after permanent MCA occlusion [19]. VEGF-A mRNA and protein were increased in another rat MCA occlusion study, with predominant expression in astrocytes [20], whereas others reported that microglia/macrophages were the main site of VEGF-A mRNA and protein expression [21]. VEGF-A protein levels and expression in neurons also rose within the first 24 h after MCA occlusion in a neonatal rat model of perinatal hypoxic-ischemic injury [22]. A photothrombotic ring model of stroke in rats revealed upregulation of not only VEGF-A, VEGFR-1, and VEGFR-2, but also of VEGF-C and VEGFR-3/Flt-4 proteins [23].

A different rat model—transient forebrain or “global” cerebral ischemia—which is often likened to hypoxic-ischemic encephalopathy following cardiac arrest, has also been used to assess the effects of ischemia on VEGF signaling. In this model, which affects the hippocampus most strikingly, VEGF-A mRNA was induced within hours in neurons and within days in astrocytes [24]. Other studies showed increased expression of VEGF-A mRNA and protein [25] and VEGFR-1 and VEGFR-2 protein [26] in hippocampal and cortical neurons over hours to days post-ischemia.

Cerebral edema

VEGF-A was identified originally based on two biological effects—angiogenesis [27] and vascular permeability [28]. The latter is associated with tissue edema which, in a closed compartment like the skull, can be lethal. For this reason, and because brain edema is a well-documented and often fatal complication of stroke, the ability of VEGF-A to cause leakage from cerebral blood vessels has received considerable attention. The mechanisms involved are thought to include transendothelial transport of small solutes via cytoplasmic fenestrations and plasmalemmal caveolae, as well as leakage of fluid and plasma proteins and extravasation of blood cells through interendothelial tight junctions [29].

A role for VEGFs in brain edema related to stroke was first shown in mice subjected to transient MCA occlusion and treated with a VEGFR-1 fusion protein that served as a VEGF-sequestering decoy receptor [30]. Compared to untreated mice, these animals showed reduced edema volume and infarct size. In a complementary study, intravenous administration of VEGF-A to rats 1 h (but not 48 h) after MCA occlusion increased extravasation of an intravenously administered contrast agent, as measured by magnetic resonance imaging, and caused an increase in infarct size [31]. Subsequent reports documented the ability to counteract the vascular permeability effect of VEGF-A in rodent models of stroke with an anti-VEGF-A neutralizing antibody [32] or the growth factor angiopoietin-1 [33].

Neuroprotection

Although VEGF-A was identified originally based on its effects on endothelium, it has since been recognized to act on several other cell types, including neurons [34]. Neurotrophic effects of VEGF-A have been described in a variety of peripheral [35, 36] and central [37–42] neuronal preparations. VEGF-A promotes neuronal survival in cell culture models of stroke involving oxygen and glucose deprivation [43] or excitotoxicity [44, 45], and has been implicated in some forms of hypoxic preconditioning in vitro [46]. Most of these direct neuronal effects of VEGF-A have been ascribed to activation of VEGFR-2, PI3 K, and ERK1/2. Another VEGF family member, PlGF, which interacts with VEGFR-1 but not VEGFR-2, was reported to be neuroprotective in a cell culture model of oxygen and glucose deprivation in one [47] but not another [43] study; however, in the former case, neuronal survival increased by only about 10 %.

Protection by VEGF-A has also been demonstrated in MCA occlusion models of stroke in vivo (Fig. 2). Topical application of VEGF-A to the cortical surface reduced [48], whereas intraventricular infusion of an anti-VEGF antibody increased [49] infarct volume in rats, suggesting a beneficial effect. In contrast, as noted above in connection with cerebral edema, effectively reducing VEGF levels by administration of a VEGF-sequestering VEGFR-1 fusion protein reduced infarct size in mice, although this seemed to be due to increased edema rather than a direct adverse effect on ischemic brain tissue [30]. As also noted above, one study described a temporally biphasic effect of intravenous VEGF-A on infarct volume in rats, with anatomic worsening when administered 1 h post-ischemia, but improved neurobehavioral function when given at 48 h [31]. Intraventricular administration of VEGF-A to rats beginning 24 h after MCA occlusion and continuing for 3 days, reduced infarct volume by approximately one-third at 1-month post-stroke, and also rescued sensorimotor and cognitive deficits, with behavioral improvement persisting for at least 2 months [50, 51]. Infarct volume was reduced to a similar extent and neurological deficits improved after MCA occlusion in VEGF-A-overexpressing transgenic mice compared to controls [52]. One important lesson from these studies is that the timing and route of VEGF-A administration are critical for achieving a desirable result [53].

Fig. 2.

VEGF effects in acute ischemic stroke. A horizontal section of the human brain shows an acute infarct within the territory of the middle cerebral artery (dotted line). VEGF-A is induced in the ischemic border zone and acts on local neurons and endothelial cells to promote neuroprotection (green arrow) and angiogenesis (red arrow). VEGF-A also stimulates neurogenesis (blue arrow) in the subventricular zone, from which new neurons migrate to the site of ischemia. Other VEGF family members, including VEGF-B and PlGF, share some of these effects

Although cerebral ischemia is most common in adults, especially the elderly, it is also an important cause of neurological disability and death in the neonatal period. Accordingly, several studies have addressed the potential for protection by VEGF-A in neonatal rat models of hypoxic-ischemic injury. Intraventricular VEGF-A, given to 7-day-old rats after unilateral common carotid artery occlusion coupled with hypoxia, reduced macroscopic and microscopic brain injury at 24 h [54]. Intracerebral administration of a VEGF-A-expressing adenoviral vector 3 days after a similar injury produced like benefit as well as neurobehavioral improvement [55]. Conversely, histological brain damage was exacerbated in 10-day-old rats subjected to transient MCA occlusion and treated for 3 days, beginning 2 days post-ischemia, with the VEGR2 tyrosine kinase inhibitor semaxanib (SU5416) [56].

The possibility that other VEGF family members have protective effects in stroke has received limited attention. However, MCA occlusion in VEGF-B-knockout mice was associated with an increase of about 40 % in infarct size and more severe neurological dysfunction compared to findings in wild-type mice [57]. Like PlGF, VEGF-B activates VEGFR-1 but not VEGFR-2 receptors. Thus, agents acting at either (or both) of these receptor subtypes on neurons might have therapeutic value in stroke. Moreover, VEGF-B [58] and PlGF [59] appear to be much less potent inducers of vascular permeability than VEGF-A, which could be advantageous in limiting stroke-related brain edema.

Neurogenesis

Neurogenesis occurs in the adult as well as the developing mammalian brain, especially in selected regions, among which the best documented are the hippocampal dentate gyrus (DG) and the zone surrounding the lateral ventricles (subventricular zone, or SVZ). Adult neurogenesis is regulated by chemical effectors such as neurotransmitters, hormones, and growth factors; by behavior; and by pathological processes. Of interest in the present context, the former include VEGFs and the latter stroke.

Stroke-induced neurogenesis (Fig. 2) has been demonstrated in a variety of rodent models [60–64] and in humans [65, 66]. An increase in neuroproliferation above physiological baseline levels is observed in both DG and SVZ, and is accompanied by migration of newborn neurons from the latter site toward the ischemic lesion [63–68]. Stroke-induced neurogenesis appears to contribute to improved anatomic and functional outcome from stroke, because postischemic outcome is worse when neurogenesis is ablated by radiation [69], cytotoxic drugs [63, 68], or genetic manipulation [70].

VEGF-A stimulated neurogenesis in embryonic rat brain cultures, as demonstrated by an increase in the number of cells co-expressing markers of cell proliferation (bromodeoxyuridine, or BrdU, labeling) and neuronal lineage (embryonic nerve cell adhesion molecule, or ENCAM) [71]. This was blocked by the VEGR-2 tyrosine kinase inhibitor SU1498, consistent with a VEGFR-2-mediated effect. Intraventricular administration of VEGF-A to rats also enhanced incorporation of BrdU into cells expressing VEGFR-2 and the neuronal lineage marker doublecortin (Dcx) in DG and SVZ in vivo. In a subsequent study [50], intraventricular administration of VEGF-A for 3 days, starting 24 h after MCA occlusion in rats, also increased postischemic BrdU labeling in neuronal (Dcx- or NeuroD-expressing) cells in DG and SVZ. Moreover, stimulation of VEGF expression has been implicated in the neurogenesis-promoting effects of therapeutic agents, such as statins [72] and antidepressants [73]. Thus, VEGF-A enhances neurogenesis not only in normal, but also in ischemic, brain, representing another way in which VEGF-A may contribute to cerebral adaptation to stroke.

VEGF-B also appears to have a role in neurogenesis [74]. Like VEGF-A, VEGF-B also increased BrdU labeling in embryonic rat brain cortical cultures, involving cells expressing the neuroepithelial cell marker nestin. Intraventricular VEGF-B also stimulated BrdU incorporation in DG and SVZ, where BrdU co-localized with the neuronal lineage marker Dcx. In addition, BrdU labeling and Dcx expression were reduced in DG and SVZ from VEGF-B-knockout mice.

Angiogenesis

VEGF-A is a principal mediator of cerebral angiogenesis [75], which is increased following stroke (Fig. 2) in rodents [76] and humans [77, 78]. Several studies have demonstrated a temporal or spatial correlation between upregulation of VEGF-A or VEGF receptors and angiogenesis following MCA occlusion in rats [17, 79, 80]. In addition, administration of VEGF-A has been shown to enhance postischemic angiogenesis. For example, intravenous VEGF-A increased microvessel density in the cortical ischemia penumbra [31] and intraventricular VEGF increased the number of von Willebrand factor-immunoreactive endothelial cells in the ischemic caudate-putamen after MCA occlusion in rats [50]. VEGF-A also appears to mediate NO-induced angiogenesis in the postischemic brain, because the NO donor, DETANONate, increased new vessel formation in the ischemic penumbra when given 24 h after MCA occlusion in rats, and an anti-VEGFR-2 neutralizing antibody inhibited capillary-like tube formation stimulated by DETANONOate in vitro [81].

Indirect evidence suggests that PlGF might also affect stroke-related angiogenesis, because PlGF-knockout mice showed a deficit in hypoxia-induced cerebral angiogenesis compared to wild-type mice [82]. However, VEGF-A levels were also lower in PlGF-knockout mice, so VEGF-A deficiency, rather than PlGF deficiency, might explain this finding.

Postischemic brain repair

Brain repair and functional recovery following stroke depend partly on brain plasticity in non-ischemic regions, such as the peri-infarct cortex and contralateral (non-ischemic) cerebral hemisphere [83, 84]. Manifestations of plasticity-related repair include changes in gene expression [85], neuronal excitability [86], axon sprouting [87], synapse formation [87], somatotopic organization [88], and intra-cortical neuronal connectivity [89, 90]. Evidence for a role of VEGF signaling in remote plasticity (Fig. 3) includes the observations that occlusion of cortical vessels supplying the primary motor (M1 hand) cortex in squirrel monkeys increased neuronal VEGF-A [91] and VEGFR-1 [92] expression in both peri-infarct cortex and remote brain regions (PMv hand and M1 hindlimb) thought to be involved in stroke recovery.

Fig. 3.

VEGF-A-mediated postischemic repair. VEGF-A appears to be a mediator of postischemic brain and vessel repair. Following cerebral infarction (blue) due to arterial occlusion (asterisk), VEGF-A expression is increased (thick arrow, top left) in nonischemic brain regions that exhibit remote plasticity (pink); VEGF-A acting here (thin arrow, top left) may help to restore brain function. Increased serum VEGF-A levels after stroke (thick arrow, bottom right) also mobilize endothelial progenitor cells (EPCs, green) from the bone marrow, enhancing repair of ischemia-damaged vessels (thin arrows, bottom right)

Postischemic vascular repair

Circulating endothelial progenitor cells (EPCs) appear to have an important role in repairing systemic blood vessels after ischemic injury [93] and VEGF-A has been implicated in mobilizing circulating EPCs in response to ischemia [94, 95] (Fig. 3). This effect is likely mediated through both VEGFR-1 and VEGFR-2 receptors, because both receptors are expressed on EPCs, antibodies against either receptor inhibit EPC mobilization or assembly into new vessels, and both VEGF-A and the VEGFR-1-selective agonist PlGF are effective [96]. Regarding the cerebral circulation, low levels of circulating EPCs are reported to correlate with increased stroke risk [97], whereas higher levels of circulating EPCs within the first week after stroke were associated with better functional outcome at 3 months [98]. In addition, serum VEGF-A levels 24 h post-stroke correlated with the growth of EPC colonies from venous blood mononuclear cells in vitro [99].

Therapeutics

In addition to the studies cited above documenting acute neuroprotective effects of VEGFs in experimental stroke [31, 48, 50, 51, 54, 55, 57], other reports have described longer-term, VEGF-mediated beneficial effects of stem cell treatment. Intravenous administration of human neural stem cells (NSCs) together with VEGF-A, beginning 24–48 h after MCA occlusion in rats, was more effective than NSCs or VEGF-A alone in reducing brain atrophy and improving behavioral recovery at 7–28 days [100]. VEGF-A-overexpressing transfected NSCs derived from fetal rat brain, transplanted into the ischemic penumbra 3 days after MCAO in rats, decreased the severity of neurological deficits to a greater extent than control NSCs at 8–12 weeks [101]. A VEGF-A-transfected human NSC clone, transplanted into ipsilesional cerebral cortex 7 days after intracerebral hemorrhage in mice, also produced better behavioral outcome at 1–9 weeks than an untransfected clone [102]. In another study, human mesenchymal stem cells transfected with a PlGF-expressing adenoviral vector, administered intravenously 3 days after MCAO in rats, were superior to cells transfected with a control vector in reducing infarct volume and behavioral impairment at 3–7 days [103].

Perhaps the most interesting studies of the role of VEGFs in neurotransplantation for experimental stroke have involved cells that have not been modified to overexpress VEGFs, but which nevertheless elicit VEGF-A-mediated improvement. In one instance, intrastriatal delivery of mouse embryonic NSCs 3 days prior to MCA occlusion in mice decreased the number of TUNEL-positive nuclei and the loss of NeuN-immunopositive neurons in the peri-infarct region; in vitro studies suggested that the neuroprotective effect of transplanted NSCs might be mediated through VEGFR-2, because it was inhibited by the VEGFR-2 tyrosine kinase receptor inhibitor SU1498 [104]. In another study, this possibility was tested directly in vivo. Human NSCs transplanted into the ipsilesional cerebral cortex 7 days after MCA occlusion in rats reduced infarct size and enhanced behavioral recovery at 2–5 weeks, and these effects were blocked by the humanized monoclonal anti-VEGF-A antibody bevacizumab [105]. Thus, at least some of the beneficial effects of neurotransplantation for stroke may be due to release of VEGF-A from transplanted cells.

Conclusions

VEGFs have a broad array of effects related to stroke. They have been implicated in the pathogenesis of atherosclerosis, a common cause of stroke, and also participate in the process of arteriogenesis underlying the formation of collateral vessels, which can protect ischemic brain from infarction and promote recanalization of occluded cerebral arteries. VEGF-A expression is induced in the ischemic penumbra during stroke and at remote sites involved in post-ischemic brain repair, and VEGFs expressed in this setting have acute neuroprotective, neurogenic, and angiogenic actions. Administration of VEGF-A or VEGF-B, or of VEGF-A together with stem cells, has therapeutic effects in animal models of stroke, and the release of VEGF-A from transplanted stem cells may be responsible for their therapeutic actions. Notwithstanding potential adverse effects of some VEGFs, including their propensity to induce vascular permeability and associated brain edema, these factors may prove to have applications in the acute or chronic treatment of stroke.