Recent developments in our understanding of the pathophysiological events that follow acute ischaemic stroke suggest an important role for angiogenesis which, through new blood vessel formation, results in improved collateral circulation and may impact on the medium-to-long term recovery of patients. Future treatment regimens may focus on optimization of this process in the ischaemic boundary zones or ‘penumbra’ region adjacent to the infarct, where partially affected neurons exposed to intermediate perfusion levels have the capability of survival if perfusion is maintained or normalized. In this review, we present evidence that angiogenesis is a key feature of ischaemic stroke recovery and neuronal post-stroke re-organization, examine the signalling mechanisms through which it occurs, and describe the therapeutic potential of treatments aimed at stimulating revascularization and neuroprotection after stroke.

ANGIOGENESIS AFTER STROKE: EVIDENCE FOR ITS OCCURRENCE AND IMPORTANCE

Folkman introduced the concept of angiogenesis as a necessity for tumour growth in 1971 [1]. Its importance in other pathological conditions, including rheumatoid arthritis, diabetes, myocardial infarction and stroke, was soon realized. Krupinski et al. [2,3] demonstrated for the first time that capillary density was increased around infarcts in post-mortem brains of patients who had survived acute ischaemic stroke for up to several weeks (Figure 1). Significantly, capillary density could be correlated with time of survival after stroke. The same group has described the revascularization process after MCAO [MCA (middle cerebral artery) occlusion] in a rat model using brain vascular casts [4]. The data suggested that new blood vessels, initiated through vascular buds, formed regular connections with intact microvessels within 1 week of ischaemia, the patterns being similar to those seen in the normal brain (Figure 2). It was also shown by this group [4] and others [5] that apoptosis within damaged ECs (endothelial cells) may be necessary to regulate the process, and that arteriolar collateral growth and new capillaries restored perfusion in the ischaemic border after ministroke [6] and in the cortical region after photothrombotic ring stroke in rats [7].

Increased microvessel density seen in the peri-infarcted tissue of a patient who survived for 1 week after stroke (A), and correlation of microvessel density with patient survival after acute ischaemic stroke (B)

Figure 1
Increased microvessel density seen in the peri-infarcted tissue of a patient who survived for 1 week after stroke (A), and correlation of microvessel density with patient survival after acute ischaemic stroke (B)

(Ai) Tissue from the contralateral hemisphere, with the box and arrow indicating the presence of a CD105 negatively stained microvessel. (Aii) Peri-infarcted tissue was stained with anti-CD105, which recognizes proliferating or active ECs, and counterstained with haematoxylin (×100; arrows point to positively stained microvessels). (B) Percentage increase in microvessels was measured against that of the contralateral hemisphere. Figures 1Aii and 1B are reproduced from Krupinski, J., Kaluza, J., Kumar, P., Kumar, S. and Wang, J.M., Role of angiogenesis in patients with cerebral ischaemic stroke, Stroke 25, 1794–1798, with permission. © (1994) Lippincott, Williams & Wilkins (http://lww.com).

Figure 1
Increased microvessel density seen in the peri-infarcted tissue of a patient who survived for 1 week after stroke (A), and correlation of microvessel density with patient survival after acute ischaemic stroke (B)

(Ai) Tissue from the contralateral hemisphere, with the box and arrow indicating the presence of a CD105 negatively stained microvessel. (Aii) Peri-infarcted tissue was stained with anti-CD105, which recognizes proliferating or active ECs, and counterstained with haematoxylin (×100; arrows point to positively stained microvessels). (B) Percentage increase in microvessels was measured against that of the contralateral hemisphere. Figures 1Aii and 1B are reproduced from Krupinski, J., Kaluza, J., Kumar, P., Kumar, S. and Wang, J.M., Role of angiogenesis in patients with cerebral ischaemic stroke, Stroke 25, 1794–1798, with permission. © (1994) Lippincott, Williams & Wilkins (http://lww.com).

Scanning electron microscopy showing vascular casts from normal rat brain and following MCAO in a rat model of stroke

Figure 2
Scanning electron microscopy showing vascular casts from normal rat brain and following MCAO in a rat model of stroke

(A and B) Leptomeningeal (large arrows) and small penetrating arterioles (small arrows) in normal brain. (C) Areas of infarction where no blood vessels are visible (arrow), and (D) stressed microvessels 24 h after MCAO. (E and F) Vascular buds were visible 3 days after MCAO (arrows), and (G and H) connections or ‘nests’ of small microvessels associating with surrounding vessels (arrows). Size is shown in the inserted bars. This Figure is reproduced from Krupinski, J., Stroemer, P., Slevin, M., Marti, E., Kumar, P. and Rubio, F., Three dimensional structure of newly formed blood vessels after focal cerebral ischaemia in rat, NeuroReport 14 (8), 1171–1176, with permission. © (2003) Lippincott, Williams & Wilkins.

Figure 2
Scanning electron microscopy showing vascular casts from normal rat brain and following MCAO in a rat model of stroke

(A and B) Leptomeningeal (large arrows) and small penetrating arterioles (small arrows) in normal brain. (C) Areas of infarction where no blood vessels are visible (arrow), and (D) stressed microvessels 24 h after MCAO. (E and F) Vascular buds were visible 3 days after MCAO (arrows), and (G and H) connections or ‘nests’ of small microvessels associating with surrounding vessels (arrows). Size is shown in the inserted bars. This Figure is reproduced from Krupinski, J., Stroemer, P., Slevin, M., Marti, E., Kumar, P. and Rubio, F., Three dimensional structure of newly formed blood vessels after focal cerebral ischaemia in rat, NeuroReport 14 (8), 1171–1176, with permission. © (2003) Lippincott, Williams & Wilkins.

These findings encouraged the hypothesis that a penumbra existed in the vicinity of the infarcted zone, comprising a partially perfused tissue capable of responding to/repairing damage caused by ischaemia followed by reperfusion. It is important to remember that the concept of penumbra is based on animal experiments in which potentially reversible functional disturbances can be observed when blood flow decreases beyond a critical threshold [8]. However, in humans, the situation is not as clear as in animals, although the concept of penumbra is possibly valid. This was confirmed by thrombolytic treatment which reversed critical ischaemia and decreased volume of final infarcts. In patients, the existence of penumbra is now generally accepted and indeed it can be visualized by studies of mismatch on MRI (magnetic resonance imaging) and PET (photon emission tomography) [9]. DWI (diffusion-weighted imaging) in combination with PWI (perfusion-weighted imaging) has become a widely accepted modality for the selection of patients for acute therapy, a mismatch between these procedures suggests viable ‘penumbral’ tissue. Currently, there is no consensus with respect to what constitutes DWI/PWI mismatch [10]. In later studies, limitations of definitions of irreversible changes have been revised, suggesting that penumbra volume is possibly overestimated by MRI. Furthermore, not all the patients with a mismatch have areas of penumbra, confirming that the mismatch does not seem to be a reliable indicator of penumbra [8].

The most recent studies expand this concept further by demonstrating that patients without DWI/PWI mismatch are equally likely to have lesion growth as those with mismatch and should not be excluded from acute stroke treatment [11]. Thus, at least in humans, we cannot identify with certainty ischaemic penumbra, and possibly tissue at risk may be salvageable far beyond 6 h [12]. Importantly, angiogenesis in the penumbra areas seems to be a natural response to ischaemia in humans [3] and animals [13]; therefore peri-infarcted tissue, as well as broader areas of tissue vulnerable to ischaemia and, where apoptosis may be present, may benefit from angiogenesis occurring over days or even weeks and months. Angiogenesis, even if initiated early, should be regarded as non-acute treatment complementary with conventional treatment, which might help in neuronal reorganization, stem cell differentiation and functional recovery, which as we know from clinical practice occurs for many months following initial stroke. The idea that the brain was capable of self-repair was not a popular one in the late 1990s and even in the early 21st century; however, adult brain stem cells have since been identified in places such as the corpus striatum and subependymal region of the lateral ventricles, giving hope of a means of long-term brain repair [14] (see the Therapeutic Potential of Angiogenesis section). Furthermore, it is apparent that central nervous system re-organization occurs after stroke, and the brain adapts to damage by attempting to preserve motor function [15]. Here, angiogenesis may help to establish this new functional architecture. Thus the concept of angiogeneis goes far beyond the idea of classical short-lived penumbra.

MECHANISMS OF ANGIOGENESIS AFTER STROKE: SIGNAL TRANSDUCTION ACTIVATION

Secretion of angiogenic factors

The angiogenic activity reported in the 1970s was beginning to be characterized in individual proteins. During the inflammatory phase of stroke, angiogenesis may be activated through release of polypeptide growth factors and cytokines originating from infiltrating macrophages, leucocytes and damaged blood platelets [16]. Specific up-regulation of the angiogenic factors, including TGF-β (transforming growth factor-β) [17], PDGF (platelet-derived growth factor) [18], VEGF (vascular endothelial growth factor) [19,20] and basic FGF (fibroblast growth factor)-2 [21], occurs in the microvessels of patients in response to ischaemic stroke (Figure 3). At least 20 small molecules and peptide growth factors are known to induce angiogenesis, but this review will concentrate on the well-characterized peptide growth factors.

Increased expression of FGF-2 (A and B), PDGF (C) and VEGF (D and E) in microvessels from peri-infarcted regions in patients after acute ischaemic stroke

Figure 3
Increased expression of FGF-2 (A and B), PDGF (C) and VEGF (D and E) in microvessels from peri-infarcted regions in patients after acute ischaemic stroke

Arrows indicate the areas of increased expression in the microvessels. Magnification, ×100 in (A, D and E), ×200 in (B), and ×400 in (C). Figures 3(A) and 3(B) are taken from Angiogenesis, volume 8, 2005, pp. 53–62, Expression of basic fibroblast growth factor mRNA and protein in the human brain following ischaemic stroke, Issa. R., AlQteishat, A., Mitsios, N., Saka, M., Krupinski, J., Tarkowski, E., Gaffney, J., Slevin, M., Kumar, S. and Kumar, P., with kind permission of Springer Science and Business Media. Figure 3(C) is reproduced from Krupinski, J., Issa, R., Bujny, T., Slevin, M., Kumar, P., Kumar, S. and Kaluza, J., A putative role for platelet derived growth factor in angiogenesis and neuroprotection after ischaemic stroke in humans, Stroke, 28, 564–573, with permission. © (1997) Lippincott, Williams & Wilkins (http://lww.com). Figures 3(D) and 3(E) are reprinted by permission from Macmillan Publishers Ltd: Laboratory Investigation [19], © (1999) (http://www.nature.com.labinvest).

Figure 3
Increased expression of FGF-2 (A and B), PDGF (C) and VEGF (D and E) in microvessels from peri-infarcted regions in patients after acute ischaemic stroke

Arrows indicate the areas of increased expression in the microvessels. Magnification, ×100 in (A, D and E), ×200 in (B), and ×400 in (C). Figures 3(A) and 3(B) are taken from Angiogenesis, volume 8, 2005, pp. 53–62, Expression of basic fibroblast growth factor mRNA and protein in the human brain following ischaemic stroke, Issa. R., AlQteishat, A., Mitsios, N., Saka, M., Krupinski, J., Tarkowski, E., Gaffney, J., Slevin, M., Kumar, S. and Kumar, P., with kind permission of Springer Science and Business Media. Figure 3(C) is reproduced from Krupinski, J., Issa, R., Bujny, T., Slevin, M., Kumar, P., Kumar, S. and Kaluza, J., A putative role for platelet derived growth factor in angiogenesis and neuroprotection after ischaemic stroke in humans, Stroke, 28, 564–573, with permission. © (1997) Lippincott, Williams & Wilkins (http://lww.com). Figures 3(D) and 3(E) are reprinted by permission from Macmillan Publishers Ltd: Laboratory Investigation [19], © (1999) (http://www.nature.com.labinvest).

VEGF

VEGF was first described as a vascular permeability factor by Dvorak and colleagues [22], then recognized as an angiogenic factor [23] and isolated by Ferrara and Henzel [24]. The VEGF family (reviewed in [25]) comprises VEGF-A, -B, -C and -D and PlGF (placental growth factor), which form homo- and hetero-dimers, and exist as several isoforms. For example, VEGF-A comprises six spliced isoforms (VEGF121, VEGF145, VEGF165, VEGF183, VEGF189 and VEGF206) and PlGF, three (PlGF1, 2 and 3). VEGF-C and -D arise by proteolytic cleavage of precursor molecules not splicing. VEGF, perhaps the most potent angiogenic growth factor, may be up-regulated by several others, including TGF-β, basic FGF, PDGF and EGF (epidermal growth factor) [26].

Issa et al. [19] were the first to demonstrate that the mRNA and protein of VEGF165, VEGF189 and flk-1 were up-regulated in human post-mortem brain after ischaemic stroke. VEGF is up-regulated within hours of stroke and has a strong influence on the growth of new blood vessels after ischaemia [27]. Angiogenesis was stimulated together with a concomitant reduction of infarct volume following intracerebroventricular injection of VEGF via an osmotic pump (10 μg/ml, 1 μl/h), beginning 24 h after focal cerebral ischaemia induced by MCAO [28]. Exercise-induced overexpression of VEGF had a similar effect, stimulating angiogenesis, in rats subjected to 2 h MCAO followed by 48 h reperfusion [29].

In an attempt to understand brain angiogenesis induced by VEGF, Stiver et al. [30] injected a vector expressing the VEGF-A164 isoform into normal adult rat striatum. They found that astrocytes took up the vector and expressed VEGF, causing increased vascular permeability and enlarged ‘mother’ vessels, in which ECs and pericytes proliferated and grew into glomeruloid bodies. As VEGF expression declined, the latter involuted to form clusters of new small vessels which remained patent for as long as 16 months. It might be that VEGF could be therapeutically beneficial in promoting angiogenesis in ischaemic brain tissue.

Recently, encapsulated cell grafts overexpressing VEGF were implanted into rat striata before induction of MCAO. Angiogenesis was significantly increased around the area of the encapsulated graft after 24 h concomitant with a reduction in infarct size, but there was no increase in cerebral blood flow at 1, 7 and 14 days after MCAO compared with control untreated animals [31].

Unfortunately, the function of VEGF as a vascular permeability factor also means that localization in the primary ischaemic core causes blood–brain barrier leakage, resulting in brain oedema [32]. Mice overexpressing human VEGF165 under a neuron-specific promoter were subjected to MCAO ischaemia. Brain microvessel density in the transgenic mice was higher than wild-type mice before ischaemia, and the increase in microvessel density 3 days after ischaemia was greater in transgenic animals than in wild-type, especially in the striatum. Transgenic mice also had less neuronal death than wild-type mice in this area. Following transient MCAO, however, [14C]iodoantipyrine autoradiography revealed that VEGF induced haemodynamic steal phenomena with reduced blood flow in ischaemic areas and increased flow outside the MCA territory, again suggesting that VEGF overexpression might worsen cerebral reperfusion after stroke.

TGF-β

Genetic studies of TGF-β in mice and humans indicate it has a pivotal role in modulation of angiogenesis. Under different conditions, TGF-β can either stimulate EC growth through activation of the ALK1 receptor or inhibit angiogenesis through the ALK5 receptor [33]. Overexpression of CD105 (endoglin), which forms a complex with TGF-β receptors, appears to inhibit the anti-angiogenic effects of TGF-β/ALK5 signalling and could affect the balance of angiogenesis in disease [34]. The role of endogenously produced TGF-β after stroke remains to be elucidated. However, overexpression of TGF-β was seen in microvascular cells from brains at autopsy after stroke [17,35] and was increased in association with NOS1 (nitric oxide synthase 1) in the serum and tissue of patients after stroke, suggesting this interaction might form part of a pro-angiogenic stimulus [36,37].

TGF-β is a paradigm to show how complicated and varied the actions of a single molecule can be (for reviews, see [38,39]). Sporn and co-workers are responsible for most of the advances in our understanding of this molecule [38]. TGF-β was first distinguished from TGF-α during their isolation from medium conditioned by Moloney-virus-transformed cells, hence its name [40]. At the same time, it was isolated from non-neoplastic tissues [41]. It had at least bifunctionality; growth promotion or inhibition depending on the context: promotion in the presence of PDGF and inhibition in the presence of EGF [42]. Its ability to induce fibrosis and angiogenesis in vitro and in vivo marked it out as an important mediator of tissue repair [43].

The multiple functions known for TGF-β have been summarized as the ‘good, the bad and the ugly’ by Wahl in 1994 [44], since its beneficial effects, which now include neuroprotection as well as angiogenesis, might be negated by the ability of exogenously administered/overexpressed TGF-β to cause inflammation and severe fibrosis, or depression of the immune system, thus reducing defence against microbial infection. Another suggestion by Sporn [38] is that TGF-β represents a molecular switch, turning on molecules that are off and vice versa. A cluster of papers (reviewed in [39]) reported that TGF-β, normally absent from many areas of adult rat brain, was up-regulated in rats after hypoxia/ischaemia, resulting in reduced infarct size and neuroprotection. Krupinski et al. [17] were the first to demonstrate up-regulation of TGF-β mRNA and protein in human post-mortem brain after acute ischaemic stroke and linked its properties to the angiogenesis they had previously described in brains of similar patients [2,3]. Further human studies demonstrated increased TGF-β in CSF (cerebrospinal fluid) but unchanged levels in serum [20]. Its neuroprotective role was realized by injecting it into animals prior to ischaemia, resulting in decreased infarct size [45]; however, it had no beneficial effect if injected after the ischaemic injury [46]. Proof of its role was obtained when injection of a selective antagonist (TβRII–Fc; TGF-β type II receptor fused with the Fc region of a human Ig) caused an increase in the infarct volume in rats following induction of cerebral focal ischaemia [47]. Ischaemia was induced by an autologous clot embolus introduced through the right internal carotid artery. TGF-β or albumin (vehicle control) was given immediately before embolization of the clot. The results suggested a potential therapeutic benefit of TGF-β treatment after stroke and this was attributed to neuroprotection, but angiogenesis was not considered [47].

FGF-2

Gospodarowicz et al. [48] were the first to purify FGF from bovine brain and compare the endothelial-growth-promoting activity of two sources of FGF activity: brain and pituitary [49]. It was concluded that both acidic and basic FGF (FGF-2) stimulated EC proliferation in vitro, but acidic FGF was 30–100-fold less potent [50]. FGF-2 was demonstrated to be angiogenic in vivo [51] and neuroprotective in vitro, increasing neuronal survival and neurite extension [52,53]. Its neuroprotective effects may confound interpretation of any beneficial effects on stroke outcome being linked to angiogenesis. In common with many peptide growth factors, FGF-2 influences the actions of other growth factors. It modulates the action of PDGF on brain O-2A progenitor cells. Whereas PDGF controls their proliferation and differentiation into oligodendrocytes, FGF-2 is necessary for proliferation, but inhibits differentiation by maintaining PDGFR (PDGF receptor)-α on the cell surface [54]. Moderate FGF-2 immunoreactivity was seen in ECs of developing capillaries 2 days after induction of rat MCAO [13]. Finklestein and co-workers, working with a focal cerebral infarction model in rats, reported that FGF-2 mRNA and protein were increased in astroglial cells [55]. FGF-2 mRNA and protein were up-regulated in the brains of patients who had died of acute ischaemic stroke and also in their serum and CSF 1–14 days after stroke [21]. FGF-2 immunoreactivity was notably increased in ECs and lamina of microvessels in the peri-infarcted regions of patients surviving from 2–43 days after stroke, confirming its importance in humans as well as in animal models.

Numerous studies have examined the effects of FGF-2 administration on infarct development and functional recovery in in vivo models of stroke. For example, FGF-2 administered 2 h after infarction in the rat reduced infarct volume [56]. Later administration did not reduce infarct volume, but still up-regulated GAP-43 necessary for neuronal sprouting and enhanced neural recovery. However, its potential effects on angiogenesis and restoration of collateral circulation have not been examined in great detail [57,58]. In addition, FGF-2 can act as a mitogenic and differentiation factor for endogenous neural progenitor cells in vivo [59].

In view of this effectiveness after treatment delay, FGF-2 was considered suitable as a therapy for human patients where treatment is often delayed, and human clinical trials have been carried out since [60]. Ren and Finklestein [61] have reviewed its use in treating human patients, suggesting that its infarct-reducing properties might be related to its induction of iNOS (inducible NOS) or up-regulation of anti-apoptotic Bcl-2, as well as its neuroprotective effects. Human clinical trials conducted in the U.S.A. were curtailed because of high dose toxicity, whereas European trials using lower doses produced no toxicity, but were curtailed because there was no clear end point to measure successful recovery. The latter trial did produce some beneficial effects; however, there has been no follow-up study to date.

PDGF

PDGF, first identified and isolated by Stiles et al. [62] and purified by Antoniades [63], exists as two isoforms PDGF-A and -B, which form homo- and hetero-dimers. PDGF-BB and PDGF-AB are angiogenic in vivo, whereas PDGF-AA is not [64]. There are direct angiogenic effects on EC migration and proliferation. Iihara et al. [65] demonstrated that ischaemia up-regulated PDGF-B chains in neurons and macrophages in rat brain, whereas Krupinski et al. [18] showed that PDGF-B mRNA and protein were increased in brain tissue of patients who had died of acute ischaemic stroke. PDGF has been shown to induce cerebral neocapillary formation in mice with a concomitant increase in microcirculation [66]. Newly formed microvessels are composed of two types of cells: ECs and pericytes. Pericytes play an important role in the maintenance of microvascular homoeostasis and capillary maturation. [67]. The mechanism of action of PDGF is thought to be via mediation of EC–pericyte interactions, which support vascular remodelling after stroke. PDGF was shown to be specifically up-regulated in vascular structures in the infarcted area, particularly associated with pericytes 48 h after mouse MCAO [68]. Recent results have shown it is essential for blood vessel maturation. As bone marrow endothelial progenitor cells differentiate into blood vessel mural cells, smooth muscle markers are induced [69]. Lehti et al. [70] have reported that recruitment of pericytes and smooth muscle cells into a vessel wall depends on MT1-MMP [membrane type-1 MMP (matrix metalloproteinase)] activity to enhance PDGF-BB/PDGFR signalling. PDGF-AA is not angiogenic, but does induce brain stem cell proliferation and differentiation (see below). Two new proteins, PDGF-C and -D, have been identified, complicating the picture of the PDGF family further [71].

Downstream signalling pathways

Activation of downstream signalling pathways passing through key second messengers, including members of the MAPK (mitogen-activated protein kinase) family [ERK1/2 (extracellular-signal regulated kinase 1/2), p38 MAPK and JNK (c-Jun N-terminal kinase)], induces EC proliferation, migration and ultimately angiogenesis (Figure 4; for a review, see [16]). Activation of MAPK may have a pivotal role in stroke-associated abrogation of apoptosis, controlling angiogenesis and promoting VEGF expression through HIF (hypoxia-inducible factor)-1 [72]. Our work has shown a transient (<24 h) increase in expression of phosphorylated p38 MAPK, ERK1/2 (Figure 5) and JNK in penumbra-associated ECs following MCAO in a rat model [73,74]. Selected overexpression of these proteins might be involved in cellular survival and revascularization [73,74].

Potential pathways of angiogenesis and EC apoptosis after stroke

Figure 4
Potential pathways of angiogenesis and EC apoptosis after stroke

NF-κB, nuclear factor κB; PLC-γ, phospholipase C-γ; PKC, protein kinase C, Grb2, growth-factor-receptor-bound protein-2; SOS, son of sevenless; MKK5, MAPK kinase 5; FAK, focal adhesion kinase.

Figure 4
Potential pathways of angiogenesis and EC apoptosis after stroke

NF-κB, nuclear factor κB; PLC-γ, phospholipase C-γ; PKC, protein kinase C, Grb2, growth-factor-receptor-bound protein-2; SOS, son of sevenless; MKK5, MAPK kinase 5; FAK, focal adhesion kinase.

Phosphorylated ERK1/2 staining in ECs (arrows) of grey matter peri-infarcted tissue from a patient 3 days after acute ischaemic stroke

Figure 5
Phosphorylated ERK1/2 staining in ECs (arrows) of grey matter peri-infarcted tissue from a patient 3 days after acute ischaemic stroke

The arrows indicate phosphorylated ERK1/2 staining. This Figure is reproduced from Slevin, M., Krupinski, J., Slowik, A., Rubio, F., Szczudlik, A. and Gaffney, J., Activation of MAP kinase (ERK-1/ERK-2) tyrosine kinase and VEGF in the human brain following acute ischaemic stroke, NeuroReport 11 (12), 2759–2764, with permission. © (2000) Lippincott, Williams & Wilkins.

Figure 5
Phosphorylated ERK1/2 staining in ECs (arrows) of grey matter peri-infarcted tissue from a patient 3 days after acute ischaemic stroke

The arrows indicate phosphorylated ERK1/2 staining. This Figure is reproduced from Slevin, M., Krupinski, J., Slowik, A., Rubio, F., Szczudlik, A. and Gaffney, J., Activation of MAP kinase (ERK-1/ERK-2) tyrosine kinase and VEGF in the human brain following acute ischaemic stroke, NeuroReport 11 (12), 2759–2764, with permission. © (2000) Lippincott, Williams & Wilkins.

Other studies using microarrays have indirectly implicated numerous potentially angiogenic proteins up-regulated in animal models of ischaemic stroke. Suppression subtractive hybridization was used to compare gene expression in cerebral capillaries from stroke-prone spontaneously hypertensive and stroke-resistant rats. Novel genes involved in activation of Rho and G-proteins (BM247), which could be associated with tight junction assembly and maintenance, and the sulfonylurea receptor SUR2B, which couples membrane excitability to cellular metabolism and may have a role in regulation of cerebral blood flow, were up-regulated in hypertensive rats [75]. DNA ‘angiogenesis profile’ microarrays containing 96 genes were used to identify changes in expression following mouse stroke-induced ischaemia. The number of blood vessels was increased within 3 days of stroke, suggesting that angiogenesis occurred immediately after injury. A total of 42, 29 and 13 genes were increased at 1 h, 1 day and 21 days respectively, including previously described growth factors and cytokines, together with members of the thrombospondin family [76]. Follow-up studies are beginning to demonstrate how these genes function in the post-ischaemic angiogenic process. For example, survivin, the inhibitor of apoptosis protein, was up-regulated uniquely in microvessels 2 days after mouse MCAO [77]. The authors went on to show that the extent of vascularization was dependent on survivin expression, since survivin-deficient mice had a significant reduction in blood vessel density. Recent microarray studies have identified numerous novel genes associated with the proliferative, adhesive and remodelling phases of angiogenesis ([78,79]; for reviews, see [80,81]).

EC apoptosis

EC apoptosis can also occur in response to hypoxic conditions associated with stroke and may inhibit the formation of new blood vessels in peri-infarcted regions. In vitro studies of vascular ECs have demonstrated that oxygen/glucose deprivation induces iNOS expression [82] and Bax translocation with concomitant cytochrome c release, leading to apoptosis [83]. Similarly, TNF-α (tumour necrosis factor-α), as well as the effects of reperfusion, can induce expression of ROS (reactive oxygen species) and subsequent cell apoptosis through Rac1 and PKC (protein kinase C) signalling intermediates [84]. Excitotoxic-induced mitochondrial damage may result in activation of caspase 9 [85], whereas increased expression of p38 MAPK during hypoxia/re-oxygenation in human microvessel cerebral ECs was associated with apoptotic cell death in association with activation of caspase 3 [86]. Although Krupinski et al. [4] have demonstrated strong TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling) and caspase 3 staining of microvessels adjacent to the infarcted region in a rat model 1–2 weeks after MCAO, this is one of few studies of this kind recorded. Figure 4 shows potential mechanisms of angiogenesis and EC apoptosis after stroke; however, the mediators and their associated second messenger pathways have not yet been fully described.

The development of large genome-spanning microarrays, the ability to choose specific genes in large-scale gene profiling real-time PCR studies (e.g. TaqMan profiling systems; Applied Biosystems), the knowledge of probable mediators of angiogenesis in disease (for reviews, see [87,88], and the ability to isolate individual disease-affected microvessels using laser-capture micro-dissection will increase our understanding of the mechanisms through which angiogenesis occurs after stroke.

THERAPEUTIC POTENTIAL OF ANGIOGENESIS

Normal brain function depends on adequate delivery of basic substances through a rich and complicated network of blood vessels. During ischaemic stroke, occlusion of blood vessels is a consequence of underlying atherosclerotic disease, mural thrombosis or cardiac embolism in the brain vasculature. Approaches to restore circulation by clot resolution or dilation of intracranial arteries by angioplasty and stenting may be limited because of the morphological properties of brain vasculature. Pharmacological treatments stimulating angiogenesis and restoration of adequately perfused cerebral tissue may become an important therapeutic option in the near future [89,90]. The immediate effects of this approach are not possible to see; however, long-term benefits may be important to help functional recovery and prevent the cognitive deterioration frequently seen in stroke patients.

Multiple pre-clinical strategies have been employed for promoting angiogenesis in ischaemic brain tissue. In animal models of chronic cerebral hypoperfusion, therapeutic angiogenesis following human VEGF or HGF (hepatocyte growth factor) gene transfer was effective, resulting in increased capillary density in the brain [91], and was associated with reduced neurological deficits [92]. However, increased leakage of blood vessels was noted, increasing the risk of subsequent brain swelling [93]. Other studies have demonstrated that VEGF overexpression may worsen, rather than improve, cerebral haemodynamics after stroke [94]. Combination therapies of VEGF and angiopoietins may overcome this problem [95]. VEGF and angiopoietin-1 promoted the formation of mature neovessels without inducing side effects on blood–brain barrier permeability [96].

Regulatory control of VEGF may be a new approach for therapeutic modulation of collateral circulation following cerebral ischaemia [97]. HIF-1 is a transcription factor that regulates the adaptive response to hypoxia in mammalian cells and promotes angiogenesis. Combination of VEGF with HIF prolyl hydroxylase inhibitors, which raise HIF-1 levels and increase expression of many hypoxia-inducible proteins including VEGF, was shown to be effective [98]. A new approach may involve modulation, rather than direct overexpression, of oxygen-sensitive HIF-response genes that, in turn, could modulate expression of other hypoxia-regulated genes [99101]. Stabilized HIF-1α by itself was found to be more effective than VEGF in inducing angiogenesis in skeletal muscle [102]. These methodologies might incorporate self-regulated physiological protection mechanisms which would help to prevent cell injury [103,104].

As described previously, the brain is a source of many pro-angiogenic factors, including VEGF, TGF-β, FGF-2, PDGF and HIF, which are overexpressed in the surviving areas of penumbra. Some currently available drugs, such as atorvastatin, induce VEGF, BDNF (brain-derived neurotrophic factor) and synaptophysin expression and promote angiogenesis and brain plasticity in animal models [105]. The time scale of administration may be crucial, since early (1 h) delivery of VEGF has been demonstrated to be detrimental, whereas later administration (48 h after stroke onset) is beneficial [106]. Potential treatment regimens may become complicated, as some factors are required in the early stages of angiogenesis. For example, a combination of VEGF and angiopoietin 2 was necessary for promoting the growth of vascular sprouts in mature mouse brain [95]. The method of administration seems to be important as only intracerebroventricularly administered VEGF protected the brain against focal ischaemia [107]. In addition VEGFR (VEGF receptor)-1 (also known as Flt-1) induces MMPs [108], as does PDGFR signalling (see above). MMPs, TIMPs (tissue inhibitors of metalloproteinases) and matrix-degrading enzymes are likely to play an important role during repair phases of ischaemia, being involved in angiogenesis and re-establishment of cerebral blood flow. However, they can also be detrimental and their improper modulation may lead to neuronal cell death [109].

Inflammation also occurs immediately after an ischaemic insult. Some inflammatory cytokines are pro-angiogenic, such as IL (interleukin)-1, TNF-α and NO, and others are anti-angiogenic, including IFN-γ (interferon-γ) and IL-2. Many peptide growth factors, such as TGF-β and VEGF, are up-regulated by IL-1 [110,111], and some ILs, such as IL-1, are up-regulated by VEGF or down-regulated by TGF-β [112]. Targeting these cytokines may be necessary to modulate angiogenesis and associated inflammation after ischaemia [100,113,114].

LINK BETWEEN ANGIOGENESIS AND NEUROGENESIS

A key objective in the effective treatment of cerebral ischaemia is to enhance both new blood vessels/collateral formation and neurogenesis. Combination therapy would not only need to secure ‘safe’ angiogenesis, but also to stimulate neurogenesis [14]. VEGF has been demonstrated to have an early neuroprotective action [115] observed significantly before recovery-promoting actions, such as angiogenesis, and possibly involving activation of the PI3K (phosphoinositide 3-kinase)/Akt pathway [107]. Hypoxia-induced PlGF, a ligand for VEGFR-1 and neuropilin-1, is involved in co-ordinated regulation of angiogenesis and neurogenesis after stroke [116]. Intraventricular injection of the human HGF gene attenuated brain ischaemic injury in a rat model, without inducing cerebral oedema, through angiogenic and neuroprotective actions, as measured by reduced apoptosis [117]. Adenovirus-mediated gene transfer of HB-EGF (heparin-binding EGF-like growth factor) enhanced neurogenesis and angiogenesis and, subsequently, functional recovery following MCAO in a rat model of stroke [118]. There is a choice of combining angiogenic/neuroprotective peptide growth factors or using them alone.

Cell-based approaches for stimulating angiogenesis in ischaemic stroke, such as multipotent embryonic stem cell transplantation, might be feasible and provide support for cell survival [119,120]. Modulation of the haematopoietic system and especially erythropoietin signalling was examined in cerebral ischaemia [121]. BMDCs (bone-marrow-derived cells) contributed to revascularization after ischaemia and were involved in blood vessel stabilization during ischaemia-induced angiogenesis [122]. BMDC therapy and administration of cells derived from the subventricular zone links angiogenesis, neurogenesis and synaptogenesis and may provide optimal concentrations of cytokines and neurotrophins necessary for functional recovery [14]. Systemic administration of human cord blood-derived CD34+ cells following stroke induced neovascularization in the ischaemic zone and provided a favourable environment for neuronal regeneration [123], stimulating VEGF, FGF-2 and IGF-1 (insulin-like growth factor-1) [115,124,125]. G-CSF (granulocyte colony-stimulating factor) stimulates neurogenesis via another angiogenic growth factor, VEGF and its receptor VEGFR-2 (also known as Flk-1) [126]. Accelerated neovessel formation seemed to be essential for enhancing endogenous neurogenesis and improving functional recovery. However, intervention efforts attempting to induce neuroprotection and angiogenesis concurrently should be approached with caution, as angiogenic molecules at low doses may be neuroprotective without initiation of angiogenesis. In contrast, high doses that induce angiogenesis may lead to neuronal damage [127].

ATHEROSCLEROSIS AND ANTI-ANGIOGENIC STRATEGIES AFTER STROKE

Angiogenesis has a complex role in atherothrombosis, whilst inflammation seems to be the leading promoter of progressing atherosclerosis both in the clinical environment and in experimental models [128,129]. Recent studies have demonstrated that pro-inflammatory markers may be involved in angiogenesis and atherosclerotic plaque destabilization [130] and subsequent growth factor signal transduction defects, leading to uncontrolled angiogenesis, plaque haemorrhagic transformation, thrombosis and stroke [131,132].

Pro-angiogenic agents may actually promote thrombosis under inflammatory conditions. Thus actions to stimulate angiogenesis in stroke patients who suffer from severe atherosclerosis of the major arteries may be counter-productive. Importantly, the response to pro-angiogenic agents may vary depending on the particular pathophysiological conditions of individual vascular beds [133].

CONCLUSIONS

This review demonstrates that there is still a need to improve our understanding of the time course of expression of angiogenic and EC apoptotic factors and their associated enzymes after ischaemic stroke. This is because non-therapeutically stimulated angiogenesis is observable only several days after stroke, which is beyond the period of reversible changes in ischaemic penumbra recognized as a therapeutic window in the ischaemic brain. Due to the complexity of physiological regulation of blood vessel formation, involving numerous critical growth factors expressed differentially in time, space and concentration, ongoing therapeutic efforts using single agents and aimed at treatment of ischaemic vascular disease are of limited potential. Optimization of therapeutic treatments might involve a complex series of interventions beginning with administration of a cocktail of drugs within the first few hours of illness to reduce inflammation, but at the same time maintaining neuronal viability with neurotrophins and stimulating growth-factor-induced angiogenesis. Perfusion pressure in the penumbra region could be increased using thrombolytic therapy, and susceptible neurons could be protected from apoptosis by viral transfer of genes such as Bcl-2 [134,135].

A large amount of pre-clinical data exists on different strategies employed in ischaemic disease, but there are no ongoing clinical trials for stroke using angiogenesis-promoting therapies [136,137]. Angiogenic gene therapy may help salvage longer surviving tissue and efficiently promote neuronal re-organization in damaged areas. One challenge would be to design therapies that are targeted to promote angiogenesis when necessary without affecting the progression of atherothrombosis. This may be feasible even with agents such as VEGF, as atherosclerotic plaque-specific isoforms exist [138], and forthcoming new imaging techniques may identify inflammatory plaque load, resulting in employment of alternative treatments.

Abbreviations

     
  • BMDC

    bone-marrow-derived cell

  •  
  • CSF

    cerebrospinal fluid

  •  
  • DWI

    diffusion-weighted imaging

  •  
  • EC

    endothelial cell

  •  
  • EGF

    epidermal growth factor

  •  
  • ERK1/2

    extracellular-signal regulated kinase 1/2

  •  
  • FGF

    fibroblast growth factor

  •  
  • HIF

    hypoxia-inducible factor

  •  
  • IL

    interleukin

  •  
  • JNK

    c-Jun N-terminal kinase

  •  
  • MAPK

    mitogen-activated protein kinase

  •  
  • MCA

    middle cerebral artery

  •  
  • MCAO

    MCA occlusion

  •  
  • MMP

    matrix metalloproteinase

  •  
  • MRI

    magnetic resonance imaging

  •  
  • NOS

    nitric oxide synthase

  •  
  • iNOS

    inducible NOS

  •  
  • PDGF

    platelet-derived growth factor

  •  
  • PDGFR

    PDGF receptor

  •  
  • PI3K

    phosphoinositide 3-kinase

  •  
  • PKC

    protein kinase C

  •  
  • PlGF

    placental growth factor

  •  
  • PWI

    perfusion-weighted imaging

  •  
  • ROS

    reactive oxygen species

  •  
  • TGF-β

    transforming growth factor-β

  •  
  • TNF-α

    tumour necrosis factor-α

  •  
  • VEGF

    vascular endothelial growth factor

  •  
  • VEGFR

    VEGF receptor

Funding of the authors' work was from the Higher Education Funding Council for England (HEFCE).

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