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VEGF and ALS
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Neuroscience Research 62 (2008) 71–77 www.elsevier.com/locate/neures
Review article
VEGF and ALS Sivakumar Sathasivam * Department of Neurology, The Walton Centre for Neurology & Neurosurgery, Lower Lane, Liverpool L9 7LJ, UK Received 22 May 2008; accepted 17 June 2008 Available online 9 July 2008
Abstract In amyotrophic lateral sclerosis (ALS), an adult-onset progressive degeneration of motor neurons occurring as sporadic and familial disease, there is emerging evidence for and against the role of vascular endothelial growth factor (VEGF), an endothelial cell mitogen crucial for angiogenesis, in its etiopathogenesis. Our understanding of the role of VEGF in ALS has come from studies of both experimental models and human cases. In this article, I have examined in detail the in vitro and in vivo evidence for and against VEGF in ALS, concluding that more compelling evidence is required before we can conclusively link VEGF to ALS in humans. # 2008 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved. Keywords: Amyotrophic lateral sclerosis; Vascular endothelial growth factor; Cell culture studies; Animal models; Human studies; Genetics
Contents 1. 2. 3. 4.
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Amyotrophic lateral sclerosis . . . . The VEGF family . . . . . . . . . . . . First link of VEGF with ALS . . . . Evidence from cell culture studies . 4.1. NSC34 cell line . . . . . . . . . 4.2. Rodent cultures . . . . . . . . . Evidence from animal models . . . . 5.1. Transgenic rodent models . . 5.1.1. Mice . . . . . . . . . . . 5.1.2. Rats . . . . . . . . . . . 5.2. Zebrafish model . . . . . . . . . Evidence from human studies . . . . 6.1. Serum or plasma . . . . . . . . 6.2. Cerebrospinal fluid . . . . . . . 6.3. Post-mortem tissue . . . . . . . 6.4. Genetics . . . . . . . . . . . . . . Conclusions. . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . References . . . . . . . . . . . . . . . . .
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Abbreviations: AMPA, a-amino-3-hydroxy-5-methyl-4-isoxazole propionate; ALS, amyotrophic lateral sclerosis; ALSFRS, amyotrophic lateral sclerosis functional rating scale; CSF, cerebrospinal fluid; Flk-1, vascular endothelial growth factor receptor-2; Flt-1, Fms-related tyrosine kinase 1; Flt-4, Fms-related tyrosine kinase 4; HRE, hypoxia-response element; IGF-1, insulin-like growth factor-1; KDR, kinase insert domain receptor; NP-1, neuropilin-1; NP-2, neuropilin-2; PlGF, placental growth factor; PI3-K, phosphatidylinositol 3-kinase; phospho-Akt, phosphorylated form of Akt; SNP, single-nucleotide polymorphism; TDP-43, TAR DNA-binding protein 43; TNF-a, tumor necrosis factor-a; SOD1, copper/zinc superoxide dismutase; VAPB, vesicle-associated membrane protein; VEGF, vascular endothelial growth factor; VEGFR, vascular endothelial growth factor receptor. * Tel.: +44 151 5298151; fax: +44 151 5295512. E-mail address: [email protected]. 0168-0102/$ – see front matter # 2008 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved. doi:10.1016/j.neures.2008.06.008
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1. Amyotrophic lateral sclerosis Amyotrophic lateral sclerosis (ALS), or Lou Gehrig disease, is a rapidly progressive adult-onset neurodegenerative disorder with an incidence of 1–2 per 100,000 and a prevalence of 4–6 per 100,000 (Cozzolino et al., 2008). Approximately 10% of cases are familial, one fifth of which are caused by mutations in the gene encoding the antioxidant copper/zinc superoxide dismutase (SOD1) on chromosome 21 (Rosen et al., 1993). Several other mutations have been identified in a small number of patients, including (a) alsin, a putative guanine nucleotide factor for GTPase (Yang et al., 2001; Hand et al., 2003), (b) senataxin, a putative DNA/RNA helicase (Chen et al., 2004), and (c) the vesicle-trafficking protein VAPB gene (Nishimura et al., 2004). However, in most familial cases, and in sporadic disease, the cause for ALS remains elusive. The pathogenesis of ALS appears to be a complex interplay of several processes, such as oxidative stress, protein aggregation, mitochondrial dysfunction, excitotoxicity, impaired axonal transport and neuroinflammation, ultimately leading to a programmed mechanism of motor neuron death (Sathasivam and Shaw, 2005; Shaw, 2005). Recently, the TAR DNA-binding protein 43 (TDP-43) was recognized as a major constituent of the neuronal cytoplasmic inclusions seen in degenerating motor neurons in ALS (Neumann et al., 2006). The exact function of TDP-43 remains unclear. TDR-43 mutations have been reported in familial and sporadic ALS in some studies (Sreedharan et al., 2008; Gitcho et al., 2008; Kabashi et al., 2008; Van Deerlin et al., 2008), but these findings are not universal (Guerreiro et al., 2008). 2. The VEGF family Vascular endothelial growth factor (VEGF), a key factor affecting vascular permeability and angiogenesis, was discovered 25 years ago (Senger et al., 1983) and cloned six years after that (Ferrara and Henzel, 1989). The VEGF family consists of six different homologous factors, VEGF-A, placental growth factor (PlGF), VEGF-B, VEGF-C, VEGFD, and VEGF-E. Although VEGF-A, PlGF, VEFG-B, VEGF-D and VEGF-E are important for the growth of blood vessels and VEGF-C mainly affects the development of lymphatic vessels, recent evidence suggests VEGF-A, VEGF-B and VEGF-C directly affect neural cells (Raab and Plate, 2007). The two main classes of receptors for the VEGF family are the tyrosine kinase and the nontyrosine kinase receptors. The former contains three structurally related receptors: VEGFR-1 (Flt-1), VEGFR-2 (KDR/Flk-1), and VEGFR-3 (Flt-4). The nontyrosine receptors consist of neuropilin-1 (NP-1) and neuropilin-2 (NP-2). The members of the VEGF family display different binding patterns to these receptors (Fig. 1) (Takahashi and Shibuya, 2005). As VEGF-A is the major member of the VEGF family that has been studied in ALS, this review will concentrate predominantly on VEGF-A. The gene encoding human VEGF-A (if the isoform designation is not stated, VEGF implies VEGF-A) is located on chromosome 6p21.5 and gives rise to many different isoforms due to alternative splicing (e.g. VEGF121, VEGF145, VEGF165)
Fig. 1. Ligands and receptors of the VEGF family. Members of the VEGF family have different binding patterns to the tyrosine kinase receptors (VEGFR-1, VEGFR-2, VEGFR-3) and nontyrosine kinase neuropilin receptors (NP-1, NP2): (1) VEGFR-1 binds to VEGF-A, VEGF-B and PlGF; (2) VEGFR-2 binds to VEGF-A, VEGF-C, VEGF-D and VEGF-E; (3) VEGFR-3 binds to VEGF-C and VEGF-D only; (4) NP-1 binds to VEGF165, VEGF-B, VEGF-E and PlGF-2; (5) NP-2 binds to the VEGF145 and VEGF165 isoforms of VEGF-A only.
(Bogaert et al., 2006). These isoforms have different molecular mass, solubility, binding affinities for extracellular matrix components and receptor subtypes and biological functions (Matsumoto and Claesson-Welsh, 2001). VEGF binds the two tyrosine kinase receptors VEGFR-1 and VEGFR-2, and the nontyrosine kinase receptors NP-1 and NP-2. VEGFR-2 is the main mediator of the angiogenic, mitogenic and permeabilityenhancing effects of VEGF (Shalaby et al., 1995). Although VEGFR-1 has a 10-fold stronger binding affinity to VEGF than VEGFR-2, the kinase activity of the former is 10-fold lower than that of the latter (Ferrara, 1999). The major function of VEGFR-1 is thought to be as a ‘decoy receptor’ that negatively regulates angiogenesis by preventing the binding of VEGF to VEGFR-2 (Fong et al., 1999; Hiratsuka et al., 1998). VEGFR-3 is not a receptor for VEGF. The other VEGF receptors, neuropilins NP-1 and NP-2, initially described as receptors for semaphorins, are thought to be involved in axon guidance (Neufeld et al., 2002). Among VEGF, NP-1 is a specific receptor for the isoform VEGF165, while NP-2 is specific for VEGF145 and VEGF165. 3. First link of VEGF with ALS The first study suggesting a role of VEGF in ALS was published in 2001 (Oosthuyse et al., 2001). Manipulation of the VEGF gene in mice resulted in a new and unexpected role for VEGF in the pathogenesis of motor neuron degeneration. Mice were generated with a homozygous deletion in the hypoxiaresponse element (HRE) site in the VEGF promoter region (VEGFd/d mice). About three fifths of the mice died before or around birth from vascular abnormalities in the lung. In the 40% of mice that survived, they begun to show symptoms of motor neuron degeneration at around five months of age. By 17 months, these mice had lost approximately 30% of motor neurons in the ventral horns of the spinal cord compared to normal controls. In addition, there was evidence of neuropathological evidence in motor neurones of fewer Nissl bodies, abnormal mitochondria and cell organelles in the early stages,
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and vacuolization, fewer free ribosomes, irregular nuclei, peripheral clumping of chromatic aggregates and marked reactive astrogliosis in the later stages. These changes were similar to those described in the G93A SOD1 transgenic mice, which is a well-established rodent model of ALS. The baseline levels and hypoxic induction of VEGF were reduced in the neural tissue (motor neurons and astrocytes) of the VEGFd/d mice. Furthermore, these mice had a 50% reduction in neural vascular perfusion. These changes in the VEGFd/d mice raised two possible non-mutually exclusive hypotheses on the possible mechanism(s) by which motor neuron degeneration may be influenced in ALS. Inadequate neurotrophic stimulation of motor neurons by VEGF may be one reason. Alternatively, vascular abnormalities due to insufficient VEGF may put motor neurons at risk of late-onset neurodegeneration brought on by chronic ischemia. 4. Evidence from cell culture studies 4.1. NSC34 cell line Physiological concentrations of the VEGF165 isoform has been shown to protect NSC34 cells, a mouse motor neuron-like cell line, against apoptosis induced by tumor necrosis factor-a (TNF-a), hypoxia, oxidative stress and serum deprivation (Oosthuyse et al., 2001). In another study of the same cell line, transfection with mutant G93A SOD1, but not vector control or wild-type SOD1, resulted in increased evidence of oxidative stress and cell death. However, when the cells were pretreated with VEGF, there was a dose-dependent resistance to oxidative injury from hydrogen peroxide, TNF-a and mutant G93A SOD1, shown to be mediated via activation of the antiapoptotic phosphatidylinositol 3-kinase (PI3-K)/Akt pathway (Li et al., 2003). 4.2. Rodent cultures VEGF has been shown to protect motor neurons from serum deprivation- and hypoxia/hypoglycaemia-induced cell death in motor neuron/glial cell cocultures from mouse and rat embryos. In addition, basal VEGF released from cultured glial cells from VEGFd/d mice was significantly lower than glial cells from control mice (Van Den Bosch et al., 2004). However, in the same study, there was no difference in basal survival and sensitivity to excitotoxicity (one of the mechanisms thought to be responsible for motor neuron death in ALS) between motor neuron/glial cell cocultures of G93A mutant SOD1 and wildtype SOD1 mice. In another study using rat spinal cord organotypic cultures, VEGF was shown to protect motor neurons against chronic glutamate-induced excitotoxicity by activating the PI3-K/Akt signal transduction pathway (Tolosa et al., 2008). This study used a model of chronic, slow excitotoxicity, in contrast to the more acute excitotoxic model of Van Den Bosch et al. (2004), which may explain the difference in vulnerability of motor neurons to excitotoxicity observed in the two studies.
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5. Evidence from animal models 5.1. Transgenic rodent models 5.1.1. Mice Crossbreeding experiments have shown that G93A SOD1 VEGFd/d mice develop muscle weakness and die earlier than their G93A SOD1 VEGF+/+ littermates. In addition, compared to wild-type mice, the G93A SOD1 VEGFd/d mice developed more severe spinal ischemia-induced paralysis, and the administration of VEGF resulted in better protection against ischemic motor neuron death in the latter group. These findings suggest that VEGF is a modifier of motor neuron degeneration in ALS (Lambrechts et al., 2003). More supportive evidence of the role of VEGF comes from another set of crossbreeding experiments which have demonstrated that overexpression of VEGF delays neurodegeneration and increases survival in ALS mice. G93A SOD1/VEGF+/+ double-transgenic mice have delayed motor neuron loss, delayed onset of motor impairment and longer survival than G93A SOD1 single transgenics (Wang et al., 2007). There also appears to be a downregulation of VEGF mRNA expression in the spinal cords of G93A SOD1 mice compared to wild-type SOD1 age-matched mice which occurs from before disease onset (Lu et al., 2007). This decrease in VEGF RNA could be due to motor neuron death or an overall decline in production. Intraperitoneal injection of VEGF has been shown to delay disease progression and increase survival in G93A SOD1 transgenic mice (Zheng et al., 2004). The same group showed that the possible mechanisms of neuroprotection may be due to reduced astrogliosis in the spinal cord or increased neuromuscular junctions in the muscles of these mice (Zheng et al., 2007). Furthermore, the injection of a VEGF-expressing lentiviral vector into muscles of the G93A SOD1 transgenic mouse model delayed disease onset and slowed disease progression in these mice. Treatment was effective even when given at the onset of paralysis, which is relevant to humans where most ALS cases occur sporadically. Extensive reporter gene expression in the spinal cord and brainstem in the mice confirmed the efficient retrograde transport of the VEGF gene (Azzouz et al., 2004). In another study, VEGF concentrations in the spinal cord of G93A SOD1 and wild-type SOD1 mice were not significantly different in normal conditions. Hypoxia induced a significant upregulation of VEGF in the mutant mice, although this did not affect survival in the mice, arguing against the importance of VEGF in ALS (Van Den Bosch et al., 2004). 5.1.2. Rats In a G93A SOD1 transgenic rat model of ALS, VEGF was shown to be reduced prior to symptom onset and progressively during the course of the disease compared to non-transgenic littermates. It is unclear if this occurrence is the underlying cause of motor neuron degeneration or a side effect of neuroinflammation in ALS (Xie et al., 2004). Intracerebroventricular delivery of VEGF in G93A SOD1 transgenic rats delays the onset of weakness, improves motor
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function and prolongs survival in these animals. This treatment resulted in delayed motor neuron degeneration in the brainstem, cervical and lumbar spinal cord of these rodents. There was also a correlation between the spatial gradient of VEGF levels and the therapeutic benefit on the disease subtype. This is important because it implies, for example, individuals with bulbar-onset ALS would benefit more from intracerebroventricular delivery of VEGF, while lumbar-onset cases would benefit more from an intrathecal infusion at the lumbar region (Storkebaum et al., 2005). The mechanism of neuroprotection appears to be, at least in part, due to attenuation of the loss of the active, phosphorylated form of Akt (phospho-Akt) in the G93A SOD1 mutant rats (Dewil et al., 2007). Upregulation of phospho-Akt induced by insulin-like growth factor-1 (IGF-1) has been shown to delay disease onset and increase survival in G93A SOD1 mice (Nagano et al., 2005). Intrathecal administration of VEGF into the lumbar region of the spinal cord resulted in the prevention of paralysis in rats which were co-infused with the glutamate receptor agonist aamino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) which is known to induce excitotoxicity, one of the mechanisms thought to be responsible for motor neuron death in ALS. The clinical effect of VEGF correlated with a greater than 75% reduction in lumbar motor neuron loss in the treated rats, suggesting neuroprotection by VEGF against excitotoxicity (Tovar-y-Romo et al., 2007). 5.2. Zebrafish model In a zebrafish model overexpressing mutant SOD1, upregulation of VEGF I the zebrafish embryo rescued the motor axonopathy induced by the mutant gene, while knockdown of VEGF expression aggravated it (Lemmens et al., 2007). Although ALS is an adult-onset disease, abnormalities of axonal transport have been described in motor neurons cultured from G93A SOD1 mouse embryos (Kieran et al., 2005). Therefore, findings in the zebrafish model may be relevant. 6. Evidence from human studies 6.1. Serum or plasma One study has shown that serum VEGF levels were significantly higher in ALS patients compared to controls. However, there was no relationship between serum VEGF levels and gender, onset type (bulbar or spinal) or the ALS Functional Rating Scale (ALSFRS). The authors of the study speculated that this increase was could be the result of increased synthesis of VEGF in skeletal muscle, which is affected in ALS, in response to hypoxia (Nygren et al., 2002). However, in another study, plasma VEGF levels were shown to be about 50% lower than patients with sporadic ALS, compared to unaffected spouses. The VEGF levels in this study did not correlate with age of onset, onset type, or disease progression (Lambrechts et al., 2003). Other studies have not found a difference in serum or plasma VEGF levels between ALS
patients and controls (Devos et al., 2004; Just et al., 2007; Moreau et al., 2006). Therefore, the usefulness of measuring serum or plasma VEGF levels in ALS is questionable, as results obtained appear variable. 6.2. Cerebrospinal fluid One study failed to detect VEGF in the cerebrospinal fluid (CSF) of patients with ALS, but this may be due to the low sensitivity of the ELISA kits used (Nygren et al., 2002). CSF VEGF levels were demonstrated to be lower in ALS patients compared to those in normal control subjects and controls with other neurological disorders, with no significant difference between the latter two groups (Devos et al., 2004). The same group found that CSF VEGF levels were significantly lower in hypoxemic ALS patients than in both normoxemic ALS patients and hypoxemic neurological controls. In addition, there was no significant difference between CSF VEGF levels in normoxemic ALS patients and normoxemic neurological controls. There was a paradoxical lack of CSF VEGF upregulation in hypoxemic ALS patients compared to hypoxemic neurological controls, pointing to a dysregulation of the hypoxia-dependent CSF VEGF response pathway (Just et al., 2007; Moreau et al., 2006). Two other studies have failed to replicate the above findings. In one, there was no difference in CSF VEGF levels between ALS patients and normal controls or controls with other neurological disorders (Nagata et al., 2007). These findings were somewhat replicated by another study which showed no difference in CSF VEGF levels when all ALS patients were compared to controls. However, subgroup analysis showed that CSF VEGF levels were significantly increased in patients with limb-onset ALS compared to patients with bulbar-onset ALS, and in patients with long disease duration compared to patients with short disease duration. However, the type of ALS onset did not significantly influence disease duration. The author of the study speculated that the increase in CSF VEGF may have a protective role against glutamate-mediated excitotoxicity and oxidative damage of motor neurons (Iłz˙ecka, 2004). The contrasting levels of VEGF detected in the CSF compared to controls in different studies may be due to the different ways in which the samples were processed or the different ELISA kits that were used. These issues need to be resolved to enable more accurate, reliable and reproducible measurements of VEGF in CSF to be obtained before we can understand its significance in patients with ALS. 6.3. Post-mortem tissue No significant differences in VEGF levels in the spinal cord was detected between ALS patients and controls in one study, but there were questions on the sensitivity of the ELISA kits used in this study (Nygren et al., 2002). An immunohistochemical study of the expression of VEGF receptors in the spinal cord reported that in control cases, VEGFR-1 was expressed on blood vessels, VEGFR-2 expression was not detected, and there was diffuse VEGFR-3 staining throughout
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the neuropil. In contrast, in ALS cases, VEGFR-1 was expressed on reactive astrocytes, VEGFR-2 at low levels on glia and blood vessels, and VEGFR-3 expression in the neuropil declined with neuronal loss (Spliet et al., 2004). In another study, the expression of VEGFR-2 in the neuropil was reduced in patients with ALS compared with control cases, and a greater proportion of anterior horn cells in ALS cases showed low expression of VEGF and VEGFR-2 compared with controls. In addition, VEGF and NP-1 were also detected on reactive astrocytes (Brockington et al., 2006). Overall, two major conclusions can be made from the above human post-mortem studies. Firstly, the reductions in the expression of VEGF and its major agonist receptor VEGFR-2 in the spinal motor neurons of ALS cases would support the hypothesis that a decline the VEGF neurotrophic effect may be important in the etiology of ALS. Secondly, the expression of VEGF, VEGFR-2 and NP-1 in reactive astrocytes support a possible role of VEGF as a driver of gliosis which is characteristically seen in ALS pathology. A decrease in the level of phospho-Akt expression in lumbar motor neurons of ALS patients compared to controls reinforces the findings in cell culture and rodent studies (discussed above) that Akt is likely to be crucial in regulating motor neuron survival (Dewil et al., 2007). 6.4. Genetics In ALS patients, haplotypes determined by three singlenucleotide polymorphisms (SNPs) ( 2578C/A, 1154G/A, and 634G/C) in the VEGF promoter/leader sequence was associated with a 1.8 times increased risk of sporadic ALS in four distinct European populations from Sweden, England and Belgium (Lambrechts et al., 2003). Another smaller follow-up association study from North America exhibited a 3-fold increased risk for ALS among individuals homozygous for the same AAG or AGG haplotypes (Terry et al., 2004). It has been postulated that this genetic susceptibility may explain why ALS is more commonly reported in airline pilots (Nicholas et al., 1998, 2001). Most airline pilots who are exposed to reduced inspired oxygen in the aircraft cabin do not suffer any untoward consequences. However, if there is an impairment in the ability of the body to adapt to these long periods of low oxygen, then hypoxia-sensitive cells such as motor neurons may be vulnerable to injury, damage and, ultimately, death (Pamphlett, 2002). However, association between VEGF haplotypes and sporadic ALS was not found in cohorts of British (Brockington et al., 2005), Dutch (Van Vught et al., 2005), North American (Chen et al., 2006), Italian (Del Bo et al., 2006), German (Ferna´ndez-Santiago et al., 2006) and Chinese (Zhang et al., 2006) patients. A recent large meta-analysis of over 7000 individuals from eight European and three North American populations confirmed that none of the three common VEGF gene variations ( 2578C/A, 1154G/A, and 634G/C) or any of their haplotype combinations were significantly associated with ALS. However, subgroup analyses by gender revealed that the 2578AA genotype increased the risk of ALS in males (Lambrechts et al., 2008).
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Interestingly, the German study discussed above suggested that the role of VEGF might be dependent on the gender of patients, with female patients at risk of ALS (Ferna´ndez-Santiago et al., 2006), which contradicts the large meta-analysis of the 11 case-control association studies (Lambrechts et al., 2008). Another Chinese study in ALS patients suggested that certain VEGF gene polymorphisms were independently associated with early onset of the disease (Chen et al., 2007). Although deletion in the HRE in the VEGF promoter was the original surprising finding in mice that sparked the interest of VEGF in ALS (Oosthuyse et al., 2001), spontaneous mutations of the HRE have not been detected in ALS patients (Lambrechts et al., 2003; Gros-Louis and Laurent, 2003; Brockington et al., 2005). Furthermore, screening of the transcriptional regulatory regions of the VEGFR-2 gene have found no association between polymorphisms in these regions and ALS (Brockington et al., 2007). There is also no evidence that epigenetic transcriptional silencing of the VEGF gene by methylation, which could affect motor neuron function, plays a role in sporadic ALS (Oates and Pamphlett, 2007). 7. Conclusions Since the original study possibly linking VEGF to ALS in 2001 (Oosthuyse et al., 2001), a huge amount of research has been done in this area. There is reasonably convincing evidence suggesting a role for VEGF in ALS from cell culture. In particular, VEGF has been shown to protect a motor neuronlike cell line (Oosthuyse et al., 2001; Li et al., 2003) and motor neurons (Van Den Bosch et al., 2003; Tolosa et al., 2008) against a variety of insults thought to be important in the pathogenesis of ALS. Likewise, there is good evidence that VEGF is important in ALS from studies in transgenic rodents. VEGF, given via a variety of methods, appears to have the neuroprotective effect of delaying disease progression and prolonging survival in these animals (Azzouz et al., 2004; Zheng et al., 2004; Storkebaum et al., 2005; Tovar-y-Romo et al., 2007). However, the evidence from studies of VEGF in humans, in particular studies of its genetic association, are much less compelling. Of major significance is the finding of a lack of association of VEGF genotypes or haplotypes with ALS in the recent large meta-analysis (Lambrechts et al., 2008), considerably weakening the argument that VEGF is a major factor in causing motor neuron degeneration in ALS. It may be possible that VEGF plays a role in patients who are already predisposed to ALS, with reduced VEGF ‘tipping the balance’ in favour of developing the disease in this susceptible subpopulation. More research is needed to conclusively prove that VEGF has a role in the pathogenesis of ALS. Competing interests None.
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Neuroscience Research 62 (2008) 71–77 www.elsevier.com/locate/neures
Review article
VEGF and ALS Sivakumar Sathasivam * Department of Neurology, The Walton Centre for Neurology & Neurosurgery, Lower Lane, Liverpool L9 7LJ, UK Received 22 May 2008; accepted 17 June 2008 Available online 9 July 2008
Abstract In amyotrophic lateral sclerosis (ALS), an adult-onset progressive degeneration of motor neurons occurring as sporadic and familial disease, there is emerging evidence for and against the role of vascular endothelial growth factor (VEGF), an endothelial cell mitogen crucial for angiogenesis, in its etiopathogenesis. Our understanding of the role of VEGF in ALS has come from studies of both experimental models and human cases. In this article, I have examined in detail the in vitro and in vivo evidence for and against VEGF in ALS, concluding that more compelling evidence is required before we can conclusively link VEGF to ALS in humans. # 2008 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved. Keywords: Amyotrophic lateral sclerosis; Vascular endothelial growth factor; Cell culture studies; Animal models; Human studies; Genetics
Contents 1. 2. 3. 4.
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Amyotrophic lateral sclerosis . . . . The VEGF family . . . . . . . . . . . . First link of VEGF with ALS . . . . Evidence from cell culture studies . 4.1. NSC34 cell line . . . . . . . . . 4.2. Rodent cultures . . . . . . . . . Evidence from animal models . . . . 5.1. Transgenic rodent models . . 5.1.1. Mice . . . . . . . . . . . 5.1.2. Rats . . . . . . . . . . . 5.2. Zebrafish model . . . . . . . . . Evidence from human studies . . . . 6.1. Serum or plasma . . . . . . . . 6.2. Cerebrospinal fluid . . . . . . . 6.3. Post-mortem tissue . . . . . . . 6.4. Genetics . . . . . . . . . . . . . . Conclusions. . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . References . . . . . . . . . . . . . . . . .
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Abbreviations: AMPA, a-amino-3-hydroxy-5-methyl-4-isoxazole propionate; ALS, amyotrophic lateral sclerosis; ALSFRS, amyotrophic lateral sclerosis functional rating scale; CSF, cerebrospinal fluid; Flk-1, vascular endothelial growth factor receptor-2; Flt-1, Fms-related tyrosine kinase 1; Flt-4, Fms-related tyrosine kinase 4; HRE, hypoxia-response element; IGF-1, insulin-like growth factor-1; KDR, kinase insert domain receptor; NP-1, neuropilin-1; NP-2, neuropilin-2; PlGF, placental growth factor; PI3-K, phosphatidylinositol 3-kinase; phospho-Akt, phosphorylated form of Akt; SNP, single-nucleotide polymorphism; TDP-43, TAR DNA-binding protein 43; TNF-a, tumor necrosis factor-a; SOD1, copper/zinc superoxide dismutase; VAPB, vesicle-associated membrane protein; VEGF, vascular endothelial growth factor; VEGFR, vascular endothelial growth factor receptor. * Tel.: +44 151 5298151; fax: +44 151 5295512. E-mail address: [email protected]. 0168-0102/$ – see front matter # 2008 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved. doi:10.1016/j.neures.2008.06.008
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1. Amyotrophic lateral sclerosis Amyotrophic lateral sclerosis (ALS), or Lou Gehrig disease, is a rapidly progressive adult-onset neurodegenerative disorder with an incidence of 1–2 per 100,000 and a prevalence of 4–6 per 100,000 (Cozzolino et al., 2008). Approximately 10% of cases are familial, one fifth of which are caused by mutations in the gene encoding the antioxidant copper/zinc superoxide dismutase (SOD1) on chromosome 21 (Rosen et al., 1993). Several other mutations have been identified in a small number of patients, including (a) alsin, a putative guanine nucleotide factor for GTPase (Yang et al., 2001; Hand et al., 2003), (b) senataxin, a putative DNA/RNA helicase (Chen et al., 2004), and (c) the vesicle-trafficking protein VAPB gene (Nishimura et al., 2004). However, in most familial cases, and in sporadic disease, the cause for ALS remains elusive. The pathogenesis of ALS appears to be a complex interplay of several processes, such as oxidative stress, protein aggregation, mitochondrial dysfunction, excitotoxicity, impaired axonal transport and neuroinflammation, ultimately leading to a programmed mechanism of motor neuron death (Sathasivam and Shaw, 2005; Shaw, 2005). Recently, the TAR DNA-binding protein 43 (TDP-43) was recognized as a major constituent of the neuronal cytoplasmic inclusions seen in degenerating motor neurons in ALS (Neumann et al., 2006). The exact function of TDP-43 remains unclear. TDR-43 mutations have been reported in familial and sporadic ALS in some studies (Sreedharan et al., 2008; Gitcho et al., 2008; Kabashi et al., 2008; Van Deerlin et al., 2008), but these findings are not universal (Guerreiro et al., 2008). 2. The VEGF family Vascular endothelial growth factor (VEGF), a key factor affecting vascular permeability and angiogenesis, was discovered 25 years ago (Senger et al., 1983) and cloned six years after that (Ferrara and Henzel, 1989). The VEGF family consists of six different homologous factors, VEGF-A, placental growth factor (PlGF), VEGF-B, VEGF-C, VEGFD, and VEGF-E. Although VEGF-A, PlGF, VEFG-B, VEGF-D and VEGF-E are important for the growth of blood vessels and VEGF-C mainly affects the development of lymphatic vessels, recent evidence suggests VEGF-A, VEGF-B and VEGF-C directly affect neural cells (Raab and Plate, 2007). The two main classes of receptors for the VEGF family are the tyrosine kinase and the nontyrosine kinase receptors. The former contains three structurally related receptors: VEGFR-1 (Flt-1), VEGFR-2 (KDR/Flk-1), and VEGFR-3 (Flt-4). The nontyrosine receptors consist of neuropilin-1 (NP-1) and neuropilin-2 (NP-2). The members of the VEGF family display different binding patterns to these receptors (Fig. 1) (Takahashi and Shibuya, 2005). As VEGF-A is the major member of the VEGF family that has been studied in ALS, this review will concentrate predominantly on VEGF-A. The gene encoding human VEGF-A (if the isoform designation is not stated, VEGF implies VEGF-A) is located on chromosome 6p21.5 and gives rise to many different isoforms due to alternative splicing (e.g. VEGF121, VEGF145, VEGF165)
Fig. 1. Ligands and receptors of the VEGF family. Members of the VEGF family have different binding patterns to the tyrosine kinase receptors (VEGFR-1, VEGFR-2, VEGFR-3) and nontyrosine kinase neuropilin receptors (NP-1, NP2): (1) VEGFR-1 binds to VEGF-A, VEGF-B and PlGF; (2) VEGFR-2 binds to VEGF-A, VEGF-C, VEGF-D and VEGF-E; (3) VEGFR-3 binds to VEGF-C and VEGF-D only; (4) NP-1 binds to VEGF165, VEGF-B, VEGF-E and PlGF-2; (5) NP-2 binds to the VEGF145 and VEGF165 isoforms of VEGF-A only.
(Bogaert et al., 2006). These isoforms have different molecular mass, solubility, binding affinities for extracellular matrix components and receptor subtypes and biological functions (Matsumoto and Claesson-Welsh, 2001). VEGF binds the two tyrosine kinase receptors VEGFR-1 and VEGFR-2, and the nontyrosine kinase receptors NP-1 and NP-2. VEGFR-2 is the main mediator of the angiogenic, mitogenic and permeabilityenhancing effects of VEGF (Shalaby et al., 1995). Although VEGFR-1 has a 10-fold stronger binding affinity to VEGF than VEGFR-2, the kinase activity of the former is 10-fold lower than that of the latter (Ferrara, 1999). The major function of VEGFR-1 is thought to be as a ‘decoy receptor’ that negatively regulates angiogenesis by preventing the binding of VEGF to VEGFR-2 (Fong et al., 1999; Hiratsuka et al., 1998). VEGFR-3 is not a receptor for VEGF. The other VEGF receptors, neuropilins NP-1 and NP-2, initially described as receptors for semaphorins, are thought to be involved in axon guidance (Neufeld et al., 2002). Among VEGF, NP-1 is a specific receptor for the isoform VEGF165, while NP-2 is specific for VEGF145 and VEGF165. 3. First link of VEGF with ALS The first study suggesting a role of VEGF in ALS was published in 2001 (Oosthuyse et al., 2001). Manipulation of the VEGF gene in mice resulted in a new and unexpected role for VEGF in the pathogenesis of motor neuron degeneration. Mice were generated with a homozygous deletion in the hypoxiaresponse element (HRE) site in the VEGF promoter region (VEGFd/d mice). About three fifths of the mice died before or around birth from vascular abnormalities in the lung. In the 40% of mice that survived, they begun to show symptoms of motor neuron degeneration at around five months of age. By 17 months, these mice had lost approximately 30% of motor neurons in the ventral horns of the spinal cord compared to normal controls. In addition, there was evidence of neuropathological evidence in motor neurones of fewer Nissl bodies, abnormal mitochondria and cell organelles in the early stages,
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and vacuolization, fewer free ribosomes, irregular nuclei, peripheral clumping of chromatic aggregates and marked reactive astrogliosis in the later stages. These changes were similar to those described in the G93A SOD1 transgenic mice, which is a well-established rodent model of ALS. The baseline levels and hypoxic induction of VEGF were reduced in the neural tissue (motor neurons and astrocytes) of the VEGFd/d mice. Furthermore, these mice had a 50% reduction in neural vascular perfusion. These changes in the VEGFd/d mice raised two possible non-mutually exclusive hypotheses on the possible mechanism(s) by which motor neuron degeneration may be influenced in ALS. Inadequate neurotrophic stimulation of motor neurons by VEGF may be one reason. Alternatively, vascular abnormalities due to insufficient VEGF may put motor neurons at risk of late-onset neurodegeneration brought on by chronic ischemia. 4. Evidence from cell culture studies 4.1. NSC34 cell line Physiological concentrations of the VEGF165 isoform has been shown to protect NSC34 cells, a mouse motor neuron-like cell line, against apoptosis induced by tumor necrosis factor-a (TNF-a), hypoxia, oxidative stress and serum deprivation (Oosthuyse et al., 2001). In another study of the same cell line, transfection with mutant G93A SOD1, but not vector control or wild-type SOD1, resulted in increased evidence of oxidative stress and cell death. However, when the cells were pretreated with VEGF, there was a dose-dependent resistance to oxidative injury from hydrogen peroxide, TNF-a and mutant G93A SOD1, shown to be mediated via activation of the antiapoptotic phosphatidylinositol 3-kinase (PI3-K)/Akt pathway (Li et al., 2003). 4.2. Rodent cultures VEGF has been shown to protect motor neurons from serum deprivation- and hypoxia/hypoglycaemia-induced cell death in motor neuron/glial cell cocultures from mouse and rat embryos. In addition, basal VEGF released from cultured glial cells from VEGFd/d mice was significantly lower than glial cells from control mice (Van Den Bosch et al., 2004). However, in the same study, there was no difference in basal survival and sensitivity to excitotoxicity (one of the mechanisms thought to be responsible for motor neuron death in ALS) between motor neuron/glial cell cocultures of G93A mutant SOD1 and wildtype SOD1 mice. In another study using rat spinal cord organotypic cultures, VEGF was shown to protect motor neurons against chronic glutamate-induced excitotoxicity by activating the PI3-K/Akt signal transduction pathway (Tolosa et al., 2008). This study used a model of chronic, slow excitotoxicity, in contrast to the more acute excitotoxic model of Van Den Bosch et al. (2004), which may explain the difference in vulnerability of motor neurons to excitotoxicity observed in the two studies.
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5. Evidence from animal models 5.1. Transgenic rodent models 5.1.1. Mice Crossbreeding experiments have shown that G93A SOD1 VEGFd/d mice develop muscle weakness and die earlier than their G93A SOD1 VEGF+/+ littermates. In addition, compared to wild-type mice, the G93A SOD1 VEGFd/d mice developed more severe spinal ischemia-induced paralysis, and the administration of VEGF resulted in better protection against ischemic motor neuron death in the latter group. These findings suggest that VEGF is a modifier of motor neuron degeneration in ALS (Lambrechts et al., 2003). More supportive evidence of the role of VEGF comes from another set of crossbreeding experiments which have demonstrated that overexpression of VEGF delays neurodegeneration and increases survival in ALS mice. G93A SOD1/VEGF+/+ double-transgenic mice have delayed motor neuron loss, delayed onset of motor impairment and longer survival than G93A SOD1 single transgenics (Wang et al., 2007). There also appears to be a downregulation of VEGF mRNA expression in the spinal cords of G93A SOD1 mice compared to wild-type SOD1 age-matched mice which occurs from before disease onset (Lu et al., 2007). This decrease in VEGF RNA could be due to motor neuron death or an overall decline in production. Intraperitoneal injection of VEGF has been shown to delay disease progression and increase survival in G93A SOD1 transgenic mice (Zheng et al., 2004). The same group showed that the possible mechanisms of neuroprotection may be due to reduced astrogliosis in the spinal cord or increased neuromuscular junctions in the muscles of these mice (Zheng et al., 2007). Furthermore, the injection of a VEGF-expressing lentiviral vector into muscles of the G93A SOD1 transgenic mouse model delayed disease onset and slowed disease progression in these mice. Treatment was effective even when given at the onset of paralysis, which is relevant to humans where most ALS cases occur sporadically. Extensive reporter gene expression in the spinal cord and brainstem in the mice confirmed the efficient retrograde transport of the VEGF gene (Azzouz et al., 2004). In another study, VEGF concentrations in the spinal cord of G93A SOD1 and wild-type SOD1 mice were not significantly different in normal conditions. Hypoxia induced a significant upregulation of VEGF in the mutant mice, although this did not affect survival in the mice, arguing against the importance of VEGF in ALS (Van Den Bosch et al., 2004). 5.1.2. Rats In a G93A SOD1 transgenic rat model of ALS, VEGF was shown to be reduced prior to symptom onset and progressively during the course of the disease compared to non-transgenic littermates. It is unclear if this occurrence is the underlying cause of motor neuron degeneration or a side effect of neuroinflammation in ALS (Xie et al., 2004). Intracerebroventricular delivery of VEGF in G93A SOD1 transgenic rats delays the onset of weakness, improves motor
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function and prolongs survival in these animals. This treatment resulted in delayed motor neuron degeneration in the brainstem, cervical and lumbar spinal cord of these rodents. There was also a correlation between the spatial gradient of VEGF levels and the therapeutic benefit on the disease subtype. This is important because it implies, for example, individuals with bulbar-onset ALS would benefit more from intracerebroventricular delivery of VEGF, while lumbar-onset cases would benefit more from an intrathecal infusion at the lumbar region (Storkebaum et al., 2005). The mechanism of neuroprotection appears to be, at least in part, due to attenuation of the loss of the active, phosphorylated form of Akt (phospho-Akt) in the G93A SOD1 mutant rats (Dewil et al., 2007). Upregulation of phospho-Akt induced by insulin-like growth factor-1 (IGF-1) has been shown to delay disease onset and increase survival in G93A SOD1 mice (Nagano et al., 2005). Intrathecal administration of VEGF into the lumbar region of the spinal cord resulted in the prevention of paralysis in rats which were co-infused with the glutamate receptor agonist aamino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) which is known to induce excitotoxicity, one of the mechanisms thought to be responsible for motor neuron death in ALS. The clinical effect of VEGF correlated with a greater than 75% reduction in lumbar motor neuron loss in the treated rats, suggesting neuroprotection by VEGF against excitotoxicity (Tovar-y-Romo et al., 2007). 5.2. Zebrafish model In a zebrafish model overexpressing mutant SOD1, upregulation of VEGF I the zebrafish embryo rescued the motor axonopathy induced by the mutant gene, while knockdown of VEGF expression aggravated it (Lemmens et al., 2007). Although ALS is an adult-onset disease, abnormalities of axonal transport have been described in motor neurons cultured from G93A SOD1 mouse embryos (Kieran et al., 2005). Therefore, findings in the zebrafish model may be relevant. 6. Evidence from human studies 6.1. Serum or plasma One study has shown that serum VEGF levels were significantly higher in ALS patients compared to controls. However, there was no relationship between serum VEGF levels and gender, onset type (bulbar or spinal) or the ALS Functional Rating Scale (ALSFRS). The authors of the study speculated that this increase was could be the result of increased synthesis of VEGF in skeletal muscle, which is affected in ALS, in response to hypoxia (Nygren et al., 2002). However, in another study, plasma VEGF levels were shown to be about 50% lower than patients with sporadic ALS, compared to unaffected spouses. The VEGF levels in this study did not correlate with age of onset, onset type, or disease progression (Lambrechts et al., 2003). Other studies have not found a difference in serum or plasma VEGF levels between ALS
patients and controls (Devos et al., 2004; Just et al., 2007; Moreau et al., 2006). Therefore, the usefulness of measuring serum or plasma VEGF levels in ALS is questionable, as results obtained appear variable. 6.2. Cerebrospinal fluid One study failed to detect VEGF in the cerebrospinal fluid (CSF) of patients with ALS, but this may be due to the low sensitivity of the ELISA kits used (Nygren et al., 2002). CSF VEGF levels were demonstrated to be lower in ALS patients compared to those in normal control subjects and controls with other neurological disorders, with no significant difference between the latter two groups (Devos et al., 2004). The same group found that CSF VEGF levels were significantly lower in hypoxemic ALS patients than in both normoxemic ALS patients and hypoxemic neurological controls. In addition, there was no significant difference between CSF VEGF levels in normoxemic ALS patients and normoxemic neurological controls. There was a paradoxical lack of CSF VEGF upregulation in hypoxemic ALS patients compared to hypoxemic neurological controls, pointing to a dysregulation of the hypoxia-dependent CSF VEGF response pathway (Just et al., 2007; Moreau et al., 2006). Two other studies have failed to replicate the above findings. In one, there was no difference in CSF VEGF levels between ALS patients and normal controls or controls with other neurological disorders (Nagata et al., 2007). These findings were somewhat replicated by another study which showed no difference in CSF VEGF levels when all ALS patients were compared to controls. However, subgroup analysis showed that CSF VEGF levels were significantly increased in patients with limb-onset ALS compared to patients with bulbar-onset ALS, and in patients with long disease duration compared to patients with short disease duration. However, the type of ALS onset did not significantly influence disease duration. The author of the study speculated that the increase in CSF VEGF may have a protective role against glutamate-mediated excitotoxicity and oxidative damage of motor neurons (Iłz˙ecka, 2004). The contrasting levels of VEGF detected in the CSF compared to controls in different studies may be due to the different ways in which the samples were processed or the different ELISA kits that were used. These issues need to be resolved to enable more accurate, reliable and reproducible measurements of VEGF in CSF to be obtained before we can understand its significance in patients with ALS. 6.3. Post-mortem tissue No significant differences in VEGF levels in the spinal cord was detected between ALS patients and controls in one study, but there were questions on the sensitivity of the ELISA kits used in this study (Nygren et al., 2002). An immunohistochemical study of the expression of VEGF receptors in the spinal cord reported that in control cases, VEGFR-1 was expressed on blood vessels, VEGFR-2 expression was not detected, and there was diffuse VEGFR-3 staining throughout
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the neuropil. In contrast, in ALS cases, VEGFR-1 was expressed on reactive astrocytes, VEGFR-2 at low levels on glia and blood vessels, and VEGFR-3 expression in the neuropil declined with neuronal loss (Spliet et al., 2004). In another study, the expression of VEGFR-2 in the neuropil was reduced in patients with ALS compared with control cases, and a greater proportion of anterior horn cells in ALS cases showed low expression of VEGF and VEGFR-2 compared with controls. In addition, VEGF and NP-1 were also detected on reactive astrocytes (Brockington et al., 2006). Overall, two major conclusions can be made from the above human post-mortem studies. Firstly, the reductions in the expression of VEGF and its major agonist receptor VEGFR-2 in the spinal motor neurons of ALS cases would support the hypothesis that a decline the VEGF neurotrophic effect may be important in the etiology of ALS. Secondly, the expression of VEGF, VEGFR-2 and NP-1 in reactive astrocytes support a possible role of VEGF as a driver of gliosis which is characteristically seen in ALS pathology. A decrease in the level of phospho-Akt expression in lumbar motor neurons of ALS patients compared to controls reinforces the findings in cell culture and rodent studies (discussed above) that Akt is likely to be crucial in regulating motor neuron survival (Dewil et al., 2007). 6.4. Genetics In ALS patients, haplotypes determined by three singlenucleotide polymorphisms (SNPs) ( 2578C/A, 1154G/A, and 634G/C) in the VEGF promoter/leader sequence was associated with a 1.8 times increased risk of sporadic ALS in four distinct European populations from Sweden, England and Belgium (Lambrechts et al., 2003). Another smaller follow-up association study from North America exhibited a 3-fold increased risk for ALS among individuals homozygous for the same AAG or AGG haplotypes (Terry et al., 2004). It has been postulated that this genetic susceptibility may explain why ALS is more commonly reported in airline pilots (Nicholas et al., 1998, 2001). Most airline pilots who are exposed to reduced inspired oxygen in the aircraft cabin do not suffer any untoward consequences. However, if there is an impairment in the ability of the body to adapt to these long periods of low oxygen, then hypoxia-sensitive cells such as motor neurons may be vulnerable to injury, damage and, ultimately, death (Pamphlett, 2002). However, association between VEGF haplotypes and sporadic ALS was not found in cohorts of British (Brockington et al., 2005), Dutch (Van Vught et al., 2005), North American (Chen et al., 2006), Italian (Del Bo et al., 2006), German (Ferna´ndez-Santiago et al., 2006) and Chinese (Zhang et al., 2006) patients. A recent large meta-analysis of over 7000 individuals from eight European and three North American populations confirmed that none of the three common VEGF gene variations ( 2578C/A, 1154G/A, and 634G/C) or any of their haplotype combinations were significantly associated with ALS. However, subgroup analyses by gender revealed that the 2578AA genotype increased the risk of ALS in males (Lambrechts et al., 2008).
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Interestingly, the German study discussed above suggested that the role of VEGF might be dependent on the gender of patients, with female patients at risk of ALS (Ferna´ndez-Santiago et al., 2006), which contradicts the large meta-analysis of the 11 case-control association studies (Lambrechts et al., 2008). Another Chinese study in ALS patients suggested that certain VEGF gene polymorphisms were independently associated with early onset of the disease (Chen et al., 2007). Although deletion in the HRE in the VEGF promoter was the original surprising finding in mice that sparked the interest of VEGF in ALS (Oosthuyse et al., 2001), spontaneous mutations of the HRE have not been detected in ALS patients (Lambrechts et al., 2003; Gros-Louis and Laurent, 2003; Brockington et al., 2005). Furthermore, screening of the transcriptional regulatory regions of the VEGFR-2 gene have found no association between polymorphisms in these regions and ALS (Brockington et al., 2007). There is also no evidence that epigenetic transcriptional silencing of the VEGF gene by methylation, which could affect motor neuron function, plays a role in sporadic ALS (Oates and Pamphlett, 2007). 7. Conclusions Since the original study possibly linking VEGF to ALS in 2001 (Oosthuyse et al., 2001), a huge amount of research has been done in this area. There is reasonably convincing evidence suggesting a role for VEGF in ALS from cell culture. In particular, VEGF has been shown to protect a motor neuronlike cell line (Oosthuyse et al., 2001; Li et al., 2003) and motor neurons (Van Den Bosch et al., 2003; Tolosa et al., 2008) against a variety of insults thought to be important in the pathogenesis of ALS. Likewise, there is good evidence that VEGF is important in ALS from studies in transgenic rodents. VEGF, given via a variety of methods, appears to have the neuroprotective effect of delaying disease progression and prolonging survival in these animals (Azzouz et al., 2004; Zheng et al., 2004; Storkebaum et al., 2005; Tovar-y-Romo et al., 2007). However, the evidence from studies of VEGF in humans, in particular studies of its genetic association, are much less compelling. Of major significance is the finding of a lack of association of VEGF genotypes or haplotypes with ALS in the recent large meta-analysis (Lambrechts et al., 2008), considerably weakening the argument that VEGF is a major factor in causing motor neuron degeneration in ALS. It may be possible that VEGF plays a role in patients who are already predisposed to ALS, with reduced VEGF ‘tipping the balance’ in favour of developing the disease in this susceptible subpopulation. More research is needed to conclusively prove that VEGF has a role in the pathogenesis of ALS. Competing interests None.
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