Vascular endothelial growth factor B (VEGF-B) is up-regulated and exogenous VEGF-B is neuroprotective in a culture model of Parkinson's disease
© Falk et al; licensee BioMed Central Ltd. 2009
Received: 3 June 2009
Accepted: 10 December 2009
Published: 10 December 2009
Parkinson's disease (PD) results from the degeneration of dopaminergic neurons in the substantia nigra and the consequent deficit of dopamine released in the striatum. Current oral dopamine replacement or surgical therapies do not address the underlying issue of neurodegeneration, they neither slow nor halt disease. Neurotrophic factors have shown preclinical promise, but the choice of an appropriate growth factor as well as the delivery has proven difficult. In this study, we used a rotenone rat midbrain culture model to identify genes that are changed after addition of the neurotoxin. (1) We challenged rat midbrain cultures with rotenone (20 nM), a pesticide that has been shown to be toxic for dopaminergic neurons and that has been a well-characterized model of PD. A gene chip array analysis demonstrated that several genes were up-regulated after the rotenone treatment. Interestingly transcriptional activation of vascular endothelial growth factor B (VEGF-B) was evident, while vascular endothelial growth factor A (VEGF-A) levels remained unaltered. The results from the gene chip array experiment were verified with real time PCR and semi-quantitative western analysis using β-actin as the internal standard. (2) We have also found evidence that exogenously applied VEGF-B performed as a neuroprotective agent facilitating neuron survival in an even more severe rotenone culture model of PD (40 nM rotenone). VEGF-B has very recently been added to the list of trophic factors that reduce effects of neurodegeneration, as was shown in an in vivo model of motor neuron degeneration, while lacking potential adverse angiogenic activity. The data of an in vivo protective effect on motor neurons taken together with the presented results demonstrate that VEGF-B is a new candidate trophic factor distinct from the GDNF family of trophic factors. VEGF-B is activated by neurodegenerative challenges to the midbrain, and exogenous application of VEGF-B has a neuroprotective effect in a culture model of PD. Strengthening this natural protective response by either adding exogenous VEGF-B or up-regulating the endogenous VEGF-B levels may have the potential to be a disease modifying therapy for PD. We conclude that the growth factor VEGF-B can improve neuronal survival in a culture model of PD.
The two most pressing therapeutic challenges in PD are to (1) provide a stable level of dopamine replacement and (2) slow disease progression [1–4]. Neurotrophic growth factors such as the glial-derived neurotrophic factor (GDNF), neurturin, FGF-2 and others, have shown great promise in experimental models of PD [5, 6]. The hope is that using these factors in human PD could provide a potent disease-modifying therapy; however, clinical development of these agents is problematic . Intracerebroventricular administration of GDNF via a micro pump  and neurturin delivery via viral vector-mediated gene transfer  ultimately failed in Phase II clinical trials. These disappointing results despite robust preclinical data could be due to problems with the delivery method or choice of neurotrophic factor.
One path to identify new potential modifiers of PD is by using gene chip arrays utilizing in vitro and in vivo models of PD. In this study, to identify candidate genes, we challenged rat midbrain cultures with rotenone, a pesticide that has been shown to be toxic for dopaminergic neurons and that has been a well-characterized model of PD [9, 10].
Timed-pregnant Sprague-Dawley rats were anesthetized by exposure to CO2 and sacrificed. Fetuses were removed at E17, anesthetized by cooling on ice, decapitated, and the midbrain was dissected. Details of the methods have been reported [11, 12]. Tissue culture media and sera were obtained from Gibco-BRL, Grand Island, NY. The procedure was approved by the IACUC at the University of Arizona and conformed to the guidelines of the National Institutes of Health. The number of animals used and their suffering was minimized. We developed protocols in vitro using rotenone (Sigma-Aldrich, St. Louis, MO) to produce damage to dopaminergic neurons by adding it at the indicated concentrations at day 6 in culture. In previous work , an initial rotenone concentration response curve was established and the LD50 for 5 day exposure was found to be 25-50 nM. We chose to look at a slightly lower concentration of rotenone (20 nM), since we were interested in changes in mRNA before the cells are lost. We isolated the mRNA of 11 day old cultures 5 days after the rotenone challenge, and of untreated control cultures, before performing a gene array analysis (n = 3 separate experiments). The RNA isolation was done with the Qiagen RNA kit (Qiagen, Valencia, CA), using the manufacturers protocol. The gene array analysis was carried out using the GeneChip Rat Genome 230 2.0 Array (Affymetrics, Santa Clara, CA) and standard procedure. Data was analysed using the limma software package [13, 14].
List of the highest up-regulated genes in the gene array after rotenone treatment
adj. P value
With all techniques we saw a significant increase of VEGF-B after rotenone challenge, while there wass no significant change in VEGF-A mRNA level. This struck our interest in light of the recently published results from Poesen et al., 2008 , proving VEGF-B to be an inducible trophic factor in a model of neurodegeneration of motor neurons.
VEGF-B is a member of the VEGF-family of trophic factors [16, 17]. VEGF-A is the best studied member due to its strong angiogenic activity and potential for cardiovascular and cancer research . VEGF-A is up-regulated in the substantia nigra but not in the striatum of PD patients . VEGF-B, on the other hand, stimulates proliferation of neuronal cultures in vitro , and has not been investigated in PD. VEGF-B has also recently been shown to be neuroprotective in motor neuron degeneration in vivo . It had previously been shown to inhibit apoptosis and having only minimal angiogenic activity  while being critical to survival of the blood vessels . This is important since efforts to use VEGF-A as a neurotrophic or a neuroprotective factor had been hampered by the strong angiogenic activity. Although neuroprotective effects of VEGF-A expressed by cells or viral vectors in models of PD were reported [23–25], they were over-shadowed by detrimental effects such as edema, ventriculomegaly  and disruption of the blood brain barrier . These negative side effects were not seen when using VEGF-B in vivo . Interestingly, the neuroprotective effect of VEGF-B in vivo was also restricted to pathological conditions. Mice lacking VEGF-B displayed normal motor behavior, but, when challenged in a model of neurodegeneration, they displayed a more severe form of motor degeneration . Loss of VEGF-B also enlarges stroke . These data suggest that VEGF-B plays a role in compensations for natural disease processes of the nervous system. It does so by binding to its only receptor VEGFR1 , a receptor with not yet delineated downstream signaling events. Further analysis of our gene array data showed the only up-regulated gene with known interaction with VEGF-B was matrix metallopeptidase 9 (MMP9). VEGFR1 signaling has been previously linked to the induction of MMP9 in lung endothelial cells  suggesting a potential role of MMP9 in the effects of VEGF-B that should be further investigated.
The fact that dopaminergic neurons make up less than 5% of the cells in our midbrain preparation argues against an up-regulation of VEGF-B only in dopaminergic neurons. We therefore hypothesize that the VEGF-B may be released by the astroglia in the preparation rather than the dopaminergic neurons themselves. Further evidence supporting this hypothesis comes from recent experiments where rotenone treatment in vivo did not cause transcriptional activation of VEGF-B in dopaminergic neurons analyzed after laser-capture microdissection . In addition, under healthy conditions, motor neurons express VEGF-B to maintain neuroprotection in an autocrine manner, whereas astrocytes may express VEGF-B after injury to maintain survival in a paracrine manner . A similar paracrine scenario is possible in the PD-like neurodegeneration induced in our model system. This hypothesis should be tested in the future. Taken together with our data showing an up-regulation of VEGF-B after rotenone challenge to rat midbrain cultures, these data lead to our hypothesis that VEGF-B may act as an endogenous trophic factor against the neurodegenerative insult in a model of PD.
Neurotrophic factors are promising agents to provide disease modification for PD. This report demonstrates that VEGF-B is a new candidate trophic factor distinct from the GDNF-family of trophic factors, and is activated by neurodegenerative challenges to the midbrain. Strengthening this natural protective response by either adding exogenous VEGF-B or up-regulating the endogenous VEGF-B levels may have the potential to be a disease modifying therapy for PD. Based on the literature, the VEGF-B186 isoform is more diffusible than VEGF-B167 in vivo [15, 31], and therefore may have a greater therapeutic potential. We conclude that the growth factor VEGF-B can improve neuronal survival in a culture model of PD.
vascular endothelial growth factor A
vascular endothelial growth factor B
vascular endothelial growth factor receptor 1
glial-derived neurotrophic factor
matrix metallopeptidase 9
Institutional Animal Care and Use Committee.
We thank Alexander D. McCourt and Brandon J. Yee for expert technical assistance; as well as Dr. David Mount at the Bioinformatics Shared Service, Arizona Cancer Center, and the Arizona Research Laboratories, Division of Biotechnology, Genomic Analysis and Technology Core Facility http://uagc.arl.arizona.edu/ for conducting the gene array analysis and the real time PCR. This work was supported by grants from the University of Arizona UPERRC program (S.J.S.) and the Arizona Biomedical Research Council (T.F.)
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