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Comparative Study
. 2011 May 25;31(21):7682-90.
doi: 10.1523/JNEUROSCI.0239-11.2011.

Spinal vascular endothelial growth factor induces phrenic motor facilitation via extracellular signal-regulated kinase and Akt signaling

Erica A Dale-Nagle  1 , Irawan Satriotomo, Gordon S Mitchell
Affiliations
Comparative Study

Spinal vascular endothelial growth factor induces phrenic motor facilitation via extracellular signal-regulated kinase and Akt signaling

Erica A Dale-Nagle et al. J Neurosci. .

Abstract

Although vascular endothelial growth factor (VEGFA-165) is primarily known for its role in angiogenesis, it also plays important neurotrophic and neuroprotective roles for spinal motor neurons. VEGFA-165 signals by activating its receptor tyrosine kinase VEGF receptor-2 (VEGFR-2). Because another growth/trophic factor that signals via a receptor tyrosine kinase (brain derived neurotrophic factor) elicits a long-lasting facilitation of respiratory motor activity in the phrenic nerve, we tested the hypothesis that VEGFA-165 elicits similar phrenic motor facilitation (pMF). Using immunohistochemistry and retrograde labeling techniques, we demonstrate that VEGFA-165 and VEGFR-2 are expressed in identified phrenic motor neurons. Furthermore, intrathecal VEGFA-165 administration at C4 elicits long-lasting pMF; intraspinal VEGFA-165 increased integrated phrenic nerve burst amplitude for at least 90 min after injection (53.1 ± 5.0% at 90 min; p < 0.001). Intrathecal VEGFA-165 increased phosphorylation (and presumed activation) of signaling molecules downstream from VEGFR-2 within the phrenic motor nucleus, including ERK (1.53 ± 0.13 vs 1.0 ± 0.05 arbitrary units in control rats; p < 0.05) and Akt (2.16 ± 0.41 vs 1.0 ± 0.41 arbitrary units in control rats; p < 0.05). VEGF-induced pMF was attenuated by the MEK/ERK inhibitor U0126 [1,4-diamino-2,3-dicyano-1,4-bis(o-aminophenylmercapto)butadiene] and was abolished by the phosphotidinositol 3 kinase/Akt inhibitor LY294002 [2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride], demonstrating that ERK mitogen-activated protein kinases and Akt are both required for full expression of VEGF-induced pMF. This is the first report that VEGFA-165 elicits plasticity in any motor system. Furthermore, because VEGFA-165 expression is hypoxia sensitive, it may play a role in respiratory plasticity after prolonged exposures to low oxygen.

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Figures

Figure 1.
Figure 1.
Representative photomicrographs of VEGF immunostaining in C4 phrenic motor neurons. A, DAB staining revealed VEGF expression in large, presumptive phrenic motor neurons (small black box) and interneurons. B, Higher magnification of small black box from A. C, CtB was injected intrapleurally to localize phrenic motor neurons (red cells in ventral horn). D–F, VEGF is expressed in phrenic motor neurons (note merged image of red CtB back-labeling and VEGF protein coexpression) but is also found in the surrounding neuropil. Sections incubated without primary or secondary antibodies served as negative controls. Scale bars: A, C, 400 μm; B, D–F, 50 μm.
Figure 2.
Figure 2.
Representative images of VEGF receptor-2 immunostaining in C4 phrenic motor neurons. A, DAB staining revealed VEGFR-2 expression in large, presumptive phrenic motor neurons (small black box) and interneurons. B, Higher magnification of small black box from A. C, Phrenic motor neurons (red cells in ventral horn) were back-labeled with CtB. D–F, VEGFR-2 is expressed in phrenic motor neurons (note merged image of red CtB back-labeling and VEGFR-2 protein coexpression) and in nearby unlabeled neurons. Sections incubated without primary or secondary antibodies served as negative controls. Scale bars: A, C, 400 μm; B, D–F, 50 μm.
Figure 3.
Figure 3.
Phospho-ERK and phospho-Akt expression in C4 phrenic motor neurons and upregulation after intrathecal VEGF injection. A, Phospho-ERK (far left panels, dark brown staining) is expressed in C4 phrenic motor neurons; staining tends to be punctate and bouton like. B, Phospho-Akt protein is colocalized with CtB-back-labeled phrenic motor neurons but appears to have a more dispersed, cytoplasmic distribution (red fluorescence). Both phospho-ERK and phospho-Akt staining increased after intrathecal VEGF injections (see Results). Sections incubated without primary or secondary antibodies served as negative controls. Data are means ± 1 SEM. *p < 0.05 versus vehicle. Scale bar, 100 μm.
Figure 4.
Figure 4.
Intrathecal VEGF elicits long-lasting phrenic motor facilitation. A, Representative, compressed phrenic neurograms showing either pMF after VEGF injection (arrow) or a lack of facilitation after vehicle injections (aCSF plus bovine serum albumen; arrowhead). B, The amplitude of integrated phrenic bursts increases above baseline after injection of 10 μl (100 ng) of VEGF (n = 10; filled circles) and is also significantly greater than vehicle controls at the same time point (10 μl; n = 9; open circles). All values are expressed as percentage changes in phrenic burst amplitude from baseline. Mean values ± 1 SEM. *p < 0.001, significantly different from baseline; †p < 0.003, significantly different from vehicle at the same time point.
Figure 5.
Figure 5.
VEGF-induced phrenic motor facilitation requires spinal ERK and Akt activation. A, Spinal VEGF elicits pMF (gray dashed line; #p < 0.001 indicates significant difference from both U0126 plus VEGF and U0126 alone, n = 10). Pretreatment with the MEK inhibitor U0126 partially blocks VEGF-induced pMF; phrenic amplitude increases versus baseline by 60 min after injection (n = 7; *p < 0.006); however, pMF is attenuated starting 30 min after VEGF (filled triangles; n = 7). U0126 alone had no effect on phrenic motor output versus baseline or vehicle controls at the same time points (open triangles; n = 3); phrenic motor output was significantly lower than U0126 plus VEGF-treated rats (†p < 0.007) at the 90 min time point. B, Gray, dashed line shows VEGF-induced pMF. After pretreatment with the PI3K inhibitor LY294002, pMF is abolished by 60 min after VEGF injection (filled squares; n = 6). LY294002 alone has no effect on phrenic nerve activity (open squares; n = 3). #p < 0.005 indicates significant difference in VEGF-injected rats versus those pretreated with inhibitor and versus inhibitor alone (all).
Figure 6.
Figure 6.
Effects of VEGF administration on phrenic burst frequency. A, Intrathecal VEGF elicits a brief facilitation in burst frequency versus baseline values (†p < 0.01; n = 10). Although frequency facilitation is demonstrated at the 30 and 60 min time points, this facilitation is absent by 90 min after VEGF injection (*p < 0.04 vs vehicle controls; n = 10). Frequency facilitation is a small and variable phenomenon, and, thus, these results are difficult to interpret. B, C, There are no significant increases in nerve burst frequency in any other group (vs baseline; all p > 0.05).
Figure 7.
Figure 7.
Working model of VEGF-induced pMF. VEGF binds its most common receptor, VEGFR-2, a receptor tyrosine kinase. Downstream signaling includes both MEK/ERK and PI3K/Akt pathways. These cascades are required for full expression of VEGF-induced pMF because PI3K (LY294002) inhibitors abolish pMF, whereas the MEK inhibitor (U0126) attenuates VEGF-induced pMF. Mechanisms downstream from Akt and ERK are unknown. However, pMF may result from increased glutamate receptor insertion on the postsynaptic membrane between premotor and phrenic motor neurons. pMF may also arise from the ability of phosphorylated ERK to alter membrane excitability, such as changes in sodium channel gating properties that increase the likelihood of phrenic motor neuron firing (Stamboulian et al., 2010).
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References

    1. Acker T, Beck H, Plate KH. Cell type specific expression of vascular endothelial growth factor and angiopoietin-1 and -2 suggests an important role of astrocytes in cerebellar vascularization. Mech Dev. 2001;108:45–57. - PubMed
    1. Atkins CM, Selcher JC, Petraitis JJ, Trzaskos JM, Sweatt JD. The MAPK cascade is required for mammalian associative learning. Nat Neurosci. 1998;1:602–609. - PubMed
    1. Bach KB, Mitchell GS. Hypoxia-induced long-term facilitation of respiratory activity is serotonin dependent. Respir Physiol. 1996;104:251–260. - PubMed
    1. Baker-Herman TL, Mitchell GS. Determinants of frequency long-term facilitation following acute intermittent hypoxia in vagotomized rats. Respir Physiol Neurobiol. 2008;162:8–17. - PMC - PubMed
    1. Baker-Herman TL, Fuller DD, Bavis RW, Zabka AG, Golder FJ, Doperalski NJ, Johnson RA, Watters JJ, Mitchell GS. BDNF is necessary and sufficient for spinal respiratory plasticity following intermittent hypoxia. Nat Neurosci. 2004;7:48–55. - PubMed

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