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VEGF is required for the maintenance of dorsal root ganglia blood vessels but not neurons during development

MATTHEW E. KUTCHER^*, MICHAEL KLAGSBRUN^*,,1 and RONI MAMLUK^*

^* Vascular Biology Program and Department of Surgery, and
Department of Pathology, Children’s Hospital and Harvard Medical School, Boston, Massachusetts, USA

¹ Correspondence: Vascular Biology Program, Children’s Hospital, Karp Family Research Laboratories Room 12.210, 300 Longwood Ave., Boston, MA 02115. E-mail: Michael.klagsbrun{at}childrens.harvard.edu

SPECIFIC AIMS

Recent reports indicate that vascular endothelial growth factor (VEGF), a potent regulator of angiogenesis and endothelial cell function, may play a previously unrecognized role in the nervous system; however, the role of VEGF in the sensory nervous system remains unclear. The aim of this study was to determine the expression and function of VEGF and its receptors in mouse dorsal root ganglia (DRG) for the purpose of discerning how capillaries, neurons, or both might differentially depend on VEGF signaling in sensory ganglia.

PRINCIPAL FINDINGS

1. VEGF is expressed by neurons and satellite cells of the DRG
Expression of the 164- and 120-amino acid isoforms of VEGF throughout embryonic and postnatal stages of development was demonstrated by RT-PCR. The cell type specificity was determined using DRG from mice expressing nuclear-targeted ß-galactosidase adjoined to the 3'UTR of the VEGF gene. X-gal staining to detect the transgene localized expression of VEGF to cells with neuronal and satellite cell-like morphology. The identity of these cells was confirmed by fluorescent double-staining using antibodies to ß-galactosidase and either ßIII tubulin (a neurofilament characteristic of all neuronal cells), glial fibrillary acidic protein (GFAP; an intermediate filament found in astrocytes, oligodendrocytes, and DRG satellite cells), CD31 (an endothelial cell marker), or desmin (a pericyte marker). Using laser-scanning confocal microscopy, ß-galactosidase/VEGF expression was detected in the nuclei of both ßIII tubulin-positive (neuronal) and GFAP-positive (satellite) cells, but was absent in CD31-positive (endothelial) and desmin-positive (pericyte) cells.

2. The VEGF receptor VEGFR-2 is specifically expressed by endothelial cells of the DRG
VEGF binds to multiple receptors expressed by endothelial cells; however, other cell types also express VEGF receptors. To determine whether other, nonendothelial cells in the DRG also express VEGF receptors, we analyzed endothelial and nonendothelial cells of the DRG as sorted by FACS. Endothelial cells (EC) were selected as CD31-positive and hematopoietic lineage marker CD45-negative (CD31⁺CD45^–, 3.54% of total viable cells; Fig. 1 A, green rectangle); the remaining cells were collected as the nonendothelial fraction (Non-EC; including both CD31^– and CD31⁺CD45⁺ populations). RT-PCR analysis for VEGF receptors and cell type-specific markers (Fig. 1B ) shows that endothelial cells of the DRG expressed VEGFR-1, VEGFR-2, and VEGFR-3, whereas both endothelial and nonendothelial populations expressed NRP1. NRP2 was not detected in either population. The expression of CD31 in the nonendothelial fraction can be attributed to CD31 expression by some hematopoietic cells of the CD31⁺CD45⁺ population, whereas the expression of the endothelial-specific marker Tie2 confirms that endothelial cells are present in the endothelial fraction only. Double-staining immunohistochemistry using highly specific antibodies for VEGFR-2 and NRP1 confirmed these results (Fig. 1C ). VEGFR-2 expression colocalized exclusively with CD31-positive endothelial cells (Fig. 1C , panel a) but not ßIII tubulin-positive neurons (Fig. 1C , panel b). In comparison, NRP1 expression was seen in both endothelial and neuronal cells (Fig. 1C , panels c and d, respectively). Similar colocalization patterns were seen in newborn and adult DRG. Thus, both RT-PCR and immunohistochemistry demonstrate that the expression of VEGFR-2 in the DRG is endothelial-specific.

View larger version (50K): [in this window] [in a new window]   Figure 1. VEGFR-2 is expressed exclusively by endothelial cells in the DRG, whereas NRP1 is expressed by both endothelial cells and neurons. A) DRG from E14.5 mice were dissected, dissociated by collagenase digestion, and separated by FACS. The CD31+CD45– population represents purified endothelial cells (EC, 3.54%); all other viable cells were pooled and analyzed as a single fraction (Non-EC). B) RT-PCR was performed on the two FACS-isolated cell fractions, showing that endothelial cells in the DRG are Tie2 positive and express VEGFR-1, VEGFR-2, and VEGFR-3. NRP1 is expressed in both the endothelial and nonendothelial fractions. C) Two-color immunostaining of frozen sections from E14.5 DRG demonstrating that VEGFR-2 staining colocalizes with CD31-positive endothelial cells (a) but is not expressed on ßIII tubulin-positive neurons (b), whereas NRP1 staining colocalizes with both endothelial cells (c) and neurons (d).

3. VEGF sequestration and VEGFR-2 kinase inhibition in DRG explants causes rapid disruption of the capillary network in embryonic but not postnatal DRG
To study the role of locally produced VEGF in the DRG, we used a tissue explant culture system. VEGF production in the cultured DRG explants was confirmed by PCR and murine VEGF-specific ELISA. Immunostaining for CD31 and VEGFR-2 revealed intact capillary network structure and continued endothelial-specific expression of VEGFR-2 after 24 h of culture. The vascular structure of cultured DRG was morphologically similar to that of uncultured DRG; the uniform structure and distribution of the capillary network was also similar in both E14.5 and P1 DRG. Thus, explant culture preserves a robust capillary network, endogenous VEGF production, and endothelial-specific expression of VEGFR-2, providing a useful model for further investigation of neuronal and satellite cell-derived VEGF action in the DRG.

To analyze the direct signaling roles of VEGF in embryonic DRG, we inhibited VEGF signaling using two complimentary approaches: sequestering VEGF from its receptors by treatment with Fc-dimerized soluble VEGFR-1 (Fc-sFlt), and inhibiting VEGFR-2 signaling using a VEGFR-2-specific kinase inhibitor, PTK787 ZK222584. CD31 immunostaining and inspection of blood vessel morphology in PTK787-treated DRG explants reveals loss of CD31-positive endothelial cells coupled with loss of vessel connectivity and a degenerate, punctate staining pattern. This disruption of the capillary network was both dose and time dependent, showing progressive disruption of capillary network integrity at doses of 50 nM and above after as little as 4 h of treatment compared with untreated controls. We also examined DRG from different stages of development to determine whether VEGF-independent vessel stability emerged (Fig. 2 ). Endothelial cells were detected by CD31 (green) and neurons by ßIII tubulin (red) immunostaining. Both inhibition of VEGFR-2 activation by PTK787 and removal of endogenous VEGF by Fc-sFlt dramatically disrupted the DRG vascular network from E14.5 to E18 with similar effect; however, there was no detectable effect on the vascular network of P1 DRG after up to 24 h of treatment.

View larger version (101K): [in this window] [in a new window]   Figure 2. DRG capillary integrity is sensitive to inhibition of VEGF during a specific developmental window. DRG from E14.5, E16, E18 mouse embryos and newborn (P1) mice were dissected and explants were cultured in DRG growth media (DGM) for 12 h followed by 12 h treatment with control DGM, 500 nM PTK787, or 100 ng/mL Fc-sFlt. Two-color immunostaining for CD31 (green) and ßIII tubulin (red) was used to visualize capillary and neuronal structure, respectively. The capillary network shows loss of both endothelial cell bulk and connectivity in both treatments at days E14.5–E18, whereas no effect was detected at P1. Neuronal structure was unaffected by the treatments in all developmental stages analyzed.

4. Inhibition of VEGF does not affect axonal outgrowth at any developmental stage analyzed
In contrast to the dramatic effect observed in the DRG vasculature, there was no detectable effect of VEGF inhibition on neuronal cells. The morphology and outgrowth of axons was similar between control and treated DRG as assessed by neurofilament staining (Fig. 2) . To quantitatively evaluate the effects of these treatments on neurons, standard axonal outgrowth measurement was performed. No difference was seen in outgrowth with either VEGF inhibition treatment at any developmental stage compared with control-treated samples.

CONCLUSIONS AND SIGNIFICANCE

The current study describes a novel use of DRG explant culture for the analysis of endothelial/neuronal interactions and vascular development (Fig. 3 ). Using this experimental system, we demonstrated that, in sensory ganglia, VEGF is produced by neurons and satellite cells while its principal signaling receptor is expressed only by endothelial cells. During development, VEGF is a strict requirement for endothelial cell survival, but has no detectable effect on neurons. This finding suggests that the role of VEGF in sensory ganglia is different from its emerging role as a direct neuroprotective factor in the central and motor nervous systems during hypoxic stress. Thus, neuronal development and maintenance may rely on distinct combinations of the vascular and nonvascular effects of VEGF in a tissue-specific fashion.

View larger version (20K): [in this window] [in a new window]   Figure 3. Schematic representation of DRG in vivo and in explant culture. In vivo, DRG neurons express VEGF and endothelial cells express VEGFR-2. In explant culture, both axonal outgrowth and capillary structure can be monitored in response to disruption of VEGF signaling by PTK787 or sequestration of VEGF by Fc-sFlt. In embryonic DRG explants, VEGF inhibition causes vascular disruption but in postnatal DRG it does not. Axonal outgrowth is not affected by either treatment.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2320fje;

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