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Nonvascular role for VEGF: VEGFR-1, 2 activity is critical for neural retinal development ¹

GREGORY S. ROBINSON², MEIHUA JU, SHU-CHING SHIH, X. XU³, GERALD MCMAHON^*, RUTH B. CALDWELL and LOIS E. H. SMITH⁴

Children’s Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA;
^* SUGEN Inc., South San Francisco, California, USA; and
Medical College of Georgia, Augusta, Georgia, USA

⁴Correspondence: Children’s Hospital, Harvard Medical School, Department of Ophthalmology, 300 Longwood Ave., Boston, MA 02115, USA. E-mail: lois.smith{at}tch.harvard.edu

SPECIFIC AIMS

VEGF is critical for angiogenesis and is commonly described as endothelial cell specific; however, there is accumulating evidence that VEGF receptors are found outside the vasculature in mouse neural retina, in Schwann cells of peripheral neurons, and other tissues. We sought to define the function of VEGFR-1 and 2 in the murine neural retina during an avascular phase of development in order to correlate neural and vascular activities.

PRINCIPAL FINDINGS

1. VEGFR-1 and VEGFR-2 are expressed in the developing avascular retina and cornea
To understand the role of VEGF in neural retina, we localized VEGFR-1 and VEGFR-2 mRNA in retinal cross sections from different developmental stages by means of in situ hybridization (Fig. 1A , B ).

View larger version (55K): [in this window] [in a new window]   Figure 1. Localization of VEGFR-1 and VEGFR-2 expression in nonvascular cells in retinal cross sections during murine retinal development. A) VEGFR-1 and B) VEGFR-2 mRNA was localized on retinal cross sections by in situ hybridization with antisense probe or sense control(*) at P5, P7, P12, P15, P17, P33. At each time point, a control retinal cross section perfused with fluorescein-dextran defines the vascular development (FITC). At P5, P7, and P12, expression of VEGFR-1 and VEGFR-2 mRNA is noted with arrows in regions of the retina that are avascular as determined by the absence of vascular outlines (FITC) after cardiac perfusion to delineate vessels. At P15, P17 and P33, the more fully developed vascular pattern is demonstrated as a yellow-green outline of vessels in an age-matched FITC perfused retina. VEGFR-1 and 2 are detected in areas not defined by the vascular pattern as well as in areas defined by a vascular pattern. Magnification: 40 x. In P17 paraffin-embedded eye cross sections, C) VEGFR-1 (left panel) and D) VEGFR-2 (left panel) mRNAs are detected by in situ hybridization in corneal stroma and epithelium, as indicated by specific blue stain and arrows not seen on sense controls (C, D , right panels). Magnification: 20 x.

VEGFR-1 mRNA (Fig. 1A ) is first detected on postnatal day 7 (P7) in avascular retina, localized to the developing ganglion cell layer and the inner nuclear layer. From P12 to P15, expression is observed in the inner nuclear layer and appears in the photoreceptor layer, consistent with the pattern of Muller cell development whose processes span the retina (arrows). VEGFR-1 mRNA expression from P17 to P33 (adult) is only partially localized in a vascular pattern, as defined at each time with fluorescein-dextran perfusion (FITC) and seen as bright spots on a black background. VEGFR-1 is also found in extravascular cells in the inner nuclear layer and ganglion cell layer. VEGFR-1 mRNA in whole retina increases 14-fold with retinal development from P3 to adult (P33) as determined by real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR).

VEGFR-2 mRNA (Fig. 1B ) is first detected at P5, localized to the inner layers of the developing avascular retina (arrow). As development progresses, (P7-P12), VEGFR-2 mRNA is observed in the inner nuclear layer (arrow) and at P15 in the inner and outer nuclear (photoreceptor) layers (arrow). At P7, neurogenesis in the retina is still incomplete, and bipolar cells and Muller cells are differentiating in the inner nuclear layer. At P5, P7, and P12, there are no vessels in these peripheral retinal sections as indicated by the absence of FITC after perfusion, which fills blood vessels. From P17 to P33 (adult), VEGFR-2 mRNA becomes more localized to blood vessels [vascular pattern is outlined with bright image (FITC)], but staining is still observed outside the vascular pattern in the ganglion cell, inner nuclear, and nerve fiber layers. This pattern is again consistent with expression in Muller cell processes that span the entire retina and in astrocytes or ganglion cells. Sense controls for VEGFR-1 and VEGFR-2 reveal the absence of nonspecific staining. VEGFR-2 mRNA decreases threefold in whole murine retina from early development to P33 as determined by qRT-PCR.

To confirm the presence of VEGFR-1 and 2 mRNA in avascular eye tissue, we examined the expression of mRNA in avascular cornea at P17. Both VEGFR-1 (Fig. 1C ) and VEGFR-2 mRNA (Fig. 1D ) are found in corneal stroma and epithelium (arrows).

2. VEGF /VEGFR-1 and 2 interactions control neural retinal development: Inhibition of VEGFR-1 and 2 in avascular and early-vascularized retina
To determine whether inhibition of VEGFR-1 and 2 affects neural retinal morphology, we treated mice with systemic administration of SU5416 (a specific antagonist of VEGFR-1 and 2) from birth to various postnatal ages (before P12) when the retina is still avascular in the periphery (Fig. 2 ). These ages were chosen to avoid the possibility that retinal development would be affected by inhibition of vascular development. Treatment of mice with SU5416 from P0 to P9 caused a significant decrease in total thickness of the avascular peripheral retina (Fig. 2A , right panel) compared with untreated control (Fig. 2A , left panel), but not in the more fully developed posterior pole (Fig. 2B ). The specific layers affected were the inner nuclear containing Muller cell nuclei, the inner plexiform and ganglion cell layers containing Muller cell processes, as well as astrocytes and ganglion cells. The outer nuclear (photoreceptor) layer was unchanged in thickness as was the choroid/ RPE. In the posterior pole (central retina near the optic nerve), where retinal development (and vascular development) is more complete during P0-P9, there was no significant decrease in thickness of any retinal layer. At the equator (midway between the periphery and optic nerve in position and in retinal development), there is a similar but less profound effect on neural inner nuclear layer retinal development with inhibition of VEGFR-1, 2. Similar effects were seen with SU5416 treatment from P0-P5, P0-P7, and P0-P12.

View larger version (40K): [in this window] [in a new window]   Figure 2. VEGFR-1, 2 Antagonist (SU5416) inhibits the development of neural retina. C57BL/6J mice were injected (s.c.) with 50 mg/kg/day SU5416 or vehicle from P0 until P5 or P7 or P9 when the peripheral retina is avascular. H&E-stained retinal cross sections from the periphery from representative control and SU5416-treated animals at P9 show thinning of the inner plexiform layer (IP), inner nuclear layer (IN), and ganglion cell layer (G); the outer nuclear (photoreceptor, ON) layer was unchanged in thickness (A, right panel) compared with untreated control (A, left panel). Similar results were seen at P5, P7 (data not shown). The widths of the inner plexiform and outer nuclear layers were measured at the periphery (avascular retina), equator, and pole (vascular retina) of the retina of SU5416-treated retinas and compared with controls (B).

Treatment with SU5416 from P12 to P17 in oxygen-treated retina when vaso-obliteration had occurred caused a 15% decrease in total retinal thickness in the periphery (P<0.01). Muller cell mitosis has ceased but cell differentiation continues at this time. Treatment with SU5416 from P12-P17 normal mice or in adult (P33) normal retinas resulted in no loss in retinal thickness or any change in the vasculature.

3. Inhibition of VEGFR-1 and VEGFR-2 activity decreases ischemia-induced retinal neovascularization
We examined the effect of inhibition of VEGFR-1 and VEGFR-2 on retinal neovascularization. Mice with induced retinal neovascularization were treated with SU5416 from P12 to P17 and showed a dose-responsive inhibition of neovascularization up to a maximum of 60% as compared with vehicle-treated controls. There was no change in the retinal vasculature in normal room air-raised mice with SU5416 treatment at P12-P17 or P33-P38. As expected, SU5416 also inhibited the developing vasculature. Neonatal mice treated with SU5416 from P0-P6 and then perfused with FITC dextran to delineate blood vessels show a significantly decreased area of vascular development, as compared with vehicle-treated controls.

4. Muller Cells express VEGFR-1, 2 and respond to VEGF through MAPK
Since both VEGFR-1 and 2 mRNA localization by in situ hybridization was consistent with expression in Muller cells, we chose this cell to study in culture. Muller cells were shown to express VEGFR-1 and 2 mRNA by qRT-PCR. The cells responded to VEGF stimulation with activation of MAPK. This response was decreased 80% with SU5416 inhibition of VEGFR-1 and 2.

CONCLUSIONS

Although VEGFR-1 and 2 are routinely described as specific to vascular endothelial cells, these receptors are expressed outside the vasculature and, specifically in the Muller cell. The function of these receptors in these nonendothelial cells has not been described before (Fig. 3 ).

View larger version (27K): [in this window] [in a new window]   Figure 3. VEGFR-1, 2 activity in vascular and nonvascular cells. VEGFR-1, 2 are shown in cells of neural and vascular origin. Neural cells: semaphorin 3A binds to the neuropilin-1 (NP-1) –plexin complex to elicit axon guidance responses (heavy lines). NP-1 interacts with VEGFR-1 whose specific role is not known (dashed line, ?). VEGF binding to VEGFR-1 and/or VEGFR-2 activates MAP kinase and may play a role in cell migration and/or proliferation. The role of VEGF on NP-1 is unknown (?). Vascular cells: VEGF binding to VEGFR-1,2 results in cellular migration and proliferation (heavy lines). The role of NP-1 in vascular cells is unclear (?), although altering the metastatic capability of the cell is hypothesized.

To study their function, a normal avascular tissue expressing VEGFR-1 and 2 is required. Inhibition of the activity of these receptors in developing avascular neural retina by the VEGFR-1- and 2-specific antagonist SU5416 results in a loss of cells in the inner nuclear layer containing Muller cell nuclei. In isolated rat Muller cells expressing VEGFR-1 and VEGFR-2 mRNA, VEGF activation of MAPK was inhibited with SU5416, suggesting VEGF-induced MAPK activation in Muller cells as one of the pathways involved in the SU5416-induced cell loss in vivo.

The greater loss of retina in the ganglion cell layer and inner plexiform layer could suggest that other cells, including astrocytes, may be VEGF dependent. Our results suggest that the development of blood vessels and neural tissue takes place in a coordinated fashion, led by development of VEGF receptors in the astrocytes or Muller cells. No neural retinal changes were seen in the deeper retinal layers served by the choroidal vasculature nor were any morphological changes seen in the choroidal vessels themselves.

VEGF is well established as being critical to vascular development. Therefore, inhibition of VEGFR-1 and 2 with SU5416, as expected, caused profound inhibition of retinal neovascularization, confirming the potential therapeutic benefit of VEGFR inhibition in disease caused by new vessel growth.

Our findings suggest that manipulation of the VEGF pathway to inhibit angiogenesis should be approached carefully in patients with developing retina or perhaps injured (and avascular or hypoxic) retinal neural tissue. Inhibition of neovascularization in mature vascularized retina does not appear to affect normal mature vessels or mature neural retina.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0598fje ; to cite this article, use FASEB J. (March 20, 2001) 10.1096/fj.00-0598fje

² Present address: Pharmacia Corporation, St. Louis, MO, USA.

³ Present address: Biogen Corporation, Boston, MA, USA.

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