Selective stimulation of VEGFR-1 prevents oxygen-induced retinal vascular degeneration in retinopathy of prematurity

Abstract

Oxygen administration to immature neonates suppresses VEGF-A expression in the retina, resulting in the catastrophic vessel loss that initiates retinopathy of prematurity. To investigate the mechanisms responsible for survival of blood vessels in the developing retina, we characterized two VEGF-A receptors, VEGF receptor-1 (VEGFR-1, also known as Flt-1) and VEGF receptor-2 (VEGFR-2, also known as Flk-1). Surprisingly, these two VEGF-A receptors differed markedly during normal retinal development in mice. At 5 days postpartum (P5), VEGFR-1 protein was colocalized with retinal vessels, whereas VEGFR-2 was detected only in the neural retina. Real-time RT-PCR identified a 60-fold induction of VEGFR-1 mRNA in retina from P3 (early vascularization) to P26 (fully vascularized), and no significant change in VEGFR-2 mRNA expression. Placental growth factor-1 (PlGF-1), which exclusively binds VEGFR-1, decreased hyperoxia-induced retinal vaso-obliteration from 22.2% to 5.1%, whereas VEGF-E, which exclusively binds VEGFR-2, had no effect on blood vessel survival. Importantly, under the same conditions, PlGF-1 did not increase vasoproliferation during (a). normal vessel growth, (b). revascularization following hyperoxia-induced ischemia, or (c). the vasoproliferative phase, indicating a selective function supporting blood vessel survival. We conclude that VEGFR-1 is critical in maintaining the vasculature of the neonatal retina, and that activation of VEGFR-1 by PlGF-1 is a selective strategy for preventing oxygen-induced retinal ischemia without provoking retinal neovascularization.

Figure 1

P5 retinal whole-mount in situ hybridization of VEGF-A, VEGFR-1, and VEGFR-2 mRNA’s. (a) VEGF-A mRNA (pink) is detected anterior to the growing vessel front as outlined by (b) FITC-dextran perfusion of vessels (yellow green). Posterior to the vessel front, VEGF-A expression is suppressed (light yellow). (c) VEGFR-1 mRNA (pink) is detected in the central retina but is not seen at the anterior edge of vessels as outlined by (d) FITC-dextran perfusion of vessels (yellow). (e) VEGFR-2 mRNA (pink) is detected in the entire retina and does not correspond to the vessels (f) outlined in yellow.

Figure 2

Real-time RT-PCR quantification of VEGFR-1 and VEGFR-2 mRNA during retinal vascular development. Copy numbers of VEGFR-1 and VEGFR-2 mRNA/10⁶ copies cyclophilin A control mRNA at specific timepoints were measured. (a) VEGFR-1 mRNA expression increases linearly with retinal vascular development; expression is 60-fold higher at P26 than at P3. (b) VEGFR-2 mRNA expression decreases modestly (<15%) during retinal vessel development. The ratio of VEGFR-2 mRNA to VEGFR-1 mRNA expression ranges from 200-fold at P3 to twofold at P26.

Figure 3

Immunohistochemical localization of VEGFR-1 and VEGFR-2 protein in P2 and P5 retinal whole mounts. On P5, VEGFR-1 protein (red) is detected in a vascular pattern (a and c) and clearly coincides with endothelial cells (blue) as detected with G. simplicifolia I isolectin (d). (e) A merged image (purple) shows coincidence. (b and f) VEGFR-2–positive signal (red) is found in the neural retina, specifically in the interstices between vessels (blue) (g) in the merged image (h), which shows little or no overlap between vessels and VEGFR-2 staining. On P2, VEGFR-1 protein signal (green) (i) coincides with endothelial cells (red) (j) detected by lectin. In a merged image (k), retinal vessels coincide with the faint VEGFR-1 staining. The retinal vessel front is defined by an arrow. Some hyaloid vasculature (H) from incomplete removal on the vitreal surface of the P2 retina is also positive for VEGFR-1 signal. (l) VEGFR-2–positive signal (green) is found in the neural retina and does not coincide with endothelial capillaries (red) (m) in the merged image (n). Arrows indicate the vessel front.

Figure 4

Immunohistochemical localization of VEGFR-1 and VEGFR-2 protein in P5 and P15 retinal cross sections. (a–f) P5 immunohistochemical localization of (a) VEGFR-1, (d) VEGFR-2, (b and e) G. simplicifolia I isolectin–stained endothelial cells, and (c and f) merged images. (a) VEGFR-1 protein (green) is seen primarily in the ganglion cell layer (GCL) and is seen to overlap with (b) endothelial cells (red) when (c) the images are merged (yellow; indicated by arrows). (d) VEGFR-2–positive signal (green) is found in the GCL, the inner plexiform layer, the inner nuclear layer (INL), and the outer nuclear layer (ONL). (e) Vascular endothelial cells (red) do not overlap with VEGFR-2–positive cells in the merged image (f) and are not coincident with vessels (arrows). (g–l) P15 immunohistochemical localization of (g) VEGFR-1 and (j) VEGFR-2 staining and (h and k) isolectin-stained endothelial cells. (i and l) Merged images. VEGFR-1 protein (green in g) completely overlaps with endothelial cells (red in h) when the images are merged (yellow in i). Some VEGFR-2 signal (green in j) overlaps with endothelial cells (k) when the images are merged (yellow in l), whereas other VEGFR-2–positive cells (indicated by arrow in d and f) span the retina and are morphologically consistent with Muller cell structure. RPE/Ch, retinal pigment epithelium/choroid.

Figure 5

PlGF-1, but not VEGF-E, prevents hyperoxia-induced retinal vessel loss, thus implicating VEGFR-1 in survival. PlGF-1: P8 FITC-dextran–perfused retinal flat-mount retina from a representative control mouse treated with room air (normoxia) (a) or a mouse given hyperoxic treatment (75% O₂ for 17 hours at P7–P8) after intravitreal injection on P7 of (b) control BSS in one eye and (c) the VEGFR-1–specific ligand PlGF-1 in the contralateral eye. Vessels delineated with FITC show that PlGF-1 confers significant protection from oxygen-induced vessel loss compared with BBS control. (d) Analysis of nonvascularized area shows a greater than fourfold difference between eyes treated with PlGF-1 (22.2% ± 3.4% vascularized area) and eyes treated with BSS (5.1% ± 1.2%) (n = 6, P < 0.001). VEGF-E: FITC-dextran–perfused retinal flat mount of P8 control retina from representative room air–treated mouse (e) or oxygen-exposed mouse after intravitreal injections at P7 of (f) control BSS in one eye and (g) VEGFR-2–specific ligand VEGF-E in the contralateral eye. (h) Analysis of nonvascularized area shows no significant difference between VEGF-E– and BSS-treated eyes (n = 6, P = 0.87). Results are representative of two independent experiments.

Figure 6

Activation of VEGFR-1 by PlGF-1 does not increase normal retinal vessel growth or revascularization. (a) Intravitreal injections at P3 of control (BSS) in one eye and PlGF-1 in the contralateral eye (n = 6). Retinal vessel growth area was measured in whole mounts at P5. PlGF-1–injected eyes had a mean of 41.67% ± 5.63% of the retina vascularized. Similarly, 42.18% ± 8.60% of the BSS-injected contralateral control eyes were vascularized (P = 0.91). Results are representative of two independent experiments. (b) Vessel revascularization was measured in P15 mice after induction of vessel loss by oxygen (P7–P12) followed by intravitreal injections of BSS at P13 in one eye and PlGF-1 in the contralateral eye. PlGF-1–injected eyes were 26.32% ± 2.62% vascularized; similarly, BSS-treated contralateral control eyes were 26.29% ± 2.86% vascularized (n = 6, P = 0.99). Results are representative of two independent experiments. (c) In eyes with oxygen-induced retinopathy, the mean number of vascular nuclei extending into the vitreous at P17 in ten retinal cross sections per eye (n = 8 eyes) was counted after intravitreal injections of BSS at P13 (after 5 days of 75% O₂ treatment, from P7 to P12) in one eye and PlGF-1 in the contralateral eye. BSS- and PlGF-1–injected eyes showed means of 9.98 and 9.96 vascular nuclei (P = 0.61), respectively, indicating no stimulation of proliferation by PlGF-1.