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Neutralizing antibody to VEGF reduces intravitreous neovascularization and may not interfere with ongoing intraretinal vascularization in a rat model of retinopathy of prematurity.

Geisen P, Peterson LJ, Martiniuk D, Uppal A, Saito Y, Hartnett ME - Mol. Vis. (2008)

Bottom Line: Neither dose caused a significant difference in avascular/total retinal area at p18 compared to control.However, at p25, the 50 ng dose caused a significant reduction in avascular/total retinal area compared to the 25 ng dose (ANOVA p=0.038; posthoc Student's t-test p=0.038).In this study, survival of already developed retinal capillaries did not appear affected.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, North Carolina.

ABSTRACT

Purpose: To study the effects of a neutralizing antibody to vascular endothelial growth factor (VEGF), given as an intravitreous injection, on intravitreous neovascularization (IVNV) and ongoing vascular development of avascular retina in a rat model relevant to human retinopathy of prematurity.

Methods: Newborn Sprague-Dawley rats were exposed to oxygen fluctuations alternating between 50% O(2) and 10% O(2) every 24 h. At postnatal day (p)12, rat pups received intravitreous injections of a neutralizing antibody to VEGF or control nonimmune rat IgG in one eye and were returned to oxygen cycling until p14, at which time they were placed into room air. At p18 (time of maximal IVNV) or p25 (time point in regression), animals were sacrificed. Their retinas were dissected, flat mounted, and stained with Alexa-isolectin for fluorescence microscopy. IVNV was measured as number of clock hours involved in injected VEGF antibody and control eyes. Mean clock hours of IVNV, avascular/total retinal areas and capillary densities within vascularized retinas were determined in injected eyes of control and treatment groups. Mean clock hours of IVNV in fellow noninjected eyes from control and treatment groups were analyzed by Student's t-tests to assess possible crossover effects from systemic absorption of antibody. Eyes from p13 rat pups were sectioned for immunohistochemistry or analyzed for VEGF receptor 2 (VEGFR2) phosphorylation by western blot. Free retinal VEGF at p13, one day following injections, was measured by ELISA.

Results: Neutralizing antibody to VEGF at 25 ng and 50 ng caused a modest but significant inhibition of IVNV compared to IgG injected controls at p18, but only the 50 ng dose decreased IVNV compared to control at p25 (one-way ANOVA p=0.003; posthoc Bonferroni t-test p=0.003). Neither dose caused a significant difference in avascular/total retinal area at p18 compared to control. However, at p25, the 50 ng dose caused a significant reduction in avascular/total retinal area compared to the 25 ng dose (ANOVA p=0.038; posthoc Student's t-test p=0.038). There was no difference in avascular/total retinal area between IgG and the 25 ng dose. At p13, qualitative analysis of immunohistochemical sections of retina showed the 50 ng dose of VEGF antibody reduced VEGFR2 phosphorylation within the retina and around blood vessels. Also at p13, there was a significant increase in free intraretinal VEGF protein in eyes that had been treated with 50 ng dose of VEGF antibody compared to IgG injected control (Student's t-test p=0.042). There were no differences in capillary densities in the vascularized retinas between eyes injected with the 50 ng dose of VEGF antibody and IgG control. There was also no difference in weight gain between treated and control groups.

Conclusions: Neutralizing antibody to VEGF at a 50 ng dose caused a significant and sustained reduction in IVNV without interfering with ongoing retinal vascularization in a rat model of ROP, whereas a lower dose of antibody did not. These data also suggest that compensatory regulatory mechanisms may lead to increased VEGF concentration after intravitreous injection of a neutralizing antibody to VEGF. Further study is necessary for safety and for determination of drug dose of VEGF antibody, since dose of treatment appears important and may vary among infants with severe ROP. In this study, survival of already developed retinal capillaries did not appear affected. Neutralizing VEGF by an intravitreous injection of antibody may offer a treatment consideration for severe ROP, which fails current standard of care management.

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Related in: MedlinePlus

Phosphorylation of VEGFR2 at p18. Phosphorylation of vascular endothelial growth factor receptor 2 (VEGFR2) in lectin-stained IVNV (arrow) in p18 in noninjected, IgG injected and 50 ng VEGFab injected eyes. There was no qualitative difference in phosphorylation between groups.
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f5: Phosphorylation of VEGFR2 at p18. Phosphorylation of vascular endothelial growth factor receptor 2 (VEGFR2) in lectin-stained IVNV (arrow) in p18 in noninjected, IgG injected and 50 ng VEGFab injected eyes. There was no qualitative difference in phosphorylation between groups.

Mentions: To determine the effects of an intravitreous injection of neutralizing antibody to VEGF on retinal VEGF and VEGF signaling through its receptors, VEGFR1 and VEGFR2, we performed immunohistochemical staining and western blot analysis on eyes that had been injected with 50 ng VEGFab or control nonimmune IgG. Cryosections were taken at p13 and were immunolabeled with antibodies to phosphorylated VEGFR-1 or phosphorylated VEGFR-2. Sections were taken centrally (toward the optic nerve) and peripherally within vascularized retina near the junction with avascular retina. In general, in both noninjected and IgG-injected eyes, VEGFR-2 phosphorylation was present mainly in the ganglion cell and nerve fiber layers (2+) and around superficial and deep retinal vessels (2+), whereas VEGFR-1 phosphorylation was present in the ganglion cell (but not nerve fiber layer) and in the outer plexiform layer (2+) (Figure 4). VEGFR-2 phosphorylation was reduced in eyes injected with VEGFab compared to control (1+) at p13 (Figure 4). There was no change observed in VEGFR1 phoshorylation from cryosections among treated control and non-injected eyes (Figure 4). At p18, there was reduced VEGFR2 phosphorylation in the ganglion cell/nerve fiber layer regions in all groups (1+) compared to p13 (Figure 5). Phosphorylation (2+) remained strong in blood vessels and in IVNV at p18 (arrow, Figure 5). There was no difference in VEGFR2 phosphorylation between noninjected, IgG injected, and 50 ng VEGFab injected eyes at p18. Despite the findings on immunohistochemistry, we were unable to detect a significant difference in phosphorylated to total VEGFR2 in whole retinas analyzed by western blot from VEGFab injected (50 ng) and control IgG injected eyes at p13 (Figure 6).


Neutralizing antibody to VEGF reduces intravitreous neovascularization and may not interfere with ongoing intraretinal vascularization in a rat model of retinopathy of prematurity.

Geisen P, Peterson LJ, Martiniuk D, Uppal A, Saito Y, Hartnett ME - Mol. Vis. (2008)

Phosphorylation of VEGFR2 at p18. Phosphorylation of vascular endothelial growth factor receptor 2 (VEGFR2) in lectin-stained IVNV (arrow) in p18 in noninjected, IgG injected and 50 ng VEGFab injected eyes. There was no qualitative difference in phosphorylation between groups.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2258217&req=5

f5: Phosphorylation of VEGFR2 at p18. Phosphorylation of vascular endothelial growth factor receptor 2 (VEGFR2) in lectin-stained IVNV (arrow) in p18 in noninjected, IgG injected and 50 ng VEGFab injected eyes. There was no qualitative difference in phosphorylation between groups.
Mentions: To determine the effects of an intravitreous injection of neutralizing antibody to VEGF on retinal VEGF and VEGF signaling through its receptors, VEGFR1 and VEGFR2, we performed immunohistochemical staining and western blot analysis on eyes that had been injected with 50 ng VEGFab or control nonimmune IgG. Cryosections were taken at p13 and were immunolabeled with antibodies to phosphorylated VEGFR-1 or phosphorylated VEGFR-2. Sections were taken centrally (toward the optic nerve) and peripherally within vascularized retina near the junction with avascular retina. In general, in both noninjected and IgG-injected eyes, VEGFR-2 phosphorylation was present mainly in the ganglion cell and nerve fiber layers (2+) and around superficial and deep retinal vessels (2+), whereas VEGFR-1 phosphorylation was present in the ganglion cell (but not nerve fiber layer) and in the outer plexiform layer (2+) (Figure 4). VEGFR-2 phosphorylation was reduced in eyes injected with VEGFab compared to control (1+) at p13 (Figure 4). There was no change observed in VEGFR1 phoshorylation from cryosections among treated control and non-injected eyes (Figure 4). At p18, there was reduced VEGFR2 phosphorylation in the ganglion cell/nerve fiber layer regions in all groups (1+) compared to p13 (Figure 5). Phosphorylation (2+) remained strong in blood vessels and in IVNV at p18 (arrow, Figure 5). There was no difference in VEGFR2 phosphorylation between noninjected, IgG injected, and 50 ng VEGFab injected eyes at p18. Despite the findings on immunohistochemistry, we were unable to detect a significant difference in phosphorylated to total VEGFR2 in whole retinas analyzed by western blot from VEGFab injected (50 ng) and control IgG injected eyes at p13 (Figure 6).

Bottom Line: Neither dose caused a significant difference in avascular/total retinal area at p18 compared to control.However, at p25, the 50 ng dose caused a significant reduction in avascular/total retinal area compared to the 25 ng dose (ANOVA p=0.038; posthoc Student's t-test p=0.038).In this study, survival of already developed retinal capillaries did not appear affected.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, North Carolina.

ABSTRACT

Purpose: To study the effects of a neutralizing antibody to vascular endothelial growth factor (VEGF), given as an intravitreous injection, on intravitreous neovascularization (IVNV) and ongoing vascular development of avascular retina in a rat model relevant to human retinopathy of prematurity.

Methods: Newborn Sprague-Dawley rats were exposed to oxygen fluctuations alternating between 50% O(2) and 10% O(2) every 24 h. At postnatal day (p)12, rat pups received intravitreous injections of a neutralizing antibody to VEGF or control nonimmune rat IgG in one eye and were returned to oxygen cycling until p14, at which time they were placed into room air. At p18 (time of maximal IVNV) or p25 (time point in regression), animals were sacrificed. Their retinas were dissected, flat mounted, and stained with Alexa-isolectin for fluorescence microscopy. IVNV was measured as number of clock hours involved in injected VEGF antibody and control eyes. Mean clock hours of IVNV, avascular/total retinal areas and capillary densities within vascularized retinas were determined in injected eyes of control and treatment groups. Mean clock hours of IVNV in fellow noninjected eyes from control and treatment groups were analyzed by Student's t-tests to assess possible crossover effects from systemic absorption of antibody. Eyes from p13 rat pups were sectioned for immunohistochemistry or analyzed for VEGF receptor 2 (VEGFR2) phosphorylation by western blot. Free retinal VEGF at p13, one day following injections, was measured by ELISA.

Results: Neutralizing antibody to VEGF at 25 ng and 50 ng caused a modest but significant inhibition of IVNV compared to IgG injected controls at p18, but only the 50 ng dose decreased IVNV compared to control at p25 (one-way ANOVA p=0.003; posthoc Bonferroni t-test p=0.003). Neither dose caused a significant difference in avascular/total retinal area at p18 compared to control. However, at p25, the 50 ng dose caused a significant reduction in avascular/total retinal area compared to the 25 ng dose (ANOVA p=0.038; posthoc Student's t-test p=0.038). There was no difference in avascular/total retinal area between IgG and the 25 ng dose. At p13, qualitative analysis of immunohistochemical sections of retina showed the 50 ng dose of VEGF antibody reduced VEGFR2 phosphorylation within the retina and around blood vessels. Also at p13, there was a significant increase in free intraretinal VEGF protein in eyes that had been treated with 50 ng dose of VEGF antibody compared to IgG injected control (Student's t-test p=0.042). There were no differences in capillary densities in the vascularized retinas between eyes injected with the 50 ng dose of VEGF antibody and IgG control. There was also no difference in weight gain between treated and control groups.

Conclusions: Neutralizing antibody to VEGF at a 50 ng dose caused a significant and sustained reduction in IVNV without interfering with ongoing retinal vascularization in a rat model of ROP, whereas a lower dose of antibody did not. These data also suggest that compensatory regulatory mechanisms may lead to increased VEGF concentration after intravitreous injection of a neutralizing antibody to VEGF. Further study is necessary for safety and for determination of drug dose of VEGF antibody, since dose of treatment appears important and may vary among infants with severe ROP. In this study, survival of already developed retinal capillaries did not appear affected. Neutralizing VEGF by an intravitreous injection of antibody may offer a treatment consideration for severe ROP, which fails current standard of care management.

Show MeSH
Related in: MedlinePlus