Association of retinal vascular endothelial growth factor with avascular retina in a rat model of retinopathy of prematurity

Abstract

Objective: To study the effects of oxygen fluctuations on rat vascular endothelial growth factor (VEGF), VEGF receptor 1(VEGFR1), and VEGFR2 in a model of retinopathy of prematurity (ROP).

Methods: Retinas at several postnatal days (p) were analyzed for VEGF splice variants, VEGFR1 and VEGFR2 messenger RNAs (mRNAs) using real-time polymerase chain reaction or for VEGF protein using enzyme-linked immunosorbent assay.

Results: Older developmental age was associated with VEGFR1 (P < .001), VEGF(120) (P < .001), and VEGF(188) (P = .03) mRNA overexpression. Expression of VEGFR2 and VEGF(164) mRNAs were associated with older age (P < .001) or exposure to the ROP model (P = .02 and P < .001, respectively). Expression of VEGF protein was greater at p14, when 30% avascular retina existed in the ROP model, compared with room air, when no avascular retina existed, and at p18, when intravitreous neovascularization existed in the model but not in room air (P < .001 for both).

Conclusions: Unlike models of oxygen-induced retinopathy that describe ROP before implementation of oxygen regulation, the ROP model re-creates oxygen stresses relevant to preterm infants with severe ROP today. Expression of VEGF(164) and VEGFR2 mRNAs and VEGF protein were increased in association with the ROP model and older developmental age and at time points when not only intravitreous neovascularization but also avascular retina were present in the ROP model and not in room air. Clinical Relevance Regulation of VEGF may have a role in the development of avascular retina and intravitreous neovascularization in some forms of severe ROP.

Figure 1

Lectin-stained retinal flat mounts created by 4 relaxing incisions showing retinal vascularization in rat. A). Room air raised pup at postnatal day (p)14 rat has retinal vascularization that extends to the ora serrata. B) ROP model at p18 following 7 cycles of oxygen fluctuations between 50% and 10% and then 4 days in room air, showing avascular retina and intravitreous neovascularization (IVNV). Optic nerve is in the center (no macula is present in rat) and peripheral avascular retina seen adjacent to the ora serrata.

Figure 2

Real time PCR mean values for VEGF Receptor (R)1 of rat pups from p0 through p18 in room air development (RA) or in the ROP model (ROP). In the ROP model, p8, p12, and p14 occur immediately following hypoxia (10% FiO₂) and p11 and p13 following hyperoxia (50% FiO₂). Following p14, pups are in room air (21% FiO₂). All values are normalized to β-actin and are compared to p0, which is the same for room air and ROP. There was a significant increase in expression associated with older developmental age (P<.0001) but not with exposure to the ROP model compared to age-matched room air pups (P=.82). The value for VEGFR1 at p0 was scaled to 1.0 to allow comparisons among VEGFR1 values in room air or ROP. The graph represents means and standard errors for each room air and ROP time point, each time point having an n=at least 5 retinas from different pups taken from at least 2 litters.

Figure 3

Real time PCR mean values for VEGF Receptor (R)2 of rat pups from p0 through p18 in room air development (RA) or in the ROP model (ROP). In the ROP model, p8, p12, and p14 occur immediately following hypoxia (10% FiO₂) and p11 and p13 following hyperoxia (50% FiO₂). Following p14, pups are in room air (21% FiO₂). All values are normalized to β-actin and are compared to p0 which is the same for room air and OIR. There was a significant increase in VEGFR2 expression associated with older developmental age (P<.0001) and with exposure to the ROP model compared to age-matched room air pups (P=.0247). The value for VEGFR2 at p0 was scaled to 1.0 to allow comparison among VEGFR2 values in room air or ROP. The graph represents means and standard errors for each room air and ROP time point, each time point having an n=at least 5 retinas from different pups taken from at least 2 litters.

Figure 4

Real time PCR mean values for VEGF splice variant VEGF₁₆₄ of rat pups from selected postnatal days (p) 0 through p18 in room air development (RA) or in the ROP model (ROP). In the ROP model, p8, p12, and p14 occur immediately following hypoxia (10% FiO₂) and p11 and p13 following hyperoxia (50% FiO₂). Following p14, pups are in room air (21% FiO₂). At p14, in room air the inner retina is vascularized to the ora serrata, whereas in the ROP model, there is 30% avascular retina. All values are normalized to β-actin and are compared to p0 which is the same for room air and OIR. There was a significant increase in expression associated with older developmental age (P<.0001) and with exposure to the ROP model compared to age-matched room air pups (P<.0001). For graphical representations, the value of VEGF₁₈₈ mRNA at p0 was scaled to 1.0 and fold expression of mRNAs of the splice variants at time points in RA and the ROP model were related to it. The graph represents means and standard errors for each room air and ROP time point, each time point having an n=at least 5 retinas from different pups taken from at least 2 litters.

Figure 5

Real time PCR mean values for VEGF splice variant VEGF₁₂₀ of rat pups from selected postnatal days (p) 0 through p18 in room air development (RA) or in the ROP model (ROP). In the ROP model, p8, p12, and p14 occur immediately following hypoxia (10% FiO₂) and p11 and p13 following hyperoxia (50% FiO₂). Following p14, pups are in room air (21% FiO₂). At p14, in room air the inner retina is vascularized to the ora serrata, whereas in the ROP model, there is 30% avascular retina. All values are normalized to β-actin and are compared to p0 which is the same for room air and OIR. There was a significant increase in expression associated with older developmental age (P<.0001) but not with exposure to the ROP model compared to age-matched room air pups (P=.614). For graphical representations, the value of VEGF₁₈₈ mRNA at p0 was scaled to 1.0 and fold expression of mRNAs of the splice variants at time points in RA and the ROP model were related to it. The graph represents means and standard errors for each room air and ROP time point, each time point having an n=at least 5 retinas from different pups taken from at least 2 litters.

Figure 6

Real time PCR mean values for VEGF splice variant VEGF₁₈₈ of rat pups from selected postnatal days (p) 0 through p18 in room air development (RA) or in the ROP model (ROP). In the ROP model, p8, p12, and p14 occur immediately following hypoxia (10% FiO₂) and p11 and p13 following hyperoxia (50% FiO₂). Following p14, pups are in room air (21% FiO₂). At p14, in room air the inner retina is vascularized to the ora serrata, whereas in the ROP model, there is 30% avascular retina. All values are normalized to β-actin and are compared to p0 which is the same for room air and OIR. There was no significant association with older developmental age or with exposure to the ROP model compared to age-matched room air pups. For graphical representations, the value of VEGF₁₈₈ mRNA at p0 was scaled to 1.0 and fold expression of mRNAs of the splice variants at time points in RA and the ROP model were related to it. The graph represents means and standard errors for each room air and ROP time point, each time point having an n=at least 5 retinas from different pups taken from at least 2 litters.

Figure 7

ELISA measurements of retinal VEGF at time points in room air development (RA) or ROP model (ROP). There was a significant increase in expression associated with older developmental age (P<.0001) and with exposure to the ROP model compared to age-matched room air pups (P<.0001). In Post-hoc testing, VEGF was significantly increased in the ROP model compared to room air at p14 and p18 (P<.0001). For each time point, an n= at least 5 retinas from different pups were analyzed from at least 2 different litters.