Comparative Effects of Early Postnatal Ibuprofen and Indomethacin on VEGF, IGF-I, and GH During Rat Ocular Development

Comparative Effects of Early Postnatal Ibuprofen and Indomethacin on VEGF, IGF-I, and GH during Rat Ocular Development

Kay D. A. Beharry,1 Houchang D. Modanlou,1 Jamal Hasan,1,2 Zahra Gharraee,1,2 Patricia Abad-Santos,2 Jeremy H. Sills,2 Amanda Jan,2 Stephen Nageotte,2 and Jacob V. Aranda3

PURPOSE. Ibuprofen and indomethacin are nonselective prosta- levels suggests decreased IGF-I bioactivity. These adverse ef- glandin synthetase inhibitors that have been shown to improve fects should be considered. (Invest Ophthalmol Vis Sci. 2006; oxygen-induced retinopathy in mice. Vascular endothelial 47:3036–3043) DOI:10.1167/iovs.06-0057 growth factor (VEGF), insulin-like growth factor (IGF)-I, and growth hormone (GH) are potent growth factors involved in ndomethacin is a nonselective cyclooxygenase (COX) inhib- retinal development. This study was conducted to examine and Iitor that leads to decreased prostaglandin (PG) synthesis. It is compare the effects of early postnatal ibuprofen and indometh- widely used in the neonatal intensive care unit during the first acin on ocular and systemic VEGF, IGF-I, and GH during rat few days of postnatal life, to treat symptomatic patent ductus ocular development. arteriosus (PDA) in premature newborn infants. Despite its METHODS. Newborn rats were treated with intraperitoneal in- effectiveness, indomethacin has significant maternal, fetal and jections of low and high doses of ibuprofen or indomethacin at neonatal adverse effects such as impaired platelet and neutro- phil function1 and cerebral, gastrointestinal, and renal hemo- birth (postnatal day [P]1) and on P2 and P3. A control group 2–4 received equivalent volumes of saline. At P14, vitreous fluid, dynamics. Indomethacin is also associated with neonatal complications such as intraventricular hemorrhage (IVH), renal retinal homogenates, and serum were analyzed for VEGF, IGF-I, 5–10 and GH protein levels. Retinal mRNA expression of VEGF dysfunction, and necrotizing enterocolitis (NEC). Similar to indomethacin, ibuprofen inhibits PG synthesis via its effects on splice variants (VEGF188, VEGF164, VEGF120), VEGF receptors (VEGFR-1, VEGFR-2, Npn-1, Npn-2), and pigment epithelium- COX. However, it has been shown to be as effective as indo- derived factor (PEDF) were also examined. methacin for closure of the ductus arteriosus with less cerebral and renal side effects.11–14 The differences between these two RESULTS. Animals treated with high-dose ibuprofen had signifi- nonsteroidal anti-inflammatory drugs (NSAIDs) may well be cantly lower somatic growth and higher serum and vitreous due to differences in their pharmacologic characteristics in the IGF-I levels. High-dose ibuprofen decreased retinal VEGF levels neonate.14 and retinal VEGF164, VEGF120, and VEGFR-2 transcripts, result- Despite their differences, animal studies have shown that ing in a significant increase in the cecal period in 87% of rats at both ibuprofen and indomethacin appear to improve oxygen- P14. Both indomethacin doses suppressed retinal VEGF164 induced retinopathy without affecting normal retinal develop- transcripts without affecting VEGF receptors. ment,15,16 perhaps because of their suppressive effects on CONCLUSIONS. Ibuprofen may be more effective than indometh- HIF-1␣17 and VEGF.18 Whereas we have shown that ibuprofen acin for suppression of retinal VEGF signaling, suggesting a enhances retinal and choroidal blood flow autoregulation in possible therapy for retinal neovascularization. However, def- newborn piglets exposed to hypertension and hypotension,19 icits in somatic growth concurrent with higher systemic IGF-I other studies have demonstrated that infants treated with indomethacin appear to be more than 1.5 times more likely to have retinopathy of prematurity (ROP) than untreated in- 20 From the 1Department of Pediatrics, Division of Neonatal-Perinatal fants. The mechanism(s) by which ibuprofen and indometh- Medicine, University of California Irvine, Irvine, California; the 2De- acin exert their effects on retinal neovascularization have not partment of Pediatrics, Division of Neonatal-Perinatal Medicine, Miller been previously studied, but may in part be related to their Children’s Hospital, Long Beach, California; and the 3Pediatric Pharma- effects on growth factors such as vascular endothelial growth cology Research Unit Network, Children’s Hospital of Michigan, De- factor (VEGF), insulin-like growth factor (IGF)-I, and/or growth troit, Michigan. hormone (GH). Presented, in part, at the 2005 Pediatric Academic Societies-Soci- VEGF is a potent endothelial cell mitogen and angiogenic ety for Pediatric Research Annual Meeting, Washington, DC, and at the factor involved in embryonic development.21 Alternative splic- 2005 European Society for Pediatric Research Annual Meeting, Siena, Italy. ing of a single gene yields three murine isoforms: VEGF188, Supported by a grant from the Long Beach Memorial Medical VEGF164 and VEGF120. VEGF164 and VEGF120 are highly in- Center Foundation, Long Beach, California. volved in angiogenesis and may constitute up to 98% of total 22 Submitted for publication January 19, 2006; revised February 22, VEGF overexpression. There is overwhelming evidence of 2006; accepted May 12, 2006. the involvement of VEGF in oxygen-induced retinopathy.23–27 Disclosure: K.D.A. Beharry, None; H.D. Modanlou, None; J. Pigment epithelium–derived factor (PEDF) is a potent naturally Hasan, None; Z. Gharraee, None; P. Abad-Santos; None; J.H. Sills, occurring angiogenic antagonist that regulates endothelial cell None; A. Jan, None; S. Nageotte, None; J.V. Aranda, None proliferation and blood vessel growth in the eye. Evidence The publication costs of this article were defrayed in part by page suggests that PEDF may be responsible for regulation of charge payment. This article must therefore be marked “advertise- 28 ment” in accordance with 18 U.S.C. §1734 solely to indicate this fact. VEGF. IGF-I, a somatic growth factor involved in ocular development and physiology, has also been implicated in ab- Corresponding author: Houchang D. Modanlou, University of 29–31 California, Irvine, Chief, Division of Neonatology, 101 The City normal retinal angiogenesis. The role of GH in ROP has Drive South, Route 81, Building 56, Suite 600, Orange, CA 92868; not been well studied; however, GH deficiency in children has [email protected]. been reported to be associated with reduced retinal vascular-

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TABLE 1. Eye Opening at P14

Ibuprofen Indomethacin

Saline Low Dose High Dose Low Dose High Dose

Male 12/16 (75) 14/16 (88) 0/12 (0) 6/12 (50) 14/14 (100) Female 8/14 (57) 8/14 (57) 4/18 (22) 6/18 (33) 14/16 (87) (93) 30/28 (40) 30/12 ء(Total 20/30 (67) 22/30 (73) 4/30 (13

Low-dose ibuprofen (10 mg/kg IP on P1 followed by 5.0 mg/kg on P2 and P3); high-dose ibuprofen (50 mg/kg IP on P1 followed by 25 mg/kg on P2 and P3); low-dose indomethacin (0.2 mg/kg IP on P1 followed by 0.1 mg/kg on P2 and P3); and high-dose indomethacin (1.0 mg/kg IP followed by 0.5 mg/kg on P2 and P3). The control group received equivalent volume saline IP on P1, P2 and P3. Data are the number of rats with open eye(s) at P14/total group (percentage). * P Ͻ 0.01 versus saline, low-dose ibuprofen, and high-dose indomethacin (n ϭ 30/group).

ization32; and high serum GH at 1 month of age was associated born rat pups delivering on the same day were pooled and randomly with the development of ROP.33 assigned to expanded litters of 15. One dam remained with the same Data emerging from our laboratory have shown that during litter for the entire study. Each pup was weighed and measured for the course of postnatal retinal development in the rat, VEGF linear growth (nose to tail length). Litters were randomly assigned to and IGF-I act synergistically to promote retinal development receive intraperitoneal injections of (1) low-dose ibuprofen (10 mg/kg) and that the vitreous fluid may be a reservoir for these growth on P1 followed by 5 mg/kg on P2 and P3; (2) high-dose ibuprofen (50 factors.34 Based on these data, as well as previously published mg/kg) on P1 followed by 25 mg/kg on P2 and P3; (3) low-dose observations,15,16,20,23–33 we examined and compared the ef- indomethacin (0.2 mg/kg) on day 1 (P1), followed by 0.1 mg/kg fects of early postnatal administration of ibuprofen and indo- indomethacin on P2 and P3; (4) high-dose indomethacin (1.0 mg/kg) methacin on systemic and ocular VEGF, IGF-I, and GH in the on P1 followed by 0.5 mg/kg on P2 and P3; or (5) equivalent volume normal, but immature retina of the newborn rat. The null saline injected intraperitoneally (n ϭ 2 litters/group). Indomethacin hypothesis was that there were no significant differences be- (Indocin IV; Merck & Co., Inc., West Point, PA) was diluted in 0.9% tween the two drugs. saline before injection. Ibuprofen IV solution was provided by Farma- con Laboratories (Westport, CT) and diluted in 0.9% saline before MATERIALS AND METHODS injection. At P14 (the time of retinal vascular maturity) the rat pups were examined for eye opening (eyes were considered opened if at All experiments were approved by the Memorial Health Services Insti- least one eye was open), weighed, measured, and killed by decapita- tutional Animal Care and Use Committee (Long Beach, CA). Animals tion. Blood samples were pooled for a total of 15 samples, collected in were managed according to the ARVO Statement for the Use of Ani- sterile tubes (Eppendorf, Fremont, CA), and placed on ice before mals in Ophthalmic and Visual Research and were treated humanely, processing. according to the guidelines outlined by the United States Department Immediately after death, both eyes from each pup were enucleated of Agriculture (USDA), and the Guide for the Care and Use of Labora- and placed in ice-cold phosphate-buffered saline (PBS, pH 7.4). Enu- tory Animals (National Research Council). Euthanasia was conducted cleation was performed with the use of iris forceps and scissors for according to the guidelines of the American Veterinary Medical Asso- separation of the eyes from the surrounding connective tissue, nerve, ciation. and muscles. The eyes were dried on sterile gauze, and the vitreous fluid was aspirated with a 0.5-mL insulin syringe and placed on ice in Experimental Design sterile tubes (Eppendorf). Vitreous fluid was pooled for a total of six Certified infection-free, timed-pregnant Sprague-Dawley rats carrying samples per group. None of the vitreous fluid samples were contami- fetuses (n ϭ 10–14) of known gestational age (19 days) were pur- nated with blood. After removal of the vitreous fluid, the corneas were chased from Charles River Laboratories (Wilmington, MA). The preg- removed, and the eye cups were placed in ice-cold phosphate-buffered nant rats were placed in USDA-approved cages and remained undis- saline (PBS, pH 7.4). The retinas were excised under a dissecting turbed until delivery (22 days gestation). The animals were housed in microscope and placed in a sterile tube containing ice-cold PBS on ice, an animal facility with a 12-hour day–night cycle and provided standard before homogenization for VEGF, IGF-I, and GH protein determination. laboratory diet and water ad libitum. Within 24 hours of birth, new- For total RNA extraction, retinas were rinsed in ice-cold PBS and placed

TABLE 2. Effect on Somatic and Linear Growth, and Serum VEGF, IGF-I, and GH Levels at P14

Ibuprofen Indomethacin

Saline Low Dose High Dose Low Dose High Dose

Body weight (g) 25.0 Ϯ 0.4 25.0 Ϯ 0.5 21.1 Ϯ 0.5*** 26.3 Ϯ 0.5 27.7 Ϯ 0.6†† Linear growth (cm) 13.2 Ϯ 0.2 13.0 Ϯ 0.1 12.4 Ϯ 0.2** 13.6 Ϯ 0.1 14.2 Ϯ 0.1††† VEGF levels (pg/mL) 141.7 Ϯ 13.0 127.1 Ϯ 12.2 142.1 Ϯ 9.6 152.4 Ϯ 9.4 101.3 Ϯ 12.1† IGF-I levels (ng/mL) 282.1 Ϯ 19.8 386.7 Ϯ 30.1‡ 474.5 Ϯ 36.6‡‡ 282.1 Ϯ 19.8 344.5 Ϯ 29.5 GH levels (ng/mL) 87.1 Ϯ 7.3 97.8 Ϯ 12.5 90.2 Ϯ 7.9 73.9 Ϯ 8.9 97.7 Ϯ 13.2

Groups are as described in Table 1. ** P Ͻ 0.01; *** P Ͻ 0.001 vs. saline, ibuprofen (low dose), indomethacin (low dose and high dose). † P Ͻ 0.05 vs. saline and indomethacin (low dose); †† P Ͻ 0.01; ††† P Ͻ 0.001 vs. saline. ‡ P Ͻ 0.05 vs. saline and indomethacin (low dose); ‡‡ P Ͻ 0.01 vs. saline, ibuprofen (low dose), indomethacin (low dose and high dose).

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FIGURE 1. Comparative effects of early postnatal ibuprofen and indomethacin on vitreous fluid (A) and retinal (B) VEGF protein levels in neonatal rats at P14 (the time of retinal maturation). VEGF protein in the retinal homogenates was standardized by total cellular protein levels. The litter size per group was 30 pups. Blood samples were pooled for a total of 15 samples. Undiluted vitreous fluid samples were pooled for a total of six samples per group. Retinas were pooled for a total of five samples per group. The saline group received intraperitoneal injections of saline on days P1, P2, and P3, low dose ibuprofen received 10.0 mg/kg on P1 followed by 5.0 mg/kg on P2 and P3 (IB-10), high dose ibuprofen received 50.0 mg/kg on P1 followed by 25.0 mg/kg on P2 and P3 (IB-50), low-dose indomethacin received 0.2 mg/kg on P1, followed by 0.1 mg/kg on P2 and P3 (IN-0.2), and high dose indomethacin received 1.0 mg/kg on P1 followed by 0.5 mg/kg on P2 and P3 (IN-1.0). Data are presented as the mean Ϯ SEM.

in reagent (TRIzol; Invitrogen, Gaithersburg, MD) before extraction of was then normalized to the density of the GAPDH bands. Identification total RNA. Retinas were pooled for a total of five samples/group for of the DNA fragments was accomplished with the use of a 100-base- retinal homogenates and five samples/group for total RNA extraction. pair DNA ladder, which consisted of 15 blunt-ended fragments between 100 and 1500 bp. The data are expressed as the mean tran- Assay for VEGF script/GAPDH ratio Ϯ SEM. VEGF levels in serum, undiluted vitreous fluid, and retinal homoge- Assay of GH nates were assayed using commercially available sandwich immunoassay kits (BioSource International, Inc., Camarillo, CA). The assay pre- GH levels in serum, undiluted vitreous fluid, and retinal homogenates

dominantly binds the monomeric VEGF165 but also detects the were determined using active ultrasensitive rat GH enzyme immuno- VEGF121 isoform. The assay recognizes the 164-amino acid splice assay kits (Diagnostic Systems Laboratories, Webster, TX). The sensi- variant of mouse VEGF and has a 98% affinity for the rat sequence. The tivities of the GH assay was 0.66 pg/mL, and the intra- and interassay assay uses a monoclonal anti-VEGF detection antibody conjugated to coefficient of variations were Ͻ10%. GH levels in the retinal homoge- horseradish peroxidase and color development with tetramethylben- nates were standardized using total cellular protein levels. zidine/hydrogen peroxide (TMB solution). All assays were performed according to the manufacturer’s protocol. VEGF levels in the sample Assay of IGF-I were determined from a linear standard curve ranging from 0 to 2000 IGF-1 levels in serum, undiluted vitreous fluid, and retinal homogenates pg/mL. The coefficient of variation from inter- and intraassay precision were determined by using commercially available, nonextraction, rat assessment was Ͻ10%. VEGF levels in the retinal homogenates were IGF-I enzyme immunoassay (EIA) kits (Diagnostic Systems Laborato- standardized by using total cellular protein levels. ries, Webster, TX). This kit uses a highly sensitive antibody method, which allows detection of extremely low levels of immunoreactive free RT-PCR IGF-I. The assay measures the true free IGF-I fraction plus the fraction Total RNA was isolated from the retinas (TRIzol reagent; Invitrogen) readily dissociated from insulin-like growth factor binding proteins according to the manufacturer’s recommendations. Integrity of the (IGFBPs), which together form the biologically active pool. Samples, ribosomal RNA bands (18S and 28S) was confirmed on 1% agarose gel standards, and controls were incubated with a free IGF-1 antibody in stained with ethidium bromide. RT-PCR was conducted with the use of microtitration wells. After incubation and washing, the wells were one-step RT-PCR kits (SuperScript; Invitrogen). Amplification of cDNA treated with an anti-free IGF-I detection antibody labeled with horse- was performed using specific sense and antisense primers for GAPDH, radish peroxidase. After the plate was washed, it was developed and VEGF isoforms, PEDF, VEGFR-1, VEGFR-2, Npn-1, and Npn-2 as previ- the absorbance measured at 450 nm. The absorbance measured is ously described.24,35 Gel electrophoresis of the PCR products was directly proportional to the free IGF-I levels. The sensitivity of the assay performed on a 1.5% agarose gel stained with ethidium bromide. The was 0.015 ng/mL and intra- and interassay coefficients of variation intensities of the bands were measured (GelDoc 1000 Darkroom Im- were Ͻ10%. IGF-I levels in the retinal homogenates were standardized ager and Molecular Analyst software; Bio-Rad Laboratories, Hercules, using total cellular protein levels. CA). The PCR fragments were identified according to their molecular mass by using a DNA mass ladder (PerkinElmer, Wellesley, MA). The Statistical Analysis amount of DNA in each specimen was semiquantitated by the inte- Categorical data were analyzed by ␹2 or Fisher exact tests for cells that grated density of the product bands within a closed rectangle, which contained five or fewer observations. One-way analysis of variance

FIGURE 2. Effects of ibuprofen and indomethacin on mRNA expression

of VEGF188 (A) and VEGF164 (B) splice variants. RT-PCR analysis was performed on whole retinas pooled for a total of ﬁve samples per group. The groups and litter size are as described in Figure 1. Data are pre-

sented as the mean ratio of VEGF188 Ϯ or VEGF164/GAPDH SEM.

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FIGURE 3. Effects of ibuprofen and indomethacin on mRNA expression

of VEGF120 splice variant (A) and PEDF (B). RT-PCR analysis was performed on whole retinas for a total of ﬁve samples per group. The groups and litter size are as described in Figure 1. Data are pre-

sented as the mean ratio of VEGF120 or PEDF/GAPDH Ϯ SEM.

(ANOVA) was used to determine differences among the groups for (pg/mL), low-dose indomethacin suppressed VEGF levels normally distributed data, and the Kruskal-Wallis test was used for (665.6 Ϯ 24.2, P Ͻ 0.01) compared with the control (906.3 Ϯ non-normally distributed data after Bartlett’s test for equality of vari- 31.1; Fig. 1A). In the retinal homogenates, VEGF levels (pg/mg ances. Post hoc analysis was performed with the Tukey and Student- protein) were suppressed at low dose (145.6 Ϯ 10.4, P Ͻ 0.05) Newman-Keuls tests for significance. Significance was set at P Ͻ 0.05, and high dose (143.6 Ϯ 11.4, P Ͻ 0.05) ibuprofen, compared and data are reported as the mean Ϯ SEM, where applicable. All with the control (191.2 Ϯ 14.7). High-dose indomethacin in- analyses were two-tailed and performed on computer (SPSS; SPSS, Inc. creased VEGF levels in the retinal homogenates compared with Chicago, IL, and Prism; GraphPad Software Inc., San Diego CA). the control (238.7 Ϯ 22.4, P Ͻ 0.05; Fig. 1B). The comparative effects of ibuprofen and indomethacin on rat systemic VEGF levels at P14 are presented in Table 2. Serum VEGF levels were RESULTS significantly lower in the high-dose indomethacin group (P Ͻ Eye Opening and Somatic Growth 0.05) than in all other groups. Pooling and randomization resulted in equal distribution of runts among the groups. No more than 15 pups were assigned Retinal VEGF Signaling to each dam, which accepted all the pups as her own, despite To determine the effects of ibuprofen and indomethacin on rat pooling from other litters. There were no differences in total retinal VEGF signaling, we examined the mRNA expression of body weight (in grams) or linear growth before treatment at three VEGF splice variants (Figs. 2, 3) as well as the mRNA P1. To determine the comparative effects of ibuprofen and expression of VEGF receptors-1 and 2 (Fig. 4) and Npn-1 and -2 indomethacin on the cecal period in rats, we documented the (data not shown). Both ibuprofen and indomethacin had no number of rat pups with at least one eye open at P14 (the time significant effect on retinal VEGF mRNA expression, al- Ͻ 188 of retinal maturation). As shown in Table 1, only 13% (P though there was a nonsignificant trend for lower expression 0.001) of pups randomized to receive the high dose of ibupro- with the high dose of indomethacin (Fig. 2A). In contrast, fen had at least one eye open compared with 93% in the group retinal VEGF164 mRNA expression was suppressed with the treated with the high dose of indomethacin. Animals random- high dose of ibuprofen (P Ͻ 0.05) and both doses of indometh- ized to receive high-dose ibuprofen had decreased total body acin (P Ͻ 0.01) compared with the control (Fig. 2B). Retinal weight and linear growth. In contrast, high-dose indomethacin VEGF120 mRNA expression was suppressed with both ibupro- resulted in significantly higher total body weights and linear fen doses, although significance was achieved only with the growth concurrent with lower serum VEGF serum levels. Both high dose (P Ͻ 0.01; Fig. 3A). Retinal PEDF mRNA expression doses of ibuprofen increased serum IGF-I levels. Ibuprofen and was unchanged with ibuprofen treatment; however, both indomethacin had no appreciable effects on serum GH levels doses of indomethacin suppressed PEDF mRNA expression (Table 2). (P Ͻ 0.01) compared with the control (Fig. 3B). Retinal VEGFR-1 (Flt-1) mRNA expression did not significantly change Ocular and Systemic VEGF Levels in response to ibuprofen or indomethacin treatment, despite a To determine the effects of ibuprofen and indomethacin on nonsignificant trend for higher expression with ibuprofen (Fig. VEGF levels in the vitreous fluid and retinal homogenates, 4A). High-dose ibuprofen suppressed VEGFR-2 (Flk-1) mRNA normal vitreous fluid and retinal VEGF levels as determined by expression (P Ͻ 0.01) compared with the control (Fig. 4B). animals randomized to the saline-treated group was compared Retinal Npn-1 and -2 remained comparable among the groups to vitreous fluid and retinal levels in animals randomized to low (data not shown). Figure 5 is a representative gel from a single and high doses of ibuprofen and indomethacin (Fig. 1). Al- sample of RT-PCR amplification of rat retinal GAPDH, VEGF though ibuprofen had no effect on vitreous fluid VEGF levels isoforms, VEGFR-1, VEGFR-2, and PEDF mRNA.

FIGURE 4. Effects of ibuprofen and indomethacin on mRNA expression of VEGFR-1 (A) and VEGFR-2 (B). RT-PCR analysis was performed on whole retinas pooled for a total of ﬁve samples per group. The groups and litter size are as described in Figure 1. Data are presented as the mean ratio of VEGFR-2 or VEGFR-2/ GAPDH Ϯ SEM.

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with the control (3.53 Ϯ 0.37; Fig. 7A). No signiﬁcant changes in retinal GH levels were detected among the groups (Fig. 7B). Similarly, ibuprofen and indomethacin had no appreciable effect on GH levels in the serum (Table 2).

DISCUSSION To our knowledge, the present study is the first to examine and compare the effects of early postnatal administration of ibuprofen and indomethacin on VEGF, IGF-I, and GH in systemic and ocular compartments during normal rat retinal development. The dosage and timing of ibuprofen and indomethacin were based on previous reports that demonstrated efficacy of the drugs for closure of the ductus in preterm infants and on pharmacokinetic data.11,35–39 Based on reported data,15,16 we examined whether the beneficial effects of ibuprofen and indomethacin on retinal neovascularization are in part due to its effects on VEGF, IGF-I, and GH. Our data showed that VEGF levels tended to be higher in the vitreous fluid and lower in the retina with ibuprofen treatment, whereas an opposite response was noted with indomethacin treatment. Examination of retinal VEGF signaling showed that high-dose ibuprofen

suppressed VEGF164, VEGF120, and VEGFR-2 transcripts, result- FIGURE 5. RT-PCR analysis was performed on whole retinas pooled ing in a significant increase in the cecal period in 87% of rats at for a total of five samples per group. A single representative gel is P14. Indomethacin treatment suppressed retinal VEGF and shown for each group. The groups and litter size are as described in 164 Figure 1. Lane 1: saline; lane 2: low-dose ibuprofen; lane 3: high-dose PEDF but had no effect on VEGF receptor transcripts. Of note, ibuprofen; lane 4: low-dose indomethacin; lane 5: high-dose indo- 93% rats treated with high-dose indomethacin had at least one methacin. Data were standardized using GAPDH. eye open at P14 compared with only 13% with high-dose ibuprofen. These findings suggest that ibuprofen may be more Ocular and Systemic IGF-I Levels effective than indomethacin for the suppression of VEGF signaling if administered during the vasoproliferative phase of In the vitreous fluid, IGF-I levels (ng/mL) were higher in the ROP. Ϯ Ͻ group that received high-dose ibuprofen (121.9 12.1, P The rat model is valid for the study of retinal vascular Ϯ 0.05) compared with saline (80.4 4.0). In contrast, high-dose development because, as in premature newborn infants, the rat Ϯ indomethacin suppressed vitreous fluid IGF-I levels (21.8 retina is immature at birth, and the maturation process contin- Ͻ 14.1, P 0.05) compared with saline (Fig. 6A). Retinal IGF-I ues until the time of eye opening. During rat retinal develop- levels remained relatively unchanged among the groups (Fig. ment, undifferentiated retinal cells are evident by 14 days of 6B). A similar response to ibuprofen treatment was noted in gestation. By postnatal day 15, the vessels are fully devel- Ͻ Ͻ the serum (Table 2). Both low (P 0.05) and high (P 0.01) oped.40 The ontogeny of the rat retina is similar to that of doses of ibuprofen increased serum IGF-I levels compared with humans, and when newborn rats are maintained in room air saline and indomethacin. after hyperoxia, their retinas show characteristics of human plus disease; abnormal vessel development, vascular leaks, Ocular and Systemic GH Levels vitreal hemorrhage, and vascular tufts.41 There is a common Vitreous fluid GH levels (ng/mL) were suppressed only with timetable for the development of the visual system related to the high dose of ibuprofen (2.0 Ϯ 0.39, P Ͻ 0.05) compared the time of eye-opening in mammals.42,43 The cecal period (the

FIGURE 6. Comparative effects of early postnatal ibuprofen and indomethacin on vitreous fluid (A) and retinal (B) IGF-I protein levels in neonatal rats at P14 (the time of retinal maturation). IGF-I protein in the retinal homogenates was standardized by total cellular protein levels. The groups and litter size are as described in Figure 1. Undiluted vitreous fluid samples were pooled for a total of six samples per group. Retinas were pooled for a total of five samples per group. Data are presented as the mean Ϯ SEM.

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FIGURE 7. Comparative effects of early postnatal ibuprofen and indomethacin on vitreous fluid (A) and retinal (B) GH protein levels in neonatal rats at P14 (the time of retinal maturation). GH protein in the retinal homogenates was standardized by total cellular protein levels. The groups and litter size are as described in Figure 1. Un- diluted vitreous fluid samples were pooled for a total of six samples per group. Retinas were pooled for a total of five samples per group. Data are presented as the mean Ϯ SEM.

time of conception to eye opening) corresponds to retinal serum levels in our animals as well as their somatic and linear maturity. The novel finding that high-dose indomethacin short- growth. A decline in body weight and linear growth started to ened the cecal period in 93% of animals compared with only emerge by 4 days after the last treatment (P7) in the group that 13% with high-dose ibuprofen, suggests increased retinal vessel received 50 mg/kg ibuprofen, an effect that persisted until P14, growth and may explain at least in part, why premature new- the time of retinal vascular maturation in rats. We were sur- born infants treated with indomethacin are 1.5 times more prised to find that serum IGF-I levels in the same group were 20 prone to development of ROP. Of note is the higher number significantly elevated. At first, the elevated serum IGF-I levels of female than male rat pups with closed eyes at P14, suggest- was difficult to reconcile with decreased somatic and linear ing that male rats are more likely to have a shorter cecal period growth because it is well documented that IGF-1 is the major resulting from exposure to NSAIDs. The significance of this stimulus for pre- and postnatal growth, and its deficiency has finding remains to be determined. been shown to cause marked fetal growth retardation.52,53 VEGF is an angiogenic and permeability-enhancing factor One possible hypothesis is that the high dose of ibuprofen may that is linked to retinal ischemia-associated neovascularization. act by cleaving IGF-I from its binding proteins thereby decreas- In the human, increased vitreous VEGF correlated with retinal ing its bioactivity. Further studies are needed to confirm this ischemia-related neovascularization in diabetic retinopathy, 44 hypothesis. It was interesting to note that despite reductions in retinal vein occlusion, and ROP. VEGF121 (VEGF120 in rats) appears to be the dominant isoform induced by IGF-I and is a serum VEGF levels with high-dose indomethacin, there was major retinal isoform.45 This isoform is bioactive, freely solu- adverse impact on body weight and linear growth. Previous studies have shown that indomethacin treatment stimulates ble, and a major mediator of angiogenesis. All the murine VEGF 54,55 isoforms were present in our rat retinas with equivalent abun- GH levels in humans. Although there was a trend for higher GH levels in that group, it was not sufficient to achieve dance. Whereas VEGF188 remained unaffected by ibuprofen statistical significance. Therefore, the increased weight accre- and indomethacin, VEGF164 was downregulated with the high dose of ibuprofen and both doses of indomethacin. However, tion is not attributable to GH, but may be due to increased

only ibuprofen suppressed VEGF120 mRNA expression. Be- uptake of VEGF. IGF-I has been shown to act synergistically cause VEGF120 is the dominant isoform induced by IGF-I, this with VEGF to increase angiogenesis. IGF-I patently increases finding suggests that the high dose of ibuprofen suppresses VEGF in retinal pigment epithelium through VEGF transcrip- 56 VEGF120 through decreased uptake of retinal IGF-I and pro- tion. Increased retinal IGF-I levels may promote retinal neo- vides further evidence that ibuprofen is more effective than vascularization through its effects on retinal VEGF gene expres- indomethacin for suppression of retinal VEGF. The cellular sion. There are abundant data to support a pathologic link effects of VEGF are mediated by VEGFR-1 and -2, which are between IGF-I and neovascularization.29–31 Data from our lab- expressed exclusively by endothelial cells. Knockout studies in oratory34 as well as others57 have shown that the vitreous fluid mice have shown that the VEGFR-1 receptor mediates organi- may act as a reservoir for retinal growth factors. Our data 46 zation of the vasculature, whereas VEGFR-2 mediates endo- showed that VEGF levels in the vitreous fluid were several 47 thelial cell differentiation and proliferation. Our data showed times higher than IGF-I and GH, with GH being close to that ibuprofen suppressed VEGFR-2, suggesting decreased subnanomolar levels. Lower VEGF and IGF-I levels in the vit- VEGF signaling and retinal vascular growth. It should be noted reous with indomethacin suggest increased uptake by the ret- that this inhibition was observed only at higher concentrations, ina and may explain the shorter cecal period with indometha- which are needed to inhibit prostaglandin synthesis. Despite cin treatment. the ability of both ibuprofen and indomethacin to inhibit COX, In summary, we have demonstrated for the first time that the differences between the two drugs were remarkable. the effects of ibuprofen and indomethacin on systemic and These differences may be due to their selectivity for COX-1 and ocular growth factors are different, and therefore we reject the COX-2. Using murine COX-null cell systems, Kirtikara et al.48 demonstrated that ibuprofen was more selective for COX-2 null hypothesis. High doses of ibuprofen delay the cecal period in rats, suggesting suppression of retinal vascular growth. This than indomethacin based on COX-2 IC50/COX-1 IC50 ratio ranking. A similar finding was reported by Uzan.49 The inter- finding is corroborated by high IGF-I levels in the vitreous fluid, action between COX-2 and VEGF is well-documented. Treat- suggesting decreased uptake by the retina. In contrast, indo- ment with VEGF increased COX-2 but not COX-1 protein in a methacin increased somatic and linear growth, possibly by dose-dependent manner in endothelial cells.50,51 Therefore, in increased uptake of VEGF. High-dose ibuprofen suppressed our study, it seems plausible to speculate that ibuprofen at high retinal VEGF164, VEGF120, and VEGFR-2, suggesting decreased doses may exert its effect on VEGF through COX-2. However, VEGF signaling, whereas, low-dose ibuprofen had no apprecia- further studies are needed to confirm or refute this specula- ble effects. These data imply that ibuprofen may be beneficial tion. if administered during the vasoproliferative phase of ROP. Considering the role of IGF-I and GH as important regula- However, the adverse effects of high doses on somatic and tors of linear and somatic growth in mammals, we measured linear growth should be considered.

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Acknowledgments 22. Whitaker GB, Limberg BJ, Rosenbaum JS. VEGF-2 and neurolipin-1 form a receptor complex that is responsible for the differential

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