Biochemistry and Molecular Biology Assessment of Plasma Cytokine Profile Changes in Bevacizumab-Treated Retinopathy of Prematurity Infants

Lingkun Kong,1 Ann B. Demny,2 Ahmar Sajjad,1 Amit R. Bhatt,1 and Sridevi Devaraj3

1Department of Ophthalmology, Baylor College of Medicine, Houston, Texas, United States 2Department of Pediatrics, Baylor College of Medicine, Houston, Texas, United States 3Department of Clinical Pathology, Baylor College of Medicine, Houston, Texas, United States

Correspondence: Lingkun Kong, De- PURPOSE. We compared changes of plasma angiogenesis cytokine profiles in infants who were partment of Ophthalmology, Baylor treated with intravitreal injection of bevacizumab (IVB) for type 1 retinopathy of prematurity College of Medicine, 6701 Fannin (ROP) with age-matched preterm non-ROP infants. Street,Suite610.25,Houston,TX 77030, USA; METHODS. Thirteen infants with type 1 ROP and 13 age-matched preterm non-ROP infants [email protected]. were included. Blood samples were collected prior to treatment (time 0) and 6 weeks after Submitted: October 28, 2015 the treatment (time 42). Plasma levels of nine cytokines from the angiogenesis growth factor Accepted: February 21, 2016 panel and seven soluble cytokine receptors were measured using a magnetic multiplex assay. Citation: Kong L, Demny AB, Sajjad A, RESULTS. Plasma cytokine profiles changed from time 0 to time 42 in both groups. In Bhatt AR, Devaraj S. Assessment of bevacizumab-treated ROP infants, the following plasma angiogenesis growth factor and plasma cytokine profile changes in soluble cytokine receptor levels decreased significantly: soluble VEGF-A (sVEGF-A; P ¼ bevacizumab-treated retinopathy of 0.0001), sVEGF-D (P ¼ 0.04), angiopoietin-2 (Ang-2; P ¼ 0.002), sVEGF receptor 1 (R1) and prematurity infants. Invest Ophthal- R2 (P ¼ 0.005), soluble IL-6 receptor (sIL-6R; P ¼ 0.002), soluble glycoprotein 130 (spg130; P mol Vis Sci. 2016;57:1649–1654. ¼ 0.0001), and soluble TNF receptor (sTNFR) I and II (P ¼ 0.0001). The following factors and DOI:10.1167/iovs.15-18528 receptors increased significantly: sVEGF-C (P ¼ 0.05), placental growth factor (PlGF; P ¼ 0.02), endothelin-1 (ET-1; P ¼ 0.0001), and FGF-1 (P ¼ 0.02). At time 42, sVEGF-A, sgp130, sIL-6R, sTNFR I, and sTNFR II were lower, and ET-1 level was higher, in bevacizumab-treated ROP infants compared to age-matched non-ROP infants.

CONCLUSIONS. The results suggest that bevacizumab treatment resulted in significant angiogenic cytokine profile changes in infants with severe ROP. The long-term clinical impact of these changes should be studied carefully. Keywords: retinopathy of prematurity, intravitreal injection, Bevacizumab, cytokine, angiogenesis

etinopathy of prematurity (ROP) is a vasoproliferative the pharmacokinetics and the short- and long-term safety to R disorder that occurs in the developing retina of preterm ocular and neurologic development. Vascular endothelial infants. It is the leading cause of blindness in children,1 with an growth factor is necessary for the development of systemic incidence rate of approximately 30% in preterm infants born at neurons and blood vessels in premature infants,12,13 and or earlier than 32 weeks gestation.2 Normal retinal vasculari- blockage of VEGF could potentially inhibit these processes. In zation begins during the 14th week of gestation. This process adults, dose-dependent adverse effects from systemic bevaci- consists of two stages: vasculogenesis followed by angiogenesis. zumab-induced VEGF blockade have been reported, which In the vasculogenesis stage, precursor cells of mesenchymal include gastrointestinal perforations, wound-healing complica- origin enter the retina through the optic disc. These cells are tions, hemorrhage, stroke or myocardial infarction, hyperten- responsible for the growth of the main retinal vessels. In the sion, proteinuria, and congestive heart failure.14 angiogenesis stage, capillary vessels increase in number and In our previous study, we described the pharmacokinetics extend out from the optic disc into the peripheral retina.3 This of bevacizumab in premature infants and showed that process is tightly regulated by a complex network of angiogenic intravitreal injection of bevacizumab reduced serum-soluble cytokines, extracellular matrix components, and growth 15 factors. Vascular endothelial growth factor (VEGF) is an VEGF-A (sVEGF-A) levels ; however, the clinical significance important component in this network and a major factor for of the reduction of sVEGF-A and its impact on other cytokines the pathogenesis of ROP.4 Therefore, the goals of treatment are are unknown. We hypothesized that the reduction of sVEGF-A to reduce the production of VEGF by the immature retina and regulates the expression of other angiogenic cytokines to eliminate the abnormal growth of new vessels. Laser secreted locally and systemically. To test this hypothesis, we photocoagulation of the peripheral avascularized retina is the assessed plasma cytokine profile changes in infants who were treatment standard for type I ROP, with a failure rate of treated with bevacizumab and compared these profiles to age- approximately 9.1%.5 Recently, intravitreal injection of the anti- matched non-ROP preterm infants. Our goal was to evaluate VEGF antibody bevacizumab has been used as an off-label the potential negative effect of systemic bevacizumab alternative therapy.6–11 However, there are concerns regarding treatment.

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TABLE 1. Plasma Cytokines Tested TABLE 2. Characteristics of Preterm Infants With and Without Retinopathy of Prematurity Growth Factor Panel Receptor Panel Type I ROP Non-ROP P Range of Range of Growth Measurement, Measurement, Birth weight, g 778 6 66 941 6 96 0.15 Factors pg/mL Receptors pg/mL Gestational age, wk 25.6 6 0.6 28.0 6 0.6 0.01 Gender, male (%) 7 (54) 6 (46) 0.7 VEGF-A 14–10,000 VEGF R1 122–500,000 PMA at first blood collection, wk 34.9 6 1.7 34.0 6 0.5 0.8 VEGF-C 7–5,000 VEGF R2 122–500,000 VEGF-D 7–5,000 VEGF R3 122–500,000 Ang-2 14–10,000 sIL-6R 12–50,000 equation (GEE) models were used to analyze the relation- ET-1 3–2,000 spg130 24–100,000 ships among variables. One-way analysis of variance (AN- BMP-9 3–2,000 sTNFR I 12–50,000 OVA) with a post-hoc multicomparison test was used to PlGF 2–1,000 sTNFR II 12–50,000 compare the three groups of data. Comparisons among FGF-1 and 2 14–10,000 variables were adjusted for GA and BW. Fisher’s exact test was used for proportions. The confidence interval level was 95%. A P value less than 0.05 was considered statistically METHODS AND MATERIALS significant. Study Subjects The study followed the tenets of the Declaration of Helsinki, RESULTS and informed consent was obtained from the parent or legal Table 2 shows the demographic data of the subjects. Thirteen guardian of infants with ROP after explaining the study and its infants with type I ROP and 13 age-matched preterm non-ROP possible consequences. The informed consent was waived for infants were included in the study. Infants with type I ROP non-ROP infants, and the research was approved by the were on average younger than non-ROP infants (P 0.01). The institutional review board of Baylor College of Medicine. ¼ BW of ROP infants was lower than non-ROP infants but was not Whole blood samples from 13 infants who were diagnosed as type I ROP16 were collected just prior to treatment (time 0) and 6 statistically significant (P ¼ 0.15). Post menstrual ages at the weeks after treatment (time 42). Plasma from 13 age-matched time blood samples were collected were similar in both premature infants who did not develop ROP (non-ROP) and were groups. not treated with anti-VEGF–related medications were collected between 32 and 34 weeks post menstrual age (PMA) (time 0) and Changes of Plasma Cytokines With Time 38 and 40 PMA (time 42) from salvaged blood at the Texas Children’s Hospital clinical pathology laboratory. For plasma Nine soluble cytokines from the angiogenesis growth factor collection, whole blood was collected in a blood collection tube panel and seven soluble cytokine receptors were quantitat- ed. In infants with ROP treated with bevacizumab, from time with 1.0 mg K2 EDTA (Microtainer; Becton, Dickinson and Company, Franklin Lakes, NJ, USA). Blood cells were removed by 0totime42,thefollowingplasmaangiogenesisgrowth centrifuging at 3000g for 15 minutes at room temperature. The factors and soluble cytokine receptor levels decreased supernatant was aliquoted into clean polypropylene tubes and significantly:sVEGF-A,sVEGF-D,Ang-2,sVEGFR1andR2, stored at 808C until analysis. sIL-6R, sgp130, and sTNFR I and sTNFR II (P values are The following clinical data were collected: ROP status at the indicatedinFig.1andFig.2).Thefollowingfactorsand time of treatment, gestational age (GA), birth weight (BW), receptors increased significantly: sVEGF-C, PlGF, ET-1, and primary diagnosis, PMA at the time of treatment, and body FGF-1 as shown in Figure 1 (growth factors) and Figure 2 weight at the time of treatment. (receptors). In non-ROP infants, a significant decrease was seenoverthetimecourseinlevelsofsVEGF-A,Ang-2,sVEGF Measurement of Plasma Cytokines and Their R3, sIL-6R, and sTNFR I, and an increase was seen in PlGF Receptors levels. All other cytokines remained at similar levels from time 0 to time 42. A multiplex assay was used to measure plasma cytokines and their receptor levels. The protocols of the manufacturer were Comparison of Plasma Cytokine Profiles in followed. The human angiogenesis/growth factor cytokine panel included the following factors: angiopoietin-2 (Ang-2), Bevacizumab-Treated Type I ROP Infants Versus fibroblast growth factor (FGF)-1, FGF-2, sVEGF-A, sVEGF-C, Non-ROP Infants sVEGF-D, endothelin-1 (ET-1), bone morphogenetic protein The plasma cytokine levels at pre- and posttreatment in both (BMP)-9, and placental growth factor (PlGF; Catalog # type I ROP and non-ROP infants are shown in Figure 1 (growth HAGP1MAG-12K, Millipore, Billerica, MA, USA). The human soluble cytokine receptor magnetic bead panel included s- factors) and Figure 2 (receptors). In bevacizumab-treated ROP interleukin (IL) 6R, soluble tumor necrosis growth factor infants, plasma levels of VEGF-A (P ¼ 0.001), sgp130 (P ¼ 0.05), receptor (sTNFR)I, sTNFRIIs, VEGF receptor (R)1, R2, R3, and sIL-6R (P ¼ 0.0001), sTNFR I (P ¼ 0.001), and sTNFR II (P ¼ soluble glycoprotein (sgp)130 (Catalog # HSCRMAG32KPX14; 0.0001) were significantly lower at time 42 compared to those Millipore, Billerica, MA, USA) (Table 1). in age-matched non-ROP infants. The plasma ET-1 level was significantly higher in bevacizumab-treated ROP infants (P ¼ 0.004). The following plasma cytokine levels were similar in Statistics both groups at time 42: VEGF-C (P ¼ 0.5), VEGF-D (P ¼ 0.1), Statistical analyses were performed using software SPSS PlGF (P ¼ 0.6), BMP-9 (P ¼ 0.1), FGF-1 and FGF-2 (P ¼ 0.4 and P version 21 (IBM, Software, Armonk, NY, USA) and GraphPad ¼ 0.2, respectively), Ang-2 (P ¼ 0.1), and VEGF R1, VEGF R2, Prism 5 (GraphPad, La Jolla, CA, USA). Generalized estimating and VEGF R3 (P ¼ 0.5, 0.2, and 0.3, respectively).

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FIGURE 1. The changes of angiogenesis growth factors in ROP and non-ROP infants. P values are indicated on the top of each paired column. Black solid column is for ROP group, and the white column is for non-ROP group.

18 DISCUSSION -C, -D, and -E, and PlGF. Our first objective was to determine whether the circulating bevacizumab reduced plasma levels of Bevacizumab is a recombinant humanized monoclonal anti- other VEGF ligands. We evaluated four VEGF family members, body that blocks angiogenesis by inhibiting VEGF-A.17 In our sVEGF-A, sVEGF-C, sVEGF-D, and PlGF (VEGF-B and -E were previous study, we showed that serum sVEGF-A levels were not measured in this study due to the availability of testing significantly reduced after bevacizumab treatment in severe beads), and their soluble receptors R1, R2, and R3. As shown in ROP patients and remained at low levels.15 The reduction of Figures 1 and 2, after bevacizumab treatment, the plasma levels sVEGF levels by bevacizumab could interrupt angiogenesis in of sVEGF-A, sVEGF-D, VEGF R1, and VEGF R2 were signifi- developing organs, raising major safety concerns regarding the cantly reduced; however, only the sVEGF-A level was use of bevacizumab in premature infants. To address this issue, significantly lower than that of the control group (P ¼ we evaluated the levels of plasma angiogenesis factors and 0.0001). Plasma levels of VEGF-C, VEGF-D, and PlGF were soluble cytokine receptors in infants with ROP before and after slightly higher in bevacizumab-treated ROP infants compared bevacizumab treatment and compared these to premature with non-ROP infants. The levels of all three soluble receptors infants who did not develop ROP as a control group. Our were similar between these two groups. These results suggest hypothesis was that developmental stages are a more that the changes of VEGF and its receptor level in the plasma significant source of variation in angiogenic cytokine levels are mainly related to the changes of ROP status (in ROP infants) than are ROP and bevacizumab treatment. and developmental status, except sVEGF-A in bevacizumab- Angiogenesis is a complex process that is regulated treated ROP infants. Our next step was to analyze the precisely by many angiogenic cytokines. Vascular endothelial differences in total plasma VEGF levels between the two growth factors are the major growth factors in this process. groups using the GEE statistical model. The results showed that The mammalian family of VEGF ligands consists of VEGF-A, -B, plasma levels of total VEGFs were similar in bevacizumab-

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FIGURE 2. The changes of angiogenesis growth factor receptors in ROP and non-ROP infants. P values are indicated on the top of each paired column. Black solid column is for ROP group, and the white column is for non-ROP group.

treated ROP infants 6 weeks after treatment as they were in the binding to VEGF R2.24 We hypothesize that the activity of other control group: 1897.7 6 363.9 pg/mL versus 2226.1 6 224.0 VEGF family members could compensate for the loss of sVEGF- pg/mL, respectively (P ¼ 0.29). These results suggest that A function and therefore that bevacizumab-treated infants systemic absorption of bevacizumab reduces plasma levels of could have a angiogenic process similar to that of the control sVEGF-A; however, the total sVEGF level remained similar to group. that in the controls. In this study, we hypothesized that reduction VEGF-A Vascular endothelial growth factors act within a complex regulates the expression of other angiogenic cytokines. To test regulatory network, binding selectively to different receptors this hypothesis, we measured plasma levels of nine soluble and activating networks that regulate angiogenesis, cell growth factors and seven soluble cytokine receptors from the growth, and survival.19,20 Vascular endothelial growth factor- human angiogenesis/growth factor panel using the magnetic A is the most prominent and founding member, and it binds to multiplex assay. The results showed that, in contrast to the both VEGF R1 and R2.21 Placental growth factor binds reduction of sVEGF-A levels, plasma ET-1 levels increased exclusively to VEGF R122 and could have comparable activity significantly in infants with type I ROP at 42 days posttreat- to VEGF.23 Vascular endothelial growth factor-C and VEGF-D ment when compared with the control group. Endothelin-1 is bind to VEGF R3, but can be proteolytically processed to allow produced by both endothelial cells and vascular smooth

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muscle cells, and its concentration is elevated in many 4. Sood BG, Madan A, Saha S, et al. Perinatal systemic tumors.25 The relationship between VEGF-A and ET-1 has been inflammatory response syndrome and retinopathy of prema- controversial. One study reported that ET-1 stimulates vaso- turity. Pediatr Res. 2010;67:394–400. proliferative processes, and at maximally effective concentra- 5. Good WV. Final results of the Early Treatment for Retinopathy tions its effects were additive to that of VEGF by functioning of Prematurity (ETROP) randomized trial. Trans Am Ophthal- through independent signaling mechanisms.26 Other studies mol Soc. 2004;102:233–250. reported that VEGF enhanced ET-1 mRNA expression and ET-1 6. Chung EJ, Kim JH, Ahn HS, Koh HJ. Combination of laser secretion in endothelial cells and vascular smooth muscle photocoagulation and intravitreal bevacizumab (Avastin) for cells.27–29 This suggests that VEGF and ET-1 have reciprocal aggressive zone I retinopathy of prematurity. Graefes Arch stimulatory interactions, resulting in concomitant proliferation Clin Exp Ophthalmol. 2007;245:1727–1730. of endothelial and vascular smooth muscle cells. However, one 7. Kong L, Mintz-Hittner HA, Penland RL, Kretzer FL, Chevez- study also reported that ET-1 and VEGF have a regulatory Barrios P. Intravitreous bevacizumab as anti-vascular endothe- relationship.30 Our results indicate that the reduction of lial growth factor therapy for retinopathy of prematurity: a sVEGF-A upregulated the expression of ET-1 in the plasma. morphologic study. Arch Ophthalmol. 2008; 126:1161–1163. The elevation of plasma ET-1 levels could be beneficial to 8. Kusaka S, Shima C, Wada K, et al. Efficacy of intravitreal infants by compensating for the loss of VEGF-A. However, this injection of bevacizumab for severe retinopathy of prematu- elevation itself could raise other concerns related to the rity: a pilot study. Br J Ophthalmol. 2008;92:1450–1455. overexpression of ET-1, such as increased smooth muscle cell 9. Lim LS, Mitchell P, Wong TY, Gole GA, Camuglia JE, Ells AL. proliferation, which is seen in persistent pulmonary hyperten- Bevacizumab for retinopathy of prematurity. Author reply. N 31 sion of newborn infants. The long-term clinical impacts Engl J Med. 2011;364:2361–2361. therefore need to be studied carefully. 10. Mintz-Hittner HA, Kennedy KA, Chuang AZ. Efficacy of A limitation of this study is the small sample size. A intravitreal bevacizumab for stage 3þ retinopathy of prematu- multicenter clinical placebo-controlled trial with a large sample rity. N Engl J Med. 2011;364:603–615. is therefore necessary to study the safety of bevacizumab, both 11. 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