Angiopoietin-like 4 is a potent angiogenic factor and a novel therapeutic target for patients with proliferative diabetic retinopathy

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1 Angiopoietinlike 4 is a potent angiogeni fator and a novel therapeuti target for patients with proliferative diabeti retinopathy Savalan abapoorfarrokhran a,, Kathleen Jee a,, rooks Puhner a, Syed Junaid Hassan a, Xiaoban Xin a, Murilo Rodrigues a, Fabiana Kashiwabuhi a, Tao Ma b,kehu a,, Monika Deshpande a, Yassine Daoud a, Sharon Solomon a, Adam Wenik a, Gerard A. Lutty a, Gregg L. Semenza d, Silvia Montaner b, and Akrit Sodhi a, a Wilmer Eye Institute, Johns Hopkins University Shool of Mediine, altimore, MD 87; b Department of Onology and Diagnosti Sienes, Shool of Dentistry, and Department of Pathology, Shool of Mediine, Greenebaum aner enter, University of Maryland, altimore, MD ; d Vasular Program, Institute for ell Engineering, and Departments of Pediatris, Mediine, Onology, Radiation Onology, iologial hemistry, and Geneti Mediine, Johns Hopkins University Shool of Mediine, altimore, MD, ; and Department of Ophthalmology, The First Affiliated Hospital of hongqing Medial University, 46 hongqing, hina Edited by George D. Yanopoulos, Regeneron Pharmaeutials, In., Tarrytown, NY, and approved May, (reeived for review Deember, 4) Diabeti eye disease is the most ommon ause of severe vision loss in the workingage population in the developed world, and proliferative diabeti retinopathy () is its most visionthreatening sequela. In, retinal ishemia leads to the upregulation of angiogeni fators that promote neovasularization. Therapies targeting vasular endothelial growth fator (VEGF) delay the development of neovasularization in some, but not all, diabeti patients, impliating additional fator(s) in pathogenesis. Here we demonstrate that the angiogeni potential of aqueous fluid from patients is independent of VEGF onentration, providing an opportunity to evaluate the ontribution of other angiogeni fator(s) to development. We identify angiopoietinlike 4 (ANGPTL4) as a potent angiogeni fator whose expression is upregulated in hypoxi retinal Müller ells in vitro and the ishemi retina in vivo. Expression of ANGPTL4 was inreased in the aqueous and vitreous of patients, independent of VEGF levels, orrelated with the presene of diabeti eye disease, and loalized to areas of retinal neovasularization. Inhibition of ANGPTL4 expression redued the angiogeni potential of hypoxi Müller ells; this effet was additive with inhibition of VEGF expression. An ANGPTL4 neutralizing antibody inhibited the angiogeni effet of aqueous fluid from patients, inluding samples from patients with low VEGF levels or reeiving antivegf therapy. olletively, our results suggest that targeting both ANGPTL4 and VEGF may be neessary for effetive treatment or prevention of and provide the foundation for studies evaluating aqueous ANGPTL4 as a biomarker to help guide individualized therapy for diabeti eye disease. diabetes neovasularization induible fator angiopoietinlike 4 vasular endothelial growth fator Diabeti eye disease is the most ommon mirovasular ompliation in the diabeti population and remains the leading ause of blindness among workingage adults in the developed world (). Diabeti retinopathy (DR) is lassified as either nonproliferative (N) or proliferative (). Although sustained hyperglyemia is the major stimulus for the development of N (), retinal ishemia is the prerequisite for and results in the upregulation of angiogeni fators that promote retinal neovasularization (). If left untreated, neovasularization an lead to vitreous hemorrhage, retinal detahment, or glauoma and result in profound and often irreversible loss of vision. For the last 4 deades, the standard of are for has been panretinal photooagulation (PRP), a proess in whih the peripheral ishemi retina is killed (burned) with a laser to protet the patient s entral vision (4). Although effetive in reduing the risk of entral vision loss, PRP results in dereased peripheral and night vision in treated patients. Moreover, an progress in patients despite appropriate treatment. This emphasizes the importane of understanding the mehanism(s) promoting the development of retinal neovasularization to identify targeted therapeuti approahes for the prevention or treatment of. In this regard, induible fators (HIFs) ativate the transription of multiple genes enoding angiogeni ytokines that promote retinal neovasularization in (). Among the angiogeni genes regulated by HIFs in, onsiderable attention has foused on the ontribution of vasular endothelial growth fator (VEGF). Several multienter randomized ontrolled linial trials have demonstrated the benefit of monthly injetions with biologi moleules direted against VEGF to treat diabeti maular edema (DME) (6). These studies have demonstrated a signifiant redution in the progression to in some but not all patients reeiving antivegf therapy (7, 8), suggesting that other mediator(s) may partiipate in the development of in many diabeti patients. Over the last deades, several angiogeni ytokines, growth fators, and inflammatory mediators have been impliated in the pathogenesis of (9). Despite these efforts, PRP treatment for Signifiane In proliferative diabeti retinopathy (), the most visionthreatening sequela of diabeti eye disease, retinal ishemia leads to inreased expression of angiogeni fators that promote neovasularization. Although therapies targeting the potent angiogeni mediator vasular endothelial growth fator have been remarkably suessful for the treatment of diabeti maular edema, this approah has not proven suffiient to prevent the development of retinal neovasularization, impliating additional angiogeni fator(s) in pathogenesis. We demonstrate here that angiopoietinlike 4 is a potent angiogeni mediator with markedly inreased expression in the eyes of patients. Our studies identify a novel therapeuti target for the treatment of oular neovasular disease and may have broad impliations for the treatment of other diseases dependent on pathologi angiogenesis. Author ontributions: S.M. and A.S. designed researh; S..F., K.J.,.P., S.J.H., X.X., M.R., F.K., T.M., K.H., and M.D. performed researh; Y.D., S.S., and A.W. ontributed new reagents/analyti tools; Y.D., S.S., and A.W. ontributed patient samples; S..F., K.J.,.P., X.X., M.R., T.M., G.A.L., G.L.S., S.M., and A.S. analyzed data; and G.L.S., S.M., and A.S. wrote the paper. The authors delare no onflit of interest. This artile is a PNAS Diret Submission. Freely available online through the PNAS open aess option. S..F. and K.J. ontributed equally to this work. To whom orrespondene should be addressed. asodhi@jhmi.edu. This artile ontains supporting information online at 7/pnas.476//DSupplemental. MEDIAL SIENES PNAS PLUS PNAS Early Edition of

2 remains unhanged. In an effort to identify an alternative therapeuti approah for the treatment of retinal neovasularization, we set out to identify an ishemiadriven mediator that diretly ontributes to the angiogeni phenotype in patients with. Results Angiogeni Potential of Aqueous Fluid from Is Elevated but Independent of VEGF Levels. The levels of angiogeni growth fators in the aqueous fluid of diabeti patients orrelate with the severity of DR (). To study the angiogeni potential of aqueous fluid from diabeti patients with and without, we assessed the ability of aqueous fluid to stimulate endothelial ell tubule formation. Aqueous fluid from ontrol patients (Fig. A, Table, and SI Appendix, Fig. SA) or diabeti patients without DR (Fig. and SI Appendix, Fig. S) did not have a signifiant effet on tubule formation. In ontrast, aqueous fluid from diabeti patients with stimulated tubule formation (Fig. and SI Appendix, Fig. S). This effet was orrelated with an approximately twofold inrease in the onentration of VEGF in aqueous fluid from patients ompared with ontrol patients or diabeti patients without DR (SI Appendix, Fig. S and Table S). These results are onsistent with the hypothesis that the inreased indution of tubule formation by aqueous fluid from patients was due to elevated levels of VEGF. However, VEGF levels in the aqueous fluid from patients demonstrated onsiderable variability (Fig. D and SI Appendix, Fig. SA); /8 (6%) of patients with had VEGF levels in the aqueous fluid that were less than the mean value measured for ontrol (nondiabeti) patients. These VEGF levels were similar to those deteted in aqueous fluid from patients who had reeived antivegf therapy within wk of sample olletion (Fig. D). Nonetheless, stimulation of tubule formation by these lowvegf aqueous fluid samples (Fig. E and SI Appendix, Fig. S) was similar to that by highvegf aqueous fluid samples (Fig. F and SI Appendix, Fig. S). Indeed, there was no orrelation between the VEGF onentration and stimulation of tubule formation by aqueous fluid samples (Fig. G and SI Appendix, Fig.SD). A D (pg/ml) E serum n= F serum DM no DR n= n= n=4 G DM no DR N antivegf (pg/ml). (pg/ml) 48 8 Y =.866X.6 R =..... (pg/ml) 6 4 n=6 Low VEGF n=4 High VEGF n=6 n= Fig.. Angiogeni potential of aqueous fluid from patients is elevated but independent of VEGF levels. (A and ) Aqueous fluid from nondiabeti patients (ontrol patients; A) and diabeti patients without DR (DM no DR; ) does not stimulate tubule formation. () Aqueous fluid from diabeti patients with ( ) stimulates tubule formation. Media without serum (ontrol) or with % (vol/vol) FS (serum) serve as ontrols. (D) Levels of VEGF in the aqueous fluid from diabeti and nondiabeti patients. Aqueous fluid levels from /8 patients measure below the average levels observed for nondiabeti patients (dashed line). Note: samples with > 6 pg/ml are not displayed to adequately demonstrate the variability within the ontrol samples. (E and F) Aqueous fluid from patients with VEGF levels below the average level for ontrol (nondiabeti) patients ( low VEGF; E) stimulate tubule formation similar to aqueous fluid from patients with VEGF levels higher than the average level for ontrol patients ( high VEGF; F). (G) Aqueous fluid stimulation of tubule formation does not orrelate with the onentration of VEGF. Oneway ANOVA. Student s t test and Pearson orrelation. of abapoorfarrokhran et al.

3 Angiogeni Potential of Aqueous Fluid from Is Not Affeted by AntiVEGF Therapy. Despite the variability in VEGF onentrations in aqueous fluid samples, the presene of VEGF in these samples ould still be responsible for the indution of tubule formation. To assess whether the angiogeni potential of aqueous fluid from patients was influened by Table. Patient samples for tubule formation assays antivegf therapy, we tested the ability of aqueous fluid from patients (with high, average, or low levels of VEGF) to stimulate tubule formation in the presene of bevaizumab at a fold higher onentration than the dose whih effetively neutralizes the highest levels of VEGF deteted in the aqueous fluid of patients (SI Appendix, Fig. S4and Table ). Treatment Patient Age, y Sex Phaki status Prior vitretomy DM type DM duration, y Prior PRP AntiVEGF within 6 wk AntiVEGF within wk 7 F P No 6 F P No 8 M P No 4 7 F P No 7 M P No 6 7 F P No 7 64 F P No 8 49 F P No 9 M P No F P No 6 F P No 7 F P No F P No 4 6 F P No 9 M P No Diabeti (no DR) 8 F P No II 9 7 M P No I 7 68 F P No II 4 76 F PP No II 6 M P No II F P No II M P No II 8 6 F P No I F P No II 6 F P No II 9 F P No II 9 Yes No No 7 F P No II No No No 46 M P No II Yes No No 4 8 M P No II No No No HighVEGF M P No I No No No 7 F P Yes I 7 Yes No No 8 M P No II No No No 4 7 F P No II No No No M P No II 4 No No No 6 M P No II Yes No No LowVEGF M P Yes II Yes No No 8 M P Yes II 9 Yes No No F PP No I 6 Yes No No 4 4 M P No I Unknown Yes No No AntiVEGF M P No II 4 No No Yes M P No I Unknown Yes No Yes F P No I No No Yes 4 F P No I Yes No Yes 4 M P No II Yes No Yes 6 68 M P No II No No Yes 7 8 F P No II Unknown Yes No Yes 8 7 M P No II Unknown Yes No Yes 9 4 M P No II Unknown Yes No Yes MEDIAL SIENES PNAS PLUS At time of sample olletion. DM, diabetes mellitus; DR, diabeti retinopathy; F, female; M, male; P, phaki;, proliferative diabeti retinopathy; PP, pseudophaki; PRP, panretinal photooagulation; VEGF, vasular endothelial growth fator. abapoorfarrokhran et al. PNAS Early Edition of

4 with bevaizumab did not signifiantly affet tubule formation stimulation by aqueous fluid from patients (Fig. A and SI Appendix, Fig. SA). Moreover, aqueous fluid from patients who reeived antivegf therapy by intravitreal injetion within wk of sample olletion had VEGF levels lower than those measured in aqueous fluid from ontrol patients and diabetis without DR (SI Appendix, Fig. S), yet these samples still stimulated tubule formation (Fig. and SI Appendix, Fig. S) and were also unaffeted by treatment with additional bevaizumab (Fig. and SI Appendix, Fig. S). HIF Promotes the Seretion of VEGF and Other Angiogeni Fators in Hypoxi Retinal ells. These results suggested that VEGF is not the only angiogeni mediator responsible for stimulation of tubule formation by aqueous fluid from patients, providing a unique opportunity to examine the ontribution of other angiogeni mediator(s) to the development of. To interrogate the role of novel angiogeni fators in mediating retinal neovasularization in, we used the oxygenindued retinopathy (OIR) model of ishemi retinal disease (). During the ishemi stage of the OIR model, ishemia in the posterior retina promotes stabilization of HIFα (Fig. A), whih results in the promotion of retinal neovasularization (Fig. ). Inhibition of HIFα aumulation by treatment with digoxin (Fig. A) prevents the development of retinal neovasularization in the OIR model (Fig. and ) (). We have previously reported that hypoxi retinal Müller ells in the ishemi inner retina play a ritial role in the expression of VEGF and the promotion of vasular permeability and angiogenesis by stabilizing HIFα (, 4). Stabilization of HIFα in hypoxi Müller ells resulted in the upregulation of VEGF expression (Fig. D and E). In turn, onditioned media from these ells were potently angiogeni, a property that was ompletely bloked following pretreatment of the Müller ells with digoxin ( nm) to inhibit HIFα aumulation (Fig. F), demonstrating the importane of this transription fator in the regulation of angiogeni genes in retinal Müller ells. These results suggested that HIFα was neessary to promote the angiogeni potential of retinal Müller ells. y ontrast, RNA interferene (RNAi) targeting VEGF (Fig. G and H) or bloking antibody to VEGF (Fig. I) only partially inhibited the angiogeni potential of onditioned media from retinal Müller ells. HIFDependent ANGPTL4 Expression Is Indued by Hypoxia in Vitro and Retinal Ishemia in Vivo. We set out to identify another andidate angiogeni mediator that may play a role in the promotion of angiogenesis in the aqueous fluid of patients with low VEGF levels. To this end, we exposed retinal Müller ells to and examined the expression of mrnas enoding inflammatory ytokines, proteases, and angiogeni ytokines previously reported to be regulated (diretly or indiretly) by HIF and proposed to promote angiogenesis. ILβ, angiogenin, PIGF, IGFP, and IGFP mrna levels were inreased within 4 h of exposure to, whereas TNFα, PAI, and bfgf were indued after 48 h of exposure to (Fig. 4A and SI Appendix, Fig. S6). However, the fold indution of these mrnas was modest ompared with VEGF mrna. Only the indution of angiopoietinlike 4 (ANGPTL4) mrna was omparable to that of VEGF mrna (Fig. 4A). HIFdependent expression of ANGPTL4 has been shown to mediate the ability of onditioned media from Kaposi s saroma and hypoxi breast aner ells to impair E E interation (, 6). We have reently reported that ANGPTL4 may play an important role in vasular permeability in ishemi retinal disease, inluding DME (). ANGPTL4 has also been impliated in the upregulation of retinal neovasularization by peroxisome proliferatorativated reeptor (PPARβ/δ) (7). However, a role for ANGPTL4 as an angiogeni fator in patients with has not been explored. We first investigated whether ANGPTL4 mrna expression was also inreased in vivo in the OIR model and found that the fold indution of ANGPTL4 mrna was similar or greater than that observed for VEGF mrna (Fig. 4). y ontrast, mrna levels of several other known HIFregulated angiogeni fators were only modestly inreased. ANGPTL4 protein expression in the OIR model was onfirmed by immunohistohemistry (Fig. 4). ANGPTL4 Is Neessary and Suffiient to Promote Angiogenesis. We next investigated whether ANGPTL4 ontributes to the angiogeni phenotype in. Reombinant human (rh)angptl4 promoted E survival and migration (SI Appendix,Fig.S7A and ) but did not affet E proliferation (SI Appendix, Fig. S7). Inreasing doses of A (pg/ml) (pg/ml) (pg/ml) (% ompared to untreated) (% ompared to untreated) 7 antivegf 4 Average AntiVEGF AntiVEGF6 AntiVEGF7 AntiVEGF8 AntiVEGF9 Average n= n=8 Fig.. Angiogeni potential of aqueous fluid from patients is unaffeted by antivegf therapy. (A) Addition of the VEGF neutralizing monolonal antibody, bevaizumab, to aqueous fluid in the tubule formation assay does not signifiantly affet the ability of aqueous fluid from patients to stimulate tubule formation, regardless of. Data presented as perent indution of tubule formation ompared with tubule formation indued by aqueous fluid in the absene of bevaizumab. () Aqueous fluid from patients who reeived an intravitreal injetion with antivegf therapy within wk of sample olletion ( antivegf) is still able to stimulate tubule formation. () Addition of bevaizumab to aqueous fluid in the tubule formation assay does not signifiantly affet the ability of aqueous fluid from antivegf patients to stimulate tubule formation. Data presented as perent indution of tubule formation ompared with tubule formation indued by aqueous fluid from antivegf patients in the absene of bevaizumab. Wiloxon test. 4of abapoorfarrokhran et al.

5 A D E HIFα P. OIR posterior HIFα GAPDH periphery P OIR P7 ontrol P7 OIR P7 OIR digoxin DAPI D 4 48 digoxin digoxin (pg/ml) P OIR digoxin % NV Tufts vs. untreated OIR P ontrol P ontrol OIR P7 OIR P7 Digoxin digoxin F... G VEGF mrna ontrol RNAi VEGF RNAi H I VEGF RNAi... bevaizumab ontrol serum ontrol (pg/ml) MEDIAL SIENES PNAS PLUS Fig.. HIF promotes the seretion of VEGF and other angiogeni fators in hypoxi retinal ells. (A) Immunohistohemial analysis demonstrates aumulation of HIFα in the retina h following return of postnatal day (P) OIR pups from hyperoxi [7% (vol/vol) O ] to normoxi [% (vol/vol) O ] onditions. Daily i.p. injetion of the HIFinhibitor digoxin inhibits HIFα protein aumulation in OIR mie. ( and ) Immunofluoresent analysis demonstrates the presene (white double arrows) or absene (red double arrows) of inner retinal vessels in the P7 ontrol vs. OIR mie, respetively (). Preretinal (pathologial) neovasularization (yellow arrows) is observed in P7 OIR mie but not in digoxintreated mie, despite the absene of inner retinal vessels (red double arrows). Inhibition of HIFα aumulation with daily i.p. injetions of digoxin prevents the development of retinal neovasularization (). (D and E) Exposure of human retinal Müller ells to % O () promotes HIFα protein aumulation (D) and VEGF seretion (E), whih are both bloked by treatment with digoxin. (F) Stimulation of tubule formation by onditioned media from retinal Müller ells exposed to is bloked with digoxin. (G and H) RNAi targeting VEGF inhibits mrna expression and protein seretion of VEGF (G) but only partially inhibits the stimulation of tubule formation by onditioned media from Müller ells exposed to (H). Similar results were obtained using a VEGF neutralizing antibody (bevaizumab; I). Oneway ANOVA. rhangptl4 potently stimulated tubule formation in vitro (Fig. A) and orneal neovasularization in vivo (Fig. and ). To diretly assess whether ANGPTL4 ontributed to the angiogeni potential of hypoxi retinal Müller ells, we transfeted Müller ells with RNAi targeting ANGPTL4 and observed a marked redution in ANGPTL4 but not VEGF mrna and protein expression (Fig. D). onditioned media from these ells had redued angiogeni potential ompared with ontrols (Fig. D). Inhibition of either ANGPTL4 or VEGF expression alone resulted in an % redution in the stimulation of tubule formation by hypoxi retinal Müller ells (Fig. E). However, inhibiting both ANGPTL4 and VEGF with RNAi resulted in an almost % redution in the stimulation of tubule formation by hypoxi retinal Müller ells (Fig. E). ANGPTL4 Is an Angiogeni Fator Expressed in the Eyes of Diabeti with. We hypothesized that ANGPTL4 may be an angiogeni fator present in lowvegf aqueous fluid from patients. To test this hypothesis, we measured levels of ANGPTL4 in aqueous fluid from diabeti patients and observed that ANGPTL4 levels were inreased in the aqueous fluid of patients ompared with the aqueous fluid of ontrol patients, diabeti patients without DR, and diabeti patients with N (Fig. 6A and SI Appendix, Table S). Levels of ANGPTL4 were inreased even in the aqueous fluid of patients who had been treated with antivegf therapy within wk of sample olletion and in whih VEGF levels were markedly redued (SI Appendix, Fig. S8), suggesting that ANGPTL4 expression was independent of VEGF expression. Immunohistohemial analysis of eyes from / patients demonstrated ANGPTL4 expression in areas of preretinal neovasularization (Fig. 6). We also observed elevated ANGPTL4 levels in the vitreous of patients ompared with ontrol patients (Fig. 6 and SI Appendix, Table S). Interestingly, we observed a lose orrelation between the aqueous and vitreous levels of ANGPTL4 in patients (Fig. 6D). abapoorfarrokhran et al. PNAS Early Edition of

6 A 4 ILb IL6 IL8 MP TNFa XL6 MMP4 TIMP UPA PAI ANG ANG ANGIOGENIN PIGF bfgf PEDF HGF PDGF IGFP IGFP ANGPTL4 VEGF mrna (fold Indution) 6 TGFb 4hr 48hr PEDF P OIR P4 OIR P4 ontrol PDFG FGF ANGPTL4 mrna (fold Indution) 4 HGF ANG VEGF PIGF ANG ANGPTL4 P P P4 Fig. 4. Hypoxia potently promotes upregulation of ANGPTL4 mrna and protein expression at levels similar to VEGF. (A and ) The mrna expression of known inflammatory ytokines, proteases, and angiogeni ytokines regulated (diretly or indiretly) by HIFα and previously reported to play a role in angiogenesis in hypoxi human retinal Müller ells (A) and in the OIR model (). Fold indution of ANGPTL4 mrna expression is omparable to the fold indution for VEGF mrna. All mrna levels were normalized to βatin (for ell ulture) or ylophilin A (for OIR) mrna and reported as fold indution ompared with ells exposed to % (vol/vol) O (ontrol). () Representative images from immunohistohemial analysis of ANGPTL4 expression in the ishemi inner retina at P and P4 in the OIR model ompared with P4 ontrol mie. Student s t test. To investigate the potential of inhibiting ANGPTL4 as a therapeuti approah for the treatment of, we identified a monolonal antibody to ANGPTL4 that ould effetively blok the stimulation of tubule formation by rhangptl4 but not by rhvegf (SI Appendix, Fig. S9 A and ). Using this ANGPTL4 neutralizing antibody, we were able to inhibit the angiogeni potential of onditioned media from hypoxi retinal Müller ells (SI Appendix, Fig. S9), similar to the VEGF neutralizing antibody, bevaizumab (see Fig. I). To assess whether bloking ANGPTL4 ould be an effetive approah for patients who do not respond adequately to antivegf therapy, we next analyzed the effet of ANGPTL4 neutralizing antibody on the ability of lowvegf aqueous fluid from patients to stimulate tubule formation and observed a potent inhibition of angiogeni potential (Fig. 6E and SI Appendix, Fig. SA). The limited volume of aqueous fluid that ould be safely removed from patients did not allow us to assess the effet of using neutralizing antibodies to VEGF and ANGPTL4 together, ompared with either antibody alone. To overome this obstale, we analyzed whether ANGPTL4 neutralizing antibody ould prevent tubule formation by aqueous fluid from patients who reeived reent antivegf therapy within wk of sample olletion. These aqueous fluid samples had markedly redued detetable VEGF levels, and their ability to stimulate tubule formation was not affeted by pretreatment with VEGF bloking antibody (see Fig. and ). ANGPTL4 neutralizing antibody redued the angiogeni potential of aqueous fluid from patients, despite treatment with antivegf therapy within wk of sample olletion (Fig. 6F and SI Appendix, Fig. S). Unlike VEGF, there was minimal overlap between the ANGPTL4 levels in the aqueous fluid from diabeti patients with and without and with diabeti patients with N (Fig. 6A and SI Appendix, Fig. S), suggesting that aqueous fluid levels of ANGPTL4 may further help define the subpopulation of patients with diabeti eye disease. Indeed, examination of the aqueous fluid levels of ANGPTL4 ompared with VEGF resulted in a lear separation between ontrol patients, diabetis without DR, and diabetis with N or (SI Appendix, Fig. S A and ). Aqueous fluid from patients ould be further stratified into four groups based on the relative levels of VEGF and ANGPTL4 (Fig. 6G and SI Appendix, Table S4). Moreover, although VEGF levels were markedly lower in the aqueous fluid from patients who underwent treatment with anti VEGF therapy within wk of sample olletion, the aqueous fluid levels of ANGPTL4 remained elevated in these patients (Fig. 6G). Disussion In a reent linial trial, it was demonstrated that although over onethird of patients with severe N progress to within y, this number was redued by up to twothirds in patients who reeive monthly antivegf therapy (8). However, for many of the patients who respond to antivegf therapy, the response may only be temporary; the number of diabeti patients with N who ontinue to progress to inreases over time (8), and this is likely to ontinue despite treatment with anti VEGF therapy. This suggests that additional fators partiipate in the development of and highlights major gaps in our understanding of the underlying biologial proesses that promote the persistene (and growth) of retinal neovasularization despite antivegf treatment. Equally troublesome are growing onerns regarding the onsequenes of hroni VEGF inhibition. VEGF produed by the retinal pigment epithelium is essential to maintaining the health of the underlying horioapillaris, the vasular bed that supplies the metabolially ative retinal photoreeptors (9 ). It is postulated that VEGF may also play a more diret role as a neurotrophi fator for the neurosensory retina (). hroni inhibition of VEGF may impair its normal physiologi roles in the eye (4, ). Indeed, it has reently been reported that anti VEGF therapy is assoiated with the development of retinal atrophy in some patients with maular degeneration (6). Reent data raise the additional onern that antivegf therapy may ontribute to the development of glauoma in vulnerable patients (7). olletively, these observations emphasize the importane of ongoing efforts to identify other angiogeni fators 6of abapoorfarrokhran et al.

7 A D ANGPTL4 mrna ontrol RNAi ANGPTL4 RNAi E ANGPTL4 mrna (fold Indution) rhangptl4 (ng/ml) ontrol RNAi ANGPTL4 RNAi VEGF RNAi [ANGPTL4] [ANGPTL4].. PS ANGPTL4 VEGF [ANGPTL4] (ng/ml) VEGF mrna Vessels per mm PS ANGPTL4 VEGF days VEGF mrna... (pg/ml) (% inhibition vs. ) 4 MEDIAL SIENES PNAS PLUS Fig.. ANGPTL4 is upregulated by and HIF in vitro and retinal ishemia in vivo and is neessary and suffiient to promote angiogenesis. (A) rhangptl4 potently stimulates tubule formation in vitro in a dosedependent manner. ( and ) Representative photographs wk following bead implantation () and quantitation of vessels per mm () demonstrating that ANGPTL4 potently stimulates orneal neovasularization in vivo, similar to VEGF. (D) RNAi targeting ANGPTL4 auses a marked redution in ANGPTL4 but not VEGF mrna and protein expression in human retinal Müller ells. This, in turn, results in redued angiogeni potential of aqueous fluid from Müller ells pretreated with RNAi targeting ANGPTL4 ompared with srambled ontrols. (E) Inhibition of ANGPTL4 or VEGF mrna and protein expression results in an % redution in the stimulation of tubule formation by hypoxi retinal Müller ells. However, ombined inhibition of both ANGPTL4 and VEGF mrna and protein expression using RNAi in hypoxi retinal Müller ells results in an almost % redution in the stimulation of tubule formation. Oneway ANOVA. that promote the development of diabeti eye disease and that may therefore serve as effetive and perhaps safer targets for patients with DR. In this regard, studies using animal models have demonstrated that expression of a onstitutively ative HIFα mutant was suffiient to promote retinal neovasularization in vivo (8), whereas expression of VEGF alone was not suffiient to mediate this effet (9 ). These results impliate additional HIFregulated angiogeni fator(s) in the promotion of retinal neovasularization in patients. Of interest, erythropoietin (EPO) the first identified HIFregulated gene has been impliated in the development of (, ). However, patients often have endstage renal disease and require parenteral injetion of rhepo on a regular basis. Additionally, in the eye, EPO has been found to redue neuronal, glial, and vasular damage or dysfuntion and to inhibit the breakdown of the blood retinal barrier in a diabeti rodent model (4 6). EPO appears to play a omplex and multifaeted role in diabeti patients, as well as in diabeti eye disease, and modulation of EPO in this population should be approahed with aution. The results of our study impliate an alternative angiogeni fator, ANGPTL4, in the promotion of retinal neovasularization in patients. ANGPTL4 has been impliated in the upregulation of retinal neovasularization by PPARβ/δ (7). We show here that HIF also plays an important role in regulating expression of ANGPTL4 in ishemi retinal disease. Expression of ANGPTL4 was markedly inreased in the aqueous and vitreous of patients, and ANGPTL4 expression was loalized to retinal neovasularization in eyes. Inhibition of ANGPTL4 redued the angiogeni potential of hypoxi retinal ells; this effet was additive, with simultaneous inhibition of VEGF. A neutralizing antibody for ANGPTL4 inhibited the angiogeni potential of aqueous fluid from patients, inluding aqueous fluid from patients who had reeived antivegf therapy within wk of sample olletion. Although we annot rule out a ontribution from other angiogeni fators in the promotion of retinal neovasularization in patients with, our results suggest that targeting both ANGPTL4 and VEGF may be neessary for effetive treatment or prevention of. An essential next step will be the development of speifi and effetive anti ANGPTL4 therapies. This will require the identifiation of the relevant endothelial ell reeptor through whih ANGPTL4 mediates its pathologial effets in the retina. Of note, as ANGPTL4 has also been impliated in the promotion of vasular permeability abapoorfarrokhran et al. PNAS Early Edition 7of

8 A [ANGPTL4] (ng/ml) 4 E [ANGPTL4] (ng/ml) % (ompared to untreated) DM No DR N Low VEGF Low VEGF Low VEGF Low VEGF4 antivegf average F [ANGPTL4] (ng/ml) % (ompared to untreated) ANGPTL ontrol AntiVEGF AntiVEGF AntiVEGF AntiVEGF4 AntiVEGF [ANGPTL4] (ng/ml) AntiVEGF6 AntiVEGF7 AntiVEGF8 average 9 8 [ANGPTL4] (ng/ml) D Aqueous [ANGPTL4] (ng/ml) G Y =.X.6999 R = Vitreous [ANGPTL4] (ng/ml) %/% antivegf 8% 4% 8%/%. 4 6 [ANGPTL4] (ng/ml) Fig. 6. ANGPTL4 is an angiogeni fator expressed in the eyes of diabeti patients with. (A) Levels of ANGPTL4 in the aqueous fluid from diabeti (with and without diabeti retinopathy) and nondiabeti patients. () Immunohistohemial analysis of eyes from patients with known demonstrates expression of ANGPTL4 in areas of preretinal neovasularization; similar results were observed in / eyes. () ANGPTL4 protein levels are elevated in the vitreous of patients ompared with ontrol patients. (D) and patient samples demonstrate a orrelation between levels of ANGPTL4 in aqueous fluid and levels of ANGPTL4 in vitreous (P =.7). (E and F) ANGPTL4 bloking antibody redues the ability of lowvegf aqueous fluid (E) or antivegf aqueous fluid (F) to stimulate tubule formation. (G) Twodimensional satter plot demonstrating the and [ANGPTL4] in the aqueous fluid from patients. The perentage of (red) or antivegf (green) eyes within eah quadrant defined by the average aqueous fluid (orange dashed line) and [ANGPTL4] (blue dashed line) levels is shown. Note: samples with [ANGPTL4] > 6 ng/ml and/or with >. ng/ml are not depited to adequately demonstrate the variability within the samples. Oneway ANOVA. Wiloxon test and Pearson orrelation. (and maular edema) in diabeti patients (), therapies targeting ANGPTL4 may also be an effetive approah for the treatment of patients with DME. These studies may have more broad impliations. The antiipated inrease in the worldwide diabeti population emphasizes the need for novel approahes to identify whih diabeti patients would benefit from aess to higher levels of ophthalmi are (7 9). Aess to eye are speialists remains limited in parts of the developing world where the diabeti population is expeted to double over the next deades (8). In this regard, we demonstrate here that aqueous fluid levels of ANGPTL4 orrelate very well with the levels of ANGPTL4 in the vitreous. Aquiring aqueous fluid from patients is relatively straightforward and poses limited risks on patients, and although there is onsiderable overlap of VEGF levels in aqueous fluid of ontrol patients, diabeti patients without DR, and diabetis with N or, ANGPTL4 levels were strongly preditive of the presene or absene of. This suggests that ANGPTL4 levels in the aqueous fluid ould serve as a diagnosti biomarker to help predit the level of diabeti eye disease in vulnerable populations. Interestingly, aqueous fluid from patients fell into four ategories: modestly high levels of both VEGF and ANGPTL4 (%), very high VEGF levels but only modestly high ANGPTL4 levels (8%), very high ANGPTL4 levels but only modestly high VEGF levels (8%), and very high VEGF and ANGPTL4 levels (4%). We speulate that the aqueous fluid levels of ANGPTL4 (and VEGF) ould help define subpopulations of patients who may be more (or less) suseptible to antivegf therapy. Indeed, the aqueous fluid levels of ANGPTL4 remain elevated despite treatment with antivegf therapy, further suggesting that aqueous fluid levels of ANGPTL4 ould be used as a biomarker in patients reeiving antivegf therapy to help guide individualized therapy. This is partiularly important given the rising ost of antivegf therapies, whih now aount for /6 of the total Mediare Part drug budget (4). This inludes the ost of treating patients who do not respond adequately to this therapeuti approah. Ongoing work is urrently foused on determining whether ANGPTL4 levels in aqueous fluid ould help predit whih patients are more (or less) likely to respond to antivegf therapy. 8of abapoorfarrokhran et al.

9 As HIF is a ritial mediator of oular neovasular disease, ANGPTL4 may play a key role in other visionthreatening diseases in whih (e.g., retinal ishemia) is a driving fore, suh as in retinal vein olusion, sikle ell retinopathy, and retinopathy of prematurity. Our results suggest that ANGPTL4 may ontribute to retinal neovasularization in these patients. Ultimately, our observations provide the foundation for studies to assess inhibition of ANGPTL4 alone or in ombination with inhibition of VEGF as a therapeuti approah for the treatment of and other oular neovasular diseases as well as for studies investigating the use of aqueous fluid ANGPTL4 as a diagnosti and/or therapeuti biomarker for these diseases. Materials and Methods onstruts and Reagents. Reombinant human ANGPTL4, VEGF, and ANGPTL4 (DuoSet) and VEGF (Quantikine) ELISA kits were purhased from R&D Systems. Predesigned ontrol (srambled), ANGPTL4, and VEGF sirna were obtained from Santa ruz. Lipofetamine RNAiMAX transfetion reagent and OptiMEM medium were obtained from Life Tehnologies. Hypoxia hambers were used to expose MIOM ells (% oxygen) and primary murine Müller ells [% (vol/vol) oxygen; exposure of primary murine Müller ells to lower oxygen onentrations resulted in ell death]. Digoxin and desferrioxamine (DFO) were obtained from Sigma. Dimethyloxalylglyine (DMOG) was obtained from ayman Pharmaeutials. ell ulture. MIOM ells were a generous gift from Astrid Limb (University ollege London Institute of Ophthalmology) and ultured with DMEM (Invitrogen) ontaining g/l gluose with % (vol/vol) FS (Invitrogen) and % peniillin/streptomyin (ellgro). Immortalized human dermal mirovasular endothelial ells (HME) were obtained from the D and ultured with DMEM ontaining 4. g/l gluose with % (vol/vol) FS and % peniillin/ streptomyin. efore treatments, the growth media were replaed with serum starvation media ontaining % FS. sirna Transfetion. ells were seeded at 6 8% onfluene at transfetion. Lipofetamine RNAiMAX reagent was diluted in OptiMEM medium. Thirty piomoles of sirna from stok of μm was diluted in OptiMEM medium. Diluted sirna was added to diluted Lipofetamine RNAiMAX reagent (: ratio) and inubated for min at room temperature. sirna lipid omplex was added to ells and inubated at 7 for 4 h. The media were then washed out, and ells were ready for experiments. Mie. Timed pregnant 7L/6 mie (E4) (harles River Laboratories) were treated in aordane with the Assoiation for Researh in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmi and Vision Researh and the guidelines of the Johns Hopkins University Animal are and Use ommittee. OIR experiments were performed as previously desribed (). riefly, 7dold (P7) 7L/6 mie and their mothers were exposed to 7% (vol/vol) oxygen for d. The mie were returned to room air at P. The mie were killed and the eyes were olleted at P ( h after return to normoxia), P, P4, and/or P7. A subset of mie was given daily i.p. injetion of vehile or mg/kg digoxin. Western lot. ells in ulture dishes were washed with PS and lysed using RIPA buffer (Sigma) with % (vol/vol) protease inhibitor mixture (Sigma). ell lysates were then solubilized in LDSsample buffer (Life Tehnologies) and inubated for min at 9. Lysates were subjeted to 4 % (wt/vol) gradient SDS/PAGE (Invitrogen). After bloking the membrane with % (wt/vol) milk (iorad), the membrane was then inubated with mouse antihifα (D, 699) or rabbit antihifα (Abam, 8) or with mouse anti GAPDH monolonal antibody (Fitzgerald) overnight at 4. After washing, the membrane was inubated with HRPonjugated antimouse or antirabbit IgG (ell Signaling) for h and then visualized with EL Super Signal West Femto (Thermo). Western blot sans are representative of at least three independent experiments. Quantitative RTPR. mrna was isolated from ultured ells or isolated retinas with RNeasy Mini Kit (Qiagen), and DNA was prepared with MuLV Reverse Transriptase (Applied iosystems). Quantitative realtime PR was performed with Power SYR Green PR Master Mix (Applied iosystems) and MyiQ RealTime PR Detetion System (iorad). ylophilin A and βatin were used for normalization of mouse tissue and ell lines or for human ell lines, respetively. Primers are listed in SI Appendix, Tables S and S6. Immunohistohemistry and Immunofluoresene. Immunohistohemial detetion of HIFα (Abam, 8) and ANGPTL4 (Abam, 798) was performed in paraffinembedded human tissue and ryopreserved mouse tissue setions using A system (Dako) as previously desribed (). D antibody was obtained from D (74). Images were aptured using a onfoal mirosope LSM 7 META (arl Zeiss). Assay. Tubule formation assay was performed using growth fatorredued Matrigel (D iosienes; 6). Sixty to eighty miroliters of Matrigel was added into a prehilled 96well plate and plaed in a 7 O inubator for min. HMEs were then ounted and plated at 4 ells per well on the Matrigel in a 96well plate. Eighteen hours later, images were aptured and analyzed using ImageJ software. Tubule formation assay with patient samples was performed with an addition of μl/well of aqueous fluid to the ell suspension before adding into the Matrigeloated wells. VEGF neutralization was performed using μg/ml of bevaizumab (Johns Hopkins University Pharmay). ANGPTL4 neutralization was performed using μg/ml of an ANGPTL4 monolonal antibody (Enzo, 84 7). Patient Samples. Institutional Review oard approval from the Johns Hopkins University Shool of Mediine was obtained for all patient samples used in this study. An a priori power analysis using a ohen s d effet size of.8 based on results from in vivo studies and preliminary results, power of.8, and of. yielded an estimated sample size of 6 ontrol and patients. Aqueous and vitreous samples were olleted from onsenting patients at the Wilmer Eye Institute undergoing atarat and/or vitretomy surgery. Vitreous samples were immediately entrifuged at 6, g for min at 4, and supernatant was removed. Aqueous and vitreous samples were then aliquoted and stored at 8 before analysis. ELISA. Aqueous diluted : and undiluted vitreous were analyzed for ANGPTL4 and VEGF with ELISAs performed aording to the manufaturer s protools (R&D Systems). Statistial Analysis. Results from ell ulture and animal models are shown as mean ± SEM from at least three independent experiments. Results from linial samples are shown as mean ± SD. Statistial differenes between groups were determined by Student s t test, Wiloxon signedrank test, and oneway ANOVA when indiated. orrelation was tested using Pearson s method. Statistial analysis was performed using Mirosoft Offie and Prism 6. software (Graph Pad). P <.; P <.; P <.; P <.;, nonsignifiant. AKNOWLEDGMENTS. This work was supported by the National Eye Institute Grant K8EY89 (to A.S.), the Mirosopy and Imaging ore Module and the Animal ore Module of the Wilmer ore Grant EY76, and an Unrestrited Grant from Researh to Prevent lindness (to A.S.). A.S. gratefully aknowledges the support he reeives from the William and Ella Owens Medial Researh Foundation, as a areer Development Award reipient from the Researh to Prevent lindness Foundation. MEDIAL SIENES PNAS PLUS. heung N, Mithell P, Wong TY () Diabeti retinopathy. 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