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Therapeutic efficacy of a novel non-peptide αvβ3 integrin antagonist for pathological retinal angiogenesis in mice
Experimental Eye Research 129 (2014) 119e126
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Therapeutic efficacy of a novel non-peptide avb3 integrin antagonist for pathological retinal angiogenesis in mice Yong-Jie Li a, 1, Xiao-Hong Li b, 1, Liang-Fen Wang a, Xi Kuang a, Zhi-Xiong Hang c, Yong Deng c, *, Jun-Rong Du a, * a
Department of Pharmacology, Key Laboratory of Drug Targeting and Drug Delivery Systems Ministry of Education, West China School of Pharmacy, Sichuan University, Chengdu, China Department of Biopharmaceutics, West China School of Pharmacy, Sichuan University, Chengdu, China c Department of Medical Chemistry, West China School of Pharmacy, Sichuan University, Chengdu, China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 21 December 2013 Received in revised form 14 September 2014 Accepted in revised form 7 November 2014 Available online 8 November 2014
avb3 integrin has been reported as a promising therapeutic target for angiogenesis. In the present study,
Keywords: Angiogenesis Integrin avb3 Oxygen-induced retinopathy Non-peptide integrin antagonist
we tested the antiangiogenic activity of 3-[3-(6-guanidino-1-oxoisoindolin-2-yl) propanamido]-3-(pyridin-3-yl) propanoic acid dihydrochloride (GOPPP), a novel non-peptide avb3 antagonist. Both human umbilical vein endothelial cells (HUVECs) and a mouse model of oxygen-induced retinopathy (OIR) were investigated separately. HUVEC adhesion, proliferation, migration, ERK1/2 and Akt phosphorylation were assessed. C57BL/6 mice were used for the studies in the OIR model. After exposure to 75% oxygen from postnatal day (PD) 7 to PD12, the mice were returned to room air, and GOPPP was intravitreally administered on PD12. Retinal neovascularization was evaluated on PD17. Hypoxia-inducible factor-1a (HIF-1a) and vascular endothelial growth factor (VEGF) protein levels and ERK1/2 phosphorylation were determined by Western blot analysis of retina proteins. GOPPP significantly inhibited the pro-angiogenic effects of vitronectin on HUVECs, including adhesion, proliferation, and migration, and inhibited ERK1/2 and Akt phosphorylation. Retinal neovascularization in the OIR model was significantly suppressed by intravitreal administration of 50 ng GOPPP. The pro-angiogenic factors HIF-1a and VEGF induced by hypoxia were significantly inhibited by GOPPP in OIR mice. GOPPP administration also inhibited ERK1/2 phosphorylation in the OIR model. These results indicate that GOPPP, a novel avb3 integrin antagonist, may have potential for the treatment of pathological retinal angiogenesis. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction Angiogenesis is characterized by the formation of new capillaries that sprout from pre-existing microvasculature. It is a normal physiological event in development and part of the pathological response in ocular neovascularization, inflammatory arthritis, and tumor growth. For endothelial cells that undergo angiogenesis, alterations in four cellular functions are defined: proliferation, migration, endothelial cell-to-cell interactions, and endothelial cell interactions with the extracellular matrix. Among the relevant molecules that regulate cell-extracellular matrix interactions are integral membrane proteins, including integrins. With regard to integrins, avb3 integrin is recognized as a key player in
* Corresponding authors. E-mail addresses: [email protected] (Y. Deng), [email protected] (J.-R. Du). 1 Equally contributed to this work. http://dx.doi.org/10.1016/j.exer.2014.11.004 0014-4835/© 2014 Elsevier Ltd. All rights reserved.
angiogenesis (Eliceiri et al., 1999; Hodivala-Dilke, 2008). The levels of avb3 integrin protein increase in angiogenic endothelial cells, and the inhibition avb3 integrin function individually with antibodies or small-molecule compounds results in the perturbation of angiogenesis (Brooks et al., 1995; Santulli et al., 2008). Integrins bind ligands by recognizing short-amino-acid stretches on exposed loops, particularly the arginineeglycineeaspartic acid (RGD) sequence. Accordingly, RGD-mimetic peptides or small molecules bind to these integrins on the surface of cells to block specific integrin-mediated signaling pathways and act as anticancer and antiangiogenic agents (Alghisi and Ruegg, 2006; Stupp and Ruegg, 2007). Yet, RGD-mimetic integrin inhibitors may have biphasic effect on avb3 integrin, with an antagonistic phase at high concentrations and agonistic phase at low concentrations (Legler et al., 2001; Reynolds et al., 2009), implying that the delivery of these agents should be designed carefully to avoid nanomolar plasma concentrations and improve their efficacy in the treatment of human
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cancers. In the present study, we designed and identified a novel non-peptide, small-molecule compound, 3-[3-(6-guanidino-1oxoisoindolin-2-yl) propanamido]-3-(pyridin-3-yl) propanoic acid dihydrochloride (GOPPP), as a potential avb3 inhibitor. This report describes the in vitro and in vivo pharmacological activity of GOPPP, which was shown to inhibit retinal neovascularization in a mouse model of oxygen-induced retinopathy (OIR).
2.4. Cell proliferation assay Cell proliferation was measured using the MTT assay as previously described (Belvisi et al., 2005). Various cells, including HUVECs and MCF-7 cells, were treated with serial dilutions of GOPPP for 48 h. Each assay was replicated three times. The results were plotted and are expressed as the mean ± SD compound concentration that inhibited 50% cell proliferation (i.e., IC50).
2. Methods 2.5. Wound healing assay 2.1. Cell cultures MCF-7 cells were obtained from the American Type Culture Collection and cultured in Dulbecco's Modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum. Human umbilical vein endothelial cells (HUVECs) were isolated from human umbilical cord veins using a standard procedure as previously described (Jaffe et al., 1973) and grown in endothelial cell medium (Sciencell, Carlsbad, California, USA). HUVECs at passages 3 to 8 were used for all of the experiments.
Monolayer HUVECs in vitronectin-coated 24-well cell plates were wounded by scratching with a pipette tip and washed with PBS. Serum-free medium that contained vehicle or different concentrations of GOPPP was added to the scratched monolayers. Images were taken using an Olympus digital camera after 24 h of cell migration. The migrated cells were quantified by manual counting, and the percentage of inhibition is expressed relative to untreated cells set to 100% (Naik and Naik, 2006). 2.6. Mouse model of oxygen-induced retinopathy
2.2. Integrin binding assay The assays were performed as described previously (MasMoruno et al., 2011; Santulli et al., 2008). Briefly, 2.0 mg/ml of vitronectin (Millipore, Billerica, MA, USA) or 5.0 mg/ml of fibronectin (Sigma, St. Louis, MO, USA) in carbonate buffer (15 mM Na2CO3 and 35 mM NaHCO3; 100 ml/well) was added to 96-well microtiter plates and incubated overnight at 4 C. The plates were then incubated with blocking solution (i.e., TS buffer: 20 mM TriseHCl, pH 7.5, 150 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, and 1 mM MnCl2) plus 2% bovine serum albumin (BSA) for an additional 1 h at room temperature. After rinsing twice with assay buffer (i.e., TS buffer plus 0.1% BSA), the plates were incubated with 1.0 mg/ml of soluble avb3 or a5b1 integrin (Millipore) in the presence of increasing concentrations of GOPPP or cyclo-Arg-Gly-Asp-D-PheVal (cRGDfV) for 1 h at room temperature, followed by washing and 1 h incubation with primary integrin antibody (Millipore) and goat anti-mouse immunoglobulin G conjugated with horseradish peroxidase (HRP; 1:200; Boster, Wuhan, China) consecutively at room temperature. After five washes with PBST buffer (10 mM Na2HPO4, pH 7.5, 150 mM NaCl, and 0.01% Tween-20), peroxidase development was performed using the substrate solution 3,3,5,50 tetramethylethylenediamine (Boster) and 3.0 M H2SO4 to stop the reaction, followed by absorbance measurement at 450 nm. Each data point is the result of the average of triplicate wells. The results were plotted and are expressed as the antagonist concentration that inhibited 50% integrin binding. 2.3. Cell adhesion assay The assays were performed as described previously (Gentilucci et al., 2010). Ninety-six-well cell plates were coated with 1.0 mg/ ml vitronectin in 0.1 M phosphate buffer, pH 7.2 (PBS), overnight at 4 C. HUVECs (5 104) were seeded in each well and allowed to adhere for 1 h at 37 C in the presence of various concentrations of GOPPP. After washed with PBS to remove non-adherent cells, adherent cells were fixed with 4% paraformaldehyde for 10 min, followed by staining with 1% toluidine blue (Aladdin, Shanghai, China) for 10 min and washing with water. Finally, the stained cells were solubilized with 1% sodium dodecyl sulfate (SDS; Amersco, OH, USA) and quantified at 600 nm. The experiments were performed in quadruplicate and repeated at least three times. The results are expressed as the mean ± SD compound concentration that inhibited 50% cell adhesion.
C57BL/6 mice were used and handled in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Mice on postnatal day 7 (PD7) were placed with their nursing dams in a 75% oxygen atmosphere for 5 days. After being returned to normoxia conditions for 5 days (i.e., on PD12), the mouse pups were intravitreally administered 0.5 ml of different concentrations of GOPPP or PBS. Five days later (i.e., on PD17), the mice were sacrificed (Mouse pups in the sham group were fostered under normoxia conditions until PD17), and the eyes were enucleated and fixed in 4% paraformaldehyde. Whole retinal flatmounts were prepared and stained with isolectin B4-Alexa Fluor 568 (Sigma) to quantify vaso-obliteration and pre-retinal neovascular tufts as described previously (Connor et al., 2009). To visualize blood vessels, dissected retinas were rinsed in 0.1 M PBS and incubated overnight at 4 C in 50 mg/ml of the isolectin in 0.1 M PBS. After incubation, the whole mounts were rinsed in 0.1 M PBS, mounted on gelatin-coated glass slides and cover-slipped with glycerine. Blood vessels were observed and photographed using fluorescence microscopy (ImageZ2; Zeiss, Deutschland). In each whole mount, the total area of pre-retinal neovascular tufts and the extent of the avascular area were measured using Image-pro Plus 6.0 System (Media Cybernetics, USA) and expressed as the percentage of the respective average in relation to total retinal area. For each experimental condition, quantitative data originated from 10 retinas. The retina was also histologically examined. Serially sectioned paraffin-embedded small pieces of retina (6 mm) were stained with hematoxylin and eosin (HE). Images were taken using light microscopy, and endothelial nuclei that extended beyond the inner limiting membrane into the vitreous were manually counted in a blinded manner (Lahdenranta et al., 2007). 2.7. Western blot analysis To determine the effects of GOPPP on the angiogenic signaling cascade, serum-starved HUVECs were plated on 2 mg/ml of vitronectin-coated dishes in medium in the presence of vehicle or different concentrations of GOPPP for 1 h. The cells were then lysed in lysis buffer that contained 1% Triton X-100 (Amersco), 1% deoxycholate (Amersco), and proteinase inhibitor cocktail (Roche, Rotkreuz, Switzerland). Protein concentrations were determined using a modified Lowry protein assay kit (Thermo, Pittsburgh PA, USA) and equalized before loading. Forty
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micrograms of cellular protein from each sample was applied to 8e12% SDS-polyacrylamide gels, and resolved proteins were transferred to nitrocellulose membrane and probed with specific antibodies, including phospho-Erk1/2 (p-Erk1/2), Erk1/2, phospho-AKT (p-AKT), and AKT (Cell Signaling Technology, Boston, USA). The blots were developed with HRP-conjugated secondary antibodies and the BeyoECL Plus enhanced chemiluminescence detection kit (Beyotime, Shanghai, China). Band density was analyzed using Quantity One software and used to determine the relative protein level. To further investigate the signaling pathway that is involved in the antiangiogenic activity of GOPPP, Western blot of OIR mouse retina proteins was also performed. After separation from the eyeballs on PD17, the retinal tissues of each group (control group: three mouse pups fostered under normoxia conditions; model group: three OIR mice intravitreally administered with PBS; GOPPP group: three OIR mice intravitreally administered with 50 ng GOPPP) were lysed together with lysis buffer for 30 min, containing protease inhibitors, followed by centrifugation at 12,000 g for 10 min at 4 C. The supernatants were collected as lysates for protein concentration determination and Western blot analysis as described above. 2.8. Statistical analysis Results were reported as mean ± SD. The data were analyzed using one-way analysis of variance (ANOVA) and SPSS 16.0 software. Differences were considered statistically significant when p < 0.05. The IC50 values were calculated using Microsoft Excel 2007. 3. Results 3.1. Identification of avb3 integrin inhibitors A library of synthetic non-peptide small molecules was screened using a solid-phase receptor assay to identify compounds that recognize avb3 integrin. The well-characterized cyclic pentapeptide integrin antagonist cRGDfV was used as a positive control. Fig. 1 shows the structure of one of the compounds of interest, GOPPP, identified by this process. Fig. 2A shows that GOPPP dosedependently inhibited vitronectin binding to avb3 integrin, with an IC50 of 7.0 nM, which is much lower than the IC50 for cRGDfV. The selectivity of this compound was demonstrated by its low capacity to compete with biotinylated fibronectin for a5b1 integrin (IC50 > 10 mM; Fig. 2B), just like cRGDfV. 3.2. Inhibition of endothelial cell adhesion, proliferation, and migration by GOPPP To assess the ability of GOPPP to act as an avb3 antagonist for endothelial cells, HUVECs were allowed to adhere to immobilized vitronectin in the presence of increasing concentrations of GOPPP
Fig. 1. Chemical structure of 3-[3-(6-guanidino-1-oxoisoindolin-2-yl)propanamido]-3(pyridin-3-yl) propanoic acid dihydrochloride (GOPPP).
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(range, 0.01e10 mM). As shown in Fig. 3A, GOPPP significantly and dose-dependently inhibited HUVEC adhesion to vitronectin, with an IC50 of 0.10 ± 0.03 mM, which was much lower than that of cRGDfV (2.52 ± 0.39 mM). To assess the anti-angiogenic activity of GOPPP in vitro, its inhibitory effects on the proliferation of HUVECs were evaluated using the MTT assay. GOPPP significantly inhibited HUVEC proliferation, with an IC50 of 4.88 ± 0.38 mM (Fig. 3B). In contrast, 10 mM GOPPP showed no significant inhibitory effect on the proliferation of MCF-7 cells, which do not express avb3 integrin (Danen et al., 1995; Doerr and Jones, 1996), suggesting low cytotoxicity (Fig. 3B). Cell migration, which is essential for endothelial cells in angiogenesis, was investigated using a wound healing assay, with vitronectin as the stimulator. As shown in Fig. 3C, vitronectin increased HUVC migration 5.06-fold compared with the control (p < 0.01). The addition of GOPPP dose-dependently inhibited vitronectin-induced HUVEC migration. Concentrations of 0.5 and 1 mM GOPPP had no significant effect on HUVEC migration compared with vehicle, whereas 5 mM GOPPP inhibited migration 2.43-fold (p < 0.01; Fig. 3C). 3.3. Effect of GOPPP on angiogenesis signaling To delineate the signaling pathway that is involved in the antiangiogenic activity of GOPPP, its effects on Akt and ERK phosphorylation were evaluated by applying Western blot analysis of HUVEC proteins after incubation with vitronectin in the presence of different concentrations of GOPPP. As shown in Fig. 4, vitronectin activated the phosphorylation of ERK and AKT in HUVECs (p < 0.01), and GOPPP dose-dependently suppressed ERK and AKT phosphorylation. GOPPP (10 mM) inhibited ERK and AKT phosphorylation by 47.6% and 42.4%, respectively, compared with vehicle (p < 0.01), suggesting that GOPPP exerts its antiangiogenic effects by antagonizing the vitronectin-mediated downstream cascade, including the Akt and MEK/ERK signaling axis. 3.4. Inhibition of retinal neovascularization in oxygen-induced retinopathy model by intravitreal administration of GOPPP To further assess the anti-angiogenic activity of GOPPP in vivo, the mouse OIR model was investigated and its effects on retinal angiogenesis were evaluated in the superficial vascular plexuses using isolectin B4 staining. Mice were exposed to 75% oxygen from PD7 to PD12, which results in extensive retinal vaso-obliteration, and then returned to room air. Such conditions made the retina relatively hypoxic, leading to the formation of retinal neovascularization (Fig. 5A, C). At PD17, in the superficial plexus of vehicle-treated retina (Fig. 5A), both an excessive vessel regrowth leading to pre-retinal neovascular tufts and the formation of a large avascular area in central retina, with only the major vessels and practically no capillary network, were detected. Intravitreal injection of GOPPP dose-dependently reduced the vessel tuft area in the superficial plexus (Fig. 5A, C). Compared with vehicle, 5 or 10 ng GOPPP had no significant effect, whereas 50 ng GOPPP reduced the vessel tufts by 60.6% (p < 0.01). The central avascular area was slightly reduced (p < 0.01; Fig. 5A, B). To further confirm the inhibitory effect of GOPPP on retinal neovascularization, vascular cell nuclei that extended beyond the internal limiting membrane were counted on HE-stained retinal tissue sections. Nuclei anterior to the internal limiting membrane were not found among the retinal tissue sections in the sham group, but a large number of neovascular nuclei were found in the eyes of OIR mice (Fig. 6AeC), 42.7% of which were suppressed by 50 ng GOPPP (p < 0.01; Fig. 6D).
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Fig. 2. GOPPP is a potent and selective inhibitor of vitronectin binding to avb3. (A) Inhibition of vitronectin binding to avb3 integrin. (B) Inhibition of fibronectin binding to a5b1 integrin. avb3 or a5b1 integrin was incubated with immobilized vitronectin or fibronectin in the presence of various concentrations of GOPPP or cRGDfV. Bound integrin was detected with primary integrin antibody and goat anti-mouse immunoglobulin G conjugated with HRP. The results are expressed as mean ± SD (n ¼ 3).
Fig. 3. GOPPP inhibits adhesion, proliferation, and migration of HUVECs. (A) GOPPP inhibits HUVEC adhesion. HUVECs were allowed to adhere to vitronectin-coated 96-well plates with different concentrations of GOPPP or cRGDfV for 1 h. After incubation, adhered cells were fixed, stained, and examined at 595 nm. The percentage of inhibition is expressed relative to HUVECs with vehicle. (B) GOPPP inhibits HUVECs proliferation. Cells were treated with various concentrations of GOPPP for 48 h. Cell viability was quantified using the MTT assay. (C) GOPPP inhibits HUVEC migration. HUVECs were allowed to grow to full confluence in 24-well plates that were uncoated or coated with 2 mg/ml vitronectin (VN). Cells were wounded with a pipette and then treated with different concentrations of GOPPP in serum-free DMEM for 24 h. After incubation, the migrated cells were quantified by manual counting. The results are expressed as mean ± SD (n ¼ 6). **p < 0.01 versus vehicle-treated VN group. Scale bar, 500 mm.
Western blot analysis of PD17 OIR murine retina proteins was performed. Hypoxia activated ERK phosphorylation in mouse retinas (p < 0.01; Fig. 7) and the expression of hypoxia-inducible factor-1a (HIF-1a) and vascular endothelial growth factor 165 (VEGF165). Intravitreal administration of 50 ng GOPPP significantly decreased ERK phosphorylation and HIF-1a and VEGF165 levels induced by relatively hypoxic conditions (p < 0.05; Fig. 7). 4. Discussion
or
Presently, three classes of integrin inhibitors are in preclinical clinical trials: monoclonal antibodies that target the
extracellular domain of the integrin heterodimer (e.g., Vitaxin), synthetic RGD motif-containing peptides (e.g., cilengitide), and peptidomimetics (e.g., S247), which are orally bioavailable nonpeptidic molecules that mimic the RGD sequence (Alghisi and Ruegg, 2006). In this study, we evaluated the in vitro and in vivo pharmacological activity of a novel nonpeptidic avb3 antagonist, GOPPP, which was shown to inhibit retinal neovascularization in a mouse OIR model. The major results were that GOPPP reduced pathologic but not developmental angiogenesis in neonatal mice. The effect of GOPPP in the OIR model appeared to be attributable to its selective action on avb3 integrin. In vitro, this compound behaved as a selective inhibitor of integrin avb3 by preventing the
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Fig. 4. GOPPP suppresses the phosphorylation of Akt and ERK induced by vitronectin in HUVECs. (A) Western blot analysis of Akt and ERK. Serum-starved HUVECs were plated on vitronectin (VN)-coated dishes in medium supplemented with various concentrations of GOPPP for 1 h and then lysed. The lysates were analyzed by Western blot. The negative control was performed without vitronectin. Blots were developed with HRP-conjugated secondary antibodies and an enhanced chemiluminescence detection kit. (B, C) Densitometry of three independent experiments. The Western blot band density was analyzed using Quantity One software and used to determine the relative protein level. Results was expressed as means ± SD (n ¼ 3). **p < 0.01, compared with vehicle-treated VN group.
Fig. 5. Quantification of vaso-obliteration and pre-retinal neovascular tufts in the murine model of OIR. (A) Representative flat mounts of mouse retina. Retinal whole-mounts from PD17 were stained for blood vessel endothelial cells using isolectin B4-Alexa Fluor 568 (red), and lectin-stained tufts show new capillary buds induced by the OIR model. Sham, mouse pups fostered under normoxia conditions. Model, OIR mice intravitreally administered with PBS. GOPPP, OIR mice intravitreally administered with various concentrations of GOPPP. (B) Quantification of vaso-obliteration. (C) Quantification of pre-retinal neovascular tufts. The results were expressed as means ± SD (n ¼ 10). **p < 0.01, compared with model group. Scale bar, 100 mm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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Fig. 6. Analysis of preretinal nuclei in HE-stained retinal tissue sections. (AeC) Sham, mouse pups fostered under normoxia conditions; model, OIR mice intravitreally administered with PBS; GOPPP, OIR mice intravitreally administered with 50 ng GOPPP. (D) Quantification of preretinal nuclei. The neovascular cell nuclei are indicated by arrows. Quantification was based on eight non-continuous retinal tissue sections per eye. The results were expressed as means ± SD (n ¼ 8). **p < 0.01, compared with model. Scale bar, 50 mm.
Fig. 7. GOPPP suppresses ERK phosphorylation and the enhancement of HIF-1a and VEGF level induced by hypoxia in OIR mouse retina. (A) Western blot of VEGF, HIF-1a, and ERK. Control, mouse pups fostered under normoxia conditions; model, OIR mice intravitreally administered with PBS; GOPPP, OIR mice intravitreally administered with 50 ng GOPPP. On PD17, all of the retinal tissues from six eyeballs in each group were lysed together and analyzed using Western blot as described in the Methods section. The blots were developed with HRP-conjugated secondary antibodies and an enhanced chemiluminescence detection kit. (B, C) Densitometry of three independent experiments. Band density was analyzed using Quantity One software and used to determine the relative protein level. Results was expressed as means ± SD (n ¼ 3), *p < 0.05, **p < 0.01, compared with model group.
binding of vitronectin to avb3 with high affinity. Additionally, it inhibited HUVEC adhesion to vitronectin with high potency. In both of the assays, GOPPP was more potent than the wellcharacterized integrin antagonist cRGDfV. Both HUVEC proliferation and migration were inhibited. Retinal angiogenesis is a major cause of blindness in ischemic retinopathies, including diabetic retinopathy (DR) and retinopathy of prematurity (ROP). VEGF has been identified as a major mediator (Adamis et al., 1994; Aiello, 2005). Angiogenesis inhibitors that target VEGF have made a significant impact on the treatment of DR, ROP, and age-related macular degeneration. In addition to growth factors, integrins have also been implicated in angiogenesis, serving
as receptors for specific extracellular matrix proteins. avb3 integrin is not generally expressed on resting or normal microvessels but is dramatically upregulated in response to angiogenic growth factors (Brooks et al., 1994). It is also thought to be a critical regulator of angiogenesis. In the well-established mouse model of OIR, av integrins are overexpressed in neovascular endothelial cells during the peak time of retinal vessel growth (Takagi et al., 2002). The efficacy of avb3 integrin inhibition has been evaluated in OIR and ROP models (Santulli et al., 2008; Wilkinson-Berka et al., 2006; Yoshida et al., 2012). Herein, the potential of GOPPP as an antiangiogenic agent for retinal neovascularization was evaluated in an OIR model. Owing to the fact that tuft formation induced by
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hypoxia only appears in the superficial vascular plexus, leaving the deep vascular plexus unaffected (Dal Monte et al., 2013; Stahl et al., 2010), we investigated the pre-retinal neovascularization only. The results revealed that intravitreal administration of 50 ng GOPPP significantly reduced pre-retinal neovascular tufts by 60.6% (Fig. 5C) and neovascular nuclei anterior to the internal limiting membrane by 42.7% (Fig. 6AeC). We observed the discrepancy of neovascularization inhibition potency of GOPPP existed between the neovascular tufts and preretinal nuclei quantitation in retina as others reported (Deliyanti et al., 2012). The potential explanation for the discrepancy observed is the fact that the quantitation of neovascular tufts on flat mounts generally counts the vascular tufts on retinal whole surface, whereas HE staining may determine the preretinal nuclei of retinal cross-sections. Moreover, the manual quantitation on flat mounts with relative low resolution of images probably disregards small capillaries in retina. In addition, it is reported that tetraiodothyroacetic acid (tetrac), a deaminated analogue of L-thyroxine (T4), blocked the pro-angiogenic actions of T4 by inhibiting its binding to avb3 integrin, and intravitreal administration of 75 ng tetrac reduced the neovascular tufts in the stained retina by 44.8% in the OIR mice (Yoshida et al., 2012). Collectively, the present study suggests that the novel avb3 antagonist GOPPP exhibits significant inhibitory action on retinal angiogenesis in the OIR mice. With regard to the promotion of angiogenesis, synergistic interactions between avb3 integrin and VEGF have been demonstrated by multiple studies (e.g., Somanath et al., 2009). Some studies offer compelling evidence that avb3 function is necessary for VEGF-mediated effects. In the monkey eye after exposure to VEGF, avb3 and avb5 are highly upregulated in migrating cells from preexisting and newly formed vessels (Witmer et al., 2004). On the other hand, integrin inhibition is reported to be accompanied with suppression of VEGF secretion (Edwards et al., 2008; Iliaki et al., 2009). And, antagonists of both avb3 and avb5 integrins block the VEGF-stimulated adhesion, proliferation, and migration of endothelial cells (Terai et al., 2001; Tsou and Isik, 2001). In our assays with OIR mice, GOPPP reduced the retinal VEGF level under the condition of relative hypoxia, suggesting that the molecular mechanism underlying therapeutic efficacy of GOPPP, a novel avb3 antagonist, is associated with its downregulation on VEGF-induced retinal angiogenesis. Clearly, additional studies will be required to define the underlying mechanism of GOPPP regulating VEGE responsiveness. Integrins mediate complex signaling events, including the regulation of cell adhesion, proliferation, survival, and migration by activating canonical pathways. Among various signaling cascades, the phosphorylation of focal adhesion kinase (FAK) and activation of the phosphatidylinositol 30 -kinase (PI3K)/Akt pathway have been shown to be involved in avb3-mediated adhesion, migration, and cell survival (Cary et al., 1996; Gilmore et al., 2000; Ilic et al., 1995). The importance of FAK in integrin signaling is well established. Integrins bind FAK and mediate FAK activation and phosphorylation. In turn, integrin-FAK can activate the MEK-ERK axis (Schlaepfer et al., 1998), which contributes to cell survival and proliferation (Frisch and Screaton, 2001), whether Src is activated or not (Bianchi-Smiraglia et al., 2013; Hood et al., 2003). Akt is a critical regulator of PI3K-mediated cell survival and migration (Shiojima and Walsh, 2002). Numerous studies have demonstrated that the constitutive activation of Akt signaling is sufficient to block cell death and migration induced by various apoptotic stimuli in various cell types. In the present study, GOPPP significantly suppressed ERK and Akt phosphorylation induced by vitronectin, suggesting that GOPPP exerted its antiangiogenic action by antagonizing the vitronectin-mediated downstream signaling cascade, including the Akt and MEK/ERK axis.
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Our studies also indicate that GOPPP decreased HIF-1a, which targets VEGF and induces its expression (Humar et al., 2002). Hypoxia, a common feature of ischemic retinopathies, can induce HIF-1a expression (Jiang et al., 1996), which in turn increases the transcription of a number of downstream target genes involved in proliferation, apoptosis and angiogenesis (Philip et al., 2013). It has been well documented that inhibition of integrin would result in the suppression of HIF-1a activation and VEGF secretion (Tang et al., 2007; Edwards et al., 2008). Our Western blot analysis of retina proteins in OIR mice suggested that intravitreal administration of 50 ng GOPPP significantly decreased the enhancement of ERK phosphorylation and HIF-1a and VEGF level induced by relatively hypoxic condition. The present findings show that GOPPP effectively reduced pathologic angiogenesis in a model of ischemic retinopathy and its beneficial effects likely involved in the inhibition of retinal VEGF. Statement of conflicts of interest The authors disclose no potential conflicts of interest. Acknowledgments This study was supported in part by the National Natural Science Foundation of China (20872099), the Research Fund for the Doctoral Program of Higher Education (20110181110079) and Sichuan University 985 project “Science and technology innovation platform for novel drug development”. References Adamis, A.P., Miller, J.W., Bernal, M.T., D'Amico, D.J., Folkman, J., Yeo, T.K., Yeo, K.T., 1994. Increased vascular endothelial growth factor levels in the vitreous of eyes with proliferative diabetic retinopathy. Am. J. Ophthalmol. 118, 445e450. Alghisi, G.C., Ruegg, C., 2006. Vascular integrins in tumor angiogenesis: mediators and therapeutic targets. Endothelium 13, 113e135. Aiello, L.P., 2005. Angiogenic pathways in diabetic retinopathy. N. Engl. J. Med. 353, 839e841. Belvisi, L., Riccioni, T., Marcellini, M., Vesci, L., Chiarucci, I., Efrati, D., Potenza, D., Scolastico, C., Manzoni, L., Lombardo, K., et al., 2005. Biological and molecular properties of a new avb3/avb5 integrin antagonist. Mol. Cancer Ther. 4, 1670e1680. Bianchi-Smiraglia, A., Paesante, S., Bakin, A.V., 2013. Integrin b5 contributes to the tumorigenic potential of breast cancer cells through the Src-FAK and MEK-ERK signaling pathways. Oncogene 32, 3049e3058. Brooks, P.C., Montgomery, A.M., Rosenfeld, M., Reisfeld, R.A., Hu, T., Klier, G., Cheresh, D.A., 1994. Integrin alpha v beta 3 antagonists promote tumor regression by inducing apoptosis of angiogenic blood vessels. Cell 79, 1157e1164. Brooks, P.C., Stromblad, S., Klemke, R., Visscher, D., Sarkar, F.H., Cheresh, D.A., 1995. Anti-integrin alpha v beta 3 blocks human breast cancer growth and angiogenesis in human skin. J. Clin. Invest. 96, 1815e1822. Cary, L.A., Chang, J.F., Guan, J.L., 1996. Stimulation of cell migration by overexpression of focal adhesion kinase and its association with Src and Fyn. J. Cell Sci. 109, 1787e1794. Connor, K.M., Krah, N.M., Dennison, R.J., Aderman, C.M., Chen, J., Guerin, K.I., Sapieha, P., Stahl, A., Willett, K.L., Smith, L.E., 2009. Quantification of oxygeninduced retinopathy in the mouse: a model of vessel loss, vessel regrowth and pathological angiogenesis. Nat. Protoc. 4, 1565e1573. Dal Monte, M., Casini, G., la Marca, G., Isacchi, B., Filippi, L., Bagnoli, P., 2013. Eye drop propranolol administration promotes the recovery of oxygen-induced retinopathy in mice. Exp. Eye Res. 111, 27e35. Danen, E.H., Aota, S., van Kraats, A.A., Yamada, K.M., Ruiter, D.J., van Muijen, G.N., 1995. Requirement for the synergy site for cell adhesion to fibronectin depends on the activation state of integrin alpha 5 beta 1. J. Biol. Chem. 270, 21612e21618. Deliyanti, D., Miller, A.G., Tan, G., Binger, K.J., Samson, A.L., Wilkinson-Berka, J.L., 2012. Neovascularization is attenuated with aldosterone synthase inhibition in rats with retinopathy. Hypertension 3, 607e613. Doerr, M.E., Jones, J.I., 1996. The roles of integrins and extracellular matrix proteins in the insulin-like growth factor I-stimulated chemotaxis of human breast cancer cells. J. Biol. Chem. 271, 2443e2447. Edwards, L.A., Woo, J., Huxham, L.A., Verreault, M., Dragowska, W.H., Chiu, G., Rajput, A., Kyle, A.H., Kalra, J., Yapp, D., et al., 2008. Suppression of VEGF
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Reynolds, A.R., Hart, I.R., Watson, A.R., Welti, J.C., Silva, R.G., Robinson, S.D., Da Violante, G., Gourlaouen, M., Salih, M., Jones, M.C., et al., 2009. Stimulation of tumor growth and angiogenesis by low concentrations of RGD-mimetic integrin inhibitors. Nat. Med. 15, 392e400. Santulli, R.J., Kinney, W.A., Ghosh, S., Decorte, B.L., Liu, L., Tuman, R.W., Zhou, Z., Huebert, N., Bursell, S.E., Clermont, A.C., et al., 2008. Studies with an orally bioavailable alpha V integrin antagonist in animal models of ocular vasculopathy: retinal neovascularization in mice and retinal vascular permeability in diabetic rats. J. Pharmacol. Exp. Ther. 324, 894e901. Schlaepfer, D.D., Jones, K.C., Hunter, T., 1998. Multiple Grb2-mediated integrinstimulated signaling pathways to ERK2/mitogen-activated protein kinase: summation of both c-Src- and focal adhesion kinase-initiated tyrosine phosphorylation events. Mol. Cell Biol. 18, 2571e2585. Shiojima, I., Walsh, K., 2002. Role of Akt signaling in vascular homeostasis and angiogenesis. Circ. Res. 90, 1243e1250. Somanath, P.R., Malinin, N.L., Byzova, T.V., 2009. Cooperation between integrin alpha v beta 3 and VEGFR 2 in angiogenesis. Angiogenesis 12, 177e185. Stahl, A., Connor, K.M., Sapieha, P., Chen, J., Dennison, R.J., Krah, N.M., Seaward, M.R., €fqvist, C., Hellstro € m, A., Willett, K.L., Aderman, C.M., Guerin, K.I., Hua, J., Lo Smith, L.E., 2010. The mouse retina as an angiogenesis model. Invest. Ophthalmol. Vis. Sci. 51, 2813e2826. Stupp, R., Ruegg, C., 2007. Integrin inhibitors reaching the clinic. J. Clin. Oncol. 25, 1637e1638. Takagi, H., Suzuma, K., Otani, A., Oh, H., Koyama, S., Ohashi, H., Watanabe, D., Ojima, T., Suganami, E., Honda, Y., 2002. Role of vitronectin receptor-type integrins and osteopontin in ischemia-induced retinal neovascularization. Jpn. J. Ophthalmol. 46, 270e278. Tang, C.H., Lu, D.Y., Tan, T.W., Fu, W.M., Yang, R.S., 2007. Ultrasound induces hypoxia-inducible factor-1 activation and inducible nitric-oxide synthase expression through the integrin/integrin-linked kinase/Akt/mammalian target of rapamycin pathway in osteoblasts. J. Biol. Chem. 282, 25406e25415. Terai, Y., Abe, M., Miyamoto, K., Koike, M., Yamasaki, M., Ueda, M., Ueki, M., Sato, Y., 2001. Vascular smooth muscle cell growth-promoting factor/F-spondin inhibits angiogenesis via the blockade of integrin avb3 on vascular endothelial cells. J. Cell Physiol. 188, 394e402. Tsou, R., Isik, F.F., 2001. Integrin activation is required for VEGF and FGF receptor protein presence on human microvascular endothelial cells. Mol. Cell Biochem. 224, 81e89. Wilkinson-Berka, J.L., Jones, D., Taylor, G., Jaworski, K., Kelly, D.J., Ludbrook, S.B., Willette, R.N., Kumar, S., Gilbert, R.E., 2006. SB-267268, a nonpeptidic antagonist of alpha (v) beta3 and alpha (v) beta5 integrins, reduces angiogenesis and VEGF expression in a mouse model of retinopathy of prematurity. Invest. Ophthalmol. Vis. Sci. 47, 1600e1605. Witmer, A.N., van Blijswijk, B.C., van Noorden, C.J., Vrensen, G.F., Schlingemann, R.O., 2004. In vivo angiogenic phenotype of endothelial cells and pericytes induced by vascular endothelial growth factor-A. J. Histochem. Cytochem. 52, 39e52. Yoshida, T., Gong, J., Xu, Z., Wei, Y., Duh, E.J., 2012. Inhibition of pathological retinal angiogenesis by the integrin avb3 antagonist tetraiodothyroacetic acid (tetrac). Exp. Eye Res. 94, 41e48.
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Experimental Eye Research journal homepage: www.elsevier.com/locate/yexer
Therapeutic efficacy of a novel non-peptide avb3 integrin antagonist for pathological retinal angiogenesis in mice Yong-Jie Li a, 1, Xiao-Hong Li b, 1, Liang-Fen Wang a, Xi Kuang a, Zhi-Xiong Hang c, Yong Deng c, *, Jun-Rong Du a, * a
Department of Pharmacology, Key Laboratory of Drug Targeting and Drug Delivery Systems Ministry of Education, West China School of Pharmacy, Sichuan University, Chengdu, China Department of Biopharmaceutics, West China School of Pharmacy, Sichuan University, Chengdu, China c Department of Medical Chemistry, West China School of Pharmacy, Sichuan University, Chengdu, China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 21 December 2013 Received in revised form 14 September 2014 Accepted in revised form 7 November 2014 Available online 8 November 2014
avb3 integrin has been reported as a promising therapeutic target for angiogenesis. In the present study,
Keywords: Angiogenesis Integrin avb3 Oxygen-induced retinopathy Non-peptide integrin antagonist
we tested the antiangiogenic activity of 3-[3-(6-guanidino-1-oxoisoindolin-2-yl) propanamido]-3-(pyridin-3-yl) propanoic acid dihydrochloride (GOPPP), a novel non-peptide avb3 antagonist. Both human umbilical vein endothelial cells (HUVECs) and a mouse model of oxygen-induced retinopathy (OIR) were investigated separately. HUVEC adhesion, proliferation, migration, ERK1/2 and Akt phosphorylation were assessed. C57BL/6 mice were used for the studies in the OIR model. After exposure to 75% oxygen from postnatal day (PD) 7 to PD12, the mice were returned to room air, and GOPPP was intravitreally administered on PD12. Retinal neovascularization was evaluated on PD17. Hypoxia-inducible factor-1a (HIF-1a) and vascular endothelial growth factor (VEGF) protein levels and ERK1/2 phosphorylation were determined by Western blot analysis of retina proteins. GOPPP significantly inhibited the pro-angiogenic effects of vitronectin on HUVECs, including adhesion, proliferation, and migration, and inhibited ERK1/2 and Akt phosphorylation. Retinal neovascularization in the OIR model was significantly suppressed by intravitreal administration of 50 ng GOPPP. The pro-angiogenic factors HIF-1a and VEGF induced by hypoxia were significantly inhibited by GOPPP in OIR mice. GOPPP administration also inhibited ERK1/2 phosphorylation in the OIR model. These results indicate that GOPPP, a novel avb3 integrin antagonist, may have potential for the treatment of pathological retinal angiogenesis. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction Angiogenesis is characterized by the formation of new capillaries that sprout from pre-existing microvasculature. It is a normal physiological event in development and part of the pathological response in ocular neovascularization, inflammatory arthritis, and tumor growth. For endothelial cells that undergo angiogenesis, alterations in four cellular functions are defined: proliferation, migration, endothelial cell-to-cell interactions, and endothelial cell interactions with the extracellular matrix. Among the relevant molecules that regulate cell-extracellular matrix interactions are integral membrane proteins, including integrins. With regard to integrins, avb3 integrin is recognized as a key player in
* Corresponding authors. E-mail addresses: [email protected] (Y. Deng), [email protected] (J.-R. Du). 1 Equally contributed to this work. http://dx.doi.org/10.1016/j.exer.2014.11.004 0014-4835/© 2014 Elsevier Ltd. All rights reserved.
angiogenesis (Eliceiri et al., 1999; Hodivala-Dilke, 2008). The levels of avb3 integrin protein increase in angiogenic endothelial cells, and the inhibition avb3 integrin function individually with antibodies or small-molecule compounds results in the perturbation of angiogenesis (Brooks et al., 1995; Santulli et al., 2008). Integrins bind ligands by recognizing short-amino-acid stretches on exposed loops, particularly the arginineeglycineeaspartic acid (RGD) sequence. Accordingly, RGD-mimetic peptides or small molecules bind to these integrins on the surface of cells to block specific integrin-mediated signaling pathways and act as anticancer and antiangiogenic agents (Alghisi and Ruegg, 2006; Stupp and Ruegg, 2007). Yet, RGD-mimetic integrin inhibitors may have biphasic effect on avb3 integrin, with an antagonistic phase at high concentrations and agonistic phase at low concentrations (Legler et al., 2001; Reynolds et al., 2009), implying that the delivery of these agents should be designed carefully to avoid nanomolar plasma concentrations and improve their efficacy in the treatment of human
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cancers. In the present study, we designed and identified a novel non-peptide, small-molecule compound, 3-[3-(6-guanidino-1oxoisoindolin-2-yl) propanamido]-3-(pyridin-3-yl) propanoic acid dihydrochloride (GOPPP), as a potential avb3 inhibitor. This report describes the in vitro and in vivo pharmacological activity of GOPPP, which was shown to inhibit retinal neovascularization in a mouse model of oxygen-induced retinopathy (OIR).
2.4. Cell proliferation assay Cell proliferation was measured using the MTT assay as previously described (Belvisi et al., 2005). Various cells, including HUVECs and MCF-7 cells, were treated with serial dilutions of GOPPP for 48 h. Each assay was replicated three times. The results were plotted and are expressed as the mean ± SD compound concentration that inhibited 50% cell proliferation (i.e., IC50).
2. Methods 2.5. Wound healing assay 2.1. Cell cultures MCF-7 cells were obtained from the American Type Culture Collection and cultured in Dulbecco's Modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum. Human umbilical vein endothelial cells (HUVECs) were isolated from human umbilical cord veins using a standard procedure as previously described (Jaffe et al., 1973) and grown in endothelial cell medium (Sciencell, Carlsbad, California, USA). HUVECs at passages 3 to 8 were used for all of the experiments.
Monolayer HUVECs in vitronectin-coated 24-well cell plates were wounded by scratching with a pipette tip and washed with PBS. Serum-free medium that contained vehicle or different concentrations of GOPPP was added to the scratched monolayers. Images were taken using an Olympus digital camera after 24 h of cell migration. The migrated cells were quantified by manual counting, and the percentage of inhibition is expressed relative to untreated cells set to 100% (Naik and Naik, 2006). 2.6. Mouse model of oxygen-induced retinopathy
2.2. Integrin binding assay The assays were performed as described previously (MasMoruno et al., 2011; Santulli et al., 2008). Briefly, 2.0 mg/ml of vitronectin (Millipore, Billerica, MA, USA) or 5.0 mg/ml of fibronectin (Sigma, St. Louis, MO, USA) in carbonate buffer (15 mM Na2CO3 and 35 mM NaHCO3; 100 ml/well) was added to 96-well microtiter plates and incubated overnight at 4 C. The plates were then incubated with blocking solution (i.e., TS buffer: 20 mM TriseHCl, pH 7.5, 150 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, and 1 mM MnCl2) plus 2% bovine serum albumin (BSA) for an additional 1 h at room temperature. After rinsing twice with assay buffer (i.e., TS buffer plus 0.1% BSA), the plates were incubated with 1.0 mg/ml of soluble avb3 or a5b1 integrin (Millipore) in the presence of increasing concentrations of GOPPP or cyclo-Arg-Gly-Asp-D-PheVal (cRGDfV) for 1 h at room temperature, followed by washing and 1 h incubation with primary integrin antibody (Millipore) and goat anti-mouse immunoglobulin G conjugated with horseradish peroxidase (HRP; 1:200; Boster, Wuhan, China) consecutively at room temperature. After five washes with PBST buffer (10 mM Na2HPO4, pH 7.5, 150 mM NaCl, and 0.01% Tween-20), peroxidase development was performed using the substrate solution 3,3,5,50 tetramethylethylenediamine (Boster) and 3.0 M H2SO4 to stop the reaction, followed by absorbance measurement at 450 nm. Each data point is the result of the average of triplicate wells. The results were plotted and are expressed as the antagonist concentration that inhibited 50% integrin binding. 2.3. Cell adhesion assay The assays were performed as described previously (Gentilucci et al., 2010). Ninety-six-well cell plates were coated with 1.0 mg/ ml vitronectin in 0.1 M phosphate buffer, pH 7.2 (PBS), overnight at 4 C. HUVECs (5 104) were seeded in each well and allowed to adhere for 1 h at 37 C in the presence of various concentrations of GOPPP. After washed with PBS to remove non-adherent cells, adherent cells were fixed with 4% paraformaldehyde for 10 min, followed by staining with 1% toluidine blue (Aladdin, Shanghai, China) for 10 min and washing with water. Finally, the stained cells were solubilized with 1% sodium dodecyl sulfate (SDS; Amersco, OH, USA) and quantified at 600 nm. The experiments were performed in quadruplicate and repeated at least three times. The results are expressed as the mean ± SD compound concentration that inhibited 50% cell adhesion.
C57BL/6 mice were used and handled in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Mice on postnatal day 7 (PD7) were placed with their nursing dams in a 75% oxygen atmosphere for 5 days. After being returned to normoxia conditions for 5 days (i.e., on PD12), the mouse pups were intravitreally administered 0.5 ml of different concentrations of GOPPP or PBS. Five days later (i.e., on PD17), the mice were sacrificed (Mouse pups in the sham group were fostered under normoxia conditions until PD17), and the eyes were enucleated and fixed in 4% paraformaldehyde. Whole retinal flatmounts were prepared and stained with isolectin B4-Alexa Fluor 568 (Sigma) to quantify vaso-obliteration and pre-retinal neovascular tufts as described previously (Connor et al., 2009). To visualize blood vessels, dissected retinas were rinsed in 0.1 M PBS and incubated overnight at 4 C in 50 mg/ml of the isolectin in 0.1 M PBS. After incubation, the whole mounts were rinsed in 0.1 M PBS, mounted on gelatin-coated glass slides and cover-slipped with glycerine. Blood vessels were observed and photographed using fluorescence microscopy (ImageZ2; Zeiss, Deutschland). In each whole mount, the total area of pre-retinal neovascular tufts and the extent of the avascular area were measured using Image-pro Plus 6.0 System (Media Cybernetics, USA) and expressed as the percentage of the respective average in relation to total retinal area. For each experimental condition, quantitative data originated from 10 retinas. The retina was also histologically examined. Serially sectioned paraffin-embedded small pieces of retina (6 mm) were stained with hematoxylin and eosin (HE). Images were taken using light microscopy, and endothelial nuclei that extended beyond the inner limiting membrane into the vitreous were manually counted in a blinded manner (Lahdenranta et al., 2007). 2.7. Western blot analysis To determine the effects of GOPPP on the angiogenic signaling cascade, serum-starved HUVECs were plated on 2 mg/ml of vitronectin-coated dishes in medium in the presence of vehicle or different concentrations of GOPPP for 1 h. The cells were then lysed in lysis buffer that contained 1% Triton X-100 (Amersco), 1% deoxycholate (Amersco), and proteinase inhibitor cocktail (Roche, Rotkreuz, Switzerland). Protein concentrations were determined using a modified Lowry protein assay kit (Thermo, Pittsburgh PA, USA) and equalized before loading. Forty
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micrograms of cellular protein from each sample was applied to 8e12% SDS-polyacrylamide gels, and resolved proteins were transferred to nitrocellulose membrane and probed with specific antibodies, including phospho-Erk1/2 (p-Erk1/2), Erk1/2, phospho-AKT (p-AKT), and AKT (Cell Signaling Technology, Boston, USA). The blots were developed with HRP-conjugated secondary antibodies and the BeyoECL Plus enhanced chemiluminescence detection kit (Beyotime, Shanghai, China). Band density was analyzed using Quantity One software and used to determine the relative protein level. To further investigate the signaling pathway that is involved in the antiangiogenic activity of GOPPP, Western blot of OIR mouse retina proteins was also performed. After separation from the eyeballs on PD17, the retinal tissues of each group (control group: three mouse pups fostered under normoxia conditions; model group: three OIR mice intravitreally administered with PBS; GOPPP group: three OIR mice intravitreally administered with 50 ng GOPPP) were lysed together with lysis buffer for 30 min, containing protease inhibitors, followed by centrifugation at 12,000 g for 10 min at 4 C. The supernatants were collected as lysates for protein concentration determination and Western blot analysis as described above. 2.8. Statistical analysis Results were reported as mean ± SD. The data were analyzed using one-way analysis of variance (ANOVA) and SPSS 16.0 software. Differences were considered statistically significant when p < 0.05. The IC50 values were calculated using Microsoft Excel 2007. 3. Results 3.1. Identification of avb3 integrin inhibitors A library of synthetic non-peptide small molecules was screened using a solid-phase receptor assay to identify compounds that recognize avb3 integrin. The well-characterized cyclic pentapeptide integrin antagonist cRGDfV was used as a positive control. Fig. 1 shows the structure of one of the compounds of interest, GOPPP, identified by this process. Fig. 2A shows that GOPPP dosedependently inhibited vitronectin binding to avb3 integrin, with an IC50 of 7.0 nM, which is much lower than the IC50 for cRGDfV. The selectivity of this compound was demonstrated by its low capacity to compete with biotinylated fibronectin for a5b1 integrin (IC50 > 10 mM; Fig. 2B), just like cRGDfV. 3.2. Inhibition of endothelial cell adhesion, proliferation, and migration by GOPPP To assess the ability of GOPPP to act as an avb3 antagonist for endothelial cells, HUVECs were allowed to adhere to immobilized vitronectin in the presence of increasing concentrations of GOPPP
Fig. 1. Chemical structure of 3-[3-(6-guanidino-1-oxoisoindolin-2-yl)propanamido]-3(pyridin-3-yl) propanoic acid dihydrochloride (GOPPP).
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(range, 0.01e10 mM). As shown in Fig. 3A, GOPPP significantly and dose-dependently inhibited HUVEC adhesion to vitronectin, with an IC50 of 0.10 ± 0.03 mM, which was much lower than that of cRGDfV (2.52 ± 0.39 mM). To assess the anti-angiogenic activity of GOPPP in vitro, its inhibitory effects on the proliferation of HUVECs were evaluated using the MTT assay. GOPPP significantly inhibited HUVEC proliferation, with an IC50 of 4.88 ± 0.38 mM (Fig. 3B). In contrast, 10 mM GOPPP showed no significant inhibitory effect on the proliferation of MCF-7 cells, which do not express avb3 integrin (Danen et al., 1995; Doerr and Jones, 1996), suggesting low cytotoxicity (Fig. 3B). Cell migration, which is essential for endothelial cells in angiogenesis, was investigated using a wound healing assay, with vitronectin as the stimulator. As shown in Fig. 3C, vitronectin increased HUVC migration 5.06-fold compared with the control (p < 0.01). The addition of GOPPP dose-dependently inhibited vitronectin-induced HUVEC migration. Concentrations of 0.5 and 1 mM GOPPP had no significant effect on HUVEC migration compared with vehicle, whereas 5 mM GOPPP inhibited migration 2.43-fold (p < 0.01; Fig. 3C). 3.3. Effect of GOPPP on angiogenesis signaling To delineate the signaling pathway that is involved in the antiangiogenic activity of GOPPP, its effects on Akt and ERK phosphorylation were evaluated by applying Western blot analysis of HUVEC proteins after incubation with vitronectin in the presence of different concentrations of GOPPP. As shown in Fig. 4, vitronectin activated the phosphorylation of ERK and AKT in HUVECs (p < 0.01), and GOPPP dose-dependently suppressed ERK and AKT phosphorylation. GOPPP (10 mM) inhibited ERK and AKT phosphorylation by 47.6% and 42.4%, respectively, compared with vehicle (p < 0.01), suggesting that GOPPP exerts its antiangiogenic effects by antagonizing the vitronectin-mediated downstream cascade, including the Akt and MEK/ERK signaling axis. 3.4. Inhibition of retinal neovascularization in oxygen-induced retinopathy model by intravitreal administration of GOPPP To further assess the anti-angiogenic activity of GOPPP in vivo, the mouse OIR model was investigated and its effects on retinal angiogenesis were evaluated in the superficial vascular plexuses using isolectin B4 staining. Mice were exposed to 75% oxygen from PD7 to PD12, which results in extensive retinal vaso-obliteration, and then returned to room air. Such conditions made the retina relatively hypoxic, leading to the formation of retinal neovascularization (Fig. 5A, C). At PD17, in the superficial plexus of vehicle-treated retina (Fig. 5A), both an excessive vessel regrowth leading to pre-retinal neovascular tufts and the formation of a large avascular area in central retina, with only the major vessels and practically no capillary network, were detected. Intravitreal injection of GOPPP dose-dependently reduced the vessel tuft area in the superficial plexus (Fig. 5A, C). Compared with vehicle, 5 or 10 ng GOPPP had no significant effect, whereas 50 ng GOPPP reduced the vessel tufts by 60.6% (p < 0.01). The central avascular area was slightly reduced (p < 0.01; Fig. 5A, B). To further confirm the inhibitory effect of GOPPP on retinal neovascularization, vascular cell nuclei that extended beyond the internal limiting membrane were counted on HE-stained retinal tissue sections. Nuclei anterior to the internal limiting membrane were not found among the retinal tissue sections in the sham group, but a large number of neovascular nuclei were found in the eyes of OIR mice (Fig. 6AeC), 42.7% of which were suppressed by 50 ng GOPPP (p < 0.01; Fig. 6D).
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Fig. 2. GOPPP is a potent and selective inhibitor of vitronectin binding to avb3. (A) Inhibition of vitronectin binding to avb3 integrin. (B) Inhibition of fibronectin binding to a5b1 integrin. avb3 or a5b1 integrin was incubated with immobilized vitronectin or fibronectin in the presence of various concentrations of GOPPP or cRGDfV. Bound integrin was detected with primary integrin antibody and goat anti-mouse immunoglobulin G conjugated with HRP. The results are expressed as mean ± SD (n ¼ 3).
Fig. 3. GOPPP inhibits adhesion, proliferation, and migration of HUVECs. (A) GOPPP inhibits HUVEC adhesion. HUVECs were allowed to adhere to vitronectin-coated 96-well plates with different concentrations of GOPPP or cRGDfV for 1 h. After incubation, adhered cells were fixed, stained, and examined at 595 nm. The percentage of inhibition is expressed relative to HUVECs with vehicle. (B) GOPPP inhibits HUVECs proliferation. Cells were treated with various concentrations of GOPPP for 48 h. Cell viability was quantified using the MTT assay. (C) GOPPP inhibits HUVEC migration. HUVECs were allowed to grow to full confluence in 24-well plates that were uncoated or coated with 2 mg/ml vitronectin (VN). Cells were wounded with a pipette and then treated with different concentrations of GOPPP in serum-free DMEM for 24 h. After incubation, the migrated cells were quantified by manual counting. The results are expressed as mean ± SD (n ¼ 6). **p < 0.01 versus vehicle-treated VN group. Scale bar, 500 mm.
Western blot analysis of PD17 OIR murine retina proteins was performed. Hypoxia activated ERK phosphorylation in mouse retinas (p < 0.01; Fig. 7) and the expression of hypoxia-inducible factor-1a (HIF-1a) and vascular endothelial growth factor 165 (VEGF165). Intravitreal administration of 50 ng GOPPP significantly decreased ERK phosphorylation and HIF-1a and VEGF165 levels induced by relatively hypoxic conditions (p < 0.05; Fig. 7). 4. Discussion
or
Presently, three classes of integrin inhibitors are in preclinical clinical trials: monoclonal antibodies that target the
extracellular domain of the integrin heterodimer (e.g., Vitaxin), synthetic RGD motif-containing peptides (e.g., cilengitide), and peptidomimetics (e.g., S247), which are orally bioavailable nonpeptidic molecules that mimic the RGD sequence (Alghisi and Ruegg, 2006). In this study, we evaluated the in vitro and in vivo pharmacological activity of a novel nonpeptidic avb3 antagonist, GOPPP, which was shown to inhibit retinal neovascularization in a mouse OIR model. The major results were that GOPPP reduced pathologic but not developmental angiogenesis in neonatal mice. The effect of GOPPP in the OIR model appeared to be attributable to its selective action on avb3 integrin. In vitro, this compound behaved as a selective inhibitor of integrin avb3 by preventing the
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Fig. 4. GOPPP suppresses the phosphorylation of Akt and ERK induced by vitronectin in HUVECs. (A) Western blot analysis of Akt and ERK. Serum-starved HUVECs were plated on vitronectin (VN)-coated dishes in medium supplemented with various concentrations of GOPPP for 1 h and then lysed. The lysates were analyzed by Western blot. The negative control was performed without vitronectin. Blots were developed with HRP-conjugated secondary antibodies and an enhanced chemiluminescence detection kit. (B, C) Densitometry of three independent experiments. The Western blot band density was analyzed using Quantity One software and used to determine the relative protein level. Results was expressed as means ± SD (n ¼ 3). **p < 0.01, compared with vehicle-treated VN group.
Fig. 5. Quantification of vaso-obliteration and pre-retinal neovascular tufts in the murine model of OIR. (A) Representative flat mounts of mouse retina. Retinal whole-mounts from PD17 were stained for blood vessel endothelial cells using isolectin B4-Alexa Fluor 568 (red), and lectin-stained tufts show new capillary buds induced by the OIR model. Sham, mouse pups fostered under normoxia conditions. Model, OIR mice intravitreally administered with PBS. GOPPP, OIR mice intravitreally administered with various concentrations of GOPPP. (B) Quantification of vaso-obliteration. (C) Quantification of pre-retinal neovascular tufts. The results were expressed as means ± SD (n ¼ 10). **p < 0.01, compared with model group. Scale bar, 100 mm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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Fig. 6. Analysis of preretinal nuclei in HE-stained retinal tissue sections. (AeC) Sham, mouse pups fostered under normoxia conditions; model, OIR mice intravitreally administered with PBS; GOPPP, OIR mice intravitreally administered with 50 ng GOPPP. (D) Quantification of preretinal nuclei. The neovascular cell nuclei are indicated by arrows. Quantification was based on eight non-continuous retinal tissue sections per eye. The results were expressed as means ± SD (n ¼ 8). **p < 0.01, compared with model. Scale bar, 50 mm.
Fig. 7. GOPPP suppresses ERK phosphorylation and the enhancement of HIF-1a and VEGF level induced by hypoxia in OIR mouse retina. (A) Western blot of VEGF, HIF-1a, and ERK. Control, mouse pups fostered under normoxia conditions; model, OIR mice intravitreally administered with PBS; GOPPP, OIR mice intravitreally administered with 50 ng GOPPP. On PD17, all of the retinal tissues from six eyeballs in each group were lysed together and analyzed using Western blot as described in the Methods section. The blots were developed with HRP-conjugated secondary antibodies and an enhanced chemiluminescence detection kit. (B, C) Densitometry of three independent experiments. Band density was analyzed using Quantity One software and used to determine the relative protein level. Results was expressed as means ± SD (n ¼ 3), *p < 0.05, **p < 0.01, compared with model group.
binding of vitronectin to avb3 with high affinity. Additionally, it inhibited HUVEC adhesion to vitronectin with high potency. In both of the assays, GOPPP was more potent than the wellcharacterized integrin antagonist cRGDfV. Both HUVEC proliferation and migration were inhibited. Retinal angiogenesis is a major cause of blindness in ischemic retinopathies, including diabetic retinopathy (DR) and retinopathy of prematurity (ROP). VEGF has been identified as a major mediator (Adamis et al., 1994; Aiello, 2005). Angiogenesis inhibitors that target VEGF have made a significant impact on the treatment of DR, ROP, and age-related macular degeneration. In addition to growth factors, integrins have also been implicated in angiogenesis, serving
as receptors for specific extracellular matrix proteins. avb3 integrin is not generally expressed on resting or normal microvessels but is dramatically upregulated in response to angiogenic growth factors (Brooks et al., 1994). It is also thought to be a critical regulator of angiogenesis. In the well-established mouse model of OIR, av integrins are overexpressed in neovascular endothelial cells during the peak time of retinal vessel growth (Takagi et al., 2002). The efficacy of avb3 integrin inhibition has been evaluated in OIR and ROP models (Santulli et al., 2008; Wilkinson-Berka et al., 2006; Yoshida et al., 2012). Herein, the potential of GOPPP as an antiangiogenic agent for retinal neovascularization was evaluated in an OIR model. Owing to the fact that tuft formation induced by
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hypoxia only appears in the superficial vascular plexus, leaving the deep vascular plexus unaffected (Dal Monte et al., 2013; Stahl et al., 2010), we investigated the pre-retinal neovascularization only. The results revealed that intravitreal administration of 50 ng GOPPP significantly reduced pre-retinal neovascular tufts by 60.6% (Fig. 5C) and neovascular nuclei anterior to the internal limiting membrane by 42.7% (Fig. 6AeC). We observed the discrepancy of neovascularization inhibition potency of GOPPP existed between the neovascular tufts and preretinal nuclei quantitation in retina as others reported (Deliyanti et al., 2012). The potential explanation for the discrepancy observed is the fact that the quantitation of neovascular tufts on flat mounts generally counts the vascular tufts on retinal whole surface, whereas HE staining may determine the preretinal nuclei of retinal cross-sections. Moreover, the manual quantitation on flat mounts with relative low resolution of images probably disregards small capillaries in retina. In addition, it is reported that tetraiodothyroacetic acid (tetrac), a deaminated analogue of L-thyroxine (T4), blocked the pro-angiogenic actions of T4 by inhibiting its binding to avb3 integrin, and intravitreal administration of 75 ng tetrac reduced the neovascular tufts in the stained retina by 44.8% in the OIR mice (Yoshida et al., 2012). Collectively, the present study suggests that the novel avb3 antagonist GOPPP exhibits significant inhibitory action on retinal angiogenesis in the OIR mice. With regard to the promotion of angiogenesis, synergistic interactions between avb3 integrin and VEGF have been demonstrated by multiple studies (e.g., Somanath et al., 2009). Some studies offer compelling evidence that avb3 function is necessary for VEGF-mediated effects. In the monkey eye after exposure to VEGF, avb3 and avb5 are highly upregulated in migrating cells from preexisting and newly formed vessels (Witmer et al., 2004). On the other hand, integrin inhibition is reported to be accompanied with suppression of VEGF secretion (Edwards et al., 2008; Iliaki et al., 2009). And, antagonists of both avb3 and avb5 integrins block the VEGF-stimulated adhesion, proliferation, and migration of endothelial cells (Terai et al., 2001; Tsou and Isik, 2001). In our assays with OIR mice, GOPPP reduced the retinal VEGF level under the condition of relative hypoxia, suggesting that the molecular mechanism underlying therapeutic efficacy of GOPPP, a novel avb3 antagonist, is associated with its downregulation on VEGF-induced retinal angiogenesis. Clearly, additional studies will be required to define the underlying mechanism of GOPPP regulating VEGE responsiveness. Integrins mediate complex signaling events, including the regulation of cell adhesion, proliferation, survival, and migration by activating canonical pathways. Among various signaling cascades, the phosphorylation of focal adhesion kinase (FAK) and activation of the phosphatidylinositol 30 -kinase (PI3K)/Akt pathway have been shown to be involved in avb3-mediated adhesion, migration, and cell survival (Cary et al., 1996; Gilmore et al., 2000; Ilic et al., 1995). The importance of FAK in integrin signaling is well established. Integrins bind FAK and mediate FAK activation and phosphorylation. In turn, integrin-FAK can activate the MEK-ERK axis (Schlaepfer et al., 1998), which contributes to cell survival and proliferation (Frisch and Screaton, 2001), whether Src is activated or not (Bianchi-Smiraglia et al., 2013; Hood et al., 2003). Akt is a critical regulator of PI3K-mediated cell survival and migration (Shiojima and Walsh, 2002). Numerous studies have demonstrated that the constitutive activation of Akt signaling is sufficient to block cell death and migration induced by various apoptotic stimuli in various cell types. In the present study, GOPPP significantly suppressed ERK and Akt phosphorylation induced by vitronectin, suggesting that GOPPP exerted its antiangiogenic action by antagonizing the vitronectin-mediated downstream signaling cascade, including the Akt and MEK/ERK axis.
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Our studies also indicate that GOPPP decreased HIF-1a, which targets VEGF and induces its expression (Humar et al., 2002). Hypoxia, a common feature of ischemic retinopathies, can induce HIF-1a expression (Jiang et al., 1996), which in turn increases the transcription of a number of downstream target genes involved in proliferation, apoptosis and angiogenesis (Philip et al., 2013). It has been well documented that inhibition of integrin would result in the suppression of HIF-1a activation and VEGF secretion (Tang et al., 2007; Edwards et al., 2008). Our Western blot analysis of retina proteins in OIR mice suggested that intravitreal administration of 50 ng GOPPP significantly decreased the enhancement of ERK phosphorylation and HIF-1a and VEGF level induced by relatively hypoxic condition. The present findings show that GOPPP effectively reduced pathologic angiogenesis in a model of ischemic retinopathy and its beneficial effects likely involved in the inhibition of retinal VEGF. Statement of conflicts of interest The authors disclose no potential conflicts of interest. Acknowledgments This study was supported in part by the National Natural Science Foundation of China (20872099), the Research Fund for the Doctoral Program of Higher Education (20110181110079) and Sichuan University 985 project “Science and technology innovation platform for novel drug development”. References Adamis, A.P., Miller, J.W., Bernal, M.T., D'Amico, D.J., Folkman, J., Yeo, T.K., Yeo, K.T., 1994. Increased vascular endothelial growth factor levels in the vitreous of eyes with proliferative diabetic retinopathy. Am. J. Ophthalmol. 118, 445e450. Alghisi, G.C., Ruegg, C., 2006. Vascular integrins in tumor angiogenesis: mediators and therapeutic targets. Endothelium 13, 113e135. Aiello, L.P., 2005. Angiogenic pathways in diabetic retinopathy. N. Engl. J. Med. 353, 839e841. Belvisi, L., Riccioni, T., Marcellini, M., Vesci, L., Chiarucci, I., Efrati, D., Potenza, D., Scolastico, C., Manzoni, L., Lombardo, K., et al., 2005. Biological and molecular properties of a new avb3/avb5 integrin antagonist. Mol. Cancer Ther. 4, 1670e1680. Bianchi-Smiraglia, A., Paesante, S., Bakin, A.V., 2013. Integrin b5 contributes to the tumorigenic potential of breast cancer cells through the Src-FAK and MEK-ERK signaling pathways. Oncogene 32, 3049e3058. Brooks, P.C., Montgomery, A.M., Rosenfeld, M., Reisfeld, R.A., Hu, T., Klier, G., Cheresh, D.A., 1994. Integrin alpha v beta 3 antagonists promote tumor regression by inducing apoptosis of angiogenic blood vessels. Cell 79, 1157e1164. Brooks, P.C., Stromblad, S., Klemke, R., Visscher, D., Sarkar, F.H., Cheresh, D.A., 1995. Anti-integrin alpha v beta 3 blocks human breast cancer growth and angiogenesis in human skin. J. Clin. Invest. 96, 1815e1822. Cary, L.A., Chang, J.F., Guan, J.L., 1996. Stimulation of cell migration by overexpression of focal adhesion kinase and its association with Src and Fyn. J. Cell Sci. 109, 1787e1794. Connor, K.M., Krah, N.M., Dennison, R.J., Aderman, C.M., Chen, J., Guerin, K.I., Sapieha, P., Stahl, A., Willett, K.L., Smith, L.E., 2009. Quantification of oxygeninduced retinopathy in the mouse: a model of vessel loss, vessel regrowth and pathological angiogenesis. Nat. Protoc. 4, 1565e1573. Dal Monte, M., Casini, G., la Marca, G., Isacchi, B., Filippi, L., Bagnoli, P., 2013. Eye drop propranolol administration promotes the recovery of oxygen-induced retinopathy in mice. Exp. Eye Res. 111, 27e35. Danen, E.H., Aota, S., van Kraats, A.A., Yamada, K.M., Ruiter, D.J., van Muijen, G.N., 1995. Requirement for the synergy site for cell adhesion to fibronectin depends on the activation state of integrin alpha 5 beta 1. J. Biol. Chem. 270, 21612e21618. Deliyanti, D., Miller, A.G., Tan, G., Binger, K.J., Samson, A.L., Wilkinson-Berka, J.L., 2012. Neovascularization is attenuated with aldosterone synthase inhibition in rats with retinopathy. Hypertension 3, 607e613. Doerr, M.E., Jones, J.I., 1996. The roles of integrins and extracellular matrix proteins in the insulin-like growth factor I-stimulated chemotaxis of human breast cancer cells. J. Biol. Chem. 271, 2443e2447. Edwards, L.A., Woo, J., Huxham, L.A., Verreault, M., Dragowska, W.H., Chiu, G., Rajput, A., Kyle, A.H., Kalra, J., Yapp, D., et al., 2008. Suppression of VEGF
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