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5
1 Integrin AntagonistDepartments of Ophthalmology and Neuroscience, The Johns Hopkins University School of Medicine, Baltimore, Maryland (N.U., H.A., P.A.C.); and Jerini Pharmaceuticals, Berlin, Germany (G.Z., D.V., R.S.)
Received November 18, 2005; accepted March 9, 2006
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Abstract |
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5
1 plays an important role in developmental angiogenesis, but its role in various types of pathologic neovascularization has not been completely defined. In this study, we found strong up-regulation of 51 in choroidal neovascularization. Implantation of an osmotic pump delivering 1.5 or 10 µg/h (
1.8 or 12 mg/kg/day) of 3-(2-{1-alkyl-5-[(pyridin-2-ylamino)-methyl]-pyrrolidin-3-yloxy}-acetylamino)-2-(alkylamino)-propionic acid (JSM6427), a selective 51 antagonist, caused significant suppression of choroidal neovascularization; the area of neovascularization was reduced by 33 to 40%. When an osmotic pump delivering 10 µg/h of JSM6427 was implanted 7 days after rupture of Bruch's membrane, there was terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining in vascular cells within the neovascularization and significant regression of the neovascularization over the next week. JSM6427 also induced apoptosis of cultured vascular endothelial cells. Fibronectin stimulates phosphorylation of extracellular signal-regulated kinase (ERK) in 51-expressing cells that is blocked by JSM6427. These data suggest that 51 plays a role in the development and maintenance of choroidal neovascularization and provides a target for therapeutic intervention.
). Vascular endothelial growth factor (VEGF) is an important hypoxia-regulated soluble signal produced by surrounding tissue that promotes neovascularization. Things other than hypoxia, such as cytokines, also up-regulate VEGF, and in many instances, this seems to be through HIF-1 or HIF-2 (Bardos et al., 2004; Haddad and Harb, 2005). Although other soluble signals undoubtedly play a role, it is clear that inhibition of VEGF is a useful strategy for treatment of neovascular diseases (Ribatti, 2005).
Another source of signals is the extracellular matrix (ECM). The ECM seems capable of providing both stabilizing signals that suppress neovascularization and stimulatory signals (Sottile, 2004). Integrins are cell surface receptors that mediate signaling from the ECM, and signaling from ECM is modulated by alterations in the integrin population on the cell surface. Therefore, it is important to determine in any given situation which integrins mediate stimulation of neovascularization and which seem to suppress it.
Integrins v3 and v5 are up-regulated on endothelial cells participating in several types of neovascularization, including tumor vessels and retinal neovascularization (Brooks et al., 1994a,b; Luna et al., 1996). Antagonists of v3 and v5 suppress tumor neovascularization and retinal neovascularization (Hammes et al., 1996; Luna et al., 1996). However, the same antagonists of v3 and v5 that suppress retinal neovascularization, have no identifiable suppressive effect on choroidal neovascularization in mice (P. A. Campochiaro, unpublished data). Gene knockout studies have shown that deletion of v, 3, and/or 5 fails to block developmental angiogenesis and in some cases may enhance angiogenesis (Hynes, 2002). Therefore, it seems that the role of integrins in neovascularization in one situation does not guarantee participation in a different vascular bed and different disease process.
Integrin 51 is also up-regulated on activated endothelial cells and tumor blood vessels (Collo and Pepper, 1999; Kim et al., 2000; Magnussen et al., 2005; Parsons-Wingerter et al., 2005). Antagonists of 51 suppress angiogenesis on chick chorioallantoic membrane induced by FGF2, tumor necrosis factor- or interleukin-8 and in murine tumor models (Kim et al., 2000; Stoeltzing et al., 2003; Magnussen et al., 2005). Integrin 51 also plays a critical role in developmental angiogenesis (Yang et al., 1993; Hynes, 2002). During vascularization of the central nervous system, angiogenic sprouts express high levels of 51, and it is markedly reduced as the vessels mature (Milner and Campbell, 2002). Thus, 51 seems to promote angiogenesis in multiple settings, but, as noted above, one cannot assume that it is proangiogenic in all tissues and pathologies. In this study, we have explored the role of 51 in choroidal neovascularization, the most common cause of severe vision loss in elderly Americans (Klein et al., 1993).
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Materials and Methods |
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). In brief, 5- to 6-week-old female C57BL/6J mice were anesthetized with ketamine hydrochloride (100 mg/kg body weight), and pupils were dilated with 1% tropicamide. Three burns of 532 nm diode laser photocoagulation (75-µm spot size, 0.1-s duration, 120 mW) were delivered to each retina with the slit lamp delivery system of an OcuLight GL diode laser (Iridex, Mountain View, CA) using a handheld cover slip as a contact lens to view the retina. Burns were performed in the 9, 12, and 3 o'clock positions of the posterior pole of the retina. Production of a bubble at the time of laser, which indicates rupture of Bruch's membrane, is an important factor in obtaining choroidal neovascularization; therefore, only burns in which a bubble was produced were included in the study.
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5 integrin subunit (Chemicon, Temecula, CA) in 1% bovine serum albumin (BSA) in TBS at 4°C overnight. For controls, nonimmune IgG (Vector Laboratories, Burlingame, CA) was substituted for primary antibody. After two rinses with TBS, sections were incubated for 30 min at room temperature with secondary antibody, 1:1000 fluorescein isothiocyanate-conjugated goat anti-rabbit IgG F(ab')2 (Jackson ImmunoResearch Laboratories Inc., West Grove, PA). Sections were counterstained with DAPI (Kirkegaard and Perry Laboratories, Gaithersburg, MD) and mounted with Aquamount (British Drug House, Poole, Dorset, UK).
Serial sections were stained with biotinylated Griffonia simplicifolia lectin B4 (GSA; Vector Laboratories) to identify choroidal neovascularization as described previously (Ozaki et al., 1998). In brief, slides were incubated in methanol/H2O2 for 10 min at 4°C, washed with 0.05 M TBS, pH 7.6, and incubated for 30 min in 10% normal porcine serum. Slides were incubated for 2 h at room temperature with biotinylated GSA. After washing, the slides were incubated in streptavidin-phosphatase and developed with HistoMark Red (Kirkegaard and Perry Laboratories) according to the manufacturer's instructions. Sections were dehydrated and mounted with Cytoseal. Stained sections were examined with a Nikon microscope and captured as digital files with a Nikon digital still camera (DXM1200; Nikon Instruments Inc., New York, NY).
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To assess the effect of JSM6427 on established choroidal neovascularization, mice had rupture of Bruch's membrane at three locations in each eye. After 7 days, 9 mice were perfused with fluorescein-labeled dextran, and the baseline area of neovascularization at each rupture site was measured. The remaining mice had implantation of osmotic minipumps containing 20 mg/ml JSM6427 in 100 mM sodium phosphate buffer, 50 mM NaCl, pH 7.4, or vehicle alone. At 7 days after implantation, some mice were euthanized for terminal terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL). The rest of the mice were perfused with fluorescein-labeled dextran 7 days after implantation, and the area of choroidal neovascularization at rupture sites was measured.
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Identification of Apoptotic Cells in Vivo by TUNEL. Eyes were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer and frozen in OCT. Serial 10-µm sections were cut through each rupture site. Sections were fixed with 1% paraformaldehyde for 10 min at room temperature, and TUNEL was done with the ApopTag Red kit (Chemicon International, Temecula, CA) according to the manufacturer's instructions. The sections were also histochemically stained with GSA as described above. Slides were also stained with DAPI and mounted in aqueous mounting medium.
Statistical Analysis. Data were analyzed using a linear mixed model accounting for possible correlations in measurements from the same mice. Dunnett's adjustment was made for multiple comparisons.
Solid Phase Binding Assay. The inhibiting activity and integrin selectivity of the integrin inhibitor was determined in a solid phase binding assay using soluble integrins and coated extracellular matrix protein. Binding of integrins was then detected by specific antibodies in an enzyme-linked immunosorbent assay. Fibronectin and vitronectin were purchased from Sigma (St Louis, MO). The integrin 51 extracellular domain Fc-fusion protein was a generous gift from M. Humphries (University of Manchester), and V3 was purchased from Chemicon (Chemicon Europe, Germany). The integrin antibodies were purchased from Pharmingen, BD Bioscience Europe (V3), and Sigma (anti-human-Fc-HRP antibody conjugate and anti-mouse-HRP conjugate).
The detection of HRP was performed using HRP substrate solution 3.3.5.5 [EC] '-tetramethylethylenediamine (Seramun Diagnostica GmbH, Dolgenbrodt, Germany) and 1 M H2SO4 for stopping the reaction. The developed color was measured at 450 nm.
51:Nunc-Immuno maxisorp plates (Nalge Nunc International, Rochester, NY) were coated over night at 4°C with fibronectin (0.25 µg/ml) in 15 mM Na2CO3, 35 mM NaHCO3, pH 9.6. All subsequent washing and binding were performed in 25 mM Tris, pH 7.6, 150 mM NaCl, 1 mM MnCl2, and 1 mg/ml BSA. The plates were blocked with 3% BSA in PBS 0.1% Tween 20 for 1 h at room temperature. Soluble integrin 51 (0.5 µg/ml) and a serial dilution of integrin inhibitor were incubated in the coated wells for 1 h at room temperature. The detection antibody (anti-human-Fc-HRP antibody conjugate) was then applied for 1 h at room temperature, and the binding was visualized as described above. For the V3 assay, plates were coated with vitronectin (1 µg/ml) and blocked as described for 51. Soluble V3 (1 µg/ml) was incubated with a serial dilution of integrin inhibitor for 1 h at room temperature. Primary (anti-V3) and secondary antibody (anti-mouse-HRP conjugate) were applied for 1 h at RT, and the binding was visualized as described above. All IC50 measurements were performed at least 30 times.
Identification of Apoptotic Cells in Vitro by TUNEL and Fluorescent Activated Cell Sorting. Human umbilical vein endothelial cells were maintained in endothelial cell growth medium (PromoCell, Heidelberg, Germany) and grown to 80% confluence, trypsinized, and preincubated for 20 min in serum-free medium alone or medium containing 100 nM, 500 nM, 1 µM, 10 µM, 25 µM, or 50 µM JSM6427 or 10 µM camptothecin. Cells (4 x 105) were plated in six-well plates coated with 10 µg/ml fibronectin (Chemicon Europe, Hampshire, UK) and grown for 48 h. Adherent and nonadherent cells were collected, pooled, and washed. Cells were fixed in 1% paraformaldehyde for 30 min on ice, washed, and carefully resuspended in 70% ice-cold ethanol. TUNEL was performed using the APO-Direct kit (BD Pharmingen, San Diego, CA). In brief, cells were incubated with TdT enzyme and fluorescein isothiocyanate-dUTP in reaction buffer for 60 min (positive and negative cells of the kit) or 180 min (probes) at 37°C. Cells were rinsed and incubated with PI-staining solution for 30 min to determine total cell numbers. Fluorescence-activated cell sorting analysis was performed using a FACSCalibur (BD Bioscience, San Diego, CA) and the CellQuest software.
Western Blotting. ARPE19 cells were incubated in serum-free Dulbecco's modified Eagle's medium/Ham's F12 (Biochrom AG, Berlin, Germany) culture medium for 16 h. Plates were coated with 10 µg/ml fibronectin or 0.002% poly-L-lysine (Sigma-Aldrich, Munich, Germany) and blocked with 1% BSA. Cells were trypsinized and held in suspension for 30 min. Subsequently, 1 x 106 cells were incubated with compounds for 10 min. Cells were allowed to adhere for 15 min on fibronectin in serum-free medium. Adherent cells and cells from supernatants were lysed in 150 µl of lysis buffer (1% Nonidet P-40, 150 mM NaCl, 50 mM Tris-HCl, pH 7.5, protease inhibitor cocktail (Sigma-Aldrich), 25 mM glycerophosphate, 50 mM NaF, 10 mM Na4P2O7, and 1 mM orthovanadate) on ice. Lysates were centrifuged, and equal amounts of protein were separated on a 4 to 15% Tris/HCl gel by SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes by semi-dry Western blotting. Membranes were blocked in 3% BSA. Phosphorylated extracellular signal regulated kinase (ERK) and total ERK were detected with a phospho-specific ERK1/2 (pThr202/pTyr204) antibody (Cell Signaling, Danvers, MA) and a p44/42 mitogen-activated protein kinase kinase antibody (Cell Signaling). Bands were visualized with peroxidase-conjugated anti-rabbit and anti-mouse antibodies (Sigma-Aldrich) and BM chemiluminescence Western blotting peroxidase substrate (Roche Applied Science, Mannheim, Germany). The luminescence signal was measured and quantified using a Lumi imager (Roche) and corresponding software.
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5 Subunit Is Increased in Vascular Cells Participating in Choroidal Neovascularization. Two weeks after laser-induced rupture of Bruch's membrane staining with GSA showed large choroidal neovascularization lesions at rupture sites (Fig. 1A, arrows). Adjacent sections immunohistochemically stained for integrin 5 subunit showed strong labeling throughout the entire choroidal neovascularization lesion (Fig. 1B, arrows). Superimposed images from DAPI-stained sections showed the overlying neural retina, confirming that the cells labeled with anti-5 are in the subretinal space (Fig. 1C). Regions of retina remote from Bruch's membrane rupture sites showed no choroidal neovascularization and no staining for 5 subunit (Fig. 1, D-F). This suggests that expression of 5 subunit in vascular cells in the eye under normal circumstances is below the sensitivity of our staining technique to detect it, but there is strong up-regulation of 5 subunit expression in vascular cells participating in choroidal neovascularization.
JSM6427 Is a Selective Antagonist of Integrin 51. The integrin inhibitor JSM6427 is an antagonist of binding to integrin 51 and is at least 1200-fold less potent in the inhibition of binding to V3, the integrin that is most closely related to 51 in structure. The selectivity against other integrins such as V5 and IIb3 is significantly higher, 3700-fold or >100,000-fold, respectively (data not shown).
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Subcutaneous Delivery of JSM6427 Causes Regression of Established Choroidal Neovascularization. Mice had laser-induced rupture of Bruch's membrane, and after 7 days, some mice were used to measure the baseline amount of choroidal neovascularization (Fig. 3A). In the remainder of the mice, an osmotic minipump was implanted that released 10 µg/h JSM6427 or vehicle. After 7 days of treatment, the mice infused with JSM6427 had small choroidal neovascularization lesions at Bruch's membrane rupture sites (Fig. 3B), whereas mice infused with vehicle had lesions that appeared larger (Fig. 3C). TUNEL showed labeling within the outer nuclear layer (ONL) of the retina overlying choroidal neovascularization in day 7 baseline eyes (Fig. 3D). This was probably a consequence of the laser photocoagulation that was used to induce rupture of Bruch's membrane, although it is possible that the presence of choroidal neovascularization is damaging to the overlying retina as a result of interference with transmission of oxygen from the choroid, interference with transmission of survival signals from the retinal pigmented epithelium, or some other reason. At 14 days after rupture of Bruch's membrane, mice infused with JSM6427 for the previous 7 days (Fig. 3E) and those infused with vehicle (Fig. 3F) showed apoptosis in the retina overlying choroidal neovascularization, but only the mice infused with JSM6427 showed apoptosis of vascular cells within the choroidal neovascularization as expected for the proposed mechanism of action and confirmed with in vitro apoptosis assays (Fig. 4). Figure 3G shows the boxed region in Fig. 3E at high magnification, providing better visualization of two yellow cells (arrows) because of colocalization of GSA and ApopTag red. Measurement of the size of choroidal neovascularization lesions by image analysis showed that mice infused with JSM6427 had significantly smaller lesions than those seen at baseline or those seen in mice infused with vehicle (Fig. 3H). This indicates that continuous systemic infusion of JSM6427 causes regression of choroidal neovascularization.
JSM6427 Causes Apoptosis of Vascular Endothelial Cells in Vitro. Human umbilical vein endothelial cells were grown in fibronectin-coated wells for 48 h in serum-free medium containing various concentrations of JSM6427. Camptothecin, a strong inducer of apoptosis, was used as a positive control. JSM6427 caused a dose-dependent increase in the percentage of TUNEL-positive cells in cultures ranging from 44% for those incubated with 50 µM to 6% for those incubated in 100 nM (Fig. 4). In cultures incubated in serum-free medium, 5% of cells were TUNEL-positive and 82% of cells were labeled in cultures incubated with 10 µM camptothecin. These data show that JSM6427 induces apoptosis of vascular endothelial cells.
JSM6427 Reduces Fibronectin-Induced Phosphorylation of ERK. The mechanism by which ECM components generate survival signals through integrins involves increased phosphorylation of intracellular messengers that increase expression of apoptosis inhibitors such as Bcl-2 (Lee and Ruoslahti, 2005). Phosphorylation of ERK plays a central role in fibronectin-mediated survival signaling through 51 in brain capillary endothelial cells (Wang and Milner, 2006). Therefore, we investigated the effect of JSM6427 on fibronectin-induced phosphorylation of ERK in ARPE19 cells, which express 51. Serum-starved ARPE19 cells were preincubated with JSM6427 for 10 min in suspension and plated for 15 min on fibronectin, poly-L-lysine, or kept in suspension. Western blots showed that for cells not preincubated in JSM6427, those plated on fibronectin had a much stronger signal for phosphorylated ERK than those plated on poly-L-lysine or those kept in suspension (Fig. 5A). Preincubation with JSM6427 before plating on fibronectin caused a dose-dependent reduction in the amount of phosphorylated ERK in the cultures. Quantification of the signal for phosphorylated ERK normalized to the total amount of ERK showed that preincubation with 10 µM JSM6427 completely eliminated the fibronectin-induced stimulation of ERK phosphorylation (Fig. 5B), and the IC50 was 0.23 µM (Fig. 5C).
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51 integrin is strongly up-regulated in choroidal neovascularization. This marked differential in expression between vascular cells participating in choroidal neovascularization and vascular cells in established choroidal vessels, suggests that 51 may play an important role in growth and maintenance of the new vessels, and blocking 51 may provide a way to selectively target choroidal neovascularization. Using a selective antagonist of 51, JSM6427, we found this to be the case. Sustained systemic delivery of JSM6427 using an osmotic minipump suppressed the development of choroidal neovascularization, and when infusion was started after the neovascularization was established, it caused it to regress. TUNEL staining showed selective apoptosis in vascular cells participating in the neovascularization in JSM6427-treated mice. This suggests that signaling through 51 provides a critical survival signal to endothelial cells in choroidal new vessels that, when blocked, triggers apoptosis. Endothelial cells in established vessels must develop alternative survival signal pathways that eliminate the dependence on 51 signaling manifested by endothelial cells in choroidal neovascular lesions. Such a switch has been noted when cerebral vessels mature and change from an angiogenic to a mature phenotype; they stop expressing 51 and 41, and begin to express 11 and 61 (Milner and Campbell, 2002). The exquisite selectivity for JSM6427-induced apoptosis for endothelial cells in choroidal neovascularization compared with mature choroidal vessels predicts a good safety profile.
Cell signaling through which integrins promote cells survival is complex (for review, see Giancotti and Ruoslahti, 1999). Binding of most integrins to a ligand in the ECM activates focal adhesion kinase, which plays a role in most integrin-mediated effects; survival signaling occurs by activation of focal adhesion kinase along with other signaling molecules that act together to increase levels of Bcl-2 or other inhibitors of apoptosis (Lee and Ruoslahti, 2005). Phosphorylation of ERK plays a central role in stimulation of brain capillary endothelial cell survival by fibronectin through 51 (Wang and Milner, 2006). JSM6427 blocks fibronectin-induced phosphorylation of ERK, which probably plays a role in its induction of apoptosis.
Despite the apparent safety of sustained systemic administration of JSM6427, local administration has the theoretical benefit of limiting exposure to the rest of the body and reducing the total amount of JSM6427 that is required for treatment. Two intravitreous injections of JSM6427 over the course of 2 weeks failed to suppress choroidal neovascularization. A possible explanation is that sustained exposure of JSM6427 to endothelial cells in choroidal neovascular lesions is necessary to perturb survival signals, and even brief lapses in blockade of 51 signaling may be sufficient to prevent apoptosis. In the future, it will be worthwhile to test this hypothesis by assessing the effect of sustained local delivery of JSM6427.
Choroidal neovascularization occurs in diseases of the retinal pigmented epithelium/Bruch's membrane complex. The most common of these is age-related macular degeneration, which is the most prevalent cause of severe vision loss in patients over the age of 60 in developed countries (Klein et al., 1993). Studies in animal models have demonstrated that VEGF is an important stimulus and that VEGF antagonists suppress the neovascularization (Kwak et al., 2000; Kryzstolik et al., 2002; Saishin et al., 2003). The role of VEGF in patients with neovascular age-related macular degeneration has now been confirmed in a clinical trial demonstrating that multiple intravitreous injections of pegaptanib, an aptamer that binds VEGF, slows the rate of visual loss (Gragoudas et al., 2004). This is a useful start to pharmacotherapy for choroidal neovascularization, but further improvements are needed. Regression of choroidal neovascularization is not achieved in patients treated with pegaptanib; the size of lesions continued to increase over the course of a year, although at a slower rate than that seen in patients that did not receive pegaptanib. Antagonism of VEGF seems to reduce excessive permeability and slows growth, but does not cause involution of choroidal neovascularization. VEGF provides some survival signals to newly developed endothelial cells (Alon et al., 1995), but survival signals from the extracellular matrix may be sufficient to allow continued growth of choroidal neovascular lesions, although at a slower rate. Combination treatment using JSM6427 with a VEGF antagonist may be extremely useful because perturbation of at least one source of matrix-derived survival signaling along with elimination of the survival signal provided by VEGF may be an effective means to promote involution of choroidal neovascularization.
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Acknowledgements |
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Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
ABBREVIATIONS: HIF-1, hypoxia inducible factor-1; VEGF, vascular endothelial growth factor; ECM, extracellular matrix; FGF2, fibroblast growth factor 2; PBS, phosphate-buffered saline; TBS, Tris-buffered saline; ERK, extracellular signal regulated kinase; ONL, outer nuclear layer; BSA, bovine serum albumin; PI, propidium iodide; GSA, Griffonia simplicifolia lectin; TUNEL, terminal deoxynucleotidyl transferase dUTP nick-end labeling; DAPI, 4,6-diamidino-2-phenylindole; HRP, horseradish peroxidase; BSA, bovine serum albumin; JSM6427, 3-(2-{1-alkyl-5-[(pyridin-2-ylamino)-methyl]-pyrrolidin-3-yloxy}-acetylamino)-2-(alkylamino)-propionic acid.
Address correspondence to: Dr. Peter A. Campochiaro, The Johns Hopkins University School of Medicine, 600 N. Wolfe Street, Baltimore, MD 21287-9277. E-mail: pcampo{at}jhmi.edu
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, p = 6 x 10-8 by Mann-Whitney U test. Scale bar, 100 µm.
