FASEB J. Avanti Polar Lipids
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Published as doi: 10.1096/fj.07-8273com.
 (The FASEB Journal. 2007;21:3272-3278.)
© 2007 FASEB
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF) Free
Right arrow All Versions of this Article:
fj.07-8273comv1
21/12/3272    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lahdenranta, J.
Right arrow Articles by Arap, W.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lahdenranta, J.
Right arrow Articles by Arap, W.

Treatment of hypoxia-induced retinopathy with targeted proapoptotic peptidomimetic in a mouse model of disease

Johanna Lahdenranta*,1, Richard L. Sidman{dagger}, Renata Pasqualini*,2 and Wadih Arap*,2

* The University of Texas, M. D. Anderson Cancer Center, Houston, Texas, USA; and

{dagger} Harvard Medical School and Department of Neurology, Beth Israel Deaconess Medical Center, Harvard Institutes of Medicine, Boston, Massachusetts, USA

2Correspondence: The University of Texas, M.D. Anderson Cancer Center, Houston, TX 77030, USA. E-mail: rpasqual{at}mdanderson.org or warap{at}mdanderson.org


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We have previously identified ligands from combinatorial peptide libraries that target tumor vasculature after in vivo selection. These ligands bind to differentially expressed receptors in angiogenic vasculature such as {alpha}vß3/{alpha}vß5 integrins, aminopeptidase N, and aminopeptidase A. We hypothesized that we can use these ligands to target angiogenic vasculature in retinopathies. Pathological retinal angiogenesis in conditions such as diabetic retinopathy, retinopathy of prematurity, and age-related macular degeneration is a major cause of blindness for which current treatments are inadequate. Here we tested whether known tumor vasculature targeting peptide ligands displayed on bacteriophage particles would home to the proliferating blood vessels of the retina in a standard mouse model of retinopathy of prematurity. We found that activated retinal blood vessels share many of the endothelial and periendothelial cell receptors expressed in tumor vasculature. Furthermore, these vascular receptors—{alpha}v integrins and aminopeptidases—are accessible through the circulation and mediate phage homing and internalization to endothelial and periendothelial cells. Treatment of mice with a peptide containing a {alpha}vß3/{alpha}vß5-integrin targeting domain fused to a proapoptotic domain significantly reduced oxygen-induced retinal angiogenesis by selectively inducing activated endothelial cell apoptosis. Targeted proapoptotic peptides may prove useful in the management of angiogenic retinal diseases.—Lahdenranta, J., Sidman, R. L., Pasqualini, R., Arap, W. Treatment of hypoxia-induced retinopathy with targeted proapoptotic peptidomimetic in a mouse model of disease.


Key Words: aminopeptidases • angiogenesis • peptide ligand • targeted therapy


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
ANGIOGENESIS, THE FORMATION of new blood vessels from preexisting vasculature, is a major component in several retinal vascular diseases causing blindness, such as retinopathy of prematurity, proliferative diabetic retinopathy, and age-related macular degeneration. Diabetes mellitus is the main cause of new cases of adult blindness. Nearly all individuals with type 1 diabetes show some symptoms of diabetic retinopathy, usually ~ 20 years after clinical recognition of diabetes (1) ; in the most advanced form of diabetic retinopathy, called proliferative diabetic retinopathy, new blood vessels grow uncontrollably on the retinal inner surface, causing hemorrhages and even retinal detachment (2) . Surgical and laser photocoagulation treatments are only partially effective and may further damage the retinal tissue. Neither pathogenetic mechanisms nor effective therapy are in hand for retinopathy of prematurity, diabetic retinopathy, or age-related macular degeneration (3 , 4) .

Angiogenesis is also a major component in tumor growth (5) , and endothelial cells in tumor vessels likewise may show different or much higher concentrations of surface markers than do the corresponding normal tissues (6 7 8) . Our hypothesis in the present study is that angiogenesis, whatever its cause, may involve an increase in the same demonstrable markers so that the extensive data available from study of tumor vessels may also apply to angiogenesis in the common retinal vascular diseases. Among such markers are receptors for vascular growth factors such as specific subtypes of VEGF and basic FGF receptors (9 10 11) , {alpha}v-integrins (12) , proteoglycans (13 , 14) , proteinases (15) , and aminopeptidases (16 17 18) . Some of these markers have already been demonstrated in vessels during retinal neovascularization (18 19 20 21 22) .

In vivo phage display technology (23) is a powerful method enabling unbiased selection of peptides capable of homing selectively to different vascular beds in mice and humans (6 , 24 , 25) . This method also allows tissue-specific targeting of angiogenesis-related molecules to tumor blood vessels (14 , 16 , 18 , 26 27 28 29) . Peptide ligands selected through phage display technology can deliver proapoptotic or other toxic peptides selectively, showing marked therapeutic efficacy in tumor-bearing mouse models (30) .

Targeted proapoptotic peptides are short peptides composed of two functional domains: a blood vessel homing motif and a programmed cell death-inducing sequence. The homing domain targets endothelial or periendothelial cells and allows for receptor-mediated internalization (30 , 31) . The proapoptotic domain D(KLAKLAK)2 is relatively nontoxic outside eukaryotic cells but promotes apoptotic death by disrupting mitochondrial membranes on cell entry (30 , 32 , 33) . In earlier work we showed that the D(KLAKLAK)2 proapoptotic peptides targeted with peptides that bind to {alpha}v integrins and to aminopeptidase N (CD13/APN), when administered systemically, home to angiogenic tumor vasculature and are selectively toxic to angiogenic endothelial cells after internalization by these receptors, thus exhibiting antitumor activity (30) .

In the present study we investigate the accessibility of known angiogenic markers to either intravenously administered peptide ligands presented on a filamentous phage particle or to antibodies. Peptide ligands include the {alpha}vß3 binding peptide ACDCRGDCFC (termed RGD-4C; ref. 34 ), aminopeptidase A (APA) binding peptide CPKVCPRECESNC (termed CPRECES; ref. 18 ), and the aminopeptidase N binding peptide CNGRC (termed NGR; ref. 16 ). In the absence of a satisfactory rodent model of diabetic retinopathy, we chose to study angiogenesis in a standard mouse model of retinopathy of prematurity (35) with which we had prior experience (18 , 36) . We find that 1) the new blood vessels in retinas exposed to oxygen share accessible endothelial and periendothelial cell receptors with tumor vasculature, 2) homing peptides can specifically target circulating phage to angiogenic endothelial cells in retinal vasculature, and 3) targeted proapoptotic peptides can reduce oxygen-induced retinal neovascularization.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Antibodies and reagents
The anti-mouse CD13 antibody R3–63 was from Serotec (Oxford, UK), anti-mouse CD61 (ß3 integrin) was from BD PharMingen (San Jose, CA, USA), anti-mouse CD31 was from Chemicon (Temecula, CA, USA), and the isotype control antibodies were from DAKO (Glostrup, Denmark). The anti-APA antibody ASD-37 has been described (37) . FITC- and Cy3-conjugated secondary antibodies were from Jackson ImmunoResearch Laboratories (West Grove, PA, USA). The peptides D(KLAKLAK)2, ACDCRGDCFC-GG-D(KLAKLAK)2, and ACDCRGDCFC (RGD-4C) were synthesized to our specifications at higher than 90% purity by high-performance liquid chromatography (Anaspec, San Jose, CA, USA). The identities of the peptides were verified by mass spectrometry.

Animals
Institutional animal care and utilization committee of the University of Texas M.D. Anderson Cancer Center approved all animal experiments. This study adhered to the Association for Research in Vision and Ophthalmology (ARVO) guidelines. Nude mice and C57BL/6 mice were obtained from Harlan (Indianapolis, IN, USA).

Mouse model of retinal neovascularization
We used a hypoxia-induced retinopathy in which mouse pups with their nursing mothers were exposed to 75% O2 from postnatal day 7 (P7) to P12. On P12, mice were returned to room air (20.8% O2) for 7 days (35) . The exposure of neonatal mice to high O2 levels causes vasoconstriction of the central retinal blood vessels and, subsequently, a decreased retinal perfusion. Returning the mice to lower oxygen levels (room air), which coincides with the rapid development of rod outer segments and an increased retinal oxygen demand, is believed to lead to relative retinal tissue hypoxia and ischemia. The relative hypoxia of the retinal tissue results in the observed retinal neovascularization (4 , 35) . For phage homing and antibody targeting assays, mice were used on P19, at which point the retinal neovessels are protruding into the vitreal space at the inner retinal surface (35 , 36) .

Immunofluorescence microscopy and intravascular imaging studies
Enucleated eyes from (P19) mouse pups that had been exposed to 75% O2 from P7 to P12 were fixed for 1–2 h by immersion in 4% paraformaldehyde in PBS, incubated for 16 h in 30% sucrose in PBS, frozen, and sectioned at 60 µm. Tissue sections were blocked with 5% normal goat serum in 0.3% Triton X-100/PBS and incubated for 16 h at room temperature with 10 µg/ml R3–63 (anti-APN/CD13), 10 µg/ml ASD-37 (anti-APA), or 1 µg/ml anti-CD61 (ß3 integrin) in combination with 10 µg/ml anti-CD31 antibody. Secondary antibodies (1:400 dilutions of FITC- or Cy3-conjugated anti-rat IgG and anti-hamster IgG) were incubated with the sections for 4 h at room temperature. After thorough washing, eye sections were mounted, cover slipped, and analyzed with a fluorescence microscope. For the intravascular imaging study, 10 µg of the anti-CD61 (ß3 integrin) antibody or a negative control antibody were injected in 50 µl of Dulbecco’s modified Eagle’s medium (DMEM) intravenously through the tail vein into P19 C57B/6 mice that had been exposed to 75% oxygen from days P7-P12 or into control P19 C57B/6 mice that had not been treated with oxygen. Antibodies were allowed to circulate for 30 min before mice were perfused through the left ventricle with 5 ml of DMEM. Eyes and the brain were collected, fixed, and sectioned at 60 µm as described previously. Tissue sections were blocked with 5% normal goat serum in 0.3% Triton X-100/PBS and incubated for 16 h at room temperature with 2 µg/ml anti-CD31 antibody. Secondary antibodies (1:400 dilutions of FITC- or Cy3-conjugated anti-rat IgG and anti-hamster IgG) were incubated with the sections for 4 h at room temperature. After thorough washing, eye sections were mounted, cover slipped, and analyzed with a fluorescence microscope.

Phage targeting
For the retina-targeting experiments, 1 x 109 transducing units (TUs) of CPRECES- (displaying the peptide insert CPKVCPRECESNC), NGR- (displaying the peptide insert CNGRC), RGD-4C- (displaying the peptide insert ACDCRGDCFC), or fd-tet-phage in 100 µl of DMEM were injected intravenously through the tail vein into either P18 C57B/6 mice that had been exposed to 75% oxygen from days P7-P12 or into P18 C57B/6 mice not treated with oxygen. Phage were allowed to circulate for 40 min, then mice were perfused with 5 ml of DMEM through the left ventricle of the heart. The eyes were surgically removed and the retinas dissected under a stereomicroscope. Retinas were homogenized in a Dounce homogenizer with ice-cold DMEM containing proteinase inhibitors aprotinin (20 µg/ml), leupeptin (1 µg/ml), and 1 mM phenylmethylsulfonyl fluoride and the retinal homogenates were washed with the above buffer. Retina-bound phage were recovered by infection of K91kan Escherichia coli with the retinal homogenates. Serial dilutions of the infected K91kan were plated on Luria-Bertani agar plates with tetracycline (40 µg/ml) and kanamycin (50 µg/ml). The number of recovered phage TUs was determined by bacterial colony counting. For the tumor-homing experiments, 1 x 109 TU of CPRECES-, NGR-, RGD-4C-, or fd-tet-phage in 100–200 µl of DMEM were injected intravenously through the tail vein into nude mice bearing tumor xenografts derived from MDA-MB-435 human breast cancer cells (27) . Phage were allowed to circulate for 5 min and the animals were perfused through the left ventricle of the heart with 10 ml of DMEM. Tumor and control organs were dissected and equal amounts of tissue were homogenized. Homogenates were washed with ice-cold DMEM containing a protease inhibitor and 0.1% bovine serum albumin. Tissue-bound phage were recovered as described above.

Systemic treatment of retinal neovascularization
Mice with hypoxia-induced retinal neovascularization received vehicle alone, 150 µg of targeted proapoptotic peptide ACDCRGDCFC-GG-D(KLAKLAK)2 (termed RGD-GG-D(KLAKLAK)2), or 150 µg of equimolar mixture of RGD-4C and D(KLAKLAK)2 control peptides on P17 (n=9 mice/group). On P21, mice were sacrificed and their eyes were enucleated, fixed in 4% paraformaldehyde in PBS, serially sectioned after embedding in paraffin, and stained with hematoxylin and eosin (H&E) for histological analysis. Endothelial cell nuclei on the vitreous side of the internal limiting membrane extending into the vitreous were counted to quantify retinal neovascularization (35 , 36) .


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Tumor targeting with APN, APA, and {alpha}vß3/{alpha}vß5 integrin binding ligands
Phage displaying peptide ligands ACDCRGDCFC (termed RGD-4C; refs. 26 , 27 ), CNGRC (27) , or CPKVCPRECESNC (termed CPRECES; ref. 18 ) were first tested for tumor homing in vivo. To confirm findings from previous studies (18 , 26 , 27) , we performed a side-by-side analysis of RGD-4C-, NGR-, CPRECES-, or a negative control phage (insertless fd-tet-phage) for their ability to home to tumor xenografts. RGD-4C-, NGR-, CPRECES-, or a negative control phage were administered intravenously into mice bearing MDA-MB-435-derived human tumor xenografts. Tumor- and control organ (brain) -bound phage were recovered and titered from the tissue homogenates after a 5 min circulation, followed by perfusion. RGD-4C-, NGR-, and CPRECES-phage were enriched in tumors with a minimum of 10-fold enrichment compared with the control organ; in contrast, negative control phage did not enrich in tumors (Fig. 1 ). These results show that APA-, APN-, and {alpha}vß3/{alpha}vß5 integrin binding phage target tumor blood vessels in vivo.


Figure 1
View larger version (16K):
[in this window]
[in a new window]

  		Figure 1. Side-by-side comparison of the tumor homing peptides in mice with MDA-MB-435 xenografts. The ability of APA binding, APN binding, and {alpha}vß3 integrin binding phage, compared with control fd-tet phage, to home to tumor vasculature was evaluated after intravenous phage administration into mice bearing human MDA-MB-435 breast carcinoma-derived tumor xenografts. Phage were recovered from tumors after perfusion. Shown are mean ± SE of TU from triplicate platings.

Receptors for the tumor-targeting peptide ligands are expressed in the proliferating retinal blood vessels
Next we wanted to determine whether the receptors for the identified tumor-homing peptide ligands are expressed during retinal neovascularization. We performed immunofluorescence staining for APA, APN, and ß3 integrin (which partners with {alpha}v integrin) on eye and brain sections from P19 mice with hypoxia-induced retinal neovascularization after an exposure to 75% O2 from P7-P12. We could detect expression of APA, APN, and ß3 integrin in the blood vessels of angiogenic retinas. We could not detect expression of ß3 integrin in quiescent brain blood vessels; in contrast, however, we did detect APA and APN expression in brain blood vessels, presumably in pericytes. Isotype control antibody served as a negative staining control (Fig. 2 ). These results show that receptors for the tumor homing peptide ligands, APA, APN, and {alpha}vß3 integrin, are expressed in blood vessels of retinas undergoing neovascularization.


Figure 2
View larger version (12K):
[in this window]
[in a new window]

  		Figure 2. {alpha}vß3 integrin, APN, and APA are expressed in the retinal vasculature during oxygen-induced retinal neovascularization. 60 µm eye and brain sections from P19 mouse pups that had been exposed to 75% O2 from P7 to P12 were stained for {alpha}vß3 integrin (anti-CD61), APN (R3–63), and APA (ASD-37). Isotype control antibody was used as a negative control. Target expression was detected by Cy3-conjugated secondary antibody. Scale bar: 100 µm.

Tumor-targeting phage home to angiogenic retina
We next evaluated the ability of tumor-targeting phage to home to angiogenic retina. Tumor-targeting phage displaying RGD-4C, NGR, or CPRECES peptide and an insertless control phage (fd-tet) were administered intravenously to mice on P18 with hypoxia-induced retinal neovascularization after exposure to 75% O2 from P7-P12 or to control mice not treated with O2. Retina-bound phage were recovered and titered from the tissue homogenates after a 40 min circulation, which was followed by perfusion. RGD-4C-, NGR-, and CPRECES-phage were significantly enriched in angiogenic retinas with 4.6 ± 0.9-, 3.5 ± 0.5-, or 5.7 ± 1.3-fold enrichment, respectively (t test, P<0.03 in each case compared with non-oxygen-treated retinas); in contrast, negative control phage did not enrich in angiogenic retinas (1.2±0.1-fold enrichment, t test, P=0.17; Fig. 3 ). These results show that APA-, APN-, and {alpha}vß3/{alpha}vß5 integrin binding phage target angiogenic retinal blood vessels in vivo.


Figure 3
View larger version (12K):
[in this window]
[in a new window]

  		Figure 3. {alpha}vß3 integrin, APN, and APA are accessible to their peptide ligands in the retinal vasculature during oxygen-induced retinal neovascularization. The ability of APA binding, APN binding, and {alpha}vß3 integrin binding phage to home to retinal neovasculature was evaluated after intravenous phage administration into P18 mice that had been exposed to 75% oxygen between days P7 and P12 or into P18 mice without oxygen treatment. Phage were recovered from retinas after perfusion. Shown are mean ± SE of TU from triplicate platings. Fd-tet served as a negative control phage.

Receptors for the tumor targeting peptide ligands in proliferating retinal blood vessels are accessible to antibodies from the circulation
Next we analyzed the accessibility of the endothelial cell receptor for the RGD-4C peptide ({alpha}vß3 integrin) after intravenous injection of anti-ß3 integrin antibody. P19 mice without oxygen treatment and with hypoxia-induced retinal neovascularization after exposure to 75% O2 from P7-P12 received 10 µg of anti-CD61 (ß3 integrin) antibody or a negative control antibody intravenously. Antibodies were allowed to circulate for 30 min before mice were perfused. The eyes and brain were collected, sectioned, and stained for the CD31 endothelial cell marker (detected with FITC-conjugated secondary antibody). Anti-CD61 and negative control antibodies were detected in the tissues by Cy3-conjugated secondary antibody. Our results (Fig. 4 ) demonstrate that ß3 integrin is accessible to circulating antibodies in retinas with proliferating blood vessels, in contrast to normal retinas without blood vessel proliferation. Control antibody showed no vascular staining. No anti-CD61 antibody staining was detected in the brain vasculature.


Figure 4
View larger version (23K):
[in this window]
[in a new window]

  		Figure 4. Accessibility of ß3-integrin in retinal blood vessels. Intravenous injection of anti-CD61 (ß3-integrin) antibody or a control antibody into P19 mice with or without hypoxia-induced retinal neovascularization illustrates that the ß3-integrin-positive cells in angiogenic vessels of the retina (arrows) are accessible to the antibodies after 30 min circulation. The control IgG did not stain tissues in any of the experimental settings. ß3-Integrin immunoreactivity is seen in red and CD31 immunoreactivity indicating the endothelium in all blood vessels is seen in green. Scale bar: 100 µm.

Targeted proapoptotic peptides reduce retinal neovascularization
Finally, we designed studies to assess the effects of {alpha}vß3/{alpha}vß5 integrin-targeting proapoptotic peptides (30) in the mouse model of hypoxia-induced retinal neovascularization. Mice received 150 µg of targeted proapoptotic [RGD-4C-GG-D(KLAKLAK)2], control [equimolar mixture of RGD-4C and D(KLAKLAK)2] peptides, or vehicle alone on P17. On P21, eyes were enucleated, fixed, embedded in paraffin, and stained with H&E. Retinal neovascularization was analyzed by counting endothelial cell nuclei protruding into the vitreous space (35 , 36) . Induction of retinal neovascularization was seen in vehicle- or control-treated mice on P21, but the number of retinal neovessels was reduced by up to 70% on systemic treatment of mice with the targeted proapoptotic peptide (48±1) endothelial cell nuclei/section (t test, P<0.01; Fig. 5 ). No endothelial cell nuclei protruding into the vitreous space were found in mice exposed only to room air (data not shown).


Figure 5
View larger version (32K):
[in this window]
[in a new window]

  		Figure 5. Treatment of hypoxia-induced retinopathy with {alpha}vß3 integrin-targeting proapoptotic peptide. P17 mice with hypoxia-induced retinal neovascularization received targeted proapoptotic [RGD-4C-GG-D(KLAKLAK)2] (right column) or control [RGD-4C and D(KLAKLAK)2 peptides separately] (middle column) on P17. Retinal neovascularization was analyzed on P21 in paraffin-embedded H&E-stained eye sections. A) Blood vessels are seen protruding from the retina into the vitreous space in eyes from mice treated with vehicle or control peptides (arrows point to endothelial cell nuclei), while few neovascular nuclei are present in eyes from mice treated with targeted proapoptotic peptide. Scale bar: 100 µm (upper panels), 50 µm (lower panels). B) The number of neovascular nuclei protruding into the vitreous space was quantified by evaluating several (≥5) serial sections of multiple (≥8) eyes. Shown are mean ± SE.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Several novel approaches have been developed to prevent and treat proliferative diabetic retinopathy. Some experimental approaches aim to block the metabolic pathways inducing hyperglycemic damage in the retina, including increased formation of advanced glycation end product, hexosamine pathway flux, polyol pathway flux, and diacylglycerol activation of a protein kinase C pathway (2 , 19 , 38) . Other approaches aim to prevent vascular leakage and neovascularization via inhibition of growth factors and integrins as well as their intracellular signaling cascades (reviewed in ref. 2 ). In the present study, we showed that endothelial cell surface markers previously demonstrated in tumor vasculature may also serve as suitable receptors in proliferating retinal blood vessels for selective targeting of phage carrying both a peptide-ligand for the receptor and a therapeutic moiety.

We previously identified APN and APA as cell surface receptors in endothelial and periendothelial cells of angiogenic vasculature that are accessible to circulating ligands such as peptides carried on phage particles. Since APN and APA enzymatic activities regulate angiogenesis, blocking their enzymatic activities genetically or biochemically significantly reduces the formation of new blood vessels in tumors and in the mouse model of retinopathy of prematurity (16 , 18) . The regulatory role for {alpha}vß3 and {alpha}vß5 integrins in angiogenesis has been established for tumor angiogenesis and angiogenesis in proliferative retinopathies (12 , 19 , 39) . We have further identified peptide ligands for APA (CPRECES peptide), APN (NGR peptide), and {alpha}vß3/{alpha}vß5 (RGD-4C peptide) integrins from combinatorial peptide libraries and have shown that these peptide ligands can target tumor vasculature (16 , 18 , 27 , 34) . We have both indirect and direct experimental evidence that the identified peptide ligands undergo receptor-mediated internalization into the target cell (18 , 30) . After identification of both RGD-4C and NGR-peptides as ligands for targeting tumor vasculature, they have been widely used for targeting an array of therapies to tumor vasculature, including cytotoxic drugs (27) , proapoptotic peptides (30) , cytokines (40) , liposomes (41) , and gene delivery vehicles (42 , 43) .

Our objective was to determine whether we can use the tumor-homing peptide ligands RGD-4C, NGR, and CPRECES for targeting the blood vessels of angiogenic retina and for intravenous delivery of therapeutic agents in proliferative retinopathies. After a side-by-side validation of the targeting properties of the CPRECES-, NGR-, and RGD-4C-peptide displaying phage to proliferating blood vessels in human breast carcinoma tumor xenografts, we analyzed the expression of APA, APN, and {alpha}v-integrins in a mouse model of retinal neovascularization using in vivo phage targeting and immunostaining. We showed by immunofluorescence microscopy the expression of APA, APN, and ß3-integrins in the blood vessels of the retina after hypoxia-induced retinal neovascularization, and we demonstrated the accessibility of these molecules to circulating ligands by phage homing experiments where phage displaying of APA, APN, and {alpha}vß3/{alpha}vß5-integrin ligands specifically targeted retinal vessels, but only in those mice that had been induced to develop neovascularization. For the therapy experiments, we chose the {alpha}vß3/{alpha}vß5 integrin binding peptide ligand RGD-4C fused to the proapoptotic moiety D(KLAKLAK)2 as a prototype-targeted peptide. We first assessed the tissue-confined accessibility of ß3-integrins to intravenously administered antibodies and showed that {alpha}-ß3-integrin antibody specifically stained the retinal vessels only after the induction of retinal neovascularization and that the staining was specific to the retina, since we did not detect staining in the control organ (brain). Intravenous treatment with the prototype-targeted peptide RGD-4C-GG-D(KLAKLAK)2 markedly reduced the number of retinal neovessels compared with controls consisting of injection of vehicle alone or of an equimolar mixture of RGD-4C and D(KLAKLAK)2 peptides. Of note, cyclic {alpha}v peptide antagonist (RGDfV; ref. 44 ) has been shown to inhibit retinal neovascularization partially when administered twice a day (20–100 µg/injection) for 5 days (19) . We did not detect a decrease in retinal neovascularization with an injection of our control peptide mixture (90 µg of RGD-4C peptide, which is also an {alpha}v-integrin antagonist), perhaps because our experimental protocol involved only a single injection. In contrast, a single injection of the RGD-4C-peptide fused to the proapoptotic moiety was sufficient to induce endothelial cell apoptosis and a subsequent reduction in retinal neovascularization.

In summary, our data indicate that APA, APN, and {alpha}vß3/{alpha}vß5 integrins are not only selectively present in oxygen-induced new retinal blood vessel endothelial cells, but are also accessible targets for therapy in at least one experimental model of a proliferative retinopathy. Since the proliferating blood vessels in the retina share common angiogenic markers with tumor vasculature, and tumor-homing peptides can specifically target circulating phage to activated endothelial and periendothelial cells in retinal vasculature, an experimental model of neovascularization such as the mouse model of retinopathy of prematurity allows more thorough and rapid studies of ligands originally isolated as tumor targeting moieties for their ability to also target angiogenic vasculature in general. In addition, we demonstrated the suitability of activated endothelial cell surface markers as receptors for targeting of therapeutic compounds to angiogenic retinal vasculature in experimental mice and propose that they may have a selective therapeutic effect against proliferative retinopathies such as human diabetic retinopathy and retinopathy of prematurity.


   ACKNOWLEDGMENTS
 
This work was partially funded by grants from the Juvenile Diabetes Research Foundation International, National Institutes of Health, and the Department of Defense. J.L. is a Robert Black Fellow of the Damon Runyon Cancer Research Foundation.


   FOOTNOTES
 
1 Current address: Department of Radiation Oncology, Massachusetts General Hospital, Boston, MA 02114, USA.

Received for publication February 27, 2007. Accepted for publication April 19, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Klein, R., Klein, B. E., Moss, S. E., Davis, M. D., DeMets, D. L. (1984) The Wisconsin epidemiologic study of diabetic retinopathy. III. Prevalence and risk of diabetic retinopathy when age at diagnosis is 30 or more years. Arch. Ophthalmol. 102,527-532[Abstract/Free Full Text]
  2. Frank, R. N. (2004) Diabetic retinopathy. N. Engl. J. Med. 350,48-58[Free Full Text]
  3. Diabetes Control and Complications Trial Research Group (1993) The effect of intensive treatment of diabetes on the development and progression of long-term complications in insulin-dependent diabetes mellitus. N. Engl. J. Med. 329,977-986[Abstract/Free Full Text]
  4. Arden, G. B., Sidman, R. L., Arap, W., Schlingemann, R. O. (2005) Spare the rod and spoil the eye. Br. J. Ophthalmol. 89,764-769[Abstract/Free Full Text]
  5. Folkman, J. (2006) Angiogenesis. Annu. Rev. Med. 57,1-18[CrossRef][Medline]
  6. Trepel, M., Arap, W., Pasqualini, R. (2002) In vivo phage display and vascular heterogeneity: implications for targeted medicine. Curr. Opin. Chem. Biol. 6,399-404[CrossRef][Medline]
  7. Luttun, A., Carmeliet, P. (2004) Angiogenesis and lymphangiogenesis: highlights of the past year. Curr. Opin. Hematol. 11,262-271[CrossRef][Medline]
  8. St. Croix, B., Rago, C., Velculescu, V., Traverso, G., Romans, K. E., Montgomery, E., Lal, A., Riggins, G. J., Lengauer, C., Vogelstein, B., Kinzler, K. W. (2000) Genes expressed in human tumor endothelium. Science 289,1197-1202[Abstract/Free Full Text]
  9. Plate, K. H., Breier, G., Millauer, B., Ullrich, A., Risau, W. (1993) Up-regulation of vascular endothelial growth factor and its cognate receptors in a rat glioma model of tumor angiogenesis. Cancer Res. 53,5822-5827[Abstract/Free Full Text]
  10. Brekken, R. A., Huang, X., King, S. W., Thorpe, P. E. (1998) Vascular endothelial growth factor as a marker of tumor endothelium. Cancer Res. 58,1952-1959[Abstract/Free Full Text]
  11. Valtola, R., Salven, P., Heikkila, P., Taipale, J., Joensuu, H., Rehn, M., Pihlajaniemi, T., Weich, H., deWaal, R., Alitalo, K. (1999) VEGFR-3 and its ligand VEGF-C are associated with angiogenesis in breast cancer. Am. J. Pathol. 154,1381-1390[Abstract/Free Full Text]
  12. Brooks, P. C., Clark, R. A., Cheresh, D. A. (1994) Requirement of vascular integrin alpha v beta 3 for angiogenesis. Science 264,569-571[Abstract/Free Full Text]
  13. Schrappe, M., Klier, F. G., Spiro, R. C., Waltz, T. A., Reisfeld, R. A., Gladson, C. L. (1991) Correlation of chondroitin sulfate proteoglycan expression on proliferating brain capillary endothelial cells with the malignant phenotype of astroglial cells. Cancer Res. 51,4986-4993[Abstract/Free Full Text]
  14. Burg, M. A., Pasqualini, R., Arap, W., Ruoslahti, E., Stallcup, W. B. (1999) NG2 proteoglycan-binding peptides target tumor neovasculature. Cancer Res. 59,2869-2874[Abstract/Free Full Text]
  15. Koivunen, E., Arap, W., Valtanen, H., Rainisalo, A., Medina, O. P., Heikkila, P., Kantor, C., Gahmberg, C. G., Salo, T., Konttinen, Y. T., et al (1999) Tumor targeting with a selective gelatinase inhibitor. Nat. Biotechnol. 17,768-774[CrossRef][Medline]
  16. Pasqualini, R., Koivunen, E., Kain, R., Lahdenranta, J., Sakamoto, M., Stryhn, A., Ashmun, R. A., Shapiro, L. H., Arap, W., Ruoslahti, E. (2000) Aminopeptidase N is a receptor for tumor-homing peptides and a target for inhibiting angiogenesis. Cancer Res. 60,722-727[Abstract/Free Full Text]
  17. Essler, M., Ruoslahti, E. (2002) Molecular specialization of breast vasculature: a breast-homing phage-displayed peptide binds to aminopeptidase P in breast vasculature. Proc. Natl. Acad. Sci. U. S. A. 99,2252-2257[Abstract/Free Full Text]
  18. Marchiò, S., Lahdenranta, J., Schlingemann, R. O., Valdembri, D., Wesseling, P., Arap, M. A., Hajitou, A., Ozawa, M. G., Trepel, M., Giordano, R. J., et al (2004) Aminopeptidase A is a functional target in angiogenic blood vessels. Cancer Cell 5,151-162[CrossRef][Medline]
  19. Hammes, H. P., Du, X., Edelstein, D., Taguchi, T., Matsumura, T., Ju, Q., Lin, J., Bierhaus, A., Nawroth, P., Hannak, D., et al (2003) Benfotiamine blocks three major pathways of hyperglycemic damage and prevents experimental diabetic retinopathy. Nat. Med. 9,294-299[CrossRef][Medline]
  20. Aiello, L. P., Pierce, E. A., Foley, E. D., Takagi, H., Chen, H., Riddle, L., Ferrara, N., King, G. L., Smith, L. E. (1995) Suppression of retinal neovascularization in vivo by inhibition of vascular endothelial growth factor (VEGF) using soluble VEGF-receptor chimeric proteins. Proc. Natl. Acad. Sci. U. S. A. 92,10457-10461[Abstract/Free Full Text]
  21. Luttun, A., Tjwa, M., Moons, L., Wu, Y., Angelillo-Scherrer, A., Liao, F., Nagy, J. A., Hooper, A., Priller, J., De Klerck, B., et al (2002) Revascularization of ischemic tissues by PlGF treatment, and inhibition of tumor angiogenesis, arthritis and atherosclerosis by anti-Flt1. Nat. Med. 8,831-840[CrossRef][Medline]
  22. Ozerdem, U., Stallcup, W. B. (2004) Pathological angiogenesis is reduced by targeting pericytes via the NG2 proteoglycan. Angiogenesis 7,269-276[CrossRef][Medline]
  23. Pasqualini, R., Ruoslahti, E. (1996) Organ targeting in vivo using phage display peptide libraries. Nature 380,364-366[CrossRef][Medline]
  24. Arap, W., Kolonin, M. G., Trepel, M., Lahdenranta, J., Cardo-Vila, M., Giordano, R. J., Mintz, P. J., Ardelt, P. U., Yao, V. J., Vidal, C. I., et al (2002) Steps toward mapping the human vasculature by phage display. Nat. Med. 8,121-127[CrossRef][Medline]
  25. George, A. J., Lee, L., Pitzalis, C. (2003) Isolating ligands specific for human vasculature using in vivo phage selection. Trends Biotechnol. 21,199-203[CrossRef][Medline]
  26. Pasqualini, R., Koivunen, E., Ruoslahti, E. (1997) Alpha v integrins as receptors for tumor targeting by circulating ligands. Nat. Biotechnol. 15,542-546[CrossRef][Medline]
  27. Arap, W., Pasqualini, R., Ruoslahti, E. (1998) Cancer treatment by targeted drug delivery to tumor vasculature in a mouse model. Science 279,377-380[Abstract/Free Full Text]
  28. Hoffman, J. A., Giraudo, E., Singh, M., Zhang, L., Inoue, M., Porkka, K., Hanahan, D., Ruoslahti, E. (2003) Progressive vascular changes in a transgenic mouse model of squamous cell carcinoma. Cancer Cell 4,383-391[CrossRef][Medline]
  29. Joyce, J. A., Laakkonen, P., Bernasconi, M., Bergers, G., Ruoslahti, E., Hanahan, D. (2003) Stage-specific vascular markers revealed by phage display in a mouse model of pancreatic islet tumorigenesis. Cancer Cell 4,393-403[CrossRef][Medline]
  30. Ellerby, H. M., Arap, W., Ellerby, L. M., Kain, R., Andrusiak, R., Rio, G. D., Krajewski, S., Lombardo, C. R., Rao, R., Ruoslahti, E., et al (1999) Anti-cancer activity of targeted pro-apoptotic peptides. Nat. Med. 5,1032-1038[CrossRef][Medline]
  31. Kolonin, M. G., Saha, P. K., Chan, L., Pasqualini, R., Arap, W. (2004) Reversal of obesity by targeted ablation of adipose tissue. Nat. Med. 10,625-632[CrossRef][Medline]
  32. Javadpour, M. M., Juban, M. M., Lo, W. C., Bishop, S. M., Alberty, J. B., Cowell, S. M., Becker, C. L., McLaughlin, M. L. (1996) De novo antimicrobial peptides with low mammalian cell toxicity. J. Med. Chem. 39,3107-3113[CrossRef][Medline]
  33. Ellerby, H. M., Martin, S. J., Ellerby, L. M., Naiem, S. S., Rabizadeh, S., Salvesen, G. S., Casiano, C. A., Cashman, N. R., Green, D. R., Bredesen, D. E. (1997) Establishment of a cell-free system of neuronal apoptosis: comparison of premitochondrial, mitochondrial, and postmitochondrial phases. J. Neurosci. 17,6165-6178[Abstract/Free Full Text]
  34. Koivunen, E., Wang, B., Ruoslahti, E. (1995) Phage libraries displaying cyclic peptides with different ring sizes: ligand specificities of the RGD-directed integrins. Bio/Technology (NY) 13,265-270[CrossRef]
  35. Smith, L. E., Wesolowski, E., McLellan, A., Kostyk, S. K., D’Amato, R., Sullivan, R., D’Amore, P. A. (1994) Oxygen-induced retinopathy in the mouse. Invest. Ophthalmol. Vis. Sci. 35,101-111[Abstract/Free Full Text]
  36. Lahdenranta, J., Pasqualini, R., Schlingemann, R. O., Hagedorn, M., Stallcup, W. B., Bucana, C. D., Sidman, R. L., Arap, W. (2001) An anti-angiogenic state in mice and humans with retinal photoreceptor cell degeneration. Proc. Natl. Acad. Sci. U. S. A. 98,10368-10373[Abstract/Free Full Text]
  37. Assmann, K. J., van Son, J. P., Dijkman, H. B., Koene, R. A. (1992) A nephritogenic rat monoclonal antibody to mouse aminopeptidase A. Induction of massive albuminuria after a single intravenous injection. J. Exp. Med. 175,623-635[Abstract/Free Full Text]
  38. Brownlee, M. (2001) Biochemistry and molecular cell biology of diabetic complications. Nature 414,813-820[CrossRef][Medline]
  39. Cheresh, D. A., Smith, J. W., Cooper, H. M., Quaranta, V. (1989) A novel vitronectin receptor integrin (alpha v beta x) is responsible for distinct adhesive properties of carcinoma cells. Cell 57,59-69[CrossRef][Medline]
  40. Curnis, F., Sacchi, A., Borgna, L., Magni, F., Gasparri, A., Corti, A. (2000) Enhancement of tumor necrosis factor alpha antitumor immunotherapeutic properties by targeted delivery to aminopeptidase N (CD13). Nat. Biotechnol. 18,1185-1190[CrossRef][Medline]
  41. Pastorino, F., Brignole, C., Marimpietri, D., Cilli, M., Gambini, C., Ribatti, D., Longhi, R., Allen, T. M., Corti, A., Ponzoni, M. (2003) Vascular damage and anti-angiogenic effects of tumor vessel-targeted liposomal chemotherapy. Cancer Res. 63,7400-7409[Abstract/Free Full Text]
  42. Hood, J. D., Cheresh, D. A. (2002) Role of integrins in cell invasion and migration. Nat. Rev. Cancer 2,91-100[CrossRef][Medline]
  43. Moffatt, S., Wiehle, S., Cristiano, R. J. (2005) Tumor-specific gene delivery mediated by a novel peptide-polyethylenimine-DNA polyplex targeting aminopeptidase N/CD13. Hum. Gene Ther. 16,57-67[CrossRef][Medline]
  44. Aumailley, M., Gurrath, M., Muller, G., Calvete, J., Timpl, R., Kessler, H. (1991) Arg-Gly-Asp constrained within cyclic pentapeptides. Strong and selective inhibitors of cell adhesion to vitronectin and laminin fragment P1. FEBS Lett. 291,50-54[CrossRef][Medline]



This article has been cited by other articles:


Home page
 Cancer Res.Home page
A. Y. Khakoo, R. L. Sidman, R. Pasqualini, and W. Arap
Does the Renin-Angiotensin System Participate in Regulation of Human Vasculogenesis and Angiogenesis?
Cancer Res., November 15, 2008; 68(22): 9112 - 9115.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
A. Corti, F. Curnis, W. Arap, and R. Pasqualini
The neovasculature homing motif NGR: more than meets the eye
Blood, October 1, 2008; 112(7): 2628 - 2635.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF) Free
Right arrow All Versions of this Article:
fj.07-8273comv1
21/12/3272    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lahdenranta, J.
Right arrow Articles by Arap, W.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lahdenranta, J.
Right arrow Articles by Arap, W.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS