0
We're unable to sign you in at this time. Please try again in a few minutes.
Retry
We were able to sign you in, but your subscription(s) could not be found. Please try again in a few minutes.
Retry
There may be a problem with your account. Please contact the AMA Service Center to resolve this issue.
Contact the AMA Service Center:
Telephone: 1 (800) 262-2350 or 1 (312) 670-7827  *   Email: subscriptions@jamanetwork.com
Error Message ......
Access to paid content on this site is currently suspended due to excessive activity being detected from your IP address 54.197.87.25. Please contact the publisher to request reinstatement.
Laboratory Sciences |

Inhibition of Choroidal Neovascularization by a Peptide Inhibitor ofthe Urokinase Plasminogen Activator and Receptor System in a Mouse Model FREE

Arup Das, MD, PhD; Nathan Boyd, BS; Terence R. Jones, PhD; Nicholas Talarico, BS; Paul G. McGuire, PhD
[+] Author Affiliations

Author Affiliations: Departments of Surgery(Drs Das and McGuire) and Cell Biology and Physiology (Drs Das and McGuireand Messrs Boyd and Talarico), University of New Mexico School of Medicine,and New Mexico VA Health Care System (Dr Das), Albuquerque; and ÅngstromPharmaceuticals, San Diego, Calif (Dr Jones).


Arch Ophthalmol. 2004;122(12):1844-1849. doi:10.1001/archopht.122.12.1844.
Text Size: A A A
Published online

Objectives  To determine the role played by the urokinase plasminogen activator(uPA) and urokinase plasminogen activator receptor (uPAR) system in choroidalneovascularization (CNV) and whether inhibition of this system can suppressthe extent of CNV in an animal model.

Methods  Choroidal neovascularization was induced in mice by laser photocoagulationusing the slitlamp delivery system. Reverse transcriptase–polymerasechain reaction and immunocytochemical analysis were performed on the retinachoroids of these animals to examine the expression of uPAR. For 2 weeks followinglaser treatment, animals were injected intraperitoneally with a novel peptideinhibitor of the uPA-uPAR system (100 mg/kg twice a day every day, every otherday, and once a week). Control laser-treated animals receive an intraperitonealinjection of phosphate-buffered saline every day. Following treatment, animalswere perfused with fluorescein-labeled dextran, eyes were removed, and theareas of new vessels were examined in the retina-choroid whole mounts by fluorescencemicroscopy and quantitated using image analysis software.

Results  In this study, uPAR was found to be up-regulated in the choroidal tissuesof mice with laser-induced CNV. The uPAR was localized to the endothelialcells of the fibrovascular tissue within the CNV complex. Systemic administrationof the peptide inhibitor of the uPA-uPAR system resulted in a significantreduction of CNV (up to 94%). The response was found to be frequency-of-dosedependent. No toxic effects or tissue destruction was noted following thepeptide treatment.

Conclusions  Our results strongly suggest that up-regulation of the uPA-uPAR systemis an important step during CNV, and significant inhibition of CNV was seenwhen cell surface–associated uPA-uPAR activity was prevented with thepeptide inhibitor.

Clinical Relevance  Inhibition of the protease system (uPA-uPAR) may prove to be a potentialnovel antiangiogenic therapy for CNV as seen in age-related macular degeneration.

Figures in this Article

Choroidal neovascularization (CNV) due to age-related macular degeneration(ARMD) is one of the most frequent causes of vision loss in people older than65 years. The exudative form of ARMD associated with all the elements of atrophicor nonexudative forms of ARMD, as well as neovascularization, results in sudden,severe central vision loss. Although the series of events that lead to thedevelopment of CNV is not totally clear, new vessels derived from the choroidpenetrate through the Bruch membrane, become leaky, and cause serious retinaldetachment and hemorrhage in the macula.

The current treatment options for CNV are conventional laser treatmentand photodynamic treatment, both of which have limitations. The former resultsin irreversible tissue damage with the resultant central scotoma and visionloss, whereas the latter causes short-term closure of new vessels as theyopen up again in a few months, and the treatment has to be repeated frequently.This fact has led to the need for a better understanding of the mechanismsof CNV formation and a search for new therapeutic methods to treat it.

A model of CNV has been developed for the mouse that uses laser photocoagulationas the primary stimulus.1 Disruption of theBruch membrane following laser treatment results in the formation of new vesselsbeneath the retina derived from the choroid. This has proved to be a usefulmodel for the identification of the mechanisms involved in subretinal neovascularizationand the development and testing of new therapeutic interventions for thismedical problem.

The angiogenic process consists of several phases, including up-regulationof angiogenic factors followed by increased expression of integrins and extracellularproteinases. The proteinases facilitate endothelial cell migration and newtube formation following the breakdown of the capillary basement membrane.One proteinase that has been shown to play an important role in angiogenesisis the serine proteinase urokinase plasminogen activator (uPA).2,3 TheuPA localizes to the surface of endothelial cells by binding to the urokinaseplasminogen activator receptor (uPAR). This interaction of uPA and uPAR facilitatescell migration through localized proteolytic and nonproteolytic regulationof cell-substrate adhesion.4,5 TheuPA-plasmin system may also interface with the matrix metalloproteinase systemto perform larger-scale proteolytic events that may change the regulatoryinformation contained within the extracellular matrix.

Studies have previously reported the increased expression of uPA alongwith the matrix metalloproteinases 2 and 9 in mice with retinal neovascularization6 and in human neovascular membranes obtained from patientswith proliferative diabetic retinopathy.7 Furthermore,experiments from this laboratory have demonstrated the up-regulation of uPARin a murine model of retinal neovascularization and the inhibition of neovascularizationin this model using a novel peptide inhibitor of the uPA-uPAR system, Å6.8 The Å6 peptide is derived from the non–receptor-bindingregion of urokinase and has been shown to inhibit the interaction of uPA withuPAR and inhibit tumor cell invasion in vitro. This peptide has antiangiogenicand antitumor activity in animal models of breast cancer and glioblastoma.9,10 Recently, transgenic mice that lackeduPA, tissue-type plasminogen activator, and plasminogen genes have been shownto be resistant to the development of experimental CNV.11 Inthe current study, we present data on the expression of uPAR in a mouse modelof CNV and the potential of the Å6 peptide to serve as an effectiveand novel antiangiogenic therapy for CNV.

ANIMAL MODEL OF CNV

All experiments were conducted in accordance with the Association forResearch in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmicand Vision Research. Specific pathogen-free C57BL/6J mice were bred at theUniversity of New Mexico Animal Research Facility. Six- to 8-week-old micewere anesthetized with ketamine hydrochloride (100 mg/kg), and pupils weredilated with 1% tropicamide eyedrops. Argon green laser photocoagulation wasperformed with the slitlamp delivery system using a coverslip as a contactlens as previously described.1 Three laserburns were placed in each eye at the 9-, 12-, and 3-o’clock positionsapproximately 2 disc diameters from the optic nerve. The power settings wereas follows: 100-μm spot size, 0.1-second duration, and 150-mW intensity.Production of a vaporization bubble during laser treatment was consideredas the end point and indicated the rupture of the Bruch membrane. Only thoseeyes with burns that resulted in formation of the vaporization bubble at all3 locations were included in this study. After 14 days, animals were killedand eyes collected for analysis.

HISTOLOGIC ANALYSIS

Fourteen days after laser treatment, mice (n = 5) were killed,and the area of new vessels at each laser site was measured in retinal pigmentepithelium (RPE)–choroidal flat mounts.12 Micewere anesthetized and perfused with 1 mL of phosphate-buffered saline (PBS)containing 50 mg/mL of fluorescein-labeled dextran (average molecular weightof 2 million; Sigma-Aldrich Co, St Louis, Mo). Eyes were removed and fixedin 10% formaldehyde for 2 hours. After removal of the cornea and lens, radialcuts were made from the edge to the equator, and whole mounts of the RPE-choroidcomplex were prepared in aqueous mounting medium (Aquamount; BDH ChemicalsLtd, Poole, England) with the sclera facing down. Whole mounts were examinedby fluorescence microscopy (Axiovert 35; Carl Zeiss, Inc, Thornwood, NY),and images of the burn areas were collected and analyzed. MetaMorph imageanalysis software (Universal Imaging Corp, Downingtown, Pa) was used to delineateand measure the area of new vessel-associated fluorescence within the burnarea. The areas occupied by new vessels were analyzed by repeated-measureanalysis of variance using the SAS statistical analysis software (Proc Mixedprocedure; SAS Institute, Cary, NC). Post hoc comparisons between treatmentswere analyzed using least squares means.

Å6 TREATMENT

Some mice were treated during the 14-day period with the inhibitor drugÅ6 peptide (Ångstrom Pharmaceuticals, San Diego, Calif). Intraperitonealinjections were given according to the following regimen: (1) 100 mg/kg twicea day once per week (n = 5 animals, 20 laser spots analyzed), (2)100 mg/kg twice a day every third day (n = 5 animals, 17 laser spotsanalyzed), and (3) 100 mg/kg twice a day every day (n = 5 animals,17 laser spots analyzed). Animals that received PBS twice a day every dayserved as controls in this experiment (n = 5 animals, 19 laser spotsanalyzed). After 14 days of drug treatment, animals were killed, and the extentof CNV was determined and quantitated in RPE-choroid whole mounts as describedherein.

IMMUNOHISTOCHEMICAL ANALYSIS

For the localization of uPAR, unfixed sections of eyes that containedlaser burns were blocked with 10% normal goat serum for 30 minutes followedby incubation with the primary rabbit anti-mouse uPAR antibody (R&D Systems,Minneapolis, Minn) for 1 hour at room temperature. Sections were washed andincubated with an alkaline phosphatase–labeled goat anti-rabbit secondaryantibody for 1 hour at room temperature. Positive staining was detected usingthe Vectastain Vector Red reagent (Vector Laboratories, Burlingame, Calif).Control sections included those without any primary antibody treatment.

REVERSE TRANSCRIPTASE–POLYMERASE CHAIN REACTION TECHNIQUE

The levels of uPAR messenger RNAs (mRNAs) in choroidal tissues weredetermined by reverse transcriptase–polymerase chain reaction (RT-PCR)analysis. Total RNA was extracted from tissues treated with or without laserand used to generate first-strand complementary DNA using Superscript reversetranscriptase (Gibco, Grand Island, NY). The relative level of uPAR mRNA wasstandardized to a coamplified invariant mRNA (18S ribosomal RNA) using standardPCR protocols. Ten microliters of each PCR was examined by agarose gel electrophoresisand ethidium bromide staining.

EXPRESSION OF uPAR DURING CNV

To determine the role of the uPA-uPAR system in CNV, we examined RPE-choroidcomplexes in the mouse model of CNV by RT-PCR analysis 14 days after lasertreatment.1 The presence of uPAR mRNA was analyzedin the RPE-choroidal tissues on days 5, 10, and 14 following laser inductionof CNV. The uPAR mRNA expression was detectable at all time points comparedwith age-matched control animals without laser treatment, which showed littleto no uPAR mRNA expression in the RPE-choroidal tissues (Figure 1).

Place holder to copy figure label and caption
Figure 1.

Expression of urokinase plasminogenactivator receptor (uPAR) during development of choroidal neovascularization.Agarose gel demonstrating the expression of uPAR (lanes 1-7) and 18S RNA (lanes8-14) in the retinal pigment epithelium–choroid complexes of mice. Age-matchedmice without laser treatment (lanes 1, 2, 8, and 9) show a low level of uPARexpression compared with mice 5 days (lanes 3, 4, 10, and 11), 10 days (lanes5, 6, 12, and 13), or 14 days (lanes 7 and 14) after laser treatment. M indicatesDNA ladder.

Graphic Jump Location
LOCALIZATION OF uPAR DURING CNV

To determine the cell types responsible for uPAR expression in thisCNV model, we then examined the tissues in control and experimental animalsby immunocytochemical analysis. The uPAR was localized to the endotheliallining of blood vessels within the fibrovascular tissue (Figure 2). Adjacent sections stained with an endothelium-specificantibody to CD31 confirmed that the staining of uPAR was limited to the endothelialcells within this fibrovascular complex of the laser burn area. Control sectionswithout the primary antibody treatment did not show any uPAR staining of thelaser burn area.

Place holder to copy figure label and caption
Figure 2.

Urokinase plasminogen activator receptor(uPAR) localization to vascular endothelial cells of new vessels. Immunocytochemicalanalysis of representative sections from the subretinal region of mice 14days after formation of laser-induced choroidal neovascularization. A, Sectionincubated with anti-mouse uPAR shows staining (pink color) of a neovascularcomplex in the subretinal space (arrowheads). B, An adjacent section incubatedwith an antibody to the endothelial cell marker CD31. The same cells thatexpress uPAR also express CD31 (arrowheads). C, An adjacent section incubatedwithout primary antibodies. The same vascular profile is indicated by thearrowheads.

Graphic Jump Location
EFFECT OF Å6 ON CNV

The ability to inhibit the development of CNV by disruption of the uPA-uPARsystem was investigated by treating animals with increasing concentrationsof the Å6 peptide. The Å6 peptide, or PBS, was given intraperitoneallyaccording to the protocol described herein. The area within the burn occupiedby new disorganized vessels was obvious microscopically and appeared to decreasewith Å6 treatment (Figure 3).At the highest dose of Å6, the burn area appeared to be completely devoidof new vessels. However, as seen at higher magnification (Figure 3E), there were small numbers of fluorescein isothiocyanate–conjugateddextran-labeled vessels present.

Place holder to copy figure label and caption
Figure 3.

Å6 treatment prevents neovascularizationin the laser-induced model of choroidal neovascularization (CNV). Representativeimages of retinal pigment epithelium–choroid whole mounts infused withfluorescein isothiocyanate–conjugated dextran 14 days after laser inductionof CNV. Images are from mice treated with phosphate-buffered saline or Å6peptide dosed at differing frequencies. The red circle roughly outlines thearea of the laser burn. The fluorescence in the center of the burn area demonstratesthe extent of the new vessel formation under the retina. The surrounding fluorescencerepresents the normal choroidal vasculature. A, Mouse treated with phosphate-bufferedsaline twice a day for 14 days. B, Mouse treated with the Å6 peptide,100 mg/kg twice a day once a week. C, Mouse treated with Å6, 100 mg/kgtwice a day every third day. D, Mouse treated with Å6, 100 mg/kg twicea day every day. E, A higher magnification image of the central region insection D. Only a few fluorescein isothiocyanate–conjugated dextran-labeledblood vessels could be seen in this area.

Graphic Jump Location

Quantitation and statistical analysis of the extent of CNV in wholemounts of RPE-choroid complexes as determined by the area of fluorescein isothiocyanate–conjugateddextran-labeled vessels following treatment indicated that there was significantinhibition of CNV in this animal model (Figure4). The effect of Å6 on inhibition of CNV development wasfound to be dependent on the frequency at which Å6 was given. The inhibitionwas minimal and nonsignificant with the less frequent doses of 100 mg/kg onceper week (P = .23). When Å6 was givenat 100 mg/kg every third day, a small but significant decrease was seen inthe extent of neovascularization (37%, P = .02).The most frequent dose of 100 mg/kg given every day produced the most significantdegree of inhibition (95%, P<.001) (Figure 4). None of the treated mice exhibited any apparent toxiceffects to the peptide during the 2-week treatment period. There were no apparenttissue toxic effects as judged by histologic analysis of the retina and choroid(not shown). In addition, there was no evidence of tissue destruction or abnormalityin the choroidal vessels outside the burn areas.

Place holder to copy figure label and caption
Figure 4.

Quantitation of the extent of choroidalneovascularization for each treatment group. Values are the mean of multiplelaser spots from at least 5 different mice in each treatment group (errorbars indicate SEM). Areas of fluorescein isothiocyanate conjugated dextranwithin each laser burn area were measured using the MetaMorph image analysissoftware. PBS (A) indicates mice treated with phosphate-buffered saline twicea day for 14 days (5 mice, 19 laser spots analyzed); Å6 (B), mice treatedwith the Å6 peptide, 100 mg/kg twice a day once a week (5 mice, 20 laserspots analyzed); Å6 (C), mice treated with Å6, 100 mg/kg twicea day every third day (5 mice, 17 laser spots analyzed); Å6 (D), micetreated with Å6, 100 mg/kg twice a day every day (5 mice, 17 laser spotsanalyzed). Asterisk indicates significantly less than PBS (A): Å6 (C)(P = .02) and Å6 (D) (P = .001).

Graphic Jump Location

Extracellular proteinases facilitate cell migration during angiogenesisby regulating cell-matrix interactions through both proteolytic and nonproteolyticmechanisms. One of these proteinases, uPA, has been implicated in the regulationof the formation of new vessels during angiogenesis and has been reportedto play an important role in tumor progression and metastasis.3,13,14 Theactive form of urokinase converts plasminogen to plasmin, which can furtherdegrade fibrin and a variety of extracellular matrix components.15 Theproteolytic activity of uPA is physiologically regulated by plasminogen activatorinhibitor 1 (PAI-1), a member of the serine proteinase inhibitor (SERPIN)family. Urokinase binds with high affinity to a cell surface receptor, uPAR,present on many cell types, including endothelial cells. The interactionsof uPA, uPAR, and PAI-1 can affect cell motility by localizing proteolyticactivity to the cell surface and regulating the activity of specific cell-matrixadhesion receptors.5,16

Results from the present study demonstrate that uPAR is significantlyup-regulated during the development of laser-induced CNV in the mouse. Thelaser-induced CNV model relies on the production of a focal injury to theBruch membrane to induce the formation of new vessels. It is conceivable thatinflammatory cells initially occupy the site of the laser burn and subsequentlyrelease cytokines and growth factors, causing an up-regulation of uPAR andextracellular proteinases by the activated endothelial cells of the surroundingchoroid. The uPAR was localized by immunocytochemical analysis specificallyto the endothelial cells within the fibrovascular area of the laser burn.The surrounding normal choroidal vessels in contrast did not demonstrate anysignificant uPAR expression. This is consistent with the fact that uPA anduPAR are not expressed by quiescent endothelium and are expressed only duringactive angiogenesis.3

Recently, the mRNAs of uPA and uPAR have been detected in choroidalneovascular membranes from patients with ARMD and animals with experimentalCNV.11 Hypoxia, a known major stimulus forangiogenesis, can increase uPAR expression in endothelial cells,17 anduPAR is up-regulated during retinal angiogenesis.8 Recentevidence indicates that there may also be a possible role of inflammatorycells in the pathogenesis of retinal neovascularization.18,19 Thesecells may have a role in regulating uPAR expression via cytokine production.However, it is not clear whether hypoxia plays any role in the formation ofCNV.20 It is possible that inflammatory cytokinesand growth factors such as vascular endothelial growth factor may be responsiblefor the increased expression of uPAR in the endothelial cells during the developmentof CNV.

In the present study, we demonstrated that by inhibiting the uPA-uPARsystem with the Å6 peptide CNV can be significantly inhibited in a dosescheduling–dependent manner. Å6, an 8–amino acid peptidefrom the connecting region of urokinase (amino acids 136-143), inhibits theuPA-uPAR interaction in a noncompetitive manner.9 Thispeptide has been shown to inhibit tumor growth and lymph node metastasis inbreast cancer and glioblastoma models without any direct cytotoxic effects.9,10 The antiangiogenic activity of theÅ6 peptide in the tumor models has been associated with a significantdecrease in the density of blood vessels in these tissues.9,21 Amechanism for this inhibition of new vessel formation was suggested to bedue to a decrease in transforming growth factor β activity and expressionof the vascular endothelial growth factor receptor Flk-1 as a direct or indirectresult of the inhibition of the uPA-uPAR system.21 Alternatively,work by Czekay et al5 has demonstrated therequirement for uPA-uPAR interactions in the PAI-1–mediated recyclingof uPAR and associated integrins that facilitates cell detachment from componentsof the extracellular matrix. Inhibition of the uPA-uPAR system by Å6might therefore be expected to disrupt this recycling process, causing increasedcell-matrix adhesion and thus rendering the cells immotile.

Similar inhibition of retinal neovascularization with the Å6 peptidein the animal model of ischemia-induced retinopathy has recently been shownin our laboratory.8 These data and the resultsfrom the present study together suggest that the uPA-uPAR system may be apotential therapeutic target for many types of abnormal ocular angiogenesis.The effect is independent of the initiating stimuli, since both CNV (inflammationor injury) and retinal neovascularization (hypoxia or ischemia) respond equallywell to Å6 treatment.

The current treatments for CNV, including conventional laser therapyand photodynamic therapy, are not optimal and have limitations. The conventionallaser therapy results in scotoma, severe vision loss, and recurrences in 50%of the cases. The photodynamic treatment does not cause permanent regressionof new vessels, since the vessels almost always grow back. This procedurerequires repeated treatments to close the new vessels,22 andthere is also collateral damage to the surrounding retina, choroid, and RPEcells.23 Numerous attempts have been made toinhibit ocular neovascularization using pharmacologic approaches. These approachesinclude inhibition of growth factor binding, integrin function, and proteinaseactivity.6,8,2430 Successfuland effective antiangiogenic therapies may be useful as an alternative oran adjunct to the current laser treatment. The effectiveness of the Å6peptide in inhibiting CNV in the mouse without any adverse effects suggeststhat inhibition of the uPA-uPAR system with this peptide may be a useful alternativetherapy in the management of CNV.

Correspondence: Arup Das, MD,PhD, Department of Surgery, University of New Mexico School of Medicine, 2211Lomas Blvd NE, Albuquerque, NM 87131 (adas@unm.edu).

Submitted for Publication: July 31, 2003; finalrevision received April 29, 2004; accepted April 29, 2004.

Funding/Support: The study was supported inpart by grant RO1-EY12604-05 (Dr Das) from the National Institutes of Health,Bethesda, Md.

Financial Disclosure: Dr Jones has a financialinterest in the drug and is affiliated with Angstrom Pharmaceuticals.

Previous Presentation: This study was presentedat the Annual Meeting of the Association for Research in Vision and Ophthalmology;May 7, 2003; Fort Lauderdale, Fla.

Tobe  TOrtega  SLuna  JD  et al.  Targeted disruption of the FGF2 gene does not prevent choroidal neovascularizationin a murine model. Am J Pathol 1998;1531641- 1646
PubMed
Pepper  MS Extracellular proteolysis and angiogenesis. Thromb Haemost 2001;86346- 355
PubMed
Pepper  MS Role of the matrix metalloproteinase and plasminogen activator-plasminsystems in angiogenesis. Arterioscler Thromb Vasc Biol 2001;211104- 1117
PubMed
Rabbani  SAMazar  AP The role of plasminogen activator system in angiogenesis and metastasis. Surg Oncol Clin N Am 2001;10393- 415
PubMed
Czekay  RPAertgeerts  KCurriden  SALoskutoff  DJ Plasminogen activator inhibitor-1 detaches cells from extracellularmatrices by inactivating integrins. J Cell Biol 2003;160781- 791
PubMed
Das  AMcLamore  ASong  WMcGuire  PG Retinal neovascularization is suppressed with a MMP inhibitor. Arch Ophthalmol 1999;117498- 503
PubMed
Das  AMcGuire  PGEriqat  C  et al.  Human neovascular membranes contain high levels of urokinase and metalloproteinaseenzymes. Invest Ophthalmol Vis Sci 1999;40809- 813
PubMed
McGuire  PGJones  TWTalarico  NWarren  EDas  A The urokinase/urokinase receptor system in retinal neovascularization:inhibition by Å6 suggests a new therapeutic target. Invest Ophthalmol Vis Sci 2003;442736- 2742
PubMed
Guo  YHigazi  AAArakelian  A  et al.  A peptide derived from the nonreceptor binding region of urokinaseplasminogen activator (uPA) inhibits tumor progression and angiogenesis andinduces tumor cell death in vivo. FASEB J 2000;141400- 1410
PubMed
Mishima  KMazar  AGown  A  et al.  A peptide derived from the nonreceptor region of urokinase plasminogenactivator inhibits glioblastoma growth and angiogenesis in vivo in combinationwith cisplatin. Proc Natl Acad Sci U S A 2000;978484- 8489
PubMed
Rakic  JMLambert  VMunaut  C  et al.  Mice without uPA, tPA, or plasminogen genes are resistant to experimentalchoroidal neovascularization. Invest Ophthalmol Vis Sci 2003;441732- 1739
PubMed
Edelman  JLCastro  MR Quantitative image analysis of laser-induced choroidal neovascularizationin rat. Exp Eye Res 2000;71523- 533
PubMed
Mignatti  PRifkin  D Biology and biochemistry of proteinases in tumor invasion. Physiol Rev 1993;73161- 195
PubMed
Andreasen  PAEgelund  RPetersen  HH The plasminogen activation system in tumor growth, invasion, and metastasis. Cell Mol Life Sci 2000;5725- 40
PubMed
Blasi  FVasalli  JDDano  K Urokinase type plasminogen activator proenzyme, receptors and inhibitors. J Cell Biol 1987;104801- 804
PubMed
Blasi  FCarmeliet  P uPAR: a versatile signaling orchestrator. Nat Rev Mol Cell Biol 2002;3932- 943
PubMed
Kroon  MEKoolwijk  Pvan der Vecht  Bvan Hinsbergh  VWM Urokinase receptor expression on human microvascular endothelial cellsis increased by hypoxia: implications for capillary-tube formation in a fibrinmatrix. Blood 2000;962775- 2783
PubMed
Ishida  SUsui  TYamashiro  K  et al.  VEGF(164) mediated inflammation is required for pathological, but notphysiological, ischemia-induced retinal neovascularization. J Exp Med 2003;198483- 489
PubMed
Ishida  SYamashiro  KUsui  T  et al.  Leukocytes mediate retinal vascular remodeling during development andvaso-obliteration in disease. Nat Med 2003;9781- 788
Campochiaro  PA Retinal and choroidal neovascularization. J Cell Physiol 2000;184301- 310
PubMed
Guo  YMazar  APLebrum  JJRabbani  SA An antiangiogenic urokinase-derived peptide combined with tamoxifendecreases tumor growth and metastasis in a syngeneic model of breast cancer. Cancer Res 2002;624678- 4684
PubMed
Treatment of Age-related Macular Degeneration with Photodynamic Therapy(TAP) Study Group, Photodynamic therapy of subfoveal choroidal neovascularization in age-relatedmacular degeneration with verteporfin: one-year results of 2 randomized clinicaltrials–TAP report. Arch Ophthalmol 1999;1171329- 1345[erratum appears in Arch Ophthalmol. 2000;118:488].
PubMed
Kramer  MMiller  JWMichaud  N  et al.  Liposomal benzoporphyrin derivative verteporfin photodynamic therapy:selective treatment of choroidal neovascularization in monkeys. Ophthalmology 1996;103427- 438
PubMed
Aiello  LPPierce  EAFoley  ED  et al.  Suppression of retinal neovascularization in vivo by inhibition ofvascular endothelial growth factor using soluble VEGF receptor chimeric proteins. Proc Natl Acad Sci U S A 1995;9210457- 10461
PubMed
Adamis  APShima  DTTolentino  MJ  et al.  Inhibition of vascular endothelial growth factor prevents ischemia-associatediris neovascularization in a non-human primate. Arch Ophthalmol 1996;11466- 71
PubMed
Ozaki  HSeo  MSOzaki  K  et al.  Blockade of vascular endothelial cell growth factor receptor signalingis sufficient to completely prevent retinal neovascularization. Am J Pathol 2000;156697- 707
PubMed
Saishin  YSaishin  YTakahashi  K  et al.  VEGF-TRAP(R1R2) suppresses choroidal neovascularization and VEGF-inducedbreakdown of the blood-retinal barrier. J Cell Physiol 2003;195241- 248
PubMed
Smith  LEKopchick  JJChen  W  et al.  Essential role of growth hormone in ischemia-induced retinal neovascularization. Science 1997;2761706- 1709
PubMed
Friedlander  MThesfeld  CLSugitta  M  et al.  Involvement of integrins avb3 and avb5 in ocular neovascular diseases. Proc Natl Acad Sci U S A 1996;939764- 9769
PubMed
Chavakis  ERiecke  BLin  J  et al.  Kinetics of integrin expression in the mouse model of proliferativeretinopathy and success of secondary intervention with cyclic RGD peptides. Diabetologia 2002;45262- 267
PubMed

Figures

Place holder to copy figure label and caption
Figure 1.

Expression of urokinase plasminogenactivator receptor (uPAR) during development of choroidal neovascularization.Agarose gel demonstrating the expression of uPAR (lanes 1-7) and 18S RNA (lanes8-14) in the retinal pigment epithelium–choroid complexes of mice. Age-matchedmice without laser treatment (lanes 1, 2, 8, and 9) show a low level of uPARexpression compared with mice 5 days (lanes 3, 4, 10, and 11), 10 days (lanes5, 6, 12, and 13), or 14 days (lanes 7 and 14) after laser treatment. M indicatesDNA ladder.

Graphic Jump Location
Place holder to copy figure label and caption
Figure 2.

Urokinase plasminogen activator receptor(uPAR) localization to vascular endothelial cells of new vessels. Immunocytochemicalanalysis of representative sections from the subretinal region of mice 14days after formation of laser-induced choroidal neovascularization. A, Sectionincubated with anti-mouse uPAR shows staining (pink color) of a neovascularcomplex in the subretinal space (arrowheads). B, An adjacent section incubatedwith an antibody to the endothelial cell marker CD31. The same cells thatexpress uPAR also express CD31 (arrowheads). C, An adjacent section incubatedwithout primary antibodies. The same vascular profile is indicated by thearrowheads.

Graphic Jump Location
Place holder to copy figure label and caption
Figure 3.

Å6 treatment prevents neovascularizationin the laser-induced model of choroidal neovascularization (CNV). Representativeimages of retinal pigment epithelium–choroid whole mounts infused withfluorescein isothiocyanate–conjugated dextran 14 days after laser inductionof CNV. Images are from mice treated with phosphate-buffered saline or Å6peptide dosed at differing frequencies. The red circle roughly outlines thearea of the laser burn. The fluorescence in the center of the burn area demonstratesthe extent of the new vessel formation under the retina. The surrounding fluorescencerepresents the normal choroidal vasculature. A, Mouse treated with phosphate-bufferedsaline twice a day for 14 days. B, Mouse treated with the Å6 peptide,100 mg/kg twice a day once a week. C, Mouse treated with Å6, 100 mg/kgtwice a day every third day. D, Mouse treated with Å6, 100 mg/kg twicea day every day. E, A higher magnification image of the central region insection D. Only a few fluorescein isothiocyanate–conjugated dextran-labeledblood vessels could be seen in this area.

Graphic Jump Location
Place holder to copy figure label and caption
Figure 4.

Quantitation of the extent of choroidalneovascularization for each treatment group. Values are the mean of multiplelaser spots from at least 5 different mice in each treatment group (errorbars indicate SEM). Areas of fluorescein isothiocyanate conjugated dextranwithin each laser burn area were measured using the MetaMorph image analysissoftware. PBS (A) indicates mice treated with phosphate-buffered saline twicea day for 14 days (5 mice, 19 laser spots analyzed); Å6 (B), mice treatedwith the Å6 peptide, 100 mg/kg twice a day once a week (5 mice, 20 laserspots analyzed); Å6 (C), mice treated with Å6, 100 mg/kg twicea day every third day (5 mice, 17 laser spots analyzed); Å6 (D), micetreated with Å6, 100 mg/kg twice a day every day (5 mice, 17 laser spotsanalyzed). Asterisk indicates significantly less than PBS (A): Å6 (C)(P = .02) and Å6 (D) (P = .001).

Graphic Jump Location

Tables

References

Tobe  TOrtega  SLuna  JD  et al.  Targeted disruption of the FGF2 gene does not prevent choroidal neovascularizationin a murine model. Am J Pathol 1998;1531641- 1646
PubMed
Pepper  MS Extracellular proteolysis and angiogenesis. Thromb Haemost 2001;86346- 355
PubMed
Pepper  MS Role of the matrix metalloproteinase and plasminogen activator-plasminsystems in angiogenesis. Arterioscler Thromb Vasc Biol 2001;211104- 1117
PubMed
Rabbani  SAMazar  AP The role of plasminogen activator system in angiogenesis and metastasis. Surg Oncol Clin N Am 2001;10393- 415
PubMed
Czekay  RPAertgeerts  KCurriden  SALoskutoff  DJ Plasminogen activator inhibitor-1 detaches cells from extracellularmatrices by inactivating integrins. J Cell Biol 2003;160781- 791
PubMed
Das  AMcLamore  ASong  WMcGuire  PG Retinal neovascularization is suppressed with a MMP inhibitor. Arch Ophthalmol 1999;117498- 503
PubMed
Das  AMcGuire  PGEriqat  C  et al.  Human neovascular membranes contain high levels of urokinase and metalloproteinaseenzymes. Invest Ophthalmol Vis Sci 1999;40809- 813
PubMed
McGuire  PGJones  TWTalarico  NWarren  EDas  A The urokinase/urokinase receptor system in retinal neovascularization:inhibition by Å6 suggests a new therapeutic target. Invest Ophthalmol Vis Sci 2003;442736- 2742
PubMed
Guo  YHigazi  AAArakelian  A  et al.  A peptide derived from the nonreceptor binding region of urokinaseplasminogen activator (uPA) inhibits tumor progression and angiogenesis andinduces tumor cell death in vivo. FASEB J 2000;141400- 1410
PubMed
Mishima  KMazar  AGown  A  et al.  A peptide derived from the nonreceptor region of urokinase plasminogenactivator inhibits glioblastoma growth and angiogenesis in vivo in combinationwith cisplatin. Proc Natl Acad Sci U S A 2000;978484- 8489
PubMed
Rakic  JMLambert  VMunaut  C  et al.  Mice without uPA, tPA, or plasminogen genes are resistant to experimentalchoroidal neovascularization. Invest Ophthalmol Vis Sci 2003;441732- 1739
PubMed
Edelman  JLCastro  MR Quantitative image analysis of laser-induced choroidal neovascularizationin rat. Exp Eye Res 2000;71523- 533
PubMed
Mignatti  PRifkin  D Biology and biochemistry of proteinases in tumor invasion. Physiol Rev 1993;73161- 195
PubMed
Andreasen  PAEgelund  RPetersen  HH The plasminogen activation system in tumor growth, invasion, and metastasis. Cell Mol Life Sci 2000;5725- 40
PubMed
Blasi  FVasalli  JDDano  K Urokinase type plasminogen activator proenzyme, receptors and inhibitors. J Cell Biol 1987;104801- 804
PubMed
Blasi  FCarmeliet  P uPAR: a versatile signaling orchestrator. Nat Rev Mol Cell Biol 2002;3932- 943
PubMed
Kroon  MEKoolwijk  Pvan der Vecht  Bvan Hinsbergh  VWM Urokinase receptor expression on human microvascular endothelial cellsis increased by hypoxia: implications for capillary-tube formation in a fibrinmatrix. Blood 2000;962775- 2783
PubMed
Ishida  SUsui  TYamashiro  K  et al.  VEGF(164) mediated inflammation is required for pathological, but notphysiological, ischemia-induced retinal neovascularization. J Exp Med 2003;198483- 489
PubMed
Ishida  SYamashiro  KUsui  T  et al.  Leukocytes mediate retinal vascular remodeling during development andvaso-obliteration in disease. Nat Med 2003;9781- 788
Campochiaro  PA Retinal and choroidal neovascularization. J Cell Physiol 2000;184301- 310
PubMed
Guo  YMazar  APLebrum  JJRabbani  SA An antiangiogenic urokinase-derived peptide combined with tamoxifendecreases tumor growth and metastasis in a syngeneic model of breast cancer. Cancer Res 2002;624678- 4684
PubMed
Treatment of Age-related Macular Degeneration with Photodynamic Therapy(TAP) Study Group, Photodynamic therapy of subfoveal choroidal neovascularization in age-relatedmacular degeneration with verteporfin: one-year results of 2 randomized clinicaltrials–TAP report. Arch Ophthalmol 1999;1171329- 1345[erratum appears in Arch Ophthalmol. 2000;118:488].
PubMed
Kramer  MMiller  JWMichaud  N  et al.  Liposomal benzoporphyrin derivative verteporfin photodynamic therapy:selective treatment of choroidal neovascularization in monkeys. Ophthalmology 1996;103427- 438
PubMed
Aiello  LPPierce  EAFoley  ED  et al.  Suppression of retinal neovascularization in vivo by inhibition ofvascular endothelial growth factor using soluble VEGF receptor chimeric proteins. Proc Natl Acad Sci U S A 1995;9210457- 10461
PubMed
Adamis  APShima  DTTolentino  MJ  et al.  Inhibition of vascular endothelial growth factor prevents ischemia-associatediris neovascularization in a non-human primate. Arch Ophthalmol 1996;11466- 71
PubMed
Ozaki  HSeo  MSOzaki  K  et al.  Blockade of vascular endothelial cell growth factor receptor signalingis sufficient to completely prevent retinal neovascularization. Am J Pathol 2000;156697- 707
PubMed
Saishin  YSaishin  YTakahashi  K  et al.  VEGF-TRAP(R1R2) suppresses choroidal neovascularization and VEGF-inducedbreakdown of the blood-retinal barrier. J Cell Physiol 2003;195241- 248
PubMed
Smith  LEKopchick  JJChen  W  et al.  Essential role of growth hormone in ischemia-induced retinal neovascularization. Science 1997;2761706- 1709
PubMed
Friedlander  MThesfeld  CLSugitta  M  et al.  Involvement of integrins avb3 and avb5 in ocular neovascular diseases. Proc Natl Acad Sci U S A 1996;939764- 9769
PubMed
Chavakis  ERiecke  BLin  J  et al.  Kinetics of integrin expression in the mouse model of proliferativeretinopathy and success of secondary intervention with cyclic RGD peptides. Diabetologia 2002;45262- 267
PubMed

Correspondence

CME
Meets CME requirements for:
Browse CME for all U.S. States
Accreditation Information
The American Medical Association is accredited by the Accreditation Council for Continuing Medical Education to provide continuing medical education for physicians. The AMA designates this journal-based CME activity for a maximum of 1 AMA PRA Category 1 CreditTM per course. Physicians should claim only the credit commensurate with the extent of their participation in the activity. Physicians who complete the CME course and score at least 80% correct on the quiz are eligible for AMA PRA Category 1 CreditTM.
Note: You must get at least of the answers correct to pass this quiz.
You have not filled in all the answers to complete this quiz
The following questions were not answered:
Sorry, you have unsuccessfully completed this CME quiz with a score of
The following questions were not answered correctly:
Commitment to Change (optional):
Indicate what change(s) you will implement in your practice, if any, based on this CME course.
Your quiz results:
The filled radio buttons indicate your responses. The preferred responses are highlighted
For CME Course: A Proposed Model for Initial Assessment and Management of Acute Heart Failure Syndromes
Indicate what changes(s) you will implement in your practice, if any, based on this CME course.
NOTE:
Citing articles are presented as examples only. In non-demo SCM6 implementation, integration with CrossRef’s "Cited By" API will populate this tab (http://www.crossref.org/citedby.html).
Submit a Comment

Multimedia

Some tools below are only available to our subscribers or users with an online account.

Related Content

Customize your page view by dragging & repositioning the boxes below.

Articles Related By Topic
Related Topics