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

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.

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.

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.

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.

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.

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.

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).

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.

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.

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.

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 ,24 - 30} 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.